MSACL 2017 EU Abstract

Quantification of Urinary Organic Acids Using Liquid Chromatography- Tandem Mass Spectrometry

Gökçe Göksu Gürsu (Presenter)
JASEM Laboratory systems and solutions

Bio: -Graduated Yıldız Technical University, PhD degree, organic chemistry department, 2011 -Employee SEM Group company, R&D department, 2011-present -Working fields; chromatography, mass spectrometry, clinical chemistry, metabolomics and food safety analyses.

Authorship: Gökçe Göksu Gürsu, Murat Emrah Maviş, Yasemin Saraç
Jasem Laboratory Systems and Solutions, Ataşehir, İstanbul,Turkey

Short Abstract

Screening the profile of urinary organic acids can be powerful reflector for inborn errors of metabolism called organic acidurias (OAs) are characterized by defections of inherited enzymes related to amino acid, carbohydrate or lipid metabolic pathways which lead to accumulation abnormal amounts of organic acids in tissues and human urine. LC–MS/MS has arisen as a significant approach including ease of sample preparation for the quantitative analysis of metabolites from human body fluids. In our present study, we have developed OA quantification method using LC-MS/MS with excellent chromatographic separation of isomers and dilute & shoot sample preparation technique in urine.

Long Abstract


Human urine is a source of valuable bioinformation which comprises myriad compounds with the inclusion of polar metabolites such as organic acids. Thus, screening the profile of urinary organic acids can be powerful reflector for metabolic disorders called organic acidurias/acidemias (OAs). OAs are inborn errors of metabolism characterized by defections of inherited enzymes related to amino acid, carbohydrate or lipid metabolic pathways which lead to accumulation abnormal amounts of organic acids in tissues and human urine causing severe health problems [1,2]. Hence, there is a remarkable concern about accurate interpretation and precise measurement of abnormally high levels urinary organic acids for the early diagnosis of OAs [3,4,5,6]. Conventionally, GC-MS is preferred due to some advantages such as sensitivity, the existence of searchable mass spectra libraries and lower matrix effects of co-eluting components. On the other hand, there are considerable disadvantages of GC-MS technique such as labour intensive extraction step from the urine specimen and then taking time consuming derivatisation step to transform sufficiently volatile species prior to GC-MS analysis [7,8,9]. Another analysis technique over traditional GC-MS is liquid chromatography tandem mass spectrometry (LC-MS/MS). LC–MS/MS has arisen as a significant and versatile approach including ease of sample preparation for the quantitative analysis of metabolites from human body fluids [10].


The new approach utilized in this work is based on establishing a simple and efficient liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method with excellent chromatographic separation allowing the analysis of isomeric urinary organic acids and simplifying sample preparation procedure by omission of extraction and chemical derivatisation. Isotopically labeled internal standards are used for the quantification of organic acids. This study is intended to appraise the use of LC-MS/MS method in order to identify biomarker metabolites of OAs and compare its specificity and sensitivity with GC-MS for patients.


Following the minimal clean-up procedure (dilute and shoot), we divided the analytical method into two panels (see List 1&2) for the successful chromatographic separation and accurate quantification of 54 urinary organic acids including isomeric forms independently with 12 minutes analysis time. In order to assess the robustness of the method, we conducted analysis of urinary organic acids for 34 children patients with different types of organic acidurias detected by GC-MS before. The findings indicated that LC-MS/MS has detected the defects of the patient samples regularly tested by GC-MS analysis, therefore we obtained precise concentrations with the calibration curve.

List 1: Panel 1-2-Methyl citrate, 2-OH-Phenylacetic acid, 2-OH-Butyric acid, 2-Oxoadipic acid, 3-Methylglutaconic acid, 3-OH-2-Methylbutanoic acid, 3-OH-2-Methylbutanoic acid, 3-OH-3-Methylglutaric acid, 3-OH-Butyric acid, 3-OH-Isobutyrate, 3-Phenyllactic acid, 4-OH-Phenylacetic acid, Fumaric acid, Glycolic acid, Hexanoylglycine, Homogentisic acid, Malic acid, Malonic acid, N-(3-phenylpropionyl)glycine, N-Acetylaspartic acid, N-Acetyltyrosine, N-isovaleryl glycine, Oxoproline, 4-OH-Phenyl lactic acid, Propionylglycine, Sebacic acid, Suberic acid, Suberylglycine and Succinylacetone.

