Issue 7 - Asian Journal of Research in Chemistry (AJRC)

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Asian Journal of
Research in Chemistry
ISSN 0974-4169 (Print)
ISSN 0974-4150 (online)
Volume 08, Issue 07, July, 2015
Abstracted in
CAB Abstracts,
Google Scholar,
EBSCO Publishing's Electronic Databases,
Indian Science Abstract.
Index Copernicus,
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Asian Journal of
ISSN 0974-4169(print) 0974-4150 (online)
www.ajrconline.org
EDITORIAL PANEL
Editor-in-Chief
Dr. Mrs. Monika S. Daharwal,
Raipur, Chhattisgarh, India
Past Editor
Dr. R. B. Saudagar,
Nashik, MS India
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Dr. U.S. Mahadeva Rao, Kuala Terengganu, Malaysia.
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Bhilai Chhattisgarh, India
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Dr.(Mrs.) Bharti Ahirwar, Bilaspur
Dr. Amit Roy, Raipur
Dr. D.K. Tripathi, Bhilai
Dr. S. J. Daharwal, Raipur
Dr. Vishal Jain, Raipur
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Dr. Ravindra Pandey, Raipur
Dr. Shekhar Verma, Raipur
Dr. S.B. Jaiswal,Vadodara
Dr. S. J. Daharwal, Raipur
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Dr. Dinesh Mishra, Ujjain
Mr. Pradeep Sahu, Raipur
Mr. Narendra Dewangan, Raipur
Dr. Manish Devgun, Karnal, Haryana
Girish Pai K., Manipal University, Manipal
Mrs. Manjusha Yeole, Nagpur
Dr. Rakesh Patel, Indore MP
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Asian Journal of
ISSN 0974-4169(print)
0974-4150 (online)
www.ajrconline.org
CONTENT
●
Comparative evaluation of marketed formulations of Metformin HCl. available in India
S. J. Daharwal, S Prakash Rao, Vijay Kumar Singh, Chandraprakash Dwivedi , Veena D. Singh…………………
●
441
Visible Spectroscopic Method for Estimation of Atenolol in Tablets
Raveendra Babu G., Sivasai Kiran B., Venkata Kumari M., Sambasiva Rao P., Madhuri P…………………………
445
Synthesis and Comparison of Phenol-Urea-Formaldehyde (PUF) Thermosetting Resin with Commercial
●
Synthetic Resins
Satish Kumar Sinha, D. P. Khali ……………………………………………………………………………………………..
449
Preparation and Characterization of Micro Crystalline Cellulose Fiber Reinforced Chitosan based
●
Polymer Composites.
Jeba Jeevitha R.S, Bella G.R, Dr. S. Avila Thanga Booshan………………………………………………………………
453
Synthesis and Biological Evaluation of Some 4-(5((1h-Benzo [D][1,2,3]Triazol-1-Yl)Methyl)-1,3,4●
Oxadiazol-2yl)-N-Benzylidenebenamine Derivative as a Anti-Microbial and Anti-Convulsant Agents
M.D. Dhanaraju, C. Gopi and V. Girija Sastry……………………………………………………………………………..
459
Development and Validation of Derivative Spectrophotometric Method for Simultaneous Estimation of
●
Lornoxicam and Eperisone in their Synthetic Mixture
Jawed Akhtar, Jatin Prajapati, ShamimAhmad, Mohammad Mujahid, Gamal Osman Elhassan…………………..
465
Quantitative planner chromatographic method development for sitagliptin phosphate monohydrate and
●
metformin hydrochloride in presence of their degradation product
Sanjay G. Walode, Avinash V. Kasture……………………………………………………………………………………….
472
Optimization of the spectrophotometric determination of Aqueous Cyanide: Application on Samira
●
(Niger) Gold Mine Groundwater Analysis
Hassane Adamou Hassane, Rabani Adamou, Maman Maazou Ahmed, Alassane Abdoulaye……………………….
●
●
481
Synthesis and Biochemical Investigation of (Thiazin, Oxadiazol, Thiadiazol )- Derivatives
Zinah Hussien Ali………………………………………………………………………………………………………............
493
Instruction to author. ………………………………………………………………………………………………………....
501
Asian Journal of
ISSN 0974-4169(print) 0974-4150 (online)
www.ajrconline.org
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Asian J. Research Chem. 8(7): July- 2015
ISSN
0974-4169 (Print)
0974-4150 (Online)
www.ajrconline.org
RESEARCH ARTICLE
Comparative evaluation of marketed formulations of Metformin HCl.
available in India
S. J. Daharwal1* , S Prakash Rao2, Vijay Kumar Singh2, Chandraprakash Dwivedi 2,
Veena D. Singh1
1
University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur-492010, Chhattisgarh, India.
2
Columbia Institute of Pharmacy, Tekari, Near Vidhan sabha Raipur, C.G. 493111, India
*Corresponding Author E-mail: [email protected]
ABSTRACT:
Metformin HCl is an oral Anti-diabetic drug belongs to the class of biguanide derivatives commonly used to
treat type 2 diabetes mellitus. The study was conducted to assess the comparative in-vitro quality control
parameters through the evaluation of mechanical strength, dissolution study in buffer solution, content and
weight uniformity between the commercially available conventional and modified (sustained release) tablets of
different brand of Metformin in India. It can be concluded that standard quality control parameters always should
be maintained not only for Metformin but also for all kinds of medicine for getting better drug products.
KEY WORDS: Metformin HCl, comparative, quality control parameters, evaluation.
INTRODUCTION:
Metformin HCl is a choice of drug to treated type 2
diabetes mellitus. This belongs to bigunide class of
drugs. Diabetes mellitus (or diabetes) is a chronic,
lifelong condition that affects your body's ability to use
the energy found in food. There are three major types of
diabetes: type 1 diabetes, type 2 diabetes, and gestational
diabetes. Metformin helps to control the amount of
glucose (sugar) in blood. It decreases the amount of
glucose absorbed from diet by suppressing glucose
production of hepatic gluconeogenesis. [1]
The type 2 diabetes patient has three times more rate of
gluconeogenesis compare to normal; treatment with
metformin reduces this by over one-third.[2]
furthermore, Metformin increases body's response to
insulin, a natural substance that controls the amount of
glucose in the blood.
Received on 10.06.2015
Accepted on 11.07.2015
Modified on 28.06.2015
© AJRC All right reserved
Asian J. Research Chem. 8(7): July- 2015; Page 441-444
DOI: 10.5958/0974-4150.2015.00070.X
Metformin comes as a liquid, a tablet, and an extendedrelease (long-acting) tablet. The regular tablet is usually
taken with meals two or three times a day. The extendedrelease tablet is usually taken once daily with the
evening meal. When used for type 2 diabetes, metformin
is often prescribed in combination with other drugs.
Several are available as fixed-dose combinations to
reduce pill burden and making administration simpler
and convenient. [3]
However, various bands of metformin tablets have been
available in the market due to the advantages behind
their regular usages. The clinical effectiveness exerted
by tablet formulation depends on availability of drug to
the body and drug must be present in the labeled amount
[4]. The main objective of tablet formulation is to deliver
the drug at certain and defined amount for producing
therapeutic effect [5]. The formulations can have a
noteworthy affect on the quality parameters such
mechanical strength, dissolution study in buffer solution,
content and weight uniformity etc. This also includes the
physiochemical properties of the active ingredients and
excipients as well as the procedures used during
manufacturing of formulations [6]. Moreover, quality
control parameters of tablet are useful tools for
maintaining consistency in batch-to-batch manufacturing
441
and it should be performed for every drug product. All of
these parameters are closely related to each other and
have effect on drug absorption, bioavailability etc. [7].
The aim of the study was to evaluate the comparative
quality control parameters between the tablets of six
different conventional and modified (Sustained release)
formulations because standard quality parameters are
essential for better quality of medicine.
EXPERIMENTAL:
percent deviation for all tablet brands was calculated by
using the mathematical equation [6].
Mechanical Strength
The mechanical strength of selected formulation was
measured by Hardness and friability test.
Hardness Test
Hardness indicates the capability of a tablet to withstand
mechanical shocks during handling in manufacturing,
packaging and shipping [10]. The acceptable range of
hardness or crushing strength of tablet is 4 to 7 kgf
(kilogram of force) [11]. During the study, hardness of
the tablet was determined using Monsanto hardness
tester for both of the formulations, six tablets of each
brand were taken and hardness of the tablets was
determined.
Design of study
The study were design to assess comparative in-vitro
quality control parameters between the commercially
available conventional and modified (sustained release)
tablets with
different brands of metformine
hydrochloride in India through the evaluation of
mechanical strength, dissolution study in buffer solution,
content and weight uniformity. The study was done by
performing various test procedures associated to the Friability Test
Friability test is essential to evaluate the physical
quality of formulations.
strength of tablets to withstand abrasion in packing,
handling and transporting. In the study, it was
Collection of Samples
To perform the study both conventional and modified determined using Roche friabilator. The value of
formulations of metformin of six different brands were friability was expressed in percentage (%). Ten tablets
purchased from the drug store of Raipur, India. All the for each brand were initially weighed and transferred
tablets of metformin were labeled to contain 500 mg of into chamber of the friabilator. The friabilator was
metfomin per tablet. The labeled shelf life of all of the operated at 25 rpm for 4 minutes (up to 100 revolutions).
tablets was three years from the date of manufacturing The tablets were weighed again and the percent (%)
and was taken for the evaluation before two years of the friability was then calculated by using following formula
[6]. Generally the considerable range of weight loss of
labeled expiry date.
conventional compressed tablet is less than 0.5 to 1%
[10].
Identification of Sample
After purchasing, all the brands were coded as A1, B1,
C1, D1 E1 and F1 for conventional tablets of six The percent friability was measured by using formula.
different manufacturers and A2, B2, C2, D2, E2 and F2 % Friability = W1-W2 /W1×100
for modified (sustained release) tablets of six different Where, W1= Weight of tablet before test
W2= Weight of tablet after test
manufacturers. Finally the coded samples were separated
as a pair of conventional and modifies and taken for
Drug Content
evaluation.
Twenty tablets were weighed and grind in a mortar with
pestle to get fine powder. Powder equivalent to the mass
Procedure for evaluation
The development and manufacture of pharmaceutical of one tablet was dissolved in distilled water and filtered
formulations involved various analytical methods and through a whatman filter paper. The filtrate was diluted
tests which are important for the evaluation, following with distilled water and measures the absorbance of the
quality control tests were performed for conventional resulting solution at 232nm in UV-Visible
spectrophotometer [9]. The content of Metformin HCl.
and modified formulations in the study.
was calculated using equation obtained from standard
Weight variation test
curve. (Y = 0.071x - 0.053., R² = 0.995)
The weight variation test is a valid method for
determining variation in the drug content [8]. The Dissolution Test
acceptable limit for the deviation of weight for tablets Dissolution test is carried out to determine drug release
having average weight of 250 mg or more should not pattern during a specific period of time [12]. Dissolution
exceed 5% [9]. Twenty tablets were selected from each test was performed using Dissolution Tester – USP
of the brand and weighed individually using electronic (Electrolab, TDT-08L Plus) for only conventional tablet.
balance. Their average weights were calculated. The 900 ml of phosphate buffer, pH 6.8 was used as
dissolution medium [9]. The process was done at a speed
442
of 50 rpm by maintaining temperature at 37±1ºC in each
test. 1.0 ml of sample was withdrawn from the
dissolution apparatus at a regular time intervals of 10
minutes, 20 mi8nute and 45 minutes. Further dilute up to
10 ml with water and measured the absorbance of the
resulting solution at 233 nm for metformin by using UV
spectrophotometer.
Data processing and analysis
Data processing and analysis after the completion of all
test procedures data for all the individual tablets were
recorded and separated on different sheets according to
the manufacturer. Finally data were analyzed by using
the above mentioned mathematical formula and MSExcel®, 2007.
RESULT AND DISCUSSION:
During the study, at first the weight variation which is
the key to controlling crushing strength and friability of
tablet was assessed [13]. The unofficial test stated that
all the samples of conventional formulation were coded
as A1, B1, C1, D1, E1,F1 and samples of modified
formulations were coded A2, B2, C2, D2, E2, F2 have
passed the weight variation uniformity test as specified
in the Indian/British Pharmacopoeia (not exceed 5%
deviation) [9]. Weight variation uniformity between two
groups of conventional and modified tablets was almost
with in limit 5% shown in table 1. Mechanical strength is
the second most important physical feature for assessing
tablet [13]. In the study, it was found that A1, B1, C1
D1, E1 and F1 brands of conventional and A2, B2, C2,
D2, E2 and F2 brands of modified group passed the test
of tablet crushing strength or hardness. The conventional
brands had acceptable crushing strength of between 6.0
kgf to 6.40 kgf. On the other hand, the modified tablet
brands had a crushing strength of between 3.8 kgf to 5.6
kgf shown in table 1. All the brands showed satisfactory
crushing strength.
In the friability test, all brands showed impressive
friability values. The friability values for conventional
tablet brands were ranged from 0.40 to 1 % whereas the
modified tablet brands showed 0.1 to 0.9% of friability.
In all formulations the percent (%) friability was less
than or near to 1% which ensures that all the tablets of
each brand of both formulations were mechanically
stable [10]. The results were shown in table 1.
Drug content was studied as important quality control
parameters which determine the uniformity of the
content of the different brands of formulations. All the
conventional brands showed 96.6 to 103% and modified
brands showed 96 to 103% of drug contents. All the
brands showed satisfactory results which were shown in
table 1.
Dissolution was another studied important quality
control parameters directly related to the absorption and
bioavailability of drug [14]. This study was performed
with only conventional formulation and which revealed
that drug release rate was better in all conventional
brands. After 45 minutes, the release rate of tablet of
conventional brands of metformin was 96 to 103% and
the results shown in table 2.
Table 1: Evaluation of different quality control parameters of conventional and modified Metformin HCl tablets.
Sample
Weight variation (%)
Hardness (kgf)
Friability (%)
Drug content (%)
(Tablet brands)
A1 Conventional
2.05
6 ± 0.42
0.45
101
B1 Conventional
1.6
6 ± 0.58
1
96.6
C1 Conventional
0.8
6.4 ± 0.76
0.8
103
D1 Conventional
0.4
6.29 ± 0.60
0.4
97
E1 Conventional
2.24
5.1 ± 0.72
7.24
98.9
F1 Conventional
0.48
6.32 ± 0.41
0.48
98
A2 Sustained Release
1.9
5.2 ± 1
5.9
103
B2 Sustained Release
1
4.2 ± 0.70
1
99
C2 Sustained Release
1
4.7 ± 0.80
1
96
D2 Sustained Release
0.4
3.8 ± 0.60
0.4
98
E2 Sustained Release
0.1
4.6 ± 0.75
0.1
96.6
F2 Sustained Release
0.9
5.6 ± 0.80
0.9
97.9
Table 2: Evaluation of dissolution profile of conventional Metformin HCl. tablets.
Sample (Tablet brands)
(%) Drug release after 10 min
A1 Conventional
32%
61%
B1 Conventional
28%
49%
C1 Conventional
30%
63%
D1 Conventional
28%
52%
E1 Conventional
35%
68%
F1 Conventional
24%
49%
443
102.1%
98%
100.5%
98.2%
103%
96.3%
CONCLUSION:
Metformin is a well established and proven oral Antidiabetic drug in the biguanide class for the treatment
of type-2 diabetes mellitus. The current pharma market
of India is flooded with various conventional and
modified preparations. With other combination and
single formulation, metformine is also now widely used
for the management of diabetis in the country.
Therapeutic response of any formulation depends on its
quality parameters. From the study it was identified that
mechanical strength, hardness and dissolution profile
during the test procedure of both conventional and
modified tablet brands complied the specification. It
should be strictly considered that an ideal tablet will
have sufficient hardness to maintain its mechanical
stability but not more. Because harder tablet can delay
disintegration time or alter dissolution profile. Finally, as
quality control parameters are related to one another
from initial step to pharmacological action of the drug, a
high-quality tablet either single or in combination should
meet all the standard quality parameter for getting its
desired therapeutic response.
12. Kishore, B.H., Venkareswararao, T., Sankar, K.R., Rao, B.S.
Studies on dissolution rate of paracetamol tablets by using
different polymers. Journal of Global Trends in Pharmaceutical
Sciences. 2011; 2(1): 1-10.
13. Tousey, M.D. Tablet pro: A tablet making training resource for
tablet making professionals. Techceuticals. 2011; 4(1):1-15.
14. Pabla, D., Akhlaghi, F., Zia, H. A comparative pH dissolution
profile study of selected commercial levothyroxine products
using inductively coupled plasma mass spectrometry. European
Journal of Pharmaceutics and Biopharmaceutics. 2009; 72: 105–
110.
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1.
Kirpichnikov D, McFarlane SI, Sowers JR. Metformin: an
update. Ann Intern Med. 2002; 137(1):25–33. doi:10.7326/00034819-137-1-200207020-00009.
2. Hundal R, Krssak M, Dufour S, Laurent D, Lebon V,
Chandramouli V, Inzucchi S, Schumann W, Petersen K, Landau
B, Shulman G. Mechanism by which metformin reduces glucose
production in type 2 diabetes. Diabetes. 2000;49(12):2063–9.
3. Bailey CJ, Day C. Fixed-dose single tablet antidiabetic
combinations. Diabetes Obes Metab. June 2009;11(6):527–33.
4. Jabeen, S., Ali, A., Hassan, F., Fatima, N. Studies on the effects
of cyclodextrin polymer as a tableting aid on some selected
analgesics. Pakistan Journal of Pharmacology. 2006; 23(1): 6771.
5. Islam, S.M.A., Islam, S., Shahriar, M., Dewan , I. Comparative in
vitro dissolution study of aceclofenac marketed tablets in two
different dissolution media by validated analytical method.
Journal of Applied Pharmaceutical Science. 2011;1(9): 87-92.
6. Kalakuntla, R., Veerlapati, U., Chepuri, M., Raparla, R. Effect of
various super disintegrants on hardness, disintegration and
dissolution of drug from dosage form. J. Adv. Sci. Res. 2010;
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superdisintegrants on formulation of taste masked fast
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Pharmaceutical Journal; 2012;1(4): 62-67.
8. Reddy, K.R, Mutalik, S., and Reddy, S. “ once-daily sustainedrelease matrix tablets of nicorandil: Formulation and invitro
evaluation”, AAps pharma sci. Tech. 2003; 4:1-9.
9. Indian Pharmacopeia, Fourth edition in 1996, P-470. British
Pharmacopoeia. (2005): Volume. 4, Appendix XII H A273,
Table: 2.9.5-1.
10. Banker, G.S. and Anderson, N.R. Tablets. In Lachman, L. and
Lieberman, H.A, The theory and practice of industrial pharmacy
CBS Publishers and Distributors Pvt. Ltd., India . 2009; 229-345.
11. Patel, R. P., Patel, M. H. Prajapati, B. G., and Baria, A. H.
“Formulation and evaluation of sustained release matrix tablet of
Tizanidine Hydrochloride by direct compression technique”, e –
Journal of Science and Technology (e-JST), pp. 69-81.
444
ISSN
0974-4169 (Print)
0974-4150 (Online)
www.ajrconline.org
RESEARCH ARTICLE
Visible Spectroscopic Method for Estimation of Atenolol in Tablets
Raveendra Babu G.1*, Sivasai Kiran B2., Venkata Kumari M2., Sambasiva Rao P3., Madhuri P4.
1
Department of Pharmaceutical Analysis, AKRG College of Pharmacy, Nallajrla-534112, A.P., India.
Department of Pharmaceutical Analysis, D.C.R.M. Pharmacy College, Inkollu-523167, A.P., India.
3
Department of Pharmaceutics, Vijaya College Pharmacy, Hayathnagar, Hyderabad, Telangana, India.
4
Department of Pharmaceutical Analysis, Vivekananda Group of Institutions, Batasingaram- 501511, Telengana,
India.
2
ABSTRACT:
A simple, accurate, cost effective and reproducible spectrophotometric method has been developed for the
estimation of atenolol in tablets. The method was based on the formation of colored chromogen (vanilline). The
λ-max of atenolol was found to be 650nm to both crude and marketed sample and is analyzed using the beerlamberts law. The percentage of recovery of atenolol ranged from (99.5 ± 0.16) in pharmaceutical dosage form.
The developed method was validated with respect to linearity, accuracy (recovery), precision and specificity.
