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User Benefits Application News Introduction Recent advances in analytical techniques have revealed the presence of D-amino acids in fermented foods and biological samples and clarified their role in the taste, preservation, and aroma of foods , and as potential disease biomarkers. Therefore, the demand for D/L separation of amino acids is increasing. Because D-amino acids are only present in trace amounts in fermented foods and biological systems, unlike L-amino acids, it is necessary to determine the concentration of each D-amino acid by separating them from the high concentrations of L- amino acids. A comprehensive method for quantitating the thirty-seven D/L- amino acids was previously reported 1) , in which two chiral derivatizing reagents were employed to provide different diastereomers for each amino acid, with the instrument automatically switching between two separation methods to analyze each sample twice. This article introduces an improved method that employs a single chiral derivatizing reagent to enable simultaneous analysis of D/L-amino acids in a shorter time. Enables high-sensitivity simultaneous fast analysis of thirty-seven D/L-amino acids using simple operations. Good reproducibility of analytical results can be achieved by keeping the derivatization reaction time constant. Statistical analysis software enables easy multivariate analysis of multiple samples. UHPLC Method for Sensitive Automatic Analysis of Thirty-Seven D/L-Amino Acids and for Liquor Profiling Natsuki Iwata High Performance Liquid Chromatograph Statistical Analysis Software Table 1 Analytical Conditions Fluorescence Derivatization of D/L-Amino Acid Diastereomers N-isobutyryl-L-cysteine (NIBC) was used for chiral derivatization. Fluorescence derivatization of D/L-amino acid diastereomers was performed by reaction with o-phthalaldehyde (OPA) in the presence of NIBC. System: Nexera X3 Column: CERI L-column 3 C18 (150 mm × 2.1 mm I.D., 2.0 µm) using pre-column filter Flowrate: 0.22 mL/min Mobile Phase: Pump A 10 mmol/L (Sodium) phosphate buffer (pH 6.9) Pump B b) Acetonitrile c) Methanol b/c = 15:85 using mobile phase blending function Time Program: 8 % B (0 - 7.27 min) → 27 % B (18.18 - 30.00 min) → 36 % B (32.00 min) → 41 % B (40.91 min) → 46 % B (54.55 min) → 50 % B (58.55 min) → 52 % B (65.28 min) → 80 % B (66.19 - 74.36 min) → 8 % B (75.27-87 min) Column Temp.: 20 °C Injection Volume: 1 µL Vial: SHIMADZU LabTotal for LC 1.5 mL, Glass* 1 Detection: RF-20AXS, Ex: 338 nm, Em: 455 nm (semimicro cell) *1: P/N: 227-34001-01 D/L-Amino Acid Analysis by UHPLC In general, LC/MS or multi-dimensional LC is used to analyze D/L-amino acids by HPLC, because it is difficult to separate proteinogenic D/L-amino acids with a single separation mode. However, it is known that LC/MS is susceptible to matrix effects and less quantitative than HPLC. It is also known that the multi- dimensional LC method requires a long analysis time and very complicated HPLC setup. Therefore, a simple operational method that provides good separation for D/L-amino acids in a short time is needed. For food analysis, a small particle column is used to achieve appropriate separation of the small amounts of D-amino acids from the large amounts of L-amino acids and co-existing contaminants. A UHPLC system (Nexera X3) was employed in this study due to the increased system pressure caused by the use of a small particle column. Table 1 shows the analytical conditions, and Fig. 1 shows the process flow diagram of the HPLC configuration for automated analysis. As in Application News No. L592, the mobile phases were prepared automatically using the mobile phase blending function of the solvent delivery pump. D/L-amino acids were derivatized using the automatic pretreatment function of the autosampler. The mobile phase blending function provided the solutions to deliver at the specified blending ratio just by setting the organic solvents at the ports of the low-pressure gradient kit integrated into the pump. The labor and the working time were reduced by this function. In addition, the automatic pretreatment function is described below. The OPA/NIBC derivatizing reagents and the target samples were set in the autosampler. This derivatization process was completed within the injection needle and the derivatized diastereomers were introduced into the column without any exposure to the external air (Table 2 and Table 3). For reference, a schematic diagram of the autosampler rack is shown in Fig. 2. On the other hand, automation of the sample-preparation step is essential due to OPA derivatization products beginning to degrade immediately after reaction initiation. The elapsed time from starting the derivatization to injection to HPLC was kept constant using the pretreatment function, resulting in good reproducibility of analysis results. In addition, the cost of consumables was reduced because vials were not required for derivatization. The automatic operations reduced the labor and approximately an hour of the working time in comparison with manual operations (Table 4). The analysis described in this article can be implemented with a simple instrument configuration, as shown in Fig. 1. Furthermore, optimization of the automated pre-column derivatization procedure enabled detection with higher sensitivity than conventional methods 2) . Method for Automatic High-Sensitivity Analysis of D/L-Amino Acids Thirty-seven D/L-amino acids were targeted (excluding D/L- prolines from proteinogenic amino acids).
