the determination of the verapamil residues on stainless steel surfaces of the equipment employed in drug manufacture is described. The cleaning validation ...
ISSN 10619348, Journal of Analytical Chemistry, 2013, Vol. 68, No. 6, pp. 545–551. © Pleiades Publishing, Ltd., 2013.
ARTICLES
Development and Validation of an HPLC Method for the Determination of Verapamil Residues in Supports of Cleaning Procedure1 ˆ
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Dragan M. Milenovica, Sne z ana P. Milo s evica, Svetlana Lj. Durica, Daniela C . Naskovica, and Sne z ana S. Miticb ˆ
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“ZdravljeActavis” company, R&D Vlajkova street 199, Leskovac, 16000 Serbia of Sciences and Mathematics, Department of Chemistry, University of Ni s Vi s egradska 33, P.O. Box 224, Ni s , 18000 Serbia
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a
b Faculty
Received March 23, 2011; in final form, June 17, 2011
Abstract—Analytical method validation, determining the recovery rate from the equipment surface, and sta bility 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 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 × 4.0 mm, 5 µm) 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 µL and detection at 278 nm. Keywords:cleaning validation, verapamil, swab analysis, residues DOI: 10.1134/S1061934813060051 1
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 sec ond 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 proce dures, and to demonstrate that execution of the clean ing 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 1 The article is published in the original.
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 prod uct. The calculation of acceptable residual limit, max imum allowable carryover, for active products in pro duction equipments should be based on therapeutical doses, toxicological index and a general limit (10 ppm) [1–4]. Verapamil, [(±)5[N(3,4dimethoxyphenethyl) Nmethylamino]2(3,4dimethoxyphenyl)2isopro pylvaleronitrile], is a calciumchannel blocker and is classified as a class IV antiarrhythmic 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 hydrochlo ride 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.
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Taking the above mentioned consideration into ac count, the aim of this study was to develop and validate simple analytical method that allows the determina tion 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 quantita tion (LOQ). The stability of verapamil samples was al so 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 extractionrecovery sam pling was done with Alpha® Swab polyester on polypropylene handle—TX714A (ITW Texwipe®, Mahwah, NJ, USA). The mobile phase was filtered through a 0.45 µm Sartorius membrane filter (Gottin gen, Germany). Equipment. The HPLC system consisted of a de gasser G1379B, a bin pump G1312A, an automatic in jector G1329A, a thermostated column compartment G1316A and a multiwavelength detector G1365B (multiwavelength), all 1200 Series, from Agilent Tech nologies controlled by an HP Chemstation software (Waldbroon, Germany). Ultrasonic bath was from Elma, Transsonic 470/H (Singen, Germany). Analytical bal ance was from Sartorius AG, CP224SOCE (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 µL. All analy ses were performed at 25°C. The separation was car ried out on a Hypersil ODS column (125 × 4.0 mm, 5 µm) from Agilent. Standard solutions preparation. Stock solution of standard was prepared by accurately weighing vera pamil hydrochloride standard (25 mg ± 0.1 mg), trans ferring it into 25 mL volumetric flask, diluting to vol ume with methanol, and sonicating for 15 min. Dilu tions were later prepared with the mobile phase to obtain the solutions for calibration (2.50 do 50 µg/mL) and standard solution for the positive swab control at three concentration levels (50, 100, and 150 µg/swab level). These solutions were filtered through a 0.45 μm regen erated cellulose filter and injected in triplicate. Sample preparation. The selected surfaces (5 × 5 cm) of stainless steel, previously cleaned and
dryed, were sprayed with 500 µL of a standard solu tions for positive swab control at all concentration lev els, and the solvent was allowed to evaporate (approx imate 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 back ground control sample was prepared from the extrac tion 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 allow able 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 cri terion being based on the therapeutical dose in this case). The calculated limit per surface area in the case of vera pamil hydrochloride was 100 µg/swab for 25 cm2. Selection of the chromatographic conditions. To ob tain the best chromatographic conditions, the wave length for detection, the column and the mobile phase composition were adequately selected. The main ob jective was to develop a liquid chromatographic meth od that, working in isocratic mode, allowed us to de termine the total verapamil hydrochloride residues collected by the swabs, without interference of impu rities 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 RP18e, 250 × 4 mm, 5 µm 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 ophosphoric acid) (40 : 40 : 20 = v/v/v), with 50 µL injection volume at 278 nm. An initial attempt
