CLIN.
CHEM.
24/2,
287-298
(1978)
Assay of Vitamins
Human
D2 and D3, and 25-Hydroxyvitamins
Plasma by High-Performance
I describe
a new assay that is capable
of measuring
vitamin
02, vitamin D3, 25-hydroxyvitamin
D2, and 25-hydroxyvitamin 03 in 2 ml of plasma
or serum.
Plasma
is extracted
by the Bligh and Dyer technique
[Can.
J. Biochem.
Physiol.
911(1959)],
the lipid component
is fractionated
by two
high-performance
liquid-chromatographic
systems
based
upon adsorption
and reversed-phase
chromatography,
and
each of the four vitamin D metabolites
is measured
by its
absorbance
at 254 nm. The method
has a sensitivity
limit
of 0.5 sg/liter
of plasma.
The identity of metabolite
peaks
was confirmed
by mass
spectrometry,
ultraviolet
absorption
spectrophotometry,
and rechromatography,
and
there was good correlation
(r = 0.84) between
plasma
25-hydroxyvitamin
0 as measured
by the present
method
and by a protein binding assay developed
in our laboratory.
Mean concentrations
of vitamin D and 25-hydroxyvitamin
D in normal adults (n = 25) in December
were 2.2 ± 1.1
(SD) and 16 ± 3.9 (SD) tg/liter,
respectively.
25-Hydroxyvitamin
D2 made up 31% of the total 25-hydroxyvitamin D. Patients
receiving
pharmacological
doses of vitamin D had values for vitamin D and 25-hydroxyvitamin
D that were 10- to 100-fold normal. This method provides
a rapid, reliable
physico-chemical
assay that appears
to
have advantages
over existing protein binding assays
and
37,
be used
AdditIonal
normal
assay
Assays
.
to measure
drawbacks
circulating
Keyphrases:
values
ultraviolet
vitamin
mass
‘
vitamin
D and
spectra
in
man,
its metabolites
protein-binding
spectrophotometry
and
D (25-OH-D)
(1, 2) and
D (1,25-(OH)2D)’
(3, 4) have
in the study
of vitamin
D me-
aids
but
existing
limitations.
binding
assays
offer
sitive to nonspecific
methods
Commonly
poor
reproducibility
interfering
substances,
have
used
be removed
by chromatography.
Alternative
chemical
assays
require
time-consuming
schemes
and
To overcome
viously
assays,
certain
protein-
and are senwhich
must
physicopurification
before
analysis.
met with
in pre-
and
protein
binding
sought
to develop
high-performance
physico-chemical
assay
based
an
upon
chromatography
(HPLC).
This
technique
resolves
all the known
metabolites
of vitamin
D2 and vitamin
D3 in a synthetic
mixture
of the compounds
(5). Because
pure metabolites
of vitamin
D
possessing
the characteristic
cis-triene
structure
were
detectable
in the low-nanogram
range with an ultraviolet detector
(5), we believed
that it should
be possible
to measure
simultaneously
many
of the metabolites
present
liquid
in a sample
of human
plasma.
D2 and
D2 and D3 in 2 ml of human
assay
for
cedure
Dyer
D3 and
plasma
vitamins
consists
of extracting
technique
tabolites
(6) and
by two
HPLC
the
sample
separating
the
by the
has
A preliminary
an
pro-
Bligh-
vitamin
D meavailable
to providing
a conveby a physicochemical
an advantage
over
avail-
in that
it proof both
25-
protein
binding
assays
on the concentrations
OH-D2
and 25-OH-D3,
and
the concentration
of vitamin
D3.
where
I report
on commercially
In addition
of 25-OH-D
procedure
able competitive
vides
information
Here,
25-hydroxyvitamin
or serum.
The
the
steps
pre-packed
columns.
nient and rapid assay
it can be used to measure
D-i.e.,
vitamins
D2 and
summary
of this
work
appears
else-
(7).
Materials and Methods
Apparatus
The
Model
chromatograph
LC 204 fitted
system,
Department
of Biochemistry,
University
of Toronto;
and the Research
Institute,
The Hospital
for Sick Children,
555 University
Ave.,
Toronto,
Ontario,
Canada
M5G
1X8.
‘Nonstandard
abbreviations
used:
25-hydroxyvitamin
D, 25OH-D;
1,25-dihydroxyvitamin
D, 1,25-(OH)2D;
high-performance
liquid
chromatography
(-ic),
HPLC;
and hydroxyalkoxypropyl
Sephadex,
HAPS.
Received
Sept.
19, 1977; accepted
Dec. 2, 1977.
chemical
derivatization
some
of the problems
reported
I have
technique,
0.
of 25-hydroxyvitamin
1,25-dihydroxyvitamin
proved
to be useful
tabolism
Liquid Chromatography
Jones
Glenville
can
D2 and D3 in
olet
U6K
fixed
injection
wavelength
ters Associates,
iments
a Model
nm)
detector
N.J.
07675)
wavelength
I used in these
studies
was
with a Model
6000
A pumping
value,
(254
Milford,
SF 770
Instrument
connected
detector.
CLINICAL
a Model
in
In either
CHEMISTRY,
440
detector
(all
Mass.
01757).
variable-wavelength
(Schoeffel
was
and
nm)
series
case,
24,
Waexper-
(200-400
Westwood,
after
dead
Vol.
ultravi-
from
In some
Corp.,
a
the
fixed-
Space
between
No.
1978
2,
287
2
ml
PLASMA
Ci/mmol)
was supplied
by New
England
Nuclear,
Boston,
Mass.
02118. [3a-3H] Vitamin
D2 (3.5 Ci/mmol)
4
Add
5000
cpm
[H]
each
25-OH.D3
and
PH]
was
D2
4
Extract
w,th
LIQUID
Zorbax
MeOHCHCI3
(21)
‘I
CHROMATOGRAPHY
SIL
5 5.,
Isoproponol
-
synthesized
by the
Ci/mmol)
tetraene
Hexane
were
biologically
homogenate
purified
CHROMATOGRAPHY
LIQUID
/
\
Containing
25 OHD3
for the high-performance
Iiquid-chromatographic
assay of vitamin D (D2/D3) and 25-hydroxyvitamin
D (02/03)
human plasma
Fig.
1. Scheme
AnalytIcal
in
were
assessed
by comparing amounts
of [3H]D2 and
InItially to those amounts collected from chromatography
recoverIes
added
[3H125-OH-03
on Zorbax.OOS
the detector
phy
was
and
the exit
minimized
less-steel
by
from
use
the liquid
chromatogra-
of 0.009-inch
(i.d.)
6.2 mm i.d.), preZorbax-SIL
or ZorbaxDu Pont
Instruments,
(22
cm
mass
spectrometer
to
a Model
620
computer
(both from Varian
Instruments,
Palo Alto,
Calif.
94303).
Samples
were introduced
by a directinsertion
probe,
temperature-programmed
from 50 to
300 #{176}C
during
500s. Ionization
voltage
was 70 eV and
background
was subtracted
by using a Varian
module
subtractor.
Scintillation
counting
was performed
on a Model LS
355 (Beckman
Instruments,
Palo Alto, Calif. 94303)
ambient-temperature
external
scintillation
counter,
fitted
with
standardization.
All solvents
Muskegon,
by the
method
and phosphate
therapy
with
were
Mich.
from
49442,
Burdick
& Jackson
“distified-in-glass”
grade.
of vitamin
D2 or D3 (1000-100
000 mt.
of Blood
To 2 ml of plasma
i
were added
5000 cpm
at 4 #{176}C
for 30 mm ensured
exogenous
and endogenous
a thorough
suggested
by the work
lipids
were extracted
gifts
from
Drs.
J. A. Campbell,
Jack Hinman,
and John Babcock
of the Upjohn
Co.,
Kalamazoo,
Mich.
49001.
