In setting the quantitation measurement through the resulting MRM transitions (321.3 > 207.2 for MPA and 514.3 > 207.2 for MPAG), it is important to distinguish that the chromatographic peak seen at the transition 514.3 > 207.2 and corresponding to MPAG is coincident with a chromatographic peak at the same retention time, but obtained through the transition (321.3 > 207.2) specific for MPA (Figure 6). This is determined to be MPAG in-source fragmenting into free MPA, making it clear that chromatographic separation is extremely important in segregating the endogenous MPA in the specimen from the MPA derived from the in-source fragmentation of MPAG.
The modified configuration described here makes it possible to include the analytes CsA, TAC, SIR, EVE, and the additional internal standard (CsD) into the method and quantitate them simultaneously whenever they are present in the same specimen. (MPA is generally analyzed in plasma while the rest of the compounds are quantitated in whole blood). Figure 7 shows the applicability of the method to real samples. In these optimized conditions, the total run time is approximately 5 minutes per sample.
Figure 8 describes the obtained linearity for the samples in the range 0.5 15 ug/mL and Figure 9 gives proof of the intra-run reproducibility on a quality control plasma sample (Level I, Recipe-Munich, Reference Value 0.48 ug/mL). Deviation from the mean value is restricted to 0.5 %.The estimated detection limit on the API 3200 using this protocol is approximately 0.03 μg/mL plasma.
Figure 10 shows the correlation obtained when MPA quantitation is compared with a commercially available HPLC-UV assay.
The modified method described in this application note (1, 2) has been demonstrated to be:<