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APP NOTE: 502 DRUG DETERMINATION Quantitation of
Immunosuppressive
Drugs Directly from
Dried Blood Spots
INTRODUCTION
Paper spray mass spectrometry (MS) allows for the
direct analysis of pharmaceuticals, drugs of abuse,
and other small molecules (up to about 1500 Da)
from blood, urine, and other biofluids (Wang et al.).
The analysis is performed by depositing the sample
directly onto a porous cellulosic substrate contained
within a disposable cartridge (VSCTM-1 shown below).
The sample is allowed to dry, and the cartridge is inserted into the Velox 360TM autosampler for analysis.
The autosampler performs all of the steps necessary
to perform the analysis, including loading the cartridge, depositing extraction solvent onto the cartridge, positioning the cartridge in front of the mass
spectrometer inlet, and applying the ionization voltage. Ions are generated from the sharp tip of the porous substrate contained within the cartridge. Analysis requires no sample preparation and takes about 1
-2 minutes per sample. More details about this method for generating ions can be found in Manicke et al.
This application note describes the use of the Velox
360 - Tandem Mass Spectrometry for quantitation of
immunosuppressive drugs from blood samples, focusing specifically on the simultaneous quantitation
of tacrolimus and cyclosporine.
EXPERIMENTAL
200 µL of the blood sample was mixed with 10 µL of
an internal standard solution containing 150 ng/mL of
[13C, 2H2]-tacrolimus and 3000 ng/mL [2H4]cyclosporine from Toronto Research Chemicals. Ten
µL of this sample was then spotted onto the paper
spray cartridge and allowed to dry either at room temperature for 90 minutes or at 40°C for 20 minutes.
Instrument parameters are shown in tables 1 and 2.
Sample analysis was carried using a prototype Velox
autosampler interfaced to a Thermo Scientific TSQ
Vantage.
Velox Sample Cartridge (VSCTM) © 2017 Prosolia, Inc.—Research Use Only. Not for diagnos c purposes.
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Table 1. Experimental Settings for Tacrolimus
Parameter
Setting
Paper spray voltage
3.4 kV
Volume of solvent
applied to cartridge
120 µl
Analysis time per
sample
2 minutes
MS inlet
Temperature
300° C
Solvent
60:40 methanol:chloroform, 0.1%
Sodium Acetate
Table 2. SRM parameters
Parent
m/z
Product
m/z
Time
(ms)
CE
(V)
Q1
PW
Q3
PW
S-Lens
826.51
826.51
443.1
616.3
0.1
0.1
44
32
0.3
0.3
0.7
0.7
182
182
1224.9
1112.9
0.1
68
0.7
0.7
195
1224.9
1084.8
0.1
62
0.7
0.7
195
RESULTS
A typical set of chronograms is shown in Figure 1.
The spray voltage was applied at about 0.1 minutes,
and signal for all of the SRM channels appears immediately. The HV was left on for 60 seconds and
then turned off. The area under the curve (AUC) for
the analyte is divided by the AUC for its internals
standard, and this ratio is used to create calibration
curves, as shown in Figure 2.
Figure 2. Calibration curves directly from
dried blood spots for tacrolimus (top) from
1.5 ng/mL to 30 ng/mL and for cyclosporine
(bottom) from 25 ng/mL to 1000 ng/mL
Figure 1. SRM Chronograms of A) m/z
826 → 616, [tacrolimus + Na]+. B) m/z 830
→ 620, [tacrolimus IS + Na]+. C) m/z 1225
→ 1113, [CsA + Na]+. D) m/z 1229 → 1113,
[CsA IS + Na]+.
© 2017 Prosolia, Inc.—Research Use Only. Not for diagnos c purposes.
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Selective detection of the analytes was confirmed by
requiring that the ratio between the quantifier and the
qualifier SRM transitions be within 20% of an authentic sample, as shown in figure 3.
CONCLUSION
This application note demonstrates the suitability of
paper spray mass spectrometry for the quantitative
analysis of tacrolimus and cyclosporine from blood.
Paper spray MS/MS maintains the excellent sensitivity and specificity of mass spectrometry while being
better suited for laboratories with limited expertise in
the maintenance and trouble-shooting of complex
HPLC-MS/MS systems. Compared to the use of immunoassays for immunosuppressive drug quantitation, paper spray MS has a lower cost per test without being more complex.
REFERENCES
He Wang; Jiangjiang Liu.; R. Graham Cooks; Zheng
Ouyang; "Paper Spray for Direct Analysis of Complex Mixtures Using Mass Spectrometry", Angewandte Chemie International Edition, 2010, 49,
877-880 .
Figure 3. SRM peaks for [tacrolimus + Na] + detected from a spiked blood standard (top) and
from an incurred sample (bottom).
The accuracy of the MS/MS assay was confirmed by
validating it against an HPLC-MS/MS assay at a reference lab. The results for 60 incurred samples,
shown in Figure 4, show excellent agreement between the two methods.
N. E. Manicke, Q. A. Yang, H. Wang, S. Oradu, Z.
Ouyang and R. G. Cooks, "Assessment of paper
spray ionization for quantitation of pharmaceuticals
in blood spots", International Journal of Mass Spectrometry, 2011, 300, 123-129.
Velox 360 is a trademark of Prosolia, Inc.
Paper Spray is a trademark of Purdue Research Founda on
Prosolia, Inc.
6500 Technology Center
Drive, Suite 200
Indianapolis, IN 46278
Tel: 866.241.0239
Fax: 317.275.5799
Figure 4. Comparison between Paper
Spray and HPLC - MS/MS
© 2017 Prosolia, Inc.—Research Use Only. Not for diagnos c purposes.
www.prosolia.com
[email protected]