Beers law was obeyed in the concentration range of 2-10μg/ml having line equation Y=0. 2071C - 0.0043 with a
correlation coefficient of 0.9999. The results of the analysis were validated statistically and by recovery study.
KEYWORDS: Atenolol, Visible Spectrophotometry, Validation, Beer’s law, Method.
INTRODUCTION:
Atenolol is (figure. 1) chemically (RS) -4-(2-hydroxy-3isopropylaminopropoxy) phenylacetamide. Atenolol is a
beta-adrenergic receptor antagonist, or a more
commonly known as a beta blocker. Atenolol is used to
treat angina, hypertension and acute myocardial
infarction, supraventricular tachycardia, ventricular
tachycardia and alcohol withdrawal symptoms1. Atenolol
was the main beta blocker identified as carrying a higher
risk of provoking type-2 diabetes2. The literature survey
reported that atenolol individually and combined with
other drugs by Spectrophotometry3-5, HPLC6-7, HPTLC89
and LC-MS10-11 methods for estimation of atenolol in
its pharmaceutical formulation. Thus the present study
was undertaken to develop and validate a simple,
sensitive, accurate, precise, and reproducible visible
methods for atenolol
Asian J. Research Chem. 8(7): July- 2015 ; Page 445-448
DOI: 10.5958/0974-4150.2015.00071.1
Figure No. 1 Structure of Atenolol
MATERIALS AND METHODS:
Instrument and materials:
The present work was carried out on ElicoSL164 UV
Visible spectrophotometer having double beam detector
configuration. Amlodipine besylate pure drug obtained
from Spectrum Labs, Hyderabad as gift sample with
99.99% w/w assay value and was used without further
purification. The absorption spectra of reference and test
solution were carried out in a 1 cm quartz cell over the
range of 400-800 nm. All chemicals of analytical grade
used as it is.
Preparation of standard stock solution:
The standard stock solution was prepared by dissolving
accurately weighed 100 mg of atenolol in methanol and
the volume was made up to 100 ml with methanol (Stock
solution-I, 1000 mcg/ml). 10 ml of solution took from
445
stock-I and then diluted to 100 ml with water (Stock
solution-II, 100 mcg/ml). 1ml of stock solution-II, 1 ml
of concentrated hydrochloride, 1ml of 1% NaNo2
solution, 1 ml of 0.1% vanilline in a10 ml volumetric
flask were added and diluted to 10 ml with distilled
water so that to produce the concentration 10 mcg/ml.
This method was done on an ice bath and maintain
temperature below at 80 C was transferred to a 10ml
volumetric flask and the final volume was diluted to 10
ml with water, so that to produce the concentration 10
mcg/ml. The absorbance of red chromogen obtained was
measured against respective blank solution in the visible
region of 400-800 NM, which shows maximum
absorbance at 650 nm.
get the solution of 100 mcg/ml. An aliquot of 1 ml of test
solution, 1 ml of concentrated hydrochloride, 1ml of 1%
NaNo2 solution, 1 ml of 0.1% β-napthol in a10 ml
volumetric flask were add and diluted to 10 ml with
distilled water so that to produce the concentration 10
mcg/ml. This method was done on an ice bath and
maintain temperature below at 80 C. The absorbance of
red chromogen obtained was measured against
respective blank solution in the visible region of 590-650
nm, which shows maximum absorbance at 650 nm.
Figure No 3- Calibration curve of atenolol (2-10mcg/ml).
Precision:
The precision of an analytical method is the degree of
agreement among individual test results when the
method is applied repeatedly to multiple samplings of
homogeneous samples. It provides an indication of
Preparation of calibration curve:
Aliquots of standard solutions of atenolol ranging from random error results and was expressed as coefficient of
0.2-1.0 ml (1 ml = 100 mg) was transferred into a series variation (CV).
of 10 ml volumetric flasks. The volume in each flask
was made up to 10 ml with distilled water and the Intra and inter-day precision:
absorbances were measured at 650 nm against solvent A variation of results within the same day (intra-day),
blank. The obtained absorbance values when plotted variation of results between days (inter-day) was
against the concentration of atenolol give the calibration analyzed. Intra-day precision was determined by
graph. The concentration of the unknown sample was analyzing atenolol for five times in the same day at
365nm. Inter-day precision was determined by analyzing
determined from the calibration graph.
the drug daily once for five days at 650 nm. The relative
standard deviation (RSD) and assay values obtained by
Preparation of sample solution:
20 tablets of one brand of atenolol were taken, and all two analysts were 0.16, 99.75 and 0.26, 100.00
the tablets were crushed to fine powder by using a pestle respectively (Table no. 4).
and mortar. Powder equivalent to 25 mg of atenolol was
weighed accurately and transferred into a 25 ml standard Accuracy (Recovery Test):
volumetric flask. The contents were dissolved in 40ml of Accuracy is the closeness of the test results obtained by
ethanol and make up to 100 ml of ethanol. Heating the the method to the true value. The recovery experiments
resulting solution to 600c and shake for 15 minutes and were performed by adding known amounts to the tablet.
cool and sonicated for five minutes. This solution was The recovery was performed at three levels, 50, 100and
filtered through 0.45 μm watchman filter paper. 10 ml of 150% of atenolol standard concentration. The recovery
the filtrate was diluted to 100 ml with distilled water to technique was performed to judge the accuracy of the
Figure No. 2 Determination of λ-max of Atenolol React with
Vanilline
446
proposed method. For this, known quantities of the
atenolol solution were mixed with definite amounts of
pre-analyzed formulations and the mixtures were
analyzed. The total amount of atenolol was determined
by using the proposed method and the amount of added
drug was calculated by the difference. The recovery
values for atenolol ranged from 99.97 ± 0.3969 (Table
no. 3) was studied by recovery experiments.
Table No.1 - Calibration curve parameter
S.No.
Concentration
Absorbance
(mg/ml)
± SD
1
2
0.410± 0.046
2
4
0.826± 0.053
3
6
1.230± 0.042
4
8
1.642± 0.064
5
10
2.080± 0.073
% Relative
standard deviation
1.4
1.8
1.6
1.5
1.1
Limit of Detection (LOD) and Limit of Quantification
(LOQ):
The LOD and LOQ of amlodipine besylate were
determined by using standard deviation of the response
and slope approach as defined in International
Conference on Harmonization (ICH) guidelines12.The
LOD and LOQ Was found to be as in table no.2.
Table No. 2 - Validation parameters
Sr.
Parameter
No
1
Absorption maxima (nm)
2
Linearity Range (mg/ml)
3
Standard Regression Equation
4
Correlation Coefficient (r2 )
5
Molar absorptivity
6
Accuracy (% Recovery ±SD)
7
Precision
Linearity:
The linearity of the response of the drug was verified at
2 to 40 _g/ml concentrations, but linearity was found to
be between 5-25 g/ml concentration. The calibration
graphs were obtained by plotting the absorbance versus
the concentration data and were treated by linear
regression analysis (Table no. 2). The equation of the
calibration curve for atenolol obtained Y = 0.0163C-0.00120, the calibration curve was found to be linear in
the precedent concentrations. The correlation coefficient
(r2) of determination was 0.9999.
8
Specificity
9
Sandell’s Sensitivity
(mg/cm2/0.001absorbance
unit)
LOD (mg/ml)
LOQ (mg/ml)
10
11
Table No. 3 - Determination of Accuracy by percentage recovery method
Ingredient
Tablet amount
Level of
Amount
(mg/ml)
addition (%)
added (mg)
Atenolol
10
50
2
10
100
4
10
150
6
Table No.4 - Determination of Precision
Sample number
1
2
3
4
5
6
Mean
RSD
Assay of Atenolol
Analyst-I (Intra-day precision)
99.71
99.83
99.84
99.51
99.61
100.10
99.75
0.16
Drug found
(mg/ml)
1.99
3.98
5.97
Result
650
2-10
Y=0.2071C - 0.0043
0.9999
0.468 X104
99.5± 0.16
99.75% (Intra-day
precision) and 100.0%
(Inter-day precision)
A 10 mg/ml solution of
candidate drug in water at
Visible detection of 650
nm will show an
absorbance value of
2.080 ± 0.073
0.0048
0.341
1.035
% Recovery
99.5
99.5
99.5
Average %
recovery
99.5± 0.16
As % labelled amount
Analyst-II (Inter-day precision)
99.79
99.85
99.78
99.93
100.23
100.42
100.0
0.26
ACKNOWLEDGEMENTS:
CONCLUSION:
From the results the method described in this paper for
the determination of Amlodipine besylate from tablet
formulation is simple, accurate, sensitive and
reproducible. The proposed method could be applied for
routine analysis in quality control laboratories.
We are thankful to Spectrum Labs at Hyderabad for
providing the gift sample of Atenolol. We would also
like to thank Dr. G. Vijay Kumar, Principle, A. K. R. G.
College of Pharmacy for providing all the facilities to
complete our work successfully.
447
REFERENCES:
1.
Agon P, Goethals P, Van HD and Kaufman JM. Permeability of
the blood brain barrier for atenolol by positron emission
tomography. J Pharm Pharmacol. 43(8); 1991: 597-600.
2. Carlberg B, Samuelsson O and Lindholm LH. Atenolol in
hypertension is it a wise choice. Lancet. 364(9446); 2004: 16849.
3. Lalitha G, Salomi P and Ravindra RK. Development of an
analytical method and its validation for the analysis of atenolol in
tablets dosage form by UV-Spectrophotometry. International
Journal of Pharmacy and Pharmaceutical Sciences. 5(3); 2013:
197-199.
4. Lalitha KV, Jyothi RK, Padma B. UV spectrophotometric method
development and validation of atenolol and losartan potassium by
q analysis. International Bulletin of Drug Research. 3(4); 2013:
54-62.
5. Dey S, Sarkar S, Malakar J and Ghosh A. Spectrophotometric
method for simultaneous of atenolol and atorvastatin in tablet
dosage forms, International Journal of Pharm Biomedicl
Research. 3(1); 2012: 40-43.
6. Vidhya BK and Sunil DR. Validated hplc method for
simultaneous quantitation of amlodipine besylate, atenolol and
aspirin in bulk drug and formulation. J P B M S. 17(9); 2012: 1-6.
7. Kavita J, Muralidharan S. Development and validation of new
method for atenolol, hydrochlorothiazide and losartan potassium
by RP-HPLC. International Journal of Chemtech Research. 2(2);
2010: 880-884.
8. Vidhya KB and Sunil RD. Validated hptlc method for
simultaneous estimation of atenolol and aspirin in bulk drug and
formulation. I S R N Analytical Chemistry. 2012, 10.5402/
609706.
9. Nikita DP, Anandkumari DC, Kreny EP. Development and
validation of hptlc method for simultaneous determination of
atenolol and losartan potassium in bulk and in pharmaceutical
dosage form. International Journal of Pharmacy and
Pharmaceutical Sciences. 5(2); 2012: 325-331.
10. Sridharan D, Thenmozhi A, Sundarananda SV. Bioanalytical
method development and validation of atenolol in human plasma
by LCMS. Asian Journal of Pharmaceutical and Clinical
Research, 3(2); 2010: 92-94.
11. Kallem RR, Mullangi R, Hotha KK, Spoorthy YN. Simultaneous
estimation of amlodipine and atenolol in human plasma a
sensitive LC-MS/MS method validation and its application to a
clinical pk study. Bioanalysis. 5(7); 2013: 827-37.
12. Text on Validation of Analytical Procedures Q2 (R1) in, I.C.H.
Harmonised Tripartite Guidelines; Nov. 1996.
448
ISSN
0974-4169 (Print)
0974-4150 (Online)
www.ajrconline.org
RESEARCH ARTICLE
Synthesis and Comparison of Phenol-Urea-Formaldehyde (PUF)
Thermosetting Resin with Commercial Synthetic Resins
Satish Kumar Sinha1*, D. P. Khali2
1
A.S.P.E.E. College of Horticulture and Forestry, Navsari Agricultural University, Navsari, Gujarat, India
2
Forest Products Division, Forest Research Institute, Dehradun, Uttarakhand, India
ABSTRACT:
Phenol-urea-formaldehyde (PUF) thermosetting resin was synthesized by reacting methylol phenol with urea
under acidic condition and its bonding strength was compared with commercial synthetic resins (PF resin, UF
resin and PUF resin made by mechanical blending of PF and UF resins). The results indicated that PUF resin
made by this process performed better bonding strength than commercial UF resin and mechanical blending of
PF and UF resins in both dry and wet conditions. Its bonding strength was found to be intermediate to UF and PF
resins.
KEYWORDS: PUF resin, PF resin, UF resin, Glue shear strength, Glue failure.
1. INTRODUCTION:
A thermosetting resin is an adhesive that hardens or sets
when heated and cannot be remolded1. Resol-type
phenol-formaldehyde (PF) and urea-formaldehyde (UF)
are the most common thermosetting resins used in the
wood composites industry. PF resins have a proved
excellent performance in producing exterior-grade wood
composites, whereas low-cost urea-formaldehyde (UF)
resins have performed well in interior applications.
Many efforts have been made by researchers to develop
a new type of resin due to potential scarcity and high
cost of phenolic resins rather than low cost, bad
durability and water resistance of UF resins2.
A common method to modify PF resin is the
introduction of urea component at the time of resin
preparation to synthesize phenol-urea-formaldehyde
(PUF) resin, in order to reduce the production cost of PF
resin and to introduce co-condensation reaction between
phenol and urea1-3. Several studies in the past have been
carried out to prepare low-cost and high-performance
PUF resins by changing the molar ratio of different
components of PUF resin2-6.
The objective of the present investigation was to
compare the bonding strength of PUF resin synthesized
by reacting methylol phenol with urea under acidic
condition with commercial synthetic resins like PF, UF
Most of the manufacturers of wood products generally and PUF resin synthesized by mechanical blending of PF
make cost effective phenol-urea-formaldehyde (PUF) and UF resin in dry and wet conditions.
resin by mechanical blending of resol-type PF resin and
UF resin, but it lacks effective co-condensation reaction 2. MATERIALS AND METHODS:
between phenol and urea during resin manufacturing2.
2.1 Materials:
Phenol (90%), formaldehyde aqueous solution (37%),
sodium hydroxide (98%), formic acid (85%), sulphuric
acid (98%) and commercial grade urea (46% N) were
used as raw materials for the preparation of resins. Sal
(Shorea robusta) veneers of 1.5 to 2 mm thickness were
used as raw materials for making plywood to check the
glue shear strength of the synthetic resins.
DOI: 10.5958/0974-4150.2015.00072.3
449
2.2 Methods:
Four types of synthetic resins viz. PF resin, UF resin,
PUF resin by mechanical blending of PF & UF resins
and PUF resin by reacting methylol phenol with urea
under acidic condition were synthesized in the
laboratory.
2.2.1 Synthesis of PF (Phenol-formaldehyde) resin:
The resol type PF resin was synthesized by taking the
ratio of formaldehyde to phenol (F/P) as 1.2/1. For this
procedure, 250 ml of phenol was charged into a round
bottom flask followed by 300 ml of formalin. Stirring
was continued throughout the reaction cycle. Required
amount of NaOH (5% of the Phenol) was dissolved in
twice amount of distilled water. The flask was kept in
the boiling water to maintain the temperature range from
85 to 950 C and the condenser was kept on the mouth of
the flask for 30 minutes. The reaction was stopped when
the viscosity of the resin reached to 100 cP and then the
resin was cooled at room temperature.
2.2.2 Synthesis of UF (Urea-formaldehyde) resin:
The UF resin was synthesized by taking the ratio of
formaldehyde to urea (F/U) as 2.5/1. For this process,
300 gm of formalin was charged into a round bottom
flask and pH of the formalin was raised to 7.2 to 7.5 by
the addition of NaOH (32-35% solution). Then, 120 gm
of urea was added and mixing was carried to get a clear
solution. The solution was allowed to boil and the
condenser was kept on the mouth of the flask for 20-30
minutes. The reaction was continued till the formation
of dimethylol urea. After cooling, the solution was made
acidic by addition of 10% of formic acid.
Approximately, 25gm of excess water from the resin
was removed by distillation till the viscosity of the resin
was reached up to 400-500 cP. The resin syrup in the
flask was again made alkaline by addition of 30-35%
solution of NaOH to store at room temperature.
2.2.3 Synthesis of PUF resin by mechanical blending
of PF and UF resin:
PUF resin was synthesized by mechanical blending of
PF and UF resins in the ratio of 1:1 prepared by the
above processes.
2.2.4 Synthesis of PUF resin by reacting methylol
phenol with urea under acidic condition:
For the manufacturing of PUF resin by reacting
methylol phenol with urea under acidic condition, the
ratio of F/P/U was taken as 2.2/1/1. For this method,
220 ml of formalin was charged in the round bottom
flask followed by 100 ml of phenol and stirring was
continued throughout the reaction cycle. 11gm of NaOH
(5% of the phenol) was dissolved in twice amount of
distilled water. The flask was kept in the boiling water
and the condenser was kept on the mouth of the flask.
The reaction was continued at 900C temperature for 30
minutes then it was cooled at room temperature and
reaction pH was brought between 3.5 to 4.5 by adding
10% conc. H2SO4. Afterward, 100 gm of urea was
dissolved into it and again kept at 900C for 25 minutes
till the viscosity was reached up to 100-200 cP. Finally,
the resin was made alkaline by adding 30 - 32% of
NaOH solution to store at room temperature.
2.2.5 Standard characteristics of synthetic resins:
Standard characteristic of synthetic resins like density,
flow time, water tolerance, ash content, pH and solid
content were evaluated according to the Bureau of
Indian Standards.
2.2.6 Testing procedure:
Glue shear strength of plywood was tested in a suitable
testing machine in dry and wet conditions by making 3ply plywood of Shorea robusta veneers glued with each
type of synthetic resin7-8. Totally six samples from each
plywood made from different types of synthetic resins
were taken to conduct the test in dry and wet conditions.
Average glue shear strength and average percentage of
glue failure in dry and wet conditions were recorded for
comparison.
Figure 1. Dimension of the test specimen of plywood
3. RESULTS AND DISCUSSION:
Four types of thermosetting resins were synthesized in
the laboratory according to the technical requirements
for the manufacturing of composite wood. The standard
characteristic features of these synthetic resins are shown
in table 1. The solid content of resins varied from 45 to
50 per cent which is prerequisite for composite wood
manufacturing. The ash content of resin is an important
parameter to evaluate the knife wear test, low ash
content shows low knife wear test and vice-versa9. The
ash content of resin should not be more than 4 per cent
of the oven dry weight of the sample10. In the present
study, the ash content of resins was less than 4 percent.
The pH value of synthetic resins was maintained above 7
at room temperature to slow down the polymerization
which increases the storage life of resins. Specific
gravity of resin is an indication of crystallinity,
molecular weight and the presence of voids in the
polymers11. In the present analysis, it was found that the
specific gravity of resins was more or less same at room
450
temperature. The water tolerance of a resin is an
indication of the miscibility of the resin with water. The
higher the water tolerance of the resin, the lower is the
molecular weight of the resin. A low molecular weight
resin has more polar end groups than a more condensed
resin12. It was found that the water tolerance of PF resin
was the highest followed by UF resin and PUF resin
synthesized by reacting methylol phenol with urea under
acidic condition. The PUF resin synthesized by
mechanical blending of PF and UF resins showed the
lowest water tolerance. It may due to low cocondensation reaction between phenol and urea. The
viscosity of synthetic resins was measured in terms of
flow time in B4 cup which showed satisfactory results
and it varied from 14 to 16 seconds.
Table 1. Standard characteristics of synthetic resins
Characteristics
Synthetic resins
PF
UF
Blending of
PF+UF
Solid content (%) at
50
48.4
48
102°C, 5 hrs.
Ash content (%) at
2.3
2.1
2.6
700°C, 5 hrs.
pH at 25°C
9.5
8.5
9.0
Specific gravity at
1.06
1.09
1.05
25°C
Water tolerance at
4.9
3.8
0.7 times
25°C
times
times
Flow time in B4
16
15
15
cup (sec.) at 25°C
PUF
45
room temperature2. In dry condition, the average glue
shear strength of plywood bonded with PUF resin
synthesized by reacting methylol phenol with urea under
acidic condition was slightly less than the UF bonded
plywood, however in wet condition after heating at
60±20C for 3hrs; the average glue shear strength was
found more than UF resin and PUF resin synthesized by
mechanical blending of PF and UF resins. Interestingly,
the glue failure of the PUF resin was less than UF resin
and mechanical blending of PF and UF resins in both dry
and wet conditions.
