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ORI GI NAL RESEARCH

Vitamin D Assays: Past and Present Debates, Difculties,


and Developments
William D. Fraser

Anna M. Milan
Received: 13 April 2012 / Accepted: 30 November 2012
Springer Science+Business Media New York 2013
Abstract Clinical interest in Vitamin D and its purported
roles not only in calcium and bone metabolism but in
several other medical conditions (diabetes, cardiovascular
disease, multiple sclerosis, cancer, psychiatric disorders,
neuro-muscular disease) has led to a surge in laboratory
requests for 25 hydroxy vitamin D and 1,25 dihydroxy
vitamin D measurement. Circulating 25 hydroxy vitamin D
concentration is routinely used as the best indicator of
vitamin D status, but measurement of other metabolites,
especially the physiologically active 1,25 dihyroxy vitamin
D, are of clinical value. Over the last 40 years the devel-
opment of assays for vitamin D and its metabolites from
early competitive binding assays through to immunoassay
and liquid chromatography aligned to mass spectrometry
have demonstrated various analytical challenges, the
advantages and disadvantages of each method are con-
stantly changing with new technological developments.
Immunoassay remains the predominant mode of measure-
ment for 25-hydroxy vitamin D although problems with
equimolar recovery of the D2 and D3 metabolites remain
an issue. Standardisation of all assays has been improved
but not resolved with the currently available reference
materials as evidenced by the international vitamin D
external quality assurance scheme, DEQAS. The choice of
method for each laboratory remains a balance mainly
between turn around time, convenience, cost and the
specicity and accuracy of the information obtained. With
increasing discussion and clinical interest surrounding
other vitamin D metabolites the vitamin D assay debate is
set to continue.
Keywords Steroid hormones: vitamin D Assay
Review
Introduction
The recent explosion of interest in vitamin D and its pos-
sible roles in both classical processes (calcium and bone
metabolism, neuromuscular function) and nonclassical
diseases (arthritis, cardiovascular, cancer, diabetes, multi-
ple sclerosis, psychiatric illness) has increased demand for
measurement of vitamin D and its metabolites. In part this
has been a chicken-and-egg situation, where the advances
in technologies in the last 20 years have resulted in the
development of assays that have improved sensitivity and
specicity while lending themselves to automation; auto-
mation has resulted in large numbers of vitamin D
metabolite measurements being performed with ease on
small sample volumes. The ability to generate a large
number of vitamin D metabolite results has expanded the
database, producing information that has resulted in the
expansion of both clinical and research interest in vitamin
D and signicant debate regarding its role in health and
disease.
There have been major advances in semiautomation
and full automation of immunoassays utilizing nonradio-
active tracers which have been incorporated into both
W. F. has a consultant/advisory role and has received funding from
IDS, Siemens, Nichols, Roche, and Abbott and has patents/
intellectual property with IDS. A. M. has stated no conict of interest.
W. D. Fraser (&)
Norwich Medical School, University of East Anglia,
Norfolk, UK
e-mail: w.fraser@uea.ac.uk
A. M. Milan
Department of Clinical Biochemistry and Metabolic Medicine,
Royal Liverpool and Broadgreen University Hospital Trust,
Liverpool, UK
1 3
Calcif Tissue Int
DOI 10.1007/s00223-012-9693-3
specialist-dedicated immunoassay systems and the large
analytical platforms of several major manufacturers.
Increasing interest in the use of mass spectrometry (MS)
detection allied to high-performance liquid chromatogra-
phy (HPLC) separation has seen many laboratories invest-
ing in tandem MS for measurement of vitamin D metab-
olites. Each methodology has its advantages and disad-
vantages, which will be discussed in this review.
Vitamin D Metabolites of Current Interest
There are well over 40 metabolites of vitamin D identied
[1], and this could potentially result in difculties in
establishing assays for specic molecules of interest. In
practice, however, the vast majority of metabolites have a
very short half-life in the circulation and thus, are currently
of minimal interest and present little challenge in assay
development. Although the parent sterol vitamin D has a
half-life of close to 24 h [2], this is relatively short com-
pared with 25-hydroxyvitamin D (25OHD), which has a
half-life of 2130 days [3, 4]. Therefore, 25OHD mea-
surement is a better indicator of vitamin D stores, whether
obtained from sunlight (ultraviolet [UV] exposure) or
dietary sources. The most potent physiologically active
circulating metabolite produced by humans is 1,25-di-
hdroxyvitamin D (1,25[OH]
2
D), which has a half-life of
415 h [58]; and while 25OHD circulates in nanomole per
liter concentrations, 1,25(OH)
2
D is present in picomole per
liter concentrations, which means that 1,25(OH)
2
D repre-
sents the greater challenge in assay development.
