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Annals of Clinical & Laboratory Science, vol. 46, no. 6, 2016 645

A Rapid and Simple Liquid-Chromatography-Tandem Mass

Spectrometry Method for Measuring 25-Hydroxyvitamin D2
and 25-Hydroxyvitamin D3 in Human Serum: Comparison
with Two Automated Immunoassays
Sanghoo Lee1,2, Joo-Hoon Kim1, Seol-A Kim1, Yeoun-Soo Sun3, Anna Lee3, Seo-Jin Park3, Yun-Tae Kim1,
Kyoung-Ryul Lee1,2, and Young-Jin Kim1
1Department of Bioanalysis, Seoul Medical Science Institute & Seoul Clinical Laboratories, Yongin, 2Companion
Biomarker Center, SCL Healthcare Inc., Yongin, and 3Department of Diagnostic Immunology, Seoul Medical Science
Institute & Seoul Clinical Laboratories, Yongin, Korea

Abstract. Background. Total 25-hydroxyvitamin D (25(OH)D) is well-known to be a reliable biomarker

of human vitamin D status, with the recognition of widespread vitamin D insufficiency in general popula-
tions. The aims of this study are to validate a fast and simple liquid chromatography-tandem mass spec-
trometry (LC-MS/MS) method for quantifying 25(OH)D2 and 25(OH)D3 in serum and to compare two
automated immunoassays with the LC-MS/MS method. Methods. Samples were prepared by protein pre-
cipitation with ethanol including 25(OH)D3-d6, followed by a liquid-liquid extraction with hexane. The
analytes were separated within a total run time of 3 min. Accuracy was evaluated with standard reference
materials (SRM) 972a. Using 150 samples, the LC-MS/MS method was compared with the LIAISON®
assay and ADVIA Centaur® assay. Results. The LC-MS/MS method had a limit of quantitation of 1 ng/mL
for the 25(OH)D2 and 25(OH)D3 with linear responses between 1 and 100 ng/mL. Intra- and inter-assay
precision were <8.8% and <13.2%, respectively. It also showed a smallest mean difference (+0.9%) for the
SRM level 1 to 3, compared to the two immunoassays. Compared to the LC-MS/MS, the mean biases of
the RIAISON and ADVIA were +2.4 and +7.9 ng/mL, respectively. Also, the agreement of the LC-MS/
MS with the RIAISON was better than that with the ADVIA. Conclusion. This study suggests that the
LC-MS/MS method traceable to the SRM can be reliably applied in routine quantification of 25(OH)D2
and 25(OH)D3.

Key words: 25-Hydroxyvitamin D2, 25-Hydroxyvitamin D3, Liquid chromatography-tandem mass spec-
trometry, Automated immunoassays

Introduction Vitamin D, which exists in two major form - vita-

Vitamin D plays essential roles in regulation of the
cell cycle such as apoptosis and differentiation [1]
as well as in bone metabolism and calcium homeo-
stasis [2]. Many studies have proposed that vitamin
D insufficiency or deficiency is related to an in-
creasing risk of many diseases including cancer, car-
diovascular disease, diabetes, infectious, and auto-
immune diseases [3-7].
Address correspondence to Young-Jin Kim, MD, PhD, Seoul Clinical
Laboratories, 24F Bldg. A, Heungdeok IT Valley, 13 Heungdeok 1-ro,
Giheung-gu, Yongin, Gyeonggi 16954, Korea; Phone: +82 2 330
2011; Fax: +82 31 660 7278; e mail: yjkim@scllab.co.kr
min D2 and D3, is metabolized in the liver to
25(OH)D) and then further hydroxylated in the
kidney to 1,25-dihydroxyvitamin D (1,25(OH)2D)
[8]. Because half-lives of vitamin D and
1,25(OH)2D are relatively short (<2 days) and
1,25(OH)2D is affected by some physiological con-
ditions [9], levels of circulating 25(OH)D which
has a half-life of 2-3 weeks in human blood, are
considered as the best indicators of vitamin D sta-
tus [10,11].
For the purpose of diagnosing or preventing human
diseases associated with vitamin D insufficiency or
deficiency, clinicians gradually are now examining

0091-7370/16/0600-645. © 2016 by the Association of Clinical Scientists, Inc.

