Analysis of Ciclosporin A in
voorbeeld
titelLC-MS/MS
Dried Titel
Blood
Spots using
J.C.G. den Burger, A.J. Wilhelm, R.M. Vos, A. Chahbouni, A. Sinjewel
VU university medical center, Laboratory of Pharmacology and Pharmacy,
PO Box 7057, 1007 MB, Amsterdam, The Netherlands.
Introduction
Validation of the assay
Ciclosporin (CsA) is an immunomodulating drug to
prevent graft versus host disease in patients undergoing
allogeneic stem cell transplantation (ASCT). Therapeutic
Drug Monitoring (TDM) of CsA is usually performed in
venous blood samples. Dried Blood Spot (DBS)
sampling is a promising, patient friendly alternative for
venous sampling and has several advantages. These
advantages are: no phlebotomist is necessary, patients
can remain at home, monitoring results are known when
visiting the hospital and monitoring can be done at any
desired time.
The assay was validated for the most common
parameters: matrix effect, linearity, between- and within
run accuracy and precision, recovery and stability.
Validation of a DBS sampling assay requires an extra
parameter to be validated which is the influence of
hematocrit (HT) on the CsA concentration. The HT is
direct proportional to the viscosity and thus the fluidity of
blood. A low fluidity of the blood would result in a poorer
blood distribution through the paper, resulting in smaller
spots. This would mean that the punched out area has a
higher CsA concentration.
Aim
Development of a sensitive, accurate and precise LCMS/MS assay for analysis of CsA in DBS samples.
Usage of DBS sampling for CsA has been described
earlier for immuno- and radioassays, however no LCMS/MS assay was described for analysis of DBS
samples.
Validated parameter
Result
Limits
Matrix effect
102%
≤ 10.0%
Recovery (efficiency)
97.1
≥ 70%
Recovery (RSD)
5.6 %
≤ 10.0%
Linearity (GOF)
Linearity (LOF)
23779
0.098
>>4,54
<4,28
Within run
Accuracy / precision
LLOQ 97.0/5.0 %
Low
91.2/5.7 %
Medium 87.7/3.6 %
High
94.2/2.5 %
Between run
Accuracy / precision
LLOQ 101.0/15.0 %
Low
88.0/5.7 %
Medium 88.8/6.1 %
High
95.1/2.9 %
Acc. (LLOQ) ± 20.0%
Prec. (LLOQ) < 20.0%
Acc. (L, M, H) ± 15.0%
Prec.(L, M, H) < 15.0%
Hematocrit
Low
High
Methods
0,20
-12 %
-9 %
DBS samples were prepared by pipetting spiked EDTA-blood
on the sampling paper until the predrawn circle was just filled.
After drying overnight a disk, with a diameter of 8 mm was
punched out using a mechanical hole puncher and collected in
microtubes. To the disk 150 µL purified water and 150 µL of
the internal standard solution (Cyclosporin D in methanol) was
added. The microtubes were then vortexed during 15 minutes
at ambient temperature, followed by placement in an ultrasonic
bath for 15 minutes. The samples were then centrifuged at
10,000 g for 10 minutes. The clear supernatant was transferred
in to vials and 20 µL was injected on the LC-MS/MS.
Chromatography was performed using gradient elution,
retention times of CsA and CsD were 1.90 and 2.00 minutes.
0,29
+1 %
-8 %
0,35
Normalized
Normalized
0,44
-7 %
0%
for more information: [email protected]
0,59
+1 %
+3 %
0,72
+14 %
+10 %
Conclusion
All validated parameters are within limits. The developed
sample pretreatment procedure has a small matrix-effect
and high extraction recovery which is, more importantly,
consistent. We did not detect a influence of HT on CsA
concentrations in the range of 0.30 HT to 0.59 HT.
The bloodspots proved to be stable during a period of at
least 17 days at ambient temperature and at least 45 days
at 4 ºCelsius (data not shown). Long term stability testing is
not completed yet. As discussed above, the method is
matrix-effect free, accurate and precise, furthermore
bloodspots proved to be stable during a appropriate period
of time. The developed method is suitable for analyzing
CsA in dried blood spot samples.