Development of High Throughput Rapid Turbidimetric Assay for Potency Determination of Gramicidin
DOI:
https://doi.org/10.29329/actanatsci.2023.353.05Keywords:
Gramicidin, Linearity, Regression, Repeatability, Robustness, SpecificityAbstract
Gramicidin is a polypeptide antibiotic composed of a mixture of antimicrobial compounds. Thus, its antibacterial activity is preferentially assessed using a microbiological assay. The aim of this study is targeting to establish and validate a microbiological potency for Gramicidin with a view to the employment of a simple method with more than two folds output per test run (if compared with symmetrical designs) using 3×1 experimental designs with reasonably statistically acceptable results. The validation criteria of gramicidin turbidimetric assay using the USP method were tested in terms of selectivity, linearity, accuracy, precision and robustness. Moreover, the consistency of the experimental groups was examined in terms of error and difference from the target labelled concentration of 0.25 mg g-1 value, in addition to the uncertainty factor. Verification of the assay suitability was evaluated statistically against reference antibiotics of known activity. Calibration of the analytical curve showed a coefficient of correlation (r) = 0.9980 with none of the relative standard deviations (RSD) values greater than three. There was no observable fixed or variable deviation in the absorbance measurement with concentration increment. The accuracy output and profile were evaluated over ranges 50%, 100% and 150% having a maximum RSD of around three with reasonable results, confidence and absence of concentration-related bias. Robustness, precision and suitability verification were evaluated with no outliers and all RSDs below five. The turbidimetric assay design of 3×1 for gramicidin showed acceptable validation parameters and could be used as a substitute design for conventional higher-level parallel line assay models.
References
Balouiri, M., Sadiki, M., & Ibnsouda, S. K. (2016). Methods for in vitro evaluating antimicrobial activity: A review. Journal of Pharmaceutical Analysis, 6(2), 71-79. https://doi.org/10.1016/j.jpha.2015.11.005
British Pharmacopoeia. (2022). Microbiological Assay of Antibiotics British Pharmacopoeia (2023 ed.). Stationery Office.
Budavari, S., O’Neal, M., Smith, A., Heckelman, P., & Kinneary, J. (1996). The Merck Index. An encyclopedia of chemicals, drugs, and biologicals. (12th ed.). Whitehouse Station, Merck & Co. Inc.
Christ, A. P., Machado, M. S., Ribas, K. G., Schwarzbold, A. V., Silva, C. D. B. D., & Adams, A. I. H. (2015). A fully validated microbiological assay for daptomycin injection and comparison to HPLC method. Brazilian Journal of Pharmaceutical Sciences, 51(4), 775-783. https://doi.org/10.1590/s1984-82502015000400003
Culture Collections. (2007). Culture collections. https://www.culturecollections.org.uk/products/bacteria/detail.jsp?+refId=NCTC+13383
Dafale, N. A., Semwal, U. P., Agarwal, P. K., Sharma, P., & Singh, G. (2015). Development and validation of microbial bioassay for quantification of Levofloxacin in pharmaceutical preparations. Journal of Pharmaceutical Analysis, 5(1), 18-26. https://doi.org/10.1016/j.jpha.2014.07.007
Dafale, N. A., Semwal, U. P., Rajput, R. K., & Singh, G. (2016). Selection of appropriate analytical tools to determine the potency and bioactivity of antibiotics and antibiotic resistance. Journal of Pharmaceutical Analysis, 6(4), 207-213. https://doi.org/10.1016/j.jpha.2016.05.006
Eissa, M., R. Rashed, E., & Eissa, D. (2021a). Validation of symmetrical two-dose parallel line assay model for nystatin potency determination in pharmaceutical product. Journal of Advanced Pharmacy Research, 5(4), 406-413. https://doi.org/10.21608/aprh.2021.86555.1138
Eissa, M., R. Rashed, E., & Eissa, D. (2021b). Statistical comparison of parallel-line symmetrical microbiological models: Analysis of agar diffusion assay in 8x8 large rectangular plates. İstatistik ve Uygulamalı Bilimler Dergisi, 2(2), 48-64. https://doi.org/10.52693/jsas.989584
Eissa, M. E., Rashed, E. R., & Eissa, D. E. (2021c). Microbiological antibiotic assay validation of gentamicin sulfate using two-dose parallel line model (PLM). HighTech and Innovation Journal, 2(4), 306-319. https://doi.org/10.28991/hij-2021-02-04-04
Eissa, D. E., Rashed, E. R., & Eissa, M. E. (2021d). Suitability system of microbiological method for nystatin potency determination in the routine analysis using agar diffusion method. SciMedicine Journal, 3(4), 302-315. https://doi.org/10.28991/scimedj-2021-0304-2
Ermer, J., & Miller, J. H. M. (Eds.). (2006). Method validation in pharmaceutical analysis: A guide to best practice. John Wiley & Sons.
