Simultaneous determination of synephrine, arecoline, and norisoboldine in Chinese patent medicine Si-Mo-Tang oral liquid preparation by strong cation exchange high performance liquid chromatography

Abstract

Context: Chinese patent medicine Si-Mo-Tang oral liquid preparation (SMT) is composed of Aucklandia luppa Decne (Compositae), Citrus aurantium Linn (Rutaceae), Lindera aggregata (Sims) Kosterm (Lauraceae), and Areca catechu Linn (Arecaceae). Studies of SMT have been impeded due to the lack of quality control methods.

Objective: This study aimed to simultaneously determine three alkaloids including synephrine, arecoline, and norisoboldine in SMT for the first time.

Materials and methods: A strong cation exchange (SCX) high performance liquid chromatography (HPLC) method was developed to simultaneously determine synephrine, arecoline, and norisoboldine in SMT, and was compared with ion-pairing chromatography using regular reversed-phase chromatography columns. System suitability parameters of synephrine, arecoline, and norisoboldine using the SCX chromatography column were investigated.

Results: Results demonstrated that good separations were achieved on an Agilent SCX (250 × 4.6 mm, 5 µm) column at 35°C. The mobile phase consisting of methanol-0.2% phosphoric acid was delivered at a constant flow of 1.0 mL min⁻¹ and the eluent was monitored at 215 nm. The HPLC method showed good linearity for the examined concentration ranges of 2.55–255.0, 1.30–208.0, and 2.06–201.6 µg mL⁻¹ for synephrine, arecoline, and norisoboldine, respectively. The limits of quantification (S/N = 10) were 2.55, 1.30, and 2.06 µg mL⁻¹, the limits of detection (S/N = 3) were 1.53, 0.78, and 1.21 µg mL⁻¹, and average recoveries were 98.99, 95.63 and 99.04%, respectively, for synephrine, arecoline, and norisoboldine.

Discussion and conclusion: This method has been successfully applied to determine synephrine, arecoline, and norisoboldine in Chinese patent medicine SMT.

Keywords::

• Alkaloids
• Chinese patent medicine
• Aucklandia luppa
• Citrus aurantium
• Lindera aggregata
• Areca catechu

Introduction

Chinese patent medicine Si-Mo-Tang oral liquid preparation (SMT) is a prescription to clear out gastrointestinal dyspeptic disease and enhance the digestive function. This prescription is mainly composed of Aucklandia luppa Decne (Compositae), Citrus aurantium Linn (Rutaceae), Lindera aggregata (Sims) Kosterm (Lauraceae), and Areca catechu Linn (Arecaceae). The combination of the four drugs could work together to promote enterocinesia and soothe the swelling and pain (Zou et al., 1999). SMT has been mainly used for the treatment of lactivorous food retention in infants and abdominal flatulence in the elderly. It also can promote recovery of gastrointestinal functions after surgery in the abdomen and postpartum, enhance gastrointestinal peristalsis, promote secretion of digestive juice, and relieve gastrointestinal indigestion (Zou et al., 1999; Liu et al., 2001).

A. luppas in the SMT contains costunolide and dehydrocostus lactone, which could stimulate large intestine excitation, strengthen the contraction, speed up the peristalsis, alleviate flatulence caused by abdominal distension, and thus promote gastric emptying and intestinal propulsion and exhibit antagonism against intestinal muscle spasms caused by acetylcholine and histamine (Zhu et al., 2000). C. aurantium and its major components synephrine and other alkaloids have certain excitatory effects on gastrointestinal smooth muscles mainly through the regulation of acetylcholine receptor, adrenergic receptor, and 5-HT receptor on cell membranes of the smooth muscles as well as direct effect on smooth muscle cells (Guan, 2004). A. catechu and its major component arecoline can stimulate M-cholinergic receptors, increase gland and digestive juice secretion, and stimulate intestinal smooth muscles, which could cure food retention, abdominal distension, and constipation, etc. (Zhou et al., 2007; Ni et al., 2004; Zhang et al., 2010). L. aggregata plays a key role in the prescription. It not only enhances the pharmacological effects of other drugs, but also stimulates gastrointestinal smooth muscle and strengthens its contraction. It also accelerates blood circulation and promotes intestinal peristalsis (Gong & Zhu, 2008; Chou et al., 1999). L. aggregata contains the main active ingredients such as isoquinoline alkaloids, norisoboldine, laurolitsine, and reticuline, etc. (Chou et al., 2005). Of these, norisoboldine has been selected as a marker to assess the quality of L. aggregata in Chinese Pharmacopoeia 2010 Edition (The State Pharmacopoeia Commission of PR China, 2010a).

