| The purpose of this study was to establish a gas chromatography-mass spectrometry?GC-MS?method for the determination of caffeine in the urine and blood of exercise horses.Three preparation methods,liquid-liquid extraction?LLE?,solid phase extraction?SPE?,QuEChERS assay was compared.Extraction solvent,pH,volume and times of LLE were optimized.Indole 13C labelled caffeine and deuterium labelled caffeine were tested as internal standard.Mass spectrometric condition including scan mode,injection solvent were also optimized.Stabilities of working solvents and spiked matrixes in different temperatures were.3mL of urine sample was first spiked with 300?L 13C caffeine as internal standard,and then adjust the pH to 8.1 by lmol/L NaOH.In plasma or serum sample,300?L of 100%trichloroacetic acid to remove proteins then adjust pH of the supernatant to 10.0.Then extract with 2mL of chloroform 3 times,chloroform layer were collected and hydrogen dried,and redissolved by 200?L of methanol to GC-MS determination?1.0 ?L injection?.hp-5ms?30m×0.25mm×0.25?m?was used and electron ionization source was used in MS detection,SIM mode of 194?197 IS?,109,82,67 m/z were selected as qualitative ion and 194 was selected as quantitative ion.Limit of detection were 30ng·mL-1 in urine 20ng·mL-1 in serum,limit of quantitative were 80ng·mL-1 in urine 60ng·mL-1 in serum,and linear range were 80ng·mL-1 to 140ng·mL-1 in urine,and 60ng·mL-1 to 120ng·mL-1 in serum.The relative recovery ranged from 96%to 101%,the absolute recovery ranged from 82%to 97%,the intraday and interday precisions give a RSD range from 0.36%to 5.71%.NIST database search found no compound with all 194,109,82,67 m/z fractions,combine the restrict of retention time give a less possibility of false positive.This method has the advantages of high extraction yield,high precision,high sensitivity,and strong specificity and applicability. |
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