Development and Validation of a Microbiological Agar Assay for Determination of Thiamphenicol in Soft Capsules

Author(s): Yugo Araújo Martins, Reginaldo dos Santos Sousa, Cristiani Lopes Capistrano Gonçalves de Oliveira*

Journal Name: Current Pharmaceutical Analysis

Volume 16 , Issue 7 , 2020

Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Background: Thiamphenicol belongs to the amphenicol class of antibiotic and possesses a broad-spectrum antimicrobial activity. An alternative microbiological assay for quantification of thiamphenicol in pharmaceutical formulations has not yet been reported in the literature.

Objective: This study aimed to develop and validate an agar diffusion method for quantification of thiamphenicol in soft capsules.

Methods: The assay was based on the inhibitory effect of thiamphenicol on the following: a strain of Kocuria rhizophila ATCC 9341, used as the test microorganism, Antibiotic 1culture medium, phosphate buffer pH 6, 0, inoculum at a concentration of 1%, as well as standard and sample solutions at the concentrations of 20.0, 40.0 and 80.0 μg mL-1.

Results: The method validation yielded good results for the parameters of linearity, precision, accuracy, robustness and selectivity. The experimental statistic results were analyzed using analysis of variance (ANOVA). The method was found to be linear (r2 = 0.9992) in the range of 20-80 μg mL-1, precise (inter-assay R.S.D = 0.09%), accurate (R.S.D. = 4.65%), specific, and robust.

Conclusion: The results demonstrated the validity of the proposed bioassay, which allows for reliable quantification of thiamphenicol in a pharmaceutical sample. An alternative methodology for thiamphenicol determination in routine quality control has been reported herein.

Keywords: Biological assay, thiamphenicol, soft capsules, quality control, validation, analytical method.

