ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
DEVELOPMENT AND VALIDATION OF AN HPLC METHOD FOR THE DETERMINATION OF VERAPAMIL RESIDUES IN SUPPORTS
OF CLEANING PROCEDURE
© 2013 г. Dragan M. Milenovic1, Snez ana P. Milosevic1, Svetlana Lj. Duric1, Daniela C. NaskoviC1, Sne z ana S. MitiC2
1 "Zdravlje-Actavis" company, R&D Vlajkova street 199, Leskovac, 16000 Serbia 2 Faculty of Sciences and Mathematics, Department of Chemistry, University of Nis Visegradska 33, P.O. Box 224, Nis, 18000 Serbia Received 23.03.2011; in final form 17.06.2011
Analytical method validation, determining the recovery rate from the equipment surface, and stability of a potential contaminant are important steps of a cleaning validation process. An HPLC method for the determination of the verapamil residues on stainless steel surfaces of the equipment employed in drug manufacture is described. The cleaning validation sample impurities as well as excipients of the commercial sample did not interfere in the analysis which proved the selectivity of the method. The validation of the method demonstrated acceptable levels of the linearity, precision and accuracy. Cotton swabs, moistened with a methanol were used to remove any residues of drugs from stainless steel surfaces, and give recoveries of above 78.59% for three diferent concentration levels. The precision of the results, reported as the relative standard deviation (RSD, %), were below 1.58%. Low quantities of the drug residues were determined by HPLC using a Hypersil ODS column (125 x 4.0 mm, 5 pm) at 25°C with the mobile phase metanol—water—triethylamine (70 : 30 : 0.2, v/v/v) at a flow rate of 0.6 mL/min, injection volume of 50 pL and detection at 278 nm.
Keywords: cleaning validation, verapamil, swab analysis, residues.
DOI: 10.7868/S0044450213060054
Pharmaceutical manufacturing equipment has to be cleaned after production in order to avoid cross contamination in the next batch of a different product. In the end of the cleaning procedure the effectiveness of the cleaning is checked using a validated analytical method suitable to investigate the traces of residues.
The cleaning validation consists of two separate steps: the first one is the development and validation of the cleaning procedure, which is used to remove drug residue from the manufacturing surfaces, and the second one involves the developing and validating of the methods for quantifing residuals from surfaces of the manufacturing equipment. It is the responsibility of the manufacturer to develop robust cleaning procedures, and to demonstrate that execution of the cleaning procedures was successful. Futhermore, many sampling points of the manufacturing equipment have to be tested for verifying occurrence of contamination. For these reasons, an analytical method for residue monitoring should also be rapid and simple [1].
The acceptable limit for residue in the equipments is not established in the current regulations. The design of a suitable sampling procedure and analytical method is very important in cleaning validation. The
technique must be appropriate for measuring the ana-lytes at and below the residue acceptable limit. According to FDA (Food and Drug Administration), the limit should be based on logical criteria, involving the risk associated with residues of a determined product. The calculation of acceptable residual limit, maximum allowable carryover, for active products in production equipments should be based on therapeutical doses, toxicological index and a general limit (10 ppm) [1 - 4].
Verapamil, [(±)-5-[N-(3,4-dimethoxyphenethyl)-N-methylamino]-2-(3,4-dimethoxyphenyl)-2-iso-pro-pyl-valeronitrile], is a calcium-channel blocker and is classified as a class IV anti-arrhythmic agent. It is used in the control of supra ventricular tachyarrhythmias, and in the management of classical and variant angina pecto-ris [5].
Numerous methods have been reported for the quantitative determination of verapamil hydrochloride in the raw materials [6—13], tablets and other solid dosage forms [5, 14—16], human plasma [17], by HPTLC or TLC [18, 19]. A literature review revealed that no validation of cleaning methods for verapamil could be found.
Taking the above mentioned consideration into account, the aim of this study was to develop and validate simple analytical method that allows the determination at trace level of residual verapamil in production area equipment and to confirm efficiency of cleaning procedure. The analytical method proposed has been validated considering selectivity, linearity, accuracy, precision and limits of detection (LOD) and quantitation (LOQ). The stability of verapamil samples was also studied [20].
EXPERIMENTAL
Chemicals and reagents. The verapamil hydrochlo-ride, working certified standard, was purchased from Recordati, Industria Chimica E Farmaceutica S.p.A. (Italy). Methanol (HPLC gradient grade) and triethyl-amine were purchased from J.T. Baker (Deventer, Holland). Purified water was obtained with a Arium Laboratory Equipment (RO, UV) by Sartorius AG (Gottingen, Germany). The extraction-recovery sampling was done with Alpha® Swab polyester on polypropylene handle — TX714A (ITW Texwipe®, Mahwah, NJ, USA). The mobile phase was filtered through a 0.45 ^m Sartorius membrane filter (Gottingen, Germany).
