title: A new HPLC method for the simultaneous determination of ascorbic acid and aminothiols in human plasma and erythrocytes using electrochemical detection.
abstract: A new, simple, economical and validated high-performance liquid chromatography linked with electrochemical detector (HPLC-ECD) method has been developed and optimized for different experimental parameters to analyze the most common monothiols and disulfide (cystine, cysteine, homocysteine, methionine, reduced (GSH) and oxidized glutathione (GSSG)) and ascorbic acid present in human plasma and erythrocytes using dopamine as internal standard (IS). Complete separation of all the targets analytes and IS at 35°C on Discovery HS C18 RP column (250 mm × 4.6mm, 5 μm) was achieved using 0.05% TFA:methanol (97:3, v/v) as a mobile phase pumped at the rate of 0.6 ml min(-1) using electrochemical detector in DC mode at the detector potential of 900 mV. The limits of detection (3 S/N) and limits of quantification (10 S/N) of the studied compounds were evaluated using dilution method. The proposed method was validated according to standard guidelines and optimization of various experimental parameters and chromatographic conditions was carried out. The optimized and validated HPLC-ECD method was successfully applied for the determination of the abovementioned compounds in human plasma and erythrocytes. The method will be quite suitable for the determination of plasma and erythrocyte profile of ascorbic acid and aminothiols in oxidative stress and other basic research studies.
authors.affiliation: Department of Pharmacy, University of Peshawar, Peshawar-25120, Pakistan.
authors.forename: Abad
authors.initials: A
authors.lastname: Khan
authors.forename: Muhammad I
authors.initials: MI
authors.lastname: Khan
authors.forename: Zafar
authors.initials: Z
authors.lastname: Iqbal
authors.forename: Yasar
authors.initials: Y
authors.lastname: Shah
authors.forename: Lateef
authors.initials: L
authors.lastname: Ahmad
authors.forename: Shabnam
authors.initials: S
authors.lastname: Nazir
authors.forename: David G
authors.initials: DG
authors.lastname: Watson
authors.forename: Jamshaid Ali
authors.initials: JA
authors.lastname: Khan
authors.forename: Fazli
authors.initials: F
authors.lastname: Nasir
authors.forename: Abbas
authors.initials: A
authors.lastname: Khan
authors.lastname: Ismail
mesh_terms: D001205:Ascorbic Acid / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004563:Electrochemistry / Q000379:methods*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D012015:Reference Standards; D013438:Sulfhydryl Compounds / Q000097:blood*
chemical_list: D013438:Sulfhydryl Compounds; D001205:Ascorbic Acid