After comparing the bonding strength of synthetic resins,
it was found that PUF resin synthesized by reacting
methylol phenol with urea under acidic condition
performed better bonding strength than commercial UF
resin and mechanical blending of PF and UF resins in
both dry and wet conditions. It may be due to high cocondensation reaction between phenol and urea2. The
performance of PF resin was still better than PUF resin
made by reacting methylol phenol with urea under acidic
condition.
2.9
8.5
1.08
2.7
times
14
The comparison of average glue shear strength and glue
failure of plywood samples bonded with different types
of thermosetting resins in dry and wet conditions are
shown in figure 2 and 3 respectively. In dry condition,
average glue shear strength of plywood bonded with PF
resin was 138 kg and in wet condition, after 8 hours of
boiling the glue shear strength was 132 Kg. These values
were much larger than the average standard value of
BWR grade plywood13. The UF bonded plywood in dry
condition met the requirement as per IS: 303-1989 and
the average glue shear strength was 111Kg, whereas in
wet condition after 3hrs of boiling the average glue shear
strength was 73 Kg which was lower than the average
standard value of MR grade plywood.
In dry condition, the average glue shear strength of
plywood bonded with PUF resin synthesized by
mechanical blending of PF and UF resin was less than
UF bonded plywood, while in wet condition after 3 hrs
heating at 60±20C the glue shear strength was slightly
more than UF bonded plywood. The glue failure of PUF
resin synthesized by mechanical blending was more than
UF resin in both dry and wet conditions which may be
due to low bonding strength of PUF resin synthesized at
Figure 2. Comparison of average glue shear strength of test
specimens composed of Shorea robusta veneers and different types
of synthetic resin in dry and wet conditions
4. CONCLUSION:
The quality of PUF resin synthesized by reacting
methylol phenol with urea under acidic condition was
found to be intermediate to UF and PF resins and it
showed better bonding strength than mechanical
blending of PF and UF resins in both dry and wet
conditions. Since phenol is costlier than urea therefore,
by altering the molar ratio of phenol, urea and
formaldehyde the quality of PUF resin can be improved
to make it cost effective. It needs further study for better
development.
451
12. Fink JK. Reactive polymers fundamentals and applications: A
concise guide to industrial polymers. William Andrew, Inc., New
York.2005.
13. Bureau of Indian Standards IS 303. Plywood for general
purposes-specification.1989.
Figure 3. Comparison of average glue failure of test specimens
composed of Shorea robusta veneers and different types of
synthetic resin in dry and wet conditions
5. ACKNOWLEDGEMENTS:
The present paper is a part of M.Sc. research work of the
first author at the Forest Research Institute (Deemed)
University, Dehradun. Facilities provided by the FRI
(Deemed) University, Dehradun are gratefully
acknowledged for carrying out the research work.
6. REFERENCES:
1.
Sinha SK. Development of phenol-urea-formaldehyde (PUF)
resin, M.Sc. dissertation, FRI Deemed University, Dehradun.
2006: pp.69.
2. Tomita B and Hse CY. Phenol-urea-formaldehyde (PUF) cocondensed wood adhesives. International Journal of Adhesion
and Adhesives. 18; 1998: 69-79.
3. Fan DB, Li GY, Qin TF and Chu FX. Synthesis and structure
characterization of phenol-urea-formaldehyde resins in presence
of magnesium oxide as catalyst. Polymers. 6, 2014: 2221-2231.
4. Fan DB, Chu FX, Qin TF and LI JZ. Effect of synthesis
conditions on the structure and curing characteristics of high-urea
content PUF resin. Journal of Adhesion. 87; 2011: 1191-1203.
5. Pizzi A, Garcia R and Wang S. On the networking mechanisms
of additives-accelerated phenol-formaldehyde polycondensates.
Journal of Applied Polymer Science.66; 1997:255-266.
6. He GB and Riedl B. Phenol-urea-formaldehyde co-condensed
resol resins: Their synthesis, curing kinetics and network
properties. Journal of Applied Polymer Science. 41; 2003: 1929–
1938.
7. Bureau of Indian Standards IS 1734 (Part 4). Methods of test for
plywood: Part 4 Determination of glue shear strength.1983.
8. Bureau of Indian Standards IS 1734 (Part 6). Methods of test for
plywood: Part 6 Determination of water resistance.1983.
9. Sahoo SC, Sill A and Pandey CN. A natural additive approaches
to enhance the performance of formaldehyde based adhesive for
plywood manufacturing. International Journal of Innovative
Science and Modern Engineering. 3(1); 2014: 22-28.
10. Bureau of Indian Standards IS 1508. Specification for extenders
for use in synthetic resin adhesive (urea-formaldehyde) for
plywood.1972.
11. Anonymous. Synthetic resins technology handbook. Asia Pacific
Business Press Inc., Delhi. 2005
452
ISSN
0974-4169 (Print)
0974-4150 (Online)
www.ajrconline.org
RESEARCH ARTICLE
Preparation and Characterization of Micro Crystalline Cellulose Fiber
Reinforced Chitosan based Polymer Composites.
Jeba Jeevitha R.S1*, Bella G.R2, Dr. S. Avila Thanga Booshan3
1
Department of Chemistry, Nesamony Memorial Christian College Marthandam, TN India.
2
Department of Chemistry, Women’s Christian College Nagercoil, TN India.
ABSTRACT:
Plastics are widely used in society due to low cost, light weight and excellent performance which varies from
soft rubbers to fibers stronger and stiffer than steel. On the other hand their non- biodegradability creates serious
environmental problems. The utilization of packaging films from bio-based compounds has received so much
attention lately due to the fact that they are readily biodegradable. The two most abundant polymers on earth like
cellulose and chitosan have attracted increasing attention as a source of renewable energy and functional
materials. Rice straw fiber is subjected to different treatment along with chitosan to form composites. The film
samples have been characterized by FTIR and SEM, thermal properties by TGA and DTA. Reinforcement of
chitosan with prepared microcrystalline cellulose from rice straw enhances the moisture resistance and strength
of chitosan film.
KEYWORDS: Chitosan, Microcrystalline cellulose, Rice straw, Dewaxed rice straw, Polymer composites.
INTRODUCTION:
Plastic materials are indispensable in our lives but they
are a threat to our environment. In order to reduce the
impact on environment, researchers have turned to bio
polymers [1]. Cellulose is the most common organic
compound and biopolymer in earth [2]. Cellulose is a
crystalline polysaccharide consisting of D- glucose [3].
Cellulose has no taste, odorless, and insoluble in water
and in most organic solvents [4]. Cellulose appears to be
an ideal support material for many enzyme systems,
being both bio sourced and biologically compatible [5].
In recent years there has been an increasing trend
towards more efficient utilization of cellulosic agroindustrial residues. Wheat, corn, oats, coir, sisal and
other crops are also used to produce fibers and
investigating to reinforce in composite area [6]. Rice
straw is one of the most abundant low cost liginocellulosic materials [7]. It consists of 43.30% cellulose,
26.40% hemicelluloses, 16.29% lignin, 26% ash and
2.18% waxes [8].
Various methods are used to remove all the
hemicelluloses, lignin, ash and waxes to get fibers [9].
Fibers are widely used in polymeric materials to improve
mechanical properties [10]. The composite materials
have advantages like light weight, high specific stiffness
and strength, easy moldable to complex forms, easy
bondable, good dumping, low electrical conductivity,
thermal expansion and good fatigue resistance [11].
Chitosan is a second abundant polymer in the earth [12].
Chitosan exhibits unique physico chemical properties
like biocompatibility [13], biodegradability and film
forming ability [14] which have attracted scientific and
industrial interest in the fields such as biotechnology,
pharmaceuticals, biomedicine, packaging etc [15]. The
films are often brittle which limits their applications
[16]. In this present study cellulose fiber reinforced
chitosan based polymer composites were prepared and
characterized.
EXPERIMENTAL METHOD:
DOI: 10.5958/0974-4150.2015.00074.7
Materials: Rice straw was collected from field of
Kuzhithurai which is located in KK Dist, Tamil Nadu.
Chitosan was purchased from India sea foods, Cochin,
Kerala. Analytical grade ethanol, H2O2, acetic acid,
toluene from S.D fine chemical was used.
453
Preparation of cellulose from Rice-straw (RS)
The rice straw (RS) was dried in the sunlight and
pulverized with a blade grinder. The chopped rice straw
was sieved through 0.8mm size screen. About 20gm of
powdered rice straw was treated with 400ml of 5%
HNO3 and refluxed for 3 hrs. The treated slurry was
washed with distilled water to remove the dissolved
substances. The fibers obtained were again refluxed with
aqueous NaOH for 2 hrs. The black slurry obtained was
filtered and washed with distilled water till the solution
become neutral.10ml of 30% H2O2 was added and
maintained at 70 to 800 C for 30 minutes and washed
with distilled water. The fiber obtained was dried in oven
at 700C for 24 hrs.
Preparation of Chitosan Films
About 12g Chitosan was dissolved in 8% acetic acid by
constant stirring and the viscous solution formed was
filtered through sieve to remove undissolved impurities
and the solution was degassed. The prepared chitosan
solution was poured in a glass mould and dried at -40C.
H2O2 was used as curing agent. A transparent, uniform
and slightly yellowish film was obtained.
Preparation of Chitosan/ Microcrystalline Cellulose
composites (CS/MCC)
Chitosan was dissolved in 8% acetic acid and stirred for
20 min to homogenize. Different percentage (1%, 3%,
and 5%) of cellulose fibers were added and stirred for 2
hrs. Added 2 to 3 drops of H2O2 as curing agent .The
prepared composites were dried at -40 C.
Thermal studies
Thermal studies of the films have been performed at a
heating rate of 200c/min in nitrogen using universal V43A instrument (model SDTQ 600). Samples were heated
from room temperature to 7000C.
Tensile strength
Tensile strength was measured with an Instron 3365
instrument. Eight samples 1cm×10cm were cut from
each film. Tensile strength was calculated by dividing
the maximum force by cross sectional area.
RESULTS AND DISCUSSION:
FTIR
FTIR spectrum of chitosan film shows a strong peak at
3510-3350cm-1 due to O-H group and N-H stretching
vibration. A bending vibration of N-H is observed at
1575cm-1. A peak at 1654cm-1 is of amide group and the
one at 2922cm-1 is of C-H vibration. The broad peaks at
1028cm-1 and 1077cm-1 and the peak at1153.4 and
898.4cm-1 correspond to saccharide structure of chitosan.
Asymmetric stretching of c-o-c is obtained at 1153cm1
.The other peaks at 2922cm-1 and 2871cm-1 can be
assigned to C-H stretching and CH3 symmetric
deformation.
The FTIR spectrum of cellulose from rice straw shows a
strong band around 3423cm-1 due to stretching of O-H
group, a weak band at 2853.48cm-1 which attributed to
the C-H stretching, band at 1636cm-1due to the absorbed
water molecule, a band at 1425cm-1 which attributed to
the CH2 bending and a strong band at 1059cm-1 due to
the stretching of C-O-C of the pyranose skeletal.
FTIR Spectroscopy
Infra red spectra were taken in a Shimadzu -FT-IR
spectrometer by KBr pellet method.
In the chitosan- 5%cellulose (CS/5MCC) composite a
broad peak observed at 3438cm-1 is due to hydrogen
Scanning electron microscopy
bonded, O-H stretching at 3400cm-1 and the NH2
The surface morphology of prepared composites was asymmetric stretching, at 3200cm-1. The shift in the peak
studied using scanning electron microscopy. A narrow confirms the intermolecular hydrogen bonding and the
primary electron beam of the order of 10Kev in energy is strong polymerization of cellulose-rice straw. A peak at
scanned across the surface of the specimen and observed 2854cm-1 refers to aliphatic C-H stretching.
under different magnification.
(a)
454
(b)
(c)
Fig.1. FTIR of (a) Chitosan film (b) Prepared cellulose (c) Reinforced chitosan film (CS/5MCC)
The peak at 1639cm-1 is due to amide (1) band and H2O
in amorphous region. C-H and N-H vibration shows a
peak at 1425cm-1. Moreover, the N-H bending vibrations
(1575cm-1) were not observed in the spectra of CS/RSC
composite. The band shifted to higher frequency. When
two components are mixed, the physical blending verses
chemical interactions are affected by changes in the
characteristic spectra peaks.
Scanning Electron Microscope
The morphology of pure chitosan and reinforced
chitosan film (CS/5MCC) are reported. The pure
chitosan film reveals that the film is non porous and the
texture is without fibers. An agglomeration is seen in
chitosan film. The reinforced composite has branch like
structure. On adding cellulose fiber with chitosan the
porosity also increases. The cellulose fiber become
thicker and also the fibers in the inner layers are clearly
visible.
455
(a)
(b)
Fig.2. SEM of (a) CS film (b) CS/5MCCcomposite
were observed. The weight loss at 10-150°C is due to the
Thermal studies
TGA thermograms and char yields of chitosan film and moisture vaporization. The weight loss at 180-380°C is
cellulose reinforced chitosan composite were shown in due to the degradation of chitosan molecule.
figure.3. In the case of pure chitosan two weights losses
(a)
456
(b)
Fig.3 TGA curve for (a) Chitosan film (b) CS/5MCC film
In TGA curve of reinforced chitosan (CS/5MCC)
composite film the weight losses at 70 to 180°C is
responsible for loss of moisture contents. The composite
film (11.4%) has lower percentage of water content
compared to chitosan film (12.6%). The weight loss at
200-400°C is due to dehydration of the saccharide rings
and depolymerization. The second degradation
temperature of chitosan is 42.3% but the composite
shows higher percentage (50.5%). Addition of cellulose
to the chitosan improves the thermal stability. The char
yield of composites at about10.9%.
Tensile strength
The tensile strength of CS film and CS based composites
were tested in dry states and the results were shown in
the figure below. The result shows that there is a small
increase of tensile strength until 3% cellulose content
and there after gets doubled for 5% chitosan composites.
On comparing the pure film with composite film the
tensile of CS/5MCC (50.5%) was greater than the other
films. This result proved that the reinforced chitosan film
act as reinforcing fillers of chitosan matrix to improve
the tensile strength of chitosan film.
Tensile strength
60
50
40
30
Tensile strength
20
10
0
CS
CS/1MCC
CS/3MCC
CS/5MCC
Fig.4. Tensile strength of chitosan film and different percentage of micro crystalline cellulose fibers added to chitosan
457
CONCLUSION:
The main aim of the above study was to prepare and
characterize microcrystalline cellulose reinforced
chitosan based polymer composites. RS being the low
density fiber and treatments on RS improve the property.
The cost of obtained composites was expected to be
significantly reduced by adding a cheap lingo-cellulosic
waste product. The composite with chitosan can be
regarded as a successful light weight engineering
material. The prepared composites were confirmed by
FTIR, SEM, Thermal studies and Tensile. From the
studies the stability of composites compared to chitosan
alone was high.
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458
ISSN
0974-4169 (Print)
0974-4150 (Online)
www.ajrconline.org
RESEARCH ARTICLE
Synthesis and Biological Evaluation of Some
4-(5((1h-Benzo [D][1,2,3]Triazol-1-Yl)Methyl)-1,3,4-Oxadiazol-2yl)-NBenzylidenebenamine Derivative as a Anti-Microbial and Anti-Convulsant
Agents
M.D. Dhanaraju1*, C. Gopi2 and V. Girija Sastry2
1
GIET School of Pharmacy, Rajahmundry-533106.
2
Research Scholar, JNTUK-Kakinada
ABSTRACT:
Drug resistant towards available drug is rapid and major worldwide problem in the present scenario. There is a
need to prepare drugs which shows less resistant and high therapeutic profile. The purpose of this research was
to solve the above issues and to find the novel anti-microbial and anti-convulsant agent of Schiff bases from
benzotriazole and ethyl chloro acetate. All the synthesized compounds were prepared by a series of reactions;
which is initiated by the action of benztriazole and ethyl chloro acetate followed by hydrazine, phosphorus
oxychloride, para amino benzoic acid and different aromatic aldehydes. All the titled compounds were
characterized by IR, H-NMR, MASS spectroscopy and Elemental analysis. Most of the compounds were
exhibited excellent anti-microbial activity and anti-convulsant activity against the standard drug.
KEYWORDS: Benzotriazole, ethyl chloro acetate, Schiff base, anti-convulsant and anti-microbial activity.
INTRODUCTION:
Life threatening disease all are facing drug resistant
problem. There is a demand to prepare a new molecule
to acquire a better therapeutic efficacy, low toxicity and
least drug resistant. In the year of 1977 united state
environmental protection agency reported that
benzotriazole molecules are to be very less toxic and low
hazard to human beings. It is a class of heterocyclic
compound having a ring system of three nitrogen atoms
fused with one benzene ring shows wide range of
biological activities such as anti-bacterial[1,2,3,4], antitubercular[5], anti-inflammatory, anti-convulsant[6],
DNA cleavage[7], herbicidal[8] and anti-viral[9]
activity. Oxadiazole is another class of heterocyclic
compound plays an important role in the medicinal
chemistry for more than a two decades. It exhibits least
drug resistant when the nucleus has properly substituted
at second and fifth position.
Asian J. Research Chem. 8(7): July- 2015; Page 459-464
DOI: 10.5958/0974-4150.2015.00075.9
Oxadiazole containing drugs act as a anti-bacterial [1015], anti-fungal[16,17], anti-inflammatory and muscle
relaxant activity[18]. So these two nucleuses are called a
perfect combination for making least resistant drug as
well as less toxic drugs for deadly diseases. Our interest
in this work is to prepare a series of novel compounds
which bears two nucleuses by systematic method from
its raw material. All the synthesized compound
structures were confirmed by its spectral results and
elemental analysis. All those compounds its biological
activity has to be screened by standard procedure with
suitable reference molecules.
MATERIAL AND METHODS:
The chemicals and reagents used in this project were of
LR and analytical grade. We were bought it from varies
company like Merck, Sigma and Ranboxy and Alrich.
All the synthesized compounds sharp melting points
were identified by open capillary tube method. In this
project each intermediates as well as final product purity
were ascertained by TLC using silica gel as a stationary
phase and mobile phase consist of a mixture of n-hexane
and ethyl acetate. IR of synthesized compounds was
done with FT-IR spectrophotometer in the range of 400
459
to 4000 cm-1. 1H-NMR was recorded on NMR this project were in excellent condition before starts our
spectrometer and chemical shift (δ) was found in parts work.
per million. Mass spectra were recorded on Shimadzu
mass spectromerter. All those apparatus which is used in
SCHEME OF SYNTHESIS:
Ar
IVa
Chemical structure of varies benzotriazole Schiff base
derivative used in this study are mentioned as follows:
IVd
OH
IVb
IVe
OCH3
IVc
N(CH3)2
460
Cl
Method of Synthesis:
Step-1
Synthesis of Ethyl 2-(1H Benzo [1, 2, 3] triazole-1YL) acetate:
A mixture of benzotriazole (1mole), ethyl chloro acetate
(1 mole) and potassium carbonate 3g taken in a beaker
contained 60 ml of acetone. The content of the flask
were mixed magnetically at 45oC, and then set a reflex
for 6 hours. The resulting hot solution was poured into
beaker containing crushed ice, the solid was filtered at
pump, and then it is recrystalised from ethanol.
