Validation of the method

While this method was derived from a previously validated method, due to changes in instrumentation and chromatographic conditions, validation of this method was attempted in terms of linearity, accuracy, precision, and limit of detection. The linear responses for AKBA and KBA in the ranges of 10-100 μg/mL of each acid were assessed by correlation coefficient values.

The accuracy was determined by using three traditional Chinese veterinary herbal formulations (Wei Qi Booster, Stomach Happy, and Liver Happy), which have been previously analyzed [17]. These herbal formulations were additionally pre-analyzed to confirm the absence of interfering peaks. For each herbal formulation, 0.9 mg standard AKBA powder, 0.9 mg standard KBA powder, and 198.2 mg of a known herb were accurately measured and combined into a 30 mL beaker, yielding 200 mg sample powder with known concentrations of each boswellic acid. MeOH (12 ml) was added to the same beaker, and the mixture was sonicated for 30 min. Contents were filtered through Whatman Filter Paper (No. 1, 11 μm) into a 50 ml volumetric flask, with an additional 18 mL MeOH added to the flask, yielding a total of 30 mL MeOH (for a known concentration of 30 μg/ml of AKBA and KBA). The sample was then filtered through 0.45 μm syringe filters, injected into the chromatographic system, and quantities of AKBA and KBA were estimated, as described for the market formulations. This was repeated in triplicate for each of the three herbs listed above.

The inter-assay precision was determined by analyzing the combination standard solution in a ratio of 1:1 over the entire calibration range for 3 consecutive days. The intra-assay precision was determined by analyzing the combination standard solution in a ratio of 1:1 over the entire calibration range 3 times in the same day using the same calibration curve. For determinations of limit detection, the concentrations of standards lower than that of the lowest point of calibration curves were injected into the HPLC and responses were measured.