By Serena Shell, Bheemraj Ramoo, C. Clinton Frazee III and Prof. Uttam Garg

Mycophenolic acid is an immunosuppressant administered to patients undergoing various organ transplants. Therapeutic drug monitoring of mycophenolic acid is important to reduce drug toxicity and organ rejection. This is accomplished using several techniques including immunoassays, high-performance liquid chromatography with an ultraviolet detector (HPLC-UV) and liquid chromatography with a mass spectrometer (LC-MS/MS). Each technique offers unique advantages and disadvantages.

Introduction

Immunosuppressants are a class of drugs given to patients undergoing a solid organ or a stem cell transplant. These drugs inhibit the body’s natural immune system response to help prevent the rejection of the transplanted organ. Blood concentrations of some immunosuppressants should be closely monitored to maintain levels within the drug’s therapeutic range. When a concentration falls outside of the therapeutic range, adjustment of the patient’s dose is necessary. If the blood concentration is below the therapeutic range, there is an increased risk for organ rejection [1]. If the blood concentration goes above the therapeutic range, the patient has an increased risk of adverse effects [1].

One common immunosuppressant drug used for kidney, liver and heart transplants is mycophenolic acid (MPA) (Fig. 1, Table 1). The mechanism of action of MPA involves noncompetitive inhibition of the enzyme inosine monophosphate dehydrogenase (IMPDH) [2]. IMPDH catalyses the production of guanosine nucleotides used to synthesize DNA in lymphocytes. Without IMPDH, lymphocytes are unable to replicate, weakening the immune system and hindering its ability to attack a transplanted organ.

Figure 1. Mycophenolic acid (MPA)

Patients undergoing MPA therapy are prescribed either the pro-drug mycophenolate mofetil or mycophenolate sodium. Treatments can be given orally or intravenously. When given intravenously, mycophenolate mofetil is rapidly converted to MPA with a half-life of less than 2 minutes [2]. When mycophenolate mofetil is given orally, MPA reaches maximum concentration

Table 1. Properties of MPA

Figure 2. Mycophenolic acid glucuronide (MPA-G)

Figure 3. Acyl glucuronide mycophenolic acid (AcMPAG)

within 1 hour [2]. 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 [3]. Administration to the pediatric population should also consider the growth and development of the child over time [4]. 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) [5]. 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.

1. Immunoassays
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 [2]. 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 [6]. 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.

2. HPLC-UV
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 [7]. 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.

Figure 4. Spectrum showing the monitored ion transitions

3. LC-MS/MS
The gold standard for monitoring MPA in biological matrices is LC-MS/MS. LC-MS/MS, like HPLC-UV, first uses a chromatographic system to separate MPA and its metabolites from other matrix components. Then a mass spectrometer fragments the individual molecular ions into smaller ions. Fragment ion masses for MPA are monitored allowing for the identification and ultimately quantitation of the compound. Since the instrument is using specific masses along with chromatographic separation, this technique has less interferences than immunoassays or HPLC-UV, and, therefore, the results obtained are more accurate. LC-MS/MS is unable to compete with the rapid turnaround times of using immunoassays, but as previously mentioned run times are typically faster than using HPLC-UV. This is because LC-MS/MS circumvents the need to fully separate MPA from other matrix components and/or chemically similar compounds which= could otherwise interfere with results. Unlike HPLC-UV, when using LC-MS/MS it is only necessary to separate MPA from other compounds with the exact same fragment masses; thus, there are fewer overall interferences.

Despite requiring expensive instrumentation operated by specialized laboratory scientists, LC-MS/MS offers the major advantage of being able to simultaneously quantify the metabolites of MPA such as MPA-G. Another advantage of using LC-MS/MS is that calibration can be completed using a deuterated internal standard. Deuterated MPA mimics the characteristics of MPA better than using a different compound as the internal standard.

Table 2. Monitored MRM transitions

As previously described, MPA and MPA-G are reliably analysed using LC-MS/MS in ~6 minutes per sample [8]. First, MPA and MPA-G are extracted from serum using a 0.1 N solution of zinc sulfate; then methanol containing isotopically labeled internal standard for both compounds. After centrifuging the samples and transferring the supernatant to an autosampler vial, they are injected into the LC-MS/MS system. MPA and MPA-G are separated using gradient elution and a Supelcosil LC-18 5 cm × 4.6 mm × 3 µm column. Multiple reaction monitoring (MRM) with electrospray ionization is used to monitor the ions listed in Table 2. Using specific instrument parameters that achieve desired separation, the MPA and MPA-G shown here have retention times of approximately 3 minutes and approximately 2.25 minutes, respectively (Figure 4).

Future improvements

Despite a variety of quantitation methods for MPA, there is still room to enhance performance and improve patient wellbeing. As many patients are simultaneously undergoing treatment with other immunosuppressant drugs, standard practice should utilize the same tube type for both analyses to reduce the amount of blood drawn. A lavender top tube (EDTA) is an ideal choice because it is commonly used for collection with other immunosuppressants. A portion of the whole blood could be spun down to separate the plasma needed to test for MPA. Another practical option is to use dried blood spot cards, thereby allowing patients to collect their sample in the convenience of their own home.

LC-MS/MS instrumentation has become more common in the clinical laboratory, so an increasing number of laboratories now have the capability to monitor the concentrations of MPA-G and AcMPAG. However, there is limited published data on the importance of these levels. Including these two metabolites in routine testing may provide additional pharmacokinetic information that would be helpful to a patient’s dosing regimen. More research is necessary to determine relationships between different MPA, MPA-G, and AcMPAG blood concentrations.

The authors

Freek van Delft¹,² Serena Shell¹ MS; Bheemraj Ramoo¹ MS; C. Clinton Frazee III¹ BS, MBA; Uttam Garg*¹,² PhD
1 Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO, USA
2 University of Missouri School of Medicine, Kansas City, MO, USA

* Corresponding author
Email: ugarg@cmh.edu

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