Simultaneous determination of ascorbic acid, aminothiols, and methionine in biological matrices using ion-pairing RP-HPLC coupled with electrochemical detector
详细信息 查看全文
- 作者：Muhammad Imran Khan ; imrankhan@upesh.edu.pk ; Zafar Iqbal
- 关键词：Column liquid-chromatography ; Vitamin C ; Aminothiols ; Methionine ; Electrochemical detection ; Optimization ; Validation
- 刊名：Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences
- 出版年：2011
- 出版时间：1 September, 2011
- 年：2011
- 卷：879
- 期：25
- 页码：2567-2575
- 全文大小：1K
文摘
A novel highly sensitive ion-pairing reversed-phase high performance liquid-chromatography/electrochemical detection method for simultaneous determination of l-ascorbic acid, aminothiols, and methionine in biological matrices was developed, optimized, and validated. Reduced forms of the analytes were extracted from the sample matrices with 10 % meta-phosphoric acid solution(aqueous). To determine the total vitamin C, the total aminothiols, and the total methionine, samples were treated with tris(2-carboxyethyl)phosphine solution in 0.05 % trifluoroacetic acid solution(aqueous) subsequent to deproteination to reduce the oxidized forms of these compounds. Various analytes were separated on a C18 (250 ¡Á 4.6 mm, 5 ¦Ìm) analytical column using methanol-0.05 % trifluoroacetic acid solution(aqueous) (05/95, v/v), containing 0.1 mM 1-octane sulphonic acid as the ion-pairing agent) as the isocratic mobile phase pumped at a flow rate of 1.5 mL min? at room temperature. The column eluents were monitored at a voltage of 0.85 V. These analytes were efficiently resolved in less than 20 min using n-acetyl cysteine as the internal standard. The present method was specific for the analysis of these analytes and demonstrated acceptable values for linearity (r2 > 0.999 in the range of 0.2-10,000 ng mL? for all the analytes), recovery (>96 % ), precision ( % RSD ?#xA0;2.0), and sensitivity (on column limit of detection: 250-400 fg and limit of quantification: 0.8-1.25 pg), indicating that the proposed method could be efficiently used for determination of these analytes in the context of clinical research.