Acta NaturaeActa NaturaeActa Naturae2075-8251Acta Naturae Ltd2770310.32607/actanaturae.27703Research ArticlesЭкспериментальные статьиResearch ArticleChiral chromatographic analysis of amino acids with pre-column derivatization by o-phthalaldehyde: improving the determination of enantiomers using ion-pair reagentsХиральный хроматографический анализ аминокислот с предколоночной модификацией о-фталевым альдегидом: улучшение определения энантиомеров с помощью ион-парных реагентовPaninNikolay V.ПанинНиколай Владимирович
Russian Federation

Belozersky Institute of Physicochemical Biology

НИИ физико-химической биологии им. А.Н. Белозерского

panin@belozersky.msu.ruPirogovIvan V.ПироговИван Викторович
Russian Federation

Faculty of Chemistry

химический факультет

ikaesovich@mail.ruGurandaDorel T.ГурандаДорел Феодорович
Russian Federation

Belozersky Institute of Physicochemical Biology

НИИ физико-химической биологии им. А.Н. Белозерского

dorel@belozersky.msu.ruŠvedasVytas K.ШвядасВитаутас Каятоно
Russian Federation

Belozersky Institute of Physicochemical Biology; Faculty of Bioengineering and Bioinformatics

НИИ физико-химической биологии им. А.Н. Белозерского; факультет биоинженерии и биоинформатики

vytas@belozersky.msu.ruLomonosov Moscow State UniversityМосковский государственный университет им. М.В. Ломоносова041220251741031092005202505082025Copyright ©; 2025, Panin N.V., Pirogov I.V., Guranda D.T., Švedas V.K.Copyright ©; 2025, Панин Н.В., Пирогов И.В., Гуранда Д.Ф., Швядас В.К.2025Panin N.V., Pirogov I.V., Guranda D.T., Švedas V.K.Панин Н.В., Пирогов И.В., Гуранда Д.Ф., Швядас В.К.https://creativecommons.org/licenses/by/4.0https://actanaturae.ru/2075-8251/article/view/27703

The development of effective and accessible methods for the chiral analysis of amino acids is an important scientific and practical necessity. One of the most common and convenient techniques is the chromatographic determination of individual enantiomers of amino acids with preliminary conversion of enantiomers into diastereomers, which can then be separated on conventional achiral columns. We have shown that by adding ion-pair reagents to the eluent and varying their structure, one can regulate the efficiency of a chiral amino acid analysis based on the chromatographic determination and resolution of the diastereomeric isoindoles obtained upon pre-column derivatization of amino acids by o-phthalaldehyde in the presence of N-acetyl-L-cysteine. The use of ion-pair reagents allows one to achieve a better resolution of diastereomeric isoindole peaks, while the analysis time can be reduced by increasing the ionic strength. Hence, adding ion-pair reagents and optimizing the mobile phase composition are useful approaches in the engineering of an amino acid chiral analysis, along with the synthesis of new chiral SH compounds and the choice of stationary phases.

Разработка эффективных и доступных методов хирального анализа аминокислот является важной научной и практической задачей. Одной из наиболее распространенных и удобных методик является хроматографическое определение отдельных энантиомеров аминокислот с предварительным превращением энантиомеров в диастереомеры, которые затем можно разделить на обычных ахиральных колонках. Нами показано, что добавление ион-парных реагентов в состав элюента и варьирование их структуры позволяют регулировать эффективность хирального анализа аминокислот, основанного на хроматографическом определении и разделении диастереомерных изоиндолов, полученных при предколоночной модификации аминокислот под действием о-фталевого альдегида в присутствии N-ацетил-L-цистеина. Использование ион-парных реагентов позволяет добиться лучшего разделения пиков диастереомерных изоиндолов, при этом время анализа можно существенно сократить за счет увеличения ионной силы. Таким образом, добавление ион-парных реагентов и оптимизация состава подвижной фазы являются важным подходом в инженерии хирального анализа аминокислот наряду с синтезом новых хиральных SH-соединений и подбором стационарных фаз.

