High-Throughput Validated Method for the Quantitation of 10,11-dihydro-10-hydroxycarbamazepine in Serum using Ultra Fast SPE-MS/MS

摘要

Oxcarbazepine (OCBZ) is approved as monotherapy and adjunctive therapy for partial seizures with and without secondary generalized seizures in adults and as adjunctive therapy for partial seizures in children. In humans, OCBZ is completely absorbed and extensively metabolized to its pharmacologically active metabolite 10, 11-dihydro-10-hydroxycarbamazepine, also known as monohyroxycarbamazepine (MHD). Optimal therapeutic effect and minimal toxicity can be achieved through monitoring MHD. Continued demands for greater efficiency and throughput have put increased stress on traditional technologies such as LC-MS/MS. Therefore, we present a method for the quantification of MHD in serum using an Ultra-fast SPE-MS/MS which has much faster sample cycle times and similar analytical results to LC-MS/MS. Methods: Calibration standards; quality controls; and patient samples were precipitated with methanol containing dueterated internal standard. After mixing and centrifugation, the supernantant was transferred to a 96 well plate. Water containing ammonium acetate, trifluroacetic Acid (TFA), and formic acid was added to each well. After mixing; sample analysis was performed at a rate of less than 15 seconds per sample using a Rapidfire 300 system coupled to an Agilent 6490 MS/MS using electrospray ionization in positive ion mode. Concentrations were calculated based on a five point calibration curve using a 1/x linear curve fit. Results: The analytical linear range was 1.0-50.0 mcg/mL with a limit of detection of 0.5 mcg/mL. The average (n=20) linear regression demonstrated the following: slope= 0.998; r~2= 0.9994; and intercept= 0.056. With-in (n=20) and between-run (n=20) precision CVs were less than 5% across the analytical range. Absence of interferences from the top 25 prescribed drugs or common drugs of abuse was observed. Minimal ion suppression or enhancement due to the matrix effect was observed. No significant carryover was seen following a sample containing 100 mcg/mL of MHD. 100 patient samples were cross validated with a current LC-MS/MS method. All patient results throughout the analytical range correlated within the acceptance criteria of ±20%. The linear regression demonstrated the following: slope= 1.016; r~2= 0.9947; and intercept= -0.063. Novel Aspect: An accurate and precise method to quantitate MHD in serum with SPE-MS/MS cycle times of <15 seconds per sample.