7.45(m,5H,Ar-H), δ7.46-7.61(m,4H,Ar-H), δ7.62-7.68
(m,2H,Ar-H),
δ7.98-7.99(q,2H,Ar-H),
δ4.8-4.9
(s,2H,CH2-H), δ8.4-8.42(s,1H,CH-H); Mass(m/z-380);
Element analysis(%)- C(69.01), H(4.21), N(22.04),
O(4.20)
4-(5((1H-BENZO[D][1,2,3]TRIAZOL-1YL)METHYL)-1,3,4-OXADIAZOL-2YL)-N-(4METHOXYBENZYLIDENE) BENAMINE (IVb):
Yield 67%, M.P-180 oC; IR (KBr) 3000 (Aromatic
proton streaching), 3200 (Aromatic proton bending),
1498 (C=N), 1324(C-N), 1523(Ar-NO2); NMR(CDCl3)δ6.47-6.68(d,2H,Ar-H), δ7.24-7.29(d,2H,Ar-H), δ7.457.56 (m,6H,Ar-H), δ7.93-7.99(q,2H,Ar-H), δ4.81STEP-2
SYNTHESIS OF (2H BEZO [1,2,3] TRIAZOLE-1- 4.83(s,2H,CH2-H), δ8.42-8.45(s,1H,CH-H); Mass(m/z410); Element analysis(%)- C(67.30), H(4.32), N(20.13),
YL ACETO HYDRAZINE):
Ethanolic solution of ethyl 2-(1H-benzo[d][1,2,3]triazol- O(7.43)
1-yl)acetate (step-I) 1 mole and hydrazine hydrate (NH2NH2.H20) 20ml were taken in a round bottom flask. The 4-(5((1H-BENZO[D][1,2,3]TRIAZOL-1content of the above flask stirred thoroughly and YL)METHYL)-1,3,4-OXADIAZOL-2YL)-N-(4refluxed it for 3 hours. The solid product obtained after (DIMETHYLAMINO) BENZYLIDENEBENAMINE
hot solution poured into cooled water. It is recrystallised (IVc):
Yield 75%, M.P-194oC; IR (KBr) 3253 (Aromatic
from ethanol.
proton N); NMR(CDCl3)- δ2.85-2.87(s,6H,NH3-H),
δ6.63-6.65(d,2H,Ar-H), δ7.32-7.35 (d,2H,Ar-H), δ7.45STEP-3
δ7.90-7.95(m,2H,Ar-H),
δ4.90SYNTHESIS OF 5-(BENZOTRIAZOLE 1-YL- 7.60(m,6H,Ar-H),
4.92(s,2H,CH
-H),
δ8.35-8.37(s,1H,CH-H);
Mass(m/zMETHYL)-2-PHENYL-1,3,4-OXADIAZOLE:
2
2-(1H-benzo[d][1,2,3]triazol-1-yl)acetohydrazide (Step 423); Element analysis(%)- C(68.02), H(5.14), N(22.97),
II) compound (1 mole) was refluxed with p-amino O(3.67).
benzoic acid (1 mole) for 6 hours in the presence of
phosphorous oxy chloride (POCl3). The hot mixture was 4-(5((1H-BENZO[D][1,2,3]TRIAZOL-1poured into ice-cold water and basified with sodium bi YL)METHYL)-1,3,4-OXADIAZOL-2YL)carbonate solution. The solid mass was separated out. PHENYLIMINO)METHYL)PHENOL (IVd):
o
Then it was filtered, washed with water and Yield 79%, M.P-174 C; IR (KBr) 3165 (Aromatic
proton stretching), 3214 (Aromatic proton bending),
recrystallised from ethanol.
1456 (C=N), 1259(C-N), 1268(Ar-OH bending);
NMR(CDCl3)δ5.20-5.22(s,1H,OH-H),
δ6.76STEP-4
GENERAL PROCEDURE FOR PREPARATION 6.80(d,2H,Ar-H), δ7.36-7.45 (d,2H,Ar-H), δ7.48δ7.91-7.96(m,2H,Ar-H),
δ8.42OF VARIES SCHIFF BASE BY USING 7.82(m,6H,Ar-H),
8.43(s,1H,CH-H), δ4.91-4.93(s,2H,CH2-H); Mass(m/zDIFFERENT AROMATIC ALDEHYDE:
A mixture of equal molar quantities of 4-(5-((1H- 396); Element analysis(%)- C(66.56), H(4.02), N(21.43),
O(8.65).
benzo[d][1,2,3]triazol-1-yl)methyl)-1,3,4-oxadiazol-2yl)benzenamine (step III) and different aromatic
aldehyde were taken in an round bottom flask. To this, 4-(5((1H-BENZO[D][1,2,3]TRIAZOL-1add 10 ml of ethanol and few drops of glacial acetic acid YL)METHYL)-1,3,4-OXADIAZOL-2YL)-N-(4and kept in water reflex for 5 hours. The hot mixture was CHLOROBENZYLIDENE)
poured into ice-cold water. The titled compounds were PHENYLIMINO)METHYL)PHENOL (IVe):
o
separated as a solid, filter it at the pumb, finally Yield 58%, M.P-210 C; IR (KBr) 3456 (Aromatic
recrystallized with DMF.
1476 (C=N), 1255(C-N), 750(chlorine); NMR(CDCl3)δ7.35-7.44 (t,4H,Ar-H), δ7.61-7.85(m,6H,Ar-H), δ7.974-(5((1H-BENZO[D][1,2,3]TRIAZOL-18.21(q,2H,Ar-H),
δ8.20-8.22(s,1H,CH-H),
δ4.87YL)METHYL)-1,3,4-OXADIAZOL-2YL)-N4.90(s,2H,CH2-H); Mass(m/z-414); Element analysis
BENZYLIDENEBENAMINE (IVa):
Yield 55%, M.P- 147oC; IR (KBr): 3945 (Aromatic (%)- C(63.60), H(3.42), Cl(8.14), N(20.19), O(3.51).
1520 (C=N), 1375(C-N); NMR (CDCl3)- δ7.41-
461
INVITRO ANTI-MICROBIAL EVALUATION:
PAPER DISC DIFFUSION METHOD:
Dish diffusion method [19] was used to find out the antimicrobal activity of the synthesized compounds from its
zone of inhibition. To find out the anti-microbial activity
of the synthesized drugs a sterilized (autoclaved at 1200C
for 30 min) culture medium (40-500C) was prepared and
poured in to the petridis to give a depth of 3-4 mm and
cool it for 15 minutes. The paper impregnated with the
synthesized compounds (IVa-e) (100µg/ml in dimethyl
formamide) and standard drugs (100µg/ml in dimethyl
formamide) was placed on solidified medium and
incubated at 37 for a minimum of 24 hours. In vitro
anti-microbial activities of IVa-e were found excellent
against varies micro organism such as E. coli, S. aureus,
B. subtilis and P. vulgaris, C. albicons and A. niger.
Compound IVe shown excellent anti-microbial activities
through its zone of inhibition (mm) against E. coli, S.
aureus, B. subtilis, P. vulgaris, C. albicons and A. niger
are 18, 20, 19, 14, 15 and 15 respectively, which is very
close result with the standard drugs. The synthesized
compounds IVe had shown almost excellent antimicrobial activity as compared to the standard drug. In
this experiment ciprofloxacin (100µg/ml) was using as a
standards for anti-bacterial activity and Ketoconazole for
anti-fungal activities, respectively. The zones of
inhibition of synthesized compounds were depicted in
table-I and Comparisons of anti-microbial activity of all
synthesized compounds with the standard drugs were
shown in Figure-I and Figure-II.
Figure: I
Figure: II
462
ANTI-CONVULSANT ACTIVITY:
All the synthesized compounds anti-convulsant activities
were screened by using pentylene tetrazole induced
technique [20] with a set up of 30-40 mg body weight of
either sex of albino rats. Before starts the test all the
animals were grouped into different category with
respect to our synthesized compounds (IVa, IVb, IVc,
IVd and IVe), reference standard and control. All the
synthesized molecules (IVa-e) were mixed with gum
acacia 5% to prepare a concentrated suspension 25%
(w/v) which was administered to the test group of albino
rats through i.p. at the dose of 100mg/kg body weight.
Keep the animals in a cage for next four hours, then
injected pentylene tetrazole (90mg/kg bv) through the
subcutaneous route to induces the convulsant activity.
The same procedure was applied to the standard drug.
Finally the activity of synthesized compounds efficacy
were compared against standard. All the compounds had
shown remarkable anti-convulsant activities when
compare to the reference drugs phenobarbitone.
Compounds IVe offered 82.15% of protection against
pentylene tetrazole induced convulsion effect. Each
synthesized molecules were exhibiting excellent anticonvulsant activity as like standard drug which is
mentioned table-II and comparisons of the anticonvulsant activity of all synthesized compounds were
demonstrated in Figure-III.
The observed Anti-Microbial Zone of Inhibition (mm) of Synthesized Compounds (IVa-e):
TABLE: I
S.NO
Zone of inhibition of anti-bacterial
Activity (100 μg/ml)
E. coli
S. aureus
B. subtilis
P. vulgaris
IVa
08
07
08
10
Ivb
06
09
10
07
Ivc
16
17
18
13
Ivd
15
13
15
11
Ive
18
20
19
14
Ciprofloxacin
22
24
20
16
Ketaconazole
Observed data of Anti-convulsant activity of synthesized compounds (IVa-e):
TABLE-II
Compounds
Dosage 25% w/v
% Protection
IVa
25
45.87
Ivb
25
53.24
Ivc
25
72.17
Ivd
25
68.90
Ive
25
82.15
phenobarbitone
25
100
Figure-III
463
Zone of inhibition of anti-fungal
Activity (100 μg/ml)
C. albincans
A. niger
08
06
07
08
14
12
12
13
15
15
17
18
Mortality
05
10
04
02
-
CONCLUSIONS:
A novel derivative of benzotriazole Schiff bases (IVa-e)
were synthesized and investigated their anti-microbial
and anti-convulsant activities against standard drugs.
The entire derivatives of benzotriazole were prepared
and obtained in an excellent yield in between 55-79% by
using systematic scheme. All the synthesized compound
characterization was verified by using IR, 1HNMR,
MASS
spectroscopy
and
elemental
analysis.
Benztriazole and oxadiazole both the nucleus has a
unique benefit in therapeutic field. Besides that, addition
of the schiff base in the above two nucleus it improves
the activity of synthesized compounds [21]. Therefore all
the mentioned synthesized compounds were exhibited
admirable anti-microbial and anti-convulsant activity.
Among the all synthesized compound, compound IVc-e
offered excellent activity due to the presence of electron
withdrawing groups were attached to the mother nucleus
made this wonder. Hence, the test compounds IVc-e
would represent a fruitful matrix for the development of
novel class of anti-microbial and anti-convulsant agent
that would be deserve further investigation.
13. Karthikeyan MS, Prasad DJ, Mahalinga M, Holl BS, Kumari NS;
Eur. J. Med Chem., 2008, 43, 25.
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1978, 55, 168.
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20. Sunny J, Anil J, Avneet G and Hemraj; Asian J Pharm Clin Res.,
2012, 5(3), 199.
ACKNOWLEDGEMENTS:
The author is thankful to Head, IIT Madras for carried
out the analytical and spectral work related to this
project. Our sincere thanks to head of the Department of
Chemistry for providing chemicals as well as Laboratory
facilities to complete the synthetic part and we would
like to thank head of the Department of Microbiology,
GIET School of Pharmacy, Rajahmundry, for biological
screening of our finished product.
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464
ISSN
0974-4169 (Print)
0974-4150 (Online)
www.ajrconline.org
RESEARCH ARTICLE
Development and Validation of Derivative Spectrophotometric Method for
Simultaneous Estimation of Lornoxicam and Eperisone in their Synthetic
Mixture
Jawed Akhtar1*, Jatin Prajapati2, ShamimAhmad1, Mohammad Mujahid1,
Gamal Osman Elhassan3
1
Translam Institute of Pharmaceutical Education and Research, Meerut, Uttar Pradesh-250001
2
Astra Life Care Pvt. Ltd, Ahmedabad.
3
Faculty of Pharmacy, Omdruman Islamic University, Khartoum, Sudan
ABSTRACT:
A simple, precise, accurate and reproducible spectrophotometric method has been developed for simultaneous
estimation of Lornoxicam (LXM) and Eperisone (EPE) by employing first order derivative zero crossing method
in methanol. The first order derivative absorption at 264.5 nm (zero cross point of LXM) was used for
quantification of EPE and 254 nm (zero cross point of EPE) for quantification of LXM. The linearity was
established over the concentration range of 2-24 µg/ml with correlation coefficient 0.9987 and 0.997,
respectively. The mean % recoveries were found to be in the range of 99.69 and 99.13 for LXM and EPE
respectively. The developed method has been validated as per ICH guidelines and successfully applied to the
estimation of LXM and EPE in their Synthetic Mixture.
.
KEYWORDS:
INTRODUCTION:
Lornoxicam (LXM) is chemically (3E)-6-chloro-3[hydroxyl (pyridin-2-ylamino) methylene]-2 methyl-2,3dihydro - 4H- thieno [2,3-e] [1,2] thiazin-4-one 1, 1dioxide.[1] Lornoxicam is an NSAID of the oxicam class
with
analgesic, anti-inflammatory and antipyretic
properties. It inhibits prostaglandin synthesis by
inhibiting both cyclo-oxygenase enzyme (COX-1 and
COX-2). EPE is chemically (2RS)-1-(4-ethylphenyl)-2methyl-3-(1-piperidyl) propane-1-one.[2] EPE is an
antispasmodic drug. It acts by relaxing both skeletal
muscles and vascular smooth muscles, and demonstrates
a variety of effects such as reduction of myotonia,
improvement of circulation, and suppression of the pain
reflex. The review of literature revealed that various
involving spectrophotometry have been reported for
LXM in single form and in combination with other
drugs.
Lornoxicam is estimated by UV and HPLC method.[3-17]
According to literature review, Eperisone is estimated by
UV, HPLC, LC-EI-MS methods.[18] There is no any
method reported for Simultaneous estimation of
Lornoxicam and Eperisone in a combination by UV and
HPLC, but individually available for each drug and in
combination with other drug. The present work describes
the development of a simple, precise, accurate and
reproducible spectrophotometric method for the
simultaneous estimation of LXM and EPE in synthetic
mixture. The developed method was validated in
accordance with ICH Guidelines and successfully
employed for the assay of LXM and EPE in synthetic
mixture.
O
H 3C
H
N
DOI: 10.5958/0974-4150.2015.00073.5
N
Structure of Eperisone
465
N
O
S
Cl
S
OH
O
O
N
Structure of Lornoxicam
repeatability, intra-day and inter-day variation in the
expected drug concentrations. After validation, the
developed methods have been applied to pharmaceutical
dosage form.
Specificity:
Commonly used excipients (starch, microcrystalline
cellulose and magnesium stearate) were spiked into a pre
weighed quantity of drugs. The D1 spectrum was
recorded by appropriate dilutions and the quantities of
drugs were determined.
Reagents and Chemicals:
Analytically pure LXM and EPE were kindly provided
by Cirex Pharmaceuticals Limited. Analytical grade Linearity:
methanol was purchased from Merck Pvt. Ltd., India.
Appropriate volume of aliquot from LXM and EPE
standard stock solution was transferred to volumetric
Instrument and Apparatus:
flask of 10 ml capacity. The volume was adjusted to the
Shimadzu-1800 UV‐Visible Spectrophotometer was mark with methanol to give a solutions containing 2-16
used for spectral measurements with spectral band width μg/ml of both LXM and EPE All D1 Spectrum were
1 nm, wavelength accuracy is 0.5 nm and 1 cm matched recorded using above spectrophotometric condition.
quartz cells. Software used was UV Probe (version D1absorbance at 254 nm and 264.5 nm were recorded
2.34). Glassware used in each procedure were soaked for LXM and EPE, respectively (n=6). Calibration
overnight in a mixture of chromic acid and sulphuric curves were constructed by plotting average absorbance
acid rinsed thoroughly with double distilled water and versus concentrations for both drugs. Straight line
dried in hot air oven.
equations were obtained from these calibration curves.
Spectrophotometric Condition:
Accuracy:
All zero order spectrums (D0) were converted to first Accuracy was assessed by determination of the recovery
derivative spectrum (D1) using delta lambda 1.
of the method by addition of standard drug to the prequantified placebo preparation at 3 different
Preparation Standard Stock Solutions:
concentration levels 80, 100 and 120 %, taking into
Accurately weighed 10mg of LXM and EPE standard consideration percentage purity of added bulk drug
were transferred to separate 100 ml volumetric flask and samples. Each concentration was analyzed 3 times and
dissolved in 50 ml 0.1 N methanolic NaOH. The flasks average recoveries were measured.
were shaken and volume was made up to the mark with
the same solvent to give solutions containing 100 μg/ml Precision:
LXM and 100 μg/ml EPE.
The repeatability was evaluated by assaying 6 times of
sample solution prepared for assay determination. The
Selection of Analytical Wavelength:
intraday and interday precision study of LXM and EPE
2-12 μg/ml solutions of both LXM and EPE were was carried out by estimating different concentrations of
prepared in methanol by appropriate dilution and LXM (0.16, 0.48, 0.96 μg/ml) and EPE(2, 6, 12 μg/ml),
spectrum was recorded between 200-500 nm and first 3 times on the same day and on 3 different days (first,
derivative spectrums were obtained using above second, third) and the results are reported in terms of
condition. The overlain first derivative spectrums of C.V.
LXM and EPE at different concentration were recorded.
The zero crossing point (ZCP) of LXM was found to be Detection limit and Quantitation limit:
264.5 nm and ZCP of EPE was found to be 254 nm.
ICH guideline describes several approaches to determine
the detection and quantitation limits. These include
METHOD VALIDATION: [19-20]
visual evaluation, signal-to-noise ratio and the use of
The proposed method has been extensively validated in standard deviation of the response and the slope of the
terms of specificity, linearity, accuracy, precision, limits calibration curve. In the present study, the LOD and
of detection (LOD) and quantification (LOQ), robustness LOQ were based on the third approach and were
and reproducibility. The accuracy was expressed in calculated according to the 3.3σ/S and 10σ/S criterions,
terms of percent recovery of the known amount of the respectively; where σ is the standard deviation of ystandard drugs added to the known amount of the intercepts of regression lines and s is the slope of the
pharmaceutical dosage forms. The precision (Coefficient calibration curve.
of Variation - C.V.) was expressed with respect to the
466
Robustness:
The sample solution was prepared and then analyzed
with change in the typical analytical conditions like
stability of analytical solution.
Reproducibility:
The absorbance readings were measured at different
laboratory for sample solution using another
spectrophotometer by analyst and the values obtained
were evaluated using t- test to verify their
reproducibility.
Determination of Lornoxicam and Eperisone in their
synthetic mixture Sample preparation:
A powder quantity equivalent to 0.8 mg LXM and 10 mg
EPE was accurately weighed and transferred to
volumetric flask of 100 ml capacity. 60 ml of 0.1 N
methanolic NaOH was transferred to this volumetric
flask and sonicated for 15 min. The flask was shaken and
volume was made up to the mark with the same solvent.
From this solution 2 ml was transferred to volumetric
flask of 100 ml capacity. Volume was made up to the
mark to give a solution containing 0.16 μg/ml of LXM
and 2 μg/ml of EPE. The resulting solution was analyzed
by proposed method. The quantitation was carried out by
keeping these values to the straight line equation of
calibration curve.
RESULTS AND DISCUSSION:
First order derivative spectrophotometric method was
developed for determination of LXM and EPE The
proposed method has been extensively validated as per
ICH guidelines. Summary of validation parameters for
proposed method was given in Table 1.
The overlain D1 spectrum of LXM and EPE at different
concentrations revealed that at 254 nm (ZCP of EPE)
TRA possesses significant D1 absorbance and at 264.5
nm (ZCP of LXM) EPE possesses significant D1
absorbance. Considering above facts, wavelength 254
nm and 264.5 nm were selected for the estimation of
LXM and EPE, respectively (figure 2). Linearity was
assessed for LXM and EPE by plotting calibration
curves of the D1 absorbance versus the concentration
over the concentration range 2-18 μg/ml for both drugs.
The correlation coefficients (r2) for LXM and EPE were
found to be 0.9986 and 0.997, respectively (Table 2).
The following equations for straight line were obtained
for LXM and EPE. Linear equation for LXM, y =
0.0014x + 0.0004 Linear equation for EPE, y = 0.0041x
+ 0.0032. The % recoveries were found to be in the
range of 99.69 % for LXM and 99.13 for EPE (Table 3).
The precision of method was determined by
repeatability, intraday and interday precision and was
expressed as the C.V. (Table 1), which indicate good
method precision. The Limit of detection for LXM and
EPE was found to be 0.144μg/ml and 1.60μg/ml
respectively. Limit of quantification for LXM and
EPEwas found to be 0.43μg/ml and 4.86μg/ml at 254 nm
and at 264.5 nm respectively (Table 1). The method was
also found to be specific, as there was no interference
observed when the drugs were estimated in presence of
excipients and robust, as there was no significant change
in absorbance up to 24 hours of preparation of solution
in methanol. The proposed spectrophotometric method
was successfully applied to LXM and EPE synthetic
mixture and its combined dosage form. The results are
shown in Table 6.
Derivative Spectra
467
Table 1 - Specificity study for synthetic mixture
Mixture
Conc. µg/ml
Max (nm.)