[1,2-3H]Vitamin
D3 (6 Ci!
mmol)
was obtained
from Amersham/Searle
Corp.,
Arlington
Heights,
Ill. 60005. [26,27-3H]25-OH-D3
(9.3
CHEMISTRY,
and
equilibration
of
form
(2/1) according
to the method
of Bligh
and
(6). To separate
phases,
I added
2.5 ml of saturated
and 2.5 ml of chloroform,
1000 X g for 10 mm.
followed
The
additional
chloroform
as
The
Dyer
KC1
by centrifugation
aqueous
(upper)
at
layer
was
re-extracted
roform.
The
with
an
combined
rated
under
extract.
This
the presence
reduced
pressure
to yield
a yellow
lipid
occasionally
appeared
cloudy,
because
of
of white
insoluble
material,
when
the ex-
with
a Swinney
zm Teflon
filter
filter
holder
6-mi portion
of chlolayers
were evapo-
containing
(organic
Associates).
The
sample
clarified
Vol.
24,
No. 2, 1978
2
to a 5-ml
conical
Co., Rockford,
of nitrogen
International
Unit
a Millipore
clarification
lipid
200 l of the isopropanol/hexane
precipitates,
sometimes
observed
CLINICAL
of [3H]D2
vitamin
D metabolites,
of Kida and Goodman
(11).
with 7.5 ml of methanol/chloro-
OH-D3
288
units/day).2
Samples
transferred
Chemical
a stream
generous
or after
doses
5000 cpm of [3H]25-OH-D3,
each in 10 l of ethanol
(Figure
1). These
labeled
metabolites
served
to monitor
analytical
recoveries
during
various
steps
of the assay.
Incubation
of the plasma
with radioactive
metabolites
ters
D metabolites:
were
et al.
metabolism
before
graded
pharmacological
Crystalline
vitamin
D2 and
vitamin
D3 were purchased
from Sigma Chemical
Co.,
St. Louis,
Mo. 63178.
Crystalline
25-OH-D2
and 25Vitamin
of Ellingboe
tract
was redissolved
in 1 ml of isopropanol/hexane
(5.5/94.5
by vol). For this reason,
before
chromatography, the solution
was filtered
through
a syringe
fitted
Materials
Solvents:
Labs., Inc.,
spectroscopic
43216)
of calcium
beginning
X
coupled
Inc.,
Hydroxyalkoxypropyl
25-OH-D3
were evaluated
by using plasma
from 25 apparently
healthy
adult laboratory
workers
at our hospital, sampled
during
December.
Some
studies
involved
the use of plasma
obtained
from children
with disorders
Extraction
columns
packed
with microparticulate
ODS,
were purchased
from
Wilmington,
Del. 19898.
A Unicam
SP 1800 Spectrophotometer
(Pye-Unicam,
Cambridge,
England
CB1 2PX) was used to measure
concentrations
of vitamin
D compounds
in solution
(e
= 18 300).
Mass
spectra
were
obtained
with
a MAT
CH-5
Ohio
08854.
metabolites
before
use (5).
supplied
by Pharmacia
Procedures
stain-
tubing.
Stainless-steel
Ci/mmol)
was
D2 by use
Plasma
samples:
For much
of the developmental
work on the assay I used a 300-ml
pool of normal
human
plasma.
Normal
ranges
for vitamin
D, 25-OH-D2,
and
containing
{3H]
(3.5
(10).
25.OH.D3
25.OH.D2
D2
[H]
lumbus,
Zorbax-ODS
91% MCOH.H20
MeOH-H20
N.J.
(11.25
Sephadex (HAPS)
was synthesized
from Sephadex
LH 20 and
Nedox
(a long-chain
olefinic
epoxide,
carbon
chain
length,
15-18)
(a gift of the Ashland
Chemical
Co., Co-
CHROMATOGRAPHY
Zorbax-ODS
985%
[3H]borohydride
3a-acetoxyergosta-3,5,7,22-
from
[3a-3H}
vitamin
incubations
(9). All
by HPLC
LH2O
was
Sephadex
Piscataway,
LIQUID
of
[3a-3H]25-OH-D2
(8).
generated
of liver
sodium
reduction
extract
screw-capped
Ill. 61105)
gas.
of vitamin
The
D
was
vial
and
residue
was
0.45
kit; Wafinally
(Pierce
evaporated
in
dissolved
in
mixture.
Flocculent
after refrigeration
=
25 ng.
of
these
extracts,
were
removed
by centrifugation
at 2000
STANDARDS
(a)
0
Xgfor2min.
Chromatography
of Lipid
As shown
fractionation
schematically
of the lipid
Zorbax-SIL
(microparticulate
vitamin
D and
Extracts
in Figure
1, the
extract
was done
silica),
25-OH-D
fractions
first stage
by HPLC
to separate
from
most
of
on
the
05-
of the
E
contaminating
lipid. The total extract,
containing
about
20 mg of lipid dissolved
in 200 il of the isopropanol!
hexane,
was injected
at a constant
flow rate of 1.5 ml/
mm
and
normal
psi)
into
a 22 cm
operating
pressure
of 8.28
MPa
6.2 mm (i.d.) Zorbax-SIL
corresponding
to “vitamin
D” (5.5-7
to remove
any
strongly
adsorbed
04
03
0
‘I,
02
01
contaminating
0 and
25-OH-D
impurities.
ml!min
of the
normal
operating
in 3 ml
ml of Aquasol
activity
was
with counting
stock
A constant
D,
recovery
solvent
of
identical
rate
of 1.5
6.9
to
MPa
that
(1000
added
13H]vitamin
D2
depicted
in Figure
1.
fraction,
dissolved
(91/9
produced
to
the
was counted
in the same mixml of Aquasol),
to assess the
of
procedure
“25-OH-D”
methanol/water
flow
a
psi).
D2, which was eluted between
13.5 and 15.5
of solvent,
was collected
and mixed with 10
(New England
Nuclear),
and the radiocounted
in a liquid
scintillation
counter
efficiency
of 30%. A 10-izl aliquot
of the
pressure
[3Hlvitamin
overall
The
by vol),
was
through
in
the
100
chromatographed
of
on
a 22 cm X 6.2 mm(i.d.)
Zorbax-ODS
column,
to separate
25-OH-D3
(14.0 mm) from 25-OH-D2
(15.3 mm) and
resolve
residual
contaminating
impurities.
A constant
solvent
flow rate of 1.5 ml/min
produced
a normal
operating
pressure
of 9.66 MPa (1400 psi). [3H]25-OH-D3,
which eluted
between
13.0 and 15.0 mm in 3 ml of effluent, was mixed directly
with 10 ml of Aquasol
and the
radioactivity
standards
alone
15
by HPLC on Zorbax-SIL
of lipid extracts
250 ng; 03: 200 ng; 25-OH.02:
(03:
825 ng; 25-OH-i)3: 920
was
counted
in
a
liquid
scintillation
counter
at a counting
efficiency
of 30%. A lO-jl aliquol
of the stock L3H125-OH-Da,
identical
to that added
to
the original
plasma
sample,
was counted
in the same
mixture
(3 ml of methanol-H20/10
ml of Aquasol),
to
assess the analytical
recovery
of [3H]25-OH-D3
through
the overall
procedure.
is a microparticulate
on to its surface.
silica
with
octadecyl
silane
of Vitamin
Rechromatography
Normal
Plasma
D Fraction
from
To measure
vitamin
D in normal
plasma
samples
it
was often necessary
to use an extra step of HPLC
after
eluting
the vitamin
D fraction
from the Zorbax-ODS
column.
Effluent
appearing
between
13.5 and 16.5 mm
and containing
both vitamin
D2 and vitamin
D3 was
collected
and evaporated
under a stream
of nitrogen
gas.
The purified
extract
was dissolved
in 100 pi of isopropanol/hexane
(1/99 by vol) and rechromatographed
on
Zorbax-SIL
(22 cm X 6.2 mm i.d.; 2 ml/min;
6.9 MPa)
in the
same
solvent.
[3H]Vitamin
D2, which
between
12.5-14.5
mm, was collected
and
under
nitrogen.