Vitamin D is derived from two major sources in
humans, with approximately 8090 % produced by the
action of sunlight (UVB) on the skin resulting in chole-
calciferol (D
3
). The other 20 % is derived from dietary
sources, which can be animal cholecalciferol (D
3
) or plant-
derived ergocalciferol (D
2
). Supplementation of foods and
health products or physician-guided treatment with either
D
2
or D
3
can increase the percentage derived from exog-
enous sources, so assay technology needs to be able to
measure both D
2
and D
3
metabolites. The production and
metabolism of vitamin D is shown in Fig. 1, and it should
be noted that a major rate-limiting step in this pathway is
25-hydroxylation, which takes place in the liver. This step
is primarily dependent on the substrate concentration
(vitamin D) [9, 10] and is the reason the well-recognized
seasonal variability related to UVB exposure exists.
1-Hydroxylation mainly takes place in the kidney, but
1a-hydroxylase activity has also been demonstrated in cells
of the placenta, bone, skin, and granuloma tissue (sarcoid,
tuberculosis) [11] and requires 25OHD
2
/D
3
(total 25OHD)
as the substrate. The rate of 1,25(OH)
2
D
2
/D
3
(total
1,25[OH]
2
D) production by the kidney can be inuenced
by prevailing calcium (Ca) and parathyroid hormone
(PTH) concentrations. For these reasons, as well as the
short half-life, total 1,25(OH)
2
D is a poor indicator of
overall vitamin D status as total 25OHD needs to decrease
to around 10 nmol/L (4 ng/mL) for total 1,25(OH)
2
D to
decrease signicantly [12].
Two other hydroxylases produce metabolites from
25OHD and 1,25(OH)
2
D. 24-Hydroxylase (renal and intes-
tinal) produces 24,25-dihydroxyvitamin D (24,25[OH]
2
D),
and 26-hydroxylase (renal and liver) produces 25,26-
dihydroxyvitamin D (25,26[OH]
2
D). Their circulating con-
centrations are not a good reection of vitamin D status, but
their role in vitamin D physiology and pathology is being
investigated.
Measurement of 25OHD
Development of 25OHD Assays
Measurement of 25OHD has proven to be a major chal-
lenge, with signicant variability in the results generated
by many differing methods. Several factors contribute to
the analytical problem, namely, 25OHD is hydrophobic
and therefore unstable in water; its lipophilic nature means
it strongly associates with vitamin D binding protein
(VDBP); endogenous lipids coextract from plasma and
serum, affecting binding and chromatographic separation;
it exists in several molecular forms; direct natural sunlight
degrades both 25OHD metabolites and internal standards
employed in some assays, and a suitable reference standard
material has not been available until very recently.
Competitive Protein Binding Assay
In the 1970s a pivotal method [13] was developed that
employed preliminary solvent extraction of samples and
chromatographic purication, resulting in elimination of
VDBP. The extract (containing unknown, unlabeled
25OHD) was incubated with a known concentration of
radioactively labeled 25OHD
3
, and they competed for an
added limited amount of binding protein. Subsequent
competitive protein binding (CPB) assays utilized either
rachitic rat, chicken-, or human-derived binding protein
and allowed correction for procedural losses during
extraction and purication with an analytical recovery of
82 3.5 % (SD). In addition, they exhibited a shorter
assay incubation time (1 h vs. 10 days) and measurement
across a wide range of concentrations [1418]. However,
these assays cross-reacted with several 25OHD metabo-
lites, suffered from instability of the binding protein
preparations, and could not be simplied by eliminating the
chromatography step [19]; and although attempts were
W. D. Fraser and A. M. Milan: Vitamin D Assays
1 3
made to automate a CPB-based assay, this proved prob-
lematic, with inaccuracy, poor detection limit, low cross-
reactivity with 25OHD
2
, poor precision, and overall
unacceptable performance [2022].