Before addition of excipients
Absorbance
Conc. µg /ml
0.64L
254
0.0012
0.57
1
8.0E
264.5
0.0331
7.29
0.8 L
254
0.0014
0.71
2
10.0E
264.5
0.0419
9.43
0.96L
254
0.0015
0.78
3
12.0E
264.5
0.0507
11.58
8.947 ± 0.17
% RSD 1.96
Mean
LXM
0.63 ± 0.0074
% RSD 1.16
EPE
After addition of excipients
Absorbance
Conc. µg/ml
0.00113
0.52
0.03291
7.24
0.00131
0.65
0.04165
9.37
0.0014
0.71
0.0504
11.51
Table 2 -Linearity
Concentration (µg/ml)
LXM
EPE
2
2
4
4
6
6
8
8
10
12
12
16
14
20
18
24
24
28
Absorbance of
LXM at 254 nm
0.084
0.163
0.256
0.353
0.375
0.453
0.552
0.696
0.891
1.026
Eperisone
Lornoxicam
468
Absorbance of
EPE at 264.5 nm
0.122
0.238
0.336
0.446
0.617
0.774
1.032
1.221
% Interference
8.75
0.63
9.0
0.64
9.09
0.63
Table 3: Accuracy - Recovery study for the synthetic mixture
Mixture
Conc.
Waveleng Absorb Conc. before
(LXM:EPE)
(µg/ml)
th (nm)
ance
spiking
(µg/ml)
1.
LXM
254
0.0015
0.78
(0.8)
264.5
0.0418
9.41
2.
+
254
0.0014
0.71
EPE
264.5
0.0417
9.39
3.
(10.0)
254
0.0014
0.71
264.5
0.0419
9.43
Reference
standard
added (µg/ml)
80 % ( 0.64 L+
8.0 E)
100% (0.8 L +
10.0 E)
120% (0.96L +
12.0 E)
Table 4: Intraday precision study for the synthetic mixture
Conc. (µg/ml)
Wavelength
Absorbance
(nm)
LXM
EPE
A
B
0.16
2.0
254
0.00062
0.00061
264.5
0.0104
0.0105
0.48
6.0
254
0.00098
0.001
264.5
0.0264
0.0263
0.96
12.0
254
0.00167
0.00168
264.5
0.0509
0.0508
C
0.00062
0.0105
0.00098
0.0265
0.00164
0.0508
Table 5: Interday precision study for the synthetic mixture
Conc. (µg/ml)
Wavelength
Absorbance
(nm)
LXM
EPE
A
B
0.16
2.0
254
0.00062
0.00061
264.5
0.0107
0.0108
0.48
6.0
254
0.001
0.001
264.5
0.0262
0.0261
0.96
12.0
254
0.00167
0.00168
264.5
0.0507
0.0506
C
0.00062
0.0107
0.00098
0.0262
0.00164
0.0505
%
Recovery
Mean
S.D.
RSD
0.000616
0.01046
0.00098
0.0264
0.00166
0.05083
5.7×10-6
5.7×10-6
1.1×10-5
1×10-4
2.1×10-5
5.7×10-5
0.936
0.551
1.17
0.378
1.25
0.113
100.44
98.47
98.21
99.75
100.44
99.18
Mean
Table 6.1: Robustness study for the synthetic mixture at 254 nm
Mixture
Conc.
Absorbance at wavelength in (nm)
LXM:EPE
(µg/ ml)
At 253
Conc. obtained
0.32
0.00089
0.28
1
0.64
0.00119
0.56
2
0.8
0.0011
0.71
3
0.35
Mean
0.0057
S.D.
1.63
R.S.D.
Table 6.2: Robustness study for the synthetic mixture at 264.5 nm
Mixture
Conc.
Absorbance at wavelength in (nm)
LXM:EPE
(µg/ml)
At 263.5
Conc. obtained
4
0.0183
3.68
1
8
0.0332
7.31
2
10
0.0421
9.48
3
3.653
Mean
0.025
S.D.
0.68
R.S.D.
Table 7: Reproducibility study for the synthetic mixture
Conc.(µg/ml)
Wavelength
Absorbance
(nm)
LXM
EPE
A
0.16
2.0
254
0.00062
264.5
0.0104
0.48
6.0
254
0.00098
264.5
0.0264
0.96
12.0
254
0.00167
264.5
0.0509
Conc. after
spiking
(µg/ml)
1.42
17.29
1.50
19.36
1.67
21.34
B
0.00061
0.0105
0.001
0.0263
0.00168
0.0508
469
0.000616
0.01046
0.000993
0.02616
0.00166
0.0506
Mean
±
SD
LXM
99.61
± 1.22
EPE
99.13
± 0.64
% RSD
LXM
1.22
EPE
0.64
RSD
S.D.
5.7×10-6
5.7×10-5
1.5×10-5
5.7×10-5
2.1×10-5
1×10-4
0.936
0.537
1.16
0.22
1.25
0.19
At 254
0.0009
0.0012
0.0014
Conc. obtained
0.35
0.57
0.71
0.57
1.01
1.75
At 255
0.00091
0.00121
0.0016
Conc. obtained
0.42
0.58
0.73
0.716
0.0115
1.61
At.264.5
0.0182
0.0331
0.0419
Conc. obtained
3.65
7.29
9.43
7.28
0.036
0.49
At 265.5
0.0181
0.0329
0.0418
Conc. obtained
3.63
7.24
9.41
9.41
0.036
0.38
Mean
S.D.
RSD
0.000616
0.01046
0.00098
0.0264
0.00166
0.05083
5.7×10-6
5.7×10-6
1.15×10-5
1×10-4
2.1×10-5
5.7×10-5
0.94
0.55
1.17
0.38
1.25
0.11
C
0.00062
0.0105
0.00098
0.0265
0.00164
0.0508
Table 8: Assay
Sr. No.
Conc. (µg/ml)
1
LXM (0.8) + EPE (10.0)
2
3
Wavelength(nm)
254
264.5
254
264.5
254
264.5
Abs.
0.0015
0.0434
0.00151
0.0436
0.0015
0.0435
Conc.
0.78
9.80
0.79
9.85
0.78
9.82
% Assay
98.21
98.04
99.107
98.53
98.21
98.29
Mean % Assay
LXM
98.51
EPE
98.29
Table 9: Result
Sr. No.
1
2
3
4
5
6
7
8
9
Parameters
Zero Crossing Point
Range (µ g/ml)
Linearity
Precision (%RSD)
1) Intraday
2) Interday
3) Reproducibility
Accuracy
Robustness
LOD
LOQ
Assay
LXM
264.5 nm
0.16-0.96
R2 = 0.9987
EPE
254 nm
2-12
R2= 0.997
0.936 -1.25
0.936 -1.25
0.936 -1.25
99.61
1.61 -1.75
0.113 - 0.551
0.19 – 0.537
0.113 - 0.551
99.13
0.38 – 0.68
98.51
98.29
CONCLUSION:
The proposed first order derivative method provides 7.
simple, specific, precise, accurate and reproducible
quantitative analysis for simultaneous determination of
LXM and EPE in synthetic mixture. The method was
validated as per ICH guidelines in terms of specificity, 8.
linearity, accuracy, precision, limits of detection (LOD)
and
quantification
(LOQ),
robustness
and
reproducibility. The proposed method can be used for
routine analysis and quality control assay of LXM and 9.
EPE in combined dosage form.
ACKNOWLEDGEMENT:
Authors are thankful to Cirex Pharmaceuticals Limited,
Hyderabad for providing gratis sample.
11.
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471
ISSN
0974-4169 (Print)
0974-4150 (Online)
www.ajrconline.org
RESEARCH ARTICLE
Quantitative planner chromatographic method development for sitagliptin
phosphate monohydrate and metformin hydrochloride in presence of their
degradation product
Sanjay G. Walode1*, Avinash V. Kasture2
1
Department of Pharmaceutical Chemistry, Sinhgad Institute of Pharmaceutical Sciences, Kusgaon (Bk),
Lonavala, Pune, 410 401, India
2
Department of Pharmaceutical Chemistry, University Department of Pharmaceutical Sciences, Nagpur – 440
010, India
*Corresponding Author E-mail: [email protected], [email protected]
ABSTRACT:
This paper presents the stability indicating method for the simultaneous analysis of sitagliptin phosphate
monohydrate and metformin hydrochloride using High Performance Thin Layer Chromatography (HPTLC) with
densitometric detection. Separation of both the drugs was performed on silica gel G 60F254 plates with detection
wavelength of 216 nm. The mobile phase is comprised of acetonitrile-methanol-glacial acetic acid (7.0:3.0:0.02
v/v/v). The Rf values were found to be 0.21 ± 0.035 and 0.53 ± 0.029 for sitagliptin phosphate monohydrate and
metformin hydrochloride, respectively. The linear regression analysis data for the calibration plots showed good
linear relationship with respect to peak area in the concentration range 8 - 64 ng/band of sitagliptin (with r =
0.9992) and 80 - 640 ng/band of metformin (with r = 0.9994). The method was validated as per International
Conference on Harmonization guideline (ICH) for accuracy, precision, robustness, and specificity, limit of
detection and limit of quantitation. Statistical analysis of results obtained proves that the method is repeatable
and selective for estimation of both the drugs. As the method could effectively separate the drug from its
degradation products, it can be employed as a stability indicating method.
KEYWORDS: Sitagliptin phosphate monohydrate; Metformin hydrochloride; HPTLC; Validation, Stability
indicating method; Degradation.
1. INTRODUCTION:
Number of individuals affected by diabetes is continuing
to increase worldwide therefore; the need for effective
management assumes ever greater urgency. Combination
therapies are found to be effective as it is well tolerated,
convenient to take with few contraindications.
Pharmacists should take various factors into
consideration, for example drug stability, possible
degradation products and potential interactions with the
excipients in concern with safety and efficacy of drug
products.
DOI: 10.5958/0974-4150.2015.00076.0
Sitagliptin phosphate monohydrate (SIT) is chemically
{(3R)-3-amino-1-[3-(trifluoromethyl)-6,8-dihydro-5H[1,2,4]triazolo[4,3-a]pyrazin-7-yl]-4-(2,4,5trifluorophenyl) butan-1-one} (Fig.1). SIT is well known
oral hypoglycemic drug of the dipeptidyl peptidase-4
(DPP-4) inhibitor class 1. DPP-4 inhibitors represent a
new therapeutic approach to the treatment of type II
diabetes that functions to stimulate glucose-dependent
insulin release and reduce glucagon level. After
extensive literature survey several methods have been
found for determination of SIT including UV-Visible
spectrophotometry2, LC-MS/MS in human plasma using
protein precipitation method3, in human plasma using
liquid-liquid extraction method4 and in human urine and
hemodialysate using turbulent flow online extraction
method5.
472
to develop an alternate accurate, rapid, specific and
reproducible method for the determination of sitagliptin
phosphate monohydrate (SIT) and metformin
hydrochloride (MET) in presence of their degradation
products for the content analysis during stability studies.
2. MATERIAL AND METHODS:
Fig. 1. Chemical structure of sitagliptin phosphate
2.1. Chemicals and reagents
Sitagliptin phosphate monohydrate (SIT) and metformin
hydrochloride (MET) were obtained as a gift sample
from Zydus Cadilla, Ahmadabad, India. Combined dose
tablet formulation containing sitagliptin phosphate
monohydrate (50 mg) and metformin hydrochloride
(500 mg), Sitar-M, manufactured by Oclare Labs
Ltd., was purchased from local market. The solvents and
chemicals used in the study were of HPLC grade
(MERCK).
Metformin hydrochloride (MET) is chemically, 1:1
dimethyl biguanidine mono hydrochloride (Fig.2) is an
anti-diabetic drug from the biguanide class of oral
hypoglycaemic agents, given in the treatment of non–
insulin-dependent diabetes mellitus6. MET is effective
in patients who lack functioning of islet cells as it act by
simulations of glycolysis in peripheral tissues 7,8. Several
analytical
methods
based
on
potentiometry,
spectrofluorimetry and UV-Visible spectrophotometry9, 2.2. Preparation of standard stock solutions
HPLC10,11 and HPTLC12 are reported for determination 2.2.1. Standard stock solution A
Accurately weighed quantity of SIT (10 mg) was
of metformin in pure form.
transferred to 10.0 mL volumetric flask, dissolved
with 5.0 mL DMSO, sonicate for 10 min and diluted to
10.0 mL with methanol. The solution was centrifuged at
2000 rpm for 5 min and 2.0 mL of supernatant was
diluted to 10.0 mL with methanol. (Concentration 200
μg/mL of SIT)
Fig. 2. Chemical structure of metformin hydrochloride
2.2.2. Standard stock solution B
Accurately weighed quantity of MET (100 mg)
transferred to 10.0 mL volumetric flask, dissolved
and diluted to the mark with methanol. The solution
was centrifuged at 2000 rpm for 5 min and 2.0 mL of
supernatant was diluted to 10.0 mL with methanol.
(Concentration 2000 μg/mL of MET)
Several methods have been reported for determination of
metformin with other antidiabetic drugs such as UVVisible spectroscopy with glibenclamide13, with
pioglitazone and glimepiride14, RP-HPLC method with
pioglitazone hydrochloride and glimepiride15, with
gliclazide and pioglitazone hydrochloride16, with
pioglitazone17,18 and glipizide, gliclazide, glibenclamide 2.2.3. Standard stock solution C
or glimperide in plasma19 and LC/(APCI)MS with Accurately weighed quantity of SIT (10 mg) and MET
(100 mg) were transferred to 10.0 mL volumetric flask,
glibenclamide in human plasma20.
dissolved with 5.0 mL DMSO, sonicate for 10 min and
Several chromatographic methods have been reported for diluted to 10.0 mL with methanol. The solution was
simultaneous estimation of MET and SIT in combination centrifuged at 2000 rpm for 5 min and 2.0 mL of
including RP-HPLC21-26 and UPLC27 in bulk as well as in supernatant was diluted to 10.0 mL with methanol.
(Concentration 200 μg/mL of SIT and 2000 μg/mL of
pharmaceutical formulation.
MET)
The advantage of High Performance Thin Layer
Chromatography (HPTLC) is that, a large number of 2.3. Selection and optimization of mobile phase
samples can be simultaneously analysed in a shorter time Aliquot portion (10 µL) of standard stock solution A, B
period. Unlike HPLC, this method utilizes less quantity and C were diluted to 1.0 mL with methanol and 10 µL
of solvents, thus lowering the cost of analysis. An ideal of resultant solution was applied on the TLC plates in the
stability indicating chromatographic method should form of band and run in different solvent systems.
estimate the drug and also be able to resolve the drug Different solvent systems using individual solvents and
from its degradation products. It was found that one in combinations were initially tried in order to
HPTLC method26 have been reported in bulk drug and determine the best condition for the effective
dosage form. However there is no any method was separation of SIT and MET. The mobile phase
stability indicating one; hence an attempt has been made comprising of acetonitrile-methanol-glacial acetic acid
473
(7.0:3.0:0.02 v/v/v) gave high resolution of SIT and
MET with improved peak shapes and hence the mobile
phase was selected for further analysis. Glacial acetic
acid was added to improve the peak shape of SIT.
phase for 10 min at room temperature (25 ± 2°C) was
employed. The length of each chromatogram run was 70
mm. After chromatographic development, plates were
air dried and densitometric scanning was performed at
216 nm with a CAMAG TLC scanner-III operated in
reflectance–absorbance mode and controlled by
WinCATS software (Version 1.4.3.6336). The slit
dimensions were 5 x 0.45 mm and the scanning speed
was 20 mm/s. The source of radiation used was a
deuterium lamp emitting continuous UV spectra between
190-400 nm. Concentrations of the compounds
chromatographed were determined from the intensity of
the diffused light. Evaluation was done by peak areas
with linear regression.
2.4.
Selection of wavelength for densitometric
evaluation of separated bands
Aliquot portion (10 µL) of standard stock solution C was
diluted to 1.0 mL with methanol and 10 µL of resultant
solution was applied on the TLC plates in the form of
band using LINOMAT-IV automatic sample applicator.
The plates were scanned densitometrically over the
wavelength range of 200-400 nm and their overlain
spectra were carried out (Fig. 3). From overlain spectra,
it was observed that both SIT and MET exhibited
significant absorbance at 216 nm, which was selected as 2.6. Development of calibration curves
the analytical wavelength for further analysis.
Aliquots portions, 20, 40, 60, 80, 100, 120, 140 and 160
μL of standard stock solution C (200 μg/mL of SIT and
2000 μg/mL of MET) were serially diluted to 2.0 mL
with methanol in different micro centrifuge tube and
vertex for 1.0 min. 4 μL of each solution was applied on
the HPTLC plate to deliver 8, 16, 24, 32, 40, 48, 56 and
64 ng/band of SIT and 80, 160, 240, 320, 400, 480, 560
and 640 ng/band of MET. This was done in triplicate.
For each concentration, the applied band bands were
evenly distributed across the plate to minimize possible
variation along the silica layer.
Given considerable extent of the calibration range (two
orders of magnitude), the homoscedasticity of the
analytical method was evaluated with Cochran’s test. In
order to achieve homoscedasticity, the Cochran C of 4
standards with 3 replicates of each standard should be
less than the critical values of 0.76828. Since the largest
and smallest values of variance usually appear at the
of the calibration curve in the
Fig. 3. Overlain spectra of sitagliptin phosphate and metformin extremities
hydrochloride
heteroscedastic case, the two lowest concentrations (8
and 16 ng/band of SIT and 80 and 160 ng/band of MET)
2.5.
Instrumentation
and
optimized and the two highest concentration (56 and 64 ng/band of
chromatographic conditions
SIT and 560 and 640 ng/band of MET) standards were
Chromatography was carried out on 10 cm x 10 cm included in the tests.
aluminium-backed HPTLC plates coated with 250nm
layer of silica gel G 60F254 (E. Merck, Darmstadt, 2.7. Assay
Germany, supplied by Merck India, Mumbai, India). The 2.7.1. Preparation of standard solution
plates were prewashed with methanol and activated at Aliquots portions, 100 μL of standard stock solution C
110⁰C for 30 min prior to chromatography. The (200 μg/mL of SIT and 2000 μg/mL of MET) was
samples were applied as bands of 6 mm length, diluted to 2.0 mL with methanol in micro centrifuge tube
under a continuous flow of nitrogen, using CAMAG and vertex for 1.0 min. (10 µg/mL of SIT and 100
LINOMAT-IV sample applicator. Samples were applied µg/mL of MET)
with a 100 µL syringe (Hamilton, Switzerland) at a
constant application rate and the distance between 2.7.2. Preparation of sample solution
adjacent bands was 8mm. The mobile phase consisting Twenty tablets were weighed accurately; average weight
of acetonitrile-methanol-glacial acetic acid (7.0:3.0:0.02 was calculated, and crushed to obtain fine powder.
v/v/v) was used for the development of the Accurately weighed quantity of tablet powder equivalent
chromatograms. For linear ascending development, a to about 10 mg of SIT was transferred to 10.0 mL
twin-trough glass chamber 10 cm × 10 cm (CAMAG , volumetric flask, dissolved with 5.0 mL DMSO, sonicate
Muttenz, Switzerland) previously saturated with mobile
474
for 10 min, diluted to 10.0 mL with methanol, mixed
well and filtered through Whatmann filter paper
No.42. The solution was centrifuged at 2000 rpm for 5
min and 2.0 mL of supernatant was diluted to 10.0 mL
with methanol. 100 μL of resultant solution diluted to
2.0 mL with methanol in micro centrifuge tube and
vertex for 1.0 min (10 µg/mL of SIT and 100 µg/mL of
MET). This solution was used as sample solution.
manner as described under analysis of the tablet
formulation.
On the TLC plate, 4 µL each, two bands of standard
solution and four bands of sample solution were applied
and the plate was developed and scanned under the
optimized chromatographic condition. Content of SIT
and MET were calculated by comparing peak areas of
sample with that of the standard. The densitogram of
tablet formulation is shown in Fig. 4.
Intermediate precision
For method precision were carried out at three different
concentration levels (24, 40 and 56 ng/band of SIT and
240, 400 and 560 ng/band of MET). Intra-day precision
was determined by repeating the assay six times at
different time interval on same day and on three
consecutive days for inter-day precision studies. Results
of intermediate precision are expressed as percent
relative standard deviation.
2.8.3. Repeatability
Six replicates of SIT-40 ng/band and MET-400 ng/band
were applied on silica gel 60F254 plate and analysed by
the proposed method for system precision studies to
determine variations due to the instrument.
