Quantitative study of fifteen components in Yinzhihuang Oral Solution by HPLC coupled with wavelength switching and gradient elution method / 中草药

ABSTRACT

Objective: To establish HPLC coupled with wavelength switching and gradient elution method (HPLC-DVD) for simultaneous determination of 14 active components (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, geniposide, 4-hydroxyacetophenone, scutellarin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, oroxylin A 7-O-glucuronide, wogonoside, baicalein, wogonin and oroxylin A and one auxiliary material (benzoic acid) in Yinzhihuang Oral Solution (YOS). Methods: The chromatographic separation was achieved on Welch Ultimate XB-C18 (250 mm × 4.6 mm, 5 μm) column with acetonitrile-0.1% phosphoric acid solution as mobile phases for gradient elution, at the flow rate of 0.8 mL/min in the first 30 min and 1.0 mL/min in the follow-up; The detection wavelength was set at 325 nm for neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid, 238 nm for geniposide, 275 nm for 4-hydroxyacetophenone, 228 nm for benzoic acid, 325 nm for scutellarin, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C, 203 nm for oroxylin A 7-O-glucuronide and wogonoside, and 274 nm for baicalein, wogonin and oroxylin A. The volume of sample injection was 10 μL. Results: The fifteen active components were well separated and showed good linearity, such as neochlorogenic acid 74.46-1 574.42 ng (r = 0.999 8), chlorogenic acid 34.58-741.1 ng (r = 0.999 6), cryptochlorogenic acid 34.65-742.62 ng (r = 0.999 7), geniposide 234.42-5 024.45 ng (r = 0.999 9), 4-hydroxyacetophenone 74.46-1 574.42 ng (r = 0.999 9), benzoic acid 321.79-6 897.1 ng (r = 0.999 8), scutellarin 44.7-958.08 ng (r = 0.999 7), isochlorogenic acid B 32.53-697.22 ng (r = 0.999 5), isochlorogenic acid A 8.6-184.38 ng (r = 0.999 6), isochlorogenic acid C 31.93-684.3 ng (r = 0.999 7), Oroxylin A 7-O-glucuronide 254.82-5 461.66 ng (r = 0.999 5), wogonoside 10.18-218.12 ng (r = 0.999 9), baicalein 92.81-1 989.33 ng (r = 0.999 6), wogonin 31.17-668 ng (r = 0.999 5), and oroxylin A 11.74-251.73 ng (r = 0.999 8). The precision was good, and RSD was not more than 0.75%. The repeatability was good, and the RSD was not more than 1.95%. The stability was good in 8 h, and RSD was not more than 1.77%. The average recoveries and corresponding RSD values were 100.69% (0.55%), 101.99% (1.78%), 99.20% (0.72%), 100.13% (0.48%), 100.96% (1.74%), 100.02% (0.46%), 101.14% (0.27%), 99.87% (0.59%), 100.21% (1.56%), 102.18% (0.33%), 99.00% (1.02%), 98.82% (0.65%), 98.31% (0.58%), 96.01% (0.44%), and 100.56 (0.71%), respectively. The content of 10 batches of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, geniposide, 4-hydroxyacetophenone, benzoic acid, scutellarin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, oroxylin A 7-O-glucuronide, wogonoside, baicalein, wogonin and oroxylin A were 0.246-0.322, 0.213-0.290, 0.203-0.267, 1.786-2.047, 0.035-0.046, 2.393-2.541, 0.263-0.392, 0.139-0.216, 0.032-0.067, 0.208-0.250, 2.120-2.648, 0.063-0.102, 0.081-1.429, 0.164-0.246, and 0.079-0.110 mg/mL. Conclusion: HPLC coupled with wavelength switching and gradient elution method has been established for simultaneous determination of 15 components in YOS. The method is simple, quick, accurate, and it can be used for content determination and quality control of YOS.