 646

Table 1. Validation results of the LC-MS/MS assay method.

Linearity Nominal Intra-assay (n=5) Inter-assay (n=5) LOQ Recovery Stability Matrix
conc.a Accuracy CV(%) Accuracy CV(%) (ng/mL) (%) (%) effect(%)
(ng/mL) (%) (%)

25(OH)D2 y = 0.02291x – 0.00369 1.0 102.0 4.1 100.2 13.1 1.0

(R 2 = 0.99950) 5.0 99.6 4.3 106.4 4.0 92.2 105.8 92.1
25.0 93.0 8.7 95.2 8.6 95.7 96.3 95.6
100.0 95.1 3.5 97.3 7.8 94.5 93.1 96.8
25(OH)D3 y = 0.11337x + 0.04093 1.0 90.4 5.7 91.6 9.0 1.0
(R 2 = 0.99986) 5.0 104.1 4.1 103.8 4.2 96.4 88.7 93.2
25.0 95.2 1.2 98.1 3.2 105.0 104.4 96.2
100.0 89.9 1.6 93.6 4.8 98.7 95.1 96.7
aThe concentrations of QC samples.

Table 2. Performance of the assay methods using NIST SRM 972a with certified values.

SRM 972aa LC-MS/MS LIAISON ADVIA Certified values (ng/mL)b

Level 1 27.67±1.37 28.13±1.37 24.96±0.21 25(OH)D3: 28.80±1.10

3-epi-25(OH)D3: 1.84±0.08
Annals of Clinical & Laboratory Science, vol. 46, no. 6, 2016

Level 2 19.91±0.23 18.03±0.50 18.40±0.44 25(OH)D2 : 0.81±0.06

1.03±0.02 (D2)c 25(OH)D3 : 18.10±0.40
18.88±0.21 (D3)d 3-epi-25(OH)D3: 1.28±0.09
Level 3 32.95±0.21 26.70±0.80 33.60±0.90 25(OH)D2: 13.30±0.30
13.11±0.11 (D2)c 25(OH)D3: 19.80±0.40
19.84±0.10 (D3)d
Level 4 56.20±1.32e 29.37±1.2f 26.10±2.08f 25(OH)D3: 29.40±0.90
3-epi-25(OH)D3: 26.00±2.2