This fraction
was counted
nol/Aquasol
Quantitation
as described
of
Vitamin
was
eluted
evaporated
in metha-
above.
D Metabolites
The triangulation
method
(12) was used to measure
peaks that had appropriate
retention
times and no peak
distortion
Zorbax-ODS
residues
bonded
tO
(mm.)
(b) standards in the presence of the lipid extract from 2 ml plasma
Arrows denote vitamin D and 25-OH-i) fractIons normally collected
for subsequent analysis on Zorbax-CIDS
Fractions
methanol/water
original
plasma
sample,
ture (3 ml of methanol/lO
analytical
5
ng)
of Vitamin
[3H]Vitamin
0
TIME
(a)
The “vitamin
D” fraction,
dissolved
in 100 iii of
methanol/water
(98.5/1.5
by vol), was subjected
to reversed-phase
chromatography
on a 22 cm X 6.2 mm
(i.d.) Zorbax-ODS3
column,
to separate
vitamin
D2 (14.6
mm) from vitamin
D3 (15.7 mm) and resolve
residual
mm
-
Fig. 2. Prepurification
plas-
components.
HPLC
LIPID
(1200
after
15 mm; thus samples
for assay were injected
successively
every
15 mm. After
running
a series
of 6 to 12
samples,
the Zorbax-SIL
column
was washed
with
10
ml of methanol,
+2,nI PLASMA
C-.’
column.
mm) and
X
(b) STANDARDS
C
Fractions
“25-OH-D”
(10.3-14
mm)
(represented
by arrows
in
Figure
2) were collected,
evaporated
under a stream
of
nitrogen
gas, and redissolved
in 100 zl of the eluting
solvent
used for reversed-phase
chromatography
on
Zorbax-ODS.
Solvent
mixtures
employed
were: “vitamin D,” methanol/water
(98.5/1.5
by vol); “25-OH-D,”
methanol/water
(91/9 by vol). No significant
ultraviolet-absorbing
peaks eluted from the Zorbax-SIL
column
ma
2$OHD
0-I-
by contaminants,
obtained
step on Zorbax
ODS. In the case
tions from normal
plasma,
peaks
CLINICAL
CHEMISTRY,
during
the
HPLC
of “vitamin
D” fracwere measured
after
Vol. 24, No. 2, 1978
289
rechromatogarphy
on Zorbax-SIL,
proved the definition
of the peaks.
directly
to the amount
of vitamin
equation:
which greatly
imPeak area was related
D metabolite
by the
xPxxBxFx10
C254
2
where
Q
=
M
=
C254
=
P
=
quantity
of vitamin
D metabolite
in peak
ng,
molecular
weight
of vitamin
D metabolite,
molar absorptivity
of vitamin
D metabolite
at254nm
16500,
peak height
in absorbance
units,
B
=
peak
F
=
flow
The
run
base
in minutes,
rate
Analytical
recoveries
from
of vitamin
by comparing
Zorbax-ODS
initially
to the
the
chromatography
plasma
sample.
and
25-OH-D2
in the
effluent
with
that
case
of “vitamin
In the
D” peaks [3H]vitammn
D2 was used to assess
both vitamin
D2 and vitamin
D3 in plasma.
[3H]25-OH-D3
was used to assess recovery
OH-D3
were
in the
added
plasma.
C
x recovery
=
in the
by the
(2)
where
concentration
in plasma
in g!liter,
quantity
in peak in ng, and
recovery
factor
were
collected
7.3 ml of gel (3 g) were
the
first
used
for
puri-
to the column
solvent
(chloro-
9 ml of effluent
was
discarded.
18 ml
of effluent
25-OH-D2
was
plus
discarded.
the
The
next
[3H]25-OH-D2
7 ml
added
as
an indicator
of recovery
of 25-OH-D2,
and the subsequent
11 ml contained
25-OH-D3
plus [3H]25-OH-D3.
Each fraction
was subjected
separately
to HPLC
on
Zorbax-ODS.
Rechromatography
of 25-OH-D
fractions
on Zorbax-SIL.
Rechromatography
of 25-OH-D
fractions
the
collection
of both
25-OH-D2
and
25-OH-D3
as a single fraction
(13-16.5
mm) from the Zorbax-ODS
HPLC
step of the regular
procedure
(Figure
1).
The sample
was evaporated
in a stream
of nitrogen
gas and redissolved
in 100 il of isopropanol/hexane
C
=
(5.5/94.5
Q
=
at a constant
flow rate of 2 mi/mm.
[3H]25-OH-D3
was
collected
(9-10.5
mm)
and
counted
in methanol!
original
recovered
dpm
dpm
added
present
Aquasol.
to plasma
-
in metabolite
peak
The basic equations,
1 and 2, were combined
simple
program
for a Model
9810A Calculator
lett-Packard,
Palo Alto, Calif. 94304).
Additional
Methods
Used
to Validate
To validate
the normal
techniques
as alternatives
schemes)
procedure,
to (e.g.,
or in addition
to (e.g.,
the
other
into a
(Hew-
mass
spectrometry.
dure were
experiments
Procedure
I used
several
purification
rechromatography
with
those
of
stituted
regular
using
purification
LH
aspects
of the
by vitamin
D and 25-OH-D
by comparison
of 25-OH-D
obtained
assay.
Alternative
Sephadex
Quantitative
assessed
and
a
steps.
20 or HAPS
proce-
recovery
results
competitive-binding
In
some
chromatography
instances
was
sub-
for the initial HPLC
on Zorbax-SIL
used in the
procedure
(Figure
1). In these cases, only fracCLINICAL CHEMISTRY,
Vol. 24, No. 2, 1978
covery
Ultraviolet
Twenty
purified
vitamin
D or 25-OH-D
peaks)
the steps of the basic
method.
Additional
evidence
of peak identity
was provided by ultraviolet
absorption
spectrophotometry
and
290
with
of lipid extracts.
After
applying
dissolved
in 250 l of eluting
contained
involved
factor
2
or 25-OH-D2
The next 10 ml of effluent,
containing
the [3H]25OH-D3 and 25-OH-D2,
was collected
for HPLC analysis
on Zorbax-ODS.
(b) HAPS
column
chromatography
was done
by
using a graduated
pipet filled to the 2.7-mi mark with
3 g of HAPS
(carbon
chain length,
15-18),
swelled
and
eluted
with chioroform/hexane
(1/9 by vol) (14). Lipid
extracts
were applied
in 250 zl of eluting solvent
and the
first
recovery
of
Similarly,
of both 25-
The concentration
of the vitamin
D metabolite
original
2-ml plasma
sample
was calculated
equation:
mark
form/hexane)
each
D metabolites
radioactivity
25-OH-D3
subjected
to Zorbax-ODS
chromatography.
(a) Sephadex
LH 20 chromatography
was performed
as originally
described
by Holick
and DeLuca
(13) by
using a 1 X 60 cm gel bed swelled
and eluted
with an
equivolume
mixture
of chloroform
and hexane.
Positions of [3H]25-OH-D2
and [3H]25-OH-D3
were monitored by taking
aliquots
of 5-mi fractions
of effluent.
Alternatively,
10-ml
graduated
pipets
filled
to the
2.7-mi
and
was checked
during
of known
concentration.
containing
and
fication
a sample
in ml/min.
validity
of this equation
by injecting
standards
measured
in
tions
by vol).
HPLC
[3H]25-OH-D3
for both
on Zorbax-SIL
was
25-hydroxy
Absorption
used
was
performed
as an indicator
of re-
metabolites.
Spectra
2-mi
aliquots
of the
by the
regular
scheme
of HPLC
normal
shown
plasma
Peaks
pool
in Figure
were
1. Vi-
tamin D and 25-OH-D
fractions
were collected
from the
Zorbax-ODS
column.
All the vitamin
D and 25-OH-D
fractions
were pooled
and evaporated
in a stream
of
nitrogen.