Equipment. The HPLC system consisted of a de-gasser G1379B, a bin pump G1312A, an automatic injector G1329A, a thermostated column compartment G1316A and a multiwavelength detector G1365B (multiwavelength), all 1200 Series, from Agilent Technologies controlled by an HP Chemstation software (Waldbroon, Germany). Ultrasonic bath was from Elma, Transsonic 470/H (Singen, Germany). Analytical balance was from Sartorius AG, CP224S-OCE (Gottingen, Germany); accuracy of the balance: ±0.0001 g.
Chromatographic conditions. All chromatographic experiments were performed in the isocratic mode. The mobile phase was constituted of methanol-wa-ter-triethylamine (70 : 30 : 0.2, v/v/v), at a flow rate of 0.6 mL/min. UV detection was made at 278 nm. The volume of injection was fixed at 50 ^L. All analyses were performed at 25°C. The separation was carried out on a Hypersil ODS column (125 x 4.0 mm, 5 ^m) from Agilent.
Standard solutions preparation. Stock solution of standard was prepared by accurately weighing verapamil hydrochlorid standard (25 mg ± 0.1 mg), transferring it into 25 mL volumetric flask, diluting to volume with methanol, and sonication for 15 min. Dilutions were later prepared with the mobile phase to obtain the solutions for calibration (2.50 do 50 ^g/mL) and standard solution for the positive swab control at three concentration levels (50, 100, and 150 ^g/swab level). These solutions were filtered through a 0.45 ^m regenerated cellulose filter and injected in triplicate.
Sample preparation. The selected surfaces (5 x 5 cm) of stainless steel, previously cleaned and
dryed, were sprayed with 500 ^L of a standard solutions for positive swab control at all concentration levels, and the solvent was allowed to evaporate (approximate time was 2 h). The surfaces were wiped with the first cotton swab soaked with methanol, passing it in various ways, to remove the residues from stainless steel. The other dry cotton swab was used to wipe the wet surfaces. The swabs were placed into the 25 mL screw cap test tubes, and 5.0 mL of the mobile phase was pipetted into adequate sample tubes. The background control sample was prepared from the extraction media. The negative swab control was prepared in the same way as the samples, using swabs, which had not been in contact with the test surface. Also, the test and excipient solutions were prepared according to the content of tablets to assure that they did not interfere with verapamil hydrochloride. After that, the tubes were placed in the ultrasonic bath for 30 min and the solutions were analysed by HPLC. FDA guidelines recommend a minimum recovery of 50%.
RESULTS AND DISCUSSION
Acceptance limit calculation. The maximum allowable carryover — MACO is acceptable transferred amount from the previous to the following product. MACO is determined on the basis of therapeutic dose, toxicity and general 10 ppm criterion. The next step is to determine the residue limit per surface area from the equipment surface area and the most stringent maximum allowable carryover (the most stringent criterion being based on the therapeutical dose in this case). The calculated limit per surface area in the case of vera-pamil hydrochloride was 100 ^g/swab for 25 cm2.
Selection of the chromatographic conditions. To obtain the best chromatographic conditions, the wavelength for detection, the column and the mobile phase composition were adequately selected. The main objective was to develop a liquid chromatographic method that, working in isocratic mode, allowed us to determine the total verapamil hydrochloride residues collected by the swabs, without interference of impurities which originated from swabs, plates, extraction media.
The wavelength of 278 nm was selected for the analysis because the drug had sufficient absorption and low quantities of verapamil hydrochloride may be detected correctly. Furthermore, the calibration curves obtained at 278 nm show good linearity.
Starting point for the development of the cleaning assay for verapamil hydrochloride was modified work on the assay method for verapamil in capsules [14] by using Purospher STAR RP-18e, 250 x 4 mm, 5 ^m column with mobile phase acetonitrile—methanol— phosphate buffer (the buffer was prepared with 0.025 M potassium dihydrogen phosphate by adjusting to pH 3.0 with o-phosphoric acid) (40 : 40 : 20 = v/v/v), with 50 ^L injection volume at 278 nm. An initial attempt
Table 1. Results of sample treatment optimization
Solvent Volume, mL Recovery % ± CI* (n = 3)
Time of extraction, min
5 15 30
Verapamil hydrochloride Mobile phase Methanol Water 5 10 5 10 5 10 91.23 ± 2.61 92.71 ± 1.15 84.41 ± 1.54 85.24 ± 2.3
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.