a Each level was measured in triplicate by each assay, and the measured values are expressed in ng/mL.
bThe target value and the uncertainty listed in the NIST certificates.
c,dThe values refer to the concentrations of 25(OH)D and 25(OH)D measured by LC-MS/MS.
2 3
eThe values refer to the sum of concentrations of the two metabolites because 25(OH)D3 and its C-3 epimer coelute under the LC-MS/MS conditions used in this study.
f The biases were assessed excluding the value for 3-epi-25(OH)D assuming a null reactivity for this metabolite.
3
 A fast and simple LC-MS/MS method for measuring 25(OH)D2 and 25(OH)D3
Figure 1. Representative MRM chromatograms of a pro-
cessed serum specimen after the sample preparation.
vitamin D status in individuals. However, the ac-
curacy of 25(OH)D assay methods still remains a
real challenge for clinical laboratories [12].
The most commonly used analytical platforms to
measure 25(OH)D in human serum are grouped
into immunochemical and chromatographic meth-
ods. The immunochemical methods based on ra-
dioactive, enzymatic, and chemiluminescence de-
tection may be susceptible to cross-reactivity
resulting from the interaction of vitamin D metab-
olites with antibodies that may lack specificity. On
the other hand, chromatographic methods have
higher specificity due to sufficient resolving power
than immunoassays. Especially, liquid chromatog-
raphy-tandem mass spectrometry (LC-MS/MS) is
receiving wide acceptance [13]. The LC-MS/MS
methods can minimize interferences and matrix ef-
fects compared to immunoassays.
However, the relatively low-throughput compared
to automated immunoassays and the high cost of
the equipment are commonly limitations to the
routine use of the LC-MS/MS methods in clinical
laboratories. Despite these limitations, the methods
are now used as a reference assay for the measure-
ment of 25(OH)D [14-18]. Therefore, the devel-
opment of rapid, simple, and specific LC-MS/MS
methods is needed to measure 25(OH)D in clinical
laboratories [19-21]. Furthermore, a validated LC-
MS/MS methodology traceable to standard refer-
ence materials (SRM) from National Institute of
Standards and Technology (NIST) is recommend-
ed to ensure the accuracy.
647
The aim of this study is to validate a LC-MS/MS
method as an in-house assay, established in our
laboratory and to compare the performances of two
automated 25(OH)D immunoassays.
Materials and Methods
Chemicals and reagents. 25(OH)D2 and 25(OH)D3
was from Sigma Chem. Co. (MO, USA). Hexadeuterated
25(OH)D3 (25-hydroxycholecalciferol-
26,26,26,27,27,27-d6) as an internal standard (IS) was
from ChromSystems Co. (Munich, Germany). Activated
charcoal and bovine serum albumin were from Sigma
and Amresco Co. (OH, USA), respectively. HPLC-grade
methanol, ethanol, hexane, formic acid, and water were
from Fisher Scientific Korea Co. (Seoul, Korea).
Sample collection. One hundred and fifty serum sam-
ples were randomly collected from patients whose sam-
ples were sent to Seoul Clinical laboratories for routine
measurement of vitamin D levels from October to
December 2012. Samples were divided into 3 aliquots,
frozen at -20°C, and analyzed in batches, with a freshly
thawed aliquot used for each analytical run. The study
was approved by the Institution Ethics Review Board of
Seoul Medical Science Institution.
Calibrator and sample preparation. A standard stock
solution including 25(OH)D2 and 25(OH)D3 was pre-
pared in ethanol at a concentration of 1 µg/mL.
Calibrators were prepared in 4% bovine serum albumin
