2.5. Validation procedures

Both methods were validated following the ICH guidelines Q2 (R1) with respect to accuracy, repeatability, intermediate precision, range, linearity and specificity [28].

Accuracy was determined by analysis of three different levels of the target concentration (50%, 100% and 150%). Each level was prepared in triplicate and their peak areas were used to calculate the average recovery using the following formula: Recovery % = (amount found/amount added) × 100. To evaluate the accuracy of the proposed method, percentage recovery and %RSD values should be ranging from 98.0% to 102.0%, and not more than 2.0%, respectively.

Linearity of the methods was determined by injecting five concentrations, covering about 50%, 75%, 100% 125% and 150% of certain vitamins. In order to prove the linearity of a method after using the method of the calibration curve, the correlation coefficient must be above 0.99.

For investigation of within-day repeatability and between-day precision, six samples of individual standard solutions at 100% level were analyzed in 1 day (repeatability) and in three consecutive days (between-day precision) to calculate the relative standard deviation, which was expected to be ≤2. The specificity is defined as the ability to assess unequivocally the analyte in the presence of components which may be expected to be present.

Specificity is the ability to distinguish the analyte in the presence of other components in the sample matrix. Specificity of the methods was detected by injecting solutions of standard, sample and blank separately. To evaluate the sensitivity, limit of detection (LOD) and limit of quantification (LOQ) were calculated from the calibration graph, according to the equations: LOD = 3.3 × (sa/S); LOQ = 10 × (sa/S), where sa is the standard deviation of the intercept a and S is the slope.