Therapeutic drug measurement of mycophenolic acid derivatives in transplant patients (original) (raw)

Therapeutic drug measurement of mycophenolic acid derivatives in transplant patients

Fawzy A. Elbarbry, Ahmed S. Shoker*
Department of Medicine, Royal University Hospital, 103 Hospital Drive, University of Saskatchewan, Saskatoon, SK, Canada S7N 0W8

Received 20 December 2006; received in revised form 3 March 2007; accepted 7 March 2007
Available online 21 March 2007

Abstract

Introduction: Mycophenolic acid, the active metabolite of the prodrug mycophenolate mofetil, is widely used as an immunosuppressive agent in transplant patients for the prophylaxis of acute rejection. Recent prospective trials suggested the need for therapeutic drug monitoring, which raises the necessity to acquire accurate methods to measure MPA and its metabolites.

Objective: Present an overview of the reasons to monitor MPA and its metabolites as well as a review of the currently available methods for their determination.

Methods: Articles published from January 1992 to December 2006 were reviewed.

Results: Most of the cited references use either chromatographic or immunoassay techniques. Basic information about biological samples used for the analysis, sample preparation, stationary phase, mobile phase, detection mode and validation data are discussed. Current information suggests the feasibility to set up method(s) to monitor MPA and its metabolites in most centers.

© 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Keywords: Mycophenolic acid; Therapeutic drug monitoring; Drug analysis; HPLC; Immunoassays; Transplantation

Contents

Introduction … 753
Pharmacokinetics of MPA and metabolites … 754
Absorption. … 754
Distribution … 755
Metabolism and elimination … 755
Analytical methods for MPA and its metabolites … 755
Literature search methods … 755
HPLC assay … 756
Sample preparation … 756
Assay of free MPA (MPA) … 756
Mobile phase … 757
Stationary phase … 757
Detection … 757

[1]

1. Abbreviations: AcMPAG, mycophenolic acid acyl glucuronide; AUC, area under the curve; CNI, calcineurin inhibitors; EMIT, enzyme-multiplied immunoassay technique; fMPA, free MPA; HPLC, high performance liquid chromatography; LLE, liquid-liquid extractions; MPA, mycophenolic acid; MPAG, mycophenolic acid glucuronide; MS, mass spectrometer; SPE, solid-phase extraction; SPME, solid-phase microextraction; TBAHS, tetrabutylammonium hydrogen sulfate; TDM, therapeutic drug monitoring; TEA, triethyl amine; TMBA, trimethyl ammonium bromide; tMPA, total MPA; UGTs, UDP-glucuronsyltransferase enzymes; UV, ultraviolet.
   • Corresponding author. Fax: +13069667996.
     E-mail address: shoker@sask.usask.ca (A.S. Shoker). ↩︎

Stability studies … 761
Other parameters … 761
Immunoassay … 761
Comparison between HPLC and immunoassay methods … 762
Conclusion … 762
References … 762

Introduction

Graft outcomes have improved over the last decade, in part, due to better immunosuppressive regimen including the introduction of mycophenolate mofetil (MMF), the ester prodrug of mycophenolic acid (MPA) or sodium mycophenolic acid (Myfortic ®{ }^{\circledR} ) [1]. MPA is a potent, selective, reversible and uncompetitive inhibitor of inosine monophosphate dehydrogenase and thus inhibits the de novo pathway of guanine nucleotide synthesis, limiting lymphocyte proliferation [2]. Mycophenolic acid (MPA) became widely accepted as an efficacious adjunctive immunosuppressant in renal transplantation following studies showing beneficial effects on rejection in cyclosporine (CsA) [3]- and tacrolimus [4,5]-based regimens.

Side effects of the prolonged use of calcineurin inhibitors (CNI), in particular nephrotoxicity, continue to hamper the longterm outcome of transplant recipients [6,7]. Consequently, the use of therapeutic regimens that involve the reduction or withdrawal of calcineurin inhibitors has been investigated in transplant patients. Published data suggest that mycophenolic acid-based CNI minimization regimens have significant clinical benefits. In fact, the use of mycophenolic acid derivative in induction protocols as well as 6 months or later after transplantation has been proven to be safe and improves renal function [1].

More recently it is becoming clear that strategies to improve renal transplant outcome require careful monitoring of the immunosuppressive dosing regiment [8]. For example, in stable renal transplant patients, CsA withdrawal under MMF was reported to be safe based on lack of short-term deterioration in graft function, although histological deterioration was observed in 50%50 \% of patients [9]. The risk of acute rejection was found elevated in the three of the four prospective randomized studies [15] performed so far, and a trend to a higher incidence of acute rejection was shown in the fourth one [8,9][8,9].

