Simultaneous Determination of Strychnine and Brucine in Herbal Formulation by RP-HPLC

Babu Ganesan¹*, Perumal Perumal¹, Vijaya Baskaran Manickam¹, Surya Rao Srikakolapu¹, Srujana Divya Gotteti¹ and Latha Sundaresan Thirumurthy²

¹Department of Pharmaceutical Analysis and Chemistry, J. K. K. Nataraja College of Pharmacy, Komarapalayam 638183 Namakkal District, TamilNadu

²Department of Pharmaceutical Chemistry, Swamy Vivekanandha College of Pharmacy, Tiruchengode 637205 Namakkal District, TamilNadu

*Corresponding Author E-mail: gbabu73@gmail.com

ABSTRACT:

A simple, precise and accurate reverse phase HPLC method has been established for simultaneous quantitative determination of strychnine and brucine in herbal formulation (tablets). The HPLC method was performed on a Phenomenex C-18 ODS column (250 mm × 4.6 mm ID, 5 µm), with isocratic elution using a mixture of acetonitrile: phosphate buffer in the ratio of 15: 85 v/v at flow rate of 1 mL/min with UV detection at 264 nm. Strychnine and brucine were well resolved on the stationary phase, and the retention times were 6.263 minutes for strychnine and 4.987 minutes for brucine. The calibration curves were found to be linear (Correlation coefficient, r = 0.9997 for strychnine and 0.9996 for brucine) in the concentration range 20-120 µg/mL for strychnine and brucine. The herbal preparation was quantified by external standard calibration method. The assay was validated for the parameters like accuracy, precision, robustness and system suitability parameters. The proposed method can be useful in the routine analysis for the determination of strychnine and brucine in herbal formulation.

KEYWORDS: Brucine, Herbal formulation, Reversed phase high performance liquid chromatography, Strychnine

INTRODUCTION:

Strychnos nux-vomica Linn. (Fam. Loganiaceae) commonly has known as Kuchila, poison nut, and semen strychnos. In herbal medicine, it is traditionally recommended for upset stomach, vomiting, and bitter stomachic. It stimulates the muscular coat of the intestine, increases peristalsis, and hence is given for constipation in an atonic condition of the intestine, problems related to menopause, and migraine headaches^1-3.The main constituents of the seeds of Strychnos nux-vomica L. are known to be strychnine and brucine. The structures of strychnine and brucine are shown in Figure 1.

The drug analysis plays an important role in the development of drugs, their manufacture and therapeutic use. These drugs may be in single component or multi-component dosage forms. The multi-component dosage forms prove to be effective due to the combined mode of action on the body.

The complexity of dosage forms including multiple drug entities possesses considerable challenge to the analytical chemist during the development of assay procedures for herbal formulations. For the estimation of drugs present in multi-component herbal formulations, reversed phase high performance liquid chromatography can be employed as it is more sensitive and gives reproducible results.

According to literature survey, only few works were reported for the determination of strychnine and brucine by HPLC⁴, TLC⁵and capillary zone electrophoresis⁶ in crude drug but not in herbal formulation. The present study deals with the development of new reverse phase HPLC method for the simultaneous estimation of strychnine and brucine in the herbal formulation and then validation of the method as per ICH guidelines (International Conference on Harmonization of Technical Requirement for Registration of Pharmaceuticals for Human use).

MATERIALS AND METHODS:

Materials:

Authentic strychnine and brucine were purchased from Sigma Aldrich, USA. Tentex Forte tablets (Himalaya Health Care) were procured from the local market. Acetonitrile, methanol and water of HPLC grade were purchased from Merck (Mumbai, India).

Figure 1 Structure of strychnine and brucine

Instrument:

The chromatographic system consisted of Shimadzu, Prominence and a manual rheodyne injector with a 20 µL fixed loop. The separation was performed on a Phenomenex C18 ODS column (250 mm × 4.6 mm ID, 5 µm) at room temperature with a UV visible detector.