(BSA, used as a blank) prepared in phosphate buffered
saline (PBS, pH 7.4) from the standard solution by serial
dilution to give concentrations of 1.0, 5.0, 10.0, 25.0,
50.0, and 100.0 ng/mL for 25(OH)D2 and 25(OH)D3.
To 200µL of serum, calibrator or controls were added
200µL of refrigerated ethanol, containing the deuterated
IS and equilibrated for 1 min. The mixture was extracted
with 1 mL of hexane to allow complete protein precipi-
tation and then centrifuged for 5 min at 13,000 rpm.
Then, 850µL of supernatant was transferred into glass
vials and evaporated to dryness under a stream of N2 gas.
The residue was re-dissolved in 120µL of 50% ethanol.
Finally, 10µL of supernatant was analyzed by LC-MS/
MS.
LC-MS/MS conditions. The system consisted of Agilent
1200 series (Palo Alto, CA, USA) coupled to an API
4000 triple-quadrupole mass spectrometer (Applied
Biosystems/MDS SCIEX, Forster city, CA, USA)
equipped with an electrospray ionization (ESI) source.
Chromatographic separation was performed using
Hypersil GOLD™ aQ C18 column (2.1 mm x 50 mm,
1.9 µm particle size) (Thermo Fisher Scientific Inc., St.
Louis, MO, USA) maintained at 35°C. The mobile
phase was a 15:85 mixture of 0.1% formic acid in
 648 Annals of Clinical & Laboratory Science, vol. 46, no. 6, 2016
methanol with 5 mM am-
monium acetate and 0.1%
formic acid at the flow rate
Table 3. Passing and Bablok regression, Bland-Altman, and concordance correlation analysis (n = 150 specimens) of the two immunoassays against the LC-MS/MS method.
of 0.3 mL/min. The total
0.876
0.949
chromatographic run time
ra
was 3 min. The ESI source
was operated in the posi-
tive ion mode. 25(OH)
CCC
0.928 (0.904-0.946)
0.737 (0.672-0.792)
D2, 25(OH)D3, and IS
were monitored in the
multiple-reaction moni-
toring (MRM) mode us-
ing the following transi-
CCC
tions: m/z
413.35→395.30,
401.37→383.20, and
Mean inaccuracy (CI 95%)
407.28→389.30, respec-
tively. The declustering
2.40 (-6.4-11.3)
potential, entrance poten-
7.90 (-4.7-20.4)
Bland-Altman
tial, collision energy, and
collision cell exit potential
were optimized at 56, 10,
13, and 14 V for 25(OH)
D2, 66, 10, 13, and 10 V
for 25(OH)D3, and 66,
10, 13, and 14 V for IS,
respectively. Collision gas,
curtain gas, ion source
75.84
52.90
Max
gases 1 and 2 were 6, 20,
60, and 45 psi, respective-
ly. Ionspray voltage and
11.85
Min
4.70
temperature were 5500 V
and 350°C, respectively.
The data were analyzed us-
ing Analyst® 1.5 data pro-
0.043 (-0.852-0.811)
Passing and Bablok regression
7.087 (5.787-7.920)
Intercept (CI 95%)
cessing software from
Applied Biosystems/MDS
SCIEX. '
Method validation. The
LC-MS/MS assay method
was validated by measur-
ing a limit of quantifica-
tion (LOQ), linearity, ac-
1.109 (1.058-1.176)
1.071(0.977-1.165)
curacy, precision
(expressed coefficient of
Slope (CI 95%)
correlation coefficient.
variation, %CV), recov-
ery, processed sample sta-
bility, matrix effect, and
carryover. The LOQ was
determined by serial dilu-
tion of the standard solu-
tion (n=9 per dilution)
LIAISON
a Pearson’s
and at the lowest concen-
Methods
ADVIA
tration where CV was
≤20%. Linearity was
determined by analyzing the 6 levels of the calibrators of
25(OH)D2 and 25(OH)D3. Accuracy and precision
were determined from in-house prepared quality control
(QC) samples by comparison of the measured value with
the calculated one. The QC samples were prepared in
4% BSA prepared in PBS to give concentrations of 1, 5,
25, and 100 ng/mL for the two 25(OH)D metabolites
using the stock solution that were independent of those
