Chiral analysis of derivatized amino acids from kefir by gas chromatography

    Chiral analysis of derivatized amino acids from kefir by gas chromatography Fiorella Menestrina, Jaiver Osorio Grisales, Cecilia B. Castells PII: DOI: Reference:

S0026-265X(16)30059-5 doi: 10.1016/j.microc.2016.05.007 MICROC 2478

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

25 February 2016 4 May 2016 11 May 2016

Please cite this article as: Fiorella Menestrina, Jaiver Osorio Grisales, Cecilia B. Castells, Chiral analysis of derivatized amino acids from kefir by gas chromatography, Microchemical Journal (2016), doi: 10.1016/j.microc.2016.05.007

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ACCEPTED MANUSCRIPT Chiral analysis of derivatized amino acids from kefir by gas chromatography

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Fiorella Menestrina, Jaiver Osorio Grisales, Cecilia B. Castells*

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Laboratorio de Investigación y Desarrollo de Métodos Analíticos (LIDMA) and División Química Analítica, Facultad de Ciencias Exactas, UNLP, CONICET, 47 and 115, La Plata (B1900AJL), Argentina

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* Corresponding author C. B. Castells E-mail: [email protected] Tel. +54 221 4228327 Abstract

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The amino acids present in the kefir samples were derivatized using different alkyl

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chloroformates combined with alcohols and chromatographed on a (3-O-butanoyl-2,6-di-Opentyl)-γ-cyclodextrin (Lipodex E) chiral gas-liquid chromatographic column. The combination

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of derivatization reagents that led to better chromatographic performance for standard amino acid solutions was critically compared. All the chloroformate/alchohol mixtures lead to fast and quantitative reactions. However, the ethyl chloroformate/ethanol mixture was selected since the number of enantioseparated amino acid derivatives was higher. The derivatization was applied

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to samples of kefir grains grown in raw and in skimmed UHT milk. The samples were previously pretreated by elution from a low pressure ion-exchange columns. Standard addition calibration curves have been used for the quantitative determinations of amino acid derivatives in both samples, and the figures of merit of the proposed method have been assessed. The L- and Dconfiguration of

D- and L-alanine, D- and L-valine, D-proline, L-threonine, aspartic and

glutamic acids, methionine and cysteine were detected and quantitatively determined.

Keywords Gas chromatography, chiral, amino acids analysis, kefir.

ACCEPTED MANUSCRIPT Introduction Hundreds of amino acids are known, and twenty of them are constitutive of proteins and peptides

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of living systems. With the exception of glycine, the other proteinogenic α-amino acids present

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an asymmetric carbon and, as a consequence, they are chiral and can exist in either of two

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enantiomeric forms, L- or D-amino acids. Natural proteins are constituted by L-amino acids, but also D-amino acids have been found in different natural samples. Some examples of matrices in which D-amino acids have been detected include: i. bacterial culture used in the production of

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food and fermented drinks [1], ii. saliva of human beings where it has been detected the presence

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of significant levels of D-alanine and D-asparagine [2]; iii. biological fluids where their presence were proposed as markers that would allow detection of some metabolic disorder [3–6]; iv. food industrial products where amino acids contribute to the food nutritional and biological value.

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Moreover, it has been found that racemization can occur during manufacturing steps of processed food, such as fermented drinks and dairy products, as well as in biotechnological

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products [7,8]. Consequently, D-amino acids in the diet deserve systematic controls [9]. Kefir is a fermented milk with demonstrated health benefits including restorative properties of

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bacterial flora and stomach mucous, reduction of the symptoms of lactose intolerance, immune system stimulation, cholesterol reduction, as well as anti-mutagenic and anti-tumor properties [10–13]. Its consumption as a dairy product has been increasingly recommended for those properties; thus, assessing its nutrient composition is very important. The determination of enantiomeric amino acids can be carried out by gas chromatography. There are two general approaches to enantiomeric separation; the indirect method, based on the chemical reaction of the racemic mixture with a chiral reagent to obtain a diastereomeric pair and their subsequent chromatographic separation in an achiral stationary phase; and the direct method, based on the the use of a chiral stationary phase which can discriminate between enantiomers.

