色谱

色谱 ›› 2015, Vol. 33 ›› Issue (2): 169-173.DOI: 10.3724/SP.J.1123.2014.10024

• 技术与应用 • 上一篇    下一篇

离子对反相高效液相色谱法同时测定尿液中复方α-酮酸片的5种有效成分

黄小雅, 钟园, 黄忠平, 金晨, 王丽丽, 潘再法   

  1. 浙江工业大学化学工程学院, 浙江 杭州 310014
  • 收稿日期:2014-10-28 修回日期:2014-11-26 出版日期:2015-02-08 发布日期:2015-01-24
  • 通讯作者: 潘再法
  • 基金资助:

    浙江省自然科学基金项目(Y4110536).

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

HUANG Xiaoya, ZHONG Yuan, HUANG Zhongping, JIN Chen, WANG Lili, PAN Zaifa   

  1. College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China
  • Received:2014-10-28 Revised:2014-11-26 Online:2015-02-08 Published:2015-01-24

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摘要：

建立了同时测定尿液中复方 α -酮酸片5种有效成分(消旋羟蛋氨酸钙(HMACa)、酮缬氨酸钙(KVCa)、消旋酮异亮氨酸钙(KILCa)、酮亮氨酸钙(KLCa)和酮苯丙氨酸钙(KPACa))的离子对反相高效液相色谱法。分别考察了离子对试剂浓度、缓冲盐浓度和流动相pH值等参数对分离情况的影响。最终采用Waters Symmetry C18色谱柱(250 mm×4.6 mm, 5 μm);以乙腈和含15 mmol/L四丁基氢氧化铵的20 mmol/L磷酸盐缓冲液(pH 7.0)为流动相进行梯度洗脱,流速为1.0 mL/min;柱温为35 ℃;检测波长为210 nm。在上述条件下,5种有效成分得到很好的分离,并且不受尿液中内源性基质的干扰,在20~200 mg/L范围内线性关系良好(r≥0.9990);HMACa、KVCa、KILCa、KLCa和KPACa的检出限(S/N=3)分别为3.0、5.0、3.6、5.7和2.5 mg/L;定量限(S/N=10)分别为9.6、16.7、12.0、19.0和8.3 mg/L;日间、日内精密度均小于7%;平均回收率在86.79%~112.00%之间,RSD小于9%(n=5)。该方法准确,灵敏度高,重现性好,适用于尿液中这5种有效成分的检测。

关键词: 复方 &alpha, 离子对反相高效液相色谱法, 尿液, -酮酸片, 有效成分

Abstract:

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.

Key words: active components, compound &alpha, human urine, ion-pair reversed-phase high performance liquid chromatography (RP-HPLC), -ketoacid tablet

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