Immunoassay
Immunoassay methods were rst reported in the 1980s with
a radioimmunoassay (RIA) described that initially utilized
a
3
H-labeled 25OHD tracer [23], which was subsequently
developed into a
125
I-labeled 25OHD assay with higher
throughput and improved performance [24]. This assay
formed the basis for a subsequent chemiluminescent
detectionbased system, which has been automated [25]. A
two-step extraction RIA [26] was produced by one com-
mercial manufacturer and subsequently developed into an
enzyme immunoassay (EIA) without the extraction step,
which could also be automated [27]. Several manufacturers
have produced 25OHD immunoassays where the solvent
extraction and chromatographic separation have been
replaced by various blocking agents that displace 25OHD
from VDBP, with varying success. This approach facili-
tates automation of these assays, but recent data suggest
that some immunoassays employing these techniques may
be affected by variations in VDBP concentrations, proba-
bly due to variable displacement of 25OHD from VDBP
and, in specic subjects, may be due to the increased
afnity of 25OHD for certain variants of VDBP, resulting
in marked variation in results [28]. Major differences in
concentrations obtained with the same samples were
reported, which serves to highlight some of the problems of
standardization of current (and previous) assays that exist
as well as VDBP effects.
An area of concern in relation to immunoassays is the
variability that exists in the detection of 25OHD
2
. Some
assays claim to have 100 % cross-reactivity with exoge-
nously added 25OHD
2
and 25OHD
3
and are therefore
equipotent for the measurement of the two metabolites.
Other assay manufacturers admit to lower cross-reactivity
with exogenous 25OHD
2
(75 % [kit insert from IDS,
Boldon, UK], 52 % [product insert from Abbott, North
Chicago, IL]), while some assays were specically
designed to measure only 25OHD
3
(product insert from
Roche, Indianapolis, IN). Reports have conrmed the
variability of commercial immunoassays to detect 25OHD
2
[21, 2931], and this is postulated to be due to differing
binding afnities to VDBP, with 25OHD
3
exhibiting a
stronger afnity than 25OHD
2
[32]. Of further concern is
the fact that some assays have been shown to have prob-
lems detecting 25OHD
2
. In a Vitamin D
2
(ergocalciferol)
supplementation study, venous sampling following oral
administration of Vitamin D
2
demonstrated a signicant
underestimation of 25OHD
2
by three immunoassays [21].
There appears to be a change in vitamin D
2
following oral
consumption and subsequent metabolism and circulation in
the blood in some individuals, which leads to altered
Fig. 1 Pathway of Vitamin D
production
W. D. Fraser and A. M. Milan: Vitamin D Assays
1 3
recognition of 25OHD
2
in vivo by the antibodies in several
immunoassays. In some populations up to 30 % of samples
can have 25OHD
2
present due to vitamin D
2
being in the
food chain or to vitamin D
2
possibly being the only rec-
ognized and approved form of supplementation. It is
important to recognize this problem as serious misclassi-
cation of patients may arise if the wrong type of immu-
noassay is utilized in a population receiving vitamin D
2
.
When interviewed, most experts agree that in an ideal
situation an immunoassay reporting total 25OHD should be
equipotent for 25OHD
2
and 25OHD
3
[33]. Currently, it
would appear that only HPLC or tandem MS methods are
able to meet this requirement.
Almost all immunoassays show a high cross-reactivity
with 24,25(OH)
2
D, which increases in concentration with
increasing sun exposure; and as 25OHD increases and/or is
metabolized to 1,25(OH)
2
D, this provides an increased
supply of the two substrates for the 24-hydroxylase
enzyme. Concentrations in the region of 1015 nmol/L
have been recorded for 24,25(OH)
2
D in serum using gas
chromatography-MS [34], with reported circulation levels
of 1015 % that of 25OHD.
HPLC and LC-MS/MS
The rst direct UV detectionbased HPLC assays for
25OHD were published in 1977 [35, 36]. A cumbersome
chloroform methanol extraction was followed by chroma-
tography on Sephadex/silica gel columns, followed by
HPLC with UV detection. Improvements in HPLC meth-
ods centered around the introduction of reversed-phase
HPLC mainly using C18 columns [37], improved internal
standard material, improved sample extraction using
chloroformmethanol [32] and methanolhexane [38], then
extraction of samples using semiautomated technology
employing acetonitrile post-sample precipitation [39]; and
a combination of these approaches decreased HPLC run
times to less than 10 min, therefore increasing sample
throughput.
