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Determination of folates in human plasma using hydrophilic interaction chromatography-tandem mass spectrometry

Determination of folates in human plasma using hydrophilic interaction chromatography-tandem mass spectrometry
Determination of folates in human plasma using hydrophilic interaction chromatography-tandem mass spectrometry
Folic acid is an essential nutrient, and folate deficiency is associated with a variety of disorders including neural tube defects (during pregnancy) and heart disease. A fast, sensitive, and robust HPLC?tandem mass spectrometry (LC?MS?MS) method was developed for the quantification of free folic acid, tetrahydrofolate, 5‘-methyltetrahydrofolate, and 5‘-formyltetrahydrofolate in human plasma. Sample preparation required only acetonitrile precipitation of proteins followed by filtration instead of solid-phase extraction or solvent?solvent extraction as in other methods. The rapid and streamlined sample handling procedure minimized degradation of the highly unstable folate species. Hydrophilic interaction chromatography was used for additional sample cleanup on-line, and baseline separation and detection of all four folate species was achieved in less than 30 min. The folate species were detected using negative ion electrospray-tandem mass spectrometry with multiple reaction monitoring of the diagnostic fragment ions of each deprotonated molecule. The predominately organic (hydrophobic) solvent system combined with the microbore flow rate (50 ?L/min) used for the chromatography resulted in enhanced electrospray signal response compared to reversed-phase HPLC using a wider bore column. The recovery of all folate species (from spiked plasma) was >97% over a concentration range from 300 pg/L to 12 mg/L with intraday precision (RSD, n = 5) of 3.7?6.5%. Stability studies were carried out for spiked samples in order to define storage and handling conditions. The folic acid limit of quantification (LOQ) in human plasma was 80 pmol/L ± 10%, and the limit of detection (LOD) was 37.5 pmol/L. The LOQ and LOD for tetrahydrofolate, 5‘-methyltetrahydrofolate, and 5‘-formyltetrahydrofolate were 1250, 400, and 360 pmol/L of plasma and 425, 165, and 140 pmol/L of plasma, respectively.
0003-2700
5358-5364
Garbis, Spiros D.
7067fd19-50c9-4d42-9611-f370289470bd
Melse-Boonstra, Alida
25597a22-8ec5-4d41-ba0c-d6330af7798a
West, Clive E.
19b714bc-3ce6-41eb-bbee-1377aa11a9f9
van Breeman, Richard B.
e91c3f2b-0c76-4a3e-82b5-53d2b643a4e3
Garbis, Spiros D.
7067fd19-50c9-4d42-9611-f370289470bd
Melse-Boonstra, Alida
25597a22-8ec5-4d41-ba0c-d6330af7798a
West, Clive E.
19b714bc-3ce6-41eb-bbee-1377aa11a9f9
van Breeman, Richard B.
e91c3f2b-0c76-4a3e-82b5-53d2b643a4e3

Garbis, Spiros D., Melse-Boonstra, Alida, West, Clive E. and van Breeman, Richard B. (2001) Determination of folates in human plasma using hydrophilic interaction chromatography-tandem mass spectrometry. Analytical Chemistry, 73 (22), 5358-5364. (doi:10.1021/ac010741y). (PMID:11816560)

Record type: Article

Abstract

Folic acid is an essential nutrient, and folate deficiency is associated with a variety of disorders including neural tube defects (during pregnancy) and heart disease. A fast, sensitive, and robust HPLC?tandem mass spectrometry (LC?MS?MS) method was developed for the quantification of free folic acid, tetrahydrofolate, 5‘-methyltetrahydrofolate, and 5‘-formyltetrahydrofolate in human plasma. Sample preparation required only acetonitrile precipitation of proteins followed by filtration instead of solid-phase extraction or solvent?solvent extraction as in other methods. The rapid and streamlined sample handling procedure minimized degradation of the highly unstable folate species. Hydrophilic interaction chromatography was used for additional sample cleanup on-line, and baseline separation and detection of all four folate species was achieved in less than 30 min. The folate species were detected using negative ion electrospray-tandem mass spectrometry with multiple reaction monitoring of the diagnostic fragment ions of each deprotonated molecule. The predominately organic (hydrophobic) solvent system combined with the microbore flow rate (50 ?L/min) used for the chromatography resulted in enhanced electrospray signal response compared to reversed-phase HPLC using a wider bore column. The recovery of all folate species (from spiked plasma) was >97% over a concentration range from 300 pg/L to 12 mg/L with intraday precision (RSD, n = 5) of 3.7?6.5%. Stability studies were carried out for spiked samples in order to define storage and handling conditions. The folic acid limit of quantification (LOQ) in human plasma was 80 pmol/L ± 10%, and the limit of detection (LOD) was 37.5 pmol/L. The LOQ and LOD for tetrahydrofolate, 5‘-methyltetrahydrofolate, and 5‘-formyltetrahydrofolate were 1250, 400, and 360 pmol/L of plasma and 425, 165, and 140 pmol/L of plasma, respectively.

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Published date: 15 November 2001
Organisations: Cancer Sciences

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Local EPrints ID: 395431
URI: https://eprints.soton.ac.uk/id/eprint/395431
ISSN: 0003-2700
PURE UUID: cb4144b4-4ef2-434e-a75d-c6f1b54f37e9

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Date deposited: 18 Jul 2016 12:52
Last modified: 17 Jul 2017 18:54

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Author: Alida Melse-Boonstra
Author: Clive E. West
Author: Richard B. van Breeman

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