The safety and efficacy of mycophenolic acid derivatives might be further enhanced with therapeutic drug monitoring (TDM) for a variety of other reasons. Firstly, MMF is known to exhibit wide inter-patient pharmacokinetic variability [8,9] and its exposure increases over the first week’s post-transplantation [10]. Secondly, recent studies have shown a relationship between plasma concentration of MPA and clinical outcomes in transplant patients receiving concomitant calcineurin inhibitors, CsA or tacrolimus, and corticosteroids [8,10-12]. These studies reported that low trough levels of MPA are associated with high risk of acute rejection episodes, while high levels are associated with risk of toxicity in both adult and pediatric transplantation. Other studies recognized that trough MPA levels between 1.0 and 3.5μ g/mL3.5 \mu \mathrm{~g} / \mathrm{mL} are associated with the least
significant complications of renal transplantation [9]. Thirdly, MPA area under the curve from 0 to 12 h(AUC0−12)12 \mathrm{~h}\left(\mathrm{AUC}_{0-12}\right) of 30−30- 60mg.h/L60 \mathrm{mg} . \mathrm{h} / \mathrm{L} in the initial phase after transplantation has been shown in both renal and heart transplants to associate with less rejection [13,14][13,14].

In support for TDM for MPA derivatives, results from the APOMYGRE French Cellcept therapeutic drug monitoring trial suggested that in renal transplant patients TIM for MMF using HPLC and a Bayesian estimator may be feasible, resulting in lower rejection rates and no increase in side effects during the first year after transplantation if dose changes are performed correctly [15]. On the other hand, the recent fixed-dose versus concentra-tion-controlled MMF dosing trial confirmed the significant relationship between early exposure on day 3 and 10 and efficacy during the first year after transplantation, highlighting the importance of the initial dosing [16-18]. This study also showed that approximately 30−50%30-50 \% of the patients have MPA exposure below the recommended 30mg.h/L30 \mathrm{mg} . \mathrm{h} / \mathrm{L} up to day 10 , suggesting the need for TDM to improve the exposure rate. Nevertheless, this target AUC can only be achieved in 50%50 \% of the time with the current recommended dosing strategy without TDM. Based on currently published information, accumulating data suggest the importance of TDM particularly in the first few days after kidney transplantation. Finally, some studies supported a relationship between MPA exposure and toxicity [17]. These findings support the need for MPA monitoring.

TDM can be seen, therefore, as an important tool to optimize the efficacy of immunosuppressive dosing including mycophenolic acid derivatives as well as a useful tool for the individualization of immunosuppressive regimens.

There is extensive literature to suggest that factors such as time after transplantation, renal function, concomitant immunosuppressive therapy influence MPA level (reviewed in [8]). For example, Kiberd et al. [17] showed that MPA-AUC on day 3 predicts efficacy but not toxicity of MMF. Furthermore, Pelletier et al. suggested that avoidance of MMF dose changes within the first year after renal transplant would result in lower rates of acute rejection and graft survival [19]. It needs emphasis however that there is no proposed target MPA ranges or applied abbreviated sampling methods for management of stable transplant patients. Considering all these published investigations in renal transplantation, we feel that MPA monitoring may be of benefit whenever there is an anticipated change in its serum level especially within the first year after transplantation. Accordingly, we propose that there is a room for measurement of MPA in the following conditions: de novo transplant patients particularly those who experience side effects, at day 3 and 90 for high immunological risk patients, with acute rejection and

whenever we anticipate changes in MPA level. Based on the above we feel that the current questions are how to measure MPA and its metabolites, the frequency of MPA monitoring, what patient population can benefit from monitoring and the relative importance of monitoring of total (and/or free) MPA versus its metabolites in different patient populations.

In an attempt to answer these questions we shall summarize a brief review of the pharmacokinetic characteristics of relevance to TDM of MPA and its measurable metabolites followed by a review of the current available monitoring methods.

