High-throughput analysis of total homocysteine and methylmalonic acid with the efficiency to separate succinic acid in serum and urine via liquid chromatography tandem mass spectrometry

Xiaoli Ma; Yutong Zou; Yueming Tang; Danchen Wang; Weiyan Zhou; Songlin Yu; Ling Qiu

doi:10.1016/j.jchromb.2022.123135

High-throughput analysis of total homocysteine and methylmalonic acid with the efficiency to separate succinic acid in serum and urine via liquid chromatography tandem mass spectrometry

J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Mar 15:1193:123135. doi: 10.1016/j.jchromb.2022.123135. Epub 2022 Feb 11.

Authors

Xiaoli Ma¹, Yutong Zou¹, Yueming Tang¹, Danchen Wang¹, Weiyan Zhou², Songlin Yu³, Ling Qiu⁴

Affiliations

¹ Department of Laboratory Medicine, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100730, China.
² National Center for Clinical Laboratories, Beijing Hospital, National Center for Gerontology, Beijing Engineering Research Center of Laboratory Medicine, Beijing, China.
³ Department of Laboratory Medicine, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100730, China. Electronic address: yusonglinpku@163.com.
⁴ Department of Laboratory Medicine, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100730, China; State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100730, China. Electronic address: lingqiubj@163.com.

PMID: 35176539
DOI: 10.1016/j.jchromb.2022.123135

Abstract

Vitamin B12 (VB12) deficiency may lead to hyperhomocysteinemia and methylmalonic acidemia development which are risk factors of cardiovascular disease and nervous system impairment, respectively. However, few analytical methods are available to simultaneously quantify total homocysteine (tHcy) and methylmalonic acid (MMA) due to complex analytical requirements, such as sensitivity at nanomolar concentration, separation performance for succinic acid (SA), an endogenous isomer of MMA, and retention properties for polar compounds. Therefore, we developed and validated a simple and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of tHcy and MMA with the efficient separation of SA in human serum and urine. The clinical performance of the assay was validated according to CLSI C62-A guidelines. The recovery for serum tHcy was 95.2-105.8%, urine tHcy was 98.1-111.5%, serum MMA was 94.6-99.4%, and urine MMA was 101.6-105.6%. In addition, the LC-MS/MS method was found to be reliable based on the value of inter-assay imprecision and total imprecision coefficient variation (CV), matrix effect, and carryover. Standards and samples were stable in -20 °C for at least 2 months. The limits of quantifications (LOQs) were 0.074 nmol/mL for tHcy and 0.040 nmol/mL for MMA, which are suitable for detecting tHcy and MMA concentrations in human serum and urine. The concentration of tHcy and MMA in samples collected from 148 subjects were measured using this method. The results suggested that the concentrations of serum tHcy and MMA considerably differed between VB12 sufficient and deficient groups. Serum tHcy and serum MMA concentrations were inversely correlated with VB12 status. Our method represents a rapid technique for estimating tHcy and MMA concentrations in serum and urine samples without the need for derivatization and may be used to assess VB12 status in clinical applications.

Keywords: Liquid chromatography-tandem mass spectrometry; Methylmalonic acid; Total homocysteine; vitamin B12 deficiency.