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Quantitative Analysis of Serum
1
α
,25-dihydroxyvitamin D by
APPI-LC-MS/MS
Xiang He, Marta Kozak; Thermo Fisher Scientific, San Jose, CA, USA
Application
Note: 544
Key Words
• TSQ Vantage
• Clinical Research
• Endocrine
Analysis
Introduction
Quantitation of 1
α
,25-dihydroxyvitamin D
2
and D
3
(1,25D) in serum is very important in clinical research but
is challenging because of the low circulating serum concen-
tration of 1,25D. Due to its high analytical specificity and
sensitivity, liquid chromatography-tandem mass spectrom-
etry (LC-MS/MS) has been used for quantitation of 1,25D.
We have previously reported the use of immunoextrac-
tion and atmospheric pressure chemical ionization (APCI)
for LC-MS/MS analysis of 1,25D in human serum
1
. Im-
munoextraction greatly simplifies the sample preparation
and efficiently removes interferences. In addition, while
APCI is good for this analysis, atmospheric pressure pho-
toionization (APPI) is a more specific ionization technique
than APCI and, therefore, further improves the analytical
sensitivity of 1,25D detection.
Goal
To develop a highly sensitive LC-MS/MS analytical method
to quantitate 1,25D with APPI using immunoextraction
that provides better sensitivity than an APCI method.
1
Methods
Sample Preparation
Serum 1,25D was purified with an immunoextraction
method using an ImmunoTube
®
immunoextraction tube
(Immundiagnostik AG, Bensheim, Germany). Briefly, sam-
ples were mixed with immobilized 1,25D antibody slurry
and incubated at room temperature for 1 hour before the
1,25D-antibody beads were washed with aqueous buffer.
Then, 1,25D
2
and 1,25D
3
were eluted with ethanol, dried,
and reconstituted for LC-MS/MS injection.
LC-MS/MS Conditions
LC-MS/MS analysis was performed on a Thermo Scientific
TSQ Vantage triple stage quadrupole mass spectrometer
coupled with a Thermo Scientific Accela UHPLC system.
A Thermo Scientific Hypersil GOLD column (150 × 1 mm,
3 μm particle size) was used. The column temperature was
maintained at 50 °C. Mobile phases were 70% methanol
in water and methanol from Fisher Chemical brand. The
LC method used a 10-minute gradient, and the LC flow
was diverted to the mass spectrometer between 2 and 5
minutes.
The mass spectrometer was equipped with an APPI
probe and operated in the positive ion mode. Selected
reaction monitoring (SRM) transitions of 1,25D
2
, 1,25D
3
,
d6-1,25D
2
and d6-1,25D
3
were monitored (see Table 1).
Table 1. SRM transitions
Q1
(m/z)
Q3
(m/z)
CE (V)
S-Lens (V)
1,25D
2
411.3
135.0
19
87
151.0
20
87
1,25D
3
399.2
135.0
21
90
151.0
22
90
d6-1,25D
2
417.3
151.0
19
95
d6-1,25D
3
405.3
151.0
20
90
Validation
The validation procedure included tests for 1) recovery,
linearity, and lower limit of quantitation (LLOQ) and 2)
precision.
Results and Discussion
1. Sample Preparation
The immobilized 1,25D antibody used in this study was
highly specific and had no cross-reactivity from other
vitamin D derivatives. Serum samples processed with im-
munoextraction showed no matrix effects or ionization
suppression.
2. Recovery, Linearity, and LLOQ
Two sets of calibrators were prepared in ethanol (solvent)
and pooled human plasma sample. Human plasma con-
tains endogenous 1,25D, so it is not an appropriate choice
to be used as the matrix for calibrators. Different levels of
1,25D were spiked into both solvent and human plasma
to evaluate the feasibility of using solvent as the calibrator
matrix. Solvent calibrators were prepared without im-
munoextraction, but with drying and reconstituting steps.
Endogenous concentrations of 1,25D in pooled plasma
were determined with solvent calibrators first. The pooled
human plasma samples were then spiked with increasing
levels of 1,25D and processed with immunoextraction.
Concentrations of total 1,25D (endogenous and spiked
concentration) in plasma were determined against solvent