used to prepare the calibrators. 25(OH)D2 and 25(OH)
D3 were measured on 5 consecutive days (inter-assay)
with 5 replicates per day (intra-assay) using fresh samples
daily. Recovery was determined by spiking known
amounts (5, 25, and 100 ng/mL) of 25(OH)D2 and
25(OH)D3 with serum samples with low concentrations
of 25(OH)D. For determination of processed sample
stability, the three QC samples were treated by sample
preparation procedure, left on an autosampler tray of the
LC system at room temperature for 24 hr, and then ana-
lyzed. To investigate the matrix effect [22], the blank
samples spiked with 25(OH)D2 and 25(OH)D3 to give
the concentrations of 5, 25, and 100 ng/mL were ex-
tracted alongside samples prepared in the mobile phase
and spiked to the same concentrations. Carryover was
determined by repeated injections of blank sample after
the highest standard (100 ng/mL) was measured.
Assay method comparison. The LC-MS/MS method
was compared with the two automated immunoassays,
DiaSorin LIAISON® 25-OH Vitamin D TOTAL Assay
(Stillwater, MN, USA) and Siemens ADVIA Centaur®
XP Vitamin D Total (VitD) assay (Tarrytown, NY,
USA), widely used for the measurement of 25(OH)D,
measuring sera collected from 150 individuals in dupli-
cate. Each immunoassay system was calibrated and per-
formed according to the manufacturer’s instruction at
Seoul Clinical Laboratories, Seoul, Korea. Method com-
parison was performed according to Clinical Laboratory
Standards Institute (CLSI) Evaluation Protocol 9 (EP9)
specifications. The two immunoassays were performed
according to manufacturer’s instructions. All serum sam-
ple testing on the automated immunoassays were per-
formed within the same day. Measuring ranges are 4.0 -
150 ng/mL for the LIAISON assay and 4.2–150 ng/mL
for the ADVIA assay. Sensitivities are 4.0 and 4.2 ng/mL
for the LIASON and the ADVIA assays, respectively.
Certified SRM 972a (levels 1-4) from NIST as indepen-
dent controls were also used to evaluate the accuracies of
all the assays including the LC-MS/MS method.
Statistical analysis. The statistical analysis of method
validation results was performed using Microsoft Office
Excel® 2013 (Microsoft Inc., Seattle, WA, USA). For
method comparison studies, the 25(OH)D measure-
ments of serum samples by the assay methods were ana-
lyzed by Passing-Bablok regression and Bland-Altman
bias plots. Agreement between the methods
 A fast and simple LC-MS/MS method for measuring 25(OH)D2 and 25(OH)D3
was evaluated by measuring Cohen’s Kappa (κ) value
(agreement: <0.4, poor; 0.4-0.75, fair to good; >0.75 ex-
cellent) [23]. Statistical analysis was performed using
MedCalc Statistical Software (ver. 11.2.1, Mariakerke,
Belgium) and Analyse-it Method Evaluation Edition
(Leeds, UK). P<0.05 was considered statistically
significant.
Results
Analytical performance evaluation of LC-MS/
MS methodology. The LC-MS/MS chromato-
grams of a serum specimen passed through the
sample preparation procedure are shown in Figure
1. Under the LC-MS/MS conditions applied, the
vitamin D metabolites and IS were adequately sep-
arated, with 25(OH)D2 eluting at 2.22 min,
25(OH)D3 at 2.04 min, and IS at 2.02 min.
Potential interfering substances such as 1α-OH-D3
were eluted between 0.7 and 1.2 min but did not
interfere with the detection of 25(OH)D2 and
25(OH)D3.
The performance characteristics of the LC-MS/MS
method are given in Table 1. The LC-MS/MS
method demonstrated a dynamic linear response
over the working range from 1.0 to 100.0 ng/mL
(r>0.999) for 25(OH)D2 and 25(OH)D3, with a