Application News Reagent Sample Derivatization Amino acid Automatic pre-column derivatization Needle Pump A Pump B Autosampler Column oven Column Detector Mobile phase blending ratio by pump B 85 % 15 % 0 % Automatic mobile phase blending Mix Derivatizing reagent Derivatizing reagent Amino acid c a b d c a b d 0 % 0.1 mol/L Borate Buffer (pH 9.1): Add 0.62 g boric acid and 0.20 g of sodium hydroxide to 100 mL of ultrapure water, and dissolve completely. OPA Reagent: Add 0.3 mL of ethanol to 10 mg of o-phthalaldehyde and dissolve completely. Then add 0.7 mL of 0.1 mol/L borate buffer (pH 9.1) and 4 mL of ultrapure water. NIBC Solution: Add 10 mg of N-isobutyryl-L-cysteine to 10 mL of 0.1 mol/L borate buffer (pH 9.1). OPA/NIBC Solutions: Mix equal volumes of OPA reagent and NIBC solution. Line Command 1 av=0.0 2 air.a 6.0,ss 3 s.aspir 3,54,2.0 4 s.aspir rn,sn,iv 5 air.a 0.1,ss 6 call f2 7 wait 1.5 8 s.load 9 s.inj 10 posttr 11 end Line Command 1 n.drain 2 for a1=1,10 3 aspir 5.5,ss 4 disp 5.5,ss 5 next a1 6 end Automatic Manual Mobile phase preparation Buffer 5 min Organic solvent ○ 0 min Note: Mobile phase blending function × 5 min Derivatizing reagent preparation 10 min Derivatization ○ 0 min Note: Automatic pre-column derivatization × 50 min Total ○ 15 min × 70 min Tray 2 Tray 3 Tray 1 1 2 1 2 ………… ………… 9 9 54 1 2 ………… 9 OPA/NIBC solution Sample Table 2 Preparation of Derivatizing Reagents Table 3 Automatic Pretreatment Program with Autosampler (When OPA/NIBC Solution Vial is Placed in Tray No. 3, Vial No. 54) Fig. 2 Schematic Diagram of Autosampler Rack (OPA/NIBC Solution Vial: Tray No. 3, Vial No. 54) Table 4 Comparison of Working Time between Automatic and Manual Operations when 20 Samples were Analyzed (b) File 2 (a) File 1 Fig. 1 Schematic Diagram of Automated System with Mobile Phase Blending and Automatic Pre-column Derivatization
Application News 0 5 10 15 20 25 30 35 40 45 50 55 60 min 0 10 20 30 40 50 60 70 80 90 100 mV Time Fluorescence intensity 3 4 2 1 5 6 7 8 9 10 11 12 15 14 13 20 21 22 24 23 25 27 28 33 34 35 36 37 16 17 19 18 26 31 32 29 30 No. Compound Conc. Range (µmol/L) r 2 No. Compound Conc. Range (µmol/L) r 2 1 L-Asp 0.5-50 0.9997 20 L-Tyr 1-50 0.9996 2 D-Asp 0.01-2 0.9999 21 D-Tyr 0.01-2 0.9999 3 L-Glu 1-50 0.9996 22 L-Val 1-50 0.9996 4 D-Glu 0.01-2 0.9999 23 L-(Cys)2 0.05-10 0.9999 5 L-Asn 0.5-50 0.9997 24 L-Met 0.01-5 0.9999 6 D-Asn 0.05-5 0.9999 25 D-(Cys)2 0.02-2 0.9999 7 L-Ser 0.2-50 0.9997 26 L-Trp 0.5-50 0.9996 8 D-Ser 0.01-5 0.9999 27 D-Met 0.01-5 0.9999 9 L-Gln 1-50 0.9997 28 D-Val 0.01-2 0.9999 10 D-Gln 0.1-5 0.9999 29 L-Ile 0.2-50 0.9997 11 L-His 1-50 0.9996 30 L-Phe 1-50 0.9996 12 L-Thr 1-50 0.9996 31 D-Trp 