ACCEPTED MANUSCRIPT Chiral stationary phases most frequently used in the enantiomeric separation of amino acids by gas chromatography are either, the commercially known as Chirasil-Val (L-Valine-tert.-

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butylamide-linked polydimethylsiloxane), introduced firstly by Frank and co-workers [14],

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which has proved to be capable of resolving most amino acids in short analysis time [15], or

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modified cyclodextrins such as (3-O-butanoyl-2,6-di-O-pentyl)-γ-cyclodextrin (Lipodex E), which has been successfully used for separation of trifluoroacetyl methyl esters of amino acids [16,18].

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For the analysis by gas chromatography, the amino acids must be derivatized to

volatile

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compounds. A widely used type of derivatization is the preparation of N(O)- trifluoroacetyl alkyl esters amino acid derivatives. This procedure involves laborious and time consuming multiple steps, including the total removal of water from the sample, esterification under heating, removal

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of the excess alcohol, followed by acylation also under heating and, finally, removal of the excess reagents. The whole derivatization technique (after drying aqueous samples) takes about 1

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h.

Another type of derivatization involves the treatment of amino acids with a mixture of alcohol-

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pyridine chloroformate to obtain N-alkyloxycarbonyl alkyl ester amino acids. This technique was introduced by Hušek [19], and then investigations were made to develop quantitative and reproducible methods of amino acids analysis in different matrices [20–22]. It offers many advantages: i. it is a one-step reaction; ii. it occurs in aqueous medium, so the analysis can be performed directly in the aqueous matrix; iii. it is practically instantaneous at room temperature under mild conditions, avoiding possible racemization. In this study, the enantiomeric characterization of the free amino acids present in kefir samples is proposed. A critical evaluation and comparison of the results obtained by using different combinations of alkyl chloroformates and alcohols to increase the chromatographic signal and to obtain the maximum number of derivatized amino acids that may be enantiomerically resolved at

ACCEPTED MANUSCRIPT the lowest possible temperature within reasonable periods of time is presented. A commercial Lipodex E (octakis(3-O-butanoyl-2,6-di-O-n-pentyl)--cyclodextrin) column was used. To the

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best of our knowledge, these amino acid derivatives have not been enantioseparated using

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cyclodextrin-based columns. Derivatization reaction and chromatographic separation conditions

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were optimized and the method was finally used for the analysis of amino acid composition in kefir. .

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Experimental

Chemicals

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The amino acids alanine (Ala), arginine (Arg) , asparagine (Asn), aspartic acid (Asp), cysteine

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(Cys), glutamine (Gln), glutamic acid (Glu), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),

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tryptophan (Trp), tyrosine (Tyr), valine (Val), were obtained from Sigma (St. Louis, MO, USA), and from The British Drug Houses (Poole, England). Ethyl chloroformate (ECF), isobutyl

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chloroformate (IbuCF), methyl chloroformate (MCF), and chloromethyl chloroformate (ClMeCF) for the derivatization reactions were purchased from Sigma-Aldrich (St. Louis, MO). The organic solvents ethanol (EtOH), 2-fluoroethanol (FetOH), 3,3,4,4,5,5,6,6,6-nonafluoro-1hexanol (PFHexOH), pyridine and tetradecane were purchased from Sigma-Aldrich (St. Louis, MO), and chloroform was obtained from Merck (Buenos Aires, Argentine). The Dowex 50WX12 cation exchange resin (200-400 mesh) was purchased to Dow Chemical Co (Midland, MI). Deionized water was obtained from a purification Milli-Q system (Simplicity, Millipore, MA).

Amino acid solutions

ACCEPTED MANUSCRIPT A stock solution containing nineteen amino acids was prepared by weighting a known amount of each amino acid (about 10 mg each) in a volumetric flask, dissolved and brought to a final

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volume of 10 mL with a solution of 60:32:8 H2O:EtOH:Pyridine.

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Gas chromatography

We used an HP 6890 gas chromatograph (Agilent, Palo Alto, CA) equipped with flameionization detection and manual-injection port. The data were acquired by means of the software

Injections were made with a split ratio 1/20, the injector

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at constant flow (1 mL/min).

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Clarity (DataApex, Czech Republic). The carrier gas was nitrogen and operation was conducted

temperature was set at 200 °C and the detector at 250 °C. Methane was used to estimate the dead time. The chiral CG column was a commercial octakis(3-O-butanoyl-2,6-di-O-n-pentyl)--

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cyclodextrin (Lipodex E) column (25 m x250 μm i. d.) (Macherey-Nagel, Düren, Germany). Chromatographic separation of the amino acids was achieved by using a temperature program. .

