Water-soluble vitamins including thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), biotin (vitamin B7), 5-methyltetrahydrofolic acid (vitamin B9), and ascorbic acid (vitamin C) are essential micronutrients functionally important for human health. Reliable and accurate quantification of the water-soluble vitamin profiles is of vital importance for evaluating their deficiency and related diseases. However, simultaneous determination of these water-soluble vitamins in complex biological fluids such as plasma is a challenge. Herein, an automated liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for quantifying water-soluble vitamins was developed using a robotic workstation. The optimized sample preparation was conducted through a two-step procedure, protein precipitation using trichloroacetic acid (TCA) and liquid–liquid extraction using methyl tert-butyl ether (MTBE). The entire sample preparation workflow was fully automated with a Hamilton robotic workstation. Eight water-soluble vitamins including B1, B2, B3, B5, B6, B7, B9, and C were simultaneously measured in 8 min by LC–MS/MS. The automated LC–MS/MS method was validated for measuring human plasma samples in clinical application. The water-soluble vitamins showed good linearity in their calibration ranges. The limits of detection (LOD) and quantification (LOQ) were 0.01–1.73 ng/mL and 0.13–7.30 ng/mL, respectively, for B-complex vitamins. For vitamin C, the LOD and LOQ were 36.2 ng/mL and 72.3 ng/mL, respectively. Satisfactory coefficients of variation (CVs) were obtained, with the intra-batch and inter-batch CVs of 0.9–5.3% and 2.5–14.8%, respectively. Recoveries were considered adequate in the range of 83.2–111.9%, 85.1–118.0%, and 76.5–112.2% for high, medium, and low concentration levels of the eight vitamins, respectively. Matrix effect factors were 80.3–116.0%, 73.2–101.7%, and 76.6–110.5% for high, medium, and low concentration levels, respectively. Robustness of the method in routine clinical practice was further demonstrated by quantifying clinical plasma samples in vitamin screening. The automated LC–MS/MS method for the simultaneous determination of water-soluble vitamins in plasma showed great promise for clinical application. Graphical abstract Eight water-soluble vitamins (B1, B2, B3, B5, B6, B7, B9, and C) in human plasma were automatically prepared by a Hamilton Microlab STAR workstation and simultaneously quantified via liquid chromatography-tandem mass spectrometry (LC–MS/MS) within 8 min.

Quantification of water-soluble vitamin profiles in human plasma via automated liquid chromatography-tandem mass spectrometry

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发表日期：

2025年11月20日

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Xiaoyi Yi,
Rongliang Liang,
Yiqi Yang,
Xiaojing Huang,
Yingsong Wu

摘要

Water-soluble vitamins including thiamine (vitamin B₁), riboflavin (vitamin B₂), niacin (vitamin B₃), pantothenic acid (vitamin B₅), pyridoxine (vitamin B₆), biotin (vitamin B₇), 5-methyltetrahydrofolic acid (vitamin B₉), and ascorbic acid (vitamin C) are essential micronutrients functionally important for human health. Reliable and accurate quantification of the water-soluble vitamin profiles is of vital importance for evaluating their deficiency and related diseases. However, simultaneous determination of these water-soluble vitamins in complex biological fluids such as plasma is a challenge. Herein, an automated liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for quantifying water-soluble vitamins was developed using a robotic workstation. The optimized sample preparation was conducted through a two-step procedure, protein precipitation using trichloroacetic acid (TCA) and liquid–liquid extraction using methyl tert-butyl ether (MTBE). The entire sample preparation workflow was fully automated with a Hamilton robotic workstation. Eight water-soluble vitamins including B₁, B₂, B₃, B₅, B₆, B₇, B₉, and C were simultaneously measured in 8 min by LC–MS/MS. The automated LC–MS/MS method was validated for measuring human plasma samples in clinical application. The water-soluble vitamins showed good linearity in their calibration ranges. The limits of detection (LOD) and quantification (LOQ) were 0.01–1.73 ng/mL and 0.13–7.30 ng/mL, respectively, for B-complex vitamins. For vitamin C, the LOD and LOQ were 36.2 ng/mL and 72.3 ng/mL, respectively. Satisfactory coefficients of variation (CVs) were obtained, with the intra-batch and inter-batch CVs of 0.9–5.3% and 2.5–14.8%, respectively. Recoveries were considered adequate in the range of 83.2–111.9%, 85.1–118.0%, and 76.5–112.2% for high, medium, and low concentration levels of the eight vitamins, respectively. Matrix effect factors were 80.3–116.0%, 73.2–101.7%, and 76.6–110.5% for high, medium, and low concentration levels, respectively. Robustness of the method in routine clinical practice was further demonstrated by quantifying clinical plasma samples in vitamin screening. The automated LC–MS/MS method for the simultaneous determination of water-soluble vitamins in plasma showed great promise for clinical application.

Graphical abstract

Eight water-soluble vitamins (B₁, B₂, B₃, B₅, B₆, B₇, B₉, and C) in human plasma were automatically prepared by a Hamilton Microlab STAR workstation and simultaneously quantified via liquid chromatography-tandem mass spectrometry (LC–MS/MS) within 8 min.