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Novel high-throughput and widely-targeted liquid chromatography–time of flight mass spectrometry method for d -amino acids in foods
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e8, 2016 www.elsevier.com/locate/jbiosc
Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods Yutaka Konya, Moyu Taniguchi, and Eiichiro Fukusaki* Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Received 8 April 2016; accepted 11 July 2016 Available online xxx
Recently, the demand for D-amino acid profiling has been drastically increasing because the significance of D-amino acid in various biological events is suggested. However, the present methodologies for D-amino acid profiling are still unsatisfactory. Therefore, a highly sensitive, robust, high-throughput, and user-friendly method for D-amino acid profiling must be developed. In this paper, we developed a novel method for D-amino acid profiling using a combination of a chiral column and time of flight mass spectrometry (TOFMS). To our knowledge, our approach has the best performance for D-amino acid analysis that includes the shortest analytical time (within 10 min), the highest enantioseparability without derivatization, and the largest coverage for analytical targets (more than one hundred targets including non-proteinogenic amino acids and amines). Thus, our novel profiling method will be instrumental in advancing the D-amino acid research in the future. Ó 2016, The Society for Biotechnology, Japan. All rights reserved. [Key words: D-Amino acids; Underivatized amino acids; Chiral amino acids; Enantioseparation; Liquid chromatographyetime of flight mass spectrometry]
Amino acids are essential and ubiquitous compounds for living organisms on Earth. L-Amino acids exist predominantly as building blocks of peptides/proteins or in free form, and perform critical roles in biological systems. In contrast, the presence of D-amino acids is comparatively very low and in the past, D-amino acids was believed to be absent in higher organisms (1). However, D-amino acids were recently found in various higher organisms (2e4) and the demand for D-amino acid profiling has been drastically increasing because the roles of D-amino acids in important diseases such as schizophrenia, amyotrophic lateral sclerosis (ALS), and renal dysfunction have been suggested (5e7). There are several reports which showed the existence of D-amino acids in various foods (8e11). D-Amino acids in fermented foods are thought to be derived from the starting materials used to prepare those foods, or they are produced during microbial fermentation (12). Therefore, there is an increasing need to develop rapid, robust and highly sensitive methods for the separation and quantification of D-amino acids (13). Consequently, many chiral analytical methods for amino acid enantiomers have been developed. Chiral and simultaneous analyses for amino acid enantiomers require high level chromatographic separation technologies. The two approaches commonly used are direct method (without derivatization) and indirect method (with derivatization) (13,14). Direct methods use chiral stationary phases or, more rarely, chiral mobile phase additives. Many chiral stationary phases for direct methods have been developed, among which the most enantioselective separations of amino acids were mainly carried out on glycopeptide
* Corresponding author. Tel./fax: þ81 6 6879 7424. E-mail address: [email protected] (E. Fukusaki).
antibiotic teicoplanin or teicoplanin aglycone chiral stationary phases. However, chiral liquid chromatographyemass spectrometry (LCeMS) analyses using a teicoplanin or its aglycone-based chiral columns could not achieve good separation of several proteinogenic amino acid enantiomers (15,16). On the other hand, indirect methods are based on the formation of derivatives by the reaction of amino acids with a chiral or achiral derivatizing agent followed by the separation of the derivatives on an achiral or chiral stationary phase. Since enantioseparation based on direct methods are relatively more difficult than indirect methods, most chiral analyses are performed with derivatization prior to analysis, such as gas chromatographyemass spectrometry (GCeMS) (17), LCeMS (3,4), liquid chromatographyefluorescence detection (LCeFLD) (2,9e11,18), and capillary electrophoresisefluorescence detection (CEeFLD) (19). However, problems using these methods are still prevalent such as the stability of derivatives, long analysis time, separations of Damino acids from enantiomers and unknown peaks. As described above, despite the progress in analytical technology, enantioselective separation and quantification of D-amino acids remain a challenge owing to the frequently incomplete separation of enantiomers and insufficient sensitivity for determination of trace amounts of D-amino acids. In this study, we aimed to develop a high-throughput and high-resolution analytical method for D-amino acids profiling. MATERIALS AND METHODS Enantiomers Proteinogenic amino acid enantiomers (DL-20 amino acids) of DL-alanine (DL-Ala), DL-arginine hydrochloride (DL-Arg), DL-asparagine monohydrate (DL-Asn), DL-aspartic acid (DL-Asp), DL-cysteine hydrochloride monohydrate (DL-Cys), DL-glutamic acid (DL-Glu), DL-glutamine (DL-Gln), DL-histidine (DL-His), DL-isoleucine (DL-Ile, mixture of four stereoisomers containing DL-allo-Ile), DL-leucine
1389-1723/$ e see front matter Ó 2016, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
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KONYA ET AL.
(DL-Leu), DL-lysine monohydrochloride (DL-Lys), DL-methionine (DL-Met), DL-phenylalanine (DL-Phe), DL-proline (DL-Pro), DL-serine (DL-Ser), DL-threonine (DL-Thr), DLtryptophan (DL-Trp), DL-tyrosine (DL-Tyr) and DL-valine (DL-Val), were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Glycine (Gly) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Non-proteinogenic amino acids and amines DL-Ethionine was obtained from Acros Organics (Geel, Belgium). DL-Pyroglutamic acid was obtained from Icn Biochemicals (CA, USA). Adenine was obtained from Kishida Chemical Co., Ltd. (Osaka, Japan). Uracil was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). DL-a-Aminoadipic acid, D-b-aminoisobutyric acid, creatinine, cystathionine (mixture of DL-, DL-allo-), cis-4-hydroxy-D-proline, DL-kynurenine and DL-pipecolic acid, were obtained from Sigma-Aldrich Japan K.K. (Tokyo, Japan). DL-2-Aminopimelic acid, DL-citrulline, cystine (DL- and meso-mixture), DL-2,4-diaminobutyric acid dihydrochloride, 3-(3,4dihydroxyphenyl)-DL-alanine (DL-Dopa), DL-homophenylalanine, DL-homoserine, DL-norvaline (DL-nor-Val) and DL-ornithine monohydrochloride were obtained from Tokyo Chemical Industry Co., Ltd. b-Alanine (b-Ala), allantoin, DL-allothreonine (DL-allo-Thr), anthranilic acid (2-Aminobenzoic acid), g-aminobutyric acid (GABA), betaine, creatine, cytosine, N,N-dimethylglycine (DMG), dopamine hydrochloride, guanine, histamine, L-hydroxyproline (trans-4-hydroxy-L-proline), hypotaurine, nicotinamide, nicotinic acid, sarcosine and thymine were obtained from Wako Pure Chemical Industries, Ltd. Wako Amino Acids Mixture Standard Solutions Wako Amino Acids Mixture Standard Solutions’ Type AN-2 and Type B were obtained from Wako Pure Chemical Industries, Ltd. Type AN-2 contained L-alanine, b-alanine, L-a-aminoadipic acid, DL-a-aminobutyric acid, DL-b-aminoisobutyric acid, L-aspartic acid, L-citrulline, L-cystathionine, L-cystine, L-glutamic acid, glycine, hydroxy-L-proline, L-isoleucine, L-leucine, L-methionine, o-phosphoserine, o-phosphoethanolamine, L-proline, L-phenylalanine, L-serine, sarcosine, taurine, L-threonine, L-tyrosine, urea and L-valine. While Type B contained g-aminobutyric acid, ammonium chloride, L-anserine, L-arginine, L-carnosine, ethanolamine, L-histidine, DL-plus allo-dhydroxylysine, L-ornithine, L-lysine, L-1-methylhistidine, L-3-methylhistidine. Reagents for extraction and mobile phase Ultrapure water for LC/MS (Water), ethanol for HPLC (EtOH), 0.1 mol/L hydrochloric acid for volumetric analysis and trifluoroacetic acid for HPLC (TFA) were obtained from Wako Pure Chemical Industries, Ltd. Methanol -Plus- for LC/MS (MeOH) and acetonitrile -Plus- for LC/MS (ACN) were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). Chloroform for HPLC was obtained from Kishida Chemical Co., Ltd. DL-Alanine-2,3,3,3-d4 were obtained from Santa Cruz Biotechnology (TX, USA). Matrices and food samples Grain vinegar (Vinegar) was obtained from Mizkan Group Corporation (Aichi, Japan). Whole cow milk (Milk) was obtained from Megmilk Snow Brand Co., Ltd. (Tokyo, Japan). Three kinds of black vinegars were obtained from AEON Co., Ltd. (Chiba, Japan), Mizkan Group Corporation, and Japan Health System Co., Ltd. (JHS) (Tokyo, Japan). Kimchi (Amakuchi) was obtained from Greenfarm Co., Ltd. (Osaka, Japan). Kimchi (Ichioshi) was obtained from Miyama Co., Ltd. (Chiba, Japan). Kimchi (Gohan-ga-Susumu; GS) was obtained from Pickles Co., Ltd. (Saitama, Japan). Yogurt drink (Bifidus SP strain, capsule) and yogurt drink (Gasseri SP strain) were obtained from Megmilk Snow Brand Co., Ltd. Yogurt drink (LB81 strain, plain) was obtained from Meiji Co., Ltd. (Tokyo, Japan). Instrument Amino acid enantiomers were analyzed on Crownpak CR-I(þ) and CR-I() columns (3.0 mm i.d. 150 mm, and 5 mm particle size, Daicel CPI, Osaka, Japan) using Nexera system (pump: LC-30AD, autosampler: SIL-30AC, degasser: DGU-20A5, column oven: CTO-30A, Shimadzu, Kyoto, Japan) coupled to TripleTOF 5600 System (AB SCIEX, Concord, Canada). Preparation of DL-20mix calibration standard solutions and ISsolution 50%MeOH was prepared by mixing MeOH and water (1/1 (v/v)). 