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Technical Briefs |
1
Centre Medico Psychologique B and
2
Clinical Pharmacology and Toxicology Laboratory, CHU G. Montpied, BP39 63003, Clermont-Ferrand Cedex 3, France
aauthor for correspondence: fax 33-4-73-751-823, e-mail fcoudore{at}chu-clermontferrand.fr
Olanzapine is an atypical antipsychotic drug now considered as a first-line agent to treat schizophrenia and psychotic mood disorders (1). Plasma concentrations indicative of a clinical response are known to be >9 µg/L (2). Knowledge of plasma concentrations, to check compliance or drug-drug interactions, is also necessary in treatment of schizophrenia (3). Moreover, olanzapine toxicity may appear at blood concentrations that are considerably lower than those observed in antidepressant-related deaths (4).
Numerous publications have described the usefulness of a HPLC silica column and aqueous methanol eluents for the analysis of many basic drugs in plasma (5)(6). The use of this type of column provides more reproducible results, making it possible to obtain a sensitive HPLC-ultraviolet (UV) procedure that is easier to perform and less expensive than electrochemical (7)(8) or mass spectrometric (9) detection. We have adapted this HPLC system for routine therapeutic drug monitoring of psy- chotropic drugs (10)(11)(12). To update this system, it is important to include new antipsychotic drugs, such as olanzapine.
The Dionex HPLC system used consisted of a Dionex injector (Model ASI-100), an isocratic pump (Model P-580A), and a photodiode array detector (Model UVD-170S). Chromatograms were processed using the ChromeleonTM chromatographic data collection and analysis system.
Blood samples were collected in tubes containing lithium heparin as an anticoagulant. After centrifugation for 10 min at 3000g, the plasma was immediately separated, supplemented with 250 g/L ascorbic acid (10 µL/mL of plasma), and stored at -20 °C until analysis.
Olanzapine was extracted from 1 mL of serum after the addition of 0.2 mL of bicarbonate buffer (pH 10.5), 20 µL of internal standard working solution (demethylated metabolite of trimipramine; 2 mg/L), and 5 mL of a mixture of hexane-isoamyl alcohol (98:2 by volume). The mixture was shaken for 15 min and centrifuged at 3000g for 5 min. The aqueous layer was discarded, and the organic layer was transferred to another glass tube and back-extracted after acidification with 1 mL of 0.2 mol/L sulfuric acid. After shaking and centrifugation, the aqueous layer was alkalinized and reextracted with the same mixture. The organic phase was evaporated to dryness under a stream of nitrogen, the residue was dissolved in 100 µL of mobile phase, and 50 µL was injected into the chromatographic system.
All analyses were performed on an Ultremex silica column [250 x 4.6 mm (i.d.); Phenomenex]. The mobile phase consisted of a methanol-deionized water mixture (70:30 by volume) containing 0.110 mL/L butylamine. The mobile phase was filtered through a 0.22 µm filter and degassed before use. The chromatography was carried out at ambient temperature at a flow rate of 1 mL/min. Peaks were monitored at 273 nm.
For calibration, a six-point calibration curve was constructed before each series of assays with calibrators prepared by adding different volumes of olanzapine working solution into drug-free serum to obtain a concentration range of 1.25–80 µg/L. Olanzapine plasma concentrations were quantified using linear regression of response (drug/internal standard peak height ratios) vs concentration. Serum samples, prepared in advance by adding 5 and 40 µg/L olanzapine to a pooled serum and then aliquoting in Eppendorf tubes and storing at -20 °C, were used as quality-control samples.
The retention times of olanzapine and the internal standard were 3.9 and 11.6 min, respectively (Fig. 1
). The limit of quantification, defined as the lowest concentration that could be calculated with a CV <10%, was 1 µg/L (n = 10). This detection limit was similar to those reported for an electrochemical detector, i.e., 1 µg/L (8) and 1.2 µg/L (3), but was lower than the limit of quantification for UV detection (6), i.e., 1.56 µg/L. However, some authors have obtained a lower quantification limit (0.25 µg/L) with electrochemical (7) and mass spectrometric detection (9).
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The linearity of the extraction procedure was verified over the calibration range by measuring drug-free plasma supplemented with known concentrations of olanzapine. The slope, y-intercept, and correlation coefficient for different calibration curves were 0.012 ± 0.004, 0.069 ± 0.269 µg/L, and 0.9962 ± 0.0037, respectively. Thus, calibration curves were linear over the range 1.25–80 µg/L.
The absolute recovery of olanzapine was obtained by comparing the peak height of extracted and nonextracted supplemented solutions. The mean extraction recovery was 67.7% ± 10.5% (n = 5). These values were comparable to those obtained by the same extraction method with tricyclic antidepressants (10)(11) or clozapine (12). The three extraction steps, although time-consuming, provided high-purity extracts and increased column longevity.
Precision was estimated from intra- and interday assay variations. The intraday assay variation was determined by analyzing six aliquots of supplemented samples containing 5 and 40 µg/L olanzapine with a calibration curve on the same day. The interday variation was determined by analyzing supplemented serum (5 and 40 µg/L) on 10 different days with an independent calibration curve on each day (Table 1
). No chromatographic interference was observed between olanzapine and commonly used psychotropic drugs (amitriptyline, clomipramine, fluoxetine, clozapine, flunitrazepam, levopromazine). The analysis method was applied to monitor plasma samples collected from a small number of patients with chronic schizophrenia. A wide concentration range was observed (3.6–89.4 µg/L) for administered doses between 10 and 20 mg. For a complete analysis, it is important to add more individual data and elements of clinical response in a larger number of patients (13).
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As illustrated in our study, the HPLC-diode array detection method with an unmodified silica column and hydrophilic eluents is a powerful and sensitive tool for the efficient separation and identification of psychotropic drugs in plasma. The HPLC analysis requires only 15 min for each sample. With the concomitant use of UV spectral analysis, this system is well suited for routine drug monitoring of multiple, coadministered medications, such as sedatives and antidepressants.
Acknowledgments
This work was supported by a grant from Eli-Lilly France.
References
The following articles in journals at HighWire Press have cited this article:
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  D. J. Newport, M. R. Calamaras, C. L. DeVane, J. Donovan, A. J. Beach, S. Winn, B. T. Knight, B. B. Gibson, A. C. Viguera, M. J. Owens, et al. Atypical Antipsychotic Administration During Late Pregnancy: Placental Passage and Obstetrical Outcomes Am J Psychiatry, August 1, 2007; 164(8): 1214 - 1220. [Abstract] [Full Text] [PDF]
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 G. Gervasini, S. Vizcaino, A. G. Herraiz, J. Benitez, and J. A. Carrillo Applicability of an Assay for Routine Monitoring of Highly Variable Concentrations of Olanzapine Based on HPLC with Mass Spectrometric Detection Clin. Chem., December 1, 2003; 49(12): 2088 - 2091. [Full Text] [PDF]
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