DRUG FORMULATIONS AND CLINICAL METHODS Quantitative Determination and Sampling of Lamivudine and Zidovudine Residues for Cleaning Validation in a Production Area MARIA INÊS ROCHA MIRITELLO SANTORO,TATIANA TATIT FAZIO,ANIL KUMAR SINGH, and ERICA ROSA MARIA KEDOR-HACKMANN Universidade de São Paulo, Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Caixa Postal 66083, CEP 05315-970, São Paulo, SP, Brazil Lamivudine (3TC) and zidovudine (AZT) are systemic antiviral substances extensively used in human immunodeficiency virus (HIV) infected patients. Nowadays, 3TC, AZT, and several other pharmacologically potent pharmaceuticals are manufactured in the same production area. To assure quality of drug products and patient safety, properly validated cleaning methodology is necessary. A carefully designed cleaning validation and its evaluation can ensure that residues of 3TC and AZT will not carry over and cross contaminate the subsequent product. The aim of this study was to validate a simple analytical method for verification of residual 3TC and AZT in equipment used in the production area and to confirm the efficiency of the cleaning procedure. The liquid chromatography method was validated using a Nova-Pak® C18 column (3.9 ´ 150 mm, 4 mm particle size) and methanol–water (20 + 80, v/v) as the mobile phase at a flow rate of 1.0 mL/min. Ultraviolet detection was made at 266 nm. The calibration curve was linear over a concentration range of 2.0–22.0 mg/mL with a correlation coefficient of 0.9998. The detection and quantitation limits were 0.36 and 1.21 mg/mL, respectively. The intra-day and interday precision expressed as relative standard deviation were below 2.0%. The mean recovery of the method was 99.19%. The mean extraction recovery from manufacturing equipment was 83.5%. L amivudine (3¢-thia-2¢,3¢-dideoxcytidine; 3TC) and zidovudine (3¢-azido-3¢-deoxythyimidine; AZT) are the most widely used substances in acquired immunodeficiency syndrome (AIDS) treatment. These antiviral agents have a potent inhibitory effect on human immunodeficiency virus (HIV; 1–4). The chemical structures of 3TC and AZT are illustrated in Figure 1. On the industrial scale, 3TC- and AZT-coated tablets are produced by a dry granulation method. For the production of 3TC and AZT, a production area of larger complexity is necessary due to the numerous risks associated to occupational exposure and cross contamination. The objective of a cleaning validation is to prove, through a validated analytical method, that the cleaning procedure is efficient in removing product residues and excipients, degradation products, cleaning substance, and other possible contaminants. In this way, cross contamination risk in the production area can be reduced substantially (5–7). During the cleaning validation, the following factors should be taken into consideration: equipment construction material, sealing part, and parts that offers the greatest risk of contamination. It is important to standardize the cleaning procedures and cleaning material, verification of chemical products residues, and postcleaning microbial load. Other factors, such as the time that the equipment can be considered clean, the sampling procedure, and analysis of contaminating residues in the equipment, should also be considered. The analysis method and selected sampling procedure should be validated and provide adequate extraction recovery to allow the analysis of possible contaminating residues (5–7). The acceptable limit for residue in the equipment is not established in the current regulations. However, the U.S. Food and Drug Administration (FDA) states that the limit should be based on logical criteria, involving the risk associated with residues of a product (6). The calculation of acceptable residual limits for active products in production equipment should be based on therapeutic doses, pharmacological activity, and toxicological index. Several mathematical formulas were proposed that can be used to establish acceptable residual limits (5). Several liquid chromatography (LC) methods have been described for 3TC and AZT determination in biological fluids (8–12) and, to a lesser extent, in raw material and pharmaceutical preparations (13). Expensive and complicated instrumentation, such as column switching, gradient chromatographic systems and mass spectrophotometric detectors, makes these methods inaccessible for routine cleaning validation studies. The aim of this study was to optimize the extraction of 3TC and AZT from a simulated production area, validate a simple analytical method and its SANTORO ET AL:JOURNAL OF AOAC INTERNATIONAL VOL. 90, NO. 3, 2007 715 Received August 23, 2006. Accepted by AH December 12, 2006. Corresponding author's e-mail: ines@usp.br Downloaded from https://academic.oup.com/jaoac/article/90/3/715/5657920 by guest on 01 March 2023