MSACL 2019 EU Abstract

Simple and Rapid Tandem Mass Spectrometry Method for the Analysis of Methylmalonic Acid in Urine

Michel Boutin (1), Tristan Martineau (1), Audrey Perreault (2), Pierrette Gaudreau (3,4), Nancy Presse (2,5), Christiane Auray-Blais (1)
(1) Division of Medical Genetics, Department of Pediatrics, Centre de Recherche-CHUS, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada (2) Research Center of Aging, CIUSSS-de-l’Estrie-CHUS, Sherbrooke, QC, Canada (3) Centre de recherche du Centre Hospitalier de l’Université de Montréal, Montréal, QC, Canada (4) Département de médecine, Faculté de médecine, Université de Montréal, Montréal, QC, Canada (5) Department of Community Health Sciences, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada

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Michel Boutin (Presenter) Université de Sherbrooke >> POSTER (PDF)

Presenter Bio: Michel Boutin is a mass spectrometry specialist. He received his master’s and Ph.D. degrees from Montreal University for research projects related to airborne monitoring of contaminants in the workplace. He did a first postdoctorate in industrial hygiene at McGill University (Montreal, QC), a second postdoctorate at the Université Pierre et Marie Curie (Paris, France) on the analysis of proteins related to cancer by mass spectrometry (ESI and MALDI), and a third postdoctorate at the Institute for Research in Immunology and Cancer (Montreal, QC) in metabolomics. He worked for 2 years for a proteomic platform (Université Laval, QC). Since 2011, he is the technical director of the Water-CHUS Expertise Centre in Clinical Mass Spectrometry (Faculty of Medicine and Health Sciences, Centre de Recherche-CHUS, Sherbrooke, QC) and adjunct teacher at Université de Sherbrooke for 2 years.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction: Vitamin B12 deficiency can lead to potentially irreversible neurological symptoms such as memory deficits and gait ataxia. Up to 40% of older adults show metabolic abnormalities due to vitamin B12 deficiency. However, most adults have normal levels of serum B12 so that this condition often remains under-recognized. Metabolic B12 deficiency causes an elevation of serum methylmalonic acid (MMA), an expensive test rarely available in clinical settings. Urine MMA also increases in cases of B12 deficiency, but it is unclear whether it correlates with serum MMA and thus be reliable for diagnostic purposes. Also, various inborn errors of metabolism lead to increased MMA levels in urine and plasma.

Objectives: The objectives of this research project were: 1) To develop and validate a multiplex UPLC-MS/MS method for the analysis of MMA and creatinine in urine without derivatization; 2) To determine MMA/creatinine levels in urine from a group of 35 older adults (>70 yrs) at different stages of B12 deficiency; 3) To compare the urine results obtained with this method with a validated GC/MS method necessitating derivatization; 4) To perform correlation studies between urine and serum MMA levels from samples collected at the same time from the same patients; 5) To analyze the levels of MMA/creatinine in urine samples collected on filter paper from newborns having B12 deficiency.

Methods: Briefly, 30 µL of urine were mixed with 60 µL of water containing MMA-D3 and creatinine-D3 internal standards. A 2-minute reverse phase chromatographic method was developed/validated, allowing the separation of MMA from succinic acid, a major isomeric interference. The Acquity I-Class UPLC system (Waters Corp.) was used. The absolute quantitation of MMA and creatinine was achieved by tandem mass spectrometry (Xevo TQ-S Micro, Waters) using the multiple reaction monitoring mode. Calibration curves with ranges up to 500 µM for MMA and 30 µM for creatinine were prepared. For newborn urine filter paper specimens, a 5 cm disk was extracted with 3 mL of NH4OH 0.01 M and analyzed in a similar manner.

Results: The validation of the method showed intra- and interday variations < 15%. Our results show that UPLC-MS/MS urine MMA/creatinine levels from 35 older adults were similar to those obtained by the GC-MS MMA method. Moreover, the correlations between MMA urine results obtained by UPLC-MS/MS with the plasma GC-MS results from the same patients were significant (Spearman r = 0.59).

Conclusion: A rapid and efficient UPLC-MS/MS method for the analysis of urine MMA and creatinine was developed and validated, requiring only a dilution of the specimen with the internal standards in a 2-min chromatographic run. This preliminary study suggests that MMA/creatinine urinary levels are equivalent to MMA plasma levels to evaluate B12 deficiency in older adults. Another method advantage is that urine specimen collection is less invasive than blood collection.