2.8.4. Limit of detection (LOD) and limit of
quantitation (LOQ)
LOD and LOQ were separately determined based on the
standard deviation of the y-intercept and mean slope of
the calibration curves.
2.8.5. Robustness
Robustness of the proposed method was studied by small
but deliberate variations in the optimized method
parameters. Variation in composition of the mobile
phase (± 0.1 mL), volume of the mobile phase (± 10 %),
chamber saturation time (± 20 %), time from bandting
Fig. 4. Densitogram of marketed formulation, Peak 1-SIT and to development (5 min, 20 min and 1 hrs) and time from
development to scanning (5 min, 20 min and 1 hr) was
Peak 2-MET
involved in this study. The effect of these changes on
both the Rf values and peak areas were evaluated by
2.8. Method validation
calculating the relative standard deviation for each
Validation of optimized HPTLC method was done with parameter.
respect to ICH guidelines.
2.8.1. Accuracy
2.8.6. Solution stability
To ascertain accuracy of the method recovery studies Sample solution was prepared and was kept at room
were performed by the standard addition method. Pre- temperature (25 ± 2°C) on a shelf protected from direct
analyzed tablet powder equivalent to about 5mg of SIT light. The solution was analyzed after 20 min, 1 hrs, 3
was weighed and transferred to 10.0 mL volumetric hrs, 8 hrs and 24 hrs. The % label claim and the relative
flask, added 3mg, 5mg and 7mg of pure SIT and 30 mg, standard deviation were calculated.
50 mg and 70 mg of pure MET to the tablet powder for
80%, 100% and 120% level of recovery. Extraction and 2.9. Forced degradation studies
dilutions were performed as described in sample Forced degradation study was carried out by attempting
solution. Solutions were prepared in triplicate and deliberate exposing the drugs to different stress
analyzed. The procedure was repeated for three conditions. A mixed stock solution of SIT (10 mg) and
consecutive days. Accuracy was determined and MET (100 mg) was prepared in 10.0 mL methanol with
expressed as percent recovery.
the aid of DMSO. This mixed stock solution was used
for forced degradation to provide an indication of the
2.8.2. Precision
stability indicating property and specificity of the
To ascertain repeatability and reproducibility of the proposed method.
method precision studies were performed. tablet sample
solution was prepared and analyzed in the similar
475
2.9.1. Acid and base induced degradation product
To 2.0 mL of mixed stock solution, 8.0 mL of 1N HCl
and 8.0 mL of 1N NaOH were added separately. These
mixtures were reflux separately for 3 h at 80⁰C. The
forced degradation study in acidic and basic media was
performed in the dark in order to leave out the possible
degradative effect of light. 100 µL of sample solution
was diluted to 2.0 mL with methanol and 4µL (40
ng/band of SIT and 400 ng/band of MET) was applied
on TLC plate.
2.9.6. Neutral hydrolysis
2.0 mL of mixed stock solution was diluted to 10.0 mL
with distilled water and was reflux for 8h. 100 µL of
each sample solution were diluted to 2.0 mL with
methanol and 4 µL was applied on TLC plate.
All the exposed samples were analyzed as discussed in
analysis of marketed formulation.
3. RESULT AND DISCUSSION:
Among the different mobile phase combinations
acetonitrile-methanol-glacial acetic acid (7.0:3.0:0.02
2.9.2. Hydrogen peroxide induced degradation v/v/v) gave better resolution and sharp peaks with Rf
product
values of 0.21 ± 0.035 and 0.53 ± 0.029 for SIT and
To 2.0 mL of mixed stock solution, 8.0 mL of hydrogen MET, respectively on densitometric scanning at 216 nm.
peroxide (30% v/v) was added. This solution was heated
in boiling water bath for 10 min to remove completely 3.1. Linearity
the excess of hydrogen peroxide and reflux for 3 h at Peak areas were found to have good linear
80°C. 100 µL of sample solution was diluted to 2.0 mL relationship with the concentration than the peak
with methanol and 4µL was applied on TLC plate.
heights. The r2 values were found to be 0.9992 and
0.9994 for SIT and MET respectively. Calibration
2.9.3. Dry heat induced degradation product
graphs were constructed in the concentration range of 8Dry heat degradation was performed by exposing the 64 ng/band for SIT and 80-640 ng/band for MET. The
powdered SIT (10 mg) and MET (100 mg) to 60°C for correlation coefficients, y-intercepts and slopes of the
24 h under dry heat condition to study the inherent regression lines of the two compounds were calculated
stability of the drugs. Dry heat exposed drugs was (Table-1).
dissolved in 10.0 mL methanol with the aid of DMSO.
2.0 mL of this solution was diluted to 10.0 mL. 100 µL
Table 1: Optical Characteristics and Validation Data of SIT and
of resultant sample solution was diluted to 2.0 mL with MET
methanol and 4µL was applied on TLC plate.
Parameters
SIT
MET
Linearity (ng mL-1)
Slope
Intercept
Regression coefficient (r2)
*Average of six determinations
8-64
80-640
529.12
448.46
2.9.4. Wet heat induced degradation product
362.87
225.56
2.0 mL of mixed stock solution was diluted to 10.0 mL
0.9992
0.9994
with methanol and reflux for 3 h at 80°C. 100 µL of
sample solution was diluted to 2.0 mL with methanol
and 4µL was applied on TLC plate.
The homoscedasticity (homogeneity of variance) of the
calibration standards was verified using a Cochran’s test.
2.9.5. Photolytic induced degradation product
The Ccalc values were 0.482 and 0.503 for SIT and MET,
2.0 mL of mixed stock solution was diluted to 10.0 mL respectively. These test statistics were smaller than the
with methanol. For photochemical stability study, 5.0 critical value, C
tab(α=0.05; k=4, n=3) = 0.768. The two
mL of resultant diluted stock solution was exposed to calibration curves pass the homoscedasticity test since
direct sunlight for 8 days on a wooden plank and kept on the Ccalc values were less than the critical value. Thus,
terrace. For UV radiation degradation study 5.0 mL of straight lines were considered adequate to describe the
resultant diluted stock solution was exposed to UV relationships between the peak area and the
radiations at 254 nm for 24 h in UV chamber. 100 µL of concentrations for each compound (Table-2).
each sample solution were diluted to 2.0 mL with
methanol and 4µL was applied on TLC plate.
Table 2: Results of Cochran’s C test
Drug
Concentration in ng/ml
8
SIT
16
56
64
80
MET
160
560
640
SD
561.84
1008.92
2087.62
3861.68
338.62
992.92
1545.52
2963.51
SD2
315664.18
1017919.57
4358157.26
14912572.42
114663.50
985890.12
2388632.07
8782391.52
476
Σ SD2
20604313.43
Cochran’s C
0.482
12271577.21
0.503
3.2. Assay
Analysis of samples of marketed tablets was carried
out and the amounts estimated were expressed as
percentage amount of the label claims. The results
obtained were closed to 100% for the drugs conclude
that the method is suitable for accurate determination of
SIT and MET without any interference of excipients
(Table-3).
Table 3: Results of Assay
Sr.
Drug
Amount of
No.
drug
estimated
(mg/tablet)*
1
49.58
SIT
2
497.32
MET
*Average of six determinations
% Label
Claim*
S.D.
(±)
C.O.V.
(%)
99.16
99.46
0.8378
0.7322
0.8299
0.7319
3.3. Accuracy
The mean percentage recovery for each compound was
calculated at each concentration level and reported with
its standard deviation. The percentage recovery at three
levels (80%, 100% and 120%) for both drugs were
studied in triplicate and the results were found to be
closed to 100% i.e. with acceptable criteria < 2 %
(Table-4). For SIT, the recoveries were found between
99.68% and 100.28% and for MET the recoveries were
found between 99.39% and 99.61%, concluded that the
method was considered to have an acceptable recovery
and accuracy.
3.4. Precision
Repeatability of sample application and measurement of
peak areas at 40 ng/band of SIT and 400 ng/band of
MET were expressed in terms of % R.S.D. and S.E. and
was found to be < 2. Intermediate precision, the
Table 4: Results of Recovery Study
Level of
Amount of pure
Amount of pure
Recovery %
drug added (mg)
drug recovered (mg)
SIT
MET
SIT
MET
80
3
30
3.008
29.817
100
5
50
4.994
49.805
120
7
70
6.978
69.594
*Denotes average of three estimations at each level of recovery.
Table 5: Results of Precision Study
Parameters
SIT
Theoretical
Amount
amount (ng)
estimated (ng)
Repeatability 40
38.96
Intra-day
24
23.68
precision
40
39.55
56
55.26
Inter-day
24
23.78
precision
40
39.11
56
55.59
*Mean of six determinations
measurement of the peak areas at three different
concentration levels (24, 40 and 56 ng/band for SIT and
240, 400 and 560 ng/band for MET) showed low value
of % R.S.D. (<2) and low value of S. E. (<2) for intraand inter-day variation (Table-5) indicating the
reproducibility of the developed method.
3.5.
Limit of detection (LOD) and limit of
quantitation (LOQ)
The limits of detection were found to be 0.5ng/band and
10.0ng/band and limits of quntitation were found to be
2.0 ng/band and 32.0 ng/band for SIT and MET
respectively.
3.6. Robustness
Robustness of the proposed method was studied by small
but deliberate variations in the optimized method
parameters. The effect of changes in the mobile phase
composition (± 0.1 mL), amount of mobile phase (±1
mL), duration of chamber saturation with mobile phase
(±20%), time from spotting to development (5 min, 20
min and 60 min) and time from development to scanning
(5 min, 20 min and 60min) on Rf value and peak area of
both drugs were examined (Table-6). The relative
standard deviation for peak area was found to be less
than 2 under all the deliberately varied method
parameters. The resolution between SIT and MET was
not significantly affected as there was no significant
change in the Rf value of both the drugs (Rf values
were within ± 0.05 Rf units of standard values).
Hence the method was found to be robust for the
determination of SIT and MET in fixed dose
combination tablet.
% Mean
Recovery*
SIT
100.28
99.87
99.68
% RSD
SE
0.7258
0.9854
0.2568
1.0458
0.8845
0.3568
0.9854
0.2568
0.2368
0.3568
0.3568
0.3565
0.2548
0.1245
477
SD (±)*
MET
99.39
99.61
99.42
SIT
0.926
1.256
1.056
MET
Theoretical
amount (ng)
400
240
400
560
240
400
560
% C.O.V.
MET
1.611
1.236
1.325
Amount
estimated (ng)
398.51
238.69
399.84
557.23
237.68
398.60
561.54
SIT
0.931
1.245
1.089
% RSD
1.2541
0.2358
0.2568
0.8544
0.8457
0.5625
0.4859
MET
1.596
1.229
1.331
SE
0.4045
0.5849
0.8457
0.5658
0.5124
0.3325
0.2566
Table 6: Robustness Testing for HPTLC method
Chromatographic Changes
Factor
Mobile phase composition (± 0.1 ml)
7.1:3.1:0.02
7.0:3.0:0.02
6.9:2.9:0.02
Amount of mobile phase (v/v) (± 1
ml)
11
10
9
Duration of Chamber Saturation (±
20 %)
12 min
10 min
8 min
Time from Spotting to development
5 min
20 min
60 min
Time from development to scanning
5 min
20 min
60 min
Level
+ 0.1
0
- 0.1
RSD
+ 0.1
0
- 0.1
RSD
+ 20%
0
- 20%
RSD
---RSD
---RSD
Peak area*
SIT
3186.66
3233.45
3262.15
1.056
SIT
MET
7562.12
7610.11
7596.16
1.368
MET
Rf Value*
SIT
0.20
0.21
0.19
MET
0.53
0.53
0.54
SIT
MET
3112.15
3285.68
2968.62
0.986
SIT
7711.15
7574.55
7682.18
1.458
MET
0.22
0.20
0.22
0.53
0.54
0.55
SIT
MET
3256.54
3189.22
3289.81
1.515
SIT
3389.54
3412.13
3378.38
1.378
SIT
3216.22
3128.65
3287.11
0.996
7722.36
7614.14
7645.28
1.688
MET
7825.44
7749.45
7811.14
1.712
MET
7516.14
7719.14
7577.88
1.145
0.22
0.21
0.23
0.54
0.53
0.51
SIT
0.23
0.22
0.19
MET
0.55
0.55
0.53
SIT
0.23
0.21
0.23
MET
0.54
0.51
0.52
*Mean of three determinations
Table 7: Result of solution stability study
Sr.
Time
Amount of drug
No.
Estimated (mg/tablet)*
SIT
MET
1
20 min
49.58
494.63
2
1 hr
49.12
503.15
3
3 hr
49.57
496.87
4
8 hr
50.48
496.36
5
24 hr
49.91
492.58
*Mean of three determinations
% Label
Claim*
SIT
99.16
98.24
99.14
100.96
99.82
S.D. (±)
MET
98.93
100.63
99.37
99.27
98.52
3.7. Stability studies
The RSD values obtained for quantitation of SIT and
MET during solution stability experiment were within 2
%. Also, the determination of SIT and MET from the
solution at various time intervals up to 24 hrs did not
show any degradation (Table-7). The results from the
solution stability experiments confirmed that the sample
solutions in methanol were stable up to 24 h during the
assay.
SIT
0.875
1.255
1.781
0.568
0.659
% C.O.V.
MET
1.323
1.458
0.983
1.054
1.563
SIT
0.869
1.246
1.763
0.576
0.652
MET
1.328
1.449
0.971
1.052
1.568
found to be stable in acidic, alkaline and oxidative,
thermal, photo degradation and neutral hydrolysis.
The results for forced degradation studies are included in
Table 8. Typical degradative induced densitograms
obtained for SIT and MET under different stress
conditions are shown in Fig. 5-7
3.8. Forced degradation studies
In the forced degradation studies, the percent recovery at
the level of 71.54% and 79.38 % and additional peaks at
different Rf value indicates, mild degradation of SIT
under basic and oxidative stress condition respectively.
However it was found to be stable to the acidic, thermal,
photo degradation and neutral hydrolysis employed.
MET was found to degrade (percent recovery 82.54%) Fig. 5. Densitogram of alkali [1N NaOH (reflux for 3 h at
80°C)] treated sample.
under UV radiation exposed condition only and was Peak 1, degradant [Rf = 0.02]; Peak 2, degradant [Rf = 0.03];
Peak 3, SIT [Rf = 0.21]; and Peak 4, MET [Rf = 0.49]
478
Fig. 6.
Densitogram of hydrogen peroxide (H2O2 -30%)
[(Reflux for 3 h at 80°C)] treated sample.
Peak 1, SIT [Rf = 0.21]; Peak 2, degradant [Rf = 0.42]; Peak 3,
MET [Rf = 0.48];
Table 8: Results of Force Degradation Study
Sr.
Exposed stress conditions
No
1
2
3
4
5
6
7
8
Acid, 8 ml (1N HCl, reflux for 3 h at 80OC)
Base, 8 ml (1N NaOH, reflux for 3 h at 80OC)
Oxide,8 ml,30%v/v H2O2 (reflux for 3 h at 80OC)
Dry heat (24 h at 60°C)
Wet heat (reflux for 3 h at 80OC)
Photo stability (Daylight, 8 days)
UV (254 nm for 24 h)
Neutral hydrolysis (reflux for 8 h)
Fig. 7.
Densitogram of UV (254 nm) light [24 h] treated
sample.
Peak 1, SIT [Rf = 0.21]; Peak 2, degradant [Rf = 0.42]; Peak 3, MET
[Rf = 0.47];
% drug estimated ± S.D.
SIT
MET
98.26 ± 3.01
71.54 ± 4.25
79.38 ± 1.98
102.52 ± 3.29
98.16 ± 3.24
97.66 ± 3.59
98.21 ± 3.33
102.22 ± 3.12
4. CONCLUSION:
Results closed to 100% for repeatability and intra-day
and inter-day precision with standard deviation <2%
conclude that the developed method is precise. The
percentage recovery at three levels (80%, 100% and
120%) with acceptable criteria < 2 %. gives indication of
accuracy method. The proposed method was studied to
check its robustness property and was found
insignificant change in percent estimation and Rf values
compare with the standard, concluded that the method is
robust and stability indicating.
96.66 ± 2.88
96.65 ± 2.98
98.55 ± 4.51
103.25 ± 3.11
96.33 ± 3.24
102.35 ± 2.45
82.54 ± 3.25
98.26 ± 2.98
Rf value of
degradation
product
Not detected
0.02, 0.03
0.42
Not detected
Not detected
Not detected
0.42
Not detected
Figure
-----Fig. 5
Fig. 6
------------------Fig. 7
-------
reported till date, the comparable data of the previously
published method are not available. Hence, it is
concluded that the proposed HPTLC method can be used
for routine analysis of combined dose tablet formulation
containing Sitagliptin phosphate and Metformin
hydrochloride.
5. REFERENCES:
1.
Validation studies indicate that the proposed method is 2.
suitable for simultaneous estimation of SIT and MET in
pharmaceutical formulation without any interference
from excipients. As the HPTLC method was able to 3.
quantitate Sitagliptin phosphate and Metformin
hydrochloride, in presence of degradation products, it
4.
can be employed as a stability indicating assay method
for determination of these drugs in fixed dose
combination tablets. As there is no HPTLC method
479
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Zeng W, et al. Determination of sitagliptin in human plasma
using protein precipitation and tandem mass spectrometry.
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Xiong Y, et al. A chemiluminescence sensor for determination of
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and metformin HCl in bulk and tablets using UV visible
spectroscopy. International Journal of ChemTech Research. 1
(4); 2009: 905-909.
Lakshmi KS, et al. Development and validation of liquid
chromatographic and UV derivative spectrophotometric methods
for the determination of metformin, pioglitazone and glimepiride
in pharmaceutical formulations. Der Pharma Chemica. 1 (1);
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hydrochloride, pioglitazone hydrochloride, and glimepiride by
RP-HPLC in tablet formulation. Journal of Chromatographic
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Havele S and Dhaneshwar S. Development and validation of a
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gliclazide and piogliglitazone hydrochloride in multicomponent
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(10); 2010:wmc001078
Lakshmi KS, et al. Simultaneous determination of metformin and
pioglitazone by reversed phase HPLC in pharmaceutical dosage
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of metformin and pioglitazone in pharmaceutical formulation.
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Abu RS, et al. The development and validation of liquid
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metformin and glipizide, gliclazide, glibenclamide or glimperide
in plasma. Journal of Chromatography B. 817 (2); 2005: 277286.
Georgita C, et al. Simultaneous assay of metformin and
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hydrochloride and sitagliptin phosphate monohydrate in bulk as
well as in pharmaceutical formulation. Der Pharmacia Sinica. 4
(4); 2013: 47-61.
Ravi PP, et al. Simultaneous estimation of metformin HCl and
sitagliptin phosphate in tablet dosage forms by RP-HPLC.
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646-649.
Shyamala M, et al. Validated RP-HPLC for simultaneous
estimation of sitagliptin phosphate and metformin in
hydrochloride in tablet dosage form. American Journal of
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monohydrate and metformin hydrochloride in bulk and
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Sankar ASK, et al. Development and validation for simultaneous
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Jadhav SB, et al. Development and validation of RP-HPLC and
HPTLC methods for simultaneous estimation of sitagliptin
phosphate and metformin hydrochloride in bulk and dosage form.
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ISSN
0974-4169 (Print)
0974-4150 (Online)
www.ajrconline.org
RESEARCH ARTICLE
Optimization of the spectrophotometric determination of Aqueous
Cyanide: Application on Samira (Niger) Gold Mine Groundwater Analysis
Hassane Adamou Hassane, Rabani Adamou*, Maman Maazou Ahmed, Alassane Abdoulaye
Faculté des Sciences et Techniques, Université Abdou Moumouni, Niamey/Niger.
ABSTRACT:
Cyanide is very toxic to humans, and its usage in gold mining potentially poses serious environmental threats. In
this paper, by revising the well-known colorimetric method based on the chlorination of cyanide with
chloramine-T and subsequent reactions with pyridine-barbituric acid reagent, we have developed a robust and
sensitive UV-Visible spectrophotometric method for cyanide determination in aqueous media.
The optimization of some analytical parameters, like the duration of the colored complex formation (50 min), the
amount of chloramine-T (1 mL) and the medium sodium hydroxide concentration (0.05 M), has allowed us to
highly improve the stability and exaltation of the colored complex absorption signal. The obtained limits of
detection (LOD) and quantification (LOQ) are respectively 0.2 and 0.7 µg L -1. In addition to this sensitivity, our
approach seems to be one of the most reliable methods for free cyanides determination in environmental
matrices. The recovery rate in spiked solutions is more than 95%.
