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Technical Briefs |
1 Department of Psychiatry, University of Mainz, Mainz, Germany;2 Division of Neurochemistry, Department of Psychiatry, Medical University of Innsbruck, Innsbruck, Austria;3 Department of Psychiatry, Heinrich-Heine-University, Duesseldorf, Germany;
aaddress correspondence to this author at: Department of Psychiatry, University of Mainz, Untere Zahlbacher Strasse 8, D-55131 Mainz, Germany; fax 49-6131-176789, e-mail hiemke{at}mail.uni-mainz.de
Aripiprazole is a novel atypical antipsychotic drug for the treatment of schizophrenia and schizoaffective disorders (1)(2)(3). The drug is metabolized by the cytochrome P450 isoenzymes 3A4 and 2D6 (4). Because of high interindividual variability in the expression of these enzymes, the aripiprazole concentration varies among healthy individuals after administration of the drug (5). In patients, insufficient response or side effects, such as somnolence, akathisia, or nausea, may result from too low or too high drug concentrations. Therapeutic drug monitoring (TDM), which is established practice for many antipsychotic drugs (6)(7), may be helpful for patients treated with aripiprazole. We measured aripiprazole by HPLC with column-switching and ultraviolet detection, based on a previously reported method for TDM of the antidepressant reboxetine (8).
Pure drug was not available; we therefore incubated tablets containing 15 mg of aripiprazole base (Abilify®; Bristol-Myers Squibb/Otsuka Pharmaceuticals) for several days in the dark at room temperature in 15 mL per tablet of methanol (LiChrosolve; Merck) or ultrapure water adjusted to pH 2 with 0.1 mol/L HCl. Before chromatographic analysis, suspensions were shaken and cleared by centrifugation (3000g for 5 min). Maximum solution was attained within the first 24 h. Because the mean drug concentrations in samples diluted to concentrations of 100, 200, or 500 µg/L differed little (2.7%; range, –7% to 10%) between methanol and water solutions, complete solution by methanol was assumed. Methanolic solutions were used for the preparation of calibrator samples with 6 concentrations between 50 and 500 µg/L and quality-control samples containing 50, 250, or 400 µg/L, prepared by supplementing drug-free plasma with aripiprazole. Perphenazine base from Sigma was used as internal standard in a final concentration of 0.0615 g/L.
Blood for serum preparation was collected from 27 schizophrenic patients who had been treated with aripiprazole. The study protocol was approved by the local ethics committee.
HPLC was performed with an Agilent 1100 series (Bio-Rad) analyzer consisting of an autosampler, a thermostated column set at 25 °C with an electric 6-port switching valve coupled to an autosampler, and 2 HPLC pumps. The variable-wavelength ultraviolet detector for monitoring absorbance was set at 210 nm. HP ChemStation software (Ver. 09.01) was used for data acquisition and integration. The analytical column [250 x 4.6 mm (i.d.)] was filled with 5-µm particles of LiChrospher CN 5 µm (MZ-Analysentechnik), and a [10 x 4 mm (i.d.)] CN column (20-µm particle size; MZ-Analysentechnik) was used as a clean-up column.
A solution of 99 µL of serum mixed with 1 µL of internal standard was injected automatically on the clean-up column. Interfering matrix compounds were washed to the waste with deionized water containing 80 mL/L acetonitrile at a flow rate of 0.8 mL/min for 5 min. After clean-up, the electric 6-port valve was switched, and the drug to be determined was eluted to the analytical column by a mobile phase consisting of 500 mL/L acetonitrile (LiChrosolve) in phosphate buffer [1.825 g/L dipotassium phosphate trihydrate (Merck) in deionized water and adjusted to pH 6.4 with 850 mL/L phosphoric acid (Merck)] at a flow rate of 1.2 mL/min. At 15 min, the switching valve was reset, and separation on the analytical column continued.
Peak heights were used for quantification. Calibrators and quality-control samples were measured twice a day over 5 days to evaluate interassay variability, assay linearity, and recovery. The sequence was as follows: drug-free plasma, control plasma with 250 µg/L supplement, sample with 50 µg/L supplement, calibration sample 1 (100 µg/L), sample with 400 µg/L supplement, calibration sample 2 (150 µg/L), calibration sample 3 (200 µg/L), sample with 400 µg/L supplement, sample with 250 µg/L supplement, calibration sample 4 (300 µg/L), sample with 50 µg/L supplement, calibration sample 5 (350 µg/L), sample with 400 µg/L supplement, sample with 250 µg/L supplement, sample with 50 µg/L supplement, and finally, calibration sample 6 (500 µg/L).
Chromatographic peaks were confirmed by tandem mass spectrometry (MS/MS) (9) on a Micromass Quattro® Ultima using mass transition 449.9
284.8 atomic mass units for aripiprazole. Samples (190 µL) were supplemented with d3-methadone as internal standard (1 mg/L in methanol) and extracted with 500 µL of cold acetonitrile (–20 °C); 100 µL of supernatant was then directly injected into the HPLC-MS/MS instrument. Chromatographic separation of the analytes was performed on a reversed-phase C18 column [Waters Sunfire®; 3.5-µm particle size; 150 x 2.1 mm (i.d.)] with a mobile phase consisting of acetonitrile, 1.5 mmol/L aqueous ammonium acetate, and formic acid (50:50:0.1 by volume) at a flow rate of 0.35 mL/min.