LOQ of 1 ng/mL. Intra-assay accuracies were
93.0% to 102.0% and 89.9% to 104.1% and inter-
assay accuracies were 95.2% to 106.4% and 91.6%
to 103.8% for 25(OH)D2 and 25(OH)D3, respec-
tively. Intra-assay CVs were 3.5% to 8.7% and
1.2% to 5.7% and inter-assay CVs were 4.0% to
13.1% and 3.2% to 9.0% for 25(OH)D2 and
25(OH)D3, respectively. Recoveries of 25(OH)D2
and 25(OH)D3 from serum samples spiked with
three different concentrations were 92.2% to
95.7% and 96.4% to 104.4%, respectively. Both
25(OH)D2 and 25(OH)D3 were found to be stable
after the QC samples were processed and left on the
autosampler tray. The mean matrix effect was
94.8% and 95.3% for 25(OH)D2 and 25(OH)D3,
respectively, indicating that a very small ion sup-
pression was observed for the two vitamin D me-
tabolites. No carryover was observed on the LC-
MS/MS chromatograms at concentrations up to
100 ng/mL.
Comparison of LC-MS/MS method and immuno-
assays. The performance of each assay method with
NIST SRM 972a is shown in Table 2. Considering
649
the estimated biases from the certified values for
25(OH)D3 or total 25(OH)D, the LC-MS/MS
method made close to or within the certified values
for level 1 through 3 but higher for level 4, and re-
peatability CVs for the four levels of SRM 972a
ranged from 0.6% to 5%. Except for level 4, the bi-
ases of level 1, 2, and 3 were -3.9%, 5.3%, and
-0.5%, respectively. For level 4, including substantial
amounts of 3-epi-25(OH)D3, the concentrations of
25(OH)D3 were within the expected value for the
sum of 25(OH)D3 and 3-epi-25(OH)D3 because
25(OH)D3 and its C-3 epimer were not separated
chromatographically under the LC-MS/MS condi-
tions used in this study. On the other hand, the two
immunoassays underestimated the concentrations of
total 25(OH)D for the four levels, except for a slight
overestimation of level 3 by the ADVIA. CVs for re-
peatability were 2.5% to 4.0% for the LIAISON and
0.9% to 8.0% for the ADVIA. The LIAISON assay
showed a significant negative bias (-19.3%) in level
3, while the ADVIA in level 1 (-13.3%) and 4
(-11.2%).
Figure 2 and Table 3 show Passing-Bablok regres-
sion, Bland-Altman difference plot, and concordance
correlation coefficient (CCC) of the automated im-
munoassays against the LC-MS/MS method. The
average values of 150 serum specimens measured by
each assay method were 19.50 ng/mL (5.24 to 50.56
ng/mL) for LC-MS/MS assay, 21.95 ng/mL (4.7 to
52.90 ng/mL) for LIAISON assay, and 27.36 ng/mL
(11.85 to 75.84 ng/mL) for ADVIA assay. Both the
immunoassays exhibited statistically equivalent
Passing-Bablok fits with uniform positive biases
throughout the whole range of concentrations com-
pared to the LC-MS/MS method. Especially, Passing-
Bablok regression analysis demonstrated that the
LIAISON assay was statistically more equivalent to
the LC-MS/MS method than the ADVIA. Bland-
Altman different plot analysis also exhibited a mean
bias of 2.4 ng/mL for the LIAISON (Figure 2B and
Table 3) and of 7.9 ng/mL for the ADVIA (Figure
2D and Table 3), both with uniform bias across the
ranges of concentrations measured. As shown in
Table 3, Pearson’s correlation analyses demonstrated
that the LC-MS/MS method correlated well with the
LIAISON assay (CCC=0.928, r=0.949) but less well
with the ADVIA assay (CCC=0.737, r=0.876).
Criteria for vitamin D insufficiency in this study was
defined as a serum concentration less than 20 ng/mL
(50 nmol/L), as previously reported [20,24-26].
 650 Annals of Clinical & Laboratory Science, vol. 46, no. 6, 2016