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Table 1. Results of sample treatment optimization (recovery % ± confidence interval; n = 3) Time of extraction, min Analite
Verapamil hydrochloride
Solvent
Volume, mL
Mobile phase Methanol Water
5
15
30
5
91.23 ± 2.61
93.34 ± 1.56
96.39 ± 0.18
10
92.71 ± 1.15
93.21 ± 2.43
97.21 ± 1.32
5
84.41 ± 1.54
85.32 ± 2.36
85.52 ± 2.11
10
85.24 ± 2.34
86.21 ± 3.31
87.51 ± 0.87
5
82.65 ± 2.12
83.74 ± 1.15
83.91 ± 2.75
10
83.24 ± 3.54
84.51 ± 2.71
84.62 ± 0.18
Table 2. Recovery (%) for different swab numbers used Sampling method Analite
Verapamil hydrochloride
Concentration, µg/swab (n = 3)
Swab wetted by methanol
Swab wetted by methanol, afterwards 1 dry swab
50
75.73 ± 2.14
93.04 ± 1.48
100
78.56 ± 2.31
95.40 ± 0.05
150
80.11 ± 4.31
96.39 ± 0.18
resulted with short retention time (about 4 min), USP tailing about 1.7, but recovery value for the lowest con centration level were above 102% (102–106%) (inter ferences with swab samples). Therefore, our work were proceeded with Hypersil ODS short column (125 × 4 mm, 5 µm) in order to get the shortest retention time, with mobile phase con taining a considerable amount of organic modifier, methanol⎯water⎯triethylamine (70 : 30 : 0.2, v/v/v). The peak symmetry was improved by the addition of a triethylamine into the mobile phase. Retention time was about 9 min, with excelent features of peaks (USP tailing about 1.0). Concerning not too long retention time and excelent features of peaks, this chromato graphic conditions were used for the rest of the work. Triethylamine acts as a competing base and minimizes solutesilanol interactions. The injection volume was set at 50 µL in order to increase the responce of the method without sacrific ing chromatographic peak shape. Also, flow rate was set at 0.6 mL/min in order to get higher responce of the metod (lower flow—lower column backpressure, higher peak area). As increase in temperature did not affect on chromatographic efficiency (number of the oretical plates), a temperature of 25°C was selected. JOURNAL OF ANALYTICAL CHEMISTRY
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Taking into account the results obtained with dif ferent columns and mobile phases assayed, finally we have chosen chromatographic conditions which were mentioned because the quantification limits obtained were the lowest, with good sensitivity and without interferences. Sample treatment optimization. Cotton swabs were spiked with 150 µg/swab of verapamil hydrochloride and were placed into a tube. After adding the different solvents (water, methanol and the mobile phase), the tube was sonicated in different times (5, 15 and 30 min) and the solutions were analyzed by HPLC. The opti mum condition was achieved with the mobile phase as an extracting solvent and the best sonication time was 30 min. Results are given in Table 1. In all cases, the best results were obtained using the two cotton swabs for sampling (the first one was wetted with methanol and the second one was dry), so this technique was used for the rest of the work. Results are given in Table 2. Surface for swabbing. Stainless steel coupon was an obvious choice for surface material, because more than 95% of manufacturing equipment surfaces are stainless steel. For practical reasons, coupon dimen sions of 5 × 5 cm were chosen. Recovery % for differ No. 6
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Table 3. Recovery (%) for different surfaces for lowest concentration level Analyte
Concentration, µg/swab
Surface
Verapamil hydrochloride
Recovery % ± CI*
Stainless steel
93.04 ± 1.48
Glass
50
86.14 ± 1.56
Polivinylplastics
84.21 ± 3.41
* Confidence interval.
ent surface areas (glass and polivinylplastics) are also investigated, regardless of their little part in total area. Results are given in Table 3.
Selectivity. Selectivity has been checked by inject ing a standard of verapamil hydrochloride, the back ground control sample containing the mobile phase, the negative swab control, the unspiked stainless steel 5 × 5 cm plate swabbed as described, the excipient mixture. In figure, it can be observed that there are no mutual interferences. Linearity. Linearity of the method was studied by analyzing the standard solutions at different concen tration levels ranging from 2.50 to 50.00 µg/mL, with triplicate determination at each level. The calibration curve was constructed by plotting the mean response area against the corresponding concentration inject ed, using the least square method. Values of the slope, the intercept and coefficient of determination of the calibration curve for verapamil hydrochloride are giv
Validation of the chromatographic method. Once the chromatographic conditions had been selected, the method was validated paying attention to selectiv ity, linearity, limit of detection, limit of quantification, precision, accuracy and sample stability.
Abs/mAU
30 25 20 15 10 5 2
Abs/mAU
0 30 25 20 15 10 5 0 30 25 20 15 10 5
2
Abs/mAU
Suitability test. System suitability testing is essen tial for the assurance of the quality performance of the chromatographic system. During performing system suitability tests, in all cases, % RSD for peak areas was 3300, the USP tailing factor was about 1.0.
