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Table 3. Summary of assay performance and therapeutic range
Therapeutic Range LOQ Linearity Range
[ng/mL] [ng/mL] [ng/mL] I.S.
Tacrolimus 2 - 15 0.13 1.3 - 46.7 Ascomycin
Sirolimus 5 - 15 0.13 1.3 - 46.9 d4-Everolimus
Everolimus 6 - 8 0.13 1.3 - 47.4 d4-Everolimus
Cyclosporin A 100 - 350 24.90 24.90 - 1264.0 Cyclosporin D
Tacrolimus
Ascomycin I.S.
Sirolimus
Everolimus
d4-Everolimus I.S.
Cyclosporin A
Cyclosporin D I.S.
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
Relative Abundance
0
50
100
0
50
100
0
50
100
RT:1.25
RT:1.24
RT:1.29
RT:1.32
RT:1.31
RT:1.42
RT:1.47
NL:1.97E3
TIC F:+cESISRMms2
821.560 [768.405-768.455] MS
ICISCal1_B
NL:5.50E4
TIC F:+cESISRMms2
809.500 [756.575-756.625] MS
ICISCal1_B
NL:5.10E2
TIC F:+cESISRMms2
931.654 [864.545-864.595] MS
ICISCal1_B
NL:4.55E2
TIC F:+cESISRMms2
975.672 [908.775-908.825] MS
ICISCal1_B
NL:3.12E3
TIC F:+cESISRMms2
979.700 [912.575-912.625] MS
ICISCal1_B
NL:1.61E2
TIC F:+cESISRMms2
1220.026 [1203.275-1203.325]
MS ICIS Cal1_B
NL:9.80E3
TIC F:+cESISRMms2
1234.000 [1216.975-1217.025]
MS ICIS Cal1_B
Figure 1: Chromatograms of the lowest calibration standard
Results and Discussion
Figure 1 displays the representative lower limit of quantifi-
cation (LLOQ) chromatograms for Tacrolimus, Sirolimus,
Everolimus, Cyclosporin A, and the internal standards.
In Table 3, the LLOQ and the linearity range for each
analyte are reported and compared to the therapeutic
range.
As shown in Tables 4 and 5, the intra- and inter-day
variabilities were excellent as well as accurate. For each
analyte, intra-day variability and accuracy were deter-
mined by performing two different extractions of each QC
sample and analyzing them two times. Inter-day variability
and accuracy were determined by repeating the intra-day
procedure on three different days. Sample extractions were
performed by different people.
Conclusion
A fast and reliable LC-MS/MS method for the quantifica-
tion of Tacrolimus, Sirolimus, Everolimus, and Cyclo-
sporin A in whole blood was validated using the RECIPE
ClinMass® Complete Kit.
This method fulfills accuracy, precision, and dynamic
range requirements of a routine method for clinical re-
search.
Table 4. Intra-day variability (%RSD) and accuracy
QC 1 QC 2 QC 3
Value %RSD %Accuracy Value %RSD %Accuracy Value %RSD %Accuracy
Tacrolimus 3.28 6.7 90.1 6.67 2.9 96.3 13.3 5.5 99.4
Sirolimus 3.64 2.7 81.7 11.20 3.8 93.6 18.9 5.2 101.8
Everolimus 3.34 7.2 90.1 10.60 7.1 97.4 18.2 7.2 101.5
Cyclosporin A 62.50 11.4 101.7 258.00 6.2 102.9 1341.0 2.8 94.6
Table 5. Inter-day variability (%RSD) and accuracy
QC 1 QC 2 QC 3
Value %RSD %Accuracy Value %RSD %Accuracy Value %RSD %Accuracy
Tacrolimus 3.28 4.7 92.5 6.67 2.1 97.4 13.3 3.3 99.4
Sirolimus 3.64 8.4 89.6 11.20 4.6 95.7 18.9 5.1 102.8
Everolimus 3.34 7.6 96.7 10.60 5.1 96.5 18.2 4.7 100.9
Cyclosporin A 62.50 15.6 103.4 258.00 6.7 99.0 1341.0 12.0 102.9