A previously published application note in this series describes an LC/MS/MS method for the simultaneous determination of four immunosuppressant drugs: Cyclosporin A (CsA), Tacrolimus (TAC, aka FK-506), Sirolimus (SIR, aka Rapamycin) and Everolimus (EVE, aka RAD-001) (1, 2). The method is based on a simple and robust hardware configuration and shows to be fast and simple, without compromising precision and accuracy. Since this method was first described, there has been considerable interest in extending it to include a fifth immunosuppressant drug, Mycophenolic Acid (MPA, see structure in Figure 1).
MPA is the pharmacologically active form of the immunosuppressant prodrug Mycophenolate Mofetil. Because its pharmacological action differs significantly from CsA, TAC, SIR, and EVE, MPA is frequently co-prescribed as part of an immunosuppressant drug cocktail. Due to the chemical structure of MPA and the strong presence of some of its metabolites -most notably the Glucuronide metabolite, MPAGseveral authors have voiced their concern about the risk of overestimating the MPA concentration. It has been shown that chromatographic separation is essential to accurately measure MPA concentration in plasma by eliminating the interfering effect of MPAG (3, 4).
In this research note, minor modifications are described that extend the original method proposed in reference (1) to include MPA and determine the entire panel of 5 immunosuppressants on the Applied Biosystems API 3200TM LC/MS/MS Mass Spectrometer. With these modifications, the same hardware configuration can be used to determine CsA, TAC, SIR, and EVE in blood and MPA in plasma, without compromising sensitivity and specificity. Quantitation of MPA metabolites (mainly MPAG) can be included if desired.
Depending on the instrumental configuration and the up-stream sample preparation step, the required time for the entire analytical procedure significantly influences the final overall sample throughput (5).
As with the original method, a fast and inexpensive on-line solid-phase extraction (SPE) is performed through a twodimensional liquid chromatography configuration. Using this configuration, sample clean up effectively minimizes any ion suppression interference during the MRM measurements. This permits the use of Cyclosporin D (CsD) as an internal standard for quantitation of all 5 immunosuppressant drugs included in the extended panel.
Samples were prepared for MPA measurements by adding 100 μL of a diluting solution* to either 50 μL of either plasma samples, calibrators or controls, This mixture was then vortex mixed for 30 seconds. After centrifugation at 13000 rpm x 5 min., 100 μL of supernatant were transferred to either an autosampler vial or a microtiter plate.
The hardware configuration included an Applied Biosystems/MDS SCIEX API 3200TM Triple Quadrupole Mass Spectrometer equipped with a Turbo VTM source. This source operates in positive ion mode at a voltage of +5500 volts and with a Turbo V gas heated at 650 C.
Multiple Reaction Monitoring (MRM) measurements were made using declustering potential (DP) and collision energy (CE) values, which were automatically optimized by the Analyst software for each of the analytes. DP values ranged between 40 and 140 V; CE values were between 30 and 65 eV.
Two-dimension chromatography was performed through a split arrangement of the modules of the LC pumping system (either a Perkin Elmer Series 200 Micro Pump, an Agilent 1100 Binary Pump with the pump heads disconnected from the mixing tee or a Shimadzu Prominence or 10Dvp pumps) and a computer-controlled Valco Valve (10-port, 2 positions) plumbed as illustrated in Figure 2. www.appliedbiosystems.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/MS is performed.(Both are dominated by the fragment ion at m/z 207.2.)
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:
Rapid. It is suitable for quantifying either MPA, and/or the other immunosuppressant drugs (CsA, TAC, SIR, EVE).
Flexible. With the same hardware setting and configuration, the method can handle whole blood, for CsA, TAC, SIR, and EVE and plasma for MPA and MPAG.
Accurate. MPAG, usually present at significant concentrations, does not interfere with the MPA quantitation results.
Fully under control of the Analyst acquisition software. All the peripheral modules are under control of the fullinclusive Acquisition Method.
Effective in reducing the solvent consumption. The programmed operation of the LC peripheral units enables the high flow-rate regime in the firstdimension LC just for the time needed for the sample clean up.
1. Applied Biosystems Application, Publication: MS-AP234.
2. T. Koal, M. Deters, B. Casetta, and V. Kaever, J. Chromatogr B, 805 (2004) 215-222.
3. B. Atcheson, P.J. Taylor, D.W. Mudge, D.W. Johnson, I. Pillans, and S.E.Tett. J. Chromatogr, B. Analyt Technol Biomed Life Sci, Jan 2004; 799(1): 157-63.
4. F. Streit, M. Shipkova, V.W. Armstrong, and M. Oellerich, Clinical Chemistry 50:1 152159 (2004).
5. T.M. Annesley, L.T. Clayton, Clinical Chemistry 51:5 872877 (2005).
Applied Biosystems/MDS SCIEX acknowledges our collaborators, Jan Lembke and Uta Ceglarek of the Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital, Leipzig (Germany), Bruno Casetta of Applied Biosystems Monza (Milano- Italy), and Daniel Blake of Applied Biosystems, Warrington (UK) for providing the method, samples, and data for this application note.