GC-MS with ethyl chloroformate derivatization for comprehensive analysis of metabolites in serum and its application to human uremia

Abstract

Abstract:An optimized method based on GC-MS with ethyl chloroformate derivatization has been developed for the comprehensive analysis of endogenous metabolites in serum. Twenty-two reference standards and serum samples were used to validate the proposed method. The correlation coefficient was higher than 0.9900 for each of the standards, and the LOD varied from 125 to 300 pg on-column. The analytical equipment exhibited good repeatability (RSD<10%) for all of the standards. Both the repeatability and the within-48-h stability of the analytical method were satisfactory (RSD<10%) for the 18 metabolites identified in the serum samples. Mean recovery was acceptable for the 18 metabolites, ranging from 70% to 120% with RSDs of less than 10%. Using the optimized protocol and a subsequent multivariate statistical technique, complete differentiation was achieved between the metabolic profile of uremic patients and that of age-and sex-matched normal subjects. Significantly decreased levels of valine, leucine, and isoleucine and increased levels of myristic acid and linoleic acid were observed in the patient group. This work demonstrated that this method is suitable for serumbased metabolic profiling studies.

A novel GC-MS method in urinary estrogen analysis from postmenopausal women with osteoporosis

“…To solve the problem, the addition of TEA and pyridine as a basic catalyst in the range of 10-40 l in 1 ml of a buffer solution was tested under the same reaction conditions (50 l of ECF, pH 9 medium, and shaking for 30 min). Although pyridine is commonly used as a suitable esterification catalyst ( 30,33,34 ), it was not selected in this study due to the diffi culty in removing the excess reagent after derivatization. Increasing the TEA amount tended to maximize the reaction yields at 30 and 40 l, and 30 l of TEA was chosen because of its rapid evaporation rate.…”

“…In alkyloxycarbonylation, an alkaline condition is essential to promote the deprotonation of the reactive species (30)(31)(32)34 ), and the pH range of 8-11 was tested (data not shown). Most estrogens showed good reaction yields (>96%) in all ranges tested, whereas yields of fi ve catechol estrogens were decreased signifi cantly at pH > 9, even though 0.2% ascorbic acid was added to prevent oxidation of the catechol moieties.…”

“…For this purpose, perfl uoroacylation and trimethylsilylation are mainly performed ( 4,(15)(16)(17)(18). As a different approach, alkoxycarbonylation, which is carried out with highly reacting agents for amino-, thiol-, imidazole-or phenolic hydroxy groups, can be suitable for direct reactions in aqueous media ( 17,(30)(31)(32)(33)(34). This direct derivatization to make analytes extractable by nonpolar organic solvents can be achieved simultaneously to provide rapid and reproducible results with excellent purifi cation ( 17,31,32 ).…”

“…Although the dansyl derivatives of estrogens show an improving detectability in LC-MS analysis ( 29 ), in this study ethoxycarbonylation (30)(31)(32)(33)(34), which is applicable to a range of compounds containing amino-or phenolic hydroxy groups of steroids, was introduced and optimized for the analysis of 19 endogenous estrogens as a simple and fast sample preparation in GC-MS analysis. Initially, the contribution of the catalyst for complete ethoxycarbonylation was examined by comparing the chromatographic intensities of the EOC derivatives in the scan range of m/z 100-650.…”

“…However, there are limitations, including a long analytical time (70 min) for the separation of the 15 estrogens, which might not be effective in large-scale applications. In our study, ethoxycarbonylation (30)(31)(32)(33)(34), which is applicable to a Supplemental Material can be found at:…”