5. Chromatographic procedure
The mixture of methanol (HPLC grade, filtered with a 0.45mm
membrane filter) with redistilled water (80:20) was used as a
mobile phase. The program of gradient elution was set for a linear
gradient starting from 50% of methanol to reach 100% at 23 min;
the injection volume was 20mL, and the detection wavelength
254 nm. The total flow-rate was 1 mL min1 and the column
temperature was 25 C. The chromatographic data were recorded
and processed by the LC solution program version 1.23 SP.
The HPLC procedure was evaluated in terms of: linearity,
precision, accuracy, detection (DL) and quantification (QL) limits.
Detection limit (DL) was calculated using equation (y + 3 sy/x)/b,
where y – the intercept of the calibration line, sy/x – standard
deviation in the y-direction of the calibration line and b – the slope
of the calibration line, whereas quantification limit (QL) was
calculated using equation 10 sy/x/b. Quantitative analysis was
performed using the calibration curve and the accuracy of the
proposed method was determined by recovery test based on the
standard solution of single BA. Obtained results were expressed as
an average value m (confidence interval, p = 95%) [39]. One-way
analysis of variance (ANOVA), followed by Duncan post test, were
performed to analyse the significant differences between obtained
results (Statistica Windows software package).
5. Chromatographic procedureThe mixture of methanol (HPLC grade, filtered with a 0.45mmmembrane filter) with redistilled water (80:20) was used as amobile phase. The program of gradient elution was set for a lineargradient starting from 50% of methanol to reach 100% at 23 min;the injection volume was 20mL, and the detection wavelength254 nm. The total flow-rate was 1 mL min1 and the columntemperature was 25 C. The chromatographic data were recordedand processed by the LC solution program version 1.23 SP.The HPLC procedure was evaluated in terms of: linearity,precision, accuracy, detection (DL) and quantification (QL) limits.Detection limit (DL) was calculated using equation (y + 3 sy/x)/b,where y – the intercept of the calibration line, sy/x – standarddeviation in the y-direction of the calibration line and b – the slopeof the calibration line, whereas quantification limit (QL) wascalculated using equation 10 sy/x/b. Quantitative analysis wasperformed using the calibration curve and the accuracy of theproposed method was determined by recovery test based on thestandard solution of single BA. Obtained results were expressed asan average value m (confidence interval, p = 95%) [39]. One-wayanalysis of variance (ANOVA), followed by Duncan post test, wereperformed to analyse the significant differences between obtainedresults (Statistica Windows software package).
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