Pharmacokinetics of MPA and metabolites

Absorption

To improve its oral absorption, MPA is administered as an ester prodrug mycophenolate mofetil (MMF) (Cellcept ®{ }^{\circledR}, Roche Pharma, Basel, Switzerland) that is completely absorbed and readily hydrolyzed by esterases to MPA (Fig. 1) or as the
enteric coated sodium salt (Myfortic ®{ }^{\circledR}, Novartis Pharma, Basel, Switzerland). In vitro studies demonstrated that the entericcoated Myfortic ®{ }^{\circledR} tablet does not release MPA under acidic conditions ( pH<5\mathrm{pH}<5 ) as in the stomach but is highly soluble in neutral pH conditions as in the intestine. Following Myfortic oral administration without food in several pharmacokinetic studies conducted in renal transplant patients, consistent with its enteric-coated formulation, the median delay (Tlag) in the rise of MPA concentration ranged between 0.25 and 1.25 h and the median time to maximum concentration (Tmax) of MPA ranged between 1.5 and 2.75 h . In comparison, following the administration of mycophenolate mofetil, the median Tmax ranged between 0.5 and 1.0 h . In stable renal transplant patients on cyclosporine, gastrointestinal absorption and absolute bioavailability of MPA following the administration of Myfortic delayed-release tablet were 93%93 \% and 72%72 \%, respectively.

Inter-individual variability in GIT physiology (gastric pH, intestinal motility, food intake) and potential differences in the activity of esterases could result in variations in systemic

Fig. 1. Metabolic biotransformation of mycophenolic acid.

bioavailability of MPA after oral administration of its prodrug ester or sodium salt.

Distribution

MPA is an acidic compound that is extensively ( >95%>95 \% ) bound to albumin at clinically relevant concentrations. The degree of protein binding has a significant intra- and interindividual variation due to factors such as serum albumin concentration, renal function and co-administration of drugs which may compete with and/or displace MPA from its protein binding sites [20].

Nowak et al. have shown that free MPA (fMPA), rather than total MPA (tMPA), is the pharmacologically active form of the drug [20]. As such, it has been suggested that measurement of fMPA is more relevant to therapeutic outcomes when compared to total MPA concentration [20]. fMPA concentration seems to be constant in patients with preserved renal function. An increase of 2-3-fold in fMPA has been demonstrated in patients with renal failure [21]. Increased fMPA concentration has been also reported in patients with liver disease, hypo-albuminemia, and severe infection. Higher levels of fMPA have been suggested to induce bone marrow suppression [22,23]. These data suggest that measurement of fMPA appears to correlate with both drug efficacies as well as drug related toxicities.

Metabolism and elimination

Subsequently MPA is extensively metabolized by UDPglucuronsyltransferase enzymes (UGTs) in the liver, gut and kidney to its inactive metabolite, phenyl mycophenolic acid glucuronide (MPAG) (Fig. 1). This metabolite exists in plasma in up to 100 -fold greater concentration than MPA. MPAG is extensively bound to serum albumin, from which it can displace MPA, and is excreted in the urine and bile [2].

Excretion of MPAG into bile allows enteric organisms with glucuronidase activity to cleave MPAG back to MPA that can undergo enterohepatic recirculation (Fig. 1). Accordingly, two MPA peaks in plasma are usually seen: one following the first absorption and occurs 0.5−1 h0.5-1 \mathrm{~h} after oral administration; and the second and smaller peak represents absorption from the distal bowel following enterohepatic recirculation occurs 4−6 h4-6 \mathrm{~h} after drug administration [24].

A second and less abundant metabolite is the acyl glucuronide (AcMPAG). Unlike MPAG, AcMPAG is pharmacologically active and cross-reacts with antibody used in the EMIT immunoassays.

In addition to the significant inter-patient variability in UGT activity, such activity can be induced or inhibited by some concomitantly administered medications, e.g. tacrolimus and corticosteroids. Also, factors affecting enterohepatic recirculation can account for variations in MPA levels. For example, antibiotic therapy reduces the enteric organisms that possess glucuronidase activity, resulting in reduced recycling of MPAG back to MPA within the bowel [11].

Cyclosporine can potentially interrupt the enterohepatic recirculation by inhibiting the multi-drug resistance protein 2
(MRP2) which excretes MPAG into bile. Such inhibitory effect of cyclosporine on the enterohepatic recirculation of MPA was supported by population pharmacokinetic studies that showed lower MPAG clearance in cyclosporine- than in tacrolimustreated patients, despite a similar renal functions [15,25]. These changes in activity could account for inter-patient variability in levels of MPA and glucuronide metabolites.

The pharmacokinetic characteristics of MPA and its metabolites are presented in Table 1.

Analytical methods for MPA and its metabolites

Literature search methods

From the aforementioned data, it becomes evident that defining a relationship between clinical factors and MPA pharmacokinetics improves drug dosing strategy. A basic requirement to achieve this goal is to establish a validated, accurate and sensitive analytical method to determine MPA (total and free fractions) with or without its metabolites. Various analytical techniques, mainly chromatographic and enzymatic methods, have been developed for the determination of MPA and its metabolites in biological fluids.

