A UPLC-MSMS method for the analysis of olanzapine in serum-with particular emphasis on drug stability testing

A method including a rapid and automated extraction of olanzapine from serum followed by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) has been developed and validated. Serum aliquots (100 μL) and internal standard (olanzapine-d3, 25 μL) were pipetted onto an OstroTM 96-well filtration plate and protein precipitated with acidic acetonitrile (300 μL) before removal of endogenous phospholipids by filtration followed by analysis. Chromatography was achieved using an HSST 3 (2.1 × 100 mm, 1.8 μm) column and gradient elution with acidic water in combination with methanol at a flow rate of 0.5 mL/min. The runtime was 1.5 min. The mass spectrometer was monitored in positive mode and multiple reaction monitoring (MRM). The m/z 313.1 > 256.1 and 313.1 > 198.0 transitions were monitored for olanzapine (m/z 316.1 > 256.1 for olanzapine-d3). The quadratic calibration curves ranged from 5 to 500 nM (R2 ≥ 0.999). Limit of quantification was 0.5 nM (CV 9.6%, accuracy 110%). Within-assay and between-assay inaccuracies were 2.6-11.9% (CV ≤ 4.8%). Recovery was 84-95% (CV ≤ 1.4%) and matrix effects ranged from 100 to 103% (CV ≤ 2.6%). Extensive stability testing showed that at ambient temperature, olanzapine in patient serum samples were stable for at least seven hours on the laboratory bench and for at least 48 h in darkness. When exposed to 3000 lux, however, significant degradation had occurred after 48 h. Notably, olanzapine in spiked serum was unstable already after four hours when exposed to 3000  lux. At 4-8 °C and exposure to 550 lux, both patient serum and spiked serum were stable for more than 48 h but less than a week, whereas in darkness, the samples were stable for at least 14 days. The cumulative light exposure causing significant degradation of olanzapine in patient serum was 50,000-100,000 lux-h. In some individual samples, however, the effect of light exposure was more pronounced. Therefore, it seems pertinent to recommend protecting all samples from light, although we found no indication that a few hours of exposure to standard indoor illumination will affect the olanzapine concentration to any significant degree.