50% MeOH-0.05 mol/L-HCl was prepared by mixing MeOH and 0.1 mol/L Hydrochloric Acid (1/1 (v/v)). A8E2 was prepared by mixing ACN and EtOH (8/2 (v/v)). To make 20 mmol/mL standard solutions, DL-Glu, DL-His & DL-Trp were dissolved in 50%MeOH-0.02 mol/L-HCl, DL-Asp and DL-Tyr were dissolved in 50%MeOH-0.05 mol/L-HCl, while the other proteinogenic amino acids were dissolved in 50%MeOH. Glycine and nineteen DL-amino acids standard solutions (20 mmol/mL) were mixed in equal amount to make 1000 nmol/mL solution (DL-20mix-1000). Then, DL-20mix1000 was diluted by 50%MeOH-0.01 mol/L-HCl to make 400, 100, 40, 10, 4 and 1 nmol/mL in 50%MeOH-0.01 mol/L-HCl (DL-20mix calibration standard solutions). DL-Alanine-2,3,3,3-d4 was dissolved in 50%MeOH to make 40 mmol/mL (ISsolution). Preparation of calibration samples Sample preparation was carried out according to LLEA (LiquideLiquid Extraction method for Amines). Briefly, a matrix (water, vinegar or milk (50 mL)), water (50 mL), DL-20mix calibration standard solutions (1000, 400, 100, 40, 10, 4, 1 or 0 nmol/mL in 50%MeOH-0.01 mol/L-HCl) (100 mL), IS-solution (20 mL) and MeOH (300 mL) were mixed, then vortexed for 10 s. After centrifugation (16,000 g, 4 C, 10 min), the supernatant (360 mL) was mixed with water (180 mL) and chloroform (360 mL), then vortexed for 10 s. After centrifugation (16,000 g, 4 C, 10 min), the MeOHeWater layer (upper layer, 50 mL) was mixed with A8E2 (200 mL) and vortexed for 10 s. After centrifugation (16,000 g, 4 C, 10 min), 1 mL of the supernatant was injected onto a Crownpak CR-I column.
J. BIOSCI. BIOENG., Preparation of black vinegar and yogurt samples Black vinegar or yogurt drink (50 mL) were mixed with water (50 mL), DL-20mix calibration standard solutions (1000 or 0 nmol/mL in 50%MeOH-0.01 mol/L-HCl) (100 mL), IS-solution (20 mL) and MeOH (300 mL), then vortexed for 10 s. After centrifugation (16,000 g, 4 C, 10 min), the supernatant (360 mL) was mixed with water (180 mL) and chloroform (360 mL), then vortexed for 10 s. After centrifugation (16,000 g, 4 C, 10 min), the MeOHewater layer (upper layer, 50 mL) was mixed with A8E2 (200 mL) and vortexed for 10 s. After centrifugation (16,000 g, 4 C, 10 min), 1 mL of the supernatant was injected onto a Crownpak CR-I column. Preparation of kimchi samples A piece of napa cabbage in kimchi (approximately 1 g) was taken as a sample and its surface was washed with water. The washed napa cabbage was crushed by a spatula and centrifuged. The resulting supernatant (50 mL) was collected and prepared in the same manner as black vinegars and yogurt samples. Preparation of mixed standard solutions of amino acids and amines Amino acid standard solutions and amine standard solutions (dissolved in 50%MeOH, or 50%MeOH-0.05 mol/L-HCl) or Wako Amino Acids Mixture Standard Solutions were mixed and diluted with the mobile phase. The combinations and concentrations of amino acids and amines in mixed standard solutions were decided based on their molecular weights and peak intensities. One microliter of each mixed standard solution was injected onto a Crownpak CR-I column. Liquid chromatographyetime of flight mass spectrometry analysis One microliter of extracted solution of calibration samples, food samples, or mixed standard solutions of amino acids and amines was injected onto a Crownpak CRI(þ) column. The mobile phase used was ACN/EtOH/water/TFA (80/15/5/0.5 (v/v/ v/v)), the flow rate was 0.4 mL/min, the autosampler temperature and the column oven temperatures were maintained at 4 C and 30 C, respectively. All analyses were performed under isocratic conditions. Instead of Crownpak CR-I(þ) column, Crownpak CR-I() column was used to calculate peak area ratios of D/Lamino acids, and to confirm the existence of D-allo-Ile in foods. Column eluent was analyzed in positive ionization mode using a TripleTOF 5600 system with the following conditions: ion source gas 1 (50 psi), ion source gas 2 (50 psi), curtain gas 1 (30 psi), temperature (600 C), ion spray voltage floating (5500 V), declustering potential (60 V), collision energy (5 V), and mass range (60e600 m/z). Data processing was performed using MultiQuant (AB SCIEX, Concord, Canada). Calculation of recovery rates of D-amino acids The recovery rates of all Damino acids (except for Gly and DL-Pro) in food samples were estimated by comparing peak area ratios (the peak area of D-amino acid/the peak area of IS) of each D-amino acid in food (1000 nmol/mL in matrics) with its corresponding peak area ratio in Water calibration samples (1000 nmol/mL in water). As several endogenous D-amino acids exist in foods, their peak area ratios were subtracted before calculating their recovery rates. Calculation of D-amino acid concentrations The regression equations (Y ¼ aX þ b; with weighting factor of 1/X2) were calculated by the least-squares method using MultiQuant. For this calculation, the peak area ratios (the peak area of D-amino acid/the peak area of IS) obtained in the measurement of the calibration samples (as Y) and the spiked concentrations (as X) were used. Damino acid concentrations in foods were calculated using Water calibration curves and then corrected by the recovery rates of D-amino acids in each food. Calculation of area ratios of D/L-amino acids The area ratios of D-form to Lform in mixed standard solutions (DL-20mix 1 and 10 nmol/mL diluted by the mobile phase) were calculated.
RESULTS AND DISCUSSION Optimization of mobile phase composition Previously, we analyzed the standard solution of mixed proteinogenic amino acid enantiomers using a combination of a Crownpak CR-I(þ) column and liquid chromatographyetime of flight mass spectrometry (TOFMS) (20). An isocratic condition of a simple mobile phase composed of ACN/Water/TFA (96/4/0.5 (v/v/v)) gave baseline separation of all underivatized amino acid enantiomers (except for Pro, secondary amine) within 15 min. However, since an extraction process was necessary for analysis of amino acids in food samples, the final composition of the extracted sample solutions was different from the composition of the standard solution. As a result, the peak shapes of several amino acids in the extracted samples significantly deteriorated compared to the standard solution. Therefore, to improve their peak shapes, the mobile phase and extracted sample solution were adjusted so that their compositions become more similar to each other while maintaining the extraction efficiency and enantioseparability.
Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
VOL. xx, 2016 Consequently, with a (80/15/5/0.5 (v/v/v/v)) ratio of ACN/EtOH/ Water/TFA, 18 proteinogenic amino acid enantiomers in the extracted samples were successfully detected and separated with good peak shapes (Fig. 1A). In addition, analyses using CR-I() column also achieved good enantioseparation (Fig. S1).
LC-TOFMS METHOD FOR D-AMINO ACIDS IN FOODS
3
Peak area ratios of D/L-amino acids As an advantage of LC isocratic condition which was adopted in our method, influence on ionization efficiency by LC mobile phase was far smaller than using LC gradient condition. We analyzed mixed standard solutions (DL20mix 1 and 10 nmol/mL in the mobile phase) using both CR-I(þ)
FIG. 1. LCeMS chromatograms of (A) water calibration sample (1000 nmol/mL in water) and (B) black vinegar (AEON) using CR-I(þ) column (extracted ion of [MþH]þ 5 mDa).
Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
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KONYA ET AL.
J. BIOSCI. BIOENG.,
and CR-I() columns. The area ratios of nearly 1.0 (Table S1).
D-form
to L-form were
Method validation For method validation, calibration curves were created using water, milk and vinegar as matrices, and the quantitativity of the method was evaluated through its recovery rate, limit of quantification, linearity, accuracy and precision. All analyses were performed using the CR-I(þ) column. The recovery rates of all D-amino acids (except for DL-Pro that is not separated) in milk and vinegar calibration samples were 72.1% or more and 89.9% or more, respectively (Table S2). The lower limit of quantification (LLOQ) was determined from the lowest concentrations with an accuracy of 80e120% and precision within 20%. For other concentrations, the accuracy was 85e115% and the precision was within 15% (Fig. S2 and Tables S3eS6).
Determination of D-amino acid concentrations in food samples All D-amino acids in blank milk and blank grain vinegar (except for D-Ala, D-Asp and D-Glu in blank grain vinegar) were under the LLOQ. Recoveries and concentrations of D-amino acids in commercially available black vinegars, kimchis and yogurt drinks were also determined. From this study, 14 D-amino acids in black vinegars, 10 D-amino acids in kimchis, and 5 D-amino acids in yogurt drinks were determined (Figs. 1B and S3 and Tables 1 and S6). Confirmation of D-allo-isoleucine (D-allo-Ile) in food samples Analysis using CR-I(þ) column could not distinguish D-allo-Ile from D-Ile because of co-elution. In contrast, CR-I() column could separate D-allo-Ile and D-Ile peaks thus, it was used to analyze the water calibration sample and black vinegar
TABLE 1. D-Amino acid concentrations in foods (nmol/mL or nmol/g). Index
Name
D-Ala
1 2 3 4
D-Ser
Grain vinegar
JHS 198.1 29.5
(1.0)b
372.6 24.7 36.6 6.6
(1.0)b (1.9)b 14.3
36.8 (3.5)b
D-Val
5 6 7 8 9 10 11 12 13 14 15 16 17 18 a b c d
D-Thr þ D-allo-Thr þD-homoserinec D-Cys d D-Ile þ D-allo-Ile D-Leu D-Asn D-Asp D-Gln D-Lys D-Glu D-Met D-His D-Phe D-Arg D-Tyr D-Trp
Black vinegar AEON
6.8 (3.6)b (1.4)b
Kimchi Mizkan
Yogurt
Amakuchi
Ichioshi
GS
Bifidus
LB81
Gasseri
41.7 16.4
175.8 7.4
(3208.4)a 21.2
172.0 7.9
49.2 Trace
97.5 4.0
111.3 Trace
7.2
7.3
(3.1)b
11.0
4.7
62.8 93.6 17.0 106.0
(2.6)b 5.9 7.4 42.5
(3.0)b 4.4 16.8 24.7
Trace 5.4
Trace 13.2
Trace 9.8
51.4
120.7
39.8
19.6
Trace
69.2 5.7 6.3 6.0 10.7 1.9
37.2 (1.5)b 5.0 7.7 (3.0)b 5.0
25.4 (1.3)b 7.0 5.1 5.8 3.1
Trace 103.4 Trace 4.4 (2.4)b 4.5 1.3
Trace 138.0 Trace 9.2 (3.5)b 11.6 2.8
96.8 Trace 6.7 4.9 4.3 1.7
14.2
32.7
27.2
(3.2)b
4.7
4.6
>upper limit of quantification (ULOQ).
FIG. 2. LCeMS chromatograms of Water calibration sample and black vinegar using CR-I(þ) and CR-I() columns for the confirmation of D-allo-Ile in food. (extracted ion of [MþH]þ 5 mDa) ¼ 132.1025. (A) Water calibration sample (100 nmol/mL in water) using CR-I(þ) column. (B) Black vinegar (AEON) using CR-I(þ) column. (C) Water calibration sample (100 nmol/mL in water) using CR-I() column. (D) Black vinegar (AEON) using CR-I() column.
Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
FIG. 3. LCeMS chromatograms of mix standard solutions of amino acids which have same molecular weight using CR-I(þ) column (extracted ion of [MþH]þ 5 mDa). (A) [MþH]þ ¼ 90.0555 (sarcosine, DL-Ala, and b-Ala). (B) [MþH]þ ¼ 104.0712 (N,N-dimethylglycine (DMG), DL-a-aminobutyric acid (DL-a-ABA), DL-b-aminoisobutyric acid (DL-b-AIBA), and g-aminobutyric acid (GABA)). (C) [MþH]þ ¼ 118.0868 (betaine, DL-Val, and DL-norvaline (DL-nor-Val)). (D) [MþH]þ ¼ 120.0661 (DL-Thr, DL-allo-Thr, and DL-homoserine). (E) [MþH]þ ¼ 132.1025 (DL-Ile, DL-allo-Ile, and DL-Leu). (F) [MþH]þ ¼ 163.1083 (DL-plus DL-allo-d-hydroxylysine (d-h-Lys-01, 02, 03, 04)). (G) [MþH]þ ¼ 223.0753 (DL-plus DL-allo-cystathionine (CST-01, 02, 03, 04)). (H) [MþH]þ ¼ 241.0317 (DD-, LL-plus meso-cystine).
FIG. 4. LCeMS chromatograms of mix standard solutions of nonproteinogenic amino acids and amines using CR-I(þ) column (extracted ion of [MþH]þ 5 mDa).
Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
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J. BIOSCI. BIOENG., TABLE 2. Extracted ion of [MþH]þ, retention time (Rt) and resolution (Rs) of amino acids and amines.
Index 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77
Formula
[MþH]þ
CH4N2O C2H7NO C2H5NO2 C3H7NO2
61.0402 62.0606 76.0399 90.0555
C4H9NO2
104.0712
C3H7NO3
106.0504
C2H7NO2S C4H5N3O C5H9N3 C4H4N2O2 C4H7N3O C5H9NO2
110.0276 112.0511 112.0875 113.0589 114.0667 116.0712
C5H11NO2
118.0868
C4H10N2O2
119.0821
C4H9NO3
120.0661
C3H7NO2S
122.0276
C6H6N2O C6H5NO2 C2H7NO3S C5H6N2O2 C5H7NO3
123.0558 124.0399 126.0225 127.0508 130.0504
C6H11NO2
130.0868
C5H9NO3
132.0661
C4H9N3O2 C6H13NO2
132.0773 132.1025
C4H8N2O3
133.0613
C5H12N2O2
133.0977
C4H7NO4
134.0453
C5H5N5 C7H7NO2 C5H10N2O3
136.0623 138.0555 147.0770
C6H14N2O2
147.1134
C5H9NO4
148.0610
C5H11NO2S
150.0589
C5H5N5O C8H11NO2 C6H9N3O2
152.0572 154.0868 156.0773
C4H6N4O3 C6H11NO4
159.0518 162.0766
Compound
Rt (min)
Urea Ethanolamine Glycine Sarcosine D-Alanine b-Alanine L-Alanine N,N-Dimethylglycine D-a-Aminobutyric acid D-b-Aminoisobutyric acid L-a-Aminobutyric acid L-b-Aminoisobutyric acid g-Aminobutyric acid D-Serine L-Serine Hypotaurine Cytosine Histamine Uracil Creatinine D-Proline L-Proline Betaine D-Valine D-Norvaline L-Valine L-Norvaline D-2,4-Diaminobutyric acid L-2,4-Diaminobutyric acid D-allo-Threonine D-Threonine D-Homoserine L-allo-Threonine L-Threonine L-Homoserine D-Cysteine L-Cysteine Nicotinamide Nicotinic acid Taurine Thymine D-Pyroglutamic acid L-Pyroglutamic acid D-Pipecolic acid L-Pipecolic acid cis-4-Hydroxy-D-proline trans-4-Hydroxy-L-proline Creatine D-allo-Isoleucine D-Isoleucine D-Leucine L-allo-Isoleucine L-Isoleucine L-Leucine D-Asparagine L-Asparagine D-Ornithine L-Ornithine D-Aspartic acid L-Aspartic acid Adenine Anthranilic acid D-Glutamine L-Glutamine D-Lysine L-Lysine D-Glutamic acid L-Glutamic acid D-Methionine L-Methionine Guanine Dopamine D-Histidine L-Histidine Allantoin D-2-Aminoadipic acid L-2-Aminoadipic acid
2.37 3.20 3.67 1.33 1.89 3.48 4.67 1.30 1.55 2.61 2.96 3.32 3.66 1.67 2.31 3.83 1.55 1.69 2.25 1.43 1.34 1.34 1.28 1.39 1.55 1.79 3.57 1.74 2.12 1.42 1.42 1.45 1.64 2.00 2.39 1.63 2.91 1.51 1.53 10.35 2.23 2.24 2.24 1.32 1.32 1.35 1.35 1.34 1.37 1.37 1.52 1.75 1.91 3.82 1.50 1.79 2.16 4.08 1.71 2.62 1.60 2.12 1.48 2.73 2.70 7.57 1.70 5.12 1.71 5.24 1.70 3.28 1.35 1.50 2.39 1.58 3.99
Rs
Remark
6.85 10.04 4.43 3.63 9.71 2.17 1.90 1.59 5.52
e
Secondary amines
1.64 2.26 2.85 11.13 3.16 e 0.38 2.55 4.54 3.43
D-allo-form/D-form
10.18
e
Secondary amines
e
Secondary amines
e
Secondary amines
e 1.98 2.81 2.02 11.59
D-allo-form/D-form
3.43 7.39 7.26
11.14 9.01 14.49 15.63
1.95
13.03
Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
VOL. xx, 2016
LC-TOFMS METHOD FOR D-AMINO ACIDS IN FOODS
7
Table 2 (continued ) Index 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110
Formula
[MþH]þ
C6H14N2O3
163.1083
d-Hydroxylysine-01 d-Hydroxylysine-02 d-Hydroxylysine-03 d-Hydroxylysine-04
C6H13NO2S
164.0745
D-Ethionine
Compound
Rt (min)
L-Ethionine
C9H11NO2
166.0868
D-Phenylalanine L-Phenylalanine
C7H11N3O2
170.0930
L-1-Methylhistidine L-3-Methylhistidine
C6H14N4O2
175.1195
D-Arginine L-Arginine
C7H13NO4
176.0923
D-2-Aminopimelic L-2-Aminopimelic
C6H13N3O3
176.1035
acid acid
D-Citrulline L-Citrulline
C10H13NO2
180.1025
D-Homophenylalanine L-Homophenylalanine
C9H11NO3
182.0817
D-Tyrosine L-Tyrosine
C9H11NO4
198.0766
D-Dopa L-Dopa
C11H12N2O2
205.0977
D-Tryptophan L-Tryptophan
C10H12N2O3
209.0926
D-Kynurenine L-Kynurenine
C7H14N2O4S
223.0753
C6H12N2O4S2
241.0317
Cystathionine-01 Cystathionine-02 Cystathionine-03 Cystathionine-04 DD-Cystine meso-Cystine LL-Cystine
2.08 2.24 4.85 5.43 1.71 5.18 1.52 2.65 1.41 1.41 1.34 1.95 1.59 3.71 1.60 3.38 1.78 5.95 1.53 2.57 1.54 2.68 1.53 2.52 1.82 3.66 1.64 2.56 4.49 10.90 1.84 3.96 16.18
Rs
Remark
1.13 7.71 1.10 15.20 9.42 e
Secondary amines
6.21 12.01 10.82 15.72 8.54 9.03 8.34 10.33 5.86 5.83 7.38 8.05 10.25
Rs, resolution of subsequent two compounds sharing a same [MþH]þ. Rs of a pair of peaks with retention times t1 and t2 (t2 > t1) was calculated using the equation Rs ¼ 1:18 ðt2 t1 Þ=ðwh1 þ wh2 Þ, where wh1 and wh2 are the corresponding peak widths measured at half the peak height.
samples. The result showed that only D-allo-Ile was detected in black vinegar samples (Fig. 2). Peak shapes and resolution of non-proteinogenic amino acids and amines Next, we applied the method using the CRI(þ) column to non-proteinogenic amino acids and amines (Figs. 3 and 4 and Table 2). All enantiomers which have primary amine in their structures were successfully separated. Because CR-I columns separate amino acid enantiomers based on the interaction between the crown ether of the solid phase and primary amines, secondary amine enantiomers such as proline and pyroglutamic acid could not be separated. Nevertheless, all of the secondary amines were detected with good peak shapes. In addition, compounds with the same molecular weight such as group-90 ([MþH]þ ¼ 90.0555: sarcosine, DL-Ala, and b-Ala) and group-104 ([MþH]þ ¼ 104.0712: N,N-dimethylglycine, DL-aaminobutyric acid, DL-b-aminoisobutyric acid, and GABA) also showed good separation. Our result showed that 110 compounds including non-proteinogenic amino acids were successfully analyzed. We determined trace D-amino acids in three kinds of foods (black vinegar, kimchi and yogurt), and our analytical method was verified to be applicable to food samples. This analytical method is classified to direct method, showed more advantages such as high enantioseparability, high throughput, and wide range of targets than not only other direct methods (15,16), but also indirect methods which need derivatization process (2e4,9e11,17e19). In conclusion, we developed a novel high throughput LC-TOFMS method for D-amino acids in foods. This method employs simpler LC conditions and shorter analysis time (10 min). Also, it does not require a derivatization process while exhibiting a higher enantioseparability compared to any previous methods for D-amino acid
analysis. Therefore, this method does not suffer from undesirable issues that may occur due to derivatization, long analytical time and fluorescence detection. Moreover, the coverage for analytical targets is wider (more than one hundred targets in total including non-proteinogenic amino acids and amines) compared to previously published protocols. Taking into account these improvements, our novel profiling method will be instrumental in advancing the D-amino acid research in the future. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2016.07.009. ACKNOWLEDGEMENTS The study represents a portion of the dissertation submitted by Yutaka Konya to Osaka University in partial fulfillment of the requirement for his Ph.D. References 1. Corrigan, J. J.: D-Amino acids in animal, Science, 164, 142e149 (1969). 2. Miyoshi, Y., Koga, R., Oyama, T., Han, H., Ueno, K., Masuyama, K., Itoh, Y., and Hamase, K.: HPLC analysis of naturally occurring free D-amino acids in mammals, Pharm. Biomed. Anal., 69, 42e49 (2012). 3. Müller, C., Fonseca, J. R., Rock, T. M., Krauss-Etschmann, S., and SchmittKopplin, P.: Enantioseparation and selective detection of D-amino acids by ultra-high-performance liquid chromatography/mass spectrometry in analysis of complex biological samples, J. Chromatogr. A, 1324, 109e114 (2014). 4. Visser, W. F., Verhoeven-Duif, N. M., Ophoff, R., Bakker, S., Klomp, L. W., Berger, R., and Koning, T. J.: A sensitive and simple ultra-high-performanceliquid chromatography-tandem mass spectrometry based method for the quantification of D-amino acids in body fluids, J. Chromatogr. A, 1218, 7130e7136 (2011). 5. Hashimoto, K., Engberg, G., Shimizu, E., Nordin, C., Lindström, L. H., and Iyo, M.: Reduced D-serine to total serine ratio in the cerebrospinal fluid of drug
Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
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KONYA ET AL. naive schizophrenic patients, Prog. Neuropsychopharmacol. Biol. Psychiatry, 29, 767e769 (2005). Sasabe, J., Miyoshi, Y., Suzuki, M., Mita, M., Konno, R., Matsuoka, M., Hamase, K., and Aiso, S.: D-amino acid oxidase controls motoneuron degeneration through D-serine, Proc. Natl. Acad. Sci. USA, 109, 627e632 (2012). Sasabe, J., Suzuki, M., Miyoshi, Y., Tojo, Y., Okamura, C., Ito, S., Konno, R., Mita, M., Hamase, K., and Aiso, S.: Ischemic acute kidney injury perturbs homeostasis of serine enantiomers in the body fluid in mice: early detection of renal dysfunction using the ratio of serine enantiomers, PLoS One, 9, e86504 (2014). Brückner, H. and Hausch, M.: Gas chromatographic detection of D-amino acids as common constituents of fermented foods, Chromatographia, 28, 487e492 (1989). Jin, D., Miyahara, T., Oe, T., and Toyo’oka, T.: Determination of D-amino acids labeled with fluorescent chiral reagents, R()- and S(þ)-4-(3isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimetylaminosulfonyl)-2,1,3,-benzoxadiazoles, in biological and food samples by liquid chromatography, Anal. Biochem., 269, 124e132 (1999). Brückner, H. and Westhauser, T.: Chromatographic determination of L- and Damino acids in plants, Amino Acids, 24, 43e55 (2003). Mutaguchi, Y., Ohmori, T., Akano, H., Doi, K., and Ohshima, T.: Distribution of D-amino acids in vinegars and involvement of lactic acid bacteria in the production of D-amino acids, Springerplus, 2, 691 (2013). Brückner, H., Becker, D., and Lüpke, M.: Chirality of amino acids of microorganisms used in food biotechnology, Chirality, 5, 385e392 (1993).