As part of the same project, the pharmacological results revealed that arecoline, synephrine, norisoboldine, and other alkaloids are the major active components in SMT (unpublished data). Thus, it is necessary to develop a simple, economical and effective method to determine the three main alkaloids in SMT for drug quality control. Liquid chromatography methods have been widely used in the analysis of Chinese herbal medicines, compound preparation, and biological specimens containing these ingredients. The methods for the determination of arecoline include high performance liquid chromatography (HPLC) (Huang & McLeish, 1989), ion-pair reversed phase HPLC (Cox et al., 2004), high performance cation exchange chromatography (Chen et al., 2002), and liquid chromatography-mass spectrometry (LC-MS) (Ding & Peng, 2008). The major methods for the measurement of synephrine include capillary electrophoresis with electrochemical detection (Peng & Ye, 2007; Avulal & Khan, 2004; Chen et al., 2002), reverse phase HPLC (Hashimoto et al., 1992), flow injection chemiluminescence (Li et al., 2006), and LC-MS (Pellegrini et al., 2007), etc. In addition, high performance liquid chromatography (HPLC) (Han et al., 2009; Chen et al., 2009) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) (Chen et al., 2011) methods have been developed for the determination of norisoboldine contained in L. aggregata and biological specimens. However, only a few HPLC methods have been developed for the determination of arecoline or synephrine in SMT (Yuan et al., 2009; Yao et al., 2009). In our previous studies, a combination method using HPLC fingerprint and multi-components analysis for quality consistency evaluation of SMT has been established for simultaneous determination of naringin, isonaringin, hesperidin, neohesperidin, norisoboldine, and potassium sorbate in SMT (Yi et al., 2011). However, due to the weak retention of arecoline and synephrine on regular reversed-phase columns, it was failed to determine arecoline and synephrine simultaneously.

In order to simultaneously determine arecoline, synephrine, and norisoboldine in SMT, a strong cation exchange column, which has specificity for alkaloids, was selected in this study for the separation of arecoline, synephrine, and norisoboldine in SMT based on the principle of high performance ion exchange chromatography. In addition, the method was compared with the one using the normal ODS reversed-phase column for the separation based on the principle of ion-pair reversed-phase HPLC.

Materials and methods

Chemicals and reagents

Synephrine (110727–200306) and arecoline hydrobromide (111684–200401) standards were provided by National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. Norisoboldine with purity >98% was provided by Shanghai Research Center of Standardization of Chinese Medicines, Shanghai, China. Phosphoric acid and methanol were of HPLC grade, and sodium dodecyl sulfate (SDS) and other reagents were of analytical grade. HPLC-grade water (>18 mΩ) was obtained from a Milli-Q water purification system (Bedford, MA). Twenty-six batches of SMT were provided by Hunan Hansen Pharmaceutical Co., Ltd. (Hunan, China) and Hunan Zhongda Wuma Pharmaceutical Co., Ltd. (Hunan, China).

Preparation of standard solutions

Standard stock solutions of arecoline hydrobromide (0.5 mg mL⁻¹), synephrine (0.5 mg mL⁻¹), and norisoboldine (0.4 mg mL⁻¹) were prepared by dissolving accurately measured standards in methanol. The mixed standard working solutions containing 50 µg mL⁻¹ arecoline hydrobromide, 50 µg mL⁻¹ synephrine, and 40 µg mL⁻¹ norisoboldine were obtained by sequential dilution of the mixture of the three standard stock solutions using methanol.

