Validated Method for the Quantification of Sodium Azide in a Range of ‘Sartan’ Drugs by Ion Chromatography
Dharmendra Kumar Kushwah*, Prakash Yashwant Kohle, Rajesh D Joshi, Bakul Rajyaguru, Rajeev Pandey and Brajesh Vishwakarma
CTX Life sciences Pvt. Ltd., 251-252, Sachin Magdalla Road, Sachin, Surat, Gujarat-394230, India
*Corresponding Author E-mail: * kushwahd@rediffmail.com / kushwahd73@gmail.com
ABSTRACT:
Sodium azide is harmful to our body organs specially heart and brain, because heart and brain use a lot of oxygen, Sodium azide prevents the cells of the body from using oxygen. For the determination of content of free Sodium azide in a range of sartan drug, a method has been developed and validated on Ion Chromatograph (IC). Strongly basic anion exchange resin column (250 x 4.0) mm is used with 50 x 4.0 mm guard column of same stationary phase. Mobile phase consisted of 2.8 ml of Sulphuric acid per litre of water. The method is validated for its Specificity, Precision, Accuracy, Linearity Ruggedness and Robustness. Sodium azide (as azide) is linear from 0.082µg/ml to 0.61 µg/ml, with Correlation Coefficient 0.99933. Limit of quantitation of the method is 0.082 µg/ml and Limit of Detection is 0.041 µg/ml.
KEYWORDS: Sodium azide, IC, Ion exchange column, Limit of Quantitation, and Limit of Detection.
INTRODUCTION:
Sodium azide is the chemical compound with the formula NaN3. This colourless azide salt is a common reagent in organic synthesis. Sodium azide is ionic. The N3− group if two strecturs of two structures, -N-N-≡N+ ↔ -N=N+=N- is centrosymmetric with N–N distances of 1.18 Å.[1]. Sodium azide is highly soluble in water, and in water forms some amounts of hydrogen azide, as in equilibrium: N3− + H2O HN3 + OH− K = 10−4.6. The azide functional group converts to an amine by reduction with lithium aluminium hydride in the Staudinger reaction, or with Raney nickel. On reaction with strong acids sodium azide gives hydrazoic acid: H2SO4+ NaN3 → HN3 + NaHSO4 2.
Sodium azides and other azides are useful for the synthesis of pharmaceuticals for [3+2] cycloedition formation of tertazole ring by reaction such as Telmisartan, Olmesartan, Irbesartan, Losartan, Candesatrtan, Alfentanil, Azosemid, Broperamol, antiviral drug Zidovudine etc. Azides act as electrophiles on the nitrogen attached to the carbon and have electron-donating character for the neighboring carbon.
Sodium azide is a tumorigen and mutagen and can be compared with cyanide in toxicity, symptoms in a poisoned individuals are same.Exposure to a large amount of sodium azide may cause some other health effects as well: convulsions, low blood pressure, low heart rate, loss of consciousness, and lung injury, respiratory failure leading to death. Sodium azide affects central nervous system, kidneys, and cardiovascular system. The National Institute for Occupational Safety and Health (NIOSH)3 and Occupational Safety and Health Administration (OSHA) has Recommended Airborne Exposure Limit (REL) 0.11 ppm as hydrazoic acid or 0.3 mg/m3 as sodium azide, which should not be exceeded at any time [4]. American Conference of Governmental Industrial Hygienists (ACGIH)5 Threshold Limit Value (TLV): 0.11 ppm as HN3, 0.29 mg/m3 as Na N3 (Ceilings), A4 Not classifiable as a human carcinogen. Oral rat LD50: 27 mg/kg Skin rabbit LD50: 20 mg/kg. Inhalation rat LC50: 37 mg/m3.
For the determination of residual azide ion in organic compounds, environmental samples, and biological samples different analytical methods such as spectrophotometry, redox titration, ion chromatography, high-performance liquid chromatography, gas chromatography capillary electrophoresis and gas chromatography-mass spectrometry are developed. [6-20] Oshima Harumi et. al. developed a method for azide determination by ion chromatography in beverages samples6, Kruszyna, R; et. al. described a method in blood samples by ion chromatography [7], Vinkovic, et. al. developed a method on Ion chromatography of azide determination in pharmaceutical protein samples with high chloride concentration using suppressed conductivity detection,8 Occupational Safety and Health Administration (OSHA) has given a method on IC by variable UV detection in air sample,9 Eva A. Terpinski, et. al.and V. Christova-Bagdassarin et. al. developed a method on Spectrophotometer,10,11 Y. Bereznitski et. al., and Vacha J et. al., have developed a method by derivatization of azide and analysis on HPLC,12,13 R. G. Clem, et. al. developed a method for determination of Azide ion by hydrogen ion titration after oxidation with nitrite,14 Nakajima Masanori, et. al., Masataka Ohashi, et. al. and Hortin, GL, et. al., developed methods in different samples by Capillary Electrophoresis,15-17 Kage, S, et. al., developed a method for the determination of azide in blood and urine by gas chromatography-mass spectrometry,18 Roger T. Echols, et. al. descrives a method for primary explosive azides in Environmental Samples by Sequential Injection Amperometry19 and a innovative chemical method is developed by Kikuchi Michio, et. al.20.