Francisco, F. L., Saviano, A. M., Pinto, T. D. J. A., & Lourenço, F. R. (2014). Development, optimization and validation of a rapid colorimetric microplate bioassay for neomycin sulfate in pharmaceutical drug products. Journal of Microbiological Methods, 103, 104-111. https://doi.org/10.1016/j.mimet.2014.05.023
Hewitt, W. (2003). Microbiological assay for pharmaceutical analysis: a rational approach. CRC Press.
Hewitt, W. (2012). Microbiological assay: An introduction to quantitative principles and evaluation. Academic Press.
Kessler, N., Schuhmann, H., Morneweg, S., Linne, U., & Marahiel, M. A. (2004). The linear pentadecapeptide gramicidin is assembled by four multimodular nonribosomal peptide synthetases that comprise 16 modules with 56 catalytic domains. Journal of Biological Chemistry, 279(9), 7413-7419. https://doi.org/10.1074/jbc.M309658200
Loureno, F. R., Kaneko, T. M., & Pinto, T. D. J. A. (2007). Validation of erythromycin microbiological assay using an alternative experimental design. Journal of AOAC INTERNATIONAL, 90(4), 1107-1110. https://doi.org/10.1093/jaoac/90.4.1107
Martins, Y. A., Dos Santos Sousa, R., & De Oliveira, C. L. C. G. (2020). Development and validation of a microbiological agar assay for determination of thiamphenicol in soft capsules. Current Pharmaceutical Analysis, 16(7), 806–813. https://doi.org/10.2174/1573412915666190328213828
Motulsky, H. (2015). Essential biostatistics. Oxford University Press.
National Library of Medicine. (2007). Gramicidin. PubChem. Retrieved on January 5, 2023, from https://pubchem.ncbi.nlm.nih.gov/#query=gramicidin
Nunes Salgado, H. R., & Gomes Tozo, G. C. (2007). Microbiological assay for cefoxitin sodium in dosage form. Journal of AOAC International, 90(2), 452-455. https://doi.org/10.1093/jaoac/90.2.452
Oppe, T. P., Menegola, J., & Schapoval, E. E. S. (2018). Microbiological assay for the determination of cefpirome in raw material and injectable preparation. Drug Analytical Research, 2(1), 29-35. https://doi.org/10.22456/2527-2616.84473
Sardella, M., Belcher, G., Lungu, C., Ignoni, T., Camisa, M., Stenver, D. I., Porcelli, P., D’Antuono, M., Castiglione, N. G., Adams, A., Furlan, G., Grisoni, I., Hall, S., Boga, L., Mancini, V., Ciuca, M., Chonzi, D., Edwards, B., Mangoni, A. A., … Le Louet, H. (2021). Monitoring the manufacturing and quality of medicines: a fundamental task of pharmacovigilance. Therapeutic Advances in Drug Safety, 12, 204209862110384. https://doi.org/10.1177/20420986211038436
Solano, A. G. R., Pereira, L. de M. C. S., Leonel, M. de F. V., & Nunan, E. de A. (2011). Development of agar diffusion method for dosage of gramicidin. Brazilian Journal of Pharmaceutical Sciences, 47(3), 564-572. https://doi.org/10.1590/S1984-82502011000300014
United States Pharmacopeia (2022). General Chapters: <81> Antibiotics-Microbial Assays (2022). USP-NF Online (44th ed.). Pharmacopeial Forum, 30(3), 1002. http://www.uspbpep.com/usp29/v29240/usp29nf24s0_c81.html
Vieira, D., Fiuza, T., & Salgado, H. (2014). Development and validation of a rapid turbidimetric assay to determine the potency of cefuroxime sodium in powder for dissolution for injection. Pathogens, 3(3), 656–666. https://doi.org/10.3390/pathogens3030656
William, H. (2003). Microbiological assay for pharmaceutical analysis. CRC Press.
WHO. (2007). Quality assurance of pharmaceuticals: a compendium of guidelines and related materials. Good manufacturing practices and inspection (Vol. 2). World Health Organization.
Zuluaga, A. F., Agudelo, M., Rodriguez, C. A., & Vesga, O. (2009). Application of microbiological assay to determine pharmaceutical equivalence of generic intravenous antibiotics. BMC Clinical Pharmacology, 9, 1. https://doi.org/10.1186/1472-6904-9-1
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Copyright (c) 2023 Mostafa Essam Eissa, Engy Refaat Rashed, Dalia Essam Eissa
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