J. BIOSCI. BIOENG., 13. Tanwar, S. and Bhushan, R. J.: Enantioresolution of amino acids: a decade’s perspective, prospects and challenges, Chromatographia, 78, 1113e1134 (2015). 14. Ilisz, I., Aranyi, A., Pataj, Z., and Péter, A.: Recent advances in the direct and indirect liquid chromatographic enantioseparation of amino acids and related compounds: a review, J. Pharm. Biomed. Anal., 69, 28e41 (2012). 15. Taujenis, L., Olsauskaite, V., and Padarauskas, A.: Enantioselective determination of protein amino acids in fertilizers by liquid chromatography-tandem mass spectrometry on chiral teicoplanin stationary phase, J. Agric. Food Chem., 62, 11099e11108 (2014). 16. Barbaro, E., Zangrando, R., Vecchiato, M., Turetta, C., Barbante, C., and Gambaro, A.: D- and L-amino acids in antarctic lakes: assessment of a very sensitive HPLC-MS method, Anal. Bioanal. Chem., 406, 5259e5270 (2014). 17. Schurig, V.: Gas chromatographic enantioseparation of derivatized-amino acids on chiral stationary phases-past and present, J. Chromatogr. B, 879, 3122e3140 (2011). 18. Gogami, Y., Okada, K., and Oikawa, T.: High-performance liquid chromatography analysis of naturally occurring D-amino acids in sake, J. Chromatogr. B, 879, 3259e3267 (2011). 19. Carlavilla, D., Moreno-Arribas, M. V., Fanali, S., and Cifuentes, A.: Chiral MEKC-LIF of amino acids in foods: analysis of vinegars, Electrophoresis, 27, 2551e2557 (2006). 20. Konya, Y., Bamba, T., and Fukusaki, E.: Extra-facile chiral separation of amino acid enantiomers by LC-TOFMS analysis, J. Biosci. Bioeng., 121, 349e353 (2016).
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Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods Yutaka Konya, Moyu Taniguchi, and Eiichiro Fukusaki* Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Received 8 April 2016; accepted 11 July 2016 Available online xxx
Recently, the demand for D-amino acid profiling has been drastically increasing because the significance of D-amino acid in various biological events is suggested. However, the present methodologies for D-amino acid profiling are still unsatisfactory. Therefore, a highly sensitive, robust, high-throughput, and user-friendly method for D-amino acid profiling must be developed. In this paper, we developed a novel method for D-amino acid profiling using a combination of a chiral column and time of flight mass spectrometry (TOFMS). To our knowledge, our approach has the best performance for D-amino acid analysis that includes the shortest analytical time (within 10 min), the highest enantioseparability without derivatization, and the largest coverage for analytical targets (more than one hundred targets including non-proteinogenic amino acids and amines). Thus, our novel profiling method will be instrumental in advancing the D-amino acid research in the future. Ó 2016, The Society for Biotechnology, Japan. All rights reserved. [Key words: D-Amino acids; Underivatized amino acids; Chiral amino acids; Enantioseparation; Liquid chromatographyetime of flight mass spectrometry]
Amino acids are essential and ubiquitous compounds for living organisms on Earth. L-Amino acids exist predominantly as building blocks of peptides/proteins or in free form, and perform critical roles in biological systems. In contrast, the presence of D-amino acids is comparatively very low and in the past, D-amino acids was believed to be absent in higher organisms (1). However, D-amino acids were recently found in various higher organisms (2e4) and the demand for D-amino acid profiling has been drastically increasing because the roles of D-amino acids in important diseases such as schizophrenia, amyotrophic lateral sclerosis (ALS), and renal dysfunction have been suggested (5e7). There are several reports which showed the existence of D-amino acids in various foods (8e11). D-Amino acids in fermented foods are thought to be derived from the starting materials used to prepare those foods, or they are produced during microbial fermentation (12). Therefore, there is an increasing need to develop rapid, robust and highly sensitive methods for the separation and quantification of D-amino acids (13). Consequently, many chiral analytical methods for amino acid enantiomers have been developed. Chiral and simultaneous analyses for amino acid enantiomers require high level chromatographic separation technologies. The two approaches commonly used are direct method (without derivatization) and indirect method (with derivatization) (13,14). Direct methods use chiral stationary phases or, more rarely, chiral mobile phase additives. Many chiral stationary phases for direct methods have been developed, among which the most enantioselective separations of amino acids were mainly carried out on glycopeptide
* Corresponding author. Tel./fax: þ81 6 6879 7424. E-mail address: [email protected] (E. Fukusaki).
antibiotic teicoplanin or teicoplanin aglycone chiral stationary phases. However, chiral liquid chromatographyemass spectrometry (LCeMS) analyses using a teicoplanin or its aglycone-based chiral columns could not achieve good separation of several proteinogenic amino acid enantiomers (15,16). On the other hand, indirect methods are based on the formation of derivatives by the reaction of amino acids with a chiral or achiral derivatizing agent followed by the separation of the derivatives on an achiral or chiral stationary phase. Since enantioseparation based on direct methods are relatively more difficult than indirect methods, most chiral analyses are performed with derivatization prior to analysis, such as gas chromatographyemass spectrometry (GCeMS) (17), LCeMS (3,4), liquid chromatographyefluorescence detection (LCeFLD) (2,9e11,18), and capillary electrophoresisefluorescence detection (CEeFLD) (19). However, problems using these methods are still prevalent such as the stability of derivatives, long analysis time, separations of Damino acids from enantiomers and unknown peaks. As described above, despite the progress in analytical technology, enantioselective separation and quantification of D-amino acids remain a challenge owing to the frequently incomplete separation of enantiomers and insufficient sensitivity for determination of trace amounts of D-amino acids. In this study, we aimed to develop a high-throughput and high-resolution analytical method for D-amino acids profiling. MATERIALS AND METHODS Enantiomers Proteinogenic amino acid enantiomers (DL-20 amino acids) of DL-alanine (DL-Ala), DL-arginine hydrochloride (DL-Arg), DL-asparagine monohydrate (DL-Asn), DL-aspartic acid (DL-Asp), DL-cysteine hydrochloride monohydrate (DL-Cys), DL-glutamic acid (DL-Glu), DL-glutamine (DL-Gln), DL-histidine (DL-His), DL-isoleucine (DL-Ile, mixture of four stereoisomers containing DL-allo-Ile), DL-leucine
1389-1723/$ e see front matter Ó 2016, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
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KONYA ET AL.
