Objective: The aim of the study was to develop an easy, sensible and rapid method for the estimation of amikacin in both pure and marketed formulation using the spectrophotometric method.Methods: Due to lack of chromophoric group in the amikacin, it was derivatized with 0.1 mmol chloranillic acid reagent. For the estimation of amikacin, Shimadzu UV-1700 model spectrophotometer with UV probe software was used. The method was based on simple charge transfer complexation of the drug with a p-chloranillic acid reagent to give a purple coloured product which was measured at 524nm against blank solution.Results: The derivatised product of amikacin was detected at a wavelength of 524 nm. Linearity was observed with the concentration range of 20-100 Âµg/ml with a regression coefficient of 0.9803. Results of all the parameters were within the acceptance criteria with % RSD less than 2.Conclusion: The spectroscopic method was validated as per ICH guidelines and was found to be applicable for routine quantitative analysis of amikacin in marketed formulations also. The results of linearity, precision, accuracy LOD and LOQ were within the specified limits. The method is highly sensitive, robust, reproducible and specific.Â

G., Surya Teja

K., Venkata Sairam

M., Gurupadayya B.

English

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 Original Article SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF AMIKACIN IN PURE AND PHARMACEUTICAL DOSAGE FORM  SURYA TEJA G., GURUPADAYYA B. M.*, VENKATA SAIRAM K. Department of Pharmaceutical Chemistry, JSS College of Pharmacy, Jagadguru Sri Shivarathreeshwara University, Mysore 570015, Karnataka, India Email: bmgurupadayya@jssuni.edu.in   Received: 21 Oct 2017, Revised and Accepted: 12 Dec 2017 ABSTRACT Objective: The aim of the study was to develop an easy, sensible and rapid method for the estimation of amikacin in both pure and marketed formulation using the spectrophotometric method. Methods: Due to lack of chromophoric group in the amikacin, it was derivatized with 0.1 mmol chloranillic acid reagent. For the estimation of amikacin, Shimadzu UV-1700 model spectrophotometer with UV probe software was used. The method was based on simple charge transfer complexation of the drug with a p-chloranillic acid reagent to give a purple coloured product which was measured at 524nm against blank solution. Results: The derivatised product of amikacin was detected at a wavelength of 524 nm. Linearity was observed with the concentration range of 20-100 µg/ml with a regression coefficient of 0.9803. Results of all the parameters were within the acceptance criteria with % RSD less than 2. Conclusion: The spectroscopic method was validated as per ICH guidelines and was found to be applicable for routine quantitative analysis of amikacin in marketed formulations also. The results of linearity, precision, accuracy LOD and LOQ were within the specified limits. The method is highly sensitive, robust, reproducible and specific.  Keywords: Amikacin, Analytical method development, ICH guidelines, Pharmaceutical dosage, Spectrophotometric. © 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ijcpr.2018v10i1.24703  INTRODUCTION Amikacin is an aminoglycoside antibiotic used for many gram-negative bacterial infections like infections in the urinary tract, infections in brain, lungs and abdomen which are resistant to gentamicin, kanamycin or tobramycin. When compared to other aminoglycosides amikacin has very narrow safety margin i.e., its therapeutic plasma concentration is 8-16 µg/ml. When it is given to renal impaired patients for over a period of time it shows ototoxicity and nephrotoxicity [1-3]. A detailed literature review indicated that there are few analytical and bioanalytical methods were reported like calorimetry [4], HPLC [5-8], LCMS [9] and immunoassay [9]. But till date, there were no reported methods for UV visible Spectroscopy by using choloranilic acid as a derivatizing agent. This method is simple, sensitive, rapid and can be possible to extend to HPLC method using similar reagent.   Fig. 1: Amikacin chemical structure  The structure of amikacin was shown in fig. 1, which has four primary amine groups, one secondary amine group, one primary OH group and seven secondary OH groups [10]. Direct UV methods are not available in literature because the drug is not absorbing in the UV region. Hence, it is essential to derivatize with 0.1% chloranilic acid using electron transfer reaction. The detailed chemical reaction between amikacin and chloranillic acid was shown in fig. 2. METHODS AND MATERIALS Equipment Absorbance and spectral were measured by using Shimadzu UV-visible spectrophotometer model 1800 with 1 cm pair quartz cells. Shimadzu electronic weighing balance was used for weighing samples. Chemicals and reagents Amikacin Sulfate was procured from Shri Chem, Mumbai and Chloranillic acid was procured from Loba chem and acetonitrile from Merck. Amikacin standard stock solution 100 mg pure drug of amikacin was taken into 100 ml volumetric flask and dissolved with distilled water and made up to mark with distilled water. Further 10 ml was taken from the above solution and diluted to 100 ml with distilled water to get 100µg/ml solution. From these serial dilutions were made to get 20,40,60,80 and 100 µg/ml solutions. 