Suitability System of Microbiological Method for Nystatin Potency Determination in the Routine Analysis Using Agar Diffusion Method

Dalia Essam Eissa, Engy Refaat Rashed, Mostafa Essam Eissa


Nystatin is a polyene macrolide antifungal active which is used for the treatment of candidiasis and obtained from some species of Streptomycesbacteria. The present work describes the statistical suitability analysis for regular monitoring of the agar diffusion bioassay in a simple, inexpensive and time-saving process before potency determination. A balanced (symmetrical) two-dose parallel line assay model was applied using the agar well diffusion method for quantification of Nystatin in raw material and finished medicinal dosage form. The routine inspection methodology yielded good results and included calculations by the linear parallel model and by means of regression analysis and verified using analysis of variance (ANOVA). The assay is based on the inhibitory effect of Nystatin upon a standard strain as described in the United States Pharmacopeia (USP). The results of the post validation regular assays were treated statistically by ANOVA and the deviations (expressed as average ± standard deviation) from both raw and column totals were 0.702 ± 0.476 and 0.865 ± 0.468, respectively. The mean value of the variance ratio for regression and parallelism squares were 534.349 ± 212.546 and 0.596 ± 0.345, respectively. The study of Nystatin's ongoing analysis showed that the microbiological assay design is satisfactory with respect to the limiting values for the determination of the potency. The established balanced parallel line assay is reasonably stable and suitable and can be used for the regular drug analysis in routine quality control testing and the quantitation of Nystatin in pharmaceutical dosage form and raw material.


Doi: 10.28991/SciMedJ-2021-0304-2

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Nystatin; Parallel Line Assay; Quality Control; Agar Diffusion; Regression; Parallelism.


Caffrey, P., De Poire, E., Sheehan, J., & Sweeney, P. (2016). Polyene macrolide biosynthesis in streptomycetes and related bacteria: recent advances from genome sequencing and experimental studies. Applied Microbiology and Biotechnology, 100(9), 3893–3908. doi:10.1007/s00253-016-7474-z.

Lyu, X., Zhao, C., Hua, H., & Yan, Z. (2016). Efficacy of nystatin for the treatment of oral candidiasis: a systematic review and meta-analysis. Drug Design, Development and Therapy, 1161. doi:10.2147/dddt.s100795.

C. Johnson; T. (2021). What is Candidiasis?. WebMD. Availble online: (accessed on April 2021).

Kaufman, D., Liken, H., & Odackal, N. J. (2019). Diagnosis, Risk Factors, Outcomes, and Evaluation of Invasive Candida Infections. Infectious Disease and Pharmacology, 69–85. doi:10.1016/b978-0-323-54391-0.00007-2.

Millsop, J. W., & Fazel, N. (2016). Oral candidiasis. Clinics in Dermatology, 34(4), 487–494. doi:10.1016/j.clindermatol.2016.02.022.

Ozturk, M. A., Gunes, T., Koklu, E., Cetin, N., & Koc, N. (2006). Oral nystatin prophylaxis to prevent invasive candidiasis in Neonatal Intensive Care Unit. Mycoses, 49(6), 484–492. doi:10.1111/j.1439-0507.2006.01274.x.

Young, G. A. R., Bosly, A., Gibbs, D. L., & Durrant, S. (1999). A double-blind comparison of fluconazole and nystatin in the prevention of candidiasis in patients with leukaemia. European Journal of Cancer, 35(8), 1208–1213. doi:10.1016/s0959-8049(99)00102-1.

National Center for Biotechnology Information (2021). PubChem Compound Summary for CID 138403272. Available online: (accessed on October 2021).

Lancelin, J. M., & Beau, J. M. (1995). Stereostructure of glycosylated polyene macrolides: the example of pimaricin. Bulletin de la Société chimique de France, 2(132), 215-223.

Martin, J. F., & McDaniel, L. E. (1977). Production of Polyene Macrolide Antibiotics. Advances in Applied Microbiology Volume 21, 1–52. doi:10.1016/s0065-2164(08)70037-6.

Keller; M. D. (2017). The effect of amphotericin B on yeast growth; & the isolation; & identification of sterols for protozoan protein X-ray crystallography (Doctoral dissertation). Available online: (accessed on October 2021).

Dafale, N. A., Semwal, U. P., Agarwal, P. K., Sharma, P., & Singh, G. N. (2015). Development and validation of microbial bioassay for quantification of Levofloxacin in pharmaceutical preparations. Journal of Pharmaceutical Analysis, 5(1), 18–26. doi:10.1016/j.jpha.2014.07.007.

Pfaller, M. A., Krogstad, D. J., Granich, G. G., & Murray, P. R. (1984). Laboratory evaluation of five assay methods for vancomycin: bioassay, high-pressure liquid chromatography, fluorescence polarization immunoassay, radioimmunoassay, and fluorescence immunoassay. Journal of Clinical Microbiology, 20(3), 311–316. doi:10.1128/jcm.20.3.311-316.1984.

Greco; G. M. (1998). Microbiological assay systems for the analysis of antibiotics in pharmaceutical formulations (Ph.D. Dissertation). The State University Of New Jersey; USA;

Pinto; T. J. A.; Lourenco; F. R.; & Kaneko; T. M. (2007). Microbiological assay of Gentamycin employing an alternative experimental design. In AOAC Annual Meeting; & Exposition. Vol. 121; p. 157.

Saviano, A. M., Francisco, F. L., & Lourenço, F. R. (2014). Rational development and validation of a new microbiological assay for linezolid and its measurement uncertainty. Talanta, 127, 225–229. doi:10.1016/j.talanta.2014.04.019.