Chiral analysis of amino acidso-phthalaldehydeN-acetyl-L-cysteinepre-column modificationHPLC conditionsion-pair reagentsseparation of diastereomeric isoindolesхиральный анализ аминокислото-фталевый альдегидN-ацетил-L-цистеинпредколоночная модификацияусловия ВЭЖХион-парные реагентыразделение диастереомерных изоиндоловПравительство РФGovernment of RF119042590056-2Knol MGE, Wulfmeyer VC, Müller RU, Rinschen ММ. Amino acid metabolism in kidney health and disease. Nat Rev Nephrol. 2024;20(12):771–788. doi: 10.1038/s41581-024-00872-8Knol MGE, Wulfmeyer VC, Müller RU, Rinschen ММ. Amino acid metabolism in kidney health and disease. Nature Reviews Nephrology. 2024;20:771–788. https://doi.org/10.1038/s41581-024-00872- 8.Chen J, Cui L, Lu S, Xu S. Amino acid metabolism in tumor biology and therapy. Cell Death Dis. 2024;15(1):42. doi: 10.1038/s41419-024-06435-wChen J, Cui L, Lu S, Xu S. Amino acid metabolism in tumor biology and therapy. Cell Death and Disease. 2024;15:42. https://doi.org/10.1038/s41419-024-06435-w.Gałęzowska G, Ratajczyk J, Wolska L. Determination of amino acids in human biological fluids by high-performance liquid chromatography: critical review. Amino Acids. 2021;53(7):993–1009. doi: 10.1007/s00726-021-03002-xGałęzowska G, Ratajczyk J, Wolska L. Determination of amino acids in human biological fluids by high-performance liquid chromatography: Critical review. Amino Acids. 2021;53:993–1009. https://doi.org/10.1007/s00726-021-03002-x.Ksenofontov AL, Boyko AI, Mkrtchyan GV, et al. Analysis of free amino acids in mammalian brain extracts. Biochemistry (Mosc). 2017;82(10):1183–1192. doi: 10.1134/S000629791710011XKsenofontov AL, Boyko AI, Mkrtchyan GV, Tashlitsky VN, Timofeeva AV, Graf AV, Bunik VI, Baratova LA. Analysis of free amino acids in mammalian brain extracts. Biochemistry Moscow. 2017;82(10):1183–1192. https://doi.org/10.1134/S000629791710011X.Pereira V, Pontes M, Câmara JS, Marques JC. Simultaneous analysis of free amino acids and biogenic amines in honey and wine samples using in loop orthophthalaldehyde derivatization procedure. J Chromatogr A. 2008;1189(1–2):435–443. doi: 10.1016/j.chroma.2007.12.014Pereira V, Pontes M, Câmara JS, Marques JC. Simultaneous analysis of free amino acids and biogenic amines in honey and wine samples using in loop orthophthalaldehyde derivatization procedure. J. Chromatogr A. 2008;1189(1–2):435–443. https://doi.org/10.1016/j.chroma.2007.12.014.Cuchiaro H, Laurens LML. Total Protein Analysis in Algae via Bulk Amino Acid Detection: Optimization of Amino Acid Derivatization after Hydrolysis with O-Phthalaldehyde 3-Mercaptopropionic Acid (OPA-3MPA). J Agric Food Chem. 2019;67(19):5672–5679. doi: 10.1021/acs.jafc.9b00884Cuchiaro H, Laurens LML. Total Protein Analysis in Algae via Bulk Amino Acid Detection: Optimization of Amino Acid Derivatization after Hydrolysis with O-Phthalaldehyde 3-Mercaptopropionic Acid (OPA-3MPA). J. Agric. Food Chem. 2019;67(19):5672–5679. https://doi.org/10.1021/acs.jafc.9b00884.Hall AE, Moraru CI. Comparative effects of high pressure processing and heat treatment on in vitro digestibility of pea protein and starch. NPJ Sci Food. 2022;6(1):2. doi: 10.1038/s41538-021-00116-0Hall AE, Moraru CI. Comparative effects of high pressure processing and heat treatment on in vitro digestibility of pea protein and starch. Science of Food. 2022;6(1):1–12. https://doi.org/10.1038/s41538-021-00116-0.Tanwar S, Bhushan R. Enantioresolution of Amino Acids: A Decade’s Perspective, Prospects and Challenges. Chromatographia. 2015;78:1113–1134. doi: 10.1007/s10337-015-2933-8Tanwar S, Bhushan R. Enantioresolution of Amino Acids: A Decade’s Perspective, Prospects and Challenges. Chromatographia. 2015;78:1113–1134. https://doi.org/10.1007/s10337-015-2933-8.Ilisz I, Péter A, Lindner W. State-of-the-art enantioseparations of natural and unnatural amino acids by high-performance liquid chromatography. Trends Anal Chem. 2016;81:11–22. doi: 10.1016/j.trac.2016.01.016Ilisz I, Péter A, Lindner W. State-of-the-art enantioseparations of natural and unnatural amino acids by high-performance liquid chromatography. Trends Anal. Chem. 2016;81:11–22. https://doi.org/10.1016/j.trac.2016.01.016.Grishin DV, Zhdanov DD, Pokrovskaya MV, Sokolov NN. D-amino acids in nature, agriculture and biomedicine. All Life. 2020;13(1):11–22. doi: 10.1080/21553769.2019.1622596Grishin DV, Zhdanov DD, Pokrovskaya MV, Sokolov NN. D-amino acids in nature, agriculture and biomedicine. All Life. 2019;13(1):11–22. https://doi.org/10.1080/21553769.2019.1622596.Uifălean A, Iacobescu M, Salantă LC, Heghes SC, Moldovan RC, Iuga C-A. Biological and Analytical Perspectives on D-Amino Acids in Cancer Diagnosis and Therapy. Pharmaceuticals(Basel). 2025;18(5):705. doi: 10.3390/ph18050705Uifălean A, Iacobescu M, Salantă LC, Heghes SC, Moldovan R-C, Iuga C-A. Biological and Analytical Perspectives on D-Amino Acids in Cancer Diagnosis and Therapy. Pharmaceuticals. 2025;18:705. https://doi.org/10.3390/ph18050705.Pollegioni L, Kustrimovic N, Piubelli L, Rosini E, Rabattoni V, Sacchi S. d-amino acids: new functional insights. FEBS J. 2025;292(17):4395–4417. doi: 10.1111/febs.70083Pollegioni L, Kustrimovic N, Piubelli L, Rosini E, Rabattoni V, Sacchi S. d-amino acids: new functional insights. The FEBS Journal. 2025. doi: 10.1111/febs.70083.Nemoto A, Horie M, Imai EI, Honda H, Hatori K, Matsuno K. Enantiomeric Excess of Amino Acids in Hydrothermal Environments. Orig Life Evol Biosph. 2005;35(2):167–174. doi: 10.1007/s11084-005-0638-yNemoto A, Horie M, Imai EI, Honda H, Hatori K, Matsuno K. Enantiomeric Excess of Amino Acids in Hydrothermal Environments. Orig. Life Evol. Biosph. 2005;35:167–174. https://doi.org/10.1007/s11084-005-0638-y.Jiang L, Dziedzic P, Spacil Z, et al. Abiotic synthesis of amino acids and self-crystallization under prebiotic conditions. Sci Rep. 2014;4:6769. doi: 10.1038/srep06769Jiang L, Dziedzic P, Spacil Z, Zhao GL, Nilsson L, Ilag LL, Córdova A. Abiotic synthesis of amino acids and self-crystallization under prebiotic conditions. Scientific Reports. 2014;4:1–6. https://doi.org/10.1038/srep06769.Kimura T, Hamase K, Miyoshi Y, et al. Chiral amino acid metabolomics for novel biomarker screening in the prognosis of chronic kidney disease. Sci Rep. 2016;6:26137. doi: 10.1038/srep26137Kimura T, Hamase K, Miyoshi Y, Yamamoto R, Yasuda K, Mita M, Rakugi H, Hayashi T, Isaka Y. Chiral amino acid metabolomics for novel biomarker screening in the prognosis of chronic kidney disease. Scientific Reports. 