Simultaneous quantification of three major sanshools in analgesic bacteriostatic gel by HPLC

Rong Rong, Meiyan Zhang, Xianying Zhou, Qili Zhang, Zhiguo Yu, Yunli Zhao＊

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China

Simultaneous quantification of three major sanshools in analgesic bacteriostatic gel by HPLC

Rong Rong, Meiyan Zhang, Xianying Zhou, Qili Zhang, Zhiguo Yu, Yunli Zhao＊

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China

A rapid and sensitive high-performance liquid chromatographic (HPLC) method had been developed and validated for the analysis of three main ingredients in analgesic bacteriostatic gel. Hydroxy-α-sanshool (HAS), hydroxyl-β-sanshool (HBS) and hydroxy-γ-sanshool (HRS) were separated on a Thermo Hypersil GOLD C18column (150 mm × 4.6 mm, 5 μm) with gradient elution system. The mobile phase consisted of acetonitrile-water (v/v) with a flow rate of 1.0 mL/min and UV detection was performed at 270 nm. The method was validated according to the Chinese Pharmacopeia (2015). Linearity (r＞ 0.9990) was observed over the concentration ranges of 3.24 58.32, 0.656 11.81 and 0.304 5.48 mg/L for HAS, HBS and HRS. The average recovery for HAS, HBS and HRS was 96.9%, 97.2% and 97.9%. The method was found simple and rapid, which can be suitable for the determination of the three main ingredients in analgesic bacteriostatic gel.

analgesic bacteriostatic gel; sanshools;·Method validation; HPLC

1 Introduction

Zanthoxylum bungeanumMaxim. (ZBM), belonging to theZanthoxylumgenus of the Rutaceae family, is widely distributed in most parts of China and Southeast Asian countries [1]. The pericarp of ZBM is not only a widely used pungent condiment [2-4], but also traditional Chinese medicinal herbal for its therapeutic properties [5, 6]. It is effective for the treatment of vomiting, toothache, stomachache, abdominal pain, diarrhea, ascariosis and inflammatory diseases [7]. Over the past few decades, more studies focus on the pharmacodynamic and phytochemistry of ZBM. Many chemical constituents had been isolated and identified from the pericarps of ZBM, such as amides, essential oil, lignans, flavonoids and alkaloids etc [8-10]. Amides, most of which were sanshools in the pericarp of ZBM, were considered as active constituents responsible for anesthetic and analgesic activities. Among the amides, hydroxyα-sanshool (HAS), hydroxyl-β-sanshool (HBS) and hydroxy-γ-sanshool (HRS) (Fig. 1) show the significant surface anesthetic activity [11]. It have been demonstrated that when HAS (25-50 μg) was applied directly to the human tongue; the tingling sensation elicited from HAS was localizable to the tongue and lasted from 10 to 20 min. Thisanesthetic effect also can be used for small surgical procedure [12].

Gel, as a novel external preparation, has multiple advantages such as the good adhesion, strong permeability. It can be used as the treatment of inflammatory diseases, analgesic, anti-bacterial and local hemostasis through the skin and mucous. In the previous study, an HPLC method for simultaneous determination of HAS, HBS and HRS in ZBM extract was established successfully [13]. In our previous work, the analgesic bacteriostatic gels containing ZBM extract had been prepared by orthogonal experiment. The aim of this research is to develop simple HPLC method for the quantitative analysis of HAS, HBS, HRS in the analgesic bacteriostatic gels, so that we could provide the reference for the quality control of ZBM analgesic bacteriostatic gel.

Fig. 1 Chemical structures of hydroxy-α-sanshool (HAS), hydroxy-β-sanshool (HBS) and hydroxy-γ-sanshool (HRS)

2 Materials and methods

2.1 Apparatuses and Reagents

Analysis was performed on an HPLC system equipped with a Shimadzu LC-10AT pump, an AT-330 column oven and a SPD-10A detector (Kyoto, Japan).

Standards of HAS, HBS and HRS (Fig. 1) were isolated from the pericarp of ZBM in our laboratory. Their structures were characterized on the basis of spectroscopic analysis (NMR, MS). The purity of each reference standard was above 98.0% determined by HPLC analysis. Analgesic bacteriostatic gels (batch number: 20151217, 20151218, 20151219) were prepared in our laboratory. Methanol, acetonitrile and glacial acetic acid of HPLC grade was bought from Concord Tech. (Tianjin, China). All the other reagents were of analytical grade. Water was purified by redistillation and passed through 0.22 μm membrane filter before use.

2.2 Analytical conditions

The HPLC analysis was carried out on a Thermo Hypersil GOLD C18column (150 mm × 4.6 mm, 5 μm) with the column temperature maintained at 30 °C. Gradient elution consisted of water (solvent A) and acetonitrile (solvent B) with a flow rate of 1.0 mL/min. It was started at 40% B, then increased linearly to 45% B over 15 min and then increased linearly to 60% B over 5 min. The UV detection was performed at 270 nm and the injection volume was 20 μL.

