A validated HPLC‐fluorescence method with a semi‐micro column for routine determination of homocysteine, cysteine and cysteamine, and the relation between the thiol derivatives in normal human plasma

Abstract

A semi-micro column HPLC-fluorescence method for routine determination of thiol derivatives such as homocysteine (Hcy), cysteine (Cys) and cysteamine (CA) is described. The thiol derivatives labeled with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) were isocratically separated within 12 min on a semi-micro ODS column (Daisopak-SP-120-5-ODS-BP) with a mixture of 25 mm acetate buffer (pH 2.00) and CH(3)CN as a mobile phase. The purity and similarity of SBD-thiols by a multi-wavelength fluorescence detector were more than 92.3 and 96.7%. The detection limits of Hcy, Cys and CA at a signal-to-noise ratio of 3 were 0.16, 0.47 and 0.03 microm, respectively. Furthermore validation parameters such as accuracy, precision and robustness of the proposed method showed satisfactory results. Almost 850 plasma sample injections (range 572-1076, n = 3) for a column could be performed without differences in retention time and peak heights of labels. As an application of the proposed method, the determination of thiol derivatives in normal human plasma (n = 103) was demonstrated. The correlation coefficients between Hcy vs Cys and Hcy vs CA were 0.38 and -0.35, respectively.

Relation of body mass index to blood folate and total homocysteine concentrations in Japanese adults

“…Erythrocyte folate concentrations were calculated using the following formula: [whole blood concentration -plasma concentration x (1 -hematocrit (%)/100)] ÷ hematocrit (%)/100. Plasma tHcy concentrations were measured by the HPLC-fluorescent detection method that was developed in our laboratory, and the inter-assay CV for the tHcy analysis ranged from 5.2 -7.8% [17]. Hemoglobin and hematocrit were measured using an automatic hematology analyzer (XE-2100, Syntex, Kobe, Japan).…”

Methodology for a rapid and simultaneous determination of total cysteine, homocysteine, cysteinylglycine and glutathione in plasma by isocratic RP-HPLC

“…Different types of labeling reagents have been used, including monobromobimane [30,37], ortho-phthaldialdehyde [38,39], N-(1-pyrenil)maleimide [40,41], 9-acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)phenyl)-3-oxo-3H-naphtho[2,1-b]pyran (ThioGlo 3) [42], N-(2-acridonyl)maleimide [1], ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F) [20,[43][44][45], and 7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide (ABD-F) [46,47]. The halogenobenzofurazans reagents (SBD-F and ABD-F) are the most often used, since they react selectively with thiol compounds (Fig.…”

HPLC‐fluorescence determination of thiol compounds in the serum of human male and female subjects using HILIC‐mode column

“…The Cys, CysGly, GSH and γ ‐GluCys concentrations in human serum (17 males and 17 females) were determined using the HPLC, and the mean ± SE values were 226 ± 4.7, 23.4 ± 1.3, 3.7 ± 0.2 and 3.2 ± 0.1 μ m , respectively ( n = 34). As regards comparison of these data with those reported previously, the order of magnitude was almost similar, but the actual concentrations slightly differed from previous reports (Ichinose et al ., ; Isokawa et al ., ; Pastore et al ., ; Valente et al ., ). For this reason, several factors such as age and race may contribute.…”