2016;6:26137. https://doi.org/10.1038/srep26137.Chang X, Zhang Y, Chen X, et al. Gut microbiome and serum amino acid metabolome alterations in autism spectrum disorder. Sci Rep. 2024;14:4037. doi: 10.1038/s41598-024-54717-2Chang X, Zhang Y, Chen X, Li S, Mei H, Xiao H, Ma X, Liu Z, Li R. Gut microbiome and serum amino acid metabolome alterations in autism spectrum disorder. Scientific Reports. 2024;14:4037. https://doi.org/10.1038/s41598-024-54717-2.Lella C, Nestor L, De Bundel D, Vander Heyden Y, Van Eeckhaut A. Targeted Chiral Metabolomics of D-Amino Acids: Their Emerging Role as Potential Biomarkers in Neurological Diseases with a Focus on Their Liquid Chromatography–Mass Spectrometry Analysis upon Chiral Derivatization. Int J Mol Sci. 2024;25(22):12410. doi: 10.3390/ijms252212410Lella C, Nestor L, De Bundel D, Vander Heyden Y, Van Eeckhaut A. Targeted Chiral Metabolomics of D-Amino Acids: Their Emerging Role as Potential Biomarkers in Neurological Diseases with a Focus on Their Liquid Chromatography–Mass Spectrometry Analysis upon Chiral Derivatization. Int. J. Mol. Sci. 2024;25(22):12410. https://doi.org/10.3390/ijms252212410.Nimura N, Kinoshita T. o-Phthalaldehyde-N-acetyl-L-cysteine as a chiral derivatization reagent for liquid chromatographic optical resolution of amino acid enantiomers and its application to conventional amino acid analysis. J Chromatogr A. 1986;352:169–177. doi: 10.1016/S0021-9673(01)83377-XNimura N, Kinoshita T. o-Phthalaldehyde - N-acetyl-L-cysteine as a chiral derivatization reagent for liquid chromatographic optical resolution of amino acid enantiomers and its application to conventional amino acid analysis. J Chromatogr A. 1986;352:169–177. https://doi.org/10.1016/S0021-9673(01)83377-X.García Alvarez-Coque MC, Medina Hernández MJ, Villanueva Camañas RM, Mongay Fernández C. Studies on the formation and stability of isoindoles derived from amino acids, o-phthalaldehyde and N-acetyl-L-cysteine. Anal Biochem. 1989;180(1):172–176. doi: 10.1016/0003-2697(89)90107-3García Alvarez-Coque MC, Medina Hernández MJ, Villanueva Camañas RM, Mongay Fernández C. Studies on the formation and stability of isoindoles derived from amino acids, o-phthalaldehyde and N-acetyl-L-cysteine. Anal. Biochem. 1989;180(1):172–176. https://doi.org/10.1016/0003-2697(89)90107-3.Švedas VJ, Galaev IJ, Borisov IL, Berezin IV. The interaction of amino acids with o-phthaldialdehyde: a kinetic study and spectrophotometric assay of the reaction product. Anal Biochem. 1980;101(1):188–195. doi: 10.1016/0003-2697(80)90059-7Švedas VJK, Galaev IJ, Borisov IL, Berezin IV. The interaction of amino acids with o-phthaldialdehyde: a kinetic study and spectrophotometric assay of the reaction product, Anal. Biochem. 1980;101:188–195. http://dx.doi.org/10.1016/ 0003-2697(80)90059-7.Meyer MW, Meyer VR, Ramseyer S. The kinetics of diastereomeric amino acids with o-phthaldialdehyde. Chirality. 