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Initially the temperature was 100 °C and it was heated at a rate of 2 °C/min to 115 °C; the temperature was kept constant for 25 minutes; then was raised again to 160 °C at 1 °C/min and

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finally it was heated from 160 °C to 185 °C at a rate of 2 °C/min.

Derivatization procedure The derivatization reaction is based on processing the amino acids with alkyl chloroformates. To 1 mg of each amino acid in a 1.5 mL Eppendorf tube, 100 μL of alcohol-water-pyridine (60:32:8),

and 6 μL of the chloroformate were added. The mixture was stirred for 5 seconds.

Finally, the derivatized analytes were extracted with 100 μL chloroform containing 1% v/v of the corresponding chloroformate [19]. After phase separation, the aqueous layer becomes opaque whereas the organic one remains clear. Aliquots of the organic phases are injected into the capillary column.

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Sample preparation Kefir grain samples were provided by researchers from CIDCA-UNLP

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(Centro de Investigación y Desarrollo en Criotecnología de Alimentos, Universidad Nacional de

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La Plata). Two different kefir grain suspensions were analyzed. Sample 1 consisted of granules

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AGK1 CIDCA grown in sterile 5% re-suspended whey permeates. 10 g of pellet was suspended in 100 mL of milk and incubated at 30 °C for 24 h. Then, the granules were strained and recovered. The measured pH was 4.25. Sample 2 was prepared with the same AGK1 CIDCA

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kefir grains grown in skimmed UHT milk. The preparation of sample 2 was the same as sample

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1 and the final pH was 4.03. Samples were pretreated by percolation through an ion-exchange column (6.5 x 1.4 cm). Previously, the strong cation exchange resin Dowex 50W-X12 was treated twice with 7 M ammonium hydroxyde for 1 h, washed with deionized water, then an

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excess of 3 M HCl was added with slow stirring. After 1 h, the resin was washed with deionized water until neutral. The treated resin was introduced into glass columns avoiding air bubbles

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[23].

Aliquots (0 - 200 μL) of standard solutions and 20 L of trichloroacetic acid were added to a 10

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mL of kefir sample and the mixture was centrifuged for 15 minutes at 4200 rpm. The supernatant was poured onto the top of the ion exchange column. The impurities were washed by passing 10 mL of deionized water through the column. The amino acids were then eluted with 10 mL of 7 M ammonium hydroxide. Then, the solutions were dried by evaporation at 40 °C and under nitrogen stream. The column was washed with deionised water and regenerated with 3 M HCl.

Results and discussion In Table 1, we reported the retention factor, separation factor and enantioresolution values of ECF/EtOH derivatives eluted from the chiral Lipodex E column. This combination of ethyl alcohol and ethyl chloroformate has been classically proposed for amino acid derivatizations.

ACCEPTED MANUSCRIPT However, our first aim was to test several combinations of chloroformate/alcohols to obtain the reagent conditions that would provide the maximum number of derivatized amino acids that can

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be enantiomerically separated. Thus, three other chloroformates combined with ethyl alcohol

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have been used: isobutyl, methyl and chloromethyl chloroformates. Derivatization of all amino

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acids were carried out and the derivatives were injected under isothermal conditions. The results of retention factor and enantioseparation factorvalues are compared with those obtained with ECF as derivatization reagent in plots gathered in Figure 1. Retention factor of IbuCF/EtOH

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derivatives increased (Figure 1A), whereas for MCF/EtOH (more volatile derivatives) decreased

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respect to the ethyl derivatives. No systematic behavior was observed for the ClMCF/EtOH derivatives. Figure 1B shows the corresponding enantioseparation factors. It can been observed that many amino acids were separated regardless the derivative formed, but this behavior does

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not apply for methionine, which was enantioseparated exclusively as ClMCF/EtOH derivative, and asparagine as ECF and IbuCF derivatives only.

