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Application Notes UHPLC - Nexera No. AD-0044 Quantitative Analysis of Twenty Amino Acids by PreColumn Derivatization with PITC Reagent and UHPLCUV Method Xing Jie, Edwin Ting Zhi Wei*, Sun Zhe, Zhan Zhaoqi, Customer Support Center, Shimadzu (Asia Pacific) Pte. Ltd. Singapore * Student from National University of Singapore for ITP Introduction: Quantitative determination of amino acids (AA) by liquid chromatography method is an important analysis in a variety of fields like in food, feed, biochemistry, clinical test and protein chemistry etc. The methods involve derivatization of AA with various reagents such as o-phthaldehyde (OPA), 9-fluoroenylmethyl chloroformate (FMOC-Cl) or phenylisothiocyanate (PITC) to convert them to derivatized AA before or after separation for high sensitivity detection by UV-Vis detection or fluorescence detection. Pre-column derivatization with PITC followed by reversed phase separation on C18 column is one of the choices, which has the advantages of using UV-VIS (254nm) detection, fast separation and high sensitivity. The analysis can be easily performed on any HPLC system with a UV-VIS detector. Derivatization of AA with PITC is carried out under alkaline conditions [1-2]. The exceed amount of PITC reagent and high concentration of triethylamine (TEA) are the main causes of poor reproducibility and short lifetime of column used. This application note shows the improvement of the pre-column PITC derivatization method for analysis of twenty naturally occurring amino acids (except cystine) using a UHPLC column on Nexera UHPLC system. . Experimental: Table 1: UHPLC system and conditions for analysis of PITC derivertized amino acids A mixed stock solution of 20 amino acids (see Table 2) was prepared from the single AA standards obtained in powder form from Sigma-Aldrich. The concentration of each AA in the mixed stock solution is 500 µmol/L, except L-Glutamic acid and LTyrosine (250 µmol/L). PITC (0.1 M) and triethylamine (1.0 M) solutions were prepared using acetonitrile as solvent. The details of PITC deritization is described in Figure 1. The system used is Nexera UHPLC with a UV-VIS detector (Shimadzu Corporation). The UHPLC column and conditions are shown in Table 1. Instrument Shimadzu Nexera UHPLC Column YMC-Triart C18, 150 x 2.0 mm ID 1.9 um Mobile Phase A : 10mM Potassium Phosphate (pH 7.0) B : Acetonitrile Gradient Elution (B Conc.) 5% (0 min) 10% (1 min) 23% (10-12 min) 95% (15-16.5 min) 5% (16.51-19 min) Flow Rate 0.40 mL/min Column Temp 35 ᴼC Detection UV 254 nm, Semi-micro flow cell Injection Vol 1.0 µL Results and Discussion: Figure 1: Procedure and conditions of PITC derivertization of mixed amino acids Amino acids are very polar molecules and could not be retained by reversed phase column. Deriverization with PITC converts amino acids into less polar products, which are suitable for reversed phase separation. Figure 2 shows the UHPLC chromatograms of twenty derivertized amino acids at lower and higher concentrations. Well separation of these derivertized amino acids were obtained under the UHPLC conditions. The great advantage of using UHPLC or fast LC [3,4] is the enhanced speed of separation due to the superior resolution of UHPLC column. Depending on the amino acid composition, the fast LC separation could be completed within 4 minutes [3] for analyzing 17 amino acids and 10 minutes for 23 amino acids [4]. The retentions of some less polar amino acids are very closed, like Asn and Ser (3.987 min and 4.101 min), Gln and Gly (4.270 min and 4.369 min) etc. Well separation of these amino acids Application Notes AD-0044 Figure 2 Chromatograms of twenty amino acids derivatized with PITC derivatization method SHIMADZU (Asia Pacific) Pte. Ltd 79 Science Park Drive, #02-01 Cintech IV Singapore Science Park I, Singapore 118264 www.shimadzu.com.sg Tel: +65-6778 6280 Fax: +65-6778 2050 Copyright © 2011 SHIMADZU (Asia Pacific) Pte. Ltd. All rights reserved. No part of this document may be reproduced in any form or by any means without permission in writing from SHIMADZU (Asia Pacific) Pte. Ltd. Application Notes AD-0044 requires use of longer column and gradient program. The current UHPLC method is optimized to analyze the 20 naturally occurring amino acids except cystine (Cys). The method can be optimized further to shorten the analysis time if samples contain less number of polar amino acids. Calibration curves of the 20 deriverized amino acids were established using mixed standard series of 50, 