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A new LC/MS/MS Research Method for Rapid Quantitation of Five Immunosuppressant Drugs, including Mycophenolic Acid (MPA)

ystems.com First dimension chromatography was accomplished via an Applied Biosystems POROS R1/20, 2.1 x 30 mm perfusion-column. Second dimension chromatography was performed by a Phenomenex Luna 5 μm Phenyl- Hexyl, 2 x 50 mm column (p.n. 00B- 4257-B0) housed in an oven at 60 C.

Diluting solution consisted of 80% methanol with 1% ZnSO4 and 200 ng/mL of Cyclosporin D as internal standard. 20 ng/mL Ascomycin is added when TAC, SIR and EVE are quantified in the same sample

Results and discussion

In relation to the other immunosuppressant drugs in the 5-drug panel, there are several analytical features unique to MPA. These are discussed below.

Despite the presence of a free acidic moiety, which should make MPA very sensitive when ionized in negative ion mode, the sensitivity in positive ion mode is higher. Several papers have demonstrated that the molecule is quite labile in the ionization process (5).

Fragmentation of the MPA precursor ion (m/z 321) results in an MS/MS spectrum as shown in Figure 3, with the most intense fragment ion at m/z 207.1.

MPA is predominantly metabolized to the glucuronide form (MPAG). This glucuronidated form is at a much higher concentration (around one order of magnitude greater) than the residual MPA in the specimen.

Despite the mild source-declustering conditions, the MPAG metabolite is prone to fragmentation in the sourceinterface region. As shown in Figure 4, the MS spectrum for the MPAG metabolite shows two prominent ions corresponding to the ammoniumadduct ion [M+NH4]+ (m/z 514.2) and the glucuronide-depleted ion, which is the original MPA protonated ion [M+H]+ (m/z 321.2) produced by in-source fragmentation.

The MS/MS spectrum of MPAG (Figure 5) is directly comparable with that of pure MPA (Figure 5 v Figure 3), since the glucuronide moiety becomes a neutral loss when MS/
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