Purpose: To develop a highly sensitive and selective UPLC-MS/MS method for simultaneous determination of paclitaxel (PTX), gemcitabine (GEM), and carboplatin (CRP) in human and animal plasma as well as various animal tissues, which could offer a rapid quantitative assay with a simple sample preparation and wide concentration ranges to support pharmacokinetic studies of three common anticancer drugs in any combination in preclinical research and clinical trials.
Methods: Using deuterium-labeled d5-PTX, d4-CRP, and 13C15N2-GEM as internal standard (IS), the analytes with their IS in plasma or homogenized animal tissues were extracted with a simple solid phase extraction (DPx Hybrid 30mg tips). The reconstituted sample was applied to a Shimadzu Nexera UPLC system connected to an AB Sciex Triple Quad 5500 using ESI source. The chromatographic separation of three compounds with IS was achieved on a Polar C18 column (Luna 1.6μm 50×2.1mm) within 2 minutes at 45°C, with a gradient profile from 7% to 100% acetonitrile at a flow rate of 0.5mL/min and acidified mobile phases of acetonitrile (B) and HPLC water with 10mM ammonium formate (A). The retention times of CRP/d4-CRP, GEM/13C15N2-GEM, and PTX/d5-PTX were 0.45, 0.70, and 1.25 minutes, respectively. The mass to charge transitions in positive mode using multiple reaction monitoring (MRM) were m/z 854.3/286.1 and 859.5/291.1 for PTX and d5-PTX, m/z 264.2/112.1 and 267.1/115.2 for GEM and 13C15N2-GEM, and m/z 372.3/294.3 and 376.3/298.3 for CRP and d4-CRP, respectively.
Results: The lower limits of quantification were 5 ng/mL for GEM and 10 ng/mL for both CRP and PTX with linear ranges up to 10,000 ng/mL (correlation coefficient ≥0.993 using 1/x2 as weighting factor). Four concentrations over the linear ranges were selected as quality control for method validation. Intra-day (n=10) and inter-day (n=5) precision (%CV) were ≤12.8% and ≤13.3% for PTX, ≤4.9% and ≤6.9 for GEM, and ≤6.4% and ≤12.7 for CRP, respectively. The accuracy of three analytes was 94.9%~102.9% for PTX, 91.0%~110.6% for GEM, and 93.4~110.4% for CRP. Recoveries for PTX/d5-PTX, GEM/13C15N2-GEM, and CRP/d4-CRP were not less than 89.8%/99.3%, 77.5%/79.1%, and 70.1%/76.0%, respectively. Matrix effects were 98.7%/94.3% for PTX/d5-PTX, 77.5%/79.1% for GEM/13C15N2-GEM, and 75.7%/82.3% for CRP/d4-CRP in plasma and homogenized tissues, respectively.
Conclusion: A sensitive and specific UPLC-MS/MS method had been developed and validated for simultaneous determination of Paclitaxel, Gemcitabine, and Carboplatin in human and animal plasma as well as various animal tissues. The main advantages of this method are: 1) Efficient: measuring three common anticancer drugs in one sample using one method for any possible combination in their pharmacokinetic evaluation and cancer therapy. 2) Convenient: assay covered wide drug concentration ranges, resulting in less sample dilution process in sample processing for both preclinical and clinical studies; 3) Rapid: the total run time is about 4 minutes; 4) Simple sample preparation: the recovery is greater than 70% and the matrix effect is minor.