ABSTRACT
Methotrexate (MTX) is an antineoplastic drug, and due to its high toxicity, the therapeutic drug mon-itoring is strictly conducted in the clinical practice. The chemometric optimization and validation of a high performance liquid chromatography (HPLC) method using core–shell particles is presented for the determination of MTX in plasma during therapeutic monitoring. Experimental design and response surface methodology (RSM) were applied for the optimization of the chromatographic system and the analyte extraction step. A Poroshell 120 EC-C18 (3.0 mm ? 75 mm, 2.7μm) column was used to obtain a fast and efficient separation in a complete run time of 4 min. The optimum conditions for the chroma-tographic system resulted in a mobile phase consisting of acetic acid/sodium acetate buffer solution (85.0 mM, pH?4.00) and 11.2%of acetonitrile at a flow rate of 0.4 mL/min. Selectivity, linearity, accuracy and precision were demonstrated in a range of 0.10–6.0 mM of MTX. The application of the optimized method required only 150 mL of patient plasma and a low consumption of solvent to provide rapid re-sults.
ABSTRACT
An efficient generic static headspace gas chromatography (HSGC) method was developed, optimized and validated for the routine determination of several residual solvents (RS) in drug substance, using a strategy with two sets of calibration. Dimethylsulfoxide (DMSO) was selected as the sample diluent and internal standards were used to minimize signal variations due to the preparative step. A gas chroma-tograph from Agilent Model 6890 equipped with flame ionization detector (FID) and a DB-624 (30 m × 0.53 mm i.d., 3.00μm film thickness) column was used. The inlet split ratio was 5:1. The influ-encing factors in the chromatographic separation of the analytes were determined through a fractional factorial experimental design. Significant variables: the initial temperature (IT), the final temperature (FT) of the oven and the carrier gas flow rate (F) were optimized using a central composite design. Response transformation and desirability function were applied to find out the optimal combination of the chromatographic variables to achieve an adequate resolution of the analytes and short analysis time. These conditions were 30 °C for IT, 158 °C for FT and 1.90 mL/min for F. The method was proven to be accurate, linear in a wide range and very sensitive for the analyzed solvents through a comprehensive validation according to the ICH guidelines.