Dafale, N. A., Agarwal, P. K., Semwal, U. P., & Singh, G. N. (2013). Development and validation of microbial bioassay for the quantification of potency of the antibiotic cefuroxime axetil. Anal. Methods, 5(3), 690–698. doi:10.1039/c2ay25848j.

Cazedey, E. C. L., & Salgado, H. R. N. (2011). Development and Validation of a Microbiological Agar Assay for Determination of Orbifloxacin in Pharmaceutical Preparations. Pharmaceutics, 3(3), 572–581. doi:10.3390/pharmaceutics3030572.

Yamamoto, C. H., & Pinto, T. J. A. (1996). Rapid Determination of Neomycin by a Microbiological Agar Diffusion Assay Using Triphenyltetrazolium Chloride. Journal of AOAC International, 79(2), 434–440. doi:10.1093/jaoac/79.2.434.

Lourenço, F. R., & Pinto, T. de J. A. (2009). Comparison of three experimental designs employed in gentamicin microbiological assay through agar diffusion. Brazilian Journal of Pharmaceutical Sciences, 45(3), 559–566. doi:10.1590/s1984-82502009000300022.

Hovstadius, B., Åstrand, B., & Petersson, G. (2009). Dispensed drugs and multiple medications in the Swedish population: an individual-based register study. BMC Clinical Pharmacology, 9(1). doi:10.1186/1472-6904-9-11.

Dafale, N. A., Semwal, U. P., Rajput, R. K., & Singh, G. N. (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. doi:10.1016/j.jpha.2016.05.006.

Nam, J.-H., Shin, J.-H., Kim, T.-H., Yu, S., & Lee, D.-H. (2019). Comparison of biological and chemical assays for measuring the concentration of residual antibiotics after treatment with gamma irradiation. Environmental Engineering Research, 25(4), 614–621. doi:10.4491/eer.2019.270.

Nahar, S., Khatun, M. S., & Kabir, M. S. (2020). Application of microbiological assay to determine the potency of intravenous antibiotics. Stamford Journal of Microbiology, 10(1), 25–29. doi:10.3329/sjm.v10i1.50729.

Hewitt; W. (2004). Microbiological assay for pharmaceutical analysis: a rational approach. CRC press. doi:10.1201/b12428.

European Pharmacopoeia Commission. (2004). Statistical analysis of results of biological assays; & tests. European Pharmacopoeia; 571-600.

United States Pharmacopoeia (USP); & National Formulary (NF). (2021). ‹81› Antibiotics-Microbial assays; USP43-NF38; US Pharmacopeial Convention Inc.; Rockville; p. 86-93.

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. doi:10.1093/jaoac/90.4.1107.

Eissa, M. E. A. (2018). Microbiological quality of purified water assessment using two different trending approaches: A case study. Sumerianz Journa l of Scientific Research, 1(3), 75-79.

Eissa, M. E., & Abid, A. M. (2018). Application of statistical process control for spotting compliance to good pharmaceutical practice. Brazilian Journal of Pharmaceutical Sciences, 54(2). doi:10.1590/s2175-97902018000217499.

Rashed; E. R.; & Eissa; M. E. (2020). Long-term monitoring of Cancer Mortality Rates in USA: A descriptive analysis using statistical process control tools. Iberoamerican Journal of Medicine; 2(2); 55-60. doi:10.5281/zenodo.3740610.

Eissa; M. E. (2018). Application of attribute control chart in the monitoring of the physical properties of solid dosage forms. Journal of Progressive Research in Modern Physics; & Chemistry (JPRMPC); 3(1); 104-113.

Mohamed; R. A.; & Mariam; H. M. (2017). Using Completely Randomized Design of Parallel Linear Model for Estimating the Biological Potency of Human Insulin Drugs: An Empirical Study. Biostatistics & Biometrics Open Access Journal; 3(4); 115-121.

Mairesse, A., Wauthier, L., Courcelles, L., Luyten, U., Burlacu, M., Maisin, D., … Gruson, D. (2021). Biological variation and analytical goals of four thyroid function biomarkers in healthy European volunteers. Clinical Endocrinology, 94(5), 845–850. doi:10.1111/cen.14356.

Cochran, W. G. (1951). Testing a Linear Relation among Variances. Biometrics, 7(1), 17. doi:10.2307/3001601.

European Pharmacopoeia (2002) 4th Ed.; Council of Europe; Strasbourg; France.

Swift, M. L. (1997). GraphPad Prism, Data Analysis, and Scientific Graphing. Journal of Chemical Information and Computer Sciences, 37(2), 411–412. doi:10.1021/ci960402j.

Motulsky; H. J. (2003). Prism 4 statistics guide—statistical analyses for laboratory & clinical researchers. GraphPad Software Inc.; San Diego; CA; 122-126.

Eissa; M. E. (2018). Variable & attribute control charts in trend analysis of active pharmaceutical components: Process efficiency monitoring & comparative study. Experimental Medicine (EM); 1(1); 32-44.

Eissa; M. (2018). Evaluation of microbiological purified water trend using two types of control chart. European Pharmaceutical Review; 23(5); 36-38.

Sullivan, J. H., & Woodall, W. H. (1996). A Control Chart for Preliminary Analysis of Individual Observations. Journal of Quality Technology, 28(3), 265–278. doi:10.1080/00224065.1996.11979677.

Fatimah, Sayuti, M., & Pertiwi, E. P. (2018). Quality control of palm kernel oil using Individual Moving Range (I-MR) chart. MATEC Web of Conferences, 204, 01006. doi:10.1051/matecconf/201820401006.

Henderson; G. R. (2011). Six Sigma quality improvement with MINITAB. John Wiley & Sons.

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DOI: 10.28991/SciMedJ-2021-0304-2


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