1991;3(6):471–475. doi: 10.1002/chir.530030611Meyer MW, Meyer VR, Ramseyer S. The kinetics of diastereomeric amino acids with o-phthaldialdehyde. Chirality. 1991;3(6):471–475. https://doi.org/10.1002/chir.530030611.Brückner H, Westhauser T, Godel H. Liquid chromatographic determination of d- and l-amino acids by derivatization with o-phthaldialdehyde and N-isobutyryl-l-cysteine applications with reference to the analysis of peptidic antibiotics, toxins, drugs and pharmaceutically used amino acids. J Chromatogr A. 1995;711(1):201–215. doi: 10.1016/0021-9673(95)00158-jBrückner H, Westhauser T, Godel H. Liquid chromatographic determination of d- and l-amino acids by derivatization with o-phthaldialdehyde and N-isobutyryl-L-cysteine applications with reference to the analysis of peptidic antibiotics, toxins, drugs and pharmaceutically used amino acids. J Chromatogr A. 1995;711(1):201–215. https://doi.org/10.1016/0021-9673(95)00158-J.Nimura N, Fujiwara T, Watanabe A, et al. A novel chiral thiol reagent for automated precolumn derivatization and high-performance liquid chromatographic enantioseparation of amino acids and its application to the aspartate racemase assay. Anal Biochem. 2003;315(2):262–269. doi: 10.1016/S0003-2697(02)00705-4Nimura N, Fujiwara T, Watanabe A, Sekine M, Furuchi T, Yohda M, Yamagishi A, Oshima T, Homma H. A novel chiral thiol reagent for automated precolumn derivatization and high-performance liquid chromatographic enantioseparation of amino acids and its application to the aspartate racemase assay, Anal. Biochem. 2003;315:262–269. https://doi.org/10.1016/S0003-2697(02)00705-4.Buck RH, Krummen K. High-performance liquid chromatographic determination of enantiomeric amino acids and amino alcohols after derivatization with o-phthaldialdehyde and various chiral mercaptans. J Chromatogr. 1987;387:255–265. doi: 10.1016/S0021-9673(01)94529-7Buck RH, Krummen K. High-performance liquid chromatographic determination of enantiomeric amino acids and amino alcohols after derivatization with ophthaldialdehyde and various chiral mercaptans. J Chromatogr A. 1987;387:255–265. http://dx.doi.org/10.1016/S0021-9673(01)94529-7.Guranda DT, Shapovalova IV, Švedas VK. A new N-acyl derivative of (S)-cysteine for quantitative determination of enantiomers of amino compounds by HPLC with a precolumn modification with o-phthalaldehyde. Bioorg Khim. 2004;30(5):451–457. doi: 10.1023/b:rubi.0000043781.72807.f5Шаповалова ИВ, Гуранда ДТ, Швядас ВК. Новое N-ацильное производное (S)-цистеина для количественного определения энантиомеров аминосоединений методом ВЭЖХ с предколоночной модификацией орто-фталевым альдегидом. Биоорганическая химия. 2004;30(5):451–457.Guranda DT, Kudryavtsev PA, Khimiuk AY, Švedas VK. Efficient enantiomeric analysis of primary amines and amino alcohols by high-performance liquid chromatography with precolumn derivatization using novel chiral SH-reagent N-(R)-mandelyl-(S)-cysteine. J Chromatogr A. 2005;1095(1–2):89–93. doi: 10.1016/j.chroma.2005.07.125Guranda DT, Kudryavtsev PA, Khimiuk AY, Švedas VK. Efficient enantiomeric analysis of primary amines and amino alcohols by high-performance liquid chromatography