100, 250 and 500 µmol/L (except Glu and Tyr which concentrations are half as stated). Excellent linearity was obtained for all amino acids (Table 2). A few calibration curves are shown as examples in Figure 3. Area 100000 Repeatability of the method including derivatization and UHPLC analysis was evaluated by preparing in parallel four samples of same concentration. The results for 250 and 500 µmol/L (Table 2) show that the RSD for RT is less than 0.6% and that of peak area is less than 1.6% except Try (2.12%). Asn 75000 150000 50000 100000 25000 50000 0 0 100 0 250 Conc. Area 125000 Ser Tyr 100000 150000 75000 100000 50000 50000 0 0 200Conc. Area 200000 The sensitivity of the method is considerably good as high S/N ratios were obtained for 50 µmol/L mixed standard sample with 1µL injection. Area 200000 Glu 25000 0 250 Conc. 0 0 100 200Conc. Figure 3: Selected calibration curves of PITC derivertized amino acids by UHPLC method. Glu and Tyr: 25~250 µmol/L; Asn and Ser: 50~500 µmol/L. Injection Vol: 1 µL Finally, the sample preparation procedure as described in Figure 1 is proven to be less harmful to the UHPLC column used. Under the conditions of this work, the YMC column has not faced any separation problem after 300 runs of the PITC-derivatized amino acid samples. Table 2: Evaluation results of 20 amino acids analysis by pre-column PITC deriverization and UHPLC method Ami no Aci d Abb Na me RT (mi n) Ca l i bra tion (4 l evel s ) RSD % (250 umol /L, n=4) RSD % (500 umol /L, n=4) Ra nge r2 RT Area RT Area L-Aspartic acid Asp 1.913 50 - 500 0.9993 0.45 0.45 0.25 0.40 L-Glutamic Acid Glu 2.246 25 - 250 0.9996 0.58 0.75 0.23 0.27 L-Hydroxy-L-Proline Hydroxy-Pro 3.358 50 - 500 0.9999 0.40 0.26 0.13 0.20 L-Asparagine Asn 3.987 50 - 500 0.9988 0.29 0.97 0.15 1.54 L-Serine Ser 4.101 50 - 500 0.9998 0.27 0.69 0.13 0.37 L-Glutamine Gln 4.270 50 - 500 0.9998 0.27 0.85 0.15 0.68 Glycine Gly 4.369 50 - 500 0.9999 0.25 0.67 0.13 0.65 L-Histidine His 4.650 50 - 500 0.9992 0.25 0.37 0.15 0.18 L-Arginine Arg 5.164 50 - 500 0.9999 0.24 0.19 0.15 0.20 L-Threonine The 5.361 50 - 500 0.9999 0.23 0.11 0.14 0.21 L-Alanine Ala 5.649 50 - 500 0.9997 0.22 0.12 0.14 0.38 L-Proline Pro 5.847 50 - 500 0.9999 0.22 0.29 0.15 0.16 L-Tyrosine Tyr 9.135 25 - 250 0.9997 0.14 0.19 0.12 0.31 L-Valine Val 9.566 50 - 500 0.9994 0.14 0.06 0.12 0.41 L-Methionine Met 10.467 50 - 500 0.9992 0.11 0.08 0.10 0.29 L-Isoleucine lle 12.355 50 - 500 0.9996 0.12 0.07 0.12 0.35 L-Leucine Leu 12.602 50 - 500 0.9997 0.12 0.19 0.12 0.32 L-Phenylalanine Phe 14.331 50 - 500 0.9999 0.05 0.25 0.03 1.42 L-Tryptophan Trp 14.482 50 - 500 0.9999 0.04 2.13 0.02 0.98 L-Lysine Lys 14.636 50 - 500 0.9999 0.04 0.40 0.02 1.08 SHIMADZU (Asia Pacific) Pte. Ltd 79 Science Park Drive, #02-01 Cintech IV Singapore Science Park I, Singapore 118264 www.shimadzu.com.sg Tel: +65-6778 6280 Fax: +65-6778 2050 Copyright © 2011 SHIMADZU (Asia Pacific) Pte. Ltd. All rights reserved. No part of this document may be reproduced in any form or by any means without permission in writing from SHIMADZU (Asia Pacific) Pte. Ltd. Application Notes AD-0044 Conclusions: This work was to set up and evaluate the method of precolumn derivatization with PITC of 20 naturally occurring amino acids except cystine following by analysis using a C18 UHPLC column (1.9 µm) and UV-VIS detection (254nm). The results show that the sample preparation, separation conditions and performance of the analysis method are reliable, sensitive and reproducible. The sample preparation procedure employed is proven to be less harmful to the UHPLC column. Under the conditions of this work, the YMC column used has not faced any separation problem after 300 runs of the PITC-derivatized amino acid samples. References: 1. 2. 3. 4. B. A. Bidlingmeyer, S. A. Cohen, T. L. Tarvin, J. Chromatorgr. 336 (1984) 93. D. R, Koop, E. T. Morgan, G. E. Tarr and M. J. Coon, J. Biol. Chem., 257, pp 8472-80 (1982). Shimadzu Corporation, Application Data Sheet, High Performance Liquid Chromatography, No. 7 (LAAN-A-LC-E075), 2007. Shimadzu Corporation, Application Data Sheet, High Performance Liquid Chromatography, No. 8 (LAAN-A-LC-E076), 2007. SHIMADZU (Asia Pacific) Pte. Ltd 79 Science Park Drive, #02-01 Cintech IV Singapore Science Park I, Singapore 118264 www.shimadzu.com.sg Tel: +65-6778 6280 Fax: +65-6778 2050 Copyright © 2011 SHIMADZU (Asia Pacific) Pte. Ltd. All rights reserved. No part of this document may be reproduced in any form or by any means without permission in writing from SHIMADZU (Asia Pacific) Pte. Ltd.