Determination of Verapamil Hydrochloride Purity Using the Acclaim PA Column
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1 Application Note 95 Determination of Hydrochloride Purity Using the Acclaim PA Column INTRODUCTION -based medications are prescribed for several heart and blood pressure indications. The fastacting formulations (verapamil hydrochloride and Isoptin ) are taken for angina, as well as irregular heartbeat and high blood pressure. The United States Pharmacopeia (USP ) has a monograph method to determine verapamil hydrochloride purity. This method separates verapamil hydrochloride and verapamil-related compound B using HPLC with a C8 (USP designation L) column. There is a recent proposal to revise the method by using a L60 column to determine verapamil hydrochloride and verapamil-related compounds B and D. The existing method can not determine compound D under the prescribed eluent. The proposed method is time consuming (6 min), and requires a special column. In this application note, we describe a new method for the fast determination of verapamil hydrochloride and verapamil-related compounds A, B, and D, using a polar-embedded reversed-phase column, the Acclaim PolarAdvantage (PA). The new method requires only about half the time of the proposed USP monograph method, provides significant eluent and therefore cost savings, and meets the resolution requirement. EQUIPMENT UltiMate 000 HPLC HPG 00 pump with SRD 00 degasser WPS 000 TSL autosampler TCC 000 thermostatted column compartment VWD-00 UV-Vis detector Chromeleon 6.80 SP Chromatography Workstation REAGENTS AND STANDARDS Water, Milli-Q water from Milli-Q Gradient A0 Acetonitrile (CH CN), Fisher, HPLC grade KH PO, reagent grade, (AR, analytical pure, grade in China) H PO, reagent grade, (AR, analytical pure, grade in China) HCl (Sigma, CAS: 5--), purity > 99.0% -related compound A (USP, P/N: 70) -related compound B (USP, P/N: 70) -related compound D (LGC Promochem GmbH, P/N: USP78) Application Note 95
2 PREPARATION OF REAGENTS AND STANDARDS Prepare two solutions for testing, consistent with the requirements of the USP monograph method. System suitability solution: Prepare a mixture of verapamil hydrochloride, verapamil-related compound B, and verapamil-related compound D where each component has a concentration of 5 µg/ml. Add an additional component not described in the USP method, verapamil-related compound A, also at 5 µg/ml. Use this solution for method development. Standard solution and test solution: Prepare a.5 mg/ml standard solution of verapamil hydrochloride. To this solution add the three related compounds to achieve a final concentration of.5 µg/ml each, which serves as the test solution to simulate real samples. CHROMATOGRAPHIC CONDITIONS Column: Acclaim PA, 5 µm,.6 50 mm (P/N 06) Mobile Phase: A: 0 mm KH PO adjust ph to.0 with H PO B: CH CN Detection: Absorbance at 78 nm Gradient Table: RESULTS AND DISCUSSION (Figure ) with two aromatic rings is ideally suited for analysis by reversed-phase HPLC. Using the proposed USP monograph method as a starting point, we developed a much faster method that requires significantly less eluent. Figure shows a separation of the system suitability solution described in the proposed revision to the USP monograph method with an addition of verapamil-related compound A. All four compounds are well resolved. The resolution of verapamil and verapamil-related compound B is >5.0 as required by the proposed method and >.5 as required by the current USP monograph method. -related compound D is eluted, as required by the proposed USP method. Table shows the repeatability for retention time and peak areas for five injections of the system suitability standard. The method described here requires only 5 min compared to the 6 min required by the proposed USP method. In addition to the 6 min savings, there is 56.5 ml of eluent savings per injection because the method described here is run at.0 ml/min rather than.5 ml/min. Chromatography of the test solution (Figure ) shows that this method easily detects 0.% quantities of verapamil-related compounds A, B, and D relative to verapamil at.5 mg/ml, making it ideal for verapamil purity analysis. OCH OCH OCH OCH Time Flow Rate Buffer (%) CH CN (%) Curve ml/min 70 0 CN N.0.0 ml/min ml/min ml/min ml/min Figure. Structure of verapamil. CH ml/min Determination of Hydrochloride Purity Using the Acclaim PA Column