with precolumn derivatization using novel chiral SH-reagent N-(R)-mandelyl-(S)-cysteine. J Chromatogr A. 2005;1095(1–2):89–93. https://doi.org/10.1016/j.chroma.2005.07.125.Chernobrovkin MG, Shapovalova EN, Guranda DT, Kudryavtsev PA, Švedas VK, Shpigun OA. Chiral high-performance liquid chromatography analysis of α-amino acid mixtures using a novel SH reagent-N-R-mandelyl-l-cysteine and traditional enantiomeric thiols for precolumn derivatization. J Chromatogr A. 2007;1175(1):89–95. doi: 10.1016/j.chroma.2007.10.034Chernobrovkin MG, Shapovalova EN, Guranda DT, Kudryavtsev PA, Švedas VK, Shpigun OA. Chiral high-performance liquid chromatography analysis of α-amino acid mixtures using a novel SH reagent-N-R-mandelyl-L-cysteine and traditional enantiomeric thiols for precolumn derivatization. J Chromatogr A. 2007;1175(1):89–95. https://doi.org/10.1016/j.chroma.2007.10.034.Lkhagva A, Tai HC. Dimethylcysteine (DiCys)/o-Phthalaldehyde Derivatization for Chiral Metabolite Analyses: Cross-Comparison of Six Chiral Thiols. Molecules. 2021;26(24):7416. doi: 10.3390/molecules26247416Lkhagva A, Tai H-C. Dimethylcysteine (DiCys)/o-Phthalaldehyde Derivatization for Chiral Metabolite Analyses: Cross-Comparison of Six Chiral Thiols. Molecules. 2021;26(24):7416. https://doi.org/10.3390/molecules26247416.Naghashian-Haghig A, Hemmateenejad B, Shamsipur M. Determination of enantiomeric excess of some amino acids by second-order calibration of kinetic-fluorescence data. Anal Biochem. 2018;550:15–26. doi: 10.1016/j.ab.2018.04.004Naghashian-Haghig A, Hemmateenejad B, Shamsipur M. Determination of enantiomeric excess of some amino acids by second-order calibration of kinetic-fluorescence data. Anal. Biochem. 2018;550:15–26. https://doi.org/10.1016/j.ab.2018.04.004.Sychev KS. Practical Guide to Liquid Chromatography. Moscow: Tekhnosfera. 2010:192–197.Сычев КС. Практическое руководство по жидкостной хроматографии. Москва: Техносфера; 2010:192–197.Cecchi T. Ion Pairing Chromatography. Crit Rev Anal Chem. 2008;38(3):161−213. doi: 10.1080/10408340802038882Cecchi T. Ion Pairing Chromatography. Crit. Rev. Anal. Chem. 2008;38(3):161−213. https://doi.org/10.1080/10408340802038882.Sagi-Kiss V, Li Y, Carey MR, et al. Ion-Pairing Chromatography and Amine Derivatization Provide Complementary Approaches for the Targeted LC-MS Analysis of the Polar Metabolome. J Proteome Res. 2022;21(6):1428−1437. doi: 10.1021/acs.jproteome.2c00030Sagi-Kiss V, Li Y, Carey MR, Grover SJ, Siems K, Cirulli F, Berry A, Musillo C, Wilson ID, Want EJ, Bundy JG. Ion-Pairing Chromatography and Amine Derivatization Provide Complementary Approaches for the Targeted LC-MS Analysis of the Polar Metabolome. J. Proteome Res. 2022;21:1428−1437. https://doi.org/10.1021/acs.jproteome.2c00030.Saprykin LV, Saprykina LV. Some aspects of the practical application of dynamic modification in HPLC on silica gel sorbents. Sorption and Chromatographic Processes. 2006;6(2):284–301.Сапрыкин ЛВ, Сапрыкина ЛВ. Некоторые аспекты практического применения динамического модифицирования в ВЭЖХ на силикагелевых сорбентах. Сорбционные и хроматографические процессы. 2006;6(2):284–301.