The techniques of sample extraction allied to chroma-
tography with MS detection methods offer increased
specicity for the molecule of interest, so the combination
of HPLC and MS in tandem, LC-MS/MS, has become a
commonly used technique in routine clinical biochemistry
laboratories. Early methods employed fast atom bom-
bardment with Cookson-type reagents [40], resulting in
derivatization of 25OHD, which improved detection of
25OHD. Isotope dilution-electrospray LC-MS/MS methods
performed on bench top analyzers became popular in the
mid-2000s with protein precipitation of the sample, liquid
liquid extraction, short run times, and computer processing
of chromatograms contributing to higher throughput and
ease of use [4143]. Deuterated 25OHD
2
and D
3
internal
standard material improves accuracy and veries recovery,
with improved extraction processes aiding in the removal
of phospholipids, thereby reducing the problem of ion
suppression [44]. Recent technical developments have
centered around reducing the manual component of sample
preparation prior to chromatography and elimination of
liquidliquid extraction using either separate automated
solid-phase extraction (SPE) [45], online solid-phase
extraction [46, 47], or online turbulent ow extraction [48].
All of these method changes contribute to increased
throughput, improved reliability, and better cost-effec-
tiveness of LC-MS/MS assays for 25OHD. A step change
in technology has recently been published that has
increased sample throughput to 300 samples/h, multiplex-
ing samples by differential mass tagging in LC-MS/MS
measurement with excellent assay performance (LLOQ
10 nmol/L [4 ng/mL] for 25OHD
2
, 5 nmol/L [2 ng/mL]
for 25OHD
3
) and CVs of 3.715.2 % across the reportable
assay range [49].
Isotope dilution LC-MS/MS is currently considered the
gold standard method for 25OHD measurement, being able
to simultaneously quantitate 25OHD
2
and 25OHD
3
, with
summation of the two values resulting in total 25OHD. One
criticism made about LC-MS/MS, however, is that there is
a multitude of home-brew or in-house methods
available using different sample preparation and extraction
methods, varying running conditions and buffers, different
HPLC systems, and multiple MS detection systems which
utilize different transitions for each molecule of interest.
This is aside from the issues relating to standards and QC
materials. A review of the International Vitamin D Exter-
nal Quality Assurance Scheme (DEQAS) results for the
LC-MS/MS group highlights the spread of results gener-
ated by these methods. While the majority of the methods
(7075 %) are positively biased against the all-laboratory
trimmed mean (ALTM), some are close to the mean
(1520 %) or negatively biased depending on the 25OHD
concentration measured (510 %). There has also been
concern raised regarding the presence of the 3-epi-25OHD
epimer of 25OHD, which because of the achiral nature of
LC-MS/MS cannot be separated from 25OHD by the
majority of current methods. An epimer is a nonsuperim-
posable/nonmirror image of a molecule that differs in
conguration at one carbon atom and has the identical
mass:charge ratio, and parent and product ion pairs fol-
lowing ionization. Although mainly seen in younger chil-
dren [50] and in specic disease states, a recent report has
claimed that 3-epi-25OHD
3
is present in 99 % of samples
tested, with concentrations ranging from 0.2 to 59.25 nmol/
L in samples from neonates to 80?-year-old adults [51].
The presence of an epimer may increase the total 25OHD
concentration measured by LC-MS/MS methods compared
to immunoassays as, apart from the Roche CPB assay,
W. D. Fraser and A. M. Milan: Vitamin D Assays
1 3
immunoassays do not cross-react with 3-epi-25OHD
3
[52].
It is important therefore to utilize an assay which does not
detect 3-epi-25OHD in neonates, and the presence of the
epimer in the reference standards (as discussed later)
should enable assay validation. The use of chiral chro-
matographic columns, cyano columns, and longer run
times can allow separation and quantication of 3-epi-
25OHD
3
[50, 5356], which should enable investigation of
the importance of this metabolite in vitamin D physiology
and pathophysiology. Isobars of 25OHD metabolites which
can cause isobaric interference, have also been reported in
a small number of subjects [54]; but their clinical impor-
tance and contribution to 25OHD measurement need to be
investigated further.
