Page 26 - 《中国药房》2021年12期

Content Determination of Pulegone in Schizonepeta tenuifolia and Its Compound Preparation by Hollow
Fiber Liquid-phase Microextraction Coupled with HPLC
LI Ping，HU Shuang，BAI Xiaohong，BI Xiaoping（Pharmaceutical Analysis Teaching and Research Section，
College of Pharmacy，Shanxi Medical University，Taiyuan 030001，China）

ABSTRACT OBJECTIVE：To establish the method 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 C18 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 μ L. 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.
KEYWORDS Hollow fiber liquid-phase microextraction； HPLC； Central composite design-response surface methodology；
Pulegone；Schizonepetae tenuifolia decoction piece；Compound pregaration

荆芥为唇形科植物荆芥Schizonepeta tenuifolia Briq. 分散液相微萃取纯化效果差和重现性差等缺点，而且还
的干燥地上部分，性微温、味辛 。现代药理研究表明， 有助于分析物的富集 [10 － 13] 。目前，尚未有研究采用
[1]
荆芥主要具有抗病毒、抗炎、镇痛、抗肿瘤、调节免疫、抗 HF-LPME 技术结合 HPLC 法对荆芥饮片及其复方制剂
菌、止血等作用，且常被加入到具有清热解毒功效的中 中胡薄荷酮进行分离、富集和定量分析。鉴于此，本研
[2]
药复方制剂中 。荆芥的主要有效成分为胡薄荷酮，其 究以荆芥饮片和本实验室自制的复方荆芥颗粒为检测
可降低白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）、 样品，在单因素实验的基础上，结合中心组合设计-响应
γ干扰素（IFN-γ）等炎性细胞因子水平，具有较强的抗炎 面法优化胡薄荷酮的 HF-LPME 条件，并建立含量测定
作用 [3－4] 。目前，胡薄荷酮常用的定量分析方法为高效 的HPLC方法，以期为荆芥饮片及其复方制剂的分析提
液相色谱法（HPLC）和气相色谱法 [5－6] 。对于荆芥饮片 供一种纯化能力强、操作简便的样品前处理方法和灵敏
及成分更为复杂的复方制剂而言，测定其中胡薄荷酮的 度高的含量测定方法。
含量时，需先进行样品的前处理，而采用传统的液液萃 1 材料
取法进行前处理存在有机溶剂用量大、提取耗时长、操 1.1 主要仪器
作繁琐、对仪器灵敏度要求相对较高等不足 [6－7] 。因此， 1200 Series型HPLC仪购自美国Agilent公司；SHB-
建立一种操作简便、纯化与富集能力强的样品前处理方 Ⅲ型循环水式多用真空泵购自郑州长城科工贸有限公
法是很有必要的。 司；KQ-50DE型超声波清洗机购自宁波新芝生物科技股
近年来，中空纤维液相微萃取（HF-LPME）技术因适 份有限公司；XS205DU 型电子天平购自瑞士 Mettler
用于复杂样品的萃取、浓缩、纯化而得以广泛应用 [8－9] 。 Toledo 公司；HJ-6B 型数显多头磁力恒温搅拌器购自金
中空纤维是一种中空、多孔的高分子材料，无毒且具有 坛市白塔金昌实验仪器厂。
很好的生物相容性，其微米级壁孔可使小分子化合物自 1.2 主要药品与试剂
由通过。中空纤维不仅可以作为保护萃取相的载体和 荆 芥 饮 片（批 号 分 别 为 20170524、20180721、
纯化样品的过滤器，克服单滴液相微萃取液滴不稳定、 20190521）购自运城市神农中药材有限公司，经山西医

·1428 · China Pharmacy 2021 Vol. 32 No. 12 中国药房 2021年第32卷第12期