Preparation of sample solutions

Five bottles of SMT were mixed and filtered through 0.45 µm microporous membrane and the filtrate was collected as sample solution.

Instrumentation and chromatographic conditions

Instruments

An Agilent 1100 series LC system consisting of a G1312A binary pump, a G1322A degasser, a G1313A autosampler, a G1316A thermostatted column compartment, a G1315A DAD detector, and HP Chemstation Software (Agilent, Palo Alto) was used to achieve HPLC separation and data analysis. BP211D electronic analytical balance (Sartorius Co.) was also used.

Chromatographic conditions

Method 1: The separation was performed on an Agilent strong cation exchange (SCX) column (250 × 4.6 mm, 5 µm) at 35°C. The mobile phase consisting of methanol-0.17% phosphoric acid solution (2 mL of 85% phosphoric acid was diluted to 1000 mL with water and pH was adjusted to 3.8 using ammonia water) (65:35) was delivered at a flow rate of 1.0 mL min⁻¹. Ten microliter of the sample solution was injected into the HPLC system and the eluent was monitored at 215 nm.

Method 2: The separation was performed on an Agilent ZORBAX SB-C₁₈ column (250 × 4.6 mm, 5 µm) at 35°C. The mobile phase consisting of methanol-0.1% SDS (50:50) was delivered by a velocity gradient (0–25 min, 0.7 mL min⁻¹; 25–43 min, 1.0 mL min⁻¹; and 43–50 min, 0.7 mL min⁻¹). Twenty microliter of the sample solution was injected into the HPLC system and the eluent was monitored at 230 nm.

Results and discussion

Chromatographic conditions and system suitability test

The structures of arecoline, synephrine, norisoboldine are shown in Figure 1. Arecoline and synephrine belong to low molecular alkaloids with high polarity, which exhibit weak retention on regular reversed-phase columns and need the addition of ion-pairing reagents to the mobile phase to improve their retention (Cox et al., 2004; Chen at al., 2002; Hashimoto et al., 1992). Considering that the general application pH ranges for the reversed-phase columns are 2–8, in order to simultaneously determine arecoline, synephrine, and norisoboldine in SMT, the effect of pH values of the mobile phase on the retention of arecoline and synephrine was investigated. When the pH value of mobile phase was low, the peak came out very early, even without retention. Thus an Agilent SCX strong cation exchange column, which is specific for alkaloid separation, was selected to simultaneously determine arecoline, synephrine, and norisoboldine in SMT. As for the mobile phase, it was found that the reported mobile phase used to determine arecoline content from A. catechu (The State Pharmacopoeia Commission of PR China, 2010b) was suitable for the separation of arecoline, synephrine, and norisoboldine from SMT with some modifications on the composition of the mobile phase, but without modification on pH value. The chromatograms of arecoline, synephrine, and norisoboldine in SMT on the Agilent ZORBAX SB-C₁₈ column and the Agilent SCX strong cation exchange column are shown in Figures 2 and 3, respectively. Table 1 shows the chromatographic parameters including resolution, theoretical plate number, symmetry factor, and peak purity of arecoline, synephrine, and norisoboldine in SMT on SCX and ZORBAX SB-C₁₈ columns, respectively.

Table 1.  Chromatographic parameters of resolution, theoretical plate number, symmetry factor, and peak purity of arecoline, synephrine, and norisoboldine in SMT on SCX and ZORBAX SB-C18 columns.

Parameters		Synephrine		Norisoboldine		Arecoline
		SCX	ZORBAX SB-C18	SCX	ZORBAX SB-C18	SCX	ZORBAX SB-C18
Resolution		4.57	1.04	1.67	14.49	8.47	2.37
Number of theoretical plates		11781	9890	9084	13994	11672	4200
Symmetry factor		0.92	0.68	0.86	0.69	0.91	0.61
Peak Purity	Purity factor	999.885	810.509	999.892	794.063	999.799	636.696
	Threshold limit	999.639	997.612	999.789	997.944	999.442	996.321

Figure 1.  Structures of arecoline, synephrine, and norisoboldine.