Application on thirteen (13) groundwater samples (drillings, piezometers and open pit mining waters), adjacent
to and around the Samira (Niger) gold mine, has shown that the obtained cyanide rates are less than the World
Health Organization (WHO) drinking-water quality guideline value (70 µg L-1). Nevertheless, the observed
values indicate a potential contamination of the ground water resources, which previously not contained this
pollutant.
KEYWORDS: Cyanide, spectrophotometric method, Samira (Niger) gold mine, groundwater.
INTRODUCTION:
Cyanides are naturally found in environmental matrices
(air, water, plants, microbes and fungi…). Observed
compounds are cyanogen, hydrogen cyanide, simple
cyanides, complex or metallocyanides and organic
cyanides1-3. The most predominant form is hydrogen
cyanide (HCN) at concentrations in the range 1.5 – 1.7 x
10-1ppbv in air, with an average life time of 2.5 years3. In
addition to natural cyanides, anthropogenic activities
result in an important environmental contamination.
Each year, approximately 2 million tons of cyanides are
produced in the world, significantly for mining, chemical
and pharmaceutical industriesneeds4. Elsewhere, plastic
waste and other nitrogen-containing materials
combustion releases a large quantity of hydrogen
cyanide in the atmosphere3.
Cyanide compounds are highly toxics to humans and
other species even in very small doses and many are
rapid-acting poisons3,5-10. Exposure to cyanides harms
the brain and heart and can even cause coma and
death3,11. The effect severity depends on the mode of
exposure and the form of cyanide. Ingestion or
inhalation of a small amount respectively of cyanide
salts or hydrogen cyanide can be deadly regardless.
Indeed, cyanide produces toxic effects at levels of 500
µg L-1 of blood, and death have occurred at levels of
3,000 µg L-1 12.Cyanide environmental impacts are
horrible. On January 2,000, when the Baia gold mine
(Roumania) cyanides spill, Tisza and Danube rivers were
polluted. Besides the ecological damage of their
ecosystem and fauna (fish, birds and carnivores), the
cyanides concentration in some places was 100 times
more than the limit value for drinking water13.
DOI: 10.5958/0974-4150.2015.00077.2
481
According to the above health effects and environmental
impacts, cyanides determination in environmental
matrices requires special attention3,13,14. Therefore, the
goal of this work is to develop a robust, sensitive and
cost-effective method for cyanides analysis in aqueous
media. Different methods are available on the market for
cyanides determination in environmental matrices. These
methods are based on voltammetry15, potentiometry15,16,
titrimetry17, spectrophotometry2,17-24, colorimetry15,
fluorometry15 techniques and are often preceded by a
time consuming distillation, chromatography or microdiffusion pretreatment steps25-29. Most of these methods
are characterized by a lack of sensitivity, reproducibility
and reagent instability3,18. The most sensitive methods
are time consuming and have a prohibitive price2,1921,25,30
. Thus, in order to allow cyanide traces analysis in
groundwater, we have revised the colorimetric method
based on the cyanide chlorination by chloramine-T and
subsequent colored complex reactions by optimizing
some analytical parameters by using the robust and
affordable UV-Visible spectrophotometer.
ethanol. Glacial acetic acid (20%) prepared in distilled
water was used to decolorize the medium.
The obtained optimum conditions were used to establish
a calibration curve which allowed cyanides contents
determination of thirteen (13) groundwater samples of
Samira (Niger) gold Mine, after ten (10) years of
activities. In order to assess the reproducibility of the
developed method, the recovery rates of different
cyanides spiked solutions were measured.
Real samples and spiked solution were collected,
preserved and storage by the Samira gold mine
groundwater sampling equips which is assisted by the
gold mining group environmental supervisor and one
responsible of our laboratory. The samples collection,
preservation and storage procedures were performed
according to the Canadian environment assessment
protocols manual for water quality sampling31.
The different solution were covered with aluminum foil
and conserved in a refrigerator at 4°C. All agents and
chemicals were obtained from Merck and are at least
analytical grade.
Samples collection preservation and storage
Groundwater samples were collected in polyethylene
containers of 500 mL, covered in aluminum foil in order
to avoid cyanides compounds photodecomposition. They
were preserved by using a sodium hydroxide (pH>12)
and conserved in a refrigerator at 4°C. Samples spiked
with potassium cyanide were prepared at the Samira gold
mine site. Spiked solutions were prepared, preserved and
storage in the same conditions as the real environmental
samples. However, neither the nature of the spiked
solutions nor their water cyanides contents are notified to
the researcher until the end of the laboratory work.
EXPERIMENTATION:
Analytical reagents
Potassium cyanide, KCN was used as a standard. The
stock solution (100 mg L-1) was prepared by dissolving
potassium cyanide in a sodium hydroxide solution (0.1
mol L-1) prepared in distilled water. Working solutions
were prepared by diluting the stock solution by the same
sodium hydroxide solution. For samples cyanide
chlorination (formation of CNCl), a solution of
chloramine-T (10 g L-1) is prepared in distilled water
using chloramine-T trihydrate (CH3C6H4SO2NClNa.
3H2O, 98%). The coloration solution was prepared by
dissolving sodium hydroxide (1.75 g), barbutiric acid
(4.2 g), pyridine-4-carboxylic acid (3.4 g) in 250 mL of
distilled water. The medium pH is adjusted to 5.2 with a
1M sodium hydroxide solution. The obtained mixture
was vigorously stirring for 1hour at 30 °C with a Velp
Scientific (Italy) heating and magnetic stirrer. After that,
it was filtered through a Whatman paper before usage. In
order to follow the coloration reaction, an indicator
solution of phenolphthalein (0.1%) was prepared in
Apparatus
All the absorbance measurements were performed at
room temperature with a double beam Thermo Evolution
300 spectrophotometer. A standard Hellma (Mulheim
Germany) quartz cuvette (path-length ℓ= 1 cm) and
micropipette Pipetman (5-50 µm or 10 – 100 µm, Gilson
–France) were used. The analytical medium pH
adjustment was done by a BASIC pH meter 20 Crison.
Principle of the method and analytical measurements
Principle of the method
The most important step of the method is the
chlorination of cyanide by chloramine-T to form
cyanogen chloride (CNCl), a relatively more stable
cyanide compound. After that, formed CNCl will react
with pyridine-barbituric acid to forma colored complex
with a maximum absorbance at 598 nm. The obtained
complex concentration is proportional to the medium
cyanides content. The two main steps of the colored
complex formation are described in figure 132, 33.
482
Figure 1-a : Cyanide chlorination with chloramine-T
Figure 1-b: Formation of the colored complex with pyridine-barbituric acid
Analytical measurements
To 20mLof a known standard working solution of
cyanide into a volumetric flask of 50 mL, 20 mL of 0.1
M NaOH solution were added. After that, two drops of
phenolphthalein (0.1%) were introduced in the medium.
A pink coloration, characteristic of phenolphthalein
color in alkaline medium, is observed. Under a smoothly
magnetic stirring, 2 mL of the glacial acetic acid solution
(20%) were required to decolorize the medium in order
to obtain an appropriate pH for the chlorination reaction.
Thereafter, in maintaining the magnetic stirring, 2mL of
chloramine-T trihydrate were added in the decolorized
solution. After only 2 minutes, 5 mL of the coloration
reagent were added. Finally, the 50 mL flask is
completed with distilled water. At the beginning, a red
staining color was observed which progressed and
stabilized finally to violet coloration.
Absorption spectra were recorded over the range 450 to
750 nm against a blank. The colored complex spectra
presented two peaks located at 525 and 598 nm. The last
wavelength (598 nm) corresponding to the height peak
of the intense and broad absorption band between 550 –
650 nm, was selected for the analytical signal
investigations (Figure 2). The absorbance values for
performing the calibration graph were acquired by
measuring the absorption signal of a series of standard
working solutions.
All the analytical measurements were carried out under
the same conditions. Measurements were performed
under incandescent light and working solutions of
different concentrations were protected from solar
ultraviolet radiations with aluminum foil in order to
avoid their photodecomposition. Curves representation
and statistical processing of data were carried out with
After a time (t), the solution absorbance measurement is the software ISIS Draw 2.4 and Microcal Origin 6.00.
performed using the spectrophotometer Evolution 300.
483
1.6
1.4
Absorbance
1.2
1.0
598
Operating conditions
Solvent: water
[NaOH] = 0.1 M
V Cl-T = 2 mL
-
-1
[ CN ] = 1,000 µg L
t = variable
0.8
0.6
525
0.4
0.2
0.0
450
500
550
600
650
700
750
Wavelength (nm)
Figure2: Absorption spectrum of the colored complex
RESULTS AND DISCUSSIONS:
Optimization of operating conditions
Optimization of each analytical parameter was
performed independently. The initially used reagent rates
were obtained from the official colorimetric reference
method
of
environmental
matrices
cyanides
determination with chloramines-T and subsequent
pyridine-barbituric acid reactions2. In parallel,
alternating variable search during the optimization
0.24
0.22
0.20
Absorbance
0.18
0.16
Solvent: water
[NaOH] = 0.1 M
V Cl-T = 2 mL
-
process was performed to found the appropriate
concentration value for each reagent.
Optimization of the colored complex formation time
Optimization of the colored complex formation time (t)
was performed with a standard concentration of cyanides
100 µg L-1. Absorbance values were measured during 0
to 90 minutes period. The obtained absorption spectra
were presented in figure 3.
598
50'
90'
-1
[ CN ] = 100 µg L
t = variable
0'
8'
0.14
0.12
525
0.10
0.08
0.06
0.04
0.02
450
Blank
500
550
600
650
Wavelength (nm)
Figure 3: Colored complex absorption spectra change in time
484
700
750
At the beginning (from 0 - 15 min), one observed, on the
colored complex formation spectra (Figure 3), the
presence of two broad absorption bands with their
maximum peaks respectively at λ1 = 525 nm and λ2 =
598 nm. Here, the most important absorption is observed
between 450 to 550 nm with a maximum peak at 525
nm. Over time, this peak is weakened and one assisted to
a bathochromic effect accompanied with an important
increase of the absorption intensity around 598 nm
(hyperchromic effect). The observed changes were
probably due to the presence of two complexes in the
medium. The firstly formed complex has an extinction
coefficient 1 =2.418 104 L mol-1cm-1with an absorption
maximum located around 525 nm. This complex is
responsible of the firstly red coloration observed in the
medium. The second complex has an extinction
coefficient 2 =5.538104 L mol-1 cm-1 and its absorption
band extend from 550 to 650 nm with a maximum
absorption peak at 598 nm. This complex is responsible
of the observed violet coloration. It’s relatively more
stable than the first formed complex which coloration
disappeared along the time. Indeed, during the used
period 0–90 min, the medium coloration progressed from
red and stabilized to violet. The above results validated
the choice of the wavelength λ2 = 598 nm for the colored
complex study after the optimized reaction time.
The representation of the complex absorption intensity
versus time will give us more information on the
optimized duration of the colored complex formation
(Figure 4).
50
0.22
0.20
Absorbance
0.18
Solvent: water
[NaOH] = 0.1 M
V Cl-T = 2 mL
0.16
0.14
8
-
-1
[ CN ] = 100 µg L
t = variable
0.12
0.10
0
20
40
60
80
100
Time (minutes)
Figure 4:Absorption of the colored complex in time at λ2 = 598 nm
An increase in the absorbance intensity of the colored
complex is observed in the used period 0 -90 minutes
(Figure4). The absorption signal increased and reached a
plateau after 50 minutes. Then, it remained almost
constant until 90 minutes. Thus, approximately fifty (50)
minutes was needed for the complete development of the
violet coloration.
In many methods used worldwide, including the
reference method for cyanides determination in
environmental matrices2, the optimal duration observed
for the colored complex formation is eight (8) minutes.
The present optimization study has shown us that this
duration is insufficient for the complete formation of the
colored complex. This is probably one of cyanides rate
under estimation in environmental matrices. Indeed, a
concentration of cyanide measured after 8 minutes
represented only the 3/5 of the exact cyanides
concentration present in the medium. Moreover, in
figure 4, the colored complex absorbance signal at just
eight (8) minutes is instable, that may partly explain
many observed errors in different laboratories for a same
cyanides sample. Hence, a small gap in the signal
measurement time will result in a different absorbance
485
value and a different cyanides rate of the sample.
Therefore, additionally to the gain of sensitivity, the
relative stability of the colored complex absorbance
signal after 50 minutes, will improve the reliability of
cyanides determination method. Hence, in this study
duration of 50 minutes is observed for the colored
complex formation before performing any absorbance
signal measurement.
Optimization of medium chloramine-T content
Chloramine-T allows the formation of cyanogen chloride
(CNCl), an important step in the medium cyanide ions
mobilization for the formation of the colored complex.
In the reference colorimetric method, 2 mL of
chloramine-T were used for the chlorination step. In this
study, in order to optimize the medium chloramine-T
content, different volumes of chloramine-T trihydrate
(VCl-T) ranged from 0 to 5 mL were used for a fixed
concentration of cyanide [CN-] =200 µg L-1.
Otherwise, in order to reduce the complete analysis
duration, all the samples series needed for the medium
chloramine-T content optimization were prepared
together. For each sample, the beginning time is noticed
in order to firmly respect the 50 minutes needed for the
complete formation of the colored complex. Before the
first sample absorbance measurement, the base line is
performed with the reference. The reference contained
also the used cyanide rate (200 µg L-1) and its
preparation respects all the colored complex formation
steps except absence of chloramine-T in the medium.
The absorption spectra of the different absorbance
measurements were shown in figure 5.
Solvent: water
0.35 [NaOH] = 0.1 M
-1
[CN ] = 200 µg L
0.30 t = 50 minutes
V Cl-T = variable
Absorbance
0.40
0.25
0.20
0.15
0.10
0.05
a (0 mL of Cl-T)
0.00
450
500
550
600
650
700
750
Wavelength (nm)
Figure 5: Absorption spectra of the medium chloramine-T content optimization
The absence of noticeable absorption at 598 nm in the
reference sample spectrum (Figure 5, spectrum a)
indicates the great importance of chloramine-T in the
process of the colored complex formation. The
remaining absorption spectra indicate that the addition of
chloramine-T in the reaction medium results in the
formation of the colored complex. However, the medium
chloramine-T contents didn’t correlate with the
absorbance signal increases. Indeed, the curve
representing the measured absorption intensity versus
added chloramine-T content shows an important
fluctuation in the studied range (Figure 6).
486
0.40
0.35
0.395
0.25
0.30
0.25
0.1
1
0.385
Absorbance
Absorbance
0.390
0.20
0.15
0.380
0.375
2
0.370
0.10
0.365
0.05
0.0
0.5
1.0
1.5
2.0
V Cl-T (mL)
0.00
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Solvent: water
[NaOH] = 0.1 M
-1
[CN ] = 200 µg L
t = 50 minutes
V Cl-T = variable
4.0
4.5
5.0
5.5
6.0
V Cl-T (mL)
Figure 6: Curve of chloramine-T volume optimization
In figure 6, the general aspect of the obtained curve
seems to show an increase of the absorbance signal
followed by a relative slight decrease respectively for the
ranges 0 to 1 mL and 1 to 5 mL. However, a focus
between 0.1 to 2 mL shows significant fluctuations of
the colored complex absorbance signal with the medium
chloramine-T content (Figure 6inset). In the used range,
the important absorption intensities were obtained for
0.1, 0.25, 1 mL of chloramine-T trihydrate. Here, the
absorption signal highly fluctuates for 0.1 and 0.25 mL
of chloramine-T while it’s relatively stable around 1
mL.
This investigation shows that a dosage of cyanides with
chloramine-T content of 0.1 or 0.25 mL, despite the
relative gain in sensitivity, will be a major source of
analytical errors. Indeed, a small uncertainty on the used
volume will give different values for the medium
cyanide concentrations. Therefore, according to the
relative high concentration of the reagent and the
potential uncertainty on the medium chloramine-T
content, the volume 1 mL seems to be the more
appropriate for the method precision improvement.
Traditionally, the used amount of chloramine-T in
cyanides analysis is 2mL. At this concentration, the
measured absorption intensity for the colored complex is
weak and less stable compared to results observed for 1
mL. Thus, the use of 1 mL of chloramine-T in the
reaction medium will give more accurate measurements
and will improve the developed method sensitivity.
Moreover, the chosen content (1 mL) will help to save
50% of used reagent at each measure.
Optimization of NaOH content in sample
preservation
Real water samples collected for cyanides analysis were
preserved in high alkaline medium, pH greater than 12.
In practice, in order to avoid samples contamination
three (3) or four (4) NaOH pellets were generally added.
This practice would be a potential source of errors in the
case that the medium NaOH content will interfere in the
colored complex formation. For this reason, we have
performed the colored complex formation with a
standard solution of cyanide (100 µg L-1) prepared in
different concentrations of sodium hydroxide. The pH
concerned in this study was ranged between 12 and 14.
Obtained absorption spectra were shown in Figure 7.
487
0.24
0.20
Solvent: water
V Cl-T = 2 mL
-
0.18
Absorbance
0.16
0.05 (pH = 12.7)
-1
[CN ] = 100 µg L
t = 50 minutes
[NaOH] = variable
0.05 M
0.22
0.2 M
0.1 M
0.15 M
0.20
0.3 M
0.14
0.01 M
0.5 M
Absorbance
0.22
0.18
0.16
0.12
0.10
0.14
0.08
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55
0.06
-1
[NaOH] (mol L )
0.04
0.02
450
500
550
600
650
700
750
Wavelength (nm)
Figure 7: Optimization of the medium NaOH concentration
In figure 7, it was observed that the preservation NaOH
content greatly influenced the colored complex
absorption intensity. Indeed, one assisted to an
hyperchromic effect, when the NaOH content of the
medium increases from 0.01 to 0.05 M (pH = 12 to 13).
Beyond this value, a general hypochromic aspect is
observed for pH = 13 to 14 (Figure 7 in set).
Therefore, it’s very important to know the used NaOH
content for cyanide samples preservation. If note, a pH
correction of the medium have to be conducted in the
laboratory prior to the colored complex formation and
analysis in order to exclude observed potential errors.
Here, NaOH 0.05 M (pH=12.7) gives the relatively
higher absorption intensity. This concentration was
chosen in order to improve the sensitivity of our method.
Calibration curve and analysis of real samples
Calibration curve
The calibration curve is performed under the following
optimal conditions obtained during the analytical
parameters optimization studies:
 The maximum absorption signal is measured at
598 nm,
 The observed duration for the colored complex
is 50 minutes,
 The volume of chloramine-T used in the
reaction medium is equal to 1mL,
 The sodium hydroxide concentration used in the
reaction medium is 0.05M.
The working standard diluted solutions were used to
perform the calibration curve. Serial dilutions between 0
to 250 µg L-1 were prepared. The working solutions were
scanned between 450 to 750 nm. All absorbance signal
measurements were carried out in triplicate and the
results were expressed as a mean values. The curves
displaying the absorbance spectra versus cyanides
concentration is shown in figure 8.
488
0.45
0.40
0.35
Absorbance
0.30
0.25
Solvent: water
[NaOH] = 0.05 M
V Cl-T = 1 mL
-1
250 µg L
-
[CN ] = variable
t = 50 minutes
0.20
-1
0 µg L
0.15
0.10
0.05
0.00
450
500
550
600
650
700
750
Wavelength (nm)
Figure 8: Spectra displaying the variation of the colored complex absorption signal versus cyanides concentration
In figure 8, it was found that, more the concentration of
cyanide increased the colored complex absorbance signal
was improved. The representation of the curve
displaying the measured absorbance versus solutions
cyanide concentration which represented the calibration
curve A = f (C) is plotted in figure 9. In the studied
cyanide concentration range (0-250 µg L-1), the
treatment of the data by linear regression analysis gives a
satisfactory correlation (R2=0.99963).