Presented method is developed on Ion Chromatograph for determination of azide in wide range of sartan drugs e.g. Telmisartan, Olmesartan, Irbesartan, Losartan, Candesatrtan etc where sodium azide is used in reaction to form tetrazole ring, which is active site of these drugs. The developed method is subsequently validated.
Sodium azide (Assay -99.6%), Merck, Sodium carbonate (AR grade) Merck, Sodium bicarbonate (AR grade) Merck, Methanol (HPLC grade) Rankem, Sulphuric acid (AR grade) Rankem, Purified water (HPLC grade) are used for analysis.
Instrumentation:
Metrohm Ion Chromatography (model IC861) with conductivity detector, Anion suppressor and IC net software, Balance: AND GR-202 and Dionex IonPac AS 10, (250 x 4.0) mm with Dionex IonPac AG 10, (50 x 4.0) mm guard column are used.
Preparation of mobile phase and other solution:
Mobile phase,:
Dissolve 190 mg of sodium carbonate and 145 mg of sodium bicarbonate in 1000 ml of water, and filter through 0.45 µm or finer porosity membrane filter and degas.
Neutralization reagent
2.8 ml per litter of Sulphuric acid is used as neutralization solution which is required for neutralize the mobile phase is system.
Diluent and blank:
Purified water is used as diluent.
Chromatographic parameters:
Injection volume: 100µl, Flow rate: 1.0 ml/min, Run time: 50 minutes, Neutralization of eluent: By continuous countercurrent circulation in a neutralizing micro membrane, performed before detection, Neutralizing solvent: 0.05M Sulphuric acid.
Prepare Sodium azide in diluent having known concentration of sodium azide about 0.625 µg/ml (equivalent azide is about 0.4 µg/ml).
Preparation of sample solution:
About 400 mg of test sample in taken in a stopper test tube, add 10.0 ml of diluent, mix and sonicate the solution for about 15 minutes. Filter this solution through Whattman filter paper No.-42 (previously washed with about 100 ml of water), discard first 1 to 2 ml of filtrate and collect the filtrate. Finally filter this solution through 0.2µm of membrane filter.
System suitability:
From the standard solution, the signal to-noise ratio for the peak due to Azide is not less than 10.
CALCULATION:
Calculate the content of Azide using formula below:
|
Content of Azide (µg/g) |
= |
AT |
X |
Ds |
X |
42.02 |
X |
P |
X |
104 |
|
AS |
DT |
65.01 |
Where, AT = Area of Azide in chromatogram obtained by sample solution.
AS = Mean area of Azide in chromatogram obtained by replicate injections of standard solution.
Ds = Dilution factor of sodium azide for standard solution i.e. weight in mg ¸ dilution in ml.
DT = Dilution factor of test sample for sample solution i.e. weight in mg ¸ dilution in ml.
P = Potency of sodium azide standard used (% w/w).
42.02 = Molecular weight of Azide.
65.01 = Molecular weight of Sodium azide.
METHOD VALIDATION:
Method validation studies are performed using different sartan drugs produced in-house10µg/g of azide is considered as specification limit for Method Validation. Developed method for the determination of azide contents in a wide range of ‘satran’ drugs is validated for its Specificity, Linearity, Precision, Accuracy (by Recovery), Ruggedness and Robustness as per ICH and USP guidelines. [21-22] Solution stability is performed in spiked sample. Standard error method is used for prediction of LOQ and LOD and precision is established on predicted levels.
RESULT AND DISCUSSION:
Specificity and selectivity:
Blank, Sodium azide individual standard, sample solution and spiked sample solution were prepared as per the method and injected separately into chromatograph. No interference from blank and peak due to any impurity was observed at the retention time of Sodium azide peaks.
