A Sensitive (5 pg/mL) Method for the Quantitative Determination of the Antihistamine, Azatadine in Human Plasma by LC-MS-MS
Authors
Klaus-Peter Adam, Christopher J.L. Buggé, Anders Ljungqvist, David B. Garcia
Purpose
Azatadine is a long acting antihistamine that is delivered in dosage forms typically containing one milligram which results in very low (pg/mL) blood concentrations. We present a very sensitive and rapid method to measure azatadine in human plasma down to 5 pg/mL by LC-MS-MS.
Method
Human plasma (0.5 mL) was extracted with ethyl acetate/cyclohexane 9:1 using trideuterated azatadine as the internal standard. The extract was injected onto a YMC CN 50 x 4 mm HPLC column with a mostly organic mobile phase of water/ethyl acetate/ TFA/MeOH 20:400:0.25:100 at 0.3 ml/min. The eluant was analyzed by a Sciex API 3000 LC-MS-MS in positive ion APCI mode with a run time of 4 min. Mass spectra were acquired in positive ion mode with multiple reaction monitoring.
Results
The linear quantitative range was established from 5 to 500 pg/mL using freshly fortified calibration samples. Precision and accuracy of the method was determined to be acceptable after evaluating LLOQ samples and QC samples over three days of validation. Precision (%CV) at the LLOQ (5 pg/mL) was 10.9% for the three days. Accuracy (% of theoretical concentration) for the LLOQ was 104%. Precision of QC samples prepared at 15, 175 and 375 pg/ml ranged from 4.3% to 9.6%, with accuracy ranging from 94. 1 % to 105%. Stability of QC samples in plasma during normal handling was determined to be acceptable after stressing with multiple freeze-thaw cycles, room temperature exposure, and an extended term in –20 ºC frozen storage. Extraction recovery was >90%. There were no matrix effects or chromatographic interferences observed.
Conclusion
This method demonstrates good ruggedness coupled with excellent sensitivity from a small volume of sample (0.5 mL). Being a simple liquid-liquid extraction, we have automated the extraction process using a robotics system (Packard Multiprobe II).