Statistical Comparison of Parallel-Line Symmetrical Microbiological Models: Analysis of Agar Diffusion Assay in 8 x 8 Large Rectangular Plates

Abstract

Abstract: (1) Background: Microbiological assay of active medicinal compounds is superior to conventional chemical means in several circumstances to date. However, ensuring the validity and suitability of the assay design proposed for the intended purpose is crucial before deriving any records or conclusions from the results of the potency determination; (2) The present work represented statistical comparison between three design models for determination of the potency of Neomycin Sulfate antibiotic using agar diffusion technique for the same test material subject under identical conditions through the application of a combination of statistical software programs, including validated programmed Microsoft Excel Workbook for the statistical testing of each assay layout; (3) Results: raw data of the three assay designs were found to be reasonably valid for further analysis of the assay suitability. Examination of the sources of variations for each design demonstrated the validity of the conducted experimentation. Variation between the computed potencies from the three designs was lower than 5 µg/mg. However, there was significant variation between the confidence windows of each type; (4) Conclusions: 2 x 4 design had the narrowest confidence range. However, improving confidence would require investigation of the assay parameters, including the modification of the number of replicates per treatment.

Keywords

• Agar Diffusion
• ANOVA
• Confidence Limit
• Homoscedasticity
• Neomycin Sulfate
• Normality
• Outlier
• PLM
• Potency
• Two-Dose Symmetrical Assay

References

1. [1] S. Gad, “Pharmaceutical manufacturing handbook: production and processes”, Wiley-Interscience, 2008.
2. [2] R. Unissa, M. Sudhakar and M. Hadi, “Lab Manual for Pharmaceutical Microbiology”, 1st ed. Ahmedabad: Nirav and Roopal Prakashan, 2011.
3. [3] V. Fedorenko et al., "Antibacterial discovery and development: from gene to product and back", BioMed Research International, vol. 2015, pp. 1-16, 2015. Available: 10.1155/2015/591349.
4. [4] Technical Guide for the elaboration of monographs, "European Pharmacopoeia", Edqm.eu, 2011. [Online]. Available: https://www.edqm.eu/medias/fichiers/technical_guide_for_the_elaboration_of_monographs_.pdf. [Accessed: 30- Aug- 2021].
5. [5] Health Ministers of the United Kingdom , “British Pharmacopoeia”, 3rd ed. London: Medicines and Healthcare Products Regulatory Agency, 2021.
6. [6] W. Hewitt, “Microbiological assay for pharmaceutical analysis: a rational approach”, Boca Raton, Fla: Interpharm/CRC, 2004.
7. [7] Pharmacopeial Forum, “The United States pharmacopoeia”, 43rd ed. North Bethesda, Maryland , United States: United States Pharmacopeial Convention, Vol. 44(6), 2021.
8. [8] W. Hewitt, “Microbiological Assay”, Saint Louis: Elsevier Science, 2014.

