Identification of glucoside and carboxyl-linked glucuronide conjugates of mycophenolic acid in plasma of transplant recipients treated with mycophenolate mofetil

Abstract

1. Mycophenolic acid (MPA), is primarily metabolized in the liver to 7-O-MPA-beta-glucuronide (MPAG). Using RP-h.p.l.c. we observed three further MPA metabolites, M-1, M-2, M-3, in plasma of transplant recipients on MMF therapy. To obtain information on the structure and source of these metabolites: (A) h.p.l.c. fractions containing either metabolite or MPA were collected and analysed by tandem mass spectrometry; (B) the metabolism of MPA was studied in human liver microsomes in the presence of UDP-glucuronic acid, UDP-glucose or NADPH; (C) hydrolysis of metabolites was investigated using beta-glucosidase, beta-glucuronidase or NaOH; (D) cross-reactivity of each metabolite was tested in an immunoassay for MPA (EMIT). 2. Mass spectrometry of M-1, M-2, MPA and MPAG in the negative ion mode revealed molecular ions of m/z 481, m/z 495, m/z 319 and m/z 495 respectively. 3. Incubation of microsomes with MPA and UDP-glucose produced M-1, with MPA and UDP-glucuronic acid MPAG and M-2 were formed, while with MPA and NADPH, M-3 was observed. 4. Beta-Glucosidase hydrolysed M-1 completely. Beta-Glucuronidase treatment led to a complete disappearance of MPAG whereas the amount of M-2 was reduced by approximately 30%. Only M-2 was labile to alkaline treatment. 5. M-2 and MPA but not M-1 and MPAG cross-reacted in the EMIT assay. 6. These results suggest that: (i) M-1 is the 7-OH glucose conjugate of MPA; (ii) M-2 is the acyl glucuronide conjugate of MPA; (iii) M-3 is derived from the hepatic CYP450 system.

Figure 1

Representative chromatogram of a plasma sample obtained from a kidney transplant recipient 75 min after administration of 0.6 g m⁻² MMF. The sample was pre-treated as described in Methods and applied to a 250 mm×4.6 mm Hypersil C-18 column maintained at 42°C. The column was eluted at a flow rate of 1.2 ml min⁻¹ with a mobile phase consisting of solution A (250 ml acetonitrile, 750 ml phosphate buffer–pH 3.0, 20 mM final concentration) and solution B (450 ml acetonitrile and 550 ml phosphate buffer–pH 6.5, 40 mM final concentration) that formed the following gradient: 7%B (0–2 min); 7–35%B (2–17 min); 35%B (17–18 min); 35–100%B (18–18.5 min); 100%B (18.5–20 min); 100–7%B (20–20.5 min). Compounds eluting from the column were monitored continuously with a diode array detector. This chromatogram illustrates the u.v. absorption at 215 nm. The 7-O glucuronide metabolite (MPAG) eluted after 3.5 min and mycophenolic acid (MPA) after 16.8 min. The metabolites M-1, M-2 and M-3 which eluted after 4.4, 6.2, and 11.2 min respectively were identified by comparison of their u.v. spectra (shown in the insets) to those of MPAG and MPA. The internal standard carboxybutoxy ether of MPA (IS) eluted after 16.3 min.

Figure 2

Comparison of the u.v. spectra of M-1 (A), M-2 (B) and M-3 (C) with those of either MPA (B and C); or MPAG (A). The spectra were determined with a diode array detector and the highest relative absorption maximum for each compound was arbitrarily set at 50%. Spectra were then normalized according to the highest relative maximum. The wavelengths of each absorption maximum are illustrated in the Figure. Values in parentheses correspond to the metabolites M-1, M-2 or M-3 and values not in parentheses either MPA or MPAG.

Figure 3

Mass spectra of MPA (A) and its metabolites MPAG (B), M-1 (C), and M-2 (D) in the negative ionization mode: Q1 scans (left) and product ions scans (right). The mass spectra were measured with a PE Sciex API 365 triple quadrople mass spectrometer using electrospray ion source. Samples were introduced into the ion source via an infusion syringe pump at a flow rate of 6 μl min⁻¹. MS-experiments (Q1-scans) were carried out at a spray voltage of +5000 or −4500 V, an orifice voltage from ±30 to ±50 V and a ring electrode voltage from ±150 to ±250 V depending on the ionization mode. Further MS/MS-experiments (product ion scans) were carried out at a collision offset voltage of ±20 to ±60 V using nitrogen as collision gas at a pressure of 3.95×10⁻³ mTorr in the collision cell. All mass spectra scans are displayed after normalization to the highest detectable mass peak in each spectrum.

Figure 4

Metabolic pathways of mycophenolic acid in humans. MMF is hydrolyzed to MPA the immunosuppressive metabolite of MMF by plasma and tissue esterases. MPA is primarily converted by glucuronidation to the major metabolite product, the phenolic MPA glucuronide (MPAG) as well as to its newly detected acyl glucuronide (M-2). In addition a phenolic 7-O-glucoside (M-1) and a CYP450 oxidation product (M-3) were observed.