0
2
(a)
4
6
8
10
12
14
10
12
14
12
14
(b)
4
6
8 (с)
4
6
8.639 Verapamil
8 Time/min
10
Chromatograms obtained for: (a) non–spiked stainless steel surface, (b) excipient mixture, (c) standard solution of verapamil hydrochloride (20 µg/mL). JOURNAL OF ANALYTICAL CHEMISTRY
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DEVELOPMENT AND VALIDATION OF AN HPLC METHOD Table 4. Linear regression data in the analysis of verapamil hydrochloride Statistical parameters
Obtained values
Concentration range (µg/mL)
2.50–50.00 y = 32.598x +10.619
Regression equation Error in slope (Sb)
0.359
Error in intercept (Sa)
9.554
Error for yest (Sy/x)
16.235
Regression sum of squares (ssreg)
2175488.610
Residual sum of squares (ssresid)
1844.964
F statistic (F)
8254.049 7.000
Degrees of freedom (dF) Coefficient of determination (r
2)
0.9992
LOD (µg/mL)
1.64
LOQ (µg/mL)
4.98
en in Table 4. The high value of the coefficient of de termination indicated a good linearity. Limits of detection and quantitation. LOD and LOQ were determined based on the standard deviation of the response (yintercept) and the slope of the cali bration curve according to the ICH guidelines. LOD and LOQ for verapamil hydrochloride were found to be 1.64 and 4.98 µg/mL, respectively (Table 4). Precision and accuracy. The precision and accura cy of the chromatographic method, reported as rela tive standard deviation (RSD %) and the recovery %, respectively were assessed by estimating the repeat ability of the results for six replicate injections at three different concentration levels. The method precision
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and accuracy was determined on the spiked and dried swabs and plates. The recovery, 95% confidence inter val, and RSD values obtained on the spiked and dried swabs and plates (Table 5) per each level illustrated good precision and accuracy of the method. These precision and recovery results are acceptable for the purpose of residue monitoring. Intermediate precision of the method was investi gated by making five consecutive injections of a stan dard solutions in two different days with different ana lysts, on two different HPLC systems. On both days the RSD % were calculated for peak area responses obtained for the verapamil hydrochloride peaks. The data obtained suggested that the method exhibited an acceptable intermediate precision with less than 2.0% RSD for the verapamil hydrochloride standard solu tion. Sample stability. The stability of the verapamil hy drochloride in the swab matrix was tested. The spiked samples at all concentration levels were stored after analyses in the injector vials in the autosampler tray at ambient temperature for 7 days. All the samples were injected into the appropriate HPLC system after 24 h, 48 h and 7 days against fresh standard solutions. No changes in the chromatography of the stored samples were found and no additional peaks appeared when compared with chromatograms of the freshly prepared samples. Results are given in Table 6. Assay of swab samples collected from different loca tions within the equipment train. Swab samples from different locations within the manufacturing equip ment train were submitted to the laboratory for the analysis of verapamil hydrochloride residual. These samples were prepared and analyzed by the proposed method. For most location (Material dispensing scoops, Turbo sieve—Bohle, Fluid bed dryer—Glatt WSG, Washer—extractor Miele, Metal detector— Lock Met 30+, Tablet press—Kilian) the residues
Table 5. Precision and accuracy of the results obtained from swabs and plates spiked with verapamil hydrochloride Sample Amount added, µg/mL Amount found, µg/mL 95% confidence interval, % Recovery, % RSD, % (n = 6)
Swabs
Plates
10.00
9.30
91.63–94.29
92.96
1.79
20.00
19.08
95.32–95.49
95.41
0.11
30.00
28.93
96.24–96.61
96.42
0.24
10.00
7.86
78.22–78.95
78.59
0.58
20.00
15.82
78.11–80.11
79.11
1.58
30.00
23.72
78.50–79.63
79.06
0.90
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Table 6. Stability results obtained from the verapamil hydrochloride swab extract samples Sample (n = 3) (µg/swab)
Mean recovery (%) ± CI*; 0 h
Mean recovery, (%) ± CI; 24 h
Mean recovery, % ± CI*; 48 h
Mean recovery (%) ± CI*; 7 days
10.00
93.68 ± 1.05
94.08 ± 1.38
94.11 ± 1.12
93.66 ± 1.67
20.00
93.99 ± 0.49
94.29 ± 0.33
94.00 ± 0.80
93.45 ± 0.92
30.00
97.45 ± 0.26
98.21 ± 0.65
97.73 ± 0.26
97.15 ± 0.18
* Confidence interval.
Table 7. Results obtained for the determination of verapamil hydrochloride in actual swab samples collected from different locations within the equipment train Equipment swabbed Material Dispensing
Scoops
High Shear Mixer—Diosna
Turbo Sieve—Bohle
Fluid bed Dryer—Glatt WSG
Location swabbed*
Verapamil hydrochloride detected, µg/swab
Internal surface