9. [9] J. Lightbown, "Biological standardisation and the Analyst. A review", The Analyst, vol. 86, no. 1021, p. 216, 1961. Available: 10.1039/an9618600216.
10. [10] W. Sokolski, C. Chidester, O. Carpenter and W. Kaneshiro, "Assay Methods for Total Neomycins B and C", Journal of Pharmaceutical Sciences, vol. 53, no. 7, pp. 826-828, 1964. Available: 10.1002/jps.2600530731.
11. [11] W. Köhler, "Who Expert Committee on Biological Standardization, 23. Report (WHO Techn. Rep. Ser. No. 463). 120. Genf 1970: World Health Organization", Zeitschrift für allgemeine Mikrobiologie, WHO/BS/70.1001., pp. 255-255, 1971. Available: 10.1002/jobm.19680080326.
12. [12] W. Wilson, G. Richard and D. Hughes, "Thin-layer chromatographic identification of the gentamicin complex", Journal of Chromatography A, vol. 78, no. 2, pp. 442-444, 1973. Available: 10.1016/s0021-9673(73)30103-0.
13. [13] L. Clontz,” Microbial limit and bioburden tests”, Boca Raton: CRC Press, 2009.
14. [14] WHO, “The International pharmacopoeia”, Geneva: World Health Organization, 2006.
15. [15] C. Sheehan, "Overview of International Harmonization through the Pharmacopeial Discussion Group", Usp.org, 2014. [Online]. Available: https://www.usp.org/sites/default/files/usp/document/get-involved/stakeholder-forums/2b-excipients-and-harmonzation-overview-2014-02-19.pdf. [Accessed: 01- Sep- 2021].
16. [16] Technical Data, "Antibiotic Assay Medium F", Himedialabs.com, 2020. [Online]. Available: https://himedialabs.com/TD/M923.pdf. [Accessed: 01- Sep- 2021].
17. [17] Pharmaceutical Secondary Standard; Certified Reference Material, "Neomycin Sulfate Pharmaceutical Secondary Standard; Certified Reference Material | Sigma-Aldrich", Sigmaaldrich.com, 2021. [Online]. Available: https://www.sigmaaldrich.com/EG/en/product/sial/phr1491. [Accessed: 01- Sep- 2021].
18. [18] C. Valgas, S. Souza, E. Smânia and A. Smânia Jr., "Screening methods to determine antibacterial activity of natural products", Brazilian Journal of Microbiology, vol. 38, no. 2, pp. 369-380, 2007. Available: 10.1590/s1517-83822007000200034.
19. [19] J. Hudzicki, "Kirby-Bauer Disk Diffusion Susceptibility Test Protocol", Asm.org, 2009. [Online]. Available: https://asm.org/getattachment/2594ce26-bd44-47f6-8287-0657aa9185ad/Kirby-Bauer-Disk-Diffusion-Susceptibility-Test-Protocol-pdf.pdf. [Accessed: 01- Sep- 2021].
20. [20] GraphPad Software LLC, "GraphPad Prism 9 User Guide - Welcome to Prism 9 User Guide", Graphpad.com, 2021. [Online]. Available: https://www.graphpad.com/guides/prism/latest/user-guide/index.htm. [Accessed: 01- Sep- 2021].
21. [21] M. Evans, G. McCabe and D. Moore, “Minitab manual for Moore and McCabe's Introduction to the practice of statistics”, third edition. New York: W.H. Freeman, 1999.
22. [22] W. Winston, “Microsoft excel 2016”, Redmond, Washington: Microsoft, 2016.
23. [23] C. Carlberg, “Statistical analysis”, Indianapolis, Indiana: Que, 2018.
24. [24] T. Oppe, J. Menegola and E. Schapoval, "MICROBIOLOGICAL ASSAY FOR THE DETERMINATION OF CEFPIROME IN RAW MATERIAL AND INJECTABLE PREPARATION", Drug Analytical Research, vol. 2, no. 1, pp. 29-35, 2018. Available: 10.22456/2527-2616.84473.
25. [25] S. Nahar, M. Khatun and M. Kabir, "Application of microbiological assay to determine the potency of intravenous antibiotics", Stamford Journal of Microbiology, vol. 10, no. 1, pp. 25-29, 2020. Available: 10.3329/sjm.v10i1.50729.
26. [26] A. Zuluaga, M. Agudelo, C. Rodriguez and O. Vesga, "Application of microbiological assay to determine pharmaceutical equivalence of generic intravenous antibiotics", BMC Clinical Pharmacology, vol. 9, no. 1, 2009. Available: 10.1186/1472-6904-9-1.
27. [27] E. Cazedey and H. Salgado, "Development and Validation of a Microbiological Agar Assay for Determination of Orbifloxacin in Pharmaceutical Preparations", Pharmaceutics, vol. 3, no. 3, pp. 572-581, 2011. Available: 10.3390/pharmaceutics3030572.
28. [28] N. Dafale, U. Semwal, P. Agarwal, P. Sharma and G. Singh, "Development and validation of microbial bioassay for quantification of Levofloxacin in pharmaceutical preparations", Journal of Pharmaceutical Analysis, vol. 5, no. 1, pp. 18-26, 2015. Available: 10.1016/j.jpha.2014.07.007.
29. [29] F. Rebello Lourenco, M. Augusto Lyrio Traple, R. Takao Okamoto and T. de Jesus Andreoli Pinto, "Development and Validation of Microbiological Assay for Ceftriaxone and its Application in Photo-stability Study", Current Pharmaceutical Analysis, vol. 9, no. 1, pp. 77-81, 2013. Available: 10.2174/1573412911309010011.
30. [30] T. Mzolo, Statistical methods for the analysis of bioassay data. Technische Universiteit Eindhoven, 2016.
31. [31] F. Lourenço and T. Pinto, "Comparison of three experimental designs employed in gentamicin microbiological assay through agar diffusion", Brazilian Journal of Pharmaceutical Sciences, vol. 45, no. 3, pp. 559-566, 2009. Available: 10.1590/s1984-82502009000300022.
32. [32] Statistical analysis, "STATISTICAL ANALYSIS OF RESULTS OF BIOLOGICAL ASSAYS AND TESTS", Uspbpep.com, 2008. [Online]. Available: http://www.uspbpep.com/ep60/5.3.%20%20statistical%20analysis%20of%20results%20of%20biological% 20assays%20and%20tests%2050300e.pdf. [Accessed: 01- Sep- 2021].
33. [33] M. Eissa, A. Mahmoud and A. Nouby, "Control Chart in Microbiological Cleaning Efficacy of Pharmaceutical Facility", Dhaka University Journal of Pharmaceutical Sciences, vol. 14, no. 2, pp. 133-138, 2016. Available: 10.3329/dujps.v14i2.28501.
34. [34] M. Eissa, "Application of Laney control chart in assessment of microbiological quality of oral pharmaceutical filterable products", Bangladesh Journal of Scientific and Industrial Research, vol. 52, no. 3, pp. 239-246, 2017. Available: 10.3329/bjsir.v52i3.34160.
35. [35] M. Essam Eissa, "Determination of the Microbiological Quality of Feed City Water to Pharmaceutical Facility: Distribution Study and Statistical Analysis", ATHENS JOURNAL OF SCIENCES, vol. 4, no. 2, pp. 143-160, 2017. Available: 10.30958/ajs.4-2-4.
36. [36] M. Essam Eissa, “Suitability system of microbiological method for nystatin potency determination in the routine analysis using agar diffusion method”, SciMedicine Journal (SMJ), in press.
37. [37] Engineering Statistics Handbook, "1.3.5.10. Levene Test for Equality of Variances", Itl.nist.gov, 2021. [Online]. Available: https://www.itl.nist.gov/div898/handbook/eda/section3/eda35a.htm. [Accessed: 01- Sep- 2021].
38. [38] M. Essam Eissa, “Validation of symmetrical two-dose parallel line assay model for nystatin potency determination in pharmaceutical product”, Journal of Advanced Pharmacy Research (JAPR), in press.