In 2011 a reference method [56] was recognized by the
Joint Committee for Traceability in Laboratory Medicine,
and a further reference method accepted by European
authorities has been published recently [55]. Comparison
with these methods in the future should allow a signicant
improvement in consistency and comparability of results
between methods.
Current 25OHD Methods
A report from DEQAS (January 2012) lists 16 categories of
methods and 1,119 users performing 25OHD measurement
of quality-assurance samples (Table 1). Immunoassay
methods dominate the scheme with 86 % of users, the
next largest group being LC-MS/MS with 11 %. A high
percentage of the immunoassay group used fully automated
methods on either dedicated or multianalyte platforms
(82 %). Evaluation of these automated assays has raised
concerns about increased variability in performance with
wider CVs [57], poor cross-reactivity with 25OHD
2
[58],
inaccuracy of results [28], interference from heterophilic
antibodies requiring a reformulation of the assay [59], and
failure to achieve minimal performance goals [60].
Standardization of 25OHD Assays
In the past the absence of a recognized international stan-
dard material for 25OHD
2
and D
3
meant that most inves-
tigators produced their own standard and calibrator
materials for HPLC and LC-MS/MS methods. The
approach adopted was to obtain the purest form of 25OHD
2
or D
3
available commercially and dissolve this in sufcient
quantity in ethanol to enable spectrophotometric estimation
of absorbance at both 265 nm (absorption maximum) and
228 nm (absorption minimum) to check for purity and then,
using the appropriate molar absorption coefcient (MAC),
calculate an accurate concentration of 25OH D
2
or D
3
in
the sample. Several problems arise from this approach
including the purity and source of the 25OHD, the need to
dissolve 25OHD in an organic solvent, the labile nature of
25OHD in water, the diluent/matrix used to make sub-
sequent standard dilutions resulting in the presence of
VDBP of different species with varying afnities for
25OHD
2
and D
3
, and the marked variability in the MACs
used. While the use of a commercially available common
standard material can improve the consistency of perfor-
mance of LC-MS/MS, the provenance of this material
remains in doubt [61] and the subsequent benet of the
approach questioned [62].
In 2009 the National Institute of Standards and Tech-
nology (NIST) produced standard reference materials (SRM
972 and SRM 2972) and certied the reference values for
25OHD
2
, 25OHD
3
, and 3-epi-25OHD
3
in four different
serum pools. Value assignment of SRM 972 was accom-
plished using a combination of three isotope-dilution MS
approaches, with measurements performed at NIST and at
the Centers for Disease Control and Prevention [63]. Three
of the four SRM 972 standards have exogenous metabolites
or are diluted with equine serum and have proved unsuitable
for standardizing commercial immunoassays. It has been
possible to align most HPLCand LC-MS/MS methods using
these standard materials, although the relatively low value
for the highest available concentration, around 40 nmol/L
(16 ng/mL), also limits the application of the material. Some
commercial manufacturers have started to use NIST-aligned
LC-MS/MS methods to calibrate their assay standards, and
this may increase the harmonization of results obtained by
different methods.
Table 1 25-Hydroxyvitamin D participants in DEQAS January 2012
Method Number
participating
CV for samples
analyzed (%)
Diasorin Liaison total 401 9.613.7
IDS EIA 136 10.211.8
IDS-iSYS 131 8.112.9
LC-MS/MS 125 8.914.9
Automated IDS EIA 106 8.610.2
Roche total 25OHD 61 8.216.9
Siemens ADVIA Centaur 46 14.019.6
Abbott Architect 34 6.810.7
HPLC 31 15.435.6
Diasorin RIA 26 14.120.7
IDS RIA 11 5.215.3
Unknown 4 9.524.3
Roche 25OHD
3
3 7.139.7
DIAsource 2 7.636.7
Diazyme 25OHVitD 1 NA
Chromatographic ligand 1 NA
Binding assay
NA not available
W. D. Fraser and A. M. Milan: Vitamin D Assays
1 3
The DEQAS scheme was established in 1989 and has
contributed signicantly to the improvement in assay per-
formance and development. Over a 15-year period an
improvement in mean interlaboratory imprecision (CV)
from 32.0 % (1994) to 15.3 % (2009) was observed, and
most major methods were giving results within plus or
minus 7.4 % of the ALTM (2009) [64]. The expansion in
LC-MS/MS technology and respondents to the scheme
have transiently stopped this trend, with most LC-MS/MS
methods positively biased against the ALTM, which is
heavily based on immunoassay users. The introduction of
SRMs for 25OHD will hopefully restore the improvement
in interlaboratory imprecision.