0.01-2 0.9999 13 D-His 0.1-10 0.9999 32 D-Phe 0.01-1 0.9999 14 D-Thr 0.01-5 0.9999 33 L-Leu 1-50 0.9996 15 Gly 1-50 0.9997 34 D-Ile 0.01-2 0.9999 16 L-Arg 1-50 0.9996 35 D-Leu 0.01-2 0.9999 17 D-Arg 0.01-2 0.9999 36 L-Lys 0.5-50 0.9997 18 L-Ala 1-50 0.9996 37 D-Lys 0.01-2 0.9999 19 D-Ala 0.01-5 0.9999 Table 5 Concentration Ranges of Calibration Curves and Coefficients of Determination (r 2 ) Repeatability The relative standard deviations (%RSD) of the peak areas based on six repeated analyses of a standard solution of D/L-amino acids (0.1 µmol/L each and 5 µmol/L each) were 1.6 % or less and 0.8 % or less, respectively. Repeatability was satisfactory by keeping the time from derivatization to the start of analysis constant. Analysis of D/L-Amino Acids Standard Solution A chromatogram of a standard solution of D/L-amino acids (1 μmol/L each) is shown in Fig. 3. The thirty-seven D/L-amino acids were separated in approximately 66 minutes. Fig. 3 Chromatogram for Mixed Standard Solution (1 µmol/L each) Calibration Curve The linearities of the calibration curves for the thirty-seven D/L- amino acids were good. Each contribution ratio (r 2 ) was 0.999 or greater (Table 5).
Application News (a) (b) 0 10 20 30 40 50 60 min 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 mV 3 4 2 1 5 67 8 9 10 11 15 13 20 22 23 33 35 36 37 16 17 19 18 26 32 29 30 3 4 2 1 5 6 7 8 9 10 11 15 13 20 22 23 33 35 36 37 16 17 19 18 26 32 29 30 beer A beer B Fluorescence intensity Time 0 10 20 30 40 50 60 min 0 250 500 750 1000 mV 3 4 2 1 5 6 7 8 9 10 11 15 13 20 22 23 33 35 36 16 17 19 18 26 30 29 30 28 29 28 33 35 36 24 22 3 4 2 1 5 6 7 8 9 10 11 15 13 20 16 17 19 18 30 29 24 22 3 4 2 1 5 6 7 8 9 10 11 15 13 20 16 19 18 12 14 33 35 36 sake red wine white wine Fluorescence intensity Time Fig. 4 Chromatograms of Liquor Samples (a) Beer A and Beer B, (b) Sake, White Wine, and Red Wine Application to Liquor Samples Two kinds of beer (Beer A and B), sake, red wine, and white wine were used as samples. Beer A, Beer B, red wine, and white wine were diluted ten-fold with 10 mmol/L hydrochloric acid and then passed through 0.2 μm membrane filters. The sake was diluted twenty-fold with 10 mmol/L hydrochloric acid and then passed through a 0.2 μm membrane filter. As shown in Fig. 4, twenty-five to twenty-eight amino acids were separated and detected in five liquor samples. Table 6 shows the D/L-amino acids contained in the liquor samples obtained from four repeated analyses. It should be noted that the concentrations of some L-amino acids were outside of the quantitation ranges. The overall ratio of D-amino acid to D/L- amino acid (%D) was found to be 6 % or less in all samples.
