title: Simultaneous determination of ascorbic acid, aminothiols, and methionine in biological matrices using ion-pairing RP-HPLC coupled with electrochemical detector.
abstract: 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 C(18) (250 × 4.6 mm, 5 μm) analytical column using methanol-0.05% trifluoroacetic acid solution((aqueous)) (05/95, v/v), containing 0.1mM 1-octane sulphonic acid as the ion-pairing agent) as the isocratic mobile phase pumped at a flow rate of 1.5 mL min(-1) 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 (r(2)>0.999 in the range of 0.2-10,000 ng mL(-1) for all the analytes), recovery (>96%), precision (%RSD ≤ 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.
authors.affiliation: Department of Pharmacy, University of Peshawar, Peshawar-25120, Pakistan. imrankhan@upesh.edu.pk
authors.forename: Muhammad Imran
authors.initials: MI
authors.lastname: Khan
authors.forename: Zafar
authors.initials: Z
authors.lastname: Iqbal
mesh_terms: D000603:Amino Acids, Sulfur / Q000032:analysis* / Q000302:isolation & purification; D001205:Ascorbic Acid / Q000032:analysis* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D055664:Electrochemical Techniques; D019803:Glutathione Disulfide / Q000032:analysis* / Q000302:isolation & purification; D016014:Linear Models; D000432:Methanol / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature; D014269:Trifluoroacetic Acid / Q000737:chemistry
chemical_list: D000603:Amino Acids, Sulfur; D014269:Trifluoroacetic Acid; D001205:Ascorbic Acid; D019803:Glutathione Disulfide; D000432:Methanol