Figure 2.  HPLC chromatograms of reference standards (A) and SMT sample (B) separated on an Agilent ZORBAX SB-C18 column (250 × 4.6 mm, 5 µm) (1: synephrine; 2: arecoline; 3: norisoboldine).

Figure 3.  HPLC chromatograms of reference standards (A) and SMT sample (B) separated on an Agilent SCX column (250 × 4.6 mm, 5 µm) (1: synephrine; 2: norisoboldine; 3: arecoline).

Figure 2 exhibited an acceptable separation of synephrine, arecoline and norisoboldine in SMT on an Agilent ZORBAX SB-C₁₈ column with SDS being added to the mobile phase. When gradient elution was performed by changing the ratio of the mobile phase, baseline drift was serious and background interference was changed with the ratio of the mobile phase, resulting in unacceptable separation effects. Thus, in order to obtain stable baseline and good separation, velocity gradient elution with fixed mobile phase consisting of methanol-0.1% SDS = 50:50 was used.

These three alkaloids exhibited different retention times on the two different types of columns. On the SCX column, the retention times for synephrine, norisoboldine, and arecoline were 13.57, 18.46, and 35.96 min, respectively, while on the ZORBAX SB-C₁₈ column, the retention times for synephrine, arecoline and norisoboldine were 15.90, 17.95, and 35.85 min, respectively, i.e., the retention times of norisoboldine and arecoline were reversed. Symmetry factors of these three alkaloids were between 0.64–0.74 on the ZORBAX SB-C₁₈ column and 0.86–0.93 on the SCX column. The number of theoretical plates for synephrine and arecoline was improved on the SCX column compared with the ZORBAX SB-C₁₈ column, which could improve the detection sensitivity. However, the number of theoretical plates was decreased for norisoboldine on the SCX column. The peak purity test indicated that the peak purity factors of those three alkaloids were all higher than their threshold limits separated on the SCX column, but not on the ZORBAX SB-C₁₈ column (Table 1).

The maximum UV absorption wavelength was 215 nm for arecoline, and 283 nm for synephrine and norisoboldine. However, synephrine and norisoboldine also had some absorption at 215 nm. In method 2, arecoline and synephrine exhibited weak retention on the ODS column without the addition of SDS in the mobile phase, which could not meet the requirement of detection. However, due to the addition of SDS, the UV background of the mobile phase was increased and the interference at 215 nm was also increased, although the retention times of arecoline and synephrine were increased. With increased detection wavelength, baseline interference was gradually reduced, but the sensitivity of detection was also reduced. Thus 215 nm and 230 nm were selected as the detection wavelength for method 1 and 2, respectively, for the simultaneous detection of synephrine, arecoline and norisoboldine.

The results of system suitability tests showed that the SCX column was more suitable for the separation and detection of synephrine, norisoboldine, and arecoline in SMT than the ZORBAX SB-C₁₈ column. Thus, the Agilent SCX column was selected for the determination of synephrine, arecoline and norisoboldine in SMT.

Robustness evaluation

Robustness test was mainly to examine the tolerability of the method itself to variable experimental conditions, i.e., the effects of column temperature and flow rate of the mobile phase on the separation of the chromatographic peaks.

Effect of column temperature

SMT sample solutions were analyzed at 30, 35 and 40°C, respectively, and the chromatograms were recorded. The column temperature between 30–40°C exhibited certain effect on the retention times of the peaks, but not on the separation of the peaks. The separation time was shortest when the column temperature was 40°C. Thus, a column temperature between 30–40°C could be selected.

Effect of flow rate

SMT sample solutions were analyzed at different flow rates of 0.8, 1.0, and 1.2 mL min⁻¹, respectively, and the chromatograms were recorded. The results showed that the flow rates exhibited little effect on peak separation, but had some effects on the retention of the peaks. The separation time was short when the flow rate was high. Thus, a flow rate of 0.8–1.2 mL min⁻¹ could be selected.