Each pooled
fraction
was redissoived
in 200
tl of isopropanol/hexane
and 1O-il aliquots
were injected
into the liquid
chromatograph
fitted
with a
Zorbax-SIL
column,
a fixed-wavelength
(254 nm) detector, and a Schoeffel
variable-wavelength
detector
set
at wavelengths
between
215 and 315 nm. Details
of the
HPLC were dictated
by the metabolite
under study and
were described
above.
Spectra
were plotted
after measuring
the peak area
at each setting
of the variable
wavelength
detector.
To
ensure
that injection
volumes
were identical
foT each
wavelength
setting
of the variable-wavelength
detector,
we also compared
peak areas obtained
with use of the
fixed-wavelength
(254 nm) detector.
Correction
factors
were applied
to the variable
wavelength
were discrepancies
in the 254-nm
peak
covered
radioactivity.
Mass
of HPLC
Spectra
plot if there
area or the re-
Peaks
Table 1. Determination of 25-OH-D3 and 25-OHD2 Concentrations In the Normal Plasma Pool by
the Present Method after Various Initial
Purification Steps
Concna
of vitamin
D
mtabolft#{149}
(In ig/Iltsr)
as
det#{149}rmlnsd by
chromatog.
on Zorbax-OCS
25-OH-D3
25-OH-D2
Forty 2-ml aliquots
of the normal
plasma
pooi were
purified
according
to the regular
scheme
shown in Figure 1. 25-OH-D
fractions
were then pooled
and rechromatographed
25-OH-D2
and
arately,
evaporated
of ethanol,
mass
on Zorbax-SIL
25-OH-D3
fractions
and
under
as described
were collected
nitrogen,
transferred
dissolved
to the
direct
above.
sepin 10 tl
probe
of the
spectrometer.
Modified
Rat-Plasma
Assay
Binding
Protein
nitrogen
and
cubation
assayed
for 25-OH-D,
being
the
those
buffers
of Beisey
and
et al.
in(15).
Under
these conditions
we observed
no interference
by
neutral
lipid or vitamin
D present
in the 25-OH-D
fraction.
Interfering
substances
remained
as a residue
on the Sephadex
columns
used here.
Results
of Plasma
Samples
Recovery
during
the chloroform/methanol
extraction
stage of the procedure
was assessed
by comparing
radioactivity
present
in lipid extracts
to radioactivity
added
to plasma
samples.
Recoveries
were 89.1 ± 4.1%
(n = 6) for [3H]25-OH-D3
and 90.5% ± 3.2% (n = 6) for
[3H] vitamin
D2.
Step
I found
necessary
of Chromatography
that
vitamin
of Lipid
chromatography
before
because
of lipid
chromatography
D metabolite
on
peaks
were
Extracts
extracts
was
Zorbax-ODS,
masked
by
overlapping
impurities
when this step was omitted.
Several
types of chfomatography
were used as the
initial
purification
step in an attempt
to find a rapid,
reliable
method
of preparing
a 25-OH-D
fraction
suitable for analysis
by HPLC
on Zorbax-ODS.
Table
1
summarizes
the results
obtained
when a single plasma
sample
was analyzed
by HPLC
on Zorbax-ODS
after
one of four different
chromatographic
procedures.
Although
values
for 25-OH-D3
determined
by each alternative
were similar,
only two methods,
HPLC
on
Zorbax-SIL
and a lengthy
“open-column”
procedure
with Sephadex
LH-20,
permitted
the determination
of
25-OH-D2.
Furthermore,
with Sephadex
LH 20, the
All
mean
values
slap
15.7 ±
LH-20
SephadeX
2.8a
3.9
16.2 ± 1.4
fl.d.b
1.1
n.d.b
15.9±
± 0.4
chromatography
conditions
Extraction
purificatIon
col.
chromatography
(b) 3g Sephadex
LH-20 col.
chromatography
(C)
3 g HAPS column
for 25-OH-D
The method
used was essentially
a modified
version
of that developed
by Belsey et al. (1). Plasma
samples
(0.5 ml) were first extracted
with methanol/chloroform
according
to Bligh and Dyer (6). Extracts
were chromatographed
on 15 cm X 6 mm columns
of Sephadex
LH 20 eluted with an equivolume
mixture
of chloroform
and hexane.
All the 25-OH-D
(25-OH-D2
and 25-OHD3) was present
in the first 9 ml of effluent
from this
column.
The 25-OH-D
fraction
was evaporated
under
Initial
Initial
(a) 169
are
expressed
±1 SD.
(c
14.7
6.2mmX22cm
Zorbax-SIL
Mean ± SD, n =4.
± 0.9
3.3 ± 0.4
HPLC
b
n.d.
=
not determined because of overlapping kru#{231}sxity
peaks.
recovery
of 25-OH-P2
could
only
be assessed
by the
use
of [3H]25-OH-D2,
whereas
[3H]25-OH-D3
acted
as a
recovery
marker
for both 25-OH-D2
and 25-OH-D3
during
HPLC
on Zorbax-SIL.
In addition
to this disadvantage,
I observed
that all of the “open-column”
procedures
resulted
in greater
ultraviolet
contamination
of the 25-OH-D3
fraction
than did HPLC
on ZorbaxSIL. Sometimes
25-OH-D3
fractions
from Sephadex
columns
were so contaminated
as to make measurements
impossible.
Thus HPLC
on Zorbax-SIL
proved
to be the most efficient
and convenient
method
of prepurification,
and it was adopted
as the method
of choice
in the scheme
shown
in Figure
1. A typical
chromatogram of the purification
of a plasma
lipid extract
on
Zorbax-SIL
is illustrated
in Figure
2, frame b. Vitamin
D2, vitamin
D3, 25-OH-D2,
and 25-OH-D3
were added
to the lipid extract
as internal
standards.
By comparison
to Figure
2, frame a, which shows the chromatogram
obtained
with standards
alone, one sees that the presence of 15-20 mg of lipid from the extraction
of 2 ml of
plasma
does not significantly
affect
the chromatographic
position
of the vitamin
D metabolites.
The excellent
reproducibility
of retention
time
from one injection
to the next facilitated
the timed
collection
of
fractions
and in addition
validated
the assumption
that
[3H]25-OH-D3
could be used to assess the recovery
of
both 25-OH-D2
and 25-OH-D3.
Chromatography
of “Vitamin
0”
Fractions
on
Zorbax-ODS
With use of a methanol/water
(98.5/1.5)
solvent
system, the Zorbax-ODS
column
packing
resolved
vitamin
D2 (14.6 mm) and vitamin
D3 (15.7 mm) very well
(Figure
3a). In plasma
extracts
from patients
receiving
pharmacological
doses of vitamin
D (Figure
3c and d)
the peak corresponding
to the type of therapy
(D2 or D3)
stands out from the background
of ultraviolet-absorbing
peaks. However,
in normal
individuals
these vitamin
D
peaks are extremely
small and are often immeasurable,
CLINICAL CHEMISTRY,
Vol. 24, No. 2, 1978
291
0,01.
(a) Standards
(c) On
03
0.025’
therapy
0.010(a) Standards
(c) On
03
therapy
0
03
C
C
LfL__jF’L
E
0.005
Sn
2S.OH.03.
E
Sn
(‘1
0.0125
5Ui
5-
U
z
w
0’
0
U
z
,
i
0
U,
0
(I,
0001
5
1015200
5
D fractions
(b) plasma
(c) plasma
5
TIME (mm.)
Fig. 5. HPLC of 25-OH-D
on Zorbax-ODS
112 ng; D3: 117 ng)
of normal
adult
extract of child treated wIth 50000 let, units of vitamin D3 daIly
(cO plasma extract
of child treated with 100000 mt. units of vitamin 03 daily
Frames
b, c, and d represent
material
purified through the Zorbax-SIL
chromatoaphy
stage of FIgure 1. Figures above peaks represent
retention
times
(a)
standards
of vitamin
5 10 15 20 0
(mm.)
