Abstract
A selective, sensitive, liquid chromatography–positive electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous quantification of fluoxetine and olanzapine in human plasma using duloxetine (IS) as an internal standard. The analytes and IS were extracted from a 0.250-mL aliquot of human plasma by liquid–liquid extraction using methyl tertiary butyl ether and n-hexane (80:20). The eluted samples were chromatographed with a run time of 3.20 min on X-terra RP8 4.6 × 50 mm, 5 µm column by using a 90:10 v/v mixture of acetonitrile and 5 mM ammonium acetate buffer (P H 5.00 ± 0.05) as an isocratic mobile phase at a flow rate of 0.4 mL/min, and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H] + ions, m/z 310.16 → 114.12 for fluoxetine, 313.19 → 256.12 for olanzapine, and 298.14→154.09 for the IS, respectively. Calibration plots were linear over the concentration range of 0.050 to 25.044 ng/mL for fluoxetine and 0.100 to 50.000 ng/mL for olanzapine. Intra-day and inter-day precision and accuracy for quality control samples of fluoxetine (0.150, 12.832, and 19.433 ng/mL) ranged between 2.95 to 10.02% and between 97.90 to 103.25% and for olanzapine (0.300, 25.000, and 38.515 ng/mL) ranged between 3.30 to 7.66% and between 100.75 to 103.11%, respectively. Matrix effect, recovery, and process efficiency experiment was conducted at five different standards, the absolute extraction efficiency was 83.32% for fluoxetine, 86.30% for olanzapine, and 77.73% for IS. This method was successfully applied on pharmacokinetic study.
ACKNOWLEDGMENT
The authors gratefully acknowledge Aurobindo Pharma Ltd., Hyderabad, India for providing necessary facilities for carrying out this study.
Notes
A, Aqueous; B, Post Extracted; C, Extracted.
a Mean area response of six replicate samples prepared in Mobile phase neat samples).
b Mean area response of six replicate samples prepared by spiking in post extracted blank.
c Mean area response of six replicate samples prepared by spiking in plasma before extraction.
d%Matrix effect: Post extracted mean response/Aqueous (Neat) mean response × 100.
e %Recovery: Extracted mean response/Post extracted mean response × 100.
f %Process efficiency: Extracted mean response/Aqueous Mean response × 100.