Measurement of 1,25(OH)
2
D
1,25(OH)
2
D is the biologically active form of vitamin D
whose production and metabolism is tightly regulated; it is
highly lipophilic, is relatively labile, and circulates at
1,000-fold lower concentration in blood than 25OHD. If
25OHD is considered a difcult analyte to measure [14],
1,25(OH)
2
D presents an even greater analytical challenge.
It is essential to perform extraction of 1,25(OH)
2
D from
serum/plasma to remove interfering substances and to
remove 1,25(OH)
2
D from VDBP and albumin. A major
problem that exists with solvent extraction methods is that
the methods used in the past were the same as those used to
extract 25OHD, so this major interferent was also present
in the extract at concentrations 10
3
greater than that of
1,25(OH)
2
D. The specicity for 1,25(OH)D is therefore
mainly dependent on the detection methods used.
CPB
The earliest published method used 20 mL of sample
extracted using methanolchloroform and subsequent
chromatographic purication [65]. Detection was performed
using intestinal vitamin D receptor freshly isolated from
chicken intestine, so this was a classical CPB assay utilizing
[
3
H]-1,25(OH)
2
D. The chromatin receptor was highly spe-
cic for 1,25(OH)
2
D, and this enabled detection with a
claimed assay sensitivity of 0.2 pmole of pure standard,
although the lowest concentration detected in plasma was
26 pmol/L (10 pg/mL). Improvements in purication and
incorporation of HPLC separation allowed simultaneous
detection of 25OHD and a signicant reduction in sample
requirement. Further developments in CPB assays came
from the use of SPE sample using C-18 Sep-Pak and silica
cartridge purication plus the move to utilizing vitamin D
receptor isolated from calf thymus glands, which was much
more stable than chick intestine preparations. This assay was
able to detect low 1,25(OH)
2
D, 27.6 3.9 pmol/L
(10.6 1.5 pg/mL), in chronic renal failure [66]. Com-
mercial methods employing the calf thymus receptor assay
became available, and a number of modications have been
made to the method over several years; but the specicity of
the thymus receptor for 1,25(OH)
2
D meant that the radio
receptor assay (RRA) has remained a standard comparator/
reference method for over 35 years.
Immunoassay
In 1978 the rst report of an RIA for 1,25(OH)
2
D was
published [67]. The assay required sample purication due
to the relatively poor specicity of the rabbit antibody, and
a tritiated tracer was employed but overall was relatively
insensitive compared to the RRA, with a detection limit of
52 pmol/L (20 pg/mL). Improved antibody technology
resulted in RIAs with better performance and detection
limits, but antibody cross-reactivity with 25OHD and
24,25(OH)
2
D meant that extensive purication of
1,25(OH)
2
D from serum was still required. By manipula-
tion of the extraction and chromatography or use of par-
ticular antibodies, assays specic for 1,25(OH)
2
D
2
and
1,25(OH)
2
D
3
were produced [6870]. A major advance
was the development and commercialization of an RIA for
1,25(OH)
2
D that required minimal sample prepurication,
did not need internal standardization, utilized calibrators in
an equivalent matrix to samples, and utilized a
125
I-labeled
tracer that allowed gamma counting of the assay. This
assay involved acetonitrile extraction, followed by treat-
ment of the extract with sodium periodate, then a second
extraction and purication of 1,25(OH)
2
D by solid-phase
chromatography (C18OH and silica cartridges), followed
by quantication by RIA. A detection limit of 6.2 pmol/L
(2.4 pg/mL) was quoted, and recovery of 1,25(OH)
2
D
2
was
6471 % and that of 1,25(OH)
2
D
3
90101 %; and by
converting the 24,25(OH)
2
D
3
and 25,26(OH)
2
D
3
to alde-
hyde and ketone forms using sodium periodate, cross-
reactivity with known metabolites was signicantly
reduced [71]. Further analytical and clinical validation of
the commercial assay (Diasorin, Stillwater, MN) was
published in 2002 [72]. A highly novel approach to sample
purication and extraction was adopted by IDS in a com-
mercial assay utilizing immunoextraction separating
1,25(OH)
2
D from delipidated samples using an antibody
bound to a mini-immunocapsule. Elution of the sample from
the capsule, evaporation to dryness, then reconstitution prior
to RIA results in a rapid, user-friendly assay [73]. The IDS
RIA has been modied to a nonisotopic format utilizing
avidin-linked horseradish peroxidase and tetramethylbenz-
idine substrate to generate a signal at 450 nm. Both the RIA
and EIA show excellent correlation with a RRA and a limit
of detection of 9.4 pmol/L (3.6 pg/mL) [74]. Concerns have
been raised about a possible contribution to 1,25(OH)
2
D
W. D. Fraser and A. M. Milan: Vitamin D Assays
1 3
measurement from other 1a-hydroxylated metabolites [75],
and cross-reactivity for 1,25(OH)
2
D
3
26,23-lactone, 1,24,25
(OH)
3
D
3
, and 1,25,26(OH)
3
D
3
has been demonstrated in
both the Diasorin and IDS assays [71].
