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 (100μL) and internal standard (olanzapine-d3, 25μL) were pipetted onto an Ostro(TM) 96-well filtration plate and protein precipitated with acidic acetonitrile (300μL) before removal of endogenous phospholipids by filtration followed by analysis. Chromatography was achieved using an HSST 3 (2.1×100mm, 1.8μm) column and gradient elution with acidic water in combination with methanol at a flow rate of 0.5mL/min. The runtime was 1.5min. 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 500nM (R(2)≥0.999). Limit of quantification was 0.5nM (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 48h in darkness. When exposed to 3000lux, however, significant degradation had occurred after 48h. Notably, olanzapine in spiked serum was unstable already after four hours when exposed to 3000 lux. At 4-8°C and exposure to 550lux, both patient serum and spiked serum were stable for more than 48h 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 50,000-100,000lux-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.