TIME
Fig. 3. HPLC
0
1015
(Di:
extract
in minutes. Flow rate = 1.5 mI/mm; solvent system methanol/water
by vol); K’t,, = 6.3: K’5, = 6.85; a
1.087; N = 4720; R5 = 1.19
(98.5/1.5
fractions
on Zorbax-ODS
standards
(25-OH.D3:
250 ng; 25-OH-02: 345 ng)
(b) plasma extract of normal
adult
(c) plasma extract of child treated with 50,000 lU. vitamin 03 daily
( plasma extract of child treated with 100,000 I.U. vitamin D2 daily
Frames
b, c, and d represent material purified through the Zorbax-SIL chromatoaphy
stage (see Figure 1). Numbers
above peaks represent
retention
times, in minutes. Flow rate
1.5 mI/mm;
solvent system methanol/water
(91/9
by vol); K’20.0,
=
6.80; K’250H.o,
=
6.05; a = 1.12; N = 4314; A5 = 1.51
(a)
E 00025C
fore vitamin
D3) is reversed
in the case of 25-hydroxy
derivatives.
Contamination
of 25-OH-D
peaks
with impurities
was not a problem.
25-OH-D3
and 25-OH-D2
peaks were
both observed
in normal
plasma
(Figure
5b), whereas
in patients
receiving
pharmacological
doses of vitamin
D the type of 25-OH-D
observed
in the plasma
corresponded
to the type of therapy
administered
(Figure
Sc
and d). In all cases, retention
times of metabolite
peaks
in plasma
extracts
were identical
to those observed
for
synthetic
standards.
Sn
5-
U.’
L
z
0
‘I)
0
2
4
6
TIME
Rechromatography
4.
FIg.
of vitamin
8
10
as in
Figure
14
D fraction
plasma
extract
Fraction from scheme shown in Figure 1 was subjected
cfromatogwphy.
Peak at 13.6 mm represents
a mixture
03. Conditions
12
16
(mm.)
from
normal
Zorbax-SL
of vitamIn D3 and vitamin
to
additional
8
owing to co-migration
with impurity
peaks (Figure
3b).
As a result
all “normal”
vitamin
D fractions
were rechromatographed
on Zorbax-SIL
as depicted
in Figure
4. The single peak shown
at 13.6 mm represents
total
vitamin
D, because
vitamin
D2 and vitamin
D3 migrate
together
in this system.
Chromatography
of “25-OH-D”
Fractions
on
Zorbax-ODS
With
from
order
a methanol/water
(91/9)
solvent
system,
the
column
well resolved
25-OH-D3
(14.0 mm)
25-OH-D2
(15.3 min) (Figure
5). Interestingly,
the
of elution
of the parent
vitamins
(vitamin
D2 be-
292
CLINICAL CHEMISTRY,
Zorbax-ODS
Vol. 24, No. 2, 1978
Overall
Analytical
Recovery
of Tracers
Overall
recovery
of radioactive
tracer,
from its addition to the original
plasma
through
the lipid extraction
and the two-column
procedure
to its final elution
from
HPLC
on Zorbax-ODS,
averaged
68.8 ± 6.5% (n = 12)
in the case of [3H]25-OH-D3
and 65.4 ± 6.0% (n = 8) in
the case of [3H]D2.
Calibration
Curve
for the
HPLC
Detector
As described
in Materials
and Methods,
vitamin
D
and its metabolites
were measured
by peak-area
measurement
by the triangulation
method
(equation
1). The
calibration
of the Waters
440 detector
at 254 nm was
checked
by injecting
solutions
of pure crystalline
vitamin D2 and 25-OH-D3
of known
concentrations
(as
determined
by ultraviolet
spectrophotometry).
Calibration
curves (not shown) were constructed
comparing
the amounts
of vitamin
or 25-OH-D3
injected
and
D2
RECOVERY
700
OF ADDED
E
a
C
/
600-
B
C
400500
.(
V
I-
“a
0‘U
I
I-
:./
0-
3-
25
.(
U)
‘I)
4
20
z
0
z
z
U’
I-
25
0.84
10
0
SLOPE
ay
0.95
1.09
z
INTERCEPT
5
-a
9
0
p
In
I
700-
25-OH-D
/
RECOVERY OF ADDED
#{244}oo25 OH D3
15
10
C”
20
HPLC
by
25
30
(ng/mI)
b
E
C
B
500-
>-
4
U)
C
V.)
4
400-
(:1
I.)
z
I‘U
0
z
0‘U
I
U-
- 27
z
0.98
LU
I-
SLOPE
0
100300200-
A.
(.12
y INTERCEPT
076
3.
/
0
-D
9
t
100
,
I
200
300
of vitamin
Recovery
U
400
OBSERvED
Fig. 6.
samples
I
500
U
600
I
700
0
I’,
(ng/ml)
(N
D metabolites
added
the amounts
determined
by peak area measurements
with the ultraviolet
detector.
There was good agreement
between
the two: for 18 such comparisons
the amount
of vitamin
D metabolite
detected
was 97.8% ± 4% (n
Precision
Plasma
Accuracy
of the
HPLC
Procedure
=
for
known
ng)
accuracy
quantities
to aliquots
of the
method
of vitamin
of a normal
was
D2 and
plasma
assessed
25-OH-D3
sample
by adding
(10-700
containing,
per liter, 2 g of vitamin
D and 16 ig of 25-OH-D
and
measuring
the vitamin
D and 25-OH-D
by the regular
two-stage
procedure.
There
was good agreement
between the observed
and theoretical
values for both D2
and 25-OH-D3
added
to plasma
(Figure
6).
by
500
400
HPLC
(ng!mI)
Fig. 7. Comparison
of plasma 25-OH-D results,
protein-binding
assay with those determined
technique
(a)
of normal
In a group
(b) In a group of children
03)
The
“within-run”
coefficient
of variation
of the
HPLC
procedure
for 25-OH-D
determined
as a single
plasma
sample,
25-OH-D
= 11.9 ± 1.1 zg/liter,
was 9%
on six repeated
determinations
on the same day. The
“between-run”
coefficient
of variation
for the HPLC
method
for 25-OH-D
determined
on the same plasma
sample
over a period
of six months
was 16% (n = 20).
The
300
200
25.OH.D
injected.
and
100
to plasma
Metabolites
(10-700
ng as determined
by ultraviolet spectrophotometry)
were
added to aliquots
of a plasma sample containing
endogenous
vitamIn 0 (2
zg/Iiter)
and 25-011-03
(16 zg/liter). Concentrations
of metabolites
were determined
by the procedure
depicted
in Figure
1
18) of that
1(
Normal
Ranges
healthy adults
receIving
0-100
000 mt.
for 25-OH-I)
and
determined
by
by the HPLC
units
of vitamIn
Vitamin
0 (03
or
D
I have applied
the new procedure
to about 100 plasma
samples
thus far. Total
25-OH-D,
determined
by the
HPLC
procedure
for a group
of apparently
healthy
laboratory
workers
in December,
was 16.0 ± 3.9 rg/liter
(n = 25), with a range of 9.1-23.9
gig/liter.
The total
25-OH-D
was composed
of 13.1 ± 10.6% 25-OH-D2,
the
remainder
appearing
as 25-OH-D3.
Total vitamin
D determined
by the HPLC
procedure
was 2.2 ± 1.1 gig/liter
(n = 24) with a range
of 0.8-4.7
ag/liter.
Comparison
HPLC
and the
of Determinations
Modified
Binding
of Total
25-OH-I)
by
Assay
Values
for total 25-OH-D
concentration
determined
by the HPLC
procedure
and those determined
by the
modified
binding
assay described
above
(1, 15) correlated well (r = 0.84) (Figure
7a). The range
of total
25-OH-D
values
determined
by the modified
binding
assay in our group of 25 healthy
laboratory
workers
was
CLINICAL
CHEMISTRY,
Vol.
