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Application Note 618
AN63992-EN 1114S
In some cases, real samples may present concentrations up
to 30 mg/L. For this reason, it was decided to evaluate the
upper limit of quantitation. A 50 mg/L solution of MPA
was prepared in bovine serum albumin (Sigma-Aldrich).
This concentration in the linear range gave accuracy
within 100 ± 15% and a CV less than 15%.
To determine the best weighting factor, concentrations
were back-calculated and the model with the lowest total
bias across the concentration range was considered the
best suited. Four-point calibration standard curves were
calculated and fitted by linear models. To determine
LLOQ, a ten-fold dilution with bovine serum albumin
was made from Calibrator 1 to get a concentration of
0.097 mg/L. At this LLOQ, the accuracy and precision
values were, respectively, 108% and 4.6% for 10
replicates.
Figure 2 shows a representative calibration curve of MPA.
Figure 2. Calibration curve of MPA.
MPA
Y = 0.664727*X R^2 = 0.9986 W: Equal
0 1 2 3 4 5 6 7 8 9 10
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
Area Ratio
Carryover
Carryover was evaluated by injecting two levels of plasma
in triplicate: a blank plasma (B) and a high-level plasma
spiked at 20 mg/L (H). The injection sequence was three
injections of the high level (H1, H2, H3 ) followed by three
injections of the low level (B 1, B2, B3). The sequence was
repeated five times and the carryover was calculated using
the following formula expressed as a percentage:
Carryover = (b1-b3) / (h-b3) x 100
b1: average concentration obtained for all B1
injections
b3: average concentration obtained for all B3
injections
h: average concentration obtained for all H
injections (from H1 to H3)
Carryover was evaluated to be less than 1.6%.
Conclusion
A simple and fast LC-MS/MS method was analytically
validated for the analysis of mycophenolic acid for
research purposes. Intraday and interday accuracy and
precision were successfully assessed in plasma-based
samples.
For Research Use Only. Not for use in diagnostic procedures.
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