This study was conducted to supplement limit of previous study. The objectives of this study were to select optimal conditions of high performance liquid chromatography(HPLC) operation for detecting urinary 2-thiothiazolidine-4-carboxylic acid(TTCA) and thiocarbamide simultaneously, and to evaluate recovery rates for various liquid-liquid extration method of these metabolites.
The results are as follows :
1. The urinary TTCA and thiocarbamide were separate sharply when flow rate is 0.7 ㎖/min, using a series C_8 and C_(18) column, 50 mM KH₂PO₄: acetonitrile (93.5 : 6.5) and pH 3.5 as a mobile phase. The retention time was TTCA, 12.07±0.11(mean±SD, n=6), thiocarbamide, 7.85±0.01(mean±SD, n=6), respectively. The calibration curve for TTCA and thiocarbamide was linear within the range 0.05 to 30 ㎍/㎖.
2. By the liquid-liquid extration, butanol extration with (NH₄)₂ as a salting-out reagent was used as a simultaneous extration method for these metabolites in acid state, and recovery rates of this method are urinary TTCA, 49.6±17.7 (mean±SD, n=16), thiocarbamide, 43.9±5.50 (mean±SD, n=16), respectively.
3. The precision(pooled coefficients of variation for 4 concentration) of the urinary thiocarbamide analysis was 0.03754 by butanol liquid-liquid extraction with (NH₄)₂ as a salting-out reagent, and TTCA was 0.04082 by ethyl acetate liquid-liquid extration with (NH₄)₂ as a salting-out reagent.
The above results show that the butanol liquid-liquid extraction with (NH₄)₂ as a salting-out reagent in acid state, and using a series C_8 and C_(18) column, 50 mM KH₂PO₄ : acetonitrile (93.5 : 6.5) and pH 3.5 as a mobile phase are suitable for the analysis of urinary TTCA and thiocarbamide simultaneously. The detection limit of TTCA and thiocarbamide was about 0.17 ㎍/㎖, 0.07 ㎍/㎖.
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