Simple, Fast, and Simultaneous Detection of Plasma Total Homocysteine, Methylmalonic Acid, Methionine, and 2-Methylcitric Acid Using Liquid Chromatography and Mass Spectrometry (LC/MS/MS)

Abstract

Cobalamin (Vitamin B12) plays an essential role both in the conversion of methylmalonyl-CoA to succinyl-CoA and in the synthesis of methionine (Met) from homocysteine (Hcy). Elevations of total homocysteine (tHcy), Met, methylmalonic acid (MMA), and 2-methylcitric acid (2MCA) are indicative of disorders in these related pathways, and can clinically present as methylmalonic acidemia, cobalamin defects or deficiency, propionic acidemia, homocystinuria, and hypermethioninemia. We have developed a fast, sensitive, and simple method for the simultaneous detection of plasma tHcy, MMA, Met, and 2MCA using liquid chromatography mass spectrometry (LC/MS/MS). All analytes were directly determined without the need of derivatization. Both positive and negative modes were used to achieve the best sensitivity and specificity. The two stereo isomers of 2MCA (2S, 3S) and (2R, 3S) were successfully separated and were designated as 2MCA1 and 2MCA2. The assays were linear up to a concentration of 800 μMol/l for tHcy, 2,000 μMol/l for Met, 80 μMol/l for MMA, 40 μMol/l for 2MCA1, and 40 μMol/l for 2MCA2 (80 μMol/l for total 2MCA), respectively. The recovery was between 84.42 % and 120.05 %. The intra-assay coefficient of variations (CVs) ranged from 2.1 % to 6.9 % (n = 20), and the inter-assay CVs ranged from 2.7 % to 11.6 % (n = 20). Reference intervals were established and verified (n = 125). A total of 15 patients with variable disorders in related pathway were successfully confirmed. The assay can be performed either in diagnostic laboratories or as second-tier, follow-up test in newborn screening laboratories.A fast, sensitive, and simple LC/MS/MS method was developed successfully for the simultaneous detection of plasma total homocysteine, methylmalonic acid, methionine, and 2-methylcitric acid for diagnosis of disorders in related pathways.

Fig. 1

Metabolic pathways involving Cobalamin (Cbl) for MMA, tHcy, and Met. The enzyme propionyl-CoA carboxylase converts propionyl-CoA into D-methylmalonyl-CoA, which is biotin dependent, and then is racemized into l-methylmalonyl-CoA and isomerized into succinyl-CoA, a Krebs cycle intermediate. Adenosylcobalamin (AdoCbl) is the cofactor of the methylmalonyl-CoA mutase (MUT) reaction; methylcobalamin (MeCbl) is the cofactor of the methionine synthase (MTR) reaction. Abbreviations used: Cbl cobalamin, OH-Cbl hydroxycobalamin, AdoCbl adenosylcobalamin, MeCbl methylcobalamin, MTR methionine synthase, MTRR methionine synthase reductase, CBS cystathionine β synthase, S-AdoMet S-adenosylmethionine, S-AdoHcy S-adenosylhomocysteine

Fig. 2

Passing and Bablok regression lines for tHcy (A) and Met (B) in the correlation study of the LC/MS/MS method with the HPLC method (n = 40)

Fig. 3

Multiple reaction monitoring chromatograms of (A) standard solution (STD); (B) a patient with propionic acidemia (PPA); (C) a normal individual (NL), and (C1) scan intensity of tHcy (blue) and internal standard D4-Hcy (red) in this normal individual, and (C2) scan intensity of MMA (blue) and internal standard, D3-MMA (red) in this normal individual; (D) a patient with cobalamin C defect (Cbl C), and (D1) scan intensity of tHcy (blue) and internal standard D4-Hcy (red) in this Cbl C defect patient, and (D2) scan intensity of MMA (blue) and internal standard D3-MMA (red) in this Cbl C defect patient. tHcy total homocysteine, D4-Hcy D4-homocysteine, MMA methylmalonic acid, D3-MMA D3-methylmalonic acid, 2MCA1 2-methylcitric acid 1, 2MCA2 2-methylcitric acid 2, SA succinic acid. The yellow line in the middle represents the switch from positive ESI mode to negative ESI mode. The run time is 6 min

Fig. 3

Multiple reaction monitoring chromatograms of (A) standard solution (STD); (B) a patient with propionic acidemia (PPA); (C) a normal individual (NL), and (C1) scan intensity of tHcy (blue) and internal standard D4-Hcy (red) in this normal individual, and (C2) scan intensity of MMA (blue) and internal standard, D3-MMA (red) in this normal individual; (D) a patient with cobalamin C defect (Cbl C), and (D1) scan intensity of tHcy (blue) and internal standard D4-Hcy (red) in this Cbl C defect patient, and (D2) scan intensity of MMA (blue) and internal standard D3-MMA (red) in this Cbl C defect patient. tHcy total homocysteine, D4-Hcy D4-homocysteine, MMA methylmalonic acid, D3-MMA D3-methylmalonic acid, 2MCA1 2-methylcitric acid 1, 2MCA2 2-methylcitric acid 2, SA succinic acid. The yellow line in the middle represents the switch from positive ESI mode to negative ESI mode. The run time is 6 min

Fig. 3

Multiple reaction monitoring chromatograms of (A) standard solution (STD); (B) a patient with propionic acidemia (PPA); (C) a normal individual (NL), and (C1) scan intensity of tHcy (blue) and internal standard D4-Hcy (red) in this normal individual, and (C2) scan intensity of MMA (blue) and internal standard, D3-MMA (red) in this normal individual; (D) a patient with cobalamin C defect (Cbl C), and (D1) scan intensity of tHcy (blue) and internal standard D4-Hcy (red) in this Cbl C defect patient, and (D2) scan intensity of MMA (blue) and internal standard D3-MMA (red) in this Cbl C defect patient. tHcy total homocysteine, D4-Hcy D4-homocysteine, MMA methylmalonic acid, D3-MMA D3-methylmalonic acid, 2MCA1 2-methylcitric acid 1, 2MCA2 2-methylcitric acid 2, SA succinic acid. The yellow line in the middle represents the switch from positive ESI mode to negative ESI mode. The run time is 6 min