Abstract
In the paper the results of the tryptophan determination in human plasma samples prepared with the novel Solvent Front Position Extraction (SFPE) technique are presented. The SFPE procedure is used for preparation of real biological sample for the first time. The results obtained using SFPE are compared with those using the classical sample preparation procedure. Under the optimal conditions, tryptophan and its internal standard were separated from other plasma compounds (matrix) as a small common zone/spot on a chromatographic plate using semiautomatic device equipped with moving pipet, which distributed developing solvent on the adsorbent layer. Tryptophan and the internal standard were evenly distributed within the small common zone from that the both substances were extracted and the solution obtained was transferred to quantitation with LC–MS and MS techniques. The determination results are satisfactory, the percentage values of relative error and RSD relative standard deviation do not exceed 5%. The procedure is characterized by simplicity, high analysis throughput, very good sample purification and seems to be easy applicable to other biological samples with these advantages mentioned.
Highlights
In the paper the results of the tryptophan determination in human plasma samples prepared with the novel Solvent Front Position Extraction (SFPE) technique are presented
The procedure is named as the Solvent Front Position Extraction (SFPE)[31] and is based on the following steps[30,31,32]: (a) the internal standard (IS) is added to the sample, (b) a drop of the sample solution is applied on the chromatographic plate, (c) the chromatogram is developed to locate the substance/s of interest and internal standard/s zones at the final solvent front position, (d) the substance/s and internal standard are extracted from this zone and transferred to the instrumental device for quantitation
We have previously shown that the SFPE procedure can be effectively used to determine selected substances in fortified serum samples
Summary
In the paper the results of the tryptophan determination in human plasma samples prepared with the novel Solvent Front Position Extraction (SFPE) technique are presented. The plasma proteins are frequently removed through pretreatment of sample with acids: perchloric acid[12,13,14], trichloroacetic acid[15,16,17], hydrochloric acid[18] or alcohols: methanol[13,19] and ethanol[20] This method has relatively poor sample cleanup, what is very unfavorable, especially in the case of LC–MS/MS analysis, where co-eluting components can interfere with ionization process of target substances (matrix effect)[11]. As with most plasma-assayed substances, the most frequently chosen sample preparation method for quantitation of tryptophan is protein precipitation As it was mentioned above, this procedure does not purify the serum samples from contaminants at a level appropriate for LC–MS analysis. The obtained results show that the procedure used can be successfully applied to the preparation of plasma samples for the quantitative determination of tryptophan, and in a perspective, for the preparation of samples for the determination of many other substances
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