Figure 2. Comparison of the immunoassays against the LC-MS/MS method using Passing-Bablock regression plots (left pan-
els) and Bland-Altman plots (right panels). (A and B) LC-MS/MS vs. LIAISON. (C and D) LC-MS/MS vs. ADVIA. In left
panels, the solid lines, dashed lines, and dotted lines are the regression lines, the confidence intervals for the regression lines,
and identity lines, respectively. In right panels, the solid lines, dashed lines, and dotted lines are the mean differences, 95%
limits of agreement, and identity lines, respectively.

Among all serum samples tested, the proportions of
those classified as insufficiency (<20 ng/mL) were
64.0% by the LC-MS/MS, 54.7% by the LIAISON
and 42.7% by the ADVIA, whereas the ADVIA, the
LIAISON and the LC-MS/MS classified 58%,
45.3% and 36% as sufficient vitamin D status, re-
spectively. As evidenced by Passing-Bablok regres-
sion and Bland-Altman difference plot analyses, in-
ter-rater agreement analysis using κ value also
demonstrated that the LC-MS/MS method exhibit-
ed a stronger agreement with the LIAISON assay
(κ=0.808, 95% CI=0.715-0.902) than with the
ADVIA assay (κ=0.579, 95% CI=0.463-0.694) for
all the serum samples tested (Table 4).
Discussion
LC–MS/MS has emerged as the latest technology to
be used to measure the metabolites of vitamin D and
to monitor clinical vitamin D status. Generally, treat-
ment of patients depend on assay results measured by
clinical laboratories. Therefore, the reliability of assay
methods may have an influence on clinician’s decision
on treatment. The LC-MS/MS method is capable of
measuring 25(OH)D2 and 25(OH)D3 simultaneous-
ly, which may be useful to monitor the efficacy of sup-
plementation especially in countries where vitamin D2
is used as a supplement [27,28]. Although a result from
vitamin D external quality assessment scheme
(DEQAS) survey proposed that major assay methods
may provide results within about 10% of the consensus
average [29], evidence of measurement accuracy is not
given by comparability between assay methods if agree-
ment between assays is not tested using SRMs or the
assays performance is not traced to reference methods.
Therefore, the performance of 25(OH)D assay meth-
ods is a real challenge in most clinical laboratories and
is an important criterion for the choice of a method.
 A fast and simple LC-MS/MS method for measuring 25(OH)D2 and 25(OH)D3
Table 4. Agreement between LC-MS/MS method and each immunoassay on vitamin D status in 150 serum samples.
LC-MS/MS LIAISON
<20 ng/mL (%) ≥20 ng/mL (%)
<20 ng/mL 82 (54.7) 14 (9.3)
≥20 ng/mL 0 (0.0) 54(36.0)
Total 82 (54.7) 68 (45.3)
Kappa (95% CI) 0.808 (0.715-0.902)
In this study, serum sample was first pretreated by
protein precipitation with ethanol including a deu-
terated IS, followed by a liquid-liquid extraction
with hexane. These steps were simple, rapid, and
were necessary to clean up the samples, to minimize
matrix effect and to maximize sensitivity.
Additionally, we tested effectiveness of protein pre-
cipitation with methanol, ethanol, and acetonitrile,
and ethanol was chosen as an optimal solvent. Our
method was distinct from a recent report in which
methanol and heptane were used as a protein pre-
cipitation and a liquid-liquid extraction solvent,
respectively [30]. The variability of LC-MS/MS
methods between laboratories may result from dif-
ferent ways of preparing samples. Furthermore,
these sample preparation methods are very impor-
tant because they can affect the accuracy and recov-
ery of the vitamin D metabolites.
In this study, we validated a fast, accurate, sensitive
LC-MS/MS method for measuring serum levels of
25(OH)D2 and 25(OH)D3 at nanomolar concen-
trations using an IS material. Method validation
demonstrated that the LC-MS/MS method meets
all technical and quality requirements for simulta-
neous quantification of 25(OH)D2 and 25(OH)
D3 in human serum (Table 1). BSA is being used
for the quantification of 25(OH)D2 and 25(OH)
D3 and proved to be an alternative matrix for cali-
bration [21,30,31].
However, how the LC-MS/MS methods are cali-
brated is a major factor to variation between-labo-
ratories or between-assays. Recently, the DEQAS
has shown differences in 25(OH)D results depend-
ing on assay methods, arousing importance for the
agreement and accuracy of different assays [29]. As
a part of this, 20 DEQAS samples were analyzed by
an LC-MS/MS method developed by as a candi-
date reference measurement procedure by the
NIST, revealing that the NIST results were
651
ADVIA Total (%)
<20 ng/mL (%) ≥20 ng/mL (%)
63 (42.7) 33 (21.3) 96 (64.0)
0 (0.0) 54 (36.0) 54 (36.0)
63 (42.7) 87 (58.0) 150 (100.0)
0.579 (0.463-0.694)
on average 11.2% lower than those measured by
routine LC-MS/MS assays. These results imply that
routine LC-MS/MS methods being used in clinical
laboratories possibly overestimate total 25(OH)D
in human serum. In this regard, LC-MS/MS meth-
ods traceable to certified materials such as NIST
SRM 972, 2972 and 972a are needed to minimize
the variations.
We demonstrated in this study that the present LC-
MS/MS method can measure 25(OH)D2 and
25(OH)D3 accurately in serum, as evidenced by
the measurements of SRM level 1 to 3 (Table 2).
Although the present LC-MS/MS method could
not resolve 3-epi-25(OH)D3 from 25(OH)D3, it
showed a smallest mean difference (+0.9%) for
SRM level 1 to 3 when compared to the two im-
munoassays (-26.2% and -14.5% for the LIAISON