0.1% mmol chloranilic acid reagent [11, 12] Solution A (1% chloranilic acid): 0.208 gms of cholranilic acid was weighed into 100 ml volumetric flask and dissolved in few ml of acetonitrile. Volume was made up to mark with acetonitrile. Working solution B (0.1% choloranilic acid): From solution A pipette out 10 ml and dilute to 100 ml using acetonitrile.   International Journal of Current Pharmaceutical Research    ISSN- 0975-7066                                                                       Vol 10, Issue 1, 2018 Gurupadayya et al. Int J Curr Pharm Res, Vol 10, Issue 1, 38-42  39 Assay procedure 1 ml of the intramuscular injection containing 250 mg was transferred into 10 ml volumetric flask. It was dissolved using distilled water. Finally, volume was made up to 10 ml using distilled water. The solution was further diluted for analysis to get a concentration of 25µg/ml. The assay results are tabulated in table 2. RESULTS AND DISCUSSION Validation of the method [13, 14] According to ICH guidelines validation of the method was carried out. Linearity, accuracy, precision, selectivity, robustness and ruggedness parameters were done. Linearity  A series of amikacin sulfate solutions were prepared in the range of 20-100µg/ml from the stock solution of 1000µg/ml. The resultant solution was measured at 524 nm against the reagent blank. The overlay graphs of absorption of the standard drug and calibration graph were shown in the fig. 2 and 3 respectively. Accuracy Accuracy is the nearness of the measured value to the obtained value of the sample.  To determine this three different standard concentrations of 50%, 100% and 150% are added to the sample which is procured from the market. The results obtained for the spiked drug are given in % recovery (94.44–106.4%) shown in the table 3. Precision The precision of the analytical method was determined by measuring the fixed concentration of the drug solution for 6 times within the Beer’s range and absorbance was found. The results of amikacin were given in the table 4-7. Limit of detection (LOD) and limit of quantitation (LOQ) The LOD and LOQ for amikacin sulfate were determined using calibration standards. The LOD and LOQ were calculated as 3.3* standard deviation/slope and 10* standard deviation/slope respectively. Robustness and ruggedness To determine the robustness of the method, reaction time and reagent concentrations were slightly altered with optimum values in spectrophotometry. To check the ruggedness, the analysis was done by four different analysts and on three different spectrophotometers using the same analyst. The robust data are expressed in % RSD. The results of amikacin were in table 8 and 9.   Fig. 2: Chemical reaction between amikacin and reagent   Sandell’s sensitivity The serial dilutions of 20-100µg/ml solutions absorbance were taken and the sensitivity is calculated using the formula: Sandell’s Sensitivity (л) = Conc. (µg/100 ml) x 0.001/D1 value. The results were given in the table 10. Selection of chloranillic acid reagent was based on the higher reactivity due to its stronger chromophore group in its structure when compared to other reagents. It enables its use for colourimetric determination of several amino groups.  The drug shows maximum absorption at 524 nm with a linearity range of 20-100 µg/ml. The method is also validated for precision, accuracy, LOD, LOQ. The precision of the method was found to be 1.78µg/ml,1.76 µg/ml and1.38 µg/ml.  The percentage recovery was ranging from 94.44% to 106.94%. And the LOD and LOQ were found to be 6.49 µg/ml and 19.68 µg/ml respectively. These data’s confirms the method is very sensitive and effectively used for quantification of Amikacin sulfate.  