Chromatographic conditions:

Chromatographic estimation was performed using an equilibrated Phenomenex ODS C18 column with a mobile phase consisting of acetonitrile: phosphate buffer in the ratio of 15: 85 v/v at flow rate of 1 mL/min with UV detection at 264 nm (Figure 2).

Preparation of Standard Solutions:

Combined standard solution of strychnine and brucine were prepared by weighing 50 mg of strychnine reference standard and 50 mg of brucine reference standard in 50 mL volumetric flask, dissolved in 20 mL of mobile phase and made up to the volume with mobile phase (Strychnine 1 mg/mL, Brucine 1 mg/mL).

Calibration curves for strychnine and brucine:

From the standard stock solution 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mL was transferred into 10ml volumetric flask and was made up to the volume with mobile phase to get a concentration in the range of 20-120 µg/mL of strychnine and brucine. Each concentration was injected. A calibration curve (peak area vs concentration) was plotted from the peak areas obtained. The correlation coefficient was found to be 0.9997 for strychnine and 0.9996 for brucine.

Application to pharmaceutical preparation (Tentex Forte Tablets):

About 20 tablets were ground to coarse powder and average weight was calculated (645.54 mg). From the tablet powder, 0.65 g was weighed and extracted with 50 mL of methanol under reflux for one hour.^[6] The solution after extraction was filtered using Whatmann filter paper no.41 and was evaporated to obtain dry residue. The residue obtained was completely dissolved in mobile phase and was injected into the column for recording of the chromatogram (Figure 3).

RESULTS AND DISCUSSION:

In the present study, a simple, precise, accurate and rapid reverse phase HPLC method has been developed and validated for the determination of strychnine and brucine in herbal formulation. The developed reverse phase HPLC method was validated in terms of precision, accuracy, LOD, LOQ, specificity, robustness, system suitability and ruggedness.

Linearity:

The standard stock solution was diluted further to get a concentration in the range of 20-120 µg/mL of strychnine and brucine. Each concentration was injected. A calibration curve was plotted using peak area vs concentration. The correlation coefficient was found to be 0.9997 for strychnine and 0.9996 for brucine^7-10_.

Precision:

The intraday and interday precisions of the proposed method were determined by estimating the corresponding response 3 times on the same day and on 6 different days over a period of 1 week for three different concentrations of 80, 100 and 120 µg/mL of strychnine and brucine. The results are reported in terms of relative standard deviation (RSD) in Table 1^7-10_.

Table 1 Summary of validation parameters of strychnine and brucine

Parameters	Results
Parameters	Strychnine	Brucine
Linearity Range Linear equation Slope (m) Intercept (C) Correlation coefficient (r) Standard deviation (SD)	20-120 Y = mx + C 13.092 33.726 0.9997 0.35	20-120 Y = mx + C 11.071 21.786 0.9996 0.25
Precision (% RSD) Intraday precision (n=3) Interday precision (n=3)	% RSD = 0.884 % RSD = 1.038	% RSD = 0.313 % RSD = 1.736
Accuracy (% Recovery)	100.52 %	100.19 %
Limit of Detection (LOD)	0.227 µg/mL	0.410 µg/mL
Limit of Quantification (LOQ)	0.758 µg/mL	1.339 µg/mL
Robustness	robust	Robust
Specificity	No degradation	No degradation

Accuracy:

The accuracy of the method was determined by calculating the recoveries of strychnine and brucine by the method of standard addition.

Known amounts of the standards (80%, 100% and 120 %) were added to the pre-analyzed sample solution, and the amounts of these standards were estimated by measuring the peak areas and by fitting these values to the straight-line equation of calibration curve^7-10_.

Sensitivity:

Limit of Detection (LOD) and Limit of Quantification (LOQ) were determined by kSD/s where k is a constant (3 for LOD and 10 for LOQ), SD is the standard deviation of the analytical signal, and s is the slope of the concentration / response graph^7-10_.