3 Column: Acclaim PA (.6 x 50 mm) Eluent: A) 0 mm KH PO adjust ph to.00 by H PO Flow Rate:.0 ml/min 8 Detection: UV (78 nm) Sample: System Suitability Solution µg/ml. related compound A 5. related compound B 5. HCI RS 5. related compound D 5 Rs= Figure. Overlay of five chromatograms of the system suitability solution Table. Repeatability of Retention Time and Peak Areas of Five Injections of the System Suitability Solution RT (min)/ Area ( min) Compound A Compound B HCI RS Compound D Injection Injection Injection Injection Injection RSD To achieve these improvements over the proposed USP monograph method, our method uses a polarembedded reversed-phase column, the Acclaim PA, rather than an L60. No current L description suitably describes the resin in the PA column. It previously proved successful in improving the USP monograph method for nevirapine, which also prescribes a L60 column. To shorten run time, achieve the best resolution, and work at a ph that will ensure the longest possible column life, the ph of the mobile phase is adjusted to.0 rather than the 7. ph recommended for the L60 column mobile phase. Column: Acclaim PA (.6 x 50 mm) Eluent: A) 0 mm KH PO Detection: UV (78 nm) adjust ph to.00 by H PO Sample: Test Solution. related compound A.5 µg/ml Flow Rate:.0 ml/min. related compound B.5 µg/ml 50. HCI RS.5 mg/ml. related compound D.5 µg/ml Figure. Chromatogram of the test solution. Application Note 95
4 Effect of mobile phase ph During method development, testing the effect of ph. to 7.0 showed that lower ph provides higher resolution of verapamil-related compound B and verapamil. However, ph values lower than. did not significantly improve resolution. To meet the USP resolution requirement (> 5.0), a 50 mm column was required (same length as the L60). If a 50-mm column is preferred and a resolution of 5.0 between verapamil and verapamil-related compound B is not required, the 50-mm column will yield a resolution of about.0. Figures and 5 show the system suitability and test solutions analyzed at ph.9,, and.. The chromatography shows that the method is rugged when challenged by small changes in ph. Column: Acclaim PA (.6 x 50 mm) Sample: Eluent: A) 0 mm KH PO adjust ph with H PO Flow Rate:.0 ml/min Detection: UV (78 nm) 8 8 A B System Suitability Solution Cgrams: A. ph =.0 B. ph =.87 C. ph =.00. related compound A 5 µg/ml. related compound B 5 µg/ml. HCI RS 5 mg/ml. related compound D 5 µg/ml ph=.0, Rs=5. Column: Acclaim PA (.6 x 50 mm) Sample: Eluent: A) 0 mm KH PO adjust ph with H PO Flow Rate:.0 ml/min Detection: UV (78 nm) A B C System Suitability Solution Cgrams: A. ph =.0 B. ph =.87 C. ph =.00. related compound A.5 µg/ml. related compound B.5 µg/ml. HCI RS.5 mg/ml. related compound D.5 µg/ml ph=.0 ph=.87 ph= Figure 5. Analysis of the test solution with different ph mobile phases. ) verapamil-related compound A (.5 µg/ml); ) verapamil-related compound B (.5 µg/ml), ) verapamil HCI RS (.5 mg/ml); ) verapamil-related compound D (.5 µg/ml). 0 C ph=.87, Rs=5. ph=.0, Rs= Figure. Overlay of chromatograms of the system suitability solution analyzed with different ph mobile phases. ) verapamilrelated compound A; ) verapamil-related compound B; ) verapamil HCI RS, ) verapamil-related compound D. Determination of Hydrochloride Purity Using the Acclaim PA Column
5 REFERENCES. United States Pharmacopeia National Formulary USP0/NF5 (007), 5.. Pharmacopeial Forum (006) () 89.. Dionex Application Note 80. Isoptin is a registered trademark of Knoll Aktiengesellschaft. USP is a registered trademark of the United States Pharmacopeia. Acclaim, Chromeleon, and UltiMate are registered trademarks of Dionex Corporation. Passion. Power. Productivity. Dionex Corporation North America Europe Asia Pacific 8 Titan Way P.O. Box 60 U.S. (87) Canada (905) Austria () Benelux () () 5 9 Denmark (5) France () Germany (9) Sunnyvale, CA Ireland (5) Italy (9) Switzerland () Taiwan (886) South America United Kingdom () Brazil (55) 7 50 (08) Australia (6) 90 5 China (85) 8 8 India (9) Japan (8) Korea (8) Singapore (65) Application Note 95 LPN 09 PDF 9/6 06 Dionex Corporation
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