High-performance liquid chromatography method for determining alendronate sodium in human plasma by detecting fluorescence: application to a pharmacokinetic study in humans.
A high-performance liquid chromatographic (HPLC) method was developed using diethylamine (DEA) solid-phase extraction (SPE), 9-fluorenylmethyl derivative (FMOC) and fluorescence detection for quantifying alendronate in human plasma. Sample preparation involved a manual protein precipitation with trichloroacetic acid, a manual coprecipitation of the bisphosphonate with calcium phosphate and derivatization with 9-fluorenylmethyl chloroformate in citrate buffer at pH 11.9. Liquid chromatography was performed on a Capcell Pak C(18) column (4.6 mm x 150 mm, 5 microm particles), using a gradient method starting with mobile phase acetonitrile/methanol-citrate/pyrophosphate buffer (32:68, v/v). The total run time was 25 min. The fluorometric detector was operated at 260 nm (excitation) and 310 nm (emission). Pamidronate was used as the internal standard. The limit of quantification was 1 ng/ml using 3 ml of plasma. The intra- and inter-day precision expressed as the relative standard deviation was less than 15%. The assay was applied to the analysis of samples from a pharmacokinetic study. Following the oral administration of 70 mg of alendronate sodium to volunteers, the maximum plasma concentration (C(max)) and elimination half-life were 40.94 +/- 19.60 ng/ml and 1.67 +/- 0.50 h, respectively. The method was demonstrated to be highly feasible and reproducible for pharmacokinetic studies including bioequivalence test of alendronate sodium in humans.
Yun
Min-Hyuk
MH
College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea.
Kwon
Kwang-il
KI
D019386:Alendronate / Q000032:analysis / Q000097:blood* / Q000493:pharmacokinetics*; D050071:Bone Density Conservation Agents / Q000032:analysis; D002138:Calibration; D002623:Chemistry Techniques, Analytical / Q000379:methods; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002853:Chromatography, Liquid; D002951:Citrates / Q000032:analysis; D004164:Diphosphonates / Q000032:analysis*; D005449:Fluorenes / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D000077268:Pamidronate; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014238:Trichloroacetic Acid / Q000737:chemistry
D050071:Bone Density Conservation Agents; D002951:Citrates; D004164:Diphosphonates; D005449:Fluorenes; D014238:Trichloroacetic Acid; D000077268:Pamidronate; D019386:Alendronate