Simultaneous determination of five active components of compound alpha-ketoacid tablet in human urine by ion-pair reversed-phase high performance liquid chromatography

A simple and sensitive method for the simultaneous determination of five active components, D, L-alpha-hydroxymethionine calcium (HMACa), alpha-ketovaline calcium (KVCa), D, L-alpha-ketoisoleucine calcium (KILCa), alpha-ketoleucine calcium (KLCa) and alpha-ketophenylala. nine calcium (KPACa) of compound alpha-ketoacid tablet in human urine by ion-pair reversed-phase high performance liquid chromatography (RP-HPLC) was developed and valida. ted. The separation conditions, such as the concentration of ion-pair reagent, the pH value of the mobile phase and the concentration of the buffer were optimized. All the five analytes were separated well on a C18 column (250 mmx4.6 mm, 5 mu m) with diode array detection at 210 nm and the column temperature of 35 degrees C. The mobile phases were acetonitrile and 20 mmol/L phosphate buffer (containing 15 mmol/L tetrabutylammonium hydroxide; pH 7) at the flow rate of 1. 0 mL/min with gradient elution. The calibration curves for the five components were linear in the range from 20 to 200 mg/L (r >= 0.999 0). The limits of detection (LODs, S/N = 3) were 3. 0, 5. 0, 3. 6, 5. 7 and 2. 5 mg/L, and the limits of quantification (LOQs, S/N = 10) were 9. 6, 16. 7, 12. 0, 19. 0 and 8. 3 mg/L for HMACa, KVCa, KILCa, KLCa and KPACa, respective. ly. The intra-day and inter-day precisions were less than 7%, and the average recoveries were between 86. 79% and 112. 00% in the human urine with RSDs lower than 9% (n = 5). The method proved precise, specific and reproducible, and can be used for the determination of the five components in urine.