3.5.3. Quantification Method Validation

In the full scan mode, the accurate mass of the precursor ion was used for quantification. Five-point calibration curves were obtained with a mixed standard solution in the concentration range of 25 to 2500 ng/mL. Linearity was evaluated using the coefficients of correlation (R2). The LOD (limit of detection) and LOQ (limit of quantification) were determined as 3.3 times and 10 times, respectively. The standard deviation of the *y*-intercept divided by the slope of the calibration curve (ICH Validation of analytical procedures: text and methodology Q2(R1), International Conference on Harmonization, 2005) [46]. The UHPLC-Q-Orbitrap mass spectrometry was validated with respect to specificity, linearity, sensitivity, and reproducibility (ICH guidelines [46]).

The precision of the established method was evaluated by intra-day and inter-day variability, and the relative standard deviations (RSD) were taken as a measure. The RSDs were used as a measure and the acceptance criterion should be within 5.0%. The intra-day and inter-day variability were measured to assess the precision of the developed method using samples of alfalfa and red clover sprouts extract in the second day of germination. The intra-day precision was evaluated by analyzing six replicates prepared from the mentioned samples, and the inter-day precision was examined over three consecutive days with six samples per day. The repeatability was determined by injection of six samples prepared by following the same procedure.
