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Letters to the Editor |
1 Université Claude Bernard Lyon 1, Institut des Sciences Pharmaceutiques, et Biologiques, Département de Pharmacie Clinique, de Pharmacocinétique, et d’Evaluation du Médicament, Lyon, France
2 Hôpital Cardio-vasculaire, et Pneumologique Louis Pradel, Laboratoire de, Pharmacocinétique Clinique, Bron, France
aAddress correspondence to this author at: Université Claude Bernard Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques Département de Pharmacie Clinique, de Pharmacocinétique et d’Evaluation du Médicament, 8 avenue Rockefeller, 69373 Lyon cedex 08, France. E-mail roselyne.boulieu{at}chu-lyon.fr.
To the Editor:
Drugs in solution may be adsorbed to the surface of containers and thus be removed from solution. We studied the loss of mycophenolic acid (MPA) and its major metabolite mycophenolic acid glucuronide (MPAG) from glass, polystyrene, and polypropylene containers based on solvent and time of contact.
MPA was from Hoffmann-La Roche and MPAG from Analytical Services International Ltd. Acetonitrile (Uvasol; purity >99.8%), methanol (Uvasol; purity >99.8%), and orthophosphoric acid (85% Suprapur; Merck) were HPLC grade. The NaCl solution (9 g/L, pH 7.4) was from Fresenius Kabi. Stock solutions of MPA, MPAG, and MPA/MPAG were prepared in acetonitrile–water (80:20 by volume) and stored in polystyrene tubes at –80 °C. The chromatographic analysis was performed as described previously (1), with a minor gradient modification.
Studies were performed on ice, which stabilizes drugs for at least 8 h (2). The MPA/MPAG stock solution was diluted with acetonitrile, methanol, or isotonic NaCl in five 6-mL glass (VSM; 69% SiO2, 13% Na2O), polystyrene (Elvetec), or polypropylene (Sarstedt) tubes to obtain MPA and MPAG concentrations of 0.5 and 5 mg/L, respectively. The solutions were vortex-mixed and then transferred to another tube of similar material, a process that was repeated 4 times. Each test sample was diluted 5-fold with isotonic NaCl in a polystyrene tube before HPLC analysis.
If a significant loss of compounds was observed with these conditions, the influence of concentration of the compounds was investigated by use of 1.25 mg/L MPA, 12.5 mg/L MPAG, or a pool of MPA/MPAG.
The influence of time of contact between the solution and the material on the adsorption of the compounds was assessed under the conditions that produced a significant loss of MPA and/or MPAG (P = 0.05, unpaired t-test). The initial MPA and MPAG concentrations were 0.5 and 5 mg/L, respectively, and concentrations were determined immediately after preparation and after 30 and 60 min of contact.
In polystyrene tubes, we observed no loss of MPA and MPAG related to the successive transfers (Mann–Whitney test) regardless of the solvent used (Table 1
). In glass tubes, compounds diluted with NaCl or methanol were not adsorbed, whereas in acetonitrile, significant decreases in concentrations occurred: after 1 transfer, 
66% of MPA and 55% of MPAG were recovered, and after 4 transfers, MPA and MPAG concentrations were below their quantification limits (0.005 and 0.05 mg/L, respectively; see Tables 1 and 2 in the Data Supplement that accompanies the online version of this letter at http://www.clinchem.org/content/vol52/issue5/). In polypropylene tubes, MPA and MPAG diluted with acetonitrile or methanol did not decrease, but in NaCl, 56% of MPA and 96% of MPAG were recovered after 1 transfer, and 34% and 89%, respectively, were recovered after 4 transfers.
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At higher concentrations of MPA (1.25 mg/L) and MPAG (12.5 mg/L) dissolved in acetonitrile in glass tubes, MPA and MPAG concentrations decreased (see Table 3 in the online Data Supplement). When the assay was performed with solutions containing only MPA or only MPAG, the MPA concentration decreased more markedly than with the MPA/MPAG mixture, whereas the loss in MPAG did not change significantly. In NaCl solutions in polypropylene tubes, the percentages of loss in MPA and MPAG concentrations were similar to those observed at lower concentrations, regardless of the number of transfers. The decrease in MPA concentration after 4 transfers was greater when both compounds were combined than with solutions containing only MPA (69% vs 44%). MPAG did not decrease with or without MPA.
The contact time interval did not affect the loss of MPA and MPAG from NaCl solutions stored in polypropylene tubes. Losses occurred during the first contact (see Table 4 in the online Data Supplement). In glass tubes with acetonitrile as the solvent, MPA and MPAG concentrations decreased significantly as a function of time of contact (P = 0.029), with losses of 18% for MPA and 40% for MPAG after 60 min. These observations are of interest for therapeutic drug monitoring of MPA and MPAG, which involves the preparation of stock solutions to establish calibration curves. Among published HPLC methods for MPA/MPAG analysis, acetonitrile (3)(4)(5), acetonitrile–water (80:20 by volume) (1)(3), and methanol(5)(6)(7)(8)(9)(10) have been used to prepare stock solutions. NaCl solutions (4)(8) have been validated as a suitable matrix for determinations of free MPA and free MPAG. Polypropylene is a very common plastic widely used in MPA and MPAG monitoring (3)(8)(9).
Although the precise mechanism leading to the observed losses is unknown, we recommend use of polystyrene tubes or methanol as solvent for MPA and MPAG calibrators and stock solutions.
References
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