I recently attempted to replicate the amino acid derivatization procedure outlined by Sobolevsky et al. 2003. While I followed the steps diligently, unfortunately, I did not achieve the expected results. I followed the following steps:

Step 1: Amino acids (Stock 1): 10 mg L-Proline, 16 mg L-Valine, 10.1 mg DL-Phenylalanine, 10 mg L-Tyrosine, and 11 mg DL-Alanine in 10 mL 0.1M HCl. (Vortexed to dissolve the amino acids)

Step 2: Amino acids (Stock 2): 100 µL of Stock 1 was added in 900 µL of 0.1M HCl to get 100 µg/mL working solution.

Step 3: 100 µL of working solution was lyophilized at -20 ºC or air dried at room temperature.

Step 4: Derivatization of the dried residue was carried out using N-tert-butyldimethylsilyl- N-methyltrifluoroacetamide (MTBSTFA) from Merck. 100 µL of HPLC-grade acetonitrile and 100 µL of MTBSTFA were added to the residue and gentle sonication was performed for 30 s (as mentioned in the article). After sonication, the mixture was heated for 30 min. at 70 ºC. Step 5: 1 mL of HPLC-grade ethyl acetate was added to the derivatized product and centrifuged at 10000 rpm for 15 min at room temperature (just to avoid any crystals) and the supernatant was injected into the GC-MS.

I have attached the column details and the result I obtained.

I greatly appreciate your efforts. Thank you.

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