Determination of D- and L-amino acids produced by cyanobacteria using gas chromatography on Chirasil-Val after derivatization with pentafluoropropyl chloroformate

Abstract

A rapid and simple method was developed for the determination of free amino acids (AAs) released from cyanobacteria. The procedure involves trapping of AAs from the centrifuged cyanobacterial culture fluid on a cation-exchange resin, their release together with the resin by direct treatment with the reaction medium, followed by immediate derivatization with a corresponding chloroformate. The extractive alkylation transfers the analytes into an organic phase, an aliquot of which is subjected to GC analysis. Identification and quantification of AAs was performed by GC/MS and GC/FID, respectively, using propyl chloroformate (PCF) as the derivatization reagent. For chiral analysis, the cyanobacteria extracts were treated with 2,2,3,3,3-pentafluoropropyl chloroformate (PFPCF) to create more volatile analytes. Separation of the AA enantiomers was accomplished on a Chirasil-Val capillary column and the D/L enantiomeric ratios were determined. AAs of cyanobacteria are considered to be important for the assessment of energy flow in an aquatic food web, nutrition value of cyanobacteria in a food web and for cell-cell communication within cyanobacteria. The highest levels of AAs were found in the summer period at the beginning of the season (July). In the September and October samples, the amount of AAs was lower, the number of D-AAs decreased and the D/L ratio was higher than in the July sample. Based on the obtained results it can be assumed that young populations excrete AAs in higher concentrations and a different composition compared to actively growing populations.

Gas chromatographic enantioseparation of derivatized α-amino acids on chiral stationary phases—Past and present

“…The alkyl chloroformate approach bears a number of advantages [220]: (i) the rapid one-step reaction can be carried out in aqueous solution without heating, (b) the cost of reagent is negligible, (iii) the derivatized amino acids can easily separated from the mixture using an organic solvent thus reducing chemical contamination and (iv) the method can easily be automated [221]. In the chiral domain, derivatization with pentafluoropropyl chloroformate has been used for the determination of d-and l-␣-amino acids on Chirasil-Val 4 [222]. The GC-MS analysis of biomarkers related to folate and cobalamin status in human serum after dimercaptopropanesulfonate reduction and heptafluorobutyl chloroformate derivatization has been described [223].…”

Comparison of derivatization and chromatographic methods for GC–MS analysis of amino acid enantiomers in physiological samples

“…Preliminary experiments were performed using enantiopure l-AA standards from Phenomenex and Fluka, respectively, to evaluate the number of the different AA derivatives amenable to detection on the Chirasil-l-Val column, which has been commonly recommended for the separation of chiral AA derivatives [8][9][10]14,17,20]. Three chloroformate and 6 anhydride derivatization reactions were tested.…”

“…The presence of d-configured AAs in prokaryotic and eukaryotic organisms is well established [8][9][10]. Especially bacteria have a distinct d-AA metabolism [1].…”

GC Analysis of Amino Acids Using Trifluoroacetylacetone and Ethyl Chloroformate as Derivatizing Reagents in Skin Samples of Psoriatic and Arsenicosis Patients

“…A number of determinations have been carried out using ECF and related compounds as derivatizing reagents [36][37][38][39][40]. The sensitivities of the determinations have been further enhanced by the use of different fluorosubstituted chloroformates and using mass-spectrometric detection devices [39,[41][42][43]. Trifluoroacetylacetone (FAA) reacts with primary amino containing compounds to form the derivatives amiable to GC [44][45][46][47].…”