HPLC, LC-MS/MS, and GC-MS
Measurement of 1,25(OH)
2
D by direct detection UV
methods is not possible due to the low concentration in
blood (picomoles per liter). 1,25(OH)
2
D has few ionizable
polar groups, so techniques to increase the ionization
efciency (namely, sample derivitization) have been
incorporated in all of the published methods.
An isotope dilution-mass fragmentography assay for
1,25(OH)
2
D was rst published in 1979 [76]. Extraction of
20 mL of serumwas performed using chloroformmethanol
after addition of [26-
2
H
3
]-1,25(OH)
2
D
3
and purication by
liquid chromatography. The puried material was converted
into the trimethylsilyl ether and analyzed by GC-MS. The
lower limit of quantication (LLOQ) was 13 pmol/L (5 pg/
mL), with a CV of 5 %; but the large sample volume limited
the general applicability of the assay. An LC/thermospray
MS technique using positive and negative ion detection after
online postcolumn Diels Alder derivitization obtained an
LLOQ of 1 nmol/L [77]. 1,25(OH)
2
D
3
in rat and pig serum
was measured by LC-MS/MS after extraction and purica-
tion using both reverse phase and normal phase on a C18
cartridge, followed by generation of an ammonium adduct
prior to positive electrospray ionization. The LLOQ was
20 pg/mL using 1 mL of sample [78]. Derivitization using
4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), a Cookson-
type reagent, of a SPE sample and measurement using
ultraperformance liquid chromatography (UPLC) electro-
spray tandem MS allowed simultaneous quantication
of a prole of vitamin D metabolites (1,25[OH]
2
D
2
,
1,25[OH]
2
D
3
, 24,25[OH]
2
D
3
, 25OHD
2
, and 25OHD
3
) with
an LLOQ of 25 pg/mL and CV of 516 % for 1,25(OH)
2
D
3
[79]. PTAD was also used in a method that quantitated the
same four metabolites with signicantly improved sensi-
tivity, 5 ng/L (12 pmol/L) for 1,25(OH)
2
D
3
, that employed
selective SPE and microow LC-MS/MS [80]. The SPE
procedure enabled high sample loading on the UPLC col-
umn, and on-column sample focusing prevented band
broadening, allowing excellent separation and eliminating
endogenous interference while minimizing ion suppression
but with a run time of 27 min. Lithium acetate has been used
to produce ionizable adducts in a method that uses a complex
online sample processing procedure with the use of a per-
fusion column followed by a chain of two monolithic col-
umns to clean and enrich the sample prior to quantication
on a highly sensitive LC-MS/MS [81]. Both 1,25(OH)
2
D
2
and 1,25(OH)
2
D
3
can be measured on 30 lL of sample with
an LLOQ for 1,25(OH)
2
D
3
of 15 ng/L (36 pmol/L), a CV of
515 % across physiological concentrations, and a total run
time per sample of 30 min. IDS immunoafnity column and
reagents were incorporated into a sample preparation pro-
cedure following protein precipitation and SPE. Lithium
acetate was used to produce adducts prior to LC-MS/MS
analysis. This method removed isobaric interferences
and matrix effects, resulting in signicantly reduced ion
suppression with the resultant LLOQs of 3.9 ng/L
(9.1 pmol/L) for 1,25(OH)
2
D
2
and 3.4 ng/L (8.2 pmol/L)
for 1,25(OH)
2
D
3
with interassay CVs of 2.57.0 % [82].
