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Vitamin C or total ascorbic acid (TAA) in fruits can be assumed as ascorbic acid (AA) plus dehydroascorbic acid (DHA) content. The aim of this work was to optimize and validate, using experimental designs, a new highperformance liquid chromatographic method for vitamin C determination in strawberries. The mobile phase (MP) consisted of a 0.03 M sodium acetate/acetic acid buffer, 5% methanol. For optimization, a Box-Behnken design was used (three factors at three levels: (a) pH of MP, 3.8-5.8; (b) wavelength, 240-270 nm; and (c) flow rate, 0.5-1.2 ml min −1 ). Responses were: AA and TAA areas, peak widths, and retention times. A global optimization was performed using the Derringer desirability function, and a value of 0.84 was reached for the combination of design factors: A05.8, B0251 nm, and C01.15 ml min −1 . Method validation, using AA standard solutions, included: linearity study, limits of detection and quantification, and calibration and analytical sensitivity quantifications. Precision and accuracy were studied in strawberry extracts. The coefficients of variation (percent) were: AA, 1.5%; TAA, 1.8%, and DHA, 4.9%. Accuracy was evaluated with AA standard spiked in 30-150% range of the expected amount of analyte in real samples. The joint confidence elliptical region test and t test were employed for the study of the difference between recoveries (percent) and the ideal 100%. The robustness was analyzed using a fractional factorial design (3 4−2 ), and an AA recovery study after slight changes in operative variables was performed. The results indicate that the optimized method was linear, sensible, precise, accurate, and robust.

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A dispersive liquid-liquid microextraction procedure was developed to extract nine fluoroquinolones in porcine blood, six of which were quantified using a univariate calibration method. Extraction parameters including type and volume of extraction and dispersive solvent and pH, were optimized using a full factorial and a central composite designs. The optimum extraction parameters were a mixture of 250 μL dichloromethane (extract solvent) and 1250 μL ACN (dispersive solvent) in 500 μL of porcine blood reached to pH 6.80. After shaking and centrifugation, the upper phase was transferred in a glass tube and evaporated under N steam. The residue was resuspended into 50 μL of water-ACN (70:30, v/v) and determined by CE method with DAD, under optimum separation conditions. Consequently, a tenfold enrichment factor can potentially be reached with the pretreatment, taking into account the relationship between initial sample volume and final extract volume. Optimum separation conditions were as follows: BGE solution containing equal amounts of sodium borate (Na B O ) and di-sodium hydrogen phosphate (Na HPO ) with a final concentration of 23 mmol/L containing 0.2% of poly (diallyldimethylammonium chloride) and adjusted to pH 7.80. Separation was performed applying a negative potential of 25 kV, the cartridge was maintained at 25.0°C and the electropherograms were recorded at 275 nm during 4 min. The hydrodynamic injection was performed in the cathode by applying a pressure of 50 mbar for 10 s.

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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 chromatograph from Agilent Model 6890 equipped with flame ionization detector (FID) and a DB-624 (30 m×0.53 mm i.d., 3.00 µm film thickness) column was used. The inlet split ratio was 5:1. The influencing 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.

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