2.4. Quantitative NMR (qNMR) Analysis of Xanthoxylin

The qNMR analysis for xanthoxylin was performed with a slight modification to a previously published analysis [8]. We used methyl 3,5-dinitrobenzoate (1⟶20) as the internal standard for absolute quantification. For identifying the purest signal and non-overlapped signals of xanthoxylin in the standard and Zanthoxylum samples, the purities of all the signals of xanthoxylin were determined using the following equation:

where Int is the integral, MW is the molecular weight, m is the mass, n is the number of protons, P is the purity expressed as %, IS is the internal standard, and F is xanthoxylin. The intraday and interday variabilities of xanthoxylin were examined by three replicate experiments at a concentration of 10 mg/ml in CDCl3 mixed with the internal standard, methyl 3,5-dinitrobenzoate (1⟶20) in one day for the intraday test and three consecutive days (1, 3, and 5 days) for the interday test. The relative standard deviations (RSD) were calculated as a measure of precision.