Rapid and simultaneous determination of twenty amino acids in complex biological and food samples by solid-phase microextraction and gas chromatography–mass spectrometry with the aid of experimental design after ethyl chloroformate derivatization

Amino acids play a vital role as intermediates in many important metabolic pathways such as the biosynthesis of nucleotides, vitamins and secondary metabolites. A sensitive and rapid analytical method has been proposed for the first time for the simultaneous determination of twenty amino acids using solid-phase microextraction (SPME). The protein samples were hydrolyzed by 6 M HCl under microwave radiation for 120 min. Then the amino acids were derivatized by ethyl chloroformate (ECF) and the ethoxy carbonyl ethyl esters of amino acids formed were extracted using SPME by direct immersion. Finally the extracted analytes on the SPME fiber were desorbed at 260 °C and analyzed by gas chromatography–mass spectrometer (GC–MS) in electron ionization mode. Factors which affect the SPME efficiency were screened by Plackett–Burmann design; most significant factors were optimized with response surface methodology. The optimum conditions for SPME are as follows: pH of 1.7, ionic strength of 733 mg, extraction time of 30 min and fiber of divinyl benzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS). The recovery of all the amino acids was found to be in the range of 89.17–100.98%. The limit of detection (LOD) of all derivatized amino acids in urine, hair and soybean was found to be in the range of 0.20–7.52 μg L−1, 0.21–8.40 μg L−1 and 0.18–5.62 μg L−1, respectively. Finally, the proposed technique was successfully applied for the determination of amino acids in complex biological (hair, urine) and food samples (soybean). The method can find wide applications in the routine analysis of amino acids in any biological as well as food samples.

► Simple, rapid, sensitive and environmentally benign method for quantitative determination of amino acids in biological and food samples.
► Simultaneous extraction, concentration and sample introduction in one single solvent free step.
► In-matrix derivatization in aqueous media without any lyophilization and pre-purification steps.
► Application of the developed method for the simultaneous determination of 20 amino acids in hair, soybean and urine samples.