Figure-1. Representative chromatogram of System suitability (S/N ratio)
Figure-2. Representative chromatogram of Sodium azide by IC and linearity Plot Retention time of Sodium azide is 42.0 minutes.
Table-1. Validation report of IC method for the determination of Azide content
|
Parameters |
Concentration |
Result |
|
Linearity Slope Intercept |
0.082 to 0.613µg/ml
|
R2 = 0.99933 9.47 0.182 |
|
Precision (a) System Precision %RSD (n=6) (b) Method precision by recovery (c) Intermediate precision by recovery (d) Ruggedness %RSD (n=12) |
0.4 µg/ml (standard)
10.0µg/g
10.0µg/g
10.0µg/g |
1.59 % ± 0.064
8.33µg/g ± 0.291
8.57µg/g ± 0.301
8.54µg/g ± 0.309 |
|
Accuracy (Mean % Recovery ± %RSD) (n =3) at 50 % level (n =3) at 100 % level (n =3) at 150 % level (n = 9) Over all recovery |
5.0 µg/g 10.0 µg/g 15.0 µg/g - |
89.22 ± 7.21 % 85.96 ± 3.40 % 85.46 ± 1.75% 86.21 ± 4.50 % |
|
Limit of Detection (%RSD) Limit of Quantitation (%RSD) |
0.041 µg/ml (1.03 µg/g) 0.082 µg/ml (2.05 µg/g) |
17.40 % 9.32 % |
n = number of determinations, limit of Sodium azide 10µg/g w.r.t sample concentration.
The peak purity plot also indicates that peak of Sodium azide is pure and do not have any co-eluting peak (Figure-1). Retention time of Sodium azide is about 42 minutes.
Linearity:
Correlation coefficient, slop and intercept were determined by injecting solutions of different concentration from 0.75µg/ml to 37.65µg/ml of Sodium azide (equivalent to azide), data is provided in Table-1.
System precision (system repeatability) by injecting standard solution six times, method precision (n=6) and intermediate precision (n=6) was studied by recovery, by spiking standard Sodium azide in sample at the 100% level of specification, and relative standard deviation of results was calculated. Ruggedness of the method was established by comparing the results obtained on different days by different analyst on different instrument and IC column data given in Table-1.
Accuracy:
Accuracy of the method was studied by recovery, standard Sodium azide was spiked with sample at different concentration levels (LOQ, 50%, 100%, and 150% level) and amount recovered was calculated. Data is given in Table-1.
Standard error and slop of linearity data is used to predict LOD and LOQ of Sodium azide and precision was established at the predicted concentrations. % Relative standard deviations are found within limits for LOQ at 0.0.082 µg/ml with (% RSD=69.32) and LOD at 0.0.041 µg/ml with (% RSD=17.40), data is given in Table-1.
Ruggedness:
The ruggedness of the method was verified by analyzing the sample of the same batch, prepared by spiking sodium azide at 100% of level of specification and analyzed separately six times by two different analysts using different column on different days. The method is rugged for analyst-to-analyst, column-to-column and day-to-day.
The retention time of azide in different variable conditions is comparable with control condition. Hence the test method is robust for ±10% variation in flow rate of mobile phase and ± 2 % (absolute) organic phase variation.
Stability of Analytical Solution:
Sample solution prepared by spiking Azide and analyzed initially and at different time intervals for about 37 hours at room temperature (i.e. 25°C ± 2). Cumulative RSD is observed 4.60% for Azide up to 37 hours. Hence the sample solution can be used at least up to 37 hours at room temperature (25°C ± 2).
Presented method was found to be sensitive enough with linearity in the concentration range of 0.082–0.613 µg/ml. Method is specific as there is no of any other component at the RT of azide peak, accuracy of method was established by recovery, and the recovery values are within acceptable limits at different concentration levels and the data in table-1. Representative chromatogram is presented in figure-1 and figure-2 with linearity plot, and the different values of validation data; linearity, Precision, Ruggedness, Robustness, Accuracy, limit of quantification and limit of detection are given in table-1.
The presented work describes that developed reveres phase IC method for the determination of Sodium azide is Specific, Rugged, Robust, Linear, Accurate, Precise and can be applied for quantification of Sodium azide in a range of ‘sartan’ drugs. With small modifications this method can also be applied to other drug products where Sodium Azide is one of residual impurities
Authors are highly thankful to the CTX Life Sciences management for granting permission for the publication of this work.
Received on 17.09.2009 Modified on 13.11.2009
Accepted on 12.12.2009 © RJPT All right reserved
Research J. Pharm. and Tech. 3(1): Jan.-Mar. 2010; Page 82-86