Content Determination of Pulegone in Schizonepeta tenuifolia and Its Compound Preparation by Hollow Fiber Liquid-phase Microextraction Coupled with HPLC / 中国药房

ABSTRACT

OBJECTIVE：To establish t he metho d for the content determination of pulegone in Schizonepetae tenuifolia decoction pieces and its compound preparation. METHODS ：Hollow fiber liquid-phase microextraction coupled with HPLC （HF-LPME-HPLC） was adopted. Based on single factor tests ，HF-LPME condition of S. tenuifolia decoction pieces and its compound preparation （taking Compound S. tenuifolia granule as an expample ） was optimized by central composite design-response surface methodology using pulegone enrichment multiple as index ，with the concentration of sample phase solution （NaCl），extraction time and stirring speed as factors. Validation test was conducted. HPLC method was adopted to determine the content of pulegone. The determination was performed on Hypersil C 18 column with mobile phase consisted of methanol- 0.3％ phosphoric acid （gradient elution ）at the flow rate of 1.0 mL/min. The detection wavelength was set at 252 nm，the column temperature was 25 ℃. The sample size was 20 μL. The feasibility of HF-LPME-HPLC method established in this study was validated by using HPLC method stated in the item of S. tenuifolia decoction pieces in 2020 edition of Chinese Pharmacopoeia （part Ⅰ）as reference. RESULTS ：The optimum HF-LPME conditions included n-nonanol as the extraction solvent ，sample phase solution with 11％ NaCl and pH value of 7，stirring speed of 800 r/min，extraction time of 36 min. Results of HPLC methodology investigation showed that linear range of pulegone were 0.05-5 μg/mL（r＝0.999 0）. The limits of detection and quantitation were 0.4 and 1.3 ng/mL，respectively. RSDs of intra-day and inter-day precision were 1.8％-4.0％ and 1.5％-4.1％（n＝3），respectively. RSDs of reproducibility and stability tests （24 h）were all lower than 8％（n＝6）. Average recoveries of S. tenuifolia decoction pieces and Compound S. tenuifolia granule were 102.6％-105.1％ and 97.2％-102.3％，respectively；RSDs were not higher than 4.1％ and 6.2％（n＝3）. The average contents of pulegone in S. tenuifolia decoction pieces determined by pharmacopoeia method and established method were 0.84 mg/g（RSD＝4.3％ ，n＝3）and 0.87 mg/g（RSD＝5.5％ ，n＝3），respectively，with no significant difference （P＞0.05）. CONCLUSIONS ：The established HF-LPME-HPLC method can enrich and concentrate pulegone ， shows strong purification ability and high sensitivity ，and can be used to determine the contents of pulegone in S. tenuifolia decoction pieces and its compound preparation.