Development and Validation of a Rapid High-Performance Liquid Chromatography⁻Tandem Mass Spectrometric Method for Determination of Folic Acid in Human Plasma.

Aref Zayed,Rana Bustami,Wafaa Alabsi,Tamam El-Elimat

doi:10.3390/ph11020052

Aref Zayed, Rana Bustami + Show 2 more

Open Access

https://doi.org/10.3390/ph11020052

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Journal: Pharmaceuticals	Publication Date: May 27, 2018
Citations: 19	License type: CC BY 4.0

Affiliation: Jordan University of Science and Technology

Abstract

There are health concerns associated with increased folic acid intake from fortified food and supplements. Existing analytical methods, however, which can be employed to carry out epidemiological and bioavailability studies for folic acid involve laborious sample preparation and/or lengthy chromatographic analysis. In this paper we describe a simple, rapid, and sensitive high-performance liquid chromatography–electrospray ionisation-tandem mass spectrometry (HPLC–ESI-MS/MS) method for determination of unmetabolised folic acid in human plasma using folic acid-d4 as an internal standard. The method required only a simple sample preparation step of protein precipitation and had a total run time of 3.5 min, which is the shortest run time reported to date for HPLC–MS/MS method employed for quantifying folic acid in plasma. The analytes were separated on a C18 column (3 µm; 50 × 3.00 mm) using an isocratic mobile phase consisting of ammonium acetate (1 mM)-acetic acid-acetonitrile (9.9:0.1:90, v/v/v). The method was fully validated in terms of accuracy, precision, linearity, selectivity, recovery, matrix effect, and stability. The short run time and the minimal sample preparation makes the method a valuable tool for performing high-throughput analyses. To demonstrate the applicability of the method in real conditions, it was applied successfully in a bioavailability study for the determination of unmetabolised folic acid levels in vivo in human plasma after oral administration of folic acid.

Highlights

Folate is a water-soluble B vitamin that is known as vitamin B9 or folic acid [1]
The synthetic folic acid vitamin, can appear in the circulation after passing unchanged from the intestine following ingesting a high dose of oral supplements or fortified food [6]
A number of high-performance liquid chromatographic (HPLC) methods were reported in the literature for quantification of folates in human plasma, such as HPLC–UV [36], HPLC–FLD [37], HPLC–ECD [38], HPLC–MS [39], and HPLC–MS/MS [40]

Summary

Introduction

Folate is a water-soluble B vitamin that is known as vitamin B9 or folic acid [1]. A number of high-performance liquid chromatographic (HPLC) methods were reported in the literature for quantification of folates in human plasma, such as HPLC–UV [36], HPLC–FLD [37], HPLC–ECD [38], HPLC–MS [39], and HPLC–MS/MS [40]. None of these methods were convenient for routine and high-throughput analysis due to several reasons, including laborious sample preparation, such as solid-phase extraction [41,42], and lengthy chromatographic methods, such as hydrophilic interaction chromatography [43] or gradient reversed-phase chromatography [40]. AAmmmmoonniiuumm aacceettaattee aanndd aacceettiicc aacciidd,, bbootthhooffAACCSSrreeaaggeennttggrraaddeess,, wweerree oobbtatainineeddfrofrmomMeMrcekrcKkGKaAGa(DAar(mDsatramdst,taGdetr,mGaenrym).aPnuyr)e. wPuatreer wwaasteprrewpaasredpruespinargetdheuSsainrtgortihues wSaartteorripuusrwifiactaetriopnusryifsitceamtio(nSasrytsotreimus,(SGaorteottriinugse, nG,oGetetrimngaenny,)G. ermany)

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Sample Preparation

Method Validation

Method Development

Optimisation of the Chromatographic Conditions

Findings

Conclusions