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Quantitative Analysis of 1,25-dihydroxy-
vitamin D
2
and D
3
using Immunoaffinity
Extraction with APCI-LC-MS/MS
Xiang He
1
, Glenn Damkroeger
2
, and Marta Kozak
1
1
Thermo Fisher Scientific, San Jose, CA , USA
2
Thermo Fisher Scientific, Dreieich, Germany
For Research Use Only. Not for use in diagnostic procedures.
Application
Note: 522
Key Words
• Endocrine Testing
• TSQ Vantage
• Accela U-HPLC
Introduction
1,25-dihydroxyvitamin D (1,25D) tests are important
in conducting clinical research in chronic renal failure
and hypoparathyroidism. Circulating 1,25D levels
are a thousand-fold less than 25-hydroxyvitamin D
levels, making it a challenging test that benefits from
immunoaffinity purification prior to analysis with liquid
chromatography coupled to tandem mass spectrometry
(LC-MS/MS). In this work, both 1,25D
2
and 1,25D
3
were extracted from human plasma using immunoaffinity
extraction and quantified with LC-MS/MS.
Goal
To validate a very sensitive LC-MS/MS method to quantify
1,25-dihydroxyvitamin D by combining immunoaffinity
extraction and highly selective atmospheric pressure
chemical ionization (APCI).
Materials
ImmunoTube
®
kits (KM1000) were purchased
from Immundiagnostik AG (Bensheim, Germany).
Immunoextraction tubes, washing and eluting buffers,
and calibrators (CAL1 and CAL2) and controls (CTRL1
and CTRL 2) were provided in the KM1000 kit. The
concentrations of the calibrators and controls are specified
in Table 1.
Table 1: Calibrators and controls in KM1000 kit
Standards
1,25D
2
(pg/mL)
1,25D
3
(pg/mL)
CAL1
33
26
CAL2
350
250
CTRL1
63-105
49-81
CTRL2
203-348
146-244
Sample Preparation
Five hundred (500) µL of plasma were spiked with
deuterated 1,25D
3
and processed with the ImmunoTube
kit. The immunoaffinity method for processing plasma was
provided in the kit.
Instrument Method
A Thermo Scientific Accela UHPLC pump and Accela
autosampler were used as the front end system. The
detector was a Thermo Scientific TSQ Vantage triple stage
quadrupole mass spectrometer run in selected reaction
monitoring (SRM) mode and equipped with an APCI
probe. The LC gradient consisted of a fast, 5-minute
method at a flow rate of 500 µL/min.
Results and Discussion
Figures 1 and 2 display the data collected for 1,25D
2
and
1,25D
3
using the calibrators and controls provided in the
ImmunoTube kit. Calibration curves were plotted without
weighting and set to include the origin of the coordinate
(x, y = 0,0).
Conclusion
In this research, ImmunoTube immunoaffinity extraction
was used to prepare human plasma prior to LC-MS/MS to
quantify 1,25D
2
and 1,25D
3
. Immunoaffinity extraction
allows for the efficient extraction of target compounds
from biological samples and almost completely eliminates
matrix effects and interferences in LC-MS/MS analysis.
The sample preparation is fast, simple, and does not
require chemical derivatization. These features make it
an ideal method in clinical research for the quantitation
of 1,25D
2
and 1,25D
3
. APCI was used for the method
validation with an ImmunoTube kit, and the lowest
concentrations tested for 1,25D
2
and 1,25D
3
in the kit
were 26 and 33 pg/mL, respectively. Based on the S/N
ratios at these concentrations, the limit of quantitation
(LOQ) of this method was estimated to be around
15 pg/mL.