and the ADVIA, respectively). Interestingly, the
present LC-MS/MS method showed the smallest
bias (-0.5%) compared to the LIAISON and the
ADVIA in level 3 with a substantial amount of
25(OH)D2 and a product from pools of human
serum with endogenous concentrations of 25(OH)
D, indicating that it has its capacity to detect
25(OH)D2 and 25(OH)D3 selectively. In contrast,
the LIAISON in level 3 showed a remarkable nega-
tive bias (-19.3%), may indicate lower recovery of
25(OH)D2, independent of 25(OH)D3 in human
serum [20].
Recently, it has been reported that the mean con-
centration of 3-epi-25(OH)D3 in adult samples is
generally lower (5.5%) than that in newborns
(27.3%) which is known to include high percent-
ages of 3-epi-25(OH)D3 [32]. For this reason, low
concentrations of the C3-epimer in adults are un-
likely to be clinically significant, although some
samples with significant amounts of the 3-epimer
may be overestimated. More recently, it has been
also reported that a LC-MS/MS assays that do not
652 Annals of Clinical & Laboratory Science, vol. 46, no. 6, 2016
separately measure the C3-epimer from other vita-
min D metabolites may potentially overestimate
total 25(OH)D levels in newborn cord blood sam-
ples but the C3-epimer concentration is unlikely to
contribute to diagnostic misclassification even in
the newborn samples [33]. Despite these disputes,
further work will be needed to investigate the im-
pact of the C3-epimer on the 25(OH)D assays and
to obtain information about the physiological role
of the C3-epimer.
In this study, the RIAISON and ADVIA assays
generally gave higher 25(OH)D measurements
than the LC-MS/MS method in serum specimens
tested. Especially, the mean bias of the ADVIA as-
say (+7.9 ng/mL) was higher than that of the
RIAISON (+2.4 ng/mL) over the range of concen-
tration measured when compared to the LC-MS/
MS method traceable to the NIST-SRM (Figure 2
and Table 3). However, compared to the ADVIA
assay (CCC=0.737), the LIAISON (CCC=0.928)
showed better concordance with LC-MS/MS re-
sults (Table 3). Recently, many of the automated
immunoassays turned out to have significant posi-
tive biases when compared to LC-MS/MS methods
[34,35]. Furthermore, it is noteworthy that most
immunoassays had difficulties measuring low lev-
els. Especially, the RIAISON and the ADVIA as-
says which turned out to show excessive mean bi-
ases of +35% and +118%, respectively, at low levels
<8 ng/mL [34]. In contrast, several studies also re-
ported that the automated RIAISON and ADVIA
demonstrated significant negative biases compared
to LC-MS/MS methods [30,36].
There are several possible explanations for the varia-
tions between the immunoassays and the LC-MS/
MS methods. First, the variations between the
methods might be caused by different calibrator
traceability. Importantly, both immunoassays and
LC-MS/MS methods may experience matrix effects
that occurs between the matrix in the calibrators
and the clinical samples, providing lower or higher
25(OH)D measurements [13]. Especially, the
25(OH)D2 and 25(OH)D3 concentrations mea-
sured by routine LC-MS/MS methods may be dif-
ferent between laboratories and show biases when
compared to those by NIST LC-MS/MS [37].
Second, all immunoassays show high cross-reactivi-
ty with other metabolites of 25(OH)D such as
24,25(OH)2D, which is known to be present in the
serum at levels of nearly 10-15 nmol/L [37], may
result in higher 25(OH)D measurements by im-
munoassays. Additionally, variations in vitamin
D-binding protein concentrations between clinical
subject groups may be a possible cause for the ob-
served differences between automated immunoas-
says and LC-MS/MS methods. For example, of the
pregnant women, 67% were vitamin D sufficient
when measured with a LC-MS/MS method, where
ADVIA categorized only 24% as vitamin D suffi-
cient [38].
Although there was a lack of agreement between
the ADVIA and RIAISON assays compared to the
LC-MS/MS method, it was proven through the
CCC and κ analysis that the agreement with the
LIAISON was better than that with the ADVIA
(Tables 3 and 4). These results were also similar to
the findings of the previous studies [25,34]. In this
study, the prevalence of vitamin D insufficiency was
higher with the RIAISON and lower with the
ADVIA, compared with the LC-MS/MS result,
suggesting that the ADVIA assay would report a
lower frequency of patients with vitamin D insuf-
ficiency or deficiency than the LIAISON or the
LC-MS/MS method. However, further studies will
be needed to establish decision limits by different
assays and to standardize existing assays for 25(OH)
measurement because our results demonstrated
that the use of uniform cut-off value by assays could
be an issue when classifying vitamin D status.
In conclusion, the present LC-MS/MS method
with a traceability to SRM 972a is a rapid, simple,
reliable, and cost effective alternative to other meth-
ods for determination of 25(OH)D in human se-
rum. Compared to the LC-MS/MS results, the
agreement with the automated RIAISON assay was
better than that of the ADVIA which showed a sub-
stantial positive bias. Thus, these results should fa-
cilitate the clinical application of the LC-MS/MS
method for separate quantification of 25(OH)D2
and 25(OH)D3.
Acknowledgment
This work was supported by Seoul Medical Science Institute &
Seoul Clinical Laboratories in 2014-2015.
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