(DL-Leu), DL-lysine monohydrochloride (DL-Lys), DL-methionine (DL-Met), DL-phenylalanine (DL-Phe), DL-proline (DL-Pro), DL-serine (DL-Ser), DL-threonine (DL-Thr), DLtryptophan (DL-Trp), DL-tyrosine (DL-Tyr) and DL-valine (DL-Val), were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Glycine (Gly) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Non-proteinogenic amino acids and amines DL-Ethionine was obtained from Acros Organics (Geel, Belgium). DL-Pyroglutamic acid was obtained from Icn Biochemicals (CA, USA). Adenine was obtained from Kishida Chemical Co., Ltd. (Osaka, Japan). Uracil was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). DL-a-Aminoadipic acid, D-b-aminoisobutyric acid, creatinine, cystathionine (mixture of DL-, DL-allo-), cis-4-hydroxy-D-proline, DL-kynurenine and DL-pipecolic acid, were obtained from Sigma-Aldrich Japan K.K. (Tokyo, Japan). DL-2-Aminopimelic acid, DL-citrulline, cystine (DL- and meso-mixture), DL-2,4-diaminobutyric acid dihydrochloride, 3-(3,4dihydroxyphenyl)-DL-alanine (DL-Dopa), DL-homophenylalanine, DL-homoserine, DL-norvaline (DL-nor-Val) and DL-ornithine monohydrochloride were obtained from Tokyo Chemical Industry Co., Ltd. b-Alanine (b-Ala), allantoin, DL-allothreonine (DL-allo-Thr), anthranilic acid (2-Aminobenzoic acid), g-aminobutyric acid (GABA), betaine, creatine, cytosine, N,N-dimethylglycine (DMG), dopamine hydrochloride, guanine, histamine, L-hydroxyproline (trans-4-hydroxy-L-proline), hypotaurine, nicotinamide, nicotinic acid, sarcosine and thymine were obtained from Wako Pure Chemical Industries, Ltd. Wako Amino Acids Mixture Standard Solutions Wako Amino Acids Mixture Standard Solutions’ Type AN-2 and Type B were obtained from Wako Pure Chemical Industries, Ltd. Type AN-2 contained L-alanine, b-alanine, L-a-aminoadipic acid, DL-a-aminobutyric acid, DL-b-aminoisobutyric acid, L-aspartic acid, L-citrulline, L-cystathionine, L-cystine, L-glutamic acid, glycine, hydroxy-L-proline, L-isoleucine, L-leucine, L-methionine, o-phosphoserine, o-phosphoethanolamine, L-proline, L-phenylalanine, L-serine, sarcosine, taurine, L-threonine, L-tyrosine, urea and L-valine. While Type B contained g-aminobutyric acid, ammonium chloride, L-anserine, L-arginine, L-carnosine, ethanolamine, L-histidine, DL-plus allo-dhydroxylysine, L-ornithine, L-lysine, L-1-methylhistidine, L-3-methylhistidine. Reagents for extraction and mobile phase Ultrapure water for LC/MS (Water), ethanol for HPLC (EtOH), 0.1 mol/L hydrochloric acid for volumetric analysis and trifluoroacetic acid for HPLC (TFA) were obtained from Wako Pure Chemical Industries, Ltd. Methanol -Plus- for LC/MS (MeOH) and acetonitrile -Plus- for LC/MS (ACN) were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). Chloroform for HPLC was obtained from Kishida Chemical Co., Ltd. DL-Alanine-2,3,3,3-d4 were obtained from Santa Cruz Biotechnology (TX, USA). Matrices and food samples Grain vinegar (Vinegar) was obtained from Mizkan Group Corporation (Aichi, Japan). Whole cow milk (Milk) was obtained from Megmilk Snow Brand Co., Ltd. (Tokyo, Japan). Three kinds of black vinegars were obtained from AEON Co., Ltd. (Chiba, Japan), Mizkan Group Corporation, and Japan Health System Co., Ltd. (JHS) (Tokyo, Japan). Kimchi (Amakuchi) was obtained from Greenfarm Co., Ltd. (Osaka, Japan). Kimchi (Ichioshi) was obtained from Miyama Co., Ltd. (Chiba, Japan). Kimchi (Gohan-ga-Susumu; GS) was obtained from Pickles Co., Ltd. (Saitama, Japan). Yogurt drink (Bifidus SP strain, capsule) and yogurt drink (Gasseri SP strain) were obtained from Megmilk Snow Brand Co., Ltd. Yogurt drink (LB81 strain, plain) was obtained from Meiji Co., Ltd. (Tokyo, Japan). Instrument Amino acid enantiomers were analyzed on Crownpak CR-I(þ) and CR-I() columns (3.0 mm i.d. 150 mm, and 5 mm particle size, Daicel CPI, Osaka, Japan) using Nexera system (pump: LC-30AD, autosampler: SIL-30AC, degasser: DGU-20A5, column oven: CTO-30A, Shimadzu, Kyoto, Japan) coupled to TripleTOF 5600 System (AB SCIEX, Concord, Canada). Preparation of DL-20mix calibration standard solutions and ISsolution 50%MeOH was prepared by mixing MeOH and water (1/1 (v/v)). 50% MeOH-0.05 mol/L-HCl was prepared by mixing MeOH and 0.1 mol/L Hydrochloric Acid (1/1 (v/v)). A8E2 was prepared by mixing ACN and EtOH (8/2 (v/v)). To make 20 mmol/mL standard solutions, DL-Glu, DL-His & DL-Trp were dissolved in 50%MeOH-0.02 mol/L-HCl, DL-Asp and DL-Tyr were dissolved in 50%MeOH-0.05 mol/L-HCl, while the other proteinogenic amino acids were dissolved in 50%MeOH. Glycine and nineteen DL-amino acids standard solutions (20 mmol/mL) were mixed in equal amount to make 1000 nmol/mL solution (DL-20mix-1000). Then, DL-20mix1000 was diluted by 50%MeOH-0.01 mol/L-HCl to make 400, 100, 40, 10, 4 and 1 nmol/mL in 50%MeOH-0.01 mol/L-HCl (DL-20mix calibration standard solutions). DL-Alanine-2,3,3,3-d4 was dissolved in 50%MeOH to make 40 mmol/mL (ISsolution). Preparation of calibration samples Sample preparation was carried out according to LLEA (LiquideLiquid Extraction method for Amines). Briefly, a matrix (water, vinegar or milk (50 mL)), water (50 mL), DL-20mix calibration standard solutions (1000, 400, 100, 40, 10, 4, 1 or 0 nmol/mL in 50%MeOH-0.01 mol/L-HCl) (100 mL), IS-solution (20 mL) and MeOH (300 mL) were mixed, then vortexed for 10 s. After centrifugation (16,000 g, 4 C, 10 min), the supernatant (360 mL) was mixed with water (180 mL) and chloroform (360 mL), then vortexed for 10 s. After centrifugation (16,000 g, 4 C, 10 min), the MeOHeWater layer (upper layer, 50 mL) was mixed with A8E2 (200 mL) and vortexed for 10 s. After centrifugation (16,000 g, 4 C, 10 min), 1 mL of the supernatant was injected onto a Crownpak CR-I column.
J. BIOSCI. BIOENG., Preparation of black vinegar and yogurt samples Black vinegar or yogurt drink (50 mL) were mixed with water (50 mL), DL-20mix calibration standard solutions (1000 or 0 nmol/mL in 50%MeOH-0.01 mol/L-HCl) (100 mL), IS-solution (20 mL) and MeOH (300 mL), then vortexed for 10 s. After centrifugation (16,000 g, 4 C, 10 min), the supernatant (360 mL) was mixed with water (180 mL) and chloroform (360 mL), then vortexed for 10 s. After centrifugation (16,000 g, 4 C, 10 min), the MeOHewater layer (upper layer, 50 mL) was mixed with A8E2 (200 mL) and vortexed for 10 s. After centrifugation (16,000 g, 4 C, 10 min), 1 mL of the supernatant was injected onto a Crownpak CR-I column. Preparation of kimchi samples A piece of napa cabbage in kimchi (approximately 1 g) was taken as a sample and its surface was washed with water. The washed napa cabbage was crushed by a spatula and centrifuged. The resulting supernatant (50 mL) was collected and prepared in the same manner as black vinegars and yogurt samples. Preparation of mixed standard solutions of amino acids and amines Amino acid standard solutions and amine standard solutions (dissolved in 50%MeOH, or 50%MeOH-0.05 mol/L-HCl) or Wako Amino Acids Mixture Standard Solutions were mixed and diluted with the mobile phase. The combinations and concentrations of amino acids and amines in mixed standard solutions were decided based on their molecular weights and peak intensities. One microliter of each mixed standard solution was injected onto a Crownpak CR-I column. Liquid chromatographyetime of flight mass spectrometry analysis One microliter of extracted solution of calibration samples, food samples, or mixed standard solutions of amino acids and amines was injected onto a Crownpak CRI(þ) column. The mobile phase used was ACN/EtOH/water/TFA (80/15/5/0.5 (v/v/ v/v)), the flow rate was 0.4 mL/min, the autosampler temperature and the column oven temperatures were maintained at 4 C and 30 C, respectively. All analyses were performed under isocratic conditions. Instead of Crownpak CR-I(þ) column, Crownpak CR-I() column was used to calculate peak area ratios of D/Lamino acids, and to confirm the existence of D-allo-Ile in foods. Column eluent was analyzed in positive ionization mode using a TripleTOF 5600 system with the following conditions: ion source gas 1 (50 psi), ion source gas 2 (50 psi), curtain gas 1 (30 psi), temperature (600 C), ion spray voltage floating (5500 V), declustering potential (60 V), collision energy (5 V), and mass range (60e600 m/z). Data processing was performed using MultiQuant (AB SCIEX, Concord, Canada). Calculation of recovery rates of D-amino acids The recovery rates of all Damino acids (except for Gly and DL-Pro) in food samples were estimated by comparing peak area ratios (the peak area of D-amino acid/the peak area of IS) of each D-amino acid in food (1000 nmol/mL in matrics) with its corresponding peak area ratio in Water calibration samples (1000 nmol/mL in water). As several endogenous D-amino acids exist in foods, their peak area ratios were subtracted before calculating their recovery rates. Calculation of D-amino acid concentrations The regression equations (Y ¼ aX þ b; with weighting factor of 1/X2) were calculated by the least-squares method using MultiQuant. For this calculation, the peak area ratios (the peak area of D-amino acid/the peak area of IS) obtained in the measurement of the calibration samples (as Y) and the spiked concentrations (as X) were used. Damino acid concentrations in foods were calculated using Water calibration curves and then corrected by the recovery rates of D-amino acids in each food. Calculation of area ratios of D/L-amino acids The area ratios of D-form to Lform in mixed standard solutions (DL-20mix 1 and 10 nmol/mL diluted by the mobile phase) were calculated.