A structured literature search was performed to identify articles in which MPA and its metabolites were assayed by high performance liquid chromatography (HPLC) and/or enzyme multiplied immunoassay technique (EMIT) methods. An internet search using the Google search engine (www.google. ca) and keywords such as: mycophenolic acid, mycophenolic acid metabolism, HPLC assay of mycophenolic acid and its metabolites, and EMIT assay of mycophenolic acid and its metabolites revealed several important web sites that offered general information about MPA, its metabolites and its pharmacological effects. Additionally, a literature search using the PubMed database of bibliographic information (http://www. ncbi.nlm.nih.gov/entrez/query.fcgi) provided a bulk of cited

Table 1
Pharmacokinetic profile of MPA and its metabolites in healthy individuals a{ }^{a}

MPA, mycophenolic acid; tMPA, total mycophenolic acid; AUC0−12(mgh/L)\mathrm{AUC}_{0-12}(\mathrm{mg} \mathrm{h} / \mathrm{L}), area under the concentration-time curve from 0 to 12 h ; Cmax, maximum plasma concentration; Tmax, time to maximum plasma concentration; dosenormalized AUC, AUC normalized to 1000 mg of mycophenolic acid; MPAG, mycophenolic acid glucuronide; AcMPAG, mycophenolic acid acyl glucuronide; fMPA, unbound mycophenolic acid; N/A, not reported.
a{ }^{a} Based on data from [12,16,26-28].
b{ }^{\mathrm{b}} Unit for fMPA is μg.h/L\mu \mathrm{g} . \mathrm{h} / \mathrm{L}.

information from the period of January 1992 to February 2007. Keyword included different combinations of MPA, free MPA, pharmacokinetics of MPA, MPA metabolites, HPLC assay, EMIT assay, and therapeutic drug monitoring. In this review, we will concentrate on the identification and quantification of MPA and its metabolites by HPLC and EMIT methods.

HPLC assay

High performance liquid chromatographic (HPLC)-based analyses are the standard methods for the determination of MPA and its metabolites. Table 2 summarizes most of these methods which can help the researchers to try out one or two method according to their laboratory facilities. Differences in hydrophilic properties of MPA (relatively non-polar, log P=3.88±0.38P=3.88 \pm 0.38 for uncharged form) and MPAG (relatively polar, log⁡P=0.49±0.52\log P=0.49 \pm 0.52 for uncharged form) make the simultaneous determination of both components in one analytical run a difficult task. Accordingly, the previously published HPLC methods used some of the following approaches for the simultaneous determination of MPA and its metabolites:
i. Use of two internal standards to ensure comparable recovery between the analytes with their respective internal standard, thereby providing higher accuracy and precision in determining the concentration of each analyte [29]. Carboxy butoxy ether derivative of MPA, naproxen and indomethacin are commonly used internal standards for MPA, while phenolphthalein glucuronic acid is a commonly used internal standard for MPAG and AcMPAG.
ii. Use of gradient elution systems [27,29-32] and Elbarbry and Shoker (unpublished work). Although this approach is important for better resolution and accurate quantification of analytes with different polarity, it is usually time-consuming. However, recent publications have reported simultaneous analysis of MPA and its metabolites using gradient elution with run time less than 6 min [33-35].
iii. Using two different sets of chromatographic conditions [36], especially when the analysis of free MPA is required. In addition of being time-consuming, this approach is not suitable when large number of samples is analyzed.

Sample preparation

Among the published methods to date sample preparation methods include:

1. Liquid-liquid extractions (LLE): although these methods have been frequently reported, they are not adequate in providing sufficient extraction yields for both MPA and its metabolites simultaneously to allow proper pharmacokinetics application due to the marked differences in polarity [37,38]. However, a recent method reported a successful extraction of both MPA and MPAG using ethyl acetate/2propanol (4:1,v/v)[39](4: 1, \mathrm{v} / \mathrm{v})[39].
2. Solid-phase extraction (SPE): these methods produce clean extracts that increase robustness and reliability of the assay and decrease the risk of column failure [29,40]. However, these methods need elution of the residue from the SPE column using large volumes of solvents resulting in 7 - to 10 fold dilution and reducing the assay sensitivity [29,40]. Also, these multi-step SPE methods are work-intensive, timeconsuming and cost-ineffective due to the use of expensive SPE columns. These drawbacks have been avoided by applying a solid-phase microextraction (SPME), a new solventless technique recently introduced for fast simultaneous extraction and pre-concentration of analytes from sample matrix [41]. However, MPAG and AcMPAG were not extracted along with MPA by this technique.
3. Protein precipitation with acidified acetonitrile or methanol [28,42-47]. Although this method does not provide clean extraction like LLE and SPE, it is much simpler, faster and less costly when compared to LLE and SPE. In addition, the use of protein precipitation can be applied universally to all types of samples, regardless of the nature of the drug being analyzed. Such advantage is important for the simultaneous separation and quantitation of MPA and its metabolites where a significant difference in polarity exists between the analytes. Previously, we have studied the effect of pH on the extraction efficiency of MPA from plasma. We concluded that the optimum pH for extraction was 3 [47]. Accordingly, protein precipitation using acetonitrile in an acidic solution is considered advantageous for clinical pharmacokinetic studies or routine drug monitoring that involve analyses of large number of patient samples.
4. Direct injection of serum or plasma samples without sample pre-treatment [48]. This approach is based on injecting samples into a precolumn where it is cleaned before elution into the analytical column. Although simple, this technique is not available in many laboratories and the total run time for MPA only is more than 18 min which makes it unsuitable for the routine run of many samples.

Assay of free MPA (fMPA)

MPAG may compete for protein binding with MPA in conditions such as impairment of renal function. Therefore interest to measure fMPA gained momentum particularly in patients with renal dysfunction. Free drug can be isolated from biological fluids by several techniques, which include equilibrium dialysis, ultrafiltration, microdialysis and ultracentrifugation.

Ultrafiltration is a simple and fast technique to isolate fMPA and it is the preferred current method used in clinical settings [29,49,50]. Ultrafiltration can be used in combination with UV or MS detectors to measure fMPA levels [29,32,50] or its glucuronide metabolite [36]. Simultaneous determination of both fMPA and MPAG has been performed by other methods [36,40,51,52]. These methods suffer, however, from certain drawbacks such as inconvenience of the indirect measurement of MPAG after hydrolysis [52], use of complicated and costly procedures [36,51] or application of time-consuming sample preparation procedures [40]. To avoid these limitations, Yau et

al. [49] developed a simple and convenient method for the simultaneous measurement of free MPA and MPAG. In this method plasma ultrafiltration is performed first to isolate the two components and then analysis is performed using a mobile phase without ion-paring reagent [49].

Mobile phase

Mobile phases are usually binary with an aqueous acidified polar solvent, such as aqueous acetic acid or phosphoric acid or low pH buffer (solvent A) and a less polar organic solvent (solvent B) such as methanol or acetonitrile, possibly acidified.

Separation and quantitation of ionic compounds such as MPA and its metabolites can be performed using ion-pair chromatography especially when there is a potential interferences from endogenous compounds in plasma or urine samples [40,44]. Addition of an ion-pairing reagent, e.g. tetrabutylammonium hydrogen sulfate (TBAHS), to the mobile phase reduces the difference in polarity between the analytes by association of the positively charged ion-pairing agent and the negatively charged anions to form non-polar, uncharged ion pairs. Accordingly, the use of ion-pairing reagents can serve to retain MPAG for improved resolution from interfering water-soluble plasma components that co-elute with MPAG [44].

Most of the ion-pairing methods for analytical assay lack reproducibility in retention times due to lack of pH control on the mobile phase. In ion-pair chromatography, it is very crucial to adjust the pH of the mobile phase to achieve maximum ionization of the analyte molecule and ion-pairing reagent molecules for the formation of the ion pair. An extensive study by Yau et al. [44] showed that adjusting pH of the buffered mobile phase to 5.5 gave the optimum results. At this pH,MPA\mathrm{pH}, \mathrm{MPA} and its metabolites ( pKa4.5\mathrm{p} K_{\mathrm{a}} 4.5 ) are 90.9%90.9 \% ionized and the analytes were separated in less than 15 min [44]. Although adjusting the pH to 7.5 results in 99.9%99.9 \% ionization, significant interfering peaks that overlapped with MPA and MPAG peaks were observed at this pH for plasma samples.

Stationary phase

Columns for the determination of MPA and its metabolites are almost exclusively reversed-phase (RP) and include both C8\mathrm{C}_{8} and C18\mathrm{C}_{18}, ranging from 150 to 300 mm in length and usually with internal diameter ranging from 3.0 to 4.6 mm . The particle size in these columns is, in most cases, either 5 or 10μ m10 \mu \mathrm{~m}.

Using a monolithic column (Chromolithin Performance RP18e) and flow rate of 3.3 mL/min3.3 \mathrm{~mL} / \mathrm{min}, MPA and MPAG were separated in an 8 -min run [53]. The Chromolithin Performance column is based on a new sol-gel process for the preparation of monolithic porous silica rods using highly pure metal free alkoxylsilanes. The highly porous skeleton of this column allows operating at higher flow rates without loss of performance and limitations due to the column backpressure. In addition to their comparable reproducibility and repeatability to
particle-packed column, the monolithic columns demonstrate a very easy handling and good stability over conventional HPLC systems [53].

Another commonly used stationary phase for the simultaneous assay of MPA and its metabolites was X-Terra RP18 (Waters Corporation) [28,44]. This stationary phase has demonstrated a longer lifetime with more than 700 biological samples without any deterioration. The advantage of using such stationary phase is its excellent stability at low pH(pH1)\mathrm{pH}(\mathrm{pH} 1) which is frequently used in mobile phases for MPA assay. Compared to the conventional reversed phase column, X-Terra stationary phase provides higher efficiency and more symmetric peaks, especially for MPAG.

Some methods keep the column at ambient temperature [28,44,47], however others, especially those used for the simultaneous determination of MPA and the metabolites, use thermostatically controlled column at temperatures 30∘C30{ }^{\circ} \mathrm{C} [50,54],42∘C[31,53][50,54], 42^{\circ} \mathrm{C}[31,53] and 62∘C[39]62^{\circ} \mathrm{C}[39].

Detection

MPA absorbs in the ultraviolet (UV) region. Due to its availability, UV detector is commonly used to detect MPA and its metabolites. UV absorption spectra show maximum absorption at 215,250 and 304 nm for MPA and 215, 251 and 295 for MPAG. Although more sensitivities were obtained at 215 nm , some investigations report high risk of interference from endogenous serum materials at this wavelength [29,30,39].

Several assay sensitivities have reported a range from 0.1μ g/mL[10,28,31,37,46,48,54]0.1 \mu \mathrm{~g} / \mathrm{mL}[10,28,31,37,46,48,54] to 1.0μ g/mL[47,53,55]1.0 \mu \mathrm{~g} / \mathrm{mL}[47,53,55] for MPA; a range from 0.25μ g/mL0.25 \mu \mathrm{~g} / \mathrm{mL} [31] to 5.0μ g/mL[55,56]5.0 \mu \mathrm{~g} / \mathrm{mL}[55,56] for MPAG and 0.05μ g/mL[27,31,54]0.05 \mu \mathrm{~g} / \mathrm{mL}[27,31,54] to 1.0μ g/mL[28,32]1.0 \mu \mathrm{~g} / \mathrm{mL}[28,32] for AcMPAG using wavelengths of 215 nm [31,32], 254 nm [30,32,37][30,32,37] and 304 nm [38,57].

Another commonly used detector is the mass spectrometer (MS). Using a thermospray interface between an HPLC system and MS, methods have been developed for the simultaneous assay of MPA and its metabolites [10,29,36,54,58][10,29,36,54,58]. When analytes are detected in the negative ionization mode, the following mass transitions are commonly used; m/z319/190.8m / z 319 / 190.8 for MPA and m/z495/319.2m / z 495 / 319.2 for both MPAG and AcMPAG. A mass transition of 356/312.2356 / 312.2 is used for indomethacin, a preferred internal standard for MS/MS analysis of MPA and its metabolites [10,29,58][10,29,58]. However, for positive ion spray, the following mass transitions are commonly used; m/z338.0/m / z 338.0 / 207.1 for MPA and m/zm / z 514.2/207.1 for both MPAG and AcMPAG [36,54]. When carboxy butoxy ether of MPA is used as an internal standard, a mass transition of 438/207438 / 207 is used [54].

A problem with this technique is the potential of fragmentation of the MPA metabolites (MPAG and AcMPAG) at the ion source to MPA. The presence of these metabolites can pose a major problem if they are not chromatographically separated. Chirag et al. [29] used indomethacin as an internal standard for the assay of MPA by LC-MS/MS instead of the commonly used internal standard, carboxy butoxy ether derivative of MPA, that undergoes in source degradation to MPA [29].

Table 2
HPLC methods for the determination of MPA and/or its metabolites in biological fluids in human

Table 2 (continued)

[1]

1. a{ }^{a} For total MPAG conditions, see #1.
   b { }^{\text {b }} Tetrabutylammonium hydrogen sulfate.
   c { }^{\text {c }} Tetrabutylammonium bromide. ↩︎

Only two methods have reported fluorescence detection of MPA using an excitation wavelength of 342 nm and emission wavelength of 425 nm[50,59]425 \mathrm{~nm}[50,59]. The advantages of this detection method are its sensitivity (LOQ 50ng/mL50 \mathrm{ng} / \mathrm{mL} for MPA), however it cannot be applied for the simultaneous determination of MPA and its metabolites in addition to its compromised specificity [50]. Methods employing fluorescence detection usually use mobile phases with alkaline pH(>9)\mathrm{pH}(>9) so that MPA becomes more highly fluorescent and less retained on the column. Changing the pH from 7 to 9.2 results in a 7 -fold increase in fluorescence intensity and a 2-fold decrease in retention time [50].

Stability studies

Stock solutions of MPA and MPAG in methanol or buffers ( pH3−9\mathrm{pH} 3-9 ) are stable at 4∘C4^{\circ} \mathrm{C} for at least 6 months and at room temperature for at least 24 h[27,39,45,50]24 \mathrm{~h}[27,39,45,50]. Furthermore, no significant degradation in plasma samples after at least three freeze-thaw cycles [27,44,49,50].

Storage of non-acidified plasma samples at room temperature or at 4∘C4^{\circ} \mathrm{C} results in a significant decrease in the concentration of AcMPAG [27,31]. This decrease corresponds to a 30%30 \% and 39%39 \% median loss in days 1 and 7 , respectively [31]. In contrast, non-significant loss of AcMPAG is observed when plasma samples are acidified ( pH 2.5 ) before storage at any temperature [31]. Freeze-thaw procedures result in degradation of AcMPAG, but such degradation is within the 15%15 \% limit accepted by FDA [27,31][27,31].

Other parameters

Chromatographic runs for the separation for MPA and its metabolites range generally from 4 to 30 min (see Table 2). Injection volume ranges from 5 to 100μ L100 \mu \mathrm{~L}. Sample volume ranges from 50 to 500μ L500 \mu \mathrm{~L}. The cost of analysis per batch shows a wide variability between laboratories based upon analytes being examined (MPA only versus MPA and one or more of its metabolites), analytical technique (HPLC-UV, HPLC-MS, HPLC-MS/MS or immunoassay), price of the reagents being used (e.g. mobile and stationary phases in HPLC, EMIT kit in immunoassay) and finally personnel wage. Based on our local experience and personal information the basic analytical cost of each run (HPLC-UV assay of MPA and its metabolites) is approximately 3.03.0\3.0 3.0 American dollars.

Immunoassay

As mentioned above, methods for determining MPA levels in biological fluids by HPLC are accurate, specific and sensitive, but they involve sample extraction using LLE, SPE or protein precipitation. These techniques require the use of expensive equipment and time-consuming sample pre-treatment, which may not be readily available for routine drug monitoring. Enzyme-multiplied immunoassay technique (EMIT) circumvents these drawbacks.

The assay depends on the conjugation of enzymes (usually glucose-6-phosphate dehydrogenase) to a hapten. Such conjugation does not disrupt the enzyme activity; however, the binding of hapten-specific antibody to haptens results in the inhibition of enzyme activity. Free haptens in the standard or sample relieve this inhibition by competing for the antibodies. Thus, enzyme activity is proportional to the concentration of free haptens.

Because of the cross-reactivity with AcMPAG, the EMIT method may overestimate MPA concentration ( 61.4%61.4 \% on average) [10] and does not accurately measure MPAG concentration. The EMIT overestimation observed in patients coadministered Sirolimus ( 19±27%19 \pm 27 \% ) is 3-fold less than that found with patients co-administered cyclosporine (61±58%)[10,33](61 \pm 58 \%)[10,33]. Analysis of quality control samples that contain no MPA metabolites does not show EMIT overestimation [10]. Such results suggest the involvement of MPA metabolites in the EMIT overestimation (Fig. 2).

Because MPAG does not show cross-reactivity in this assay, it has been suggested that the epitope recognized by the antiMPA antibody includes the free hydroxyl group at position 7 of the phenol ring of the molecule [31]. Because AcMPAG has a pharmacological activity in vitro, the EMIT assay is expected to provide a better reflection of the immunosuppressive activity during MMF treatment. It is worth noticing that several points have to be considered before the interpretation of values determined by the EMIT technique they include:

1. Cross-reactivity of AcMPAG with the EMIT assay is concentration-dependent [31]. Such increased cross-reactivity may be due to the flexibility of the antibody to undergo conformational changes and accommodates different antigens [61].
2. The pharmacokinetics of AcMPAG is different from that of the parent drug, MPA. The former compound reaches its

Fig. 2. Principle of EMIT assay. Binding of the antibody to the antigen (analyte) conjugated with the enzyme inhibits the activity of the enzyme (usually glucose-6phosphate dehydrogenase, G6PD). In the assay, enzyme activity is proportional to concentration of the analyte. Adapted from [60].

peak concentration (Cmax) 1−3 h1-3 \mathrm{~h} after MPA [27]. Therefore, the impact of AcMPAG on the MPA concentration is low in the first hour but increases 3−5 h3-5 \mathrm{~h} after MMF administration.
3. Inter-individual variability of MPA pharmacokinetics [26,27] is also a factor that should be kept into consideration before interpreting the EMIT assay data.
4. Finally, the limited stability of acyl glucuronide has to consider carefully. Its stability depends on pH , temperature, and the nature of the aglycon [62]. Shipkova et al. reported that AcMPAG undergoes hydrolysis when plasma samples were stored at physiological pH up to 24 h at room temperature or up to 30 days at 4∘C4^{\circ} \mathrm{C} or −20∘C-20^{\circ} \mathrm{C} [31]. However, immediate acidification of plasma samples to pH 2.5 after collection followed by storage at −20∘C-20^{\circ} \mathrm{C} provides satisfactory stabilization for a longer time [31].

A recent study by Wesley et al. reported the effect of transplant type on plasma MPA concentrations determined by immunoassay [63]. Data from heart transplantation showed a greater bias, but were more strongly correlated than those from renal transplant recipients [63]. In renal transplantation, the resultant percentage cross-reactivity decreased with increasing MPA concentrations potentially suggests that time of sampling may affect the cross-reactivity [63].

Comparison between HPLC and immunoassay methods

Although HPLC-MS is more sensitive and specific than HPLC-UV, both techniques were demonstrated to give identical results for the analysis of MPA and/or its metabolites [33,54]. Both EMIT and HPLC-based assays were found to give no significant differences in measuring concentrations of MPA in quality control samples provided with EMIT kits or distributed by the International Mycophenolic Acid Testing Scheme. However, EMIT overestimation over HPLC-based methods was observed when measuring MPA concentration in patients’ samples [10,15,31,57,63]. The magnitude of EMIT overestimation over HPLC depends on graft type, sampling time, associated calcineurin inhibitors (e.g. cyclosporine versus tacrolimus or Sirolimus) and statistical test used [15]. In renal transplant populations, a positive bias of 0.59%0.59 \% to 95%95 \% for EMIT over HPLC-UV was observed [64]. Schutz et al. reported a 30%−35%30 \%-35 \% overestimation by EMIT compared to HPLC in liver transplant population [65]. A positive bias of 7%7 \% and 25%25 \% with EMIT was observed in heart transplant recipients under tacrolimus or cyclosporine, respectively [65].

Conclusion

Recent studies support the notion that TDM of MPA and possibly its metabolites improve monitoring of transplant patients on calcineurin inhibitor minimization or withdrawal protocols. The frequency of MPA monitoring remains to be decided based on several factors including the availability of accurate and simple to use TDM methods. A variety of analytical techniques have been described for the analysis of

MPA and its metabolites in different biological fluids. Both the HPLC and EMIT based techniques have their appeal and drawbacks. Based on the availability of equipment either method can be considered based on each center experience. Unfortunately little is published on the cost of either method. We are aware of pharmaceutical clinical trials exploring a decisive answer for the role of TDM of MPA and its metabolites in transplantation. Therefore we considered this review as a desk reference for researchers interested in MPA monitoring, which we anticipate to be the routine at least in transplant subpopulations if not in all transplant recipients.

These methods have been reported to be accurate and reproducible and, in case of HPLC, very specific and sensitive. RP-HPLC methods are still the most commonly used techniques for the separation and quantitation of MPA and its metabolites. Although both LLE and SPE are commonly used methods protein precipitation using acidified acetonitrile has the added advantage to simultaneously determine analytes with different polarity. Immunoassay is also commonly used for the assay of MPA; however several reports have documented crossreactivity between MPA and its active metabolite AcMPAG. Users of immunoassays must interpret the results with caution and not assume that the metabolite fraction is constant in recipients of the same organ type or in different organ transplant populations. The differences between the two analytical methods should be taken into account for appropriate TDM based on MPA exposure.

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