List 2: Panel 2-2-OH-Glutaric acid, 2-OH-3-Methylpentanoic acid, 2-OH-Isocaproic acid, 2-OH-Isovaleric acid, 3-OH-Glutaric acid, 3-OH-Propanoic acid, 3-Methylcrotonyl glycine, 3-Methylglutaric acid, 3-Methyl-2-oxovaleric acid, 3-OH-Isovaleric acid, 3-OH-pentanoic acid, 4-Methyl-2-oxovaleric acid, 4-OH-Phenylpyruvic acid, Adipic acid, 2-Ketoglutaric acid, Citric acid, Ethylmalonic acid, Glutaconic acid, Glutaric acid, Lactic acid, Methylmalonic acid, Orotic acid, Phenylpyruvic acid, Pyruvic acid, Succinic acid and Tiglylglycine.

Conclusions & Discussion

The mainstay of this study was to develop almost effortless sample preparation process and straightforward method for accurately quantifying of urinary organic acids with principles of LC-MS/MS. It is required for scientifically relevant calibration curve to separate isomers in chromatographic method. We have spent most effort during the development chromatographic method for separation of adipic acid & 3-methylglutaric acid isomers, lactic acid & 3-OH-propanoic acid isomers, 2-OH-isocaproic & 2-OH-3-methylpentanoic acid isomers, 2-OH-isovaleric & 3-OH-isovaleric & 3-OH-pentanoic acid isomers, respectively. The proposed method was successfully applied for chromatographic separation of crucial isomers and measurement of the concentrations the targeted organic acids. Indeed, LC–MS/MS with Jasem organic acid method has proved to be a facile tool in the identification and quantification of urinary organic acids instead of challenging GC-MS method.

References & Acknowledgements:

[1]-Kannan Vaidyanathan, M. P. Narayanan, D. M. Vasudevan, ”Organic Acidurias:An Updated Review”, Indian Journal of Clinical Biochemistry, 26(4):319–325, 2011.

[2]-Joanna Kaluzna-Czaplinska, “Current Applications of Gas Chromatography/Mass Spectrometry in the Study of Organic Acids in Urine”, Critical Reviews in Analytical Chemistry, 41:114–123, 2011.

[3]-Chrysoula Christou, Helen. G. Gika, Nikolaos Raikos, Georgios Theodoridis, ”GC-MS analysis of organic acids in human urine in clinical settings: A study of derivatization and other analytical parameters”, Journal of Chromatography B, 964, 195-201, 2014.

[4]-El-Mesallamy H, Gouda A, Fateen E, Elbaz A, “Detection of Urinary Organic Acids in high risk Egyptian Children by Electrospray Tandem Mass Spectrometry”, Journal of Applied Sciences Research, 9(3): 1909-1916, 2013.

[5]-Joanna Kału˙zna-Czapli´nska, Ewa˙Zurawicz, Wiktoria Struck, Michał Markuszewski, “Identification of organic acids as potential biomarkers in the urine of autistic children using gas chromatography/mass spectrometry”, Journal of Chromatography B, 966, 70-76, 2014.

[6]-Moacir Wajner, Daniella de Moura Coelho, Rafaela Ingrassia, Anderson Büker de Oliveira, Estela Natacha Brandt Busanello, Kimiyo Raymond, Ricardo Flores Pires, Carolina Fischinger Moura de Souza, Roberto Giugliani, Carmen Regla Vargas,” Selective screening for organic acidemias by urine organic acid GC–MS analysis in Brazil: Fifteen-year experience”, Clinica Chimica Acta 400,77–81, 2009.

[7]-Dina A. Ghoraba, Magdy M. Mohamed, Osama K. Zaki, “Screening of diseases associated with abnormal metabolites for evaluation of HPLC in organic aciduria profiling”, The Egyptian Journal of Medical Human Genetics, 15, 69–78, 2014.

[8]-Petr Husek, Zdenek Svagera, Dagmar Hanzlíková, Lucie Rimnácová, Helena Zahradnícková, Iva Opekarová, Petr Simek, “Profiling of urinary amino-carboxylic metabolites by in-situheptafluorobutyl chloroformate mediated sample preparation and gas chromatography–mass spectrometry”, Journal of Chromatography A 1443,211-232, 2016.

[9]-Katsuhiro Nakagawa, Shuichi Kawana, Yuki Hasegawa, Seiji Yamaguchi, “Simplified method for the chemical diagnosis of organic aciduria using GC/MS”, Journal of Chromatography B, 878, 942–948, 2010.

[10]-Susen Becker, Linda Kortz, Christin Helmschrodt, Joachim Thiery, Uta Ceglarek, “LC–MS-based metabolomics in the clinical laboratory”, Journal of Chromatography B, 883–884,68-75, 2012.

Financial Disclosure

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