RESULTS AND DISCUSSION Optimization of mobile phase composition Previously, we analyzed the standard solution of mixed proteinogenic amino acid enantiomers using a combination of a Crownpak CR-I(þ) column and liquid chromatographyetime of flight mass spectrometry (TOFMS) (20). An isocratic condition of a simple mobile phase composed of ACN/Water/TFA (96/4/0.5 (v/v/v)) gave baseline separation of all underivatized amino acid enantiomers (except for Pro, secondary amine) within 15 min. However, since an extraction process was necessary for analysis of amino acids in food samples, the final composition of the extracted sample solutions was different from the composition of the standard solution. As a result, the peak shapes of several amino acids in the extracted samples significantly deteriorated compared to the standard solution. Therefore, to improve their peak shapes, the mobile phase and extracted sample solution were adjusted so that their compositions become more similar to each other while maintaining the extraction efficiency and enantioseparability.
Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
VOL. xx, 2016 Consequently, with a (80/15/5/0.5 (v/v/v/v)) ratio of ACN/EtOH/ Water/TFA, 18 proteinogenic amino acid enantiomers in the extracted samples were successfully detected and separated with good peak shapes (Fig. 1A). In addition, analyses using CR-I() column also achieved good enantioseparation (Fig. S1).
LC-TOFMS METHOD FOR D-AMINO ACIDS IN FOODS
3
Peak area ratios of D/L-amino acids As an advantage of LC isocratic condition which was adopted in our method, influence on ionization efficiency by LC mobile phase was far smaller than using LC gradient condition. We analyzed mixed standard solutions (DL20mix 1 and 10 nmol/mL in the mobile phase) using both CR-I(þ)
FIG. 1. LCeMS chromatograms of (A) water calibration sample (1000 nmol/mL in water) and (B) black vinegar (AEON) using CR-I(þ) column (extracted ion of [MþH]þ 5 mDa).
Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
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J. BIOSCI. BIOENG.,
and CR-I() columns. The area ratios of nearly 1.0 (Table S1).
D-form
to L-form were
Method validation For method validation, calibration curves were created using water, milk and vinegar as matrices, and the quantitativity of the method was evaluated through its recovery rate, limit of quantification, linearity, accuracy and precision. All analyses were performed using the CR-I(þ) column. The recovery rates of all D-amino acids (except for DL-Pro that is not separated) in milk and vinegar calibration samples were 72.1% or more and 89.9% or more, respectively (Table S2). The lower limit of quantification (LLOQ) was determined from the lowest concentrations with an accuracy of 80e120% and precision within 20%. For other concentrations, the accuracy was 85e115% and the precision was within 15% (Fig. S2 and Tables S3eS6).
Determination of D-amino acid concentrations in food samples All D-amino acids in blank milk and blank grain vinegar (except for D-Ala, D-Asp and D-Glu in blank grain vinegar) were under the LLOQ. Recoveries and concentrations of D-amino acids in commercially available black vinegars, kimchis and yogurt drinks were also determined. From this study, 14 D-amino acids in black vinegars, 10 D-amino acids in kimchis, and 5 D-amino acids in yogurt drinks were determined (Figs. 1B and S3 and Tables 1 and S6). Confirmation of D-allo-isoleucine (D-allo-Ile) in food samples Analysis using CR-I(þ) column could not distinguish D-allo-Ile from D-Ile because of co-elution. In contrast, CR-I() column could separate D-allo-Ile and D-Ile peaks thus, it was used to analyze the water calibration sample and black vinegar
TABLE 1. D-Amino acid concentrations in foods (nmol/mL or nmol/g). Index
Name
D-Ala
1 2 3 4
D-Ser
Grain vinegar
JHS 198.1 29.5
(1.0)b
372.6 24.7 36.6 6.6
(1.0)b (1.9)b 14.3
36.8 (3.5)b
D-Val
5 6 7 8 9 10 11 12 13 14 15 16 17 18 a b c d
D-Thr þ D-allo-Thr þD-homoserinec D-Cys d D-Ile þ D-allo-Ile D-Leu D-Asn D-Asp D-Gln D-Lys D-Glu D-Met D-His D-Phe D-Arg D-Tyr D-Trp
Black vinegar AEON
6.8 (3.6)b (1.4)b
Kimchi Mizkan
Yogurt
Amakuchi
Ichioshi
GS
Bifidus
LB81
Gasseri
41.7 16.4
175.8 7.4
(3208.4)a 21.2
172.0 7.9
49.2 Trace
97.5 4.0
111.3 Trace
7.2
7.3
(3.1)b
11.0
4.7
62.8 93.6 17.0 106.0
(2.6)b 5.9 7.4 42.5
(3.0)b 4.4 16.8 24.7
Trace 5.4
Trace 13.2
Trace 9.8
51.4
120.7
39.8
19.6
Trace
69.2 5.7 6.3 6.0 10.7 1.9
37.2 (1.5)b 5.0 7.7 (3.0)b 5.0
25.4 (1.3)b 7.0 5.1 5.8 3.1
Trace 103.4 Trace 4.4 (2.4)b 4.5 1.3
Trace 138.0 Trace 9.2 (3.5)b 11.6 2.8
96.8 Trace 6.7 4.9 4.3 1.7
14.2
32.7
27.2
(3.2)b
4.7
4.6
>upper limit of quantification (ULOQ).
FIG. 2. LCeMS chromatograms of Water calibration sample and black vinegar using CR-I(þ) and CR-I() columns for the confirmation of D-allo-Ile in food. (extracted ion of [MþH]þ 5 mDa) ¼ 132.1025. (A) Water calibration sample (100 nmol/mL in water) using CR-I(þ) column. (B) Black vinegar (AEON) using CR-I(þ) column. (C) Water calibration sample (100 nmol/mL in water) using CR-I() column. (D) Black vinegar (AEON) using CR-I() column.
Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
FIG. 3. LCeMS chromatograms of mix standard solutions of amino acids which have same molecular weight using CR-I(þ) column (extracted ion of [MþH]þ 5 mDa). (A) [MþH]þ ¼ 90.0555 (sarcosine, DL-Ala, and b-Ala). (B) [MþH]þ ¼ 104.0712 (N,N-dimethylglycine (DMG), DL-a-aminobutyric acid (DL-a-ABA), DL-b-aminoisobutyric acid (DL-b-AIBA), and g-aminobutyric acid (GABA)). (C) [MþH]þ ¼ 118.0868 (betaine, DL-Val, and DL-norvaline (DL-nor-Val)). (D) [MþH]þ ¼ 120.0661 (DL-Thr, DL-allo-Thr, and DL-homoserine). (E) [MþH]þ ¼ 132.1025 (DL-Ile, DL-allo-Ile, and DL-Leu). (F) [MþH]þ ¼ 163.1083 (DL-plus DL-allo-d-hydroxylysine (d-h-Lys-01, 02, 03, 04)). (G) [MþH]þ ¼ 223.0753 (DL-plus DL-allo-cystathionine (CST-01, 02, 03, 04)). (H) [MþH]þ ¼ 241.0317 (DD-, LL-plus meso-cystine).
FIG. 4. LCeMS chromatograms of mix standard solutions of nonproteinogenic amino acids and amines using CR-I(þ) column (extracted ion of [MþH]þ 5 mDa).
Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
6
KONYA ET AL.