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Another assays using fluorinated alcohols were planned in order to obtain more volatile derivatives. Thus, ethanol was replaced by either, FEtOH or PFHexOH. In Figure 2, we

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compared the chromatographic results (retention and enantioseparation factors) obtained for those derivatives. Retention of fluoroderivatives were was lower for all the amino acids, so lower temperatures could be set for eluting those derivatives. However, separation factors were similar or worse than those achieved for the derivatives synthesized using ethyl alcohol. The exceptions were isoleucine and valine as fluoroderivatives, which had barely larger separation factors as compared with the ethyl derivatives. Some general observations from the previous assays can be highlighted: i. The enantiomeric pair of proline could be separated in all their derivative forms except when the combination MCF/EtOH is used, in contrast with results obtained with the more widely known Chirasil-Val column [20]; ii. Arginine could not be detected in any case, which might be attributed to lower

ACCEPTED MANUSCRIPT yields in the derivatization reaction due to the low reactivity of the guanidine group under the derivatization conditions [22,24]; iii. Similarly, lysine, and the more hydrophobic tyrosine and volatile

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tryptophan derivatives were not eluted, even at higher temperatures and using

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fluoroalcohols as derivatization reagents.

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For Ala, Ile, and Thr, resolution factors were higher for the combination IbuCF/EtOH due to the higher enantioseparation factors; also for Pro and Val, the resolution was larger for the IbuCF/EtOH derivatives because the peaks were more symmetric. For Asn, Asp and Ser

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derivatives, higher resolutions were achieved for the ECF/EtOH combination due to larger

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enantioseparation factors . Since there are no significant differences in enantioresolution and selectivity for the aforementioned solvent combinations, we selected the ECF/EtOH mixture to determine the enantiomeric composition of amino acids in kefir. The main advantage is the

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reduction in retention times of the obtained derivatives and, thus, reduction in analysis time. In Figure 3 and Figure 4, the chromatograms obtained for Pro and Val using ECF/EtOH and

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iBuCF/EtOH, respectively are shown as an example. For the same chromatographic conditions, the analysis time decreased from approximately 40 to 20 min for proline and 37 to 15 min for

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valine.

Method validation and application to kefir samples The following figures of merit for the analytical method were examined: linear range , accuracy, precision, and limits of detection (LOD) and quantification (LOQ) [25]. Quantitative determinations were carried out by using the standard addition method , Aliquots of the standard solutions were spiked at five different concentration levels ( by triplicate). Tables 2 and 3 show the least square regression parameters , LOD and LOQ obtained for kefir samples 1 and 2, respectively. These figures of merit were calculated using the peak area ratios of each amino acid derivative against n-tetradecane peak area, used as injection internal standard.

ACCEPTED MANUSCRIPT Classical ANOVA tests were applied to check for linearity

(lack-of-fit) in the regression

equations. Results indicate that, even though the kefir granules were the same, after growing in

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different media the obtained samples were different and thus, standard addition method for

precision of the determinations was calculated by the intra-day reproducibility

of

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The

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quantitative analytical information was selected.

quantitative results expressed as the relative standard deviation (%RSD) from triplicates. For that purpose, a standard solution and also the samples were spiked with 150 L amino acid standard

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solution. These results are gathered in Table 4. For the spiked samples, the RSD% values were

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larger than 15%. This relatively poor precision is attributed to the analyte isolation and preconcentration from the dairy samples, which involves many steps. On the other hand, the

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precision (RSD%) obtained with the standard solution of amino acids was smaller than 10%.

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In order to evaluate the method accuracy, recovery tests were made by spiking sample 1 and 2 with 50 and 150 μL of amino acid standard solution (low and high concentration level,

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approximately 250 and 750 ppm respectively) . The relative recovery were 79% (s.d.=6.7) and 94% (s.d.=4.5) at the low and the high concentration levels for sample 1, and of 84% (s.d.=4.1)

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and 93% (s.d=4,3) at low and high concentration levels for sample 2, respectively.

Kefir samples

In Figures 5 and 6, two typical chromatograms of kefir samples 1 and 2 (without spiking) are shown. Alanine, valine, proline, threonine, aspartic and glutamic acids, methionine and cysteine could be accurately detected. Other peaks clearly observed in both samples did not correspond with any of the other amino acid derivatives. The results of the free amino acid composition in both samples are reported in Table 5. It can be seen that in both samples , proteinogenic amino acids are present. Furthermore the presence of D-amino acids in kefir is important since previous research had demonstrated that they are used to cover metabolic demand against the complete

ACCEPTED MANUSCRIPT absence of the L-form. For instance, the use of D-amino acids in infants was investigated and it

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was found that both the D-Ala and D-Val were used for protein synthesis [9].

