Simultaneous determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human serum by ultra performance liquid chromatography: An economical and validated method with bovine serum albumin

Research output: Contribution to journalArticle

Abstract

A simple and economical method has been developed for simultaneous determination of human serum 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) using Ultra Performance Liquid Chromatography (UPLC). Non-human matrix of 4% BSA was used to construct the calibration curve and in quality control samples’ preparation to avoid interference of the endogenous 25-hydroxyvitamin D (25OHD) present in the human serum. 25OHD2, 25OHD3 and dodecanophenone (internal standard, IS) were separated on a CORTECS solid-core particle column and monitored by photodiode array detector at wavelength of 265 nm within five min run time. The relationship between 25OHD concentration and peak area ratio (25OHD:IS) was linear over the range of 12.5 – 200 nM with mean correlation coefficients (r2) >0.998. The limit of detection (LOD) for 25OHD2 and 25OHD3 was 3.00 nM and 3.79 nM, while the lower limit of quantification (LLOQ) was 9.11 nM and 11.48 nM, respectively. High repeatability was obtained for both isomers with intra-day CV% <5.6% and <5.3% for inter-day assay. This method was further tested with a commercial lyophilized serum control with an accuracy of 92.87–108.31% and applied on 214 human serum samples. In summary, this validated method with BSA can be reliably applied for routine quantification of 25OHD in adults.

Original languageEnglish
Pages (from-to)60-66
Number of pages7
JournalClinica Chimica Acta
Volume485
DOIs
Publication statusPublished - 1 Oct 2018

Fingerprint

25-Hydroxyvitamin D 2
Calcifediol
Liquid chromatography
Bovine Serum Albumin
Liquid Chromatography
Serum
Photodiodes
Isomers
Quality control
Assays
Calibration
Quality Control
Detectors
Limit of Detection
Wavelength

Keywords

  • 25-Hydroxyvitamin D
  • bovine serum albumin (BSA)
  • serum
  • UPLC
  • validation

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

@article{f2aeb0c31a354295b5073f846a99563e,
title = "Simultaneous determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human serum by ultra performance liquid chromatography: An economical and validated method with bovine serum albumin",
abstract = "A simple and economical method has been developed for simultaneous determination of human serum 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) using Ultra Performance Liquid Chromatography (UPLC). Non-human matrix of 4{\%} BSA was used to construct the calibration curve and in quality control samples’ preparation to avoid interference of the endogenous 25-hydroxyvitamin D (25OHD) present in the human serum. 25OHD2, 25OHD3 and dodecanophenone (internal standard, IS) were separated on a CORTECS solid-core particle column and monitored by photodiode array detector at wavelength of 265 nm within five min run time. The relationship between 25OHD concentration and peak area ratio (25OHD:IS) was linear over the range of 12.5 – 200 nM with mean correlation coefficients (r2) >0.998. The limit of detection (LOD) for 25OHD2 and 25OHD3 was 3.00 nM and 3.79 nM, while the lower limit of quantification (LLOQ) was 9.11 nM and 11.48 nM, respectively. High repeatability was obtained for both isomers with intra-day CV{\%} <5.6{\%} and <5.3{\%} for inter-day assay. This method was further tested with a commercial lyophilized serum control with an accuracy of 92.87–108.31{\%} and applied on 214 human serum samples. In summary, this validated method with BSA can be reliably applied for routine quantification of 25OHD in adults.",
keywords = "25-Hydroxyvitamin D, bovine serum albumin (BSA), serum, UPLC, validation",
author = "Chin, {Siok Fong} and Junaida Osman and {A. Jamal}, {A. Rahman}",
year = "2018",
month = "10",
day = "1",
doi = "10.1016/j.cca.2018.06.024",
language = "English",
volume = "485",
pages = "60--66",
journal = "Clinica Chimica Acta",
issn = "0009-8981",
publisher = "Elsevier",

}

TY - JOUR

T1 - Simultaneous determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human serum by ultra performance liquid chromatography

T2 - An economical and validated method with bovine serum albumin

AU - Chin, Siok Fong

AU - Osman, Junaida

AU - A. Jamal, A. Rahman

PY - 2018/10/1

Y1 - 2018/10/1

N2 - A simple and economical method has been developed for simultaneous determination of human serum 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) using Ultra Performance Liquid Chromatography (UPLC). Non-human matrix of 4% BSA was used to construct the calibration curve and in quality control samples’ preparation to avoid interference of the endogenous 25-hydroxyvitamin D (25OHD) present in the human serum. 25OHD2, 25OHD3 and dodecanophenone (internal standard, IS) were separated on a CORTECS solid-core particle column and monitored by photodiode array detector at wavelength of 265 nm within five min run time. The relationship between 25OHD concentration and peak area ratio (25OHD:IS) was linear over the range of 12.5 – 200 nM with mean correlation coefficients (r2) >0.998. The limit of detection (LOD) for 25OHD2 and 25OHD3 was 3.00 nM and 3.79 nM, while the lower limit of quantification (LLOQ) was 9.11 nM and 11.48 nM, respectively. High repeatability was obtained for both isomers with intra-day CV% <5.6% and <5.3% for inter-day assay. This method was further tested with a commercial lyophilized serum control with an accuracy of 92.87–108.31% and applied on 214 human serum samples. In summary, this validated method with BSA can be reliably applied for routine quantification of 25OHD in adults.

AB - A simple and economical method has been developed for simultaneous determination of human serum 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) using Ultra Performance Liquid Chromatography (UPLC). Non-human matrix of 4% BSA was used to construct the calibration curve and in quality control samples’ preparation to avoid interference of the endogenous 25-hydroxyvitamin D (25OHD) present in the human serum. 25OHD2, 25OHD3 and dodecanophenone (internal standard, IS) were separated on a CORTECS solid-core particle column and monitored by photodiode array detector at wavelength of 265 nm within five min run time. The relationship between 25OHD concentration and peak area ratio (25OHD:IS) was linear over the range of 12.5 – 200 nM with mean correlation coefficients (r2) >0.998. The limit of detection (LOD) for 25OHD2 and 25OHD3 was 3.00 nM and 3.79 nM, while the lower limit of quantification (LLOQ) was 9.11 nM and 11.48 nM, respectively. High repeatability was obtained for both isomers with intra-day CV% <5.6% and <5.3% for inter-day assay. This method was further tested with a commercial lyophilized serum control with an accuracy of 92.87–108.31% and applied on 214 human serum samples. In summary, this validated method with BSA can be reliably applied for routine quantification of 25OHD in adults.

KW - 25-Hydroxyvitamin D

KW - bovine serum albumin (BSA)

KW - serum

KW - UPLC

KW - validation

UR - http://www.scopus.com/inward/record.url?scp=85048955449&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85048955449&partnerID=8YFLogxK

U2 - 10.1016/j.cca.2018.06.024

DO - 10.1016/j.cca.2018.06.024

M3 - Article

C2 - 29935177

AN - SCOPUS:85048955449

VL - 485

SP - 60

EP - 66

JO - Clinica Chimica Acta

JF - Clinica Chimica Acta

SN - 0009-8981

ER -