Journal of Pharmaceutical Investigation

The Korean Society of Pharmaceutical Sciences and Technology (한국약제학회)
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Development and Validation of HPLC Method for Pharmacokinetic Study of Promethazine in Human

염산프로메타진 체내동태 연구를 위한 혈청 중 프로메타진의 HPLC 정량법 개발 및 검증

  • Cho, Hae-Young (Institute of Bioequivalence and Bridging Study, College of Pharmacy, Chonnam National University, Clinical Trial Center, Chonnam National University Hospital) ;
  • Kang, Hyun-Ah (Institute of Bioequivalence and Bridging Study, College of Pharmacy, Chonnam National University, Clinical Trial Center, Chonnam National University Hospital) ;
  • Lee, Hwa-Jeong (College of Pharmacy, Ewha Womans University) ;
  • Choi, Hoo-Kyun (College of Pharmacy, Chosun University) ;
  • Lee, Yong-Bok (Institute of Bioequivalence and Bridging Study, College of Pharmacy, Chonnam National University, Clinical Trial Center, Chonnam National University Hospital)
  • 조혜영 (전남대학교 약학대학 부속 생물학적동등성 및 가교시험연구소, 전남대학교 병원 임상시험센터) ;
  • 강현아 (전남대학교 약학대학 부속 생물학적동등성 및 가교시험연구소, 전남대학교 병원 임상시험센터) ;
  • 이화정 (이화여자대학교 약학대학) ;
  • 최후균 (조선대학교 약학대학) ;
  • 이용복 (전남대학교 약학대학 부속 생물학적동등성 및 가교시험연구소, 전남대학교 병원 임상시험센터)
  • Published : 2006.02.20
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Abstract

A rapid, selective and sensitive reversed-phase HPLC method for the determination of promethazine in human serum was developed, validated, and applied to the pharmacokinetic study of promethazine. Promethazine and internal standard, chlorpromazine, were extracted from human serum by liquid-liquid extraction with n-hexane containing 0.8% isopropanol and analyzed on a Capcell Pak CN column with the mobile phase of acetonitrile-0.2 M potassium dihydrogen phosphate (42:58, v/v, adjusted to pH 6.0 with 1 M NaOH). Detection wavelength of 251 nm and flow rate of 0.9 mL/min were fixed for the study. The assay robustness for the changes of mobile phase pH, organic solvent content, and flow rate was confirmed by $3^{3}$ factorial design using a fixed promethazine concentration (10 ng/mL) with respect to its peak area and retention time. In addition, the ruggedness of this method was investigated at three different laboratories using same quality control (QC) samples. This method showed linear response over the concentration range of 1-40 ng/mL with correlation coefficients greater than 0.999. The lower limit of quantification using 1 mL of serum was 1 ng/mL, which was sensitive enough for pharmacokinetic studies. The overall accuracy of the quality control samples ranged from 96.15 to 105.40% for promethazine with overall precision (% C.V.) being 6.70-11.22%. The relative mean recovery of promethazine for human serum was 63.54%. Stability (freeze-thaw and short-term) studies showed that promethazine was stable during storage, or during the assay procedure in human serum. However, the storage at $-80^{\circ}C$ for 4 weeks showed that promethazine was not stable. Extracted serum sample and stock solution were not allowed to stand at ambient temperature for 12 hr prior to injection. The peak area and retention time of promethazine were not significantly affected by the changes of mobile phase pH, organic solvent content, and flow rate under the conditions studied. This method showed good ruggedness (within 15% C.V.) and was successfully used for the analysis of promethazine in human serum samples for the pharmacokinetic studies of orally administered Himazin tablet (25 mg as promethazine hydrochloride) at three different laboratories, demonstrating the suitability of the method.

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References

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