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Conclusion

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The study of amino acid derivatization method by using chloroformate/alcohol in aqueous medium has been extended to the use of different alkyl chloroformate and alcohols. These reactions provide an extremely fast and simple route for derivatization that can be carried out

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from aqueous samples in only one step. Enantioseparations of nine amino acid derivatives with

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the combinations ECF/EtOH and IbuCF/EtOH, were obtained. ECF/EtOH derivatives eluted faster than IbuCF/EtOH derivatives . D- and L-Proline could be baseline enantioseparated, and in fact, both enantiomers were detected and quantified in kefir samples.

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The derivatization method was successfully applied to the determination of free amino acids in a fermented dairy product such as kefir grains grown in raw and in skimmed UHT milk. The

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kefir samples.

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presence of L- and D- Ala, D- and L-Val, D-Pro, L-Threo, Glu, Met and Cys was found in both

Acknowledgments

The authors kindly acknowledge to Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), and Universidad Nacional de La Plata (UNLP) for financial support.

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ACCEPTED MANUSCRIPT Figure Captions Figure 1. Comparison of chromatographic retention (A) and enantioseparation factors (B) of

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amino acids derivatized whit ECF/EtOH, IbuCF/EtOH, MCF/EtOH and ClMCF/EtOH.

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Column: Lipodex-E, 25 m. Temperature: Ala, Leu and Val= 120°C, except for MCF

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derivatives, run at 110°C; Asp, AspOH, Isoleu, Met, Ser and Threo = 150°C; Pro= 130°C and ClMCF at 150°C.

Figure 2. Comparison of chromatographic retention (A) and enantioseparation factors (B) of

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amino acids derivatized whit ECF/EtOH, ECF/Fluoroethanol and ECF/perfluorohexanol.

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Column Lipodex E, 25 m. Temperatures: Ala=120°C; Asp, and Isoleu=150°C; AspOH= 150°C(EtOH), 120°C(F-EtOH) and 130°C(PFHxOH); Cys=160°C(EtOH), 130°C(FEtOH) and 120°C(PFHxOH); Glu= 150°C(EtOH), and 130°C(PFHxOH); Leu=120°C

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(EtOH and PFHxOH), and 110°C(F-EtOH); Phe= 140°C(F-EtOH) and 150°C(PFHxOH); Pro=130°C(EtOH), 110°C(F-EtOH) and 120°C(PFHxOH); Threo=150°C(EtOH), 130°C

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(F-EtOH) and 130°C(PFHxOH); Val=120°C(EtOH), 110°C(F-EtOH) and 100°C (PFHxOH).

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Figure 3. Chromatograms obtained for proline derivatives using A) ECF/EtOH B) IbuCF/EtOH. Column: Lipodex E (25 m x 0.25 cm i.d.). Temperature: 130°C. Carrier flow-rate: 1 mL/min.

Figure 4. Chromatograms obtained for valine derivatives using A) ECF/EtOH B) IbuCF/EtOH. Column: Lipodex E (25 m x 0.25 cm i.d.). Temperature: 130°C. Carrier flow-rate: 1 mL/min. Figure 5. Gas chromatogram of amino acid ECF/EtOH derivatives of kefir grains in whey permeates (sample 1). Temperature program: Initially 100 °C, heated at a rate of 2 °C/min to 115 °C, then, temperature was kept constant for 25 minutes, heating from 115 °C to 160 °C at 1 °C/min, and finally it was heated from 160°C to 185 °C at 2 °C/min. Carrier flow-

ACCEPTED MANUSCRIPT rate: 1 mL/min. Figure 6. Gas chromatogram of amino acid ECF/EtOH derivatives of kefir grains in skimmed

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UHT milk (sample 2). Temperature program: Initially 100 °C, heated at a rate of 2 °C/min

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to 115 °C, then, temperature was kept constant for 25 minutes, heating from 115 °C to 160

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°C at 1 °C/min, and finally it was heated from 160°C to 185 °C at 2 °C/min. Carrier flow-

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rate: 1 mL/min.

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Table 1. Retention factors, separation factors and enantioresolutions of ECF/EtOH derivatives using isothermal conditions.

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Amino acid T ºC k1 α Rs Ala 120 5.5 1.02 0.55 Arg ne Asn 150 11.55 1.04 0.86 Asp 150 10.43 1.05 1.3 Cys 160 24.11 1 Gln ne Glu 150 17.73 1 His 190 35.28 1 Ile 150 3 1.07 1.5 Leu 120 13.99 1.03 0.63 Lys ne Met 150 16.35 1 Phe ne Pro 130 11.14 1.05 1.22 Ser 150 10.82 1.06 1.36 Thr 150 8.18 1.12 2.89 Trp ne Tyr ne Val 120 8.74 1.04 0.62 Separation factor α=k2/k1, and resolution determined at isothermal temperature T (ºC); ne= not eluted.