A very similar approach using the IDS columns for immu-
noextraction but derivitizing using PTAD prior to UPLC
MS/MS resulted in improved sensitivity with LLOQs of
0.65 ng/L (1.5 pmol/L) for 1,25(OH)
2
D
2
and 1.25 ng/L
(3.0 pmol/L) for 1,25(OH)
2
D
3
and interassay CVs of
8.013.0 %. All of the LC-MS/MS methods described above
are fairly labor-intensive, with manual workows and lim-
ited throughput (Strathmann et al. [83] quote 7.5 h to process
21 samples). Future developments will likely incorporate
more online processing, automated sample preparation, and
mass tagging [49] to increase throughput.
A comparison of LC-MS/MS measurement of
1,25(OH)
2
D and the IDS immunoassay has highlighted
differences in recognition of 1,25(OH)
2
D
2
when high-dose
vitamin D
2
(300,000 IU) is administered intramuscularly.
The LC-MS/MS method detected a signicant increase
in total 1,25(OH)
2
D, mainly due to an increase in
1,25(OH)
2
D
2
; but the immunoassay failed to detect this
increase in 1,25(OH)D [84]. This appears to be very similar
to the situation observed with 25OHD immunoassays,
discussed previously, where antibodies incorporated in the
assay system are not able to recognize the 1,25(OH)
2
D
2
synthesized in vivo after exogenous administration of
vitamin D
2
.
Current 1,25(OH)
2
D Methods
A recent report from DEQAS (January 2012) lists seven
categories of methods and 123 users performing
Table 2 1,25-Dihydroxyvitamin D participants in DEQAS January
2012
Method Number
participating
CV for samples
analyzed (%)
IDS RIA 63 15.119.2
DiaSorin RIA 28 22.324.1
IDS EIA 21 21.722.9
LC-MS/MS 5 14.723.9
Biosource CT (ELISA) 3 2.714.9
AMP RIA 2 2.721.7
In-house RIA 1 NA
NA not available
W. D. Fraser and A. M. Milan: Vitamin D Assays
1 3
1,25(OH)
2
D measurement of quality-assurance samples
(Table 2). Immunoassay methods dominate the scheme
with 96 % of users: 68 % are performing an IDS method,
and 4 % report results by LC-MS/MS. The CVs reported
are wide for all the methods at around 20 % for most
samples circulated, and this reects the difculty of mea-
surement and a lack of availability of an international
standard material.
Standardization
There is no international standard material available, so
most methods utilize in-house standards, produced as
previously discussed for 25OHD using solvent dissolution,
spectrophotometric analysis at 264 nm, and a MAC of
19,400 to calculate the concentration. Human serum
stripped of 1,25(OH)
2
D
3
by activated charcoal can then be
used to make calibrators of varying concentrations by
adding dissolved 1,25(OH)
2
D
3
[71]. Some authors report
using substrate addition technology to calculate concen-
trations, and commercial standard material with a consen-
sus mean concentration attached is also available.
Conclusions
Measurement of vitamin D and its metabolites is difcult.
Many methods have been developed over the years, with
immunoassay becoming the most popular method for both
25OHD and 1,25(OH)
2
D due to a combination of avail-
ability, ease of use, relative cost, high throughput using a
small sample volume, and rapid turnaround. Recently,
questions have been asked regarding immunoassays, with
problems of accuracy and specicity being demonstrated
and the recognition of uctuations of assay performance
noted in long-term surveys such as the National Health and
Nutrition Examination Survey [85]. Many clinical labora-
tories have moved to LC-MS/MS technology, with poten-
tial greater specicity and accuracy of measurement. The
recent generation of international SRM, the promise of
human serumbased SRMs in 2012, and the development
and acceptance of reference method procedures allied to
the technical innovations in sample processing and analysis
should all contribute to an improvement in the accuracy,
precision, and harmonization of results generated by all
methods in use currently and those developed in the future.
Acknowledgment The authors thank Dr. Graham Carter for per-
mission to use the synopsis of data from the January 2012 DEQAS
report.
Disclosures WD Fraser has given lectures on vitamin D measure-
ments for all the companies listed in this manuscript currently
involved in measuring vitamin D and provides advice on vitamin D
measurement to IDS, Siemens, Roche and Nichols Diagnostics.
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