24,
No. 2, 1978
293
005
0025
(a) Standard
B
(b)
03
C
In
I-
4
Ui
LI
Z
0025
,
‘0.625’
Cc) On
Cd) On
therapy
D3
02
(a)
Cc) On 03
therapy
0.01
Standards
02
23.OH.D3
JOD2
1
0,0125
0025
0.025
Stondord
E
0.0125
C
In
(N
I-
4
Ui
LI
0’
z
(b)
00025
4
0.025
Nor,nol
Cd) On
02 therapy
pIosno
therapy
extroct
0
Id,
4
0125
0.00125
0 0125’
LLt
0,
‘A
0
I
0246810121416
U
representing
fractions
(a)
solvent system isopropanol/hexane
2 mI/mm;
5.40; N
=
(1/99 by vol); K’0,
the
Metabolic
Peaks
Were
(a)
Rechromatography
samples
Table
on
from
fractions
of vitamin
2. Comparison
Represenadults
and
of normal
were
outlined
fractions
from
patients
vitamins
they
adult
25-OH-D
I
I
I
6
8
10
Total
(mm.)
of pooled
fractions
analyzed
by the
usual
two-step
in Figure
1. Then
vitamin
D and
were separately
rechromatographed
D, rerun
8. Though
Zorbax-SIL,
receiving
on Zorbax-SIL,
of
25-011-03
iotal
50 000
--
25-OH-D
25-OH-D2
pharmacological
are shown
doses
in Figure
D2 and D3 are not separated
on
have
slightly
different
retention
do
receIving
mt. unIts
Os/day
ChIld
Blood bank
March; Toronto
25-OH-D,
I
4
on Zorbax-SIL
of Vitamin D and 25-OH-D Concentrations
as Determined
Figure 1 a and by Methods Involving Further
Purification
Normal
I
2
The results
of rechromatography
of vitamin
D fractions
from
normal
plasma
have
already
been
given
(Figure
4). Assay
results
for vitamin
D-containing
Correctly
Zorbax-SIL.
a variety
0
on Zorbax-SIL.
Identified
tative
patients
procedure
25-OH-D
7b).
That
‘I
10
25-OH-D2/25-OH-D3
peaks
(25-OH-02:
166 ng; 25-OH-D3: 184 ng)
standards
treated
8.7 to 23.6 ag/liter
(mean,
16.3 ± 4.4 tg/liter).
The good
correlation
between
the HPLC
procedure
and the protein-binding
assay extends
across the range of 25-OH-D
values
(10-500
tg/liter)
observed
in children
receiving
0 to 100 000 mt. units
of vitamin
D per day (Figure
Evidence
8
(b) 25.OH-0
fraction
from
normal
adult
(c) 25-OH-D
fraction
from
child treated
with 50 000 mt. units vitamin
03
daily
( 25-OH-0 fraction from child treated with 100 000 mt. units vitamin 02
daily
Frames b, c, and d represent
rechromatography
of the fractions
depicted
in
frames b, c and d of Figure 5. Numbers above peaks represent
retention times,
in minutes.
Flow rate = 2 mI/mm;
solvent
system
isopropanol/hex.ar,e
(5.5/94.5
by vol); K’25.OH.02 = 2.36; K’2oH.o3
=
2.84: o = 1.203; N = 9733: and R5
2.92
11 660
=
6
Fig. 9. Rechromatography
of pooled
with 50 000 mt. units of vitamin
D3
daily
(
vitamin
D fraction
of child treated with 100 000 mt. units of vitamIn 02
daily
Frames c and d represent rechromatography
of the fractions
depicted in frames
c and dof Figure 3. Numbers above peaks represent retention times, In minutes.
Flow rate =
=
5.48; K’0,
4
TIME
(mm.)
vitamin
02 (240 ng)
0 fraction
of child treated
vitamin
(C)
2
UI
D3 (524 ng)
vitamin
(b) standard
I
810121416
Fig. 8. Rechromatography
on Zorbax-SIL
representing
vitamin D2/D3 peaks
standard
U
0246
TIME
(a)
I
by the Method
Depicted
In
b.c
Child
100
25-OH-D
25-OH-D2
receiving
mt. units
of 03/day
--
000
25-OH-D3
Total
25-OH-D
gfllier
Regular
assay
25-OH-D
Rechromatography
12.9
1.6
a
of 25-OH-D
on
1.6
of 25-OH-D
on
1.9
0
14.5
11.9
8
13.5
376
376
371
379
480
463
10
7
490
470
Zorbax-SIL6
Rechromatography
Zorbax-ODS
b
0,
03
Total
D assay
Rechromatography
Regular
Further
of 0 on ZorbaxSILt)
Rechromatography
C on Zorbax-SIL
The two-cohnin
procedure as in the scheme
shown in Figures 3, 4, 5, 8, and 9.
294
CLINICAL
CHEMISTRY,
Vol.
24,
No.
in Figure
2, 1978
1.
b
Ttwee-colurm
procedure.
D
D2
D3
zg,inor
Total
0
D2
Total
03
2.8
0
772
772
583
0
583
1.4
1.8
0
790
790
570
0
570
C
Fow.cokmvl
procedure.
These
data represent
results
obtained
D
from ctwomatograms
100
118’
136
>-
I-
90
C,)
z
LU
z
80
Ui
58
>
H
I-
4
70
Ui
LU
341
ILL._L
LI
Z
L#{192}
60
4
v:
367400
II’I
CoO
50
300
200
400
me
Fig. 11. Mass spectrum
plasma
40
80 ml of normal
which was pooled
mass spectrometry
30
20
necessary
0
215
2s5
255
275
WAVELENGTH
Fig. 10. Ultraviolet
resenting
tracts
absorption
25-OH-D3
Metabolite
peaks
and
represent
25-OH-D2
materCa)
and
in patients
of either
vitamin
type of vitamin
retention
295
of pooled
peaks
rep-
plasma
ex-
from
subjected
to the usual purification
shown
into a Zorbax-SIL
column connected to a
from
receiving
215 to 305 nm
count5
(16)
Zorbax-ODS
D2 or D3 it was possible
had received
of the
vitamin
for this column
system
and,
detection
was
rechromatography
completely
doses
to identify
the
on the basis of
D fraction
rerun
on
was greater
as a result,
increased.
of
consistent
with
than that of the
the sensitivity
of
Again,
identities
obtained
the
25-OH-D
peaks
were
those
ascribed
to the
peaks
on use of the two-column
procedure.
Table
2 presents
the quantitation
of the peaks obtained by rechromatography
of vitamin
D and 25-OH-D
fractions.
The data confirm
that the specific
activity
(ng/dpm)
for each peak was constant
after the regular
two-stage
procedure
and that additional
purification
did not decrease
the value obtained
for the concentration of the metabolite
in the original
plasma.
Had the
“vitamin
D metabolite”
peak been contaminated
with
other ultraviolet-absorbing
impurities
the specific
activity
(ng/dpm),
and hence
the concentration
of meIn the legend
to Figure
9, chromatographic
by the following
symbols:
K’
represented
a
=
plasma, processed
as In FIgure 1. yIelded 700 ng of 25-OH.03,
and rechromatographed
on Zorbax-SIL before dIrect-probe
selectivity;
N
=
theoretical
plates
per
parameters
=
relative
column;
to reach
retention
Rn
=
constant
specific
activity
measure-
(b) Ultraviolet
spectra.
Ultraviolet
spectra
of 25OH-D
fractions
from representative
normal
plasma
samples
were plotted
as were the spectra
of the vitamin
D and 25-OH-D
fractions
from patients
treated
with
pharmacological
doses of vitamin
D2 or D3. Figure
10
depicts
pharmacological
the patient
time
fractions
Zorbax-SIL.
In all cases this identification
was consistent with that ascribed
from Zorbax-ODS
chromatography.
On rechromatography
of 25-OH-D
fractions
on
Zorbax-SIL,
25-OH-D2
(8.4 mm) and 25-OH-D3
(9.6
mm) were well resolved
(Figure
9).
The theoretical
plate
the
by
from normal hta’nan
ments.
(nm)
spectra
in Figure 1, pooled and reinjected
Schoeffel detector set at wavelengths
the
isolated
tabolite
in plasma
(pig/liter),
would
have been falsely
high. It is most likely that these values would have decreased
after rechromatography.