ACCEPTED MANUSCRIPT

0.051±0.007

48

L- Ala

8.6±1.6

0.038±0.009

49

D-Val

3.0±0.3

0.001±0.002

21

L-Val

2.2±5.2

0.058±0.004

D-Pro

5.9±1.0

0.081±0.006

L-Pro

7.0±0.8

0.001±0.004

D-Thr

3.5±0.4

0.002±0.002

L-Thr

3.6±0.4

D-Asp

162 163

IP

6.9±1.2

SC R

D- Ala

T

Table 2. Calibration line coefficients, LOD and LOQ for amino acids in kefir sample 1. Sample 1 calibration line parametersa LODb LOQb Amino acid Intercept (x105) (ppm) (ppm) slope

69

166

30

91

17

56

18

59

0.009±0.002

20

60

2.7±0.4

0.002±0.002

21

69

L-Asp

2.5±0.3

0.003±0.002

18

59

DL-Glu

1.1±0.2

0.009±0.003

78

241

DL-Met

1.0±0.2

0.004±0.002

50

168

DL-Cys

3.2±0.9

112

344

D

MA

NU

54

TE

0.023±0.012

a: Least squares regression coefficients of the calibration line. Triplicates of the sample with five

CE P

spiked amounts: 0, 25, 50, 75 and 100 g of each enantiomer. .

AC

b: Calculated according to IUPAC recommendations [25].

ACCEPTED MANUSCRIPT Table 3. Calibration line coefficients, LOD and LOQ for amino acids in kefir sample 2 LODb (ppm)

LOQb (ppm)

T

amino acid

sample 2 calibration line parametersa Intercept (x105) slope

59

4.3±0.5

0.011±0.003

18

L- Ala

5.1±0.6

0.010±0.004

19

64

D-Val

1.3±0.1

0.010±0.001

23

75

L-Val

2.8±0.6

0.027±0.005

45

149

D-Pro

1.9±0.4

0.230±0.022

32

106

L-Pro

3.7±0.5

0.004±0.003

22

75

D-Thr

1.8±0.3

0.002±0.002

36

121

L-Thr

3.4±0.8

0.013±0.004

24

82

D-Asp

2.6±0.5

0.0002±0.0029

30

101

L-Asp

1.0±0.4

0.003±0.002

60

200

DL-Glu

1.5±0.2

0.017±0.003

53

175

DL-Met

3.5±0.7

0.066±0.008

63

210

DL-Cys

1.5±0.5

0.448±0.056

104

348

TE

D

MA

NU

SC R

IP

D- Ala

a: Least squares regression coefficients of the calibration line. Triplicates of sample with five

CE P

spiked amounts: 0, 25, 50, 75 and 100 g of each enantiomer. .

AC

b: Calculated according to IUPAC recommendations [25].

ACCEPTED MANUSCRIPT Table 4. Relative standard deviation RSD% for two spiked samples

MA

NU

SC R

IP

T

RSD% (n=3) sample 1 RSD% (n=3) sample 2 28 18 19 16 20 21 21 19 8 16 11 14 14 10 9 18 15 12 12 16 19 13 12 11 15 22

AC

CE P

TE

D

Amino acid D- Ala L- Ala D-Val L-Val D-Pro L-Pro D-Thr L-Thr D-Asp L-Asp DL-Glu DL-Met DL-Cys

ACCEPTED MANUSCRIPT Table 5. Free amino acids content in two kefir samples.

T

IP

SC R

AC

CE P

TE

D

MA

NU

Amino acid Ala Val Pro Thr Asp Glua Metb Cysc

Sample 1 Sample 2 L (ppm) D (ppm) L (ppm) D (ppm) det det 2 3 27 Det. 10 8 14 12 2 4 9 12 19 7 30 a det.= detectable, not quantifiable, DL Glu, bDL Met, cDL Cys

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Highlights – Analysis of amino acids in kefir samples by chiral gas chromatography was performed – Several combinations of alkyl chloroformate/alcohols derivatives were synthesized – Ethyl chloroformate/ethanol mixture lead to the optimum compromise separation/time – D- and L-Proline could be baseline enantioseparated and quantified in kefir samples