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 SymmetryC18色谱柱（250mm×4．6mm，5μm）；以乙腈和含15mmol／L四丁基氢氧化铵的20mmol／L磷酸盐缓冲液（pH7．0）为流动相进行梯度洗脱，流速为1．0mL／min；柱温为35℃；检测波长为210nm。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20～200mg／L范围内线性关系良好（r≥0．9990）；HMACa、KV-Ca、KILCa、KLCa和KPACa的检出限（S/N=3）分别为3．0、5．0、3．6、5．7和2．5mg／L；定量限（S/N=10）分别为9．6、16．7、12．0、19．0和8．3mg／L；日间、日内精密度均小于7％；平均回收率在85．79％～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。
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-ketophenylala- nine 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 min×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. 999 0）. The limits of detection （LODs, S/N= 3） were 3.0, 5.0, 3.5, 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 meth-od proved precise, specific and reproducible, and can be used for the determination of the five components in urine.
出处
《色谱》
CAS
CSCD
北大核心
2015年第2期169-173,共5页
Chinese Journal of Chromatography
基金
浙江省自然科学基金项目(Y4110536)
关键词
离子对反相高效液相色谱法
有效成分
尿液
复方Α-酮酸片
ion-pair reversed-phase high performance liquid chromatography （RP-HPLC）
active components
human urine
compound α-ketoacid tablet