Belda Junior, W.; Siqueira, L.F.G.; Fagundes, L.J. Thiamphenicol in the treatment of chancroid. A study of 1,128 cases. Rev. Inst. Med. Trop. São Paulo, 2000, 42(3), 133-135.
[] [PMID: 10887371]
European Pharmacopoeia, 9th ed; Council of Europe: Strasbourg, 2017.
Tullio, V.; Cuffini, A.; Mandras, N.; Roana, J.; Banche, G.; Ungheri, D.; Carlone, N. Influence of thiamphenicol on the primary functions of human polymorphonuclear leucocytes against Streptococcus pyogenes. Int. J. Antimicrob. Agents, 2004, 24(4), 381-385.
[] [PMID: 15380265]
Wang, Z.; Yang, H.; Sun, W.; Huang, C-K.; Cui, X.; Qiu, X-J.; Lian, Q-Q.; Wang, Z-S. UPLC-MS/MS determination of thiamphenicol in human plasma and its application to a pharmacokinetic study. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 2014, 967, 235-239.
[] [PMID: 25129408]
Fodey, T.L.; George, S.E.; Traynor, I.M.; Delahaut, P.; Kennedy, D.G.; Elliott, C.T.; Crooks, S.R.H. Approaches for the simultaneous detection of thiamphenicol, florfenicol and florfenicol amine using immunochemical techniques. J. Immunol. Methods, 2013, 393(1-2), 30-37.
[] [PMID: 23587555]
Li, J.; Chen, H.; Chen, H.; Ye, Y. Selective determination of trace thiamphenicol in milk and honey by molecularly imprinted polymer monolith microextraction and highperformance liquid chromatography. J. Sep. Sci., 2012, 35(1), 137-144.
[] [PMID: 22102397]
Pfenning, A.P.E.; Roybal, J.E.; Rupp, H.S.; Turnipseed, S.B.; Gonzales, S.A.; Hurlbut, J.A. Simultaneous determination of residues of chloramphenicol, florfenicol, florfenicol amine, and thiamphenicol in shrimp tissue by gas chromatography with electron capture detection. J. AOAC Int., 2000, 83(1), 26-30.
[] [PMID: 10693001]
Xie, X.; Wang, B.; Pang, M.; Zhao, X.; Xie, K.; Zhang, Y.; Wang, Y.; Guo, Y.; Liu, C.; Bu, X.; Wang, R.; Shi, H.; Zhang, G.; Zhang, T.; Dai, G.; Wang, J. Quantitative analysis of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in eggs via liquid chromatographyelectrospray ionization tandem mass spectrometry. Food Chem., 2018, 269, 542-548.
[] [PMID: 30100471]
Wang, F.; Wang, W.; Yuan, S.; Wang, W.; Hu, Z. Comparison of UV/H2O2 and UV/PS processes for the degradation of thiamphenicol in aquous solution. J. Photochem. Photobiol. Chem., 2017, 348, 79-88.
Shen, J.; Xia, X.; Jiang, H.; Li, C.; Li, J.; Li, X.; Ding, S. Determination of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in poultry and porcine muscle and liver by gas chromatography-negative chemical ionization mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 2009, 877(14-15), 1523-1529.
[] [PMID: 19395324]
Ran, L.; Ze-Jia, L.; Zhen-Yi, Y.; Zhen-li, X.; Hong, W.; Hong-Tao, L.; Yuan-Ming, S.; Yu-Dong, S. An indirect competitive enzyme-linked immunosorbent assay for simultaneous determination of florfenicol and thiamphenicol in animal meat and urine. Chin. J. Anal. Chem., 2018, 46, 1321-1328.
Guidi, L.R.; Tette, P.A.S.; Gloria, M.B.A.; Fernandes, C. A simple and rapid LC-MS/MS method for the determination of amphenicols in Nile tilapia. Food Chem., 2018, 262, 235-241.
[] [PMID: 29751915]
Schmidt, C.A.; Agarrayua, D.A.; Laporta, L.V.; Machado, J.C.; Manfio, M.L.; Bittencourt, C.F. Development and validation of a microbiological agar assay for determination of cefuroxime sodium in pharmaceutical preparations. J. Microbiol. Methods, 2009, 77(3), 308-315.
[] [PMID: 19344741]
Lourenco, F.R.; Traple, M.A.L.; Okamoto, R.T.; Pinto, T.J.A. Development and validation of microbiological assay for ceftriaxone and its application in photo-stability study. Curr. Pharm. Anal., 2013, 9, 77-81.
Curbete, M.M.; Salgado, H.R.N. Rapid turbidimetric assay for quantification of fusidic acid in a dermatological cream. Talanta, 2016, 153, 51-56.
[] [PMID: 27130089]
da Silva, L.M.; Salgado, H.R.N. Rapid turbidimetric assay to potency evaluation of tigecycline in lyophilized powder. J. Microbiol. Methods, 2015, 110, 49-53.
[] [PMID: 25619746]
Mahmoudi, A.; Fourar, R.E-A.; Boukhechem, M.S.; Zarkout, S. Microbiological assay for the analysis of certain macrolides in pharmaceutical dosage forms. Int. J. Pharm., 2015, 491(1-2), 285-291.
[] [PMID: 26112961]
Lopes, C.C.G.O.; Salgado, H.R.N. Development and validation of a stability-indicative agar diffusion assay to determine the potency of linezolid in tablets in the presence of photodegradation products. Talanta, 2010, 82(3), 918-922.
[] [PMID: 20678646]
Schmidt, C.A.; Carazzo, M.; Laporta, L.V.; Bittencourt, C.F.; Santos, M.R.; Friedrich, M. Development and validation of an agar diffusion assay for determination of ceftazidime in pharmaceutical preparations. J. AOAC Int., 2008, 91(1), 59-66.
[] [PMID: 18376586]
De Marco, B.A.; Salgado, H.R.N. Rapid stability-indicative turbidimetric assay to determine the potency of cefadroxil monohydrate capsules. Anal. Methods, 2018, 10, 660-666.
Suke, S.G.; Chahande, A.D.; Kasliwal, R.H.; Asnani, A.J. Roxithromycin potency quantification in pharmaceutical preparation by applying a validated bioassay method and comparison with HPLC analysis. Ann. Pharm. Fr., 2015, 73(5), 340-350.
[] [PMID: 26021576]
Saviano, A.M.; Francisco, F.L.; Lourenço, F.R. Rational development and validation of a new microbiological assay for linezolid and its measurement uncertainty. Talanta, 2014, 127, 225-229.
[] [PMID: 24913880]
Pedroso, T.M.; Nunes Salgado, H.R. Development and validation of a microbiological assay by turbidimetry to determine the potency of cefazolin sodium in the lyophilized powder form. Anal. Methods, 2014, 6, 1391-1396.
Dafale, N.A.; Semwal, U.P.; Agarwal, P.K.; Sharma, P.; Singh, G.N. Quantification of ceftriaxone sodium in pharmaceutical preparations by a new validated microbiological bioassay. Anal. Methods, 2012, 4, 2490-2498.
Saviano, A.M.; Francisco, F.L.; Ostronoff, C.S.; Lourenço, F.R. Development, optimization, and validation of a microplate bioassay for relative potency determination of linezolid using a design space concept, and its measurement uncertainty. J. AOAC Int., 2015, 98(5), 1267-1275.
[] [PMID: 26525245]
Farmacopeia Brasileira, 5th ed; Brasília, 2010.
Hewitt, W. Microbiological Assay for Pharmaceutical Analysis: A Rational Approach; Interpharm / CRC Press: Boca Raton, 2003.
international conference on harmonization. ich. validation of analytical procedures: text and methodology q2 r1. geneva: international conference on harmonization; 2005. availa-ble from
Horwitz, W. Official Methods of Analysis of AOAC International, 17th ed; AOAC International: Arlington, 2000.
Lourenço, F.R.; Pinto, T.J.A. Comparison of three experimental designs employed in gentamicin microbiological assay through agar diffusion. Braz. J. Pharm. Sci., 2009, 45, 559-566.
Shah, V.P.; Midha, K.K.; Findlay, J.W.A.; Hill, H.M.; Hulse, J.D.; McGilveray, I.J.; McKay, G.; Miller, K.J.; Patnaik, R.N.; Powell, M.L.; Tonelli, A.; Viswanathan, C.T.; Yacobi, A. Bioanalytical method validation- a revisit with a decade of progress. Pharm. Res., 2000, 17(12), 1551-1557.
[] [PMID: 11303967]
Hodges, N.A. Pharmaceutical Applications of Microbiological Techniques. Pharmaceutics: The Science of Dosage Form Design; Churchill Livingstone: London, 2001, pp. 623-643.
United States Pharmacopeia. National Formulary, 26th ed; Rockville, 2007.
Baird, R.M.; Hodges, N.A.; Denyer, S. P. Handbook of Microbiological Quality Control; Taylor & Francis: London, 2000.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2020
Published on: 17 August, 2020
Page: [806 - 813]
Pages: 8
DOI: 10.2174/1573412915666190328213828
Price: $65

Article Metrics

PDF: 30
PRC: 1