HPLC ANALYSIS OF AZITHROMYCIN SUSPENSIONS AND TABLETS Alex Okaru, M.Pharm. Department of Pharmaceutical Chemistry, SOP, UoN

Co-Authors: K.O. Abuga, PhD., F.N. Kamau, PhD. and S.N. Ndwigah, PhD. Department of Pharmaceutical Chemistry, SOP, UoN

Introduction Azithromycin Azithromycin (AZT) is a dibasic 15-membered ring macrolide derivative pKa1 (8.1-8.85) -C3’-N(CH3)2 of desosamine pKa2 (8.8-9.45)-C9a –NCH3 Figure 1: Structure of AZT

Study justification Azithromycin is indicated for the management of ENT, eye and atypical infections among others.  AZT is listed as an essential drug by WHO and MoH, Kenya Shortcomings of existing analytical methods gave impetus for research. Need for a better validated LC method for quality control of AZT products

Objectives Develop and validate a LC method for the analysis of AZT Apply the developed method in the assay of some commercial samples of AZT in the city of Nairobi.

Optimized LC chromatographic conditions of the developed method Column: Waters XTerra® 5 μm C18, 250×4.6mm ID. Mobile Phase: ACN-0.1M Potassium phosphate pH 6.5-0.1M TBAOH-Water (25:15:1:59, % v/v/v/v). Column temperature: 43 °C, Detection λ: 215 nm, Flow rate: 1.0 ml/min; Inj. Vol. : 20μl

DAZT-DecladinosylAZT, AZA-Azathromycin, AZT-Azithromycin, NDMAZT-N-demetylazithromycin, EAIE-Erythromycin A iminoether, EAO-Erythromycin A oxime Figure 2: Typical chromatogram of the working mixture obtained under optimized chromatographic conditions

Sample preparation procedure Tablet powder equivalent to 250 mg AZT was transferred into a 50 ml volumetric flask followed by addition of 25 ml acetonitrile then sonicated for 15 min, filled to the mark with distilled water and filtered through a 0.45 µm membrane filter. Suspension equivalent to 40 mg/ml weighed in 5 ml volumetric flasks. This was followed by addition of 2.5 ml of acetonitrile then sonicated for 15 min, filled to the mark with distilled water and filtered . Azithromycin raw material was prepared using the procedure described for the standard solution in the USP (2012).

Analysis of samples The online drug PPB register listed 77 products with only two brands of AZT manufactured locally Two brands (all tablets) did not appear on the online register Test samples randomly purchased from pharmacies within Nairobi CBD. Samples analysed using the developed and validated LC method. Nine AZT suspensions analysed -5 premixed, 4-powder. 13 tablet brands analysed Zithromax® donated by Pfizer, Raw material-Skylight Chemicals Ltd

Assay results Product Code Formulation % Content Remarks I Tablet 93.6 (1.55) C 94.8 (1.04) II 110.3(0.16) 109.1(0.12) III 98.5 (0.46 99.2 (0.31) IV 97.2 (0.19) 97.6 (0.19) V 102.0 (1.51)

VI Tablet 109.9 (0.32) C 107.3 (0.26) VII 105.1 (1.60) 104.3 (1.28) VIII Suspension (M) 110.0 (0.61) Suspension(M) 109.3 (0.47) IX 96.4 (0.72 96.1 (0.18) X 69.3 (1.68) NC XI 89.3 (0.39) XII 81.4 (0.46) XIII 99.8 (0.31) XIV 83.3 (0.15) XV 87.0 (1.13)

P-Powder for reconstitution, M-Premixed, C-Complied, NC- Did not comply XVI Tablet 103.3 (1.33) C XVII Suspension (P) 88.6 (0.16) NC XVIII 105.5 (0.36) XIX Suspension (M) 87.79 (1.32) XX 98.7 (0.66) Raw material Powder 94.7 (0.09) Zithromax® 99.6 (0.71) 96.4 (1.48)

Summary of results Formulation Number Failure (%) Tablets 13 23.1 Suspension 9 44.4

II XIX Figure 3: Typical chromatograms of samples analysed (II=Tablet sample, XIX= Suspension sample)

Analytical findings Content for premixed suspensions was relatively lower compared dry powders for reconstitution. One of the 3 tablet brands that did not meet the assay specifications was listed in the online PPB register. Need for post market surveillance to ensure good products are in circulation Limitations: Batch to batch comparison not possible, limited sample area, suspicion, convenience sampling.

Conclusion and recommendations A simple, fast, isocratic LC method for the analysis of AZT was developed and validated. Market has poor quality AZT is a matter of public health concern Post market surveillance to curb poor quality products and counterfeits

Acknowledgements Director NQCL Mr. Rotich (NQCL)and Mr. Mugo (UoN) Pfizer Laboratories: Dr. Maina, Ms. Winnie, Mr. Seroney, Dr. Epaphrodite. Skylight Chemicals: Dr. Weru Drug Analysis Research Unit staff Chairman and staff at The Department of Pharmaceutical Chemistry, UoN.