Application News Beer A Beer B Sake D L %D* 5 D L %D* 5 D L %D* 5 Asp 6.9 (0.4) 38.9 (0.4) 15.0 3.3 (1.2) 9.5 (0.9) 25.6 2.0 (3.6) 311.7 (2.9) 0.6 Glu 11.6 (0.3) 92.1 (0.3) 11.2 4.9 (1.1) 27.1 (1.3) 15.4 2.6 (1.1) 779.6 (1.3) 0.3 Asn 16.4 (0.4) 21.3 (0.6) 43.6 8.8 (1.1) 7.8 (0.7) 53.1 3.7 (1.4) 502.8 (1.1) 0.7 Ser 11.0 (0.5) 17.2 (0.3) 39.0 5.6 (1.1) 8.6 (1.2) 39.4 1.5 (1.3) 439.7 (0.9) 0.3 Gln 15.7 (0.5) 33.1 (0.5) 32.1 8.5 (1.2) 16.1 (0.7) 34.6 3.0 (0.6) 46.5 (1.3) 6.1 His 94.1 (0.4) 432.5 (0.4) 17.9 52.8 (1.2) 327.6 (1.2) 13.9 6.3 (1.3) 169.9 (1.6) 3.3 Thr n.d. n.d. n.d. n.d. 1.3 (1.8) 214.4 (2.1) 0.1 Gly 439.9 (0.4) 180.5 (1.2) 1498.3* 4 (0.9) Arg 9.4 (1.2) 336.3 (0.4) 2.7 2.8 (1.5) 30.0 (1.2) 8.6 n.d. 796.5 (1.2) Ala 24.4 (0.5) 1228.0* 4 (0.6) 1.9 10.1 (1.2) 447.8 (1.2) 2.2 11.8 (0.8) 2762.5* 4 (0.9) 0.4 Tyr n.d. 443.1 (0.4) n.d. 251.8 (1.2) n.d. 407.4 (1.1) Val n.d. 620.6* 4 (0.3) n.d. 183.9 (1.2) n.d. 538.9 (1.1) Met n.d. n.d. n.d. n.d. n.d. 8.0 (0.9) (Cys)2 n.d. 68.1 (0.4) n.d. 35.4 (1.1) n.d. 48.1 (1.6) Trp n.d. 170.4 (0.3) n.d. 89.6 (1.1) n.d. 12.9 (0.8) Ile n.d. 120.4 (0.3) n.d. 20.9 (1.1) n.d. 273.5 (1.1) Phe 1.6 (1.6) 398.1 (0.3) 0.4 0.7 (1.5) 148.3 (1.3) 0.5 n.d. 240.0 (1.1) Leu 4.9 (1.6) 296.1 (0.4) 1.6 2.3 (1.8) 36.2 (1.2) 5.9 0.4 (1.5) 646.6 (1.0) 0.1 Lys 6.2 (1.8) 63.2 (0.2) 8.9 2.1 (3.2) 8.7 (0.9) 19.1 n.d. 259.8 (1.1) Total 202.1 4819.2 4.0 101.9 1830 5.3 32.5 9957.1 0.3 Red wine White wine D L %D* 5 D L %D* 5 Asp 6.0 (0.8) 14.1 (0.3) 30.0 2.4 (0.3) 22.8 (0.3) 9.6 Glu 12.5 (0.4) 81.8 (0.4) 13.3 3.9 (0.7) 76.0 (0.6) 4.9 Asn 2.5 (0.5) 104.6 (0.4) 2.3 3.3 (1.0) 140.8 (1.0) 2.3 Ser 1.5 (0.8) 30.3 (0.4) 4.7 1.2 (1.4) 28.4 (1.1) 4.1 Gln 13.6 (0.9) 114.1 (0.6) 10.6 3.8 (1.3) 36.2 (0.9) 9.6 His 5.9 (0.4) 131.6 (0.5) 4.3 4.4 (1.1) 130.8 (1.2) 3.3 Thr n.d. n.d. n.d. n.d. Gly 77.0 (0.6) 110.5 (1.4) Arg 7.1 (0.3) 1260.8* 4 (0.5) 0.6 2.2 (1.2) 234.7 (1.2) 0.9 Ala 17.8 (0.8) 497.8 (0.5) 3.5 6.5 (1.5) 891.4* 4 (1.1) 0.7 Tyr n.d. 12.6 (0.4) n.d. 13.4 (0.8) Val 3.1 (0.4) 11.3 (0.9) 21.7 2.8 (0.3) 36.2 (0.7) 7.2 Met n.d. n.d. n.d. 4.4 (0.9) (Cys)2 n.d. n.d. n.d. n.d. Trp n.d. n.d. n.d. n.d. Ile n.d. 3.9 (0.4) n.d. 9.8 (1.1) Phe n.d. 10.5 (0.7) n.d. 24.2 (2.0) Leu 3.2 (0.9) 11.9 (0.3) 21.0 1.5 (1.0) 36.7 (1.1) 3.9 Lys n.d. 17.0 (0.5) n.d. 97.8 (1.1) Total 73.2 2379.1 3.0 32.0 1894.1 1.7 (a) (b) *2: n.d. indicates “not detected.” *3: Numbers in parentheses indicate %RSDs. *4: Concentrations of some L-amino acids were outside of the quantitation ranges. *5: Overall ratio of D-amino acid to D/L-amino acid. Table 6 Contents of D/L-Amino Acids in Liquor Samples* 2, * 3 (µmol/L, n = 4) (a) Beer A and Beer B, (b) Sake, Red Wine, and White Wine
