A novel automated multi-cycle magnetic solid-phase extraction coupled to LC-MS/MS to study the disorders of six functional B vitamins in patients with gastroenterology and hyperhomocysteinemia.

B vitamins are essential for human life and their disorders can cause a variety of diseases. Solid-phase extraction (SPE) coupled to LC-MS/MS is a preferred technique for determining multiple B vitamins, however, their complexity in real biological matrices makes it hard to achieve satisfactory recovery and accuracy when simultaneous detection. In this study, a novel automated multi-cycle magnetic SPE (MSPE) coupled to the LC-MS/MS method was established using a mixed-mode anion exchange magnetic adsorbent for the simultaneous extraction of six functional B vitamins, including methylmalonic acid, riboflavin, pantothenic acid, 4-pyridoxic acid, folic acid, and 5-methyltetrahydrofolate. After three consecutive MSPE cycles, the recoveries of all analytes were between 51.5% and 89.6%. The method exhibited excellent sensitivity and linearity, with a dynamic range of 200-fold (R > 0.99 for all analytes), exceptional accuracy (ranging between 95.4% and 105.6%) and precision (with RSDs ≤ 6.2%) without significant matrix effects or interferences. Compared to manual SPE method, the automated multi-cycle MSPE method has better feasibility and greater vitamin coverage. It shows a high correlation with the manual method for the detection of 5-methyltetrahydrofolate and folate (R > 0.99). A study of patients from the gastroenterology department showed that those undergoing surgery and those with malignancies may be at risk of folate deficiency. In addition, patients with hyperhomocystinemia had higher levels of methylmalonic acid and lower levels of 5-methyltetrahydrofolate, which correlated with homocysteine levels (R = 0.404 and -0.311, respectively) and showed dose-response relationships. This method is highly automated and cost-effective, with minimal systematic error, making it suitable for the analysis of clinical samples.

Evaluation of the predictive values of elevated serum L-homoarginine and dimethylarginines in preeclampsia.

L-homoarginine (hARG) is involved in nitric oxide biosynthesis, but its role and concentration in preeclampsia (PE) have not been fully revealed. The purpose of this study was to develop and validate a feasible clinical assay to quantify serum hARG, arginine (ARG), asymmetric (ADMA) and symmetric dimethylarginines (SDMA) levels by LC-MS/MS and investigate their differences at different stages of pregnancy with or without preeclampsia. Serum samples were collected from 84 pregnant women without complications (controls), 84 with mild preeclampsia (MPE), and 81 with severe preeclampsia (SPE) at various gestation stages (before the 20th week, during the 20th-28th week or after the 28th week of gestation). No significant difference in ARG levels was observed between PE and controls at any stage (P > 0.05). The serum hARG levels and hARG/ADMA ratios of MPE before the 20th week were higher than those of controls (P < 0.001). ADMA levels of MPE were higher than those of controls during the 20th-28th week (P < 0.01). SDMA levels of SPE were higher than those of MPE (P < 0.01) and controls (P < 0.05) after the 28th week. Elevated serum hARG before the 20th week was identified as an independent predictor for PE (OR = 1.478, 95% CI 1.120-1.950). ROC curve analysis showed serum hARG before the 20th week had a good potential to predict MPE (AUC = 0.875, 95% CI 0.759-0.948). In conclusion, our study indicated that elevated serum hARG and dimethylarginine levels detected by LC-MS/MS might serve as potential biomarkers for the early prediction of PE.

Application of a micro plasma collection card for the detection of homocysteine by liquid chromatography with tandem mass spectrometry.

Generally, it is recommended that blood samples for homocysteine detection should be centrifuged immediately to separate plasma in order to avoid continuous synthesis by blood cells. The use of a micro plasma collection card may improve sample stability and result accuracy by offering automatic and instant plasma separation. We compare a micro plasma collection method with routine wet plasma to explore applications of the dried plasma spots for homocysteine determination by using liquid chromatography with tandem mass spectrometry. The method was validated for both dried plasma spots and wet plasma. The assay was linear from 0.5-45 µmol/L with good precisions and accuracies. The extraction recovery and matrix effect for dried plasma spots were >97% and 0.98 after internal standard normalization, respectively. It was reproducible for retaining homocysteine in dried plasma spots and kept stable for 30 days. The plasma conversion factor was 7.77 ± 0.7% by calculating the ratio of homocysteine concentration between dried plasma spots and wet plasma (n = 165). Neither hematocrit nor homocysteine concentration affected the plasma conversion factor as long as the hematocrit was within the normal range. The results support the clinical usefulness of the dried plasma spots as a convenient and stable biological matrix for testing homocysteine.