An HPLC-fluorescence (FL) method for determination of sulfur-containing amino acids such as homocysteine (Hcy), methionine (Met) and cysteine (Cys) in human plasma was developed. The sulfur-containing amino acids were labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F). Calibration curves in the range of 1-100 µm (Hcy and Met) and 5-500 µm (Cys) indicated good linearities (r ≥ 0.998). The limits of detection at a signal-to-noise ratio of 3 were 0.13 (Hcy), 0.02 (Met) and 0.11 µm (Cys), respectively. Acceptable results for accuracy and precision of intra- and inter-day measurements were obtained. The results of Hcy and Cys obtained by the proposed method indicated good correlations with the conventional method (r > 0.911, n = 20). Furthermore, the method was applied to determination of the sulfur-containing amino acids in maternal plasma (n = 200) after delivery. The concentrations of Hcy, Met and Cys as a median (inter quartile range, Q1 and Q3 ) were 5.37 (3.32-7.79) μm, 25.20 (20.10-31.06) μm and 147.25 (102.81-189.31) μm, respectively.

Sulfur-containing amino acids (SAAs), homocysteine (Hcy), methionine (Met) and cysteine (Cys) in blood are related to homocystinuria, an inborn error of metabolism. In this study, an assay method with HPLC-fluorescence detection to quantify the SAAs in a dried blood spot was established and applied to samples from newborn babies (n=200). Sample pretreatment involving reduction, derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole, and liquid-liquid extraction with ethyl acetate gave the separation of the derivatives with retention times within 12 min. The method was enough sensitive to determine the SAAs in a dried blood spot with 0.04-0.14 µm as the limit of detection at a signal-to-noise ratio of 3. However, the absolute recoveries were very low (5.7% for Hcy, 4.6% for Cys) except for Met (105.4%) owing to inefficient recovery of Hcy and Cys from the blood matrix. Other validation parameters such as accuracy (93.5-106.2%) and intra- (≤ 9.0%) and inter-day precisions (≤ 8.7%) were acceptable. The reliability of a dried blood spot as an analytical sample was estimated. Furthermore, the proposed method was successfully applied to dried blood spots prepared from newborn babies.

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