Three‐phase hollow‐fiber microextraction combined with ion‐pair high‐performance liquid chromatography for the simultaneous determination of five components of compound α‐ketoacid tablets in human urine

The determination of α‐ketoacid concentration is demanded to evaluate the absorption and metabolic behavior of compound α‐ketoacid tablets taken by chronic kidney disease patients. To eliminate the interference of endogenous substance of urine and enrich the analytes, a three‐phase hollow‐fiber liquid‐phase microextraction combined with ion‐pair high‐performance liquid chromatography method was established for the determination of d ,l ‐α‐hydroxymethionine calcium, d ,l ‐α‐ketoisoleucine calcium, α‐ketovaline calcium, α‐ketoleucine calcium, and α‐ketophenylalanine calcium of compound α‐ketoacid tablets in human urine samples. The extraction parameters, such as organic solvent, pH of donor phase and acceptor phase, stirring rate, and extraction time were optimized. Under the optimal conditions, the obtained enrichment factors were up to 11‐, 110‐, 198‐, 202‐, and 50‐fold, respectively. The calibration curves for these analytes were linear over the range of 0.1–10 mg/L for α‐ketovaline calcium, d ,l ‐α‐ketoisoleucine calcium, and α‐ketoleucine calcium, 0.5–10 mg/L for d ,l ‐α‐hydroxymethionine calcium, and α‐ketophenylalanine calcium with r > 0.99. The relative standard deviations (n = 5) were less than 6.27% and the LODs were 100.7, 10.0, 5.8, 7.8, and 8.6 μg/L (based on S/N = 3), respectively. Good recoveries from spiked urine samples (92–118%) were obtained. The proposed method demonstrated excellent sample clean‐up and analytes enrichment to determine the five components in human urine.