The automated HPLC method with column-switching enabled the analysis of serum samples containing aripiprazole and perphenazine, which had been added as internal standard, within <25 min (Fig. 1
). The on-line clean-up procedure efficiently removed matrix constituents. In samples supplemented with 50, 200, and 1000 µg/L aripiprazole, the absolute recoveries for aripiprazole were 94.7%–111.5%, and the analytical recoveries were 96.7%–115.5%. To calculate assay precision, samples containing concentrations of 50, 250, and 400 µg/L were analyzed. Mean (SD) measured concentrations were 50 (6), 253 (20), and 400 (39) µg/L, respectively. Within-run imprecision (as CV) was 9.1%, 8.1%, and 7.8%; between-day imprecision was 7.0%, 3.0%, and 4.7%; and between-run imprecision was 8.0%, 3.4%, and 4.8%, respectively. In accordance with the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) guidelines (10), the limit of quantification was <50 µg/L (see Fig. 1B
) because this is the lowest concentration at which the CV was <15% (10). When samples supplemented with 50–1000 µg/L aripiprazole were used, the method was linear with a correlation coefficient (r2) >0.998. The equation for the linear regression line was: y = 0.0026x + 0.032 µg/L. To test interference, drugs that might interfere with the measurement of aripiprazole were added to drug-free plasma. Among the 48 psychoactive drugs tested for interference (see Table 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue9/), reboxetine, pipamperone, and norclozapine had retention times similar to that for aripiprazole.
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For stability measurements, plasma supplemented with 500 µg/L aripiprazole was stored for 14 days at room temperature in the dark or exposed to daylight. Concentrations were measured in duplicate on day 0 and reanalyzed on days 1, 3, 7, and 14. The results were as follows: 500 µg/L on day 0, 503 µg/L (101%) on day 1, 548 µg/L (110%) on day 3, 495 µg/L (99%) on day 7, and 485 µg/L (97%) on day 14. Freezing and thawing did not lead to significant changes in drug concentrations.
Aripiprazole was found in measurable quantities in patient serum samples. HPLC fractions containing peaks that corresponded to aripiprazole were collected and analyzed by MS/MS to confirm the identity, using the mass transition 449.9284.8 atomic mass units for aripiprazole. Serum samples from 27 patients (12 male; age range, 19–69 years) treated with daily doses of 10–30 mg (mean, 18.4 mg/day; median, 15.0 mg/day) showed trough steady-state serum aripiprazole concentration variations among individual patients ranging from 105 to 549 µg/L. The median aripiprazole concentration was 219 µg/L, and the 25th and 75th percentiles were 146 and 254 µg/L, respectively. In patient serum samples, an additional peak with a retention time of 16 min was observed (Fig. 1C
) and was confirmed by MS/MS to be the active metabolite dehydroaripiprazole (4). Quantification of the suggested metabolite, however, was not possible because reference material was not available.
The column-switching technique described here makes it possible to run the procedure automatically with minimal sample preparation before chromatographic analysis. The results of the assay validation met CLSI requirements (10). Reboxetine (17.0 min), pipamperone (17.8 min), and the clozapine metabolite norclozapine (18.0 min) had retention times similar to that for aripiprazole (17.6 min) and thus interfered with aripiprazole measurements. These interferences should be taken into account when patients are treated with combinations of these drugs. In clinical practice, this TDM method revealed the expected interindividual variations in serum concentrations attributable to variations in patient compliance and variable metabolic capacities. The observed mean concentrations were similar to those reported in a pharmacokinetic study on healthy persons receiving steady-state doses of aripiprazole (5). Target concentrations for optimal aripiprazole response, however, remain unclear. More studies are needed to define the therapeutic window of this novel antipsychotic drug. Future studies should also include analyses of the pharmacologically active metabolite because its contribution to clinical effects is unclear. Positron emission tomography imaging studies to quantify dopamine D2 receptor occupancy gave evidence that therapeutic doses of aripiprazole are associated with 90% dopamine receptor occupancy and blood concentrations of 100–500 µg/L (11). Our analyses of patients treated with therapeutic doses of aripiprazole revealed an interquartile range (25th–75th percentiles) of 146–254 µg/L. As long as more valid data are lacking, serum concentrations of 146–254 µg/L can be considered as a preliminary target range for TDM of aripiprazole. Moreover, it should be stressed that because pure and authentic reference materials for aripiprazole and its metabolite were not available, the accuracy of the method described here remains uncertain.
Acknowledgments
We thank Sandra Heller for skillful technical assistance.
Note Added in Proof: The manufacturer (Bristol-Myers Squibb) has subsequently provided us with pure aripiprazole. We prepared samples containing 50, 250, 500, and 1000 µg/L and assayed them with our method. The results obtained were 49, 240, 488, and 995 µg/L.
References
The following articles in journals at HighWire Press have cited this article:
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  G. Grunder, C. Fellows, H. Janouschek, T. Veselinovic, C. Boy, A. Brocheler, K. M. Kirschbaum, S. Hellmann, K. M. Spreckelmeyer, C. Hiemke, et al. Brain and Plasma Pharmacokinetics of Aripiprazole in Patients With Schizophrenia: An [18F]Fallypride PET Study Am J Psychiatry, August 1, 2008; 165(8): 988 - 995. [Abstract] [Full Text] [PDF]
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