For analysis
of vitamin
D in normal
plasma,
the three-column
procedure
was
10
times
of 25-OH-03
(16) are
time;
resolution.
examples
of the
characteristic
violet spectrum
(absorption
maximum
minimum
at 228 nm Xmar:Xmin -4.9:1)
25-OH-D2
and 25-OH-D3
from normal
cis-triene
at 265
ultranm
obtained
plasma.
and
with
25-OH-D2.
The
(Figure
11) posthe expected
molecular
ion ,(m/e 400) and all the
fragments
observed
previously
(17): [382
(c) Mass
spectra
of 25-OH-D3
and
mass spectrum
of 700 ng of 25-OH-D3
sessed
major
367 (-CH3
and
of side chain);
(-H20);
H20);
341
253 (271
(C24-C25
cleavage);
271
(loss
H20);
158; 136 (cis-triene
cleavage);
and 118 (136
H2O)]. The mass spectrum
of
-100
ng of 25-OH-D2
(not illustrated)
was not sufficiently
intense
to exhibit
the molecular
ion at 412, but
showed
the major fragments
generated
by most vitamin
D compounds
Ernie 136 and 1181.
-
-
Discussion
I have described
a procedure
for using HPLC
to simultaneously
determine
vitamin
D2, vitamin
D3, 25OH-D2,
and 25-OH-D3
in 2 ml of human
plasma
or
serum.
It differs
from other
physico-chemical
assays
that have been developed
for 25-OH-D3
(18, 19) in that
it requires
no derivatization
of the metabolite
under
study, involves
a much simpler
purification
scheme,
and
measures
both 25-OH-D2
and 25-OH-D3.
Furthermore,
the assay is similar
in terms
of speed,
sensitivity,
and
reproducibility
to existing
competitive
protein-binding
assays for estimation
of total 25-OH-D,
but it has several additional
advantages.
Only rarely
(20, 21) have
protein-binding
assays
been designed
to measure
more
than a single variable,
whereas
the present
assay proCLINICAL
CHEMISTRY,
Vol.
24, No.
2, 1978
295
vides information
on the ratio of 25-OH-D2/25-OH-D3,
and determines
the concentration
of vitamin
D in
plasma.
Knowledge
of the type of 25-OH-D
in plasma
provides
an opportunity
to determine
the relative
ployed
by Eisman
et al. (22) (1600 plates per column),
makes possible
the routine
detection
of metabolites
in
only 2 ml of plasma
as opposed
to the 4-mi sample
used
contributions
6.2-mm
of diet
in experimental
and
animals
are receiving
assessing
the
exposure
and
supplements
response
to ultraviolet
the
effects
light
in humans
who
of vitamin
D2, and it aids
of human
subjects
to vitamin
in
D
procedure
is simple
in design;
it consists
of lipid
extraction
and two chromatographic
steps. Extraction
with chloroform/methanol
was shown
to be a reliable
method
for recovering
lipid from plasma
protein.
Our
indicate
that
chloroform/methanol
Eisman
the use
solvent
variations
often
not
present
extraction
form/methanol/water
and
Dyer
procedure
of the
(2/2/1.8)
in their
original
proportions
can
of chloro-
recommended
by Bligh
description
of the
method
The
Zorbax-SIL
of the
column
lipid
extract
by HPLC
proved
of plasma
on
a
to be far more
satisfactory
than
any of the other
alternatives
we have
tested.
Eisman
et al. (22) recently
devised
a method
for
determining
25-OH-D2
and 25-OH-D3
in 4 ml of human
plasma,
based
upon
a procedure
involving
two “open”
Sephadex
columns
The misconceptions
necessary
plasma
and
before
that
sufficient
are
onstrated
that
column
can
let-absorbing
and a single HPLC
step on silica.
that complex
prepurification
is
HPLC
can
the loading
be applied
for assaying
capacity
of HPLC
is in-
dispelled
by these
experiments.
a 22 cm X 6.2 mm microparticulate
separate
lipid
most
of the extraneous
from
the vitamin
D and
I dem-
silica
ultravio25-OH-D
fractions
of whole-lipid
extracts
of plasma.
Use of
HPLC
as a preparative
tool offers numerous
advantages
over open-column
procedures.
Precise
sample
injection,
constant
flow elution,
and timed
collection
of each
plasma
extract
permit
better
reproducibility.
Detector
monitoring
of column
effluent
makes it feasible
to regularly
check
elution
positions
by use of standards.
The
theoretical
disadvantage
that
all samples
must
be run
through
the same
HPLC
column
rather
than
simultaneously
by the
through
a set of similar
“open”
short
time
(15 mm)
required
columns
is offset
for analysis
on
Zorbax-SIL.
Use
cently
I have
that
of Zorbax-ODS
been described
demonstrated
a 22 cm
X 6.2
for
HPLC
of 25-OH-D3
has
re(23).
by Koshy and VanDerSlik
by using methanol/water
mixtures
mm
Zorbax-ODS
column
will
ade-
quately
resolve
25-OH-D2
from 25-OH-D3
and improve
the resolution
of vitamin
D2 and vitamin
D3 reported
by others
(24). The use here of a highly efficient
HPLC
column
(N5 greater
than
4000 plates
per column)
offering
superior
sensitivity
and resolution
to that
em296
CLINICAL CHEMISTRY,
Vol. 24, No. 2, 1978
the
exceeded
of peak
of vitamin
D
10 tg/liter.
with
How-
vitamin
D con-
normal
plasmas
fell into
to use a three-column
chromatography
step
values
of two
the
and
on the peaks
were
fractions
and did
analysis
plasmas
to obtain
that
volume,
in measurement
with
combination
noted
as the sample
to minimize
the
time.
“Shoulders”
analyzing
25-OH-D
from
on Zorbax-SIL
be
injection
subsequently
that helped
centrations
<10 g/iiter
(all
this category)
it was necessary
method
involving
an additional
for total
different
vitamin
types
D.
of chroma-
adsorption
on Zorbax-SIL
and reversed-phase
chromatography
on Zorbax-ODS,
prob-
accounts
for
the
successful
purification
of nano-
gram quantities
of vitamin
D metabolites
consisting
of milligram
quantities
of lipid.
from a matrix
Furthermore,
though
an extraction
and
Prepurification
should
fixed
encountered
in fractions
ably
chloroform/methanol
to non-use
It
the
if concentrations
ever,
success
the
(22).
problems
fractions
tography,
liquid-liquid
with
al.
column,
of eluting
solvent
were all measures
recovers
90% of radioactively
labeled
vitamin
D or
25-OH-D
added
to plasma.
Recently,
it was suggested
that extraction
with chloroform/methanol
was inferior
to a procedure
involving
dichloromethane
(4) in its
ability
to extract
radioactive
vitamin
D metabolites
from plasma,
but I believe
that the reported
lack of
be attributed
et
(i.d.)
area and retention
not observed
when
therapy.
The
results
by
the
two
original
probably
amounts
procedure
HPLC
for plasma
steps,
involves
it recovers
about
two-thirds
of the
amount
of vitamin
D metabolites.
Losses
due to problems
of manipulating
of lipid extract
and semipurified
fractions.
precision
of the
present
assay
is comparable
were
small
The
to that
of
existing
protein-binding
assays
(25).
Much evidence
from recovery
experiments,
rechromatography,
ultraviolet
absorption,
and mass spectral
studies
indicates
that the HPLC
assay procedure
for
25-OH-D
and vitamin
D actually
measures
the ascribed
metabolite
correctly
and
accurately.
16 ig/iiter
for the concentration
normal
adult
human
plasma
that
obtained
techniques
27), but
by some
in which
agrees
well
Our
of total
is somewhat
workers
using
chromatography
with that obtained
mean
value
of
25-OH-D
in
lower than
protein-binding
is omitted
(15, 26,
by others
using
protein-binding
techniques
mnvolvmng
chromatography
(2,28).
