Citation: HUANG Xiaoya, ZHONG Yuan, HUANG Zhongping, JIN Chen, WANG Lili, PAN Zaifa. Simultaneous determination of five active components of compound α -ketoacid tablet in human urine by ion-pair reversed-phase high performance liquid chromatography[J]. Chinese Journal of Chromatography, ;2015, 33(2): 169-173. doi: 10.3724/SP.J.1123.2014.10024 shu

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

1.
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China

Corresponding author: PAN Zaifa,
Received Date: 28 October 2014
Available Online: 26 November 2014
Fund Project: 浙江省自然科学基金项目(Y4110536). (Y4110536)

A simple and sensitive method for the simultaneous determination of five active components, D,L-α-hydroxymethionine calcium (HMACa), α -ketovaline calcium (KVCa), D, L-α-ketoisoleucine calcium (KILCa), α -ketoleucine calcium (KLCa) and α-ketophenylalanine calcium (KPACa) of compound α -ketoacid tablet in human urine by ion-pair reversed-phase high performance liquid chromatography (RP-HPLC) was developed and validated. 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 mm×4.6 mm, 5 μm) with diode array detection at 210 nm and the column temperature of 35 ℃. 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≥0.9990). 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, respectively. 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.
- ion-pair reversed-phase high performance liquid chromatography (RP-HPLC),
- active components,
- human urine,
- compound α -ketoacid tablet
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沈阳化工大学材料科学与工程学院沈阳 110142