2.3 Preparation of calibration standards

The stock standard solutions of HAS, HBS and HRS were separately prepared in methanol to obtain final concentrations of 810 mg/L, 164 mg/L and 76 mg/L, respectively. Mixed working standard solution of HAS, HBS and HRS was prepared by the appropriate dilution of the corresponding stockstandard solutions in methanol. Mixed calibration standard solutions at six concentration levels for HAS, HBS and HRS were obtained by the appropriate dilution of the mixed working standard solution in methanol.

2.4 Sample preparation

Analgesic bacteriostatic gel (0.1 g) was accurately weighed and extracted under ultrasonic with 10 mL methanol for 30 min. Then the solution was centrifuged at 12000 rpm for 5 min and filtered by 0.45 μm membrane filter. Then an aliquot of 2.5 mL filtration was diluted to 10 mL with methanol to obtain sample solution.

The blank sample solution was also prepared by analgesic bacteriostatic gel (0.1 g) without ZBM extract using the same procedure as above.

2.5 Validation Procedure

The developed method was validated in terms of linearity, precision, repeatability, accuracy and stability according to the Chinese Pharmacopeia (2015).

The linearity was tested in order to demonstrate the proportional relationship between the concentration and the analytical response at six different concentration levels. The precision was assessed by carrying out a standard solution of the three analytes on six successive occasions. The repeatability was evaluated by analyzing six replicate samples prepared as described above. Both precision and repeatability were evaluated according to the relative standard deviation (RSD, %) values. Accuracy was measured by spiking standard solutions of each analyte to known real samples. The accuracy was determined by calculating the average recoveries of the three analytes from the spiked standard solutions.

3 Results and Discussion

3.1 Optimization of the Chromatographic Procedure

Different mobile phases can affect the separation, peak shape and detection sensitivity of the analytes. During the optimization of chromatographic conditions, acetonitrile was found to provide better resolution and lower background noise than methanol. Then various ratios of water and acetonitrile were tested to obtain a better separation as well as proper retention time. Initially, isocratic elution was attempted, but the retention time of HRS was too long. So a gradient elution was optimized to elute the three analytes rapidly as well as obtain a satisfactory separation. The detection wavelength of 270 nm was chosen as it was close to the maximum absorption of the three analytes.

System suitability test was assessed to ensure the system performance. All the parameters of theoretical plates, symmetry factors, resolutions and RSD values of retention times for HAS, HBS and HRS were shown in Table 1. The blank sample, the standard substances and the sample solution were analyzed. A satisfactory separation of the analytes was observed with no interferences from the blank (Fig. 2).

Table 1 System suitability test parameters

3.2 Calibration curves and linearity

Linearity was studied by analyzing the calibration standard solutions at six concentration levels. The linear regressions, expressed by the equationY= aX+ b, whereXis the concentration of the analyte andYmeans the corresponding corrected peak area, were obtained withr＞ 0.9990 for both analytes. Results demonstrated that HAS, HBS and HRS showed a wide linearity over the concentration range test. The linear regression equations, correlation coefficients (r) and linear ranges were detailed in Table 2.

Table 2 The regression equations, correlation coefficient (r) and linear ranges for the determination of the analytes

3.3 Precision, repeatability and stability

The instrument precision was determined by analysis six consecutive injections of the standard solution containing the three analytes used above. The RSD values in terms of peak area were 0.6%, 1.3%, 0.7% for HAS, HBS and HRS, respectively.

Fig. 2 Representative HPLC chromatograms of the blank sample (A); the standard substances (B) and the sample solution (C)

Repeatability of the method was verified by analysis of six replicate samples prepared with the same procedure as described under Section 2.3.3. Values of RSD were 1.2% for HAS, 1.5% for HBS and 1.7% for HRS, respectively. It demonstrated the good repeatability of the method.

The stability was checked using the same sample solution injected six times within 12 h. The stability of the peak area with RSD 1.1% for HAS, 1.2% for HBS and 1.8% for HRS was observed during 12 h of the solution's storage at room temperature.

3.4 Recovery test

Average recoveries of the three analytes were calculated to evaluate the accuracy. The results were summarized in Table 3. The average recoveries of the three analytes were 96.9%, 97.2% and 97.6% respectively and all of the RSD values were less than 2.0%. The results demonstrated that the assay was accurate.

Table 3 Method recoveries (n=6)

3.5 Application

The established and validated method was applied to simultaneously determine HAS, HBS and HRS in analgesic bacteriostatic gel from different production batches. The results were presented in Table 4.

Table 4 Contents of the three analytes in the gel

4 Conclusion

A simple, rapid and sensitive HPLC method was developed and validated for determination of HAS, HBS and HRS in analgesic bacteriostatic gel. Validation results confirmed that the method was linear, precise, accurate, and robust. Thus, the proposed method is suitable for the quality control of analgesic bacteriostatic gel.

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* Author to whom correspondence should be addressed. Address: School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning 110016, China; Tel: +86-24-23986295; E-mail: yunli76@163.com

Received: 2015-03-23 Accepted: 2016-04-08