MSACL 2017 EU Abstract

Development and Validation of an LC-MS/MS Method for Measurement of Methylmalonic Acid and Homocysteine in Human Plasma

Forough Bahadory (Presenter)
SEALS, Department of Clinical Chemistry

Bio: I have completed a PhD in clinical pharmacology (UNSW 2011- Oct 2015), a post-graduate degree in medical biotechnology (Flinders university, Adelaide 2009-2010) and in laboratory medicine (2002-2004), a bachelor’s degree in laboratory medicine (Kerman, Iran 1998-2002). I have more than ten years work experience in various fields including biochemistry, haematology, blood bank, immunology, physiology and pharmacology in both research and diagnostic laboratories. I have been presenting my work at several national and international conferences, and won several awards. I have been learning and practicing LC-MS for the last one year. I have gained expertise for both HPLC and LC-MS techniques working with various instruments including Q TRAP 3200/4000/5500/6500. I have conducted a project to measure methylmalonic acid in plasma, and will be presenting my data at the MSACL conference.

Authorship: Forough Bahadory, Barbara Hiley, Ben Matthews, Rita (Andrea) Horvath
SEALS North, Department of Clinical Chemistry and Endocrinology, Prince of Wales Hospital, Sydney, Australia

Short Abstract

Methylmalonic acid (MMA) is considered to be the most specific marker for vitaminB12 deficiency and methylmalonic acidaemia. VitaminB12 or folic acid deficiency can lead to hyperhomocysteinaemia. Traditionally analysis of MMA and homocysteine has been performed separately, limited by interfering substances (succinate), and required extensive sample pre-treatment. Here we present a simple, specific and time efficient LC-MS/MS assay to measure homocysteine and MMA simultaneously in plasma, free from interferences. Using protein precipitation techniques followed by a selective chromatographic separation, we have developed a fast, reliable assay readily amenable to automation into high throughput laboratories.

Long Abstract


Methylmalonic acid (MMA) is considered to be the most specific marker for vitamin B12 deficiency, and is monitored in patients with methylmalonic acidaemia. Vitamin B12 or folic acid deficiency can lead to hyperhomocysteinaemia, megaloblastic anaemia and neuropathy. Several LC-MS methods have been developed to measure MMA. However, complicated extraction methods including liquid-liquid extraction and derivatization were required. The other major difficulty for measurement of MMA in biological fluids is the interference from its naturally occurring isomer, succinic acid. Here we present a simple, fast and reliable assay using liquid chromatography tandem mass spectrometry (LC-MS/MS) to measure both MMA and homocysteine in plasma.


We employed a simple protein precipitation technique using trichloroacetic acid (TCA), followed by a reduction step using tris 2-carboxyethyl phosphine (TCEP) to reduce potential di-sulphide interactions from the thiol group in homocysteine. The analytical steps involve a simple, fast (five minute) chromatographic method using a Ascentis RP-amide column (10cm x 3.0mm, 2.7µm) and gradient mobile phase solution of 3% to 90% v/v methanol with 0.1% formic acid at a flow rate of 0.5 ml/min. Analysis was performed on Sciex 6500 QTRAP mass spectrometer using polarity switching with the following MRMs for MMA 117-73 in negative mode and homocysteine 135.9-89.8 in positive mode.


Our results showed a significant separation between homocysteine (retention time 0.86 min) MMA (retention time 1.44 min) and succinate (retention time 1.2 min) to abrogate any isomeric interference with MMA quantitation. The calibrators were linear in a range of 0.025 to 10µmol/L. The inter-run and intra-run imprecisions were 5% and 10%, respectively.

Conclusions & Discussion

Here we have provided a fast, specific and sensitive assay readily amenable to a high throughput clinical pathology environment as a quick and cost effective tool for the diagnosis of Vitamin B12 deficiency.

References & Acknowledgements:

1- Schloss et al., 2015

2- Shetty et al., 2015

3- Kushnir et al., 2001

4- Schmedes & Brandslund, 2006

5- Ekaterina et al., 2015

6- Schroder et al., 2016

Financial Disclosure

Board Memberno

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above: