| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1
Abteilung Klinische Chemie, Georg-August-Universität Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany;
a author for correspondence: fax 49-551-398551, e-mail ewieland{at}med.uni-goettingen.de
Mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF), is being used increasingly in immunosuppressant therapy after solid organ transplantation (1)(2)(3). Its immunosuppressive activity is thought to reside in the inhibition of inosine monophosphate dehydrogenase (IMPDH), leading to a suppression of purine nucleotide synthesis in lymphocytes, thereby suppressing cell mitosis (3)(4). MPA is converted in the liver mainly to the 7-O-glucuronide metabolite, mycophenolic acid glucuronide (MPAG), which does not have immunosuppressant activity (3)(5). During MMF therapy, MPAG peak concentrations observed in plasma of patients (5–600 mg/L) can be up to 300-fold higher than those of MPA (0.1–50 mg/L). Whether drug monitoring of MPA or MPAG is required to improve the therapeutic efficacy or to minimize adverse side effects is still under investigation (3)(6). For monitoring of MPA concentrations in human plasma, several HPLC procedures as well as one immunoassay (Emit; Behring-Syva) are available (3)(6).
In the present investigation we addressed the question as to whether MPA and MPAG are stable in human plasma because it is possible that deglucuronidation of MPAG might take place in vitro during shipping and sample storage, thus leading to false high MPA concentrations. For this purpose, we added three different MPA and MPAG concentrations to three different pools of human plasma and followed both MPA and MPAG plasma concentrations for 7 days at three different storage temperatures. MPA and MPAG concentrations were measured using an HPLC method developed recently in our laboratory (7).
For this study, MPA, MPAG, and the carboxy butoxy ether of MPA (MPAC) were a gift of Hoffmann-La Roche (Grenzach-Wyhlen, Germany). Sodium tungstate dihydrate, potassium dihydrogen phosphate, sodium hydroxide, phosphoric acid, and perchloric acid were from Merck. Acetonitrile (HPLC grade) was obtained from S.T. Baker B.V. Stock solutions of MPA and MPAC (1 g/L) were prepared in acetonitrile and stored at -20 °C. The MPAG stock solution (5 g/L) was prepared in 800 mL/L acetonitrile–200 mL/L water and was stored in the same way.
EDTA blood from patients not undergoing MMF therapy and sent to our laboratory for routine blood cell counting was pooled, and plasma was obtained by centrifugation (3000g for 15 min). Three different plasma pools were prepared, and each pool was divided into two equal parts. MPA was added to the one set of three pools to yield the following final concentrations: 1, 10, or 50 mg/L. MPAG was added to the other set of three pools to give the following final concentrations: 50, 200, or 500 mg/L. Because the MPAG was contaminated with MPA, the plasma pools to which MPAG was added also contained MPA at concentrations of ~0.15, 0.6, and 1.5 mg/L, respectively. This MPA contamination has been observed before (5) and was determined chromatographically in our MPAG stock solution by the ultraviolet spectrum and retention time. The resulting six plasma pools were further split into three parts, which were stored separately at -20 °C, 4 °C, or at room temperature. MPA and MPAG concentrations were determined immediately after addition of the substances, and after 1 day, 3 days, and 1 week of storage. MPA was also determined at the same time intervals in the pools to which only MPA was added. In addition, 14 specimens of EDTA plasma from renal (n = 9) and liver (n = 5) transplant recipients undergoing MMF therapy and sent to the laboratory for routine determination of MPA were stored at room temperature. MPA concentrations were analyzed before storage and again after storage for 7 days.
MPA and MPAG were quantified according to a procedure developed recently in our laboratory (7). Briefly, for the determination of plasma concentrations of MPAG and MPA, 200 µL of plasma and 100 µL of acetonitrile containing the internal standard MPAC (15 mg/L) were mixed by vortex-mixing in a 1.5-mL polypropylene tube for 5 s. This was followed by the sequential addition of 20 µL of 150 g/L perchloric acid and 20 µL of 250 g/L sodium tungstate. The tube was mixed by vortex-mixing for 15 s after each addition. The sample was then centrifuged for 5 min at 10 000g, and 50 µL of the supernatant was removed for chromatography.
The HPLC system consisted of a chromatographic pump (M450), an automatic injector (GINA 50), a diode array detector (UVD 340S), a computer interface system controller linked to a personal computer (Gynkotek), and a 250 x 4.6 mm Symmetry C18 reversed-phase column (Waters Associates). The column was maintained at 38 °C. The mobile phase consisted of solution A (250 mL/L acetonitrile–750 mL/L phosphate buffer, pH 3.0; final phosphate buffer concentration, 20 mmol/L) and solution B (700 mL/L acetonitrile–300 mL/L phosphate buffer, pH 6.5; final phosphate buffer concentration, 20 mmol/L), which formed the following gradient: 0–4.5 min, 97% A–3% B; 5–12 min, 70% A–30% B; 12.5–14.5 min, 100% B. The flow rate was 1.2 mL/min, and the compounds were quantified at 215 nm. Calculations were made in the internal standard mode, using peak-area ratios. Calibration and quality control were performed by use of accurately weighed in-house plasma samples.
MPA and MPAG concentrations after days 1 and 3, and after 1 week at different storage conditions were compared with the initial concentrations, using the Wilcoxon test for paired samples. P <0.05 was considered significant. For regression analysis, the Spearman rank correlation was used.
Storage of plasma pools containing MPAG at concentrations (50, 200, and
500 mg/L) observed in plasma specimens from patients undergoing MMF
therapy produced a significant increase in apparent concentrations
even after only 1 day of storage at room temperature. When the
concentrations of MPA in plasma pools stored at room temperature were
compared with their initial values (0.13–1.44 mg/L), the
concentrations were increased to 0.15–1.65 mg/L on day 1, to
0.16–1.78 mg/L on day 3, and to 0.18–2.66 mg/L on day 7 (Table 1
). This corresponds to an overall mean increase at room
temperature to 113.0% ± 6.5% at day 1 (n = 9; P
<0.05), 122.5% ± 13.1% at day 3 (n = 9; P <0.05),
and 161.3% ± 24.0% (n = 9; P <0.05) after 1 week in
the three different plasma pools. Formation of MPA from MPAG was not
observed during the 1-week storage period when plasma pools with added
MPA were stored at either -20 or 4 °C (Fig. 1
A).
|
|
There was a positive and significant correlation (r =
0.87; n = 27; P <0.05) between the MPAG concentration
added to the plasma pools and the amount of MPA formed during storage
at room temperature. However, whereas 0.77 mg/L MPA was formed from 500
mg/L MPAG during 1 week at room temperature in one pool, 1.29 mg/L was
formed in another pool. This variability explains the relatively high
SD observed in MPA values obtained from pools stored at room
temperature (Fig. 1A
). This suggests that there are matrix-related
effects such as the activity of glucuronidases and possibly other
hydrolases in addition to a simple cleavage of MPAG. This is further
supported by the results obtained from the storage of 14 specimens from
patients undergoing MMF therapy for 1 week at room temperature, which
caused a variable but overall significant increase (P
<0.05) of MPA plasma concentrations ranging from 0.06 to 0.82 mg/L
(median, 0.17 mg/L), corresponding to a median increase of 27.9%
(range, 4.8–97.7%).
A major influence on the magnitude of the error in the MPA
determination will certainly be the ratio between MPAG and MPA
concentrations in the plasma sample. The relative overestimation of MPA
will be greater if the initial MPA concentrations are lower and the
MPAG concentrations are higher. Because of the large differences
between MPA and MPAG concentrations, with MPAG being up to 300-fold
higher than MPA, the conversion of MPAG to MPA was not apparent when
MPAG concentrations were measured. The increase in MPA at room
temperature was therefore accompanied by a nonsignificant loss of MPAG
(Fig. 1B
). When MPA was added at therapeutic concentrations (1, 10, or
50 mg/L) to three plasma pools that did not contain MPAG, the
concentrations remained stable for 7 days at all temperatures
investigated (Fig. 1C
).
These results show that storage and shipment of nonrefrigerated plasma samples containing MPAG concentrations such as those in patients undergoing MMF therapy may cause falsely increased MPA results. Plasma samples should therefore be shipped and stored refrigerated if drug monitoring of MPA is to be performed.
Acknowledgments
This work was supported in part by a grant from the Volkswagenstiftung (M.S.). We thank Hoffmann-La Roche for financial support of the study.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  F. Streit, M. Shipkova, V. W. Armstrong, and M. Oellerich Validation of a Rapid and Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for Free and Total Mycophenolic Acid Clin. Chem., January 1, 2004; 50(1): 152 - 159. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. R. J. Kuypers, K. Claes, P. Evenepoel, B. Maes, W. Coosemans, J. Pirenne, and Y. Vanrenterghem Long-Term Changes in Mycophenolic Acid Exposure in Combination with Tacrolimus and Corticosteroids Are Dose Dependent and Not Reflected by Trough Plasma Concentration: A Prospective Study in 100 De Novo Renal Allograft Recipients J. Clin. Pharmacol., August 1, 2003; 43(8): 866 - 880. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 4 °C (
), and room temperature (
).