Fig. 3: Linearity plot for amikacin sulfate Gurupadayya et al. Int J Curr Pharm Res, Vol 10, Issue 1, 38-42  40  Fig. 4: Overlay absorption spectra of amikacin sulphate  Table 1: Optical parameters of the method Parameters Method ƛmax 524 nm Beers law limits µg/ml 20-100 Regression equation y=mx+c Y=0.0021x-0.0075 Slope,m 0.0021 Intercept,c -0.0075 LOD, µg/ml 6.49 LOQ, µg/ml 19.68 Correlation coefficient(r2) 0.9803 Sandell’s Sensitivity, µg/cm2/0.001 A. U. 0.23 Molar absorpitivity, cm-1M-1 0.122*104  Table 2: Assay procedures Brand name Available form Label claim Amount found Assay MIKACIN IM injection 250 mg/ml 248.97 mg/ml 99.58  Table: 3 Accuracy/% recovery Level of recovery Amount of formulation (µg/ml) Amount of pure drug (µg/ml) Total amount of drug (µg/ml) Absorbance Difference  % recovery Mean  50 40 20 60 0.113 0.073 101.39 100.46 40 20 60 0.111 0.071 98.61 40 20 60 0.113 0.073 101.39  100 40 40 80 0.143 0.071 98.61 100.92 40 40 80 0.149 0.077 106.94 40 40 80 0.142 0.07 97.22  150 40 60 100 0.189 0.075 104.17 99.07 40 60 100 0.182 0.068 94.44 40 60 100 0.185 0.071 98.61  Table 4: Method precision (intraday) Concentration µg/ml Absorbance Concentration µg/ml Absorbance Concentration µg/ml Absorbance  20 0.0401  60 0.11  100 0.203 0.0409 0.109 0.201 0.042 0.112 0.21 0.0405 0.107 0.209 0.0409 0.11 0.201 0.04 0.106 0.203 Avg 0.0407 Avg 0.109 Avg 0.2045 SD 0.0006 SD 0.002 SD 0.003 % RSD 1.63 %RSD 1.83 %RSD 1.77 Avg: Average SD: Standard Deviation RSD: Relative Standard Deviation Gurupadayya et al. Int J Curr Pharm Res, Vol 10, Issue 1, 38-42  41 Table 5: Method precision (interday) Concentration µg/ml Absorbance Concentration µg/ml Absorbance Concentration µg/ml Absorbance  20 0.041  60 0.109  100 0.209 0.04 0.11 0.21 0.042 0.113 0.205 0.04 0.112 0.203 0.041 0.11 0.207 0.04 0.115 0.213 Avg 0.0406 Avg 0.115 Avg 0.2078 SD 0.0007 SD 0.002 SD 0.003 %RSD 1.83 %RSD 1.84 %RSD 1.58 Avg: Average SD: Standard Deviation RSD: Relative Standard Deviation  Table 6: System precision (intraday) Concentration µg/ml Absorbance Concentration µg/ml Absorbance Concentration µg/ml Absorbance 20 0.04 60 0.109 100 0.199 0.04 0.11 0.203 0.042 0.114 0.201 0.04 0.112 0.2 0.04 0.109 0.197 0.0401 0.113 0.203 Avg 0.0405 Avg 0.111 Avg 0.2005 SD 0.0007 SD 0.001 SD 0.002 %RSD 1.86 %RSD 1.75 %RSD 1.06 Avg: Average SD: Standard Deviation RSD: Relative Standard Deviation  Table 7: System precision (interday) Concentration µg/ml Absorbance Concentration µg/ml Absorbance Concentration µg/ml Absorbance  20 0.0401  60 0.112  100 0.201 0.04 0.114 0.199 0.0403 0.109 0.203 0.042 0.11 0.2 0.0399 0.109 0.201 0.0401 0.11 0.206 Avg 0.0405 Avg 0.11 Avg 0.201 SD 0.0007 SD 0.001 SD 0.002 %RSD 1.82 %RSD 1.62 %RSD 1.13 Avg: Average SD: Standard Deviation RSD: Relative Standard Deviation  Table 8: Robustness data of the developed method Wavelength (nm) Concentration µg/ml Absorbance Wavelength (nm) Concentration µg/ml Absorbance 523 60 0.114 525 60 0.114 60 0.112 60 0.111 60 0.113 60 0.113 Avg 0.113 Avg 0.112 St Dev 0.001 St Dev 0.001 %RSD 0.88 %RSD 1.35  Table 9: Ruggedness of the developed method Concentration µg/ml Linearity absorbance Change in instrument absorbance Mean SD %RSD 0 0 0 0 0 0 20 0.04 0.041 0.04 0.0007 1.74 40 0.072 0.074 0.073 0.001 1.94 60 0.114 0.112 0.113 0.001 1.25 80 0.179 0.176 0.177 0.002 1.98 100 0.201 0.205 0.203 0.003 1.39 SD: Standard Deviation RSD: Relative Standard Deviation  Gurupadayya et al. Int J Curr Pharm Res, Vol 10, Issue 1, 38-42  42 Table 10: Sandell’s sensitivity S. No. Concentration(µg/ml) Absorbance Sensitivity Mean sensitivity 1 20 0.04 0.5  0.23  2 40 0.072 0.278 3 60 0.114 0.175 4 80 0.179 0.112 5 100 0.201 0.010  CONCLUSION  The developed spectrophotometric method was easy, responsive and authentic with good precision and accuracy. The procedure did not involve any critical steps; hence it can be used routinely for determination of amikacin in pure and in the marketed formulation. AUTHORS CONTRIBUTIONS All the author have contributed equally CONFLICT OF INTERESTS  Declared none REFERENCES 1. Feng CH, Lin SJ, Wu HL, Chen SH. Trace analysis of amikacin in commercial preparation by derivatization and HPLC. J Liq Chroma Related Tech 2001;24:381-92. 2. Ovalles JF, Brunetto Mdel R, Gallignani M. 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Int J Chem Tech Res 2012;4:79-91. 12. Darwish IA, Refaat IH. Spectrophotometric analysis of selective serotonin reuptake inhibitors based on the formation of charge-transfer complexes with tetracyanoquinodimethane and chloranilic acid. J AOAC Int 2006;89:326-33. 13. ICH, Q2B, Validation of Analytical Procedures: Methodology, International Conference on Harmonization, Geneva; 1996. p. 1. 14. ICH, Q2(R1), Validation of analytical procedures: Text and Methodology; 1995.  

SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF AMIKACIN IN PURE AND PHARMACEUTICAL DOSAGE FORM

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SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF AMIKACIN IN PURE AND PHARMACEUTICAL DOSAGE FORM

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