Method validation

Standard curves and linearity

Standards stock solutions of synephrine, arecoline hydrobromide, and norisoboldine were mixed and diluted with methanol to get standard working solutions with a serial concentrations of synephrine (2.55, 5.10, 25.5, 51.0, 102.0, 153.0, and 255.0 µg mL⁻¹), arecoline hydrobromide (1.30, 2.60, 13.0, 26.0, 52.0, 154.0, and 208.0 µg mL⁻¹), and norisoboldine (2.02, 4.03, 20.16, 40.32, 80.64, 120.96, and 201.6 µg mL⁻¹). Ten microliter of these standard working solutions were injected into the HPLC system and analyzed under conditions as described in method 1.

Linear regression of chromatographic peak areas of synephrine, arecoline hydrobromide, and norisoboldine and corresponding concentrations was performed, resulting in regression equations of standard curves. The standard curves for synephrine, arecoline hydrobromide, and norisoboldine were y = 18027X + 12.397, r = 0.9999; y = 20543X + 10.220, r = 0.9999; and y = 46596X −1.189, r = 1, respectively. Good linearity was observed for synephrine, arecoline hydrobromide, and norisoboldine within the ranges of 2.55–255.0, 1.30–208.0, and 2.02–201.60 µg mL⁻¹, respectively.

Limit of detection and limit of quantification

One microliter of the mixed standard working solution of synephrine, arecoline hydrobromide, and norisoboldine was diluted with methanol to different concentrations desired. Ten microliter was injected into the HPLC system. Limits of quantitation for synephrine, norisoboldine, and arecoline hydrobromide were 2.55, 2.02, and 1.30 µg mL⁻¹, respectively with S/N = 10, whereas limits of detection for synephrine, norisoboldine, and arecoline hydrobromide were 1.53, 1.21, and 0.78 µg mL⁻¹, respectively, with S/N = 3.

Precision

Mixed standard solutions of synephrine (25.5, 51.0, 102.0 µg mL⁻¹), norisoboldine (20.16, 40.32, 80.64 µg mL⁻¹), and arecoline hydrobromide (13.0, 26.0, 52.0 µg mL⁻¹) at three concentrations were injected repeatedly for six times, with 10 µL each time, and the peak areas were recorded. Intra-day RSD% values at three concentrations were 0.20, 0.16, and 0.26 for synephrine, 0.22, 0.54, and 0.34 for norisoboldine, and 0.33, 1.03 and 1.14 for arecoline hydrobromide, respectively. Inter-day RSD% values obtained by continuous injection for three days were 1.00, 1.56 and 1.38 for synephrine, 1.27, 1.58, 1.56 for norisoboldine, and 0.81, 1.63, and 1.68 for arecoline hydrobromide at three concentrations. The intra-day and inter-day RSD values of synephrine, arecoline hydrobromide, and norisoboldine were all less than 2%, which met the requirements of sample analysis (ICH, 2005).

Sample stability

SMT sample was processed according to the above method and 10 µL sample solution was injected at 0, 2, 4, 8, 12, 24, 48 h. Peak areas were recorded and the stability of synephrine, arecoline, and norisoboldine was assessed by comparing the peak areas. The results showed that RSD% of the peak areas of synephrine, arecoline, and norisoboldine were 1.57, 0.53, and 1.95, respectively. Thus, these three components in SMT were stable within 48 h.

Repeatability

Six SMT sample solutions were prepared according to the above methods and 10 µL of each was injected into the HPLC system, with peak areas recorded. The RSD% of the peak areas of synephrine, arecoline, and norisoboldine were 1.60, 1.04, and 0.85%, respectively. Thus, this method exhibited good repeatability.

Accuracy and recovery

SMT (2.5 mL) (Lot # 090436), of which the contents of synephrine, arecoline, and norisoboldine were accurately determined, was put into 5 mL volumetric flasks, followed by the addition of standard stock solutions containing 80, 100, 120% of each component. Methanol was added to make the total volume of 5 mL and the solution was mixed, filtered and the filtrate was injected into the HPLC system. The accuracy and recovery were calculated. The average recoveries for synephrine, arecoline and norisoboldine were 98.99, 95.63 and 99.04% with RSD of 2.64, 1.07, and 1.51%, respectively (Table 2).