0.45
0.40
0.35
Absorbance
0.30
Solvent: water
[NaOH] = 0.05 M
V Cl-T = 1 mL
-
[CN ] = variable
t = 50 minutes
0.25
0.20
0.15
-3
0.10
-
-2
A = 1.43 10 [CN ] + 4.322 10
2
Correlation Cofficient, R = 0.99963
-1
Range: 0 - 250 µg L
0.05
0.00
0
25
50
75
100
125
-
150
-1
[CN ] (µg L )
Figure 9: Calibration curve
489
175
200
225
250
275
Analytical figures of merit of our method
This curve is used to determine the limits of detection
(LOD) and quantification (LOQ), which are respectively
given by the following formulas: LOD= 3 s and LOQ=
10 s, with S the standard deviation of 10 replica of a
relatively low solution concentration of 1 µg L-1
cyanides37. After that, the absolute limit of detection
(ALOD), calculating using 2.5 mL is determined. To
assess the relevance of these two values, we have
calculated the ratio of conformity (R') of the method
which is expressed by the formula,
, where
is the average calculate concentration value34. The
obtained ratio of conformity R' is 6. A value of R’
between 4 and 10 means that the used concentration is
adequate for the LOD and LOQ determination.
(Pt1, Pt2, Pt3, Pt4, Pt5) open pit mining waters issued for
the mineral extraction and used as industrial water. All
these installations are adjacent to and around the gold
mine.
In order to assess the accuracy of the developed method,
two (2) kind of spiked samples were analyzed. The
cyanides concentrations obtained for the control
samples, RV= 0 µg L-1 and RF= 2.101 mg L-1. The
obtained results are almost identical to the used spiked
concentrations. Indeed, the two solutions were prepared
with a commercial drinking water called Rarhous. The
RV sample (Rharous Virgo) not contained cyanides and
the second sample RF (Fortified Rharous) contained
2 mg L-1 of cyanide issued from potassium cyanide
pellets.
The performances of our method are listed in Table1.
Table 1: Merit of our method
Parameters
Absorption maximum (nm)a
Linearity range (µg L-1)b
Correlation coefficient (R2)c
Recovery rate (%)d
Limit of detection (µg L-1)e
Limit of quantification (µg L-1)f
ALOD (µg)g
Ratio of conformityh
Precisioni
Obtained absorbance intensities for real samples were
used in the calibration curve to determine their cyanides
concentrations. The calculated cyanide contents were
shown in Table 2.
Results
598
0–250
0.99963
>95
0.2
0.7
0.5
6
99,6% (intra-day),
98,2% inter-day
a
Wavelength chosen of the analysis, bRange linear dynamic
concentration, cCorrelation coefficient, dRecovery rate, eThe limit of
detection of the method, fThe limit of quantification of the method,
g
Absolute limite of detection, hRatio of conformity, iprecision of intraday analysis (n=6) and inter-days (one week).
The obtained method with a recovery rate superior to
95% and a very weak relative standard deviation (%
RSD = ±0.5 %, n = 6) has a very significant accuracy.
The intra-day and inter-days analysis results comparison
have shown that the method precision is excellent (>
98%). The observed limits of detection (LOD) and
quantification (LOQ) which are respectively 0.2 and 0.7
µg L-1 indicate the very high sensitivity of the method,
more than others colorimetric methods19,20. Therefore,
the revised UV-Visible spectrophotometric method will
be suitable for cyanides traces analysis in aqueous
media.
Application
The developed method was applied to determine
cyanides content in groundwater samples of Samira
(Niger) gold mine. Investigations were focused on the
area around industrial process residues tailings. The
groundwater sampling concerned three (D1, D2, D3)
drillings which are used as drinking water supply for the
mine and surrounding villages, five (P1, P2, P3, P4, P5)
piezometers developed to control the industrial process
residues tailing impacts on the groundwater and five
Table 2: Concentrations given in free cyanides samples
Ground water source
Absorbance
Measured
cyanide
(µg L-1)
P1 (FCD-P1)
0,053
6,8
P2 (FCD-P2)
0,047
2,6
Piezometers
P3 (FCD-P3)
0,048
3,3
P4 (FCD-P4)
0,041
0
P5 (FCD-P5)
0,043
0
Pt1 (MLNW)
0,042
0
Pt2 (MLB)
0,044
0,5
Pits
Pt3 (MBD)
0,044
0,5
Pt4 (MSM)
0,044
0,5
Pt5 (LP)
0,047
2,6
D1 (FS)
0,04
0
Drillings
D2 (Tw02)
0,044
0,5
D3 (Tw05)
0,042
0
Before the mining activities Samira region groundwater
sources were free of cyanides. Therefore, cyanide traces
detected in the analyzed ground water would come from
interactions between the gold mining activities and the
ground water resources. However, the maximum content
cyanide observed (6.8 µg L-1) is lower than the limit
values in drinking water allowed in the matter by the
World Health Organization (WHO)
standards
(70 µg L-1)35 and the European Union (EU) (50 µgL-1)36.
CONCLUSION:
The use of UV-Visible spectrophotometric instrument
and the optimization of the main analytical parameters
allowed us to overcome many short comings regarding
the instability and inhibition of the absorbance signal of
the colored complex observed in current methods used
for cyanides analysis in environmental matrices. The
developed method is robust, cost-effective and covers a
large range of concentrations.
490
The method analytical figures of merit show that it could 14. Dzombak DA, Ghosh RS, Wong-Chong GM. Cyande in water
and soil: Chemistry, Risk, and Management. Taylor and Francis:
be a simple and cheaper alternative to gain sensitivity
Boca Raton London New York; 2006.
and reliability, which traditionally requires a heavy and 15. Ma J and Dasgupta PK. Recent developments in cyanide
expensive instrumentation. The application of our
detection: A review. Anal ChimActa. 673(2); 2010: 117–125.
method on spiked solutions and real gold mining 16. United States Environmental Protection Agency. Potentiometric
determination of cyanide in aqueous samples and distillates with
groundwater gives satisfactory results.
ACKNOWLEDGMENTS:
This research was founded trough a SOPAMIN (Société 17.
de Patrimoine des Mines du Niger) research grant.
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492
ISSN
0974-4169 (Print)
0974-4150 (Online)
www.ajrconline.org
RESEARCH ARTICLE
Synthesis and Biochemical Investigation of
(Thiazin, Oxadiazol, Thiadiazol )- Derivatives
Zinah Hussien Ali
Assistant Lecturer, Department of Pharmaceutical Chemistry, College of Pharmacy,
*Corresponding Author E-mail: zenaa.77 @yahoo.com
ABSTRACT:
The study involved synthesis of six and five- membered rings from hetero cyclic compounds containing sulfur
and nitrogen with oxygen atoms such as (thiazine, oxadiazol, thiadiazol)- derivatives., some of compounds were
prepared from condensation reaction and other by chalcone. The synthesized compounds [1-10] were
characterized by many methods {FT.IR- spectra, H.NMR- spectra, (C.H.N)- analysis} and tested for their
potential antibacterial {Gram(+) positive and Gram(-) negative} and melting points .
KEYWORDS: Thiazine, chemical, oxygen.
INTRODUCTION:
The oxadiazol, thiazine and thiadiazole nucleus are a
useful structure for research and development of new
pharmaceutical molecules ,it found in several natural and
non- natural products., most of sulfur and nitrogen hetero
cycles derivatives are marketed as anti-Psychotic drugs,
antifungal, anti-thelmintic, antibacterial, anticancer,
HIV- Inhibitors, anti- hypertensive, anti- allergic,
anticonvulsant, anti-tubercular, anti- inflammatory
activity, and some of derivatives have been found to
possess some interesting bioactivities such as anti
diabetic activity(1-4) .
Melting points were determined by electro thermal 9300,
LTD, FT.IR- Shimadzu 8300, KBr-disc, H.NMRspectra in DMSO- solvent and (C.H.N)- analysis in
Kashan University in Iran, biological tests in Bio-Lab,
Biology Department in College of Education .
These derivatives exhibit adverse biological activities
possibly due the present of (N-C-S) moiety, which are
very interesting compounds for their applications in
pharmaceutical and analytical fields(5-7). In addition,
these derivatives have been used for the preparation of
structures in polymers .
Synthesis of Compounds [1 - 3]:
According to procedures(8,9), a mixture of 2-amino
imidazole (0.01mole) and chloro ethyl acetate
(0.02mole) was reacted in presence of ethanol with
potassium hydroxide under magnetic stirrer., then
filtered and recrystallized to produce (84%)
of
compound [1], which (0.01mole) refluxed with
(0.02mol) of thiosemicarbazide in presence of absolute
ethanol for (3hrs), after filtered and recrystallized to
yield (84%) of compound [2], which cyclized by
addition (POCl3) and refluxed in ethanol for (5hrs) to
yield (82% ) of compound [3] .
Synthesis of Compounds [4 , 5]:
According to procedure(9), a mixture of compound [1]
(0.01mol) and semicarbazide (0.02mole) was refluxed in
present of absolute ethanol for (3hrs) ,then filtered and
recrystallized to yield (86%) of compound [4]., which
(0.01mole) cyclized in presence of POCl3 with ethanol to
produce (82%) of compound [5] .
DOI: 10.5958/0974-4150.2015.00078.4
493
Synthesis of Compounds [6 , 7] :
According to procedure(9), a mixture of (0.02 mol) of 2amino thiazol and (0.01mol) acetyl acetone was refluxed
in presence of absolute ethanol and drops of glacial
acetic acid for (2hrs) ,then filtered and recrystallized
with ethanol to yield (84%) of compound [6], which
(0.01mol) reacted at room temperature with (0.01mole)
benzadehyde in presence of (10% NaOH) to yield (82%)
of compound [7] .
Synthesis of Compounds [8 -10]:
According to procedure(9) ., a mixture of (0.02mol) Phydroxyl benzaldehyde and (0.01mol) chloro acetyl
chloride was reacted in presence of basic medium
(KOH)., the solid filtered and dried, recrystallized to
yield (82% ) from compound [8], which (0.01mol)
reacted with (0.02mol) acetophenone
at room
temperature in presence of ethanol with (10% Na OH) to
yield (84%) compound [9], which (0.01mol) refluxed
with (0.02mol) of thiourea in presence of ethanol with
5ml (HCl), then filtered and dried, recrystallized to yield
(84%) compound [10].
PoCl3
N
N
H 2N
N-N
S
CH 2
N-N
NH-CH2
NH 2
S
[3]
Bis(thiadiazol derivative)
N
N
NH-CH2 -CO-NH-NH-CS-NH 2
2(NH2 -NH-CS-NH2 )
CH 2-CO-NH-NH-CS-NH2
[2]
N
N
N
NH 2 + 2ClCH 2 COOC2 H 5
NH-CH2 -COOC2 H5
N
CH 2
H
COOC 2H 5
[1]
N
N
NH-CH2 -CO-NH-NH-CO-NH2
CH 2-CO-NH-NH-CO-NH 2
[4]
PoCl3
N
N
N-N
N-N
H 2N
O
NH-CH2
CH 2
[5]
Bis(oxadiazol derivative)
494
O
NH 2
CO-CH 3
N
2
N=C
NH 2 +
S
CH 3
CH 3
N
C=N
N
S
S
CO-CH 3
N
S
CH 3
Ph-C HO
[6]
CH 3
N=C
N
C=N
S
[7]
CHO + ClCO- CH2 Cl
2 HO
OHC
O-CO-CH 2-O
CHO
[8]
O
O
2(Ph-COCH 3 )
Ph
OCO-CH2 -O
Ph
[9]
NH 2
N
Ph
NH 2
S
N
2(NH2 -CS-NH2 )
S
OCO-CH2 -O
Ph
[10]
Bis(Thiazine derivative )
495
RESULTS AND DISCUSSIONS:
This study involved, synthesis of heterocyclic
compounds (five and six) –membered rings such as
thiadiazol and oxadiazol with thiazin rings, these
compounds [1-10] contain imidazole and thiazol in
their structures which cause biological activity. All
synthesized compounds [1-9] have been characterized by
spectrophotometer chemical methods [FT.IR, H.NMR,
(C.H.N)- analysis], melting points
and
physical
properties with biological study :
The FT.IR- spectrum : showed an absorption band at
1722 cm-1 due to carbonyl of ester (-COO-) in compound
[1], which disappeared and other bands appeared such as
1690 cm-1 for carbonyl of amide (CO-NH), bands at
(3290 cm-1, 3310 cm-1) for amine group (NH2) in
compound [3] ,band at (1686)cm-1 for carbonyl of amide
Table (1): FT.IR-data (cm-1) of Compounds [1-10]
Comp.
(C=N)
NH , NH2
No.
endocycle
1610
3190
[1]
1608
3205
[2]
1605, 1618
3290,3310
[3]
1610
3220
[4]
1604, 1618
3280,3300
[5]
1612
/
[6]
1608
/
[7]
/
/
[8]
(CO-NH) in compound [4], bands at (1604, 1618)cm-1
for (C=N) end o cycle of oxadiazol rings and (3280,
3300) cm-1 for primary amine group (NH2) in compound
[5] . other bands at 1630 cm-1 for (C=N) and 782 cm-1 for
(C-S) of thiazol ring in compound [6], band at 3102 cm-1
for (=CH) alkene in compound [7]., bands at {1710 cm-1
for carbonyl of aldehyde (CO-H), 1728 cm-1 for carbonyl
of ester, 1230 cm-1 for (C-O-C) ether in compound [8].,
bands at 1728 cm-1 for carbonyl of ester, 1687 cm-1 for
carbonyl of chalcone, 1235 cm-1 for (C-O-C) ether, 3110
cm-1 for (CH=CH) alkene in compound [9], bands at
(3280, 3300) cm-1 for primary amine group (-NH2), 1725
cm-1 for carbonyl of ester, 3105 cm-1 for (=CH) alkene,
1235 cm-1 for (C-O-C) ether, 795 cm-1 for (C-S) in
thiazine ring in compound [10]., and other bands(10-13)
listed in table (1) .
(-COO)
Other groups
1722
/
/
/
/
/
/
1728
(CH)aliph : 2982
(CH) aliph : 2955., (CO-NH) amide: 1690
(CH) aliph: 2975
(CH) aliph: 2982., (CO-NH) amide: 1686
(CH) aliph: 2998
(CH) aliph: 2981., (C=N): 1626 (C-S): 782
(CH)aliph: 2973., (C=N): 1630., (=CH) alkene: 3102
(CO-H) carbonyl of aldehyde: 1710., (C.O.C)ether: 1230.,
(CH)aliph: 2965., (CH) arom: 3080
(CO-CH=CH) chalcone: 1687., (C-O-C)ether: 1235.,
(CH)aliph: 2990., (CH=CH): 3110
(=CH)alkene: 3105., (C-O-C)ether: 1242., (CH) aliph: 2982.,
(CH)arom: 3040., (C-S): 795
[9]
/
/
1720
[10]
1614
3280,3300
1725
Fig ( 1 ) : FT.IR of Compound [ 2 ]
496
497
H.NMR- spectrum: showed signals at δ (3.8-4.30) for
(COOC2H5) ethyl of ester in compound [1], which
disappeared and other signals appeared at δ(5.0 , 5.25 ,
5.34) for (NH2 , NH) , δ 10.02 for (NH-CO) amide in
compound [2], signals at δ(4.09, 5.20) for (NH 2 , NH)
groups in compound [3] ,signals at δ(5.10, 5.25) for
(NH) groups, signals at δ(10.04- 10.28) for amide groups
(NH-CO) in compound [4],signals at δ(5.21, 5.03) for
(NH, NH2) amine groups in compound [5], signals at
δ(7.93) for protons of thiazol ring in compound [6],
signal at δ 6.04 for alkene (C=CH), signal at δ 7.10 for
Table (2): 1H.NMR - data (δ, ppm) of compounds [1-10]
Comp.
NH , NH2
(COOCH2-)
No.
ester
5.02
(3.8- 4.30)
[1]
5.0 ,
/
[2]
5.25 ,
5.34
5.09,
/
[3]
5.20
5.10 ,
/
[4]
5.25
5.21,
/
[5]
5.03
/
/
[6]
/
/
[7]
/
3.98
[8]
/
3.86
[9]
5.08
3.94
[10]
protons of phenyl group., signals at δ 3.98 for ester
(COOCH2-), signal at δ 11.82 for proton of aldehyde
group (CO-H), signals at δ (6.9- 7.4) for protons of
phenyl groups in compound [8]., signals at δ 3.86 for
ester (COOCH2-), signals at δ (6.52- 7.63) for protons of
phenyl groups, signals at δ (5.72 , 5.85) for alkene
(CH=CH) in compound [9], signals at δ 5.08 for (NH 2),
signal at δ 3.94 for ester (COOCH2-) ,signals at δ(6.76 7.54) for protons of phenyl groups., and other signals for
functional groups(14-17) are listed in table (2) .
(CH2),
(CH3)
0.95, 1.15
1.0 , 1.15
Other groups
0.98, 1.18
Protons of imidazole ring : 7.91
1.0 , 1.20
0.96, 1.13
Protons of imidazole ring : 7.84., (CO-NH) amide and (CONH2): (10.04-10.28)
1.04, 1.21
1.03
/
/
/
Protons of thiazol ring : 7.93
Protons of thiazol ring : 7.78., (C=CH): 6.04., Phenyl ring: 7.10 .
(HC=O) aldehyde: 11.82., Phenyl rings (6.9- 7.4)
Phenyl rings : 6.52- 7.63., (CH=CH) chalcone: 5.72, 5.85
Phenyl rings : 6.76- 7.54 .
Fig ( 5 ) : H.NMR of Compound [ 8 ]
498
Protons of imidazole ring : 7.86.,
(CO-NH) amide: 10.02
The (C.H.N)- analysis : the microanalytical of carbon, and other physical properties are listed in tables (3) and
Hydrogen , Nitrogen atoms, melting points, solubility (4).
Table (3): Physical properties and (C.H.N)- analysis of compounds [1-10]
Comp.
M.F
M.P(Co)
Calc. /found
No.
C%
C11H17N3O4
134
51.76
[1]
51.42
C9H15N9O2S2
174
31.30
[2]
31.18
C9H11N9S2
208
34.95
[3]
34.74
C9H15N9O4
166
34.50
[4]
34.28
C9H11N9O2
200
38.98
[5]
38.77
C11H12N4S2
180
50.00
[6]
49.82
C18H16N4S2
190
61.36
[7]
61.19
C16H12O5
182
67.60
[8]
67.39
C32H24O5
220
78.68
[9]
78.40
C34H28N4O3S2
232
67.54
[10]
67.31
Table (4): Analytical properties of compounds
Comp.
Color
No.
Yellow
[1]
Yellow
[2]
Yellow
[3]
Pale yellow
[4]
Yellowish orange
[5]
Orange
[6]
Yellowish Orange
[7]
Yellow
[8]
Yellowish Orange
[9]
Orange
[10]
Product %
84
84
82
86
82
84
82
82
84
84
499
H%
6.66
6.41
4.34
4.21
3.55
3.33
4.79
4.64
3.97
3.80
4.54
4.31
4.54
4.30
4.22
4.09
4.91
4.68
4.63
4.38
Solubility in solvents
(Good solvents )
Ethanol , DMSO
Ethanol , DMSO
Ethanol , DMSO
Ethanol , DMSO
Ethanol , DMSO
Ethanol , DMSO
Ethanol , DMSO
Ethanol , DMSO
Ethanol , DMSO
Ethanol , DMSO
N%
16.47
16.23
36.52
36.36
40.77
40.59
40.25
40.14
45.48
45.25
21.21
21.10
15.90
15.72
/
/
/
/
9.27
9.12
Biological Study: (8 , 9)
Bacteria supplied from bio-Lab in college of Education
.,antimicrobial activity was tested by the filtered paper
disc diffusion method against gram (+) positive bacteria
(Staphylococcus aureus ) and gram (-) negative bacteria
(E-coli), (0.1mol) of the bacterial suspensions was
seeded on agar .To determine minimum inhibitory
concentration (MIC) for each compounds [1-10] were
performed with two replicates .
REFERENCES:
1.
2.
3.
4.
5.
6.
7.
Generally, the results showed that the compounds [1-10]
have good inhibitory effect against tested bacteria.
Table (5) showed the zone of inhibition of the
compounds [1-10] in this study ranged (from 32 to 10)
mm . from results , we noted the compounds [3, 5, 7, 10]
have higher antibacterial activity against two type of
bacteria (G+ and G-) due to their structures (consist of
thiazole and imidazole rings with thiazine rings)
consequently ,which it become more effective in
precipitating proteins on bacteria.
Table (5): Antibacterial Activity of Compounds [1-10]
Compounds
Diameter of zone (MM)
G+ :
G- :
Staphylococcus aureus
E-coil
16
14
Compound [1]
22
16
Compound [2]
32
28
Compound [3]
18
12
Compound [4]
30
24
Compound [5]
24
16
Compound [6]
26
20
Compound [7]
14
10
Compound [8]
14
8
Compound [9]
28
20
Compound [10]
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
500
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