The value is in excellent
agreement
with dur own
protein-binding
assay, which includes
an initial
chromatographic
step
was
step.
We
essential
before
found
that
carrying
such
out
a purification
the
competitive
protein
binding,
to remove
interfering
substances
that
increase
the reaction
value by two- to three-fold
above
the value obtained
when the chromatographic
step is
incorporated
into the protein
binding
assay. Although
seasonal
(29), racial (30), and geographical
(28) factors
can influence
population,
results
counted
tography
published
which
range
human
tamed
serum
I believe
obtained
25-OH-D
that some
in various
concentrations
of the disparities
laboratories
can
in the
in the
be
ac-
for by omission
or inadequate
use of chromaby some
workers
(15, 26, 27). There
are no
assays
for vitamin
D in human
plasma
with
to compare
for the
our results
concentration
plasma,
by others
by HPLC.
of vitamin
However,
D in normal
our
adult
0.9 to 4.7 zg/liter,
is similar
to that ohwith a protein-binding
assay (E. Deivin
and F. Glorieux,
personal
communication)
and a gas
chromatography/selected
ion monitoring
procedure
(31).
Because
of the low concentration
of total vitamin
D in the samples,
it was impossible
to apply many of the
analytical
tools at our disposal
to establish
the identity
and purity
of the vitamin
D in norrnal
plasma.
One must
accept
the
measured
value
of
vitamin
D in
normal
plasma
(2.2 tg/liter)
as the maximum
possible
concentration.
The true value
could
be lower if further
refinements
in methodology
revealed
contamination
of
the small peak with extraneous
ultraviolet-absorbing
substances.
Nevertheless,
the view
rather
that
little
these
vitamin
is transported
observations
D circulates
rapidly
to the
liver
support
in plasma,
but
for storage
(32,
My
observation
plasma
istration
that
that
there
concentration
of pharmacological
the
assay
will
is a dramatic
of vitamin
doses
be useful
increase
D during
of vitamin
in
6.
silica
Bligh,
traction
E. G.,
and
(200-500
sg/liter)
Fukushima
plasma.
hydroxylation
of vitamin
different
liver
tamin
concentrations
et al.
enzymes:
specific
25-hydroxylase
la-hydroxyvitamin
substrate.
It
hydroxylase
25-OH-D
uses
in an
D3
a non-regulated
can also
use
dihydrotachysterol
that
this
high
by two
specific
vi-
non-regulated
a
25-
9. Tucker,
G., Gagnon, R. E., and Haussler,
Tissue
occurrence and apparent
Biophys.
155,47
of vitamin
dosage
with
D are
vitamin
increased
by
ex-
and iden1250
14,
M. R., Vitamin
D3-25Arch.
lack of regulation.
(1973).
11. Kida,
K., and Goodman,
and of 25-hydroxyvitamin
485 (1976).
Sjovall,
and
J., Liquid-gel
derivatives.
Sephadex
D. S., Studies
chroma-
J. Lipid
on the transport
of vitamin
plasma.
J. Lipid Res. 17,
D in human
Kirkland,
J. J., Introduction
Wiley
& Sons, New York,
F., and
D3 and its
13. Holick,
for vitamin
metabolite.
15.
M.
Liquid
1974, pp 435-
to Modern
N.Y.,
J. Lipid
DeLuca,
H. F., A new chromatographic
system
metabolites:
Resolution
of a new vitamin
D3
Res. 12,460
(1971).
G., Schnoes,
H. K., and DeLuca,
D2 hydroxylases
in the chick.
Belsey,
16. Ref.
R. E., DeLuca,
H. F., and
D3 without
H. F., An in-vitro
J. Biol.
Potts,
preparative
17. Blunt,
J. T., Jr.,
J., DeLuca,
Hughes,
M.
A rapid
assay
J. Clin.
chromatography.
H. F., and Schnoes,
active
metabolite
H. K., 25-Hydroxycholeof vitamin
Biochemistry
Determination
of 25gas chromatog-
and
I., A novel
Acta
D3.
specific
assay
of 25-
68,215 (1976).
R., Baylink,
D. J., Jones, P. G., and Haussler,
M. R.,
assay for 25-hydroxyvitamin
D21r)3 and 1,25D21D3. J. Clin. Invest.
58,61(1976).
receptor
dihydroxyvitamin
D.
study
251, 24
38, 1046 (1974).
19. Bj#{246}rkhem, I., and Holmberg,
hydroxy-vitamin
D3. Clin. Chim.
Radioligand
high-
Chem.
12, p 29.
calciferol.
A biologically
7,3317
(1968).
20.
vitamin
D as a substrate
to generate
uncontrolled
manner
when
plasma
concentrations
lipid
37, 911 (1959).
500.
18. Sklan,
D., Budowski,
P., and Katz,
M.,
hydroxycholecalciferol
by combined
thin-layer
raphy.
Anal. Biochem.
56,606 (1973).
unsynthetic
as
of total
Physiol.
440.
in their
that
25-
out
regulated
and
that
and
is possible
therapy
(35)
D can be carried
(36)
very
of 25-OH-D
have
shown
a tightly
D-25-hydroxylase
with
method
Can. J. Biochem.
8. Jones, G., Schnoes, H. K., and DeLuca, H. F., Isolation
tification
of 1,25-dihydroxyvitamin
D2. Biochemistry
(1975).
for 25-OH-vitamin
Endocrinol.
Metab.
patients
on small
G., Application
ofhigh-pressure
liquid
chromatography
for
vitamin
D; metabolites.
In Vitamin
D; Biochemical,,Chemical
and Clinical Aspects Related
to Calcium
Metabolism.
A. W. Norman
et al.,Eds.,
Walter
de Gruyter, Berlin, New York, 1977, pp 491-
Chromatography.
that the
of treated
chromatogra-
and D3
(1975).
16, 448
J., A rapid
D2
assayof
hydroxylase
in
Res.
W.
liquid
of vitamins
7. Jones,
14. Jones,
of vitamin
(1976).
is seen
Dyer,
purification.
patients
may be the result of mobilization
of extrahepatic stores
of vitamin
D (34). The fact that the concentration
of vitamin
D in plasma
remains
high despite
discontinuation
of therapy
may help explain
the apparent
lack of inhibition
of the liver vitamin
D 25that
J. Lipid
columns.
and
H. F., High-pressure
metabolites
12. Snyder, L. R., and
response
of patients
to therapy.
I accept
the suggestion
high concentration
of vitamin
D in the plasma
particle
of the
D
admin-
the
separation
10. Ellingboe,
J., Nystrom,
E.,
tography
on lipophilic-hydrophobic
Res. 11,266 (1970).
D suggests
in assessing
phy:
hydroxylase:
Biochem.
33).
the
5. Jones, G., and DeLuca,
21. Preece, M. A., Tomlinson,
S., Ribot, C. A. et al., Studies
deficiency
in man. Q. J. Med. 44, 575 (1975).
of vitamin
22. Eisman, J. A., Shepard,
R. M., and DeLuca, H. F. Determination
of 25-hydroxyvitamin
D2 and 25-hydroxyvitamin
D3 in human plasma
using high pressure liquid chromatography.
Anal. Biochem. 80,298
I
Beth Byrnes
and David
Duthie
for their technical
help, and
Fraser and Sang Whay Kooh for the blood samples
used
study
and their encouragement
and helpful discussion during
thank
Drs. Donald
in this
the development
provided
useful
Dr. Louis
Marai
of the assay. Drs. Ingeborg
Radde
suggestions
during
the preparation
operated
the mass spectrometer.
and Graham
Ellis
of the manuscript.
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554
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.2. Haddad,
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Brumbaugh,
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24. Dupont
Instruments
Lab. Report LC A95441. The separation
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Du Pont Instruments,
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25. Bouillon,
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of 25-hydroxy-vitamin
R., DeLuca,
for
(1977).
23. Koshy,
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Lawson,
D. E. M., and Kodicek,
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Clin. Chim. Acta 54, 235
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Frederich,
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