Table 2.  Accuracy and recovery of synephrine, arecoline and norisoboldine at different concentrations (n = 3).

Compounds	Background amount (mg)	Added amount (mg)	Observed amount (mg)	RSD (%)	Accuracy (Relative error, %)	Recovery (%)	Average recovery (%)	RSD (%)
Synephrine	0.0559	0.0255	0.0821	0.84	−3.53	96.30
		0.0510	0.1081	0.61	−0.78	99.14	98.99	2.64
		0.0612	0.1196	1.27	1.47	101.52
Arecoline	0.0879	0.0821	0.1674	0.64	−3.29	96.65
		0.1027	0.1862	0.12	−4.38	95.63	95.63	1.07
		0.1283	0.2098	0.05	−5.38	94.60
		0.0605	0.1587	0.39	−0.50	99.55
Norisoboldine	0.0985	0.0968	0.1927	0.59	−2.69	97.36	99.04	1.51
		0.1210	0.2197	0.14	0.16	100.22

Sample determinations

Five bottles of SMT were mixed and filtered through 0.45 µm membrane. The filtrate (10 µL) and 10 µL of the standard solutions were injected into the HPLC system. The average contents of synephrine, arecoline, and norisoboldine in 26 batches of SMT samples from two manufactures were analyzed using one-point external method. The average contents of synephrine, norisoboldine, and arecoline were 0.024 ± 0.005, 0.041 ± 0.009, and 0.029 ± 0.007 mg mL⁻¹, respectively (Calculated from dada in Table 3).

Table 3.  Determination of synephrine, arecoline and norisoboldine from 26 batches of SMT (n = 3).

No.	Batch	Manufacturer*	Content (mg mL^−1) (mean ± SD)**
			Synephrine	Norisoboldine	Arecoline
1	070109	A	0.021	0.021	0.026
2	070419	A	0.024	0.024	0.036
3	070706	A	0.031	0.042	0.024
4	071017	A	0.022	0.028	0.022
5	080121	A	0.021	0.036	0.026
6	080421	A	0.018	0.040	0.023
7	080712	A	0.022	0.041	0.025
8	081036	A	0.027	0.038	0.027
9	090225/731	A	0.027	0.044	0.030
10	090628/477	A	0.027	0.056	0.043
11	091026/470	A	0.034	0.054	0.035
12	091243/448	A	0.040	0.049	0.037
13	090424	A	0.024	0.044	0.021
14	090425	A	0.025	0.046	0.033
15	090426	A	0.024	0.046	0.028
16	090435	A	0.027	0.046	0.042
17	090436	A	0.022	0.039	0.035
18	20060311	A	0.020	0.040	0.023
19	20060601	B	0.015	0.042	0.036
20	20060905	B	0.019	0.046	0.037
21	20061203	B	0.022	0.042	0.029
22	20070308	B	0.022	0.039	0.026
23	20070624	B	0.028	0.043	0.019
24	20070918	B	0.021	0.021	0.028
25	20071201	B	0.019	0.042	0.022
26	20080306	B	0.026	0.043	0.028

*A: Hunan Hansen Pharmaceutical Co., Ltd. Hunan; B: Hunan Zhongda Wuma Pharmaceutical Co., Ltd.

**The standard deviation (SD) values for all data were smaller than 0.002.

Conclusion

Based on system suitability study, cation exchange high performance chromatography resulted in improved peak separation, symmetry, and theoretical plate number, and could detect synephrine, arecoline, and norisoboldine at low UV region (215 nm), resulting in improved sensitivity. The system robustness test and method validation demonstrated that the established method could simultaneously determine synephrine, arecoline, and norisoboldine in SMT.

Declaration of interest

This work was partially supported by the grants from National Basic Research Program of China (2009CB523002), National Action of Technology Personnel Servicing Enterprise Program of China (2009FJ5049, 2009GJD20014), Foundation of Hunan Science and Technology Committee (2009XK6032, 2009-152), Foundation of Hunan Educational Committee (09CY001).