within 1 hour . Mycophenolate sodium is only given orally as an enteric-coated tablet. MPA is metabolized in the liver to its major inactive metabolite mycophenolic acid glucuronide (MPA-G) (Fig. 2) and to a lesser extent, the active metabolite acyl glucuronide mycophenolic acid (AcMPAG) (Fig. 3). Prior to excretion, MPA-G can undergo enterohepatic recirculation followed by bacterial deconjugation in the intestine causing a second concentration peak maxima that can be observed during a pharmacokinetic profile.
Trough levels are routinely monitored for MPA. However, patients who do not show the expected dose–concentration response, a complete pharmacokinetic profile may be necessary for therapeutic drug monitoring. Variances between individuals such as gastric pH, liver and renal function, time post-transplant, co-administered drugs (especially other immunosuppressants) and comorbidities cause differences in adsorption, metabolism and excretion; thus, overall blood concentration . Administration to the pediatric population should also consider the growth and development of the child over time . Consequently, it is important to monitor the MPA concentration for each patient to createan individualized and evolving treatment plan, and not use a generic dosage regime.
Current therapeutic drug monitoring techniques used for MPA
The matrix of choice for therapeutic drug monitoring of MPA is plasma or serum. There are many published quantitative techniques to determine the concentration of MPA including immunoassays, high pressure liquid chromatography coupled with ultraviolet detection (HPLC-UV), and liquid chromatography tandem mass spectrometry (LC-MS/MS) . Slightly over 60 labs participated in the 2023 MPA proficiency survey issued by the College of American Pathologists (CAP) with approximately 50% using an immunoassay, 6% using HPLC-UV, and 40% using LC-MS/MS. The subsequent sections provide a brief description and discussion of each method.
Immunoassays evaluate a drug’s concentration by measuring drug-antibody or drug-enzyme interactions through spectrophotometry. Changes in absorbance for patient samples are compared to absorbance changes for known standards. Several immunoassay kits for the quantitation of MPA are commercially available. These kits are either semi or fully automated, require little or no sample preparation, and have relatively low start-up costs because the immunoassay instrumentation necessary to use the kits is commonly found in a clinical laboratory and can often be used. Results are reported within minutes . One significant drawback of immunoassays is that other molecules chemically similar to the compound of interest (MPA, in this case) can cross-react with the antibody or enzyme resulting in erroneous results. Unfortunately, the metabolite AcMPAG is one known compound which cross-reacts in this way and will produce a falsely elevated result . Owing to differences between individuals as discussed earlier, it is difficult, if not impossible, to correct for cross-reactivity. Immunoassay kits are also not available for MPA-G limiting the scope of the assay.
HPLC-UV was the first accepted method for quantifying MPA. Briefly MPA and desired metabolites are extracted from patient samples and injected into the HPLC instrument. Samples move through a column where compounds of interest adhere to the stationary phase of the column with different affinities based on their unique physical and chemical properties. The compounds are eluted from the column using mobile phases that have a carefully selected polarity or pH, and then travel towards the UV detector at different speeds. The UV detector calculates the absorbance at a wavelength specific to MPA such as 210 nm, 254 nm, or 305 nm . Concentrations of MPA in a patient sample are calculated by comparing the absorbances to absorbances of one or more MPA target standards with known concentrations.
HPLC-UV offers more specificity than immunoassays because it can separate the metabolites from the parent compound. However, it requires more sample preparation and has a longer turnaround time. When compared to LC-MS/MS, HPLC-UV generally has longer instrument run times, but the instrumentation itself is much less expensive. Multi-point calibration with or without an internal standard can be completed using HPLC-UV. However, labelled internal standards, which help to improve accuracy and precision, cannot be used since they elute from the column at a very similar time as the analyte and cannot be identified as separate compound. This technique is falling from popularity in favour of other techniques.