Case study: Agilent customer collaboration delivers robust LC-QQQ method to quantify vitamin B12 absorption
Sarita. D St. John’s Research Institute, Research Fellow
Siji Joseph Agilent Application Scientist
Vitamin B12, also known as cobalamin, is a water-soluble micronutrient that serves as a coenzyme for methionine synthase (MS) and for methylmalonyl-CoA mutase (MCM) . Deficiency of this vitamin is widespread in India, affecting all age groups . Scientists at St. John’s Research Institute have taken advantage of Agilent Triple Quadrupole LC/MS technology to measure vitamin B12 absorption.
Deficiencies of vitamin B12 may cause hematological and neurological complications, and inadequate functioning of the methionine cycle, resulting in high plasma homocysteine levels (a risk factor for cardiovascular disease) [3,4]. Another area of concern is the potential impact of vitamin B12 deficiency on epigenetic regulation during early conception and the related risk of subsequent adult chronic disease . Two areas of investigation are required: one relates to the development of sensitive methods to measure vitamin B12 absorption, and the second relates to sensitive biomarkers of vitamin B12 deficiency.
While various techniques exist to measure vitamin B12 absorption, St. John’s has adopted a 13C stable isotopic labeling method. The key to using this labeled vitamin is to measure its metabolites in plasma after it is fed, as an index of its absorption.
Figure 1. Researchers at St. John’s Research Institute use Agilent Triple Quadrupole LC/MS systems for vitamin B12 absorption studies.
For this study, St. John’s researchers employed Agilent GC/MS instruments to measure plasma methylmalonic acid (MMA) and homocysteine (Hcy) concentrations as indicators of low levels of vitamin B12 (Figure 1). Hcy levels can increase with low folate levels which complicates detection of vitamin B12 and necessitates identification of other biomarkers . One strategy is to monitor the accumulation of propionyl carnitine (C3), a marker of methylmalonic aciduria and propionic academia that occurs during vitamin B12 deficiency . However, the challenge is to measure these biomarkers from complex biological samples.
Agilent LC-QQQ for sensitive biomarker quantitation
St. John’s used microorganisms to synthesize the stable 13C labeled vitamin B12 and characterize it with an Agilent LC-QQQ. Having used this tracer in a rodent model, they are in the process of characterizing the concentration of the vitamin and its metabolites in plasma.
Figure 2. MS/MS spectra show the product ions of the molecular ions (M+H)+ of B12 and 13C-B12.
Engaged in a long-standing collaboration with Agilent, St. John’s leverages Agilent’s application support and systems to study these trace metabolites with minimal sample preparation. They use an Agilent 1290 Infinity LC system coupled with an Agilent 6460 Triple Quadrupole LC/MS equipped with Agilent Jet Stream technology to characterize the synthesized isotopically labeled vitamin B12 for chemical integrity and isotopic distribution. Product ion spectra of labeled and unlabeled vitamin B12 obtained from LC-QQQ experiments are shown in Figure 2. St. John’s has successfully developed the methods and actively establishing an accurate, reliable metabolic signature-based approach to detect vitamin B12 absorption.
Eighty percent of pregnant women had low levels of vitamin B12
St. John’s Research Lab, as part of St. John’s Medical Hospital, has focused on the problem of vitamin B12 deficiency during conception. Researchers have measured the plasma vitamin B12 levels in more than 200 pregnant women at each of the three trimesters (but most importantly in the first trimester) and observed that approximately 80 percent of the pregnant women had low levels (<150 pmol/L). It is important to evaluate additional biomarkers like propionylcarnitine (C3) and its ratios with acetylcarnitine (C3/C2) and palmitoylcarnitine (C3/C16). These ratios exhibit a negative correlation with maternal levels of vitamin B12 in the first trimester of pregnancy. 
They are currently setting up characteristic signatures of all of these metabolites to help us understand vitamin B12 deficiency so mothers can take prompt corrective measures. At an early stage of pregnancy, it is very important to have this metabolome-based approach established to predict deficiency and to understand the response to supplementation.
Future plans: additional studies with an even more sensitive QQQ
To complement their metabolomics approach, St. John’s Research Lab is procuring an Agilent 6495 Triple Quadrupole LC/MS system for further biomarker characterization. The goal is to develop a more sensitive, precise method to identify and quantify these biomarkers to better understand the vitamin B12 deficiency pattern.
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If your lab must accurately and precisely quantitate biomarkers with attogram to zeptomole limits of detection, watch this video about the Agilent 6495 Triple Quadrupole LC/MS system with iFunnel technology. Then contact your Agilent representative for all of the details.
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Luciana Hannibal, Vegard Lysne, Anne-Lise Bjørke-Monsen, Sidney Behringer, Sarah C. Grüner, Ute Spiekerkoette, Donald W. Jacobsen, Henk J. Blom. “Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency”. Front Mol Biosci. 2016; 3: 27. Published online 2016 Jun 27. doi: 10.3389/fmolb.2016.00027
Samuel T.M, Duggan C, Thomas T, Bosch R, Rajendran R, Virtanen S.M, Srinivasan K, Kurpad A.V, “Vitamin B12 Intake and Status in Early Pregnancy among Urban South Indian Women”, Ann Nutr. Metab.2013;62:113–122.