J. BIOSCI. BIOENG., TABLE 2. Extracted ion of [MþH]þ, retention time (Rt) and resolution (Rs) of amino acids and amines.
Index 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77
Formula
[MþH]þ
CH4N2O C2H7NO C2H5NO2 C3H7NO2
61.0402 62.0606 76.0399 90.0555
C4H9NO2
104.0712
C3H7NO3
106.0504
C2H7NO2S C4H5N3O C5H9N3 C4H4N2O2 C4H7N3O C5H9NO2
110.0276 112.0511 112.0875 113.0589 114.0667 116.0712
C5H11NO2
118.0868
C4H10N2O2
119.0821
C4H9NO3
120.0661
C3H7NO2S
122.0276
C6H6N2O C6H5NO2 C2H7NO3S C5H6N2O2 C5H7NO3
123.0558 124.0399 126.0225 127.0508 130.0504
C6H11NO2
130.0868
C5H9NO3
132.0661
C4H9N3O2 C6H13NO2
132.0773 132.1025
C4H8N2O3
133.0613
C5H12N2O2
133.0977
C4H7NO4
134.0453
C5H5N5 C7H7NO2 C5H10N2O3
136.0623 138.0555 147.0770
C6H14N2O2
147.1134
C5H9NO4
148.0610
C5H11NO2S
150.0589
C5H5N5O C8H11NO2 C6H9N3O2
152.0572 154.0868 156.0773
C4H6N4O3 C6H11NO4
159.0518 162.0766
Compound
Rt (min)
Urea Ethanolamine Glycine Sarcosine D-Alanine b-Alanine L-Alanine N,N-Dimethylglycine D-a-Aminobutyric acid D-b-Aminoisobutyric acid L-a-Aminobutyric acid L-b-Aminoisobutyric acid g-Aminobutyric acid D-Serine L-Serine Hypotaurine Cytosine Histamine Uracil Creatinine D-Proline L-Proline Betaine D-Valine D-Norvaline L-Valine L-Norvaline D-2,4-Diaminobutyric acid L-2,4-Diaminobutyric acid D-allo-Threonine D-Threonine D-Homoserine L-allo-Threonine L-Threonine L-Homoserine D-Cysteine L-Cysteine Nicotinamide Nicotinic acid Taurine Thymine D-Pyroglutamic acid L-Pyroglutamic acid D-Pipecolic acid L-Pipecolic acid cis-4-Hydroxy-D-proline trans-4-Hydroxy-L-proline Creatine D-allo-Isoleucine D-Isoleucine D-Leucine L-allo-Isoleucine L-Isoleucine L-Leucine D-Asparagine L-Asparagine D-Ornithine L-Ornithine D-Aspartic acid L-Aspartic acid Adenine Anthranilic acid D-Glutamine L-Glutamine D-Lysine L-Lysine D-Glutamic acid L-Glutamic acid D-Methionine L-Methionine Guanine Dopamine D-Histidine L-Histidine Allantoin D-2-Aminoadipic acid L-2-Aminoadipic acid
2.37 3.20 3.67 1.33 1.89 3.48 4.67 1.30 1.55 2.61 2.96 3.32 3.66 1.67 2.31 3.83 1.55 1.69 2.25 1.43 1.34 1.34 1.28 1.39 1.55 1.79 3.57 1.74 2.12 1.42 1.42 1.45 1.64 2.00 2.39 1.63 2.91 1.51 1.53 10.35 2.23 2.24 2.24 1.32 1.32 1.35 1.35 1.34 1.37 1.37 1.52 1.75 1.91 3.82 1.50 1.79 2.16 4.08 1.71 2.62 1.60 2.12 1.48 2.73 2.70 7.57 1.70 5.12 1.71 5.24 1.70 3.28 1.35 1.50 2.39 1.58 3.99
Rs
Remark
6.85 10.04 4.43 3.63 9.71 2.17 1.90 1.59 5.52
e
Secondary amines
1.64 2.26 2.85 11.13 3.16 e 0.38 2.55 4.54 3.43
D-allo-form/D-form
10.18
e
Secondary amines
e
Secondary amines
e
Secondary amines
e 1.98 2.81 2.02 11.59
D-allo-form/D-form
3.43 7.39 7.26
11.14 9.01 14.49 15.63
1.95
13.03
Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009
VOL. xx, 2016
LC-TOFMS METHOD FOR D-AMINO ACIDS IN FOODS
7
Table 2 (continued ) Index 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110
Formula
[MþH]þ
C6H14N2O3
163.1083
d-Hydroxylysine-01 d-Hydroxylysine-02 d-Hydroxylysine-03 d-Hydroxylysine-04
C6H13NO2S
164.0745
D-Ethionine
Compound
Rt (min)
L-Ethionine
C9H11NO2
166.0868
D-Phenylalanine L-Phenylalanine
C7H11N3O2
170.0930
L-1-Methylhistidine L-3-Methylhistidine
C6H14N4O2
175.1195
D-Arginine L-Arginine
C7H13NO4
176.0923
D-2-Aminopimelic L-2-Aminopimelic
C6H13N3O3
176.1035
acid acid
D-Citrulline L-Citrulline
C10H13NO2
180.1025
D-Homophenylalanine L-Homophenylalanine
C9H11NO3
182.0817
D-Tyrosine L-Tyrosine
C9H11NO4
198.0766
D-Dopa L-Dopa
C11H12N2O2
205.0977
D-Tryptophan L-Tryptophan
C10H12N2O3
209.0926
D-Kynurenine L-Kynurenine
C7H14N2O4S
223.0753
C6H12N2O4S2
241.0317
Cystathionine-01 Cystathionine-02 Cystathionine-03 Cystathionine-04 DD-Cystine meso-Cystine LL-Cystine
2.08 2.24 4.85 5.43 1.71 5.18 1.52 2.65 1.41 1.41 1.34 1.95 1.59 3.71 1.60 3.38 1.78 5.95 1.53 2.57 1.54 2.68 1.53 2.52 1.82 3.66 1.64 2.56 4.49 10.90 1.84 3.96 16.18
Rs
Remark
1.13 7.71 1.10 15.20 9.42 e
Secondary amines
6.21 12.01 10.82 15.72 8.54 9.03 8.34 10.33 5.86 5.83 7.38 8.05 10.25
Rs, resolution of subsequent two compounds sharing a same [MþH]þ. Rs of a pair of peaks with retention times t1 and t2 (t2 > t1) was calculated using the equation Rs ¼ 1:18 ðt2 t1 Þ=ðwh1 þ wh2 Þ, where wh1 and wh2 are the corresponding peak widths measured at half the peak height.
samples. The result showed that only D-allo-Ile was detected in black vinegar samples (Fig. 2). Peak shapes and resolution of non-proteinogenic amino acids and amines Next, we applied the method using the CRI(þ) column to non-proteinogenic amino acids and amines (Figs. 3 and 4 and Table 2). All enantiomers which have primary amine in their structures were successfully separated. Because CR-I columns separate amino acid enantiomers based on the interaction between the crown ether of the solid phase and primary amines, secondary amine enantiomers such as proline and pyroglutamic acid could not be separated. Nevertheless, all of the secondary amines were detected with good peak shapes. In addition, compounds with the same molecular weight such as group-90 ([MþH]þ ¼ 90.0555: sarcosine, DL-Ala, and b-Ala) and group-104 ([MþH]þ ¼ 104.0712: N,N-dimethylglycine, DL-aaminobutyric acid, DL-b-aminoisobutyric acid, and GABA) also showed good separation. Our result showed that 110 compounds including non-proteinogenic amino acids were successfully analyzed. We determined trace D-amino acids in three kinds of foods (black vinegar, kimchi and yogurt), and our analytical method was verified to be applicable to food samples. This analytical method is classified to direct method, showed more advantages such as high enantioseparability, high throughput, and wide range of targets than not only other direct methods (15,16), but also indirect methods which need derivatization process (2e4,9e11,17e19). In conclusion, we developed a novel high throughput LC-TOFMS method for D-amino acids in foods. This method employs simpler LC conditions and shorter analysis time (10 min). Also, it does not require a derivatization process while exhibiting a higher enantioseparability compared to any previous methods for D-amino acid
analysis. Therefore, this method does not suffer from undesirable issues that may occur due to derivatization, long analytical time and fluorescence detection. Moreover, the coverage for analytical targets is wider (more than one hundred targets in total including non-proteinogenic amino acids and amines) compared to previously published protocols. Taking into account these improvements, our novel profiling method will be instrumental in advancing the D-amino acid research in the future. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2016.07.009. ACKNOWLEDGEMENTS The study represents a portion of the dissertation submitted by Yutaka Konya to Osaka University in partial fulfillment of the requirement for his Ph.D. References 1. Corrigan, J. J.: D-Amino acids in animal, Science, 164, 142e149 (1969). 2. Miyoshi, Y., Koga, R., Oyama, T., Han, H., Ueno, K., Masuyama, K., Itoh, Y., and Hamase, K.: HPLC analysis of naturally occurring free D-amino acids in mammals, Pharm. Biomed. Anal., 69, 42e49 (2012). 3. Müller, C., Fonseca, J. R., Rock, T. M., Krauss-Etschmann, S., and SchmittKopplin, P.: Enantioseparation and selective detection of D-amino acids by ultra-high-performance liquid chromatography/mass spectrometry in analysis of complex biological samples, J. Chromatogr. A, 1324, 109e114 (2014). 4. Visser, W. F., Verhoeven-Duif, N. M., Ophoff, R., Bakker, S., Klomp, L. W., Berger, R., and Koning, T. J.: A sensitive and simple ultra-high-performanceliquid chromatography-tandem mass spectrometry based method for the quantification of D-amino acids in body fluids, J. Chromatogr. A, 1218, 7130e7136 (2011). 5. Hashimoto, K., Engberg, G., Shimizu, E., Nordin, C., Lindström, L. H., and Iyo, M.: Reduced D-serine to total serine ratio in the cerebrospinal fluid of drug
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Please cite this article in press as: Konya, Y., et al., Novel high-throughput and widely-targeted liquid chromatographyetime of flight mass spectrometry method for D-amino acids in foods, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.07.009