Application News www.shimadzu.com/an/ Shimadzu Corporation © Shimadzu Corporation, 2026 For Research Use Only. Not for use in diagnostic procedures. This publication may contain references to products that are not available in your country. Please contact us to check the availability of these products in your country. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. See https://www.shimadzu.com/about/trademarks/index.html for details. Third party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether or not they are used with trademark symbol “TM” or “”. Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. First Edition: Jun. 2026 01-01169-EN Nexera and eMSTAT Solution are trademarks of Shimadzu Corporation or its affiliated companies in Japan and/or other countries. Conclusion A method was developed for simultaneous separation of OPA/NIBC diastereomers for thirty-seven D/L-amino acids using a simple UHPLC system in approximately half the time of the conventional methods. By optimizing the derivatization reaction, all compounds were determined sensitively, accurately, and precisely. Separation of the target compounds from the contaminants was good in the liquor samples when evaluated using real liquor samples. It is expected to be easily applicable to D/L-amino acid profiling in food and beverage samples without using expensive MS detection or complicated multi- dimensional HPLC configurations as long as samples are prepared properly. Moreover, D/L-amino acid profiling could be used to research and develop foods containing D-amino acids intended to help to maintain and improve the intestinal environment, and could help in the early detection of diseases and determining optimal treatment strategies through biomarker discovery. L-Arg D-Val L-Gln D-His D-Lys D-Phe L-Thr D-Asp D-Glu D-Ala L-Met L-Asn L-Ser L-Asp L-Glu L-Lys D-Thr L-Ala L-Leu L-Ile L-Val L-Tyr L-Phe L-Trp L-(Cys) 2 Gly D-Asn L-His D-Ser D-Leu D-Gln D-Arg Beer A Beer B Red wine White wine Sake Wine Beer Fig. 5 Principal Component Analysis Result (n = 4) (a) Score Plot, (b) Loading Plot Liquor Profiling Principal component analysis (PCA) was performed using the statistical analysis software eMSTAT Solution to determine the content of each compound in liquor samples obtained from four repeated analyses (Fig. 5). As a result of the PCA, Beer A and Beer B were plotted close together on the score plot, as well as the red and white wines (Fig. 5a). It was also found that sake showed different characteristics from the other liquor samples. The loading plot suggested that the two kinds of beers and the red and white wines contained relatively larger amounts of D- isomers, whereas the sake contained relatively larger amounts of L-isomers (Fig. 5b). Specifically, L-Trp, D-His, and D-Lys contributed significantly to the two kinds of beers, D-Val contributed to the red and white wines, and L-Thr contributed to sake. The first principal component (PC1) reflects differences among the liquor types, while the second principal component (PC2) suggests that each liquor is characterized by differences in amino acid stereoisomer composition (D-isomers versus L- isomers). (b) Loading Plot (a) Score Plot References 1) Iwata N., Watabe Y., Horie S., and Hayakawa Y., Chromatography, 42, 133-141 (2021) 2) Iwata N. and Kobayashi M., Chromatography, 45, 63-72 (2024) Related Applications 1. Automated Analysis of Thirty-seven D/L-amino Acids using Liquid Chromatography with Fluorescence Detection and Its Application to Liquor Samples, Application News No. L592
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