Robustness:

Robustness of the proposed method was evaluated by changing the column to a Phenomenex ODS, C18 column. The effect of change in temperature was studied^7-10_.

System suitability:

Standard solution was injected 6 times for each change. System suitability parameters and RSD were calculated for each peak. Recoveries and % RSDs were calculated for each component during each change^7-10_.

A simple HPLC method was adopted for the simultaneous determination of strychnine and brucine in herbal formulation. To optimize the proposed HPLC method, all of the experimental conditions were investigated. For the choice of stationary phase, reversed-phase separation was preferred due to the drawbacks of the normal phase. To optimize the mobile phase, different systems were tried for chromatographic separation of the components; the best resolution was achieved using a mobile phase consisting of acetonitrile: phosphate buffer in the ratio of 15: 85 v/v, which gave good resolution and sensitivity of both drugs. The calibration curves constructed for the markers were linear over the concentration range of 20-120 µg/mL for strychnine and brucine. Peak areas of the markers were plotted versus the concentration and linear regression analysis performed on the resultant curve with the correlation coefficients 0.9997 and 0.9996 for strychnine and brucine respectively (Table 1). The precision result of the solution at medium concentration (Table 1) indicate that the RSD values of retention time were less than 1%, while the RSD values of peak area were less than 2% both for intra-day assay and inter-day assay precision (Intra 2 h six injections, inter 3 days). The LOD was found to be 0.2275 µg/mL and 0.410 µg/mL respectively for strychnine and brucine. The LOQ was found to be 0.758 µg/mL and 1.339 µg/mL for strychnine and brucine respectively. Satisfactory results were obtained, indicating the high specificity of the proposed method for the determination of the markers in ternary mixture and formulations. No interferences were observed as shown in overlaid chromatograms of standard solution containing the two compounds. The robustness study indicated that the selected factors remained unaffected by small variations of parameters. The recovery obtained individually was 100.52 % for strychnine and 100.19 % for brucine. Therefore, it can be concluded that the method is consistent for selected column and solvent brand. A system suitability test was performed to evaluate the chromatographic parameters (capacity factor, separation factor, column efficiency, number of theoretical plates, HETP asymmetry of the peaks and resolution between two consecutive peaks) before the validation runs (Table 1). The standard peaks of strychnine and brucine were well resolved and with high efficiency. There was no interference with the standard peak in presence of interferences in the solution indicating the method to be specific.

Table 2 Assay results of herbal formulation

Formulation	Strychnine (mg/tablet)	Brucine (mg/tablet)
Tentex Forte Tablets	2.67	2.05

CONCLUSION:

In the present study, a simple and reproducible method for the Simultaneous determination of strychnine and brucine in herbal formulations by reverse phase HPLC method is developed. The strychnine and brucine content in Tentex forte herbal tablet was quantified. The proposed method being precise, sensitive and reproducible can be used for quantitative determination of strychnine and brucine in crude drugs and its herbal formulations. The advantage of the method lies in the simplicity of the sample preparation, less run time and the low costs of reagents used. The validated parameters indicate that the developed method is quick, selective and cheap. Hence the developed method is more suitable for the simultaneous determination of strychnine and brucine in crude drug as well as in multi-component herbal formulation.

REFERENCES:

1. Bensky D and Gamble A. Chinese Herbal Medicine, Eastland Press, Seattle. 1986, 646.

2. Samulesson G. Drugs of Natural Origin, Swedish Pharmaceutical Press, Stockholm. 1992, 282.

3. http://www.ayurveda-recipes.com/kuchala.html

4. Wang Z, Zhao J, Xing J, He Y. Analysis of strychnine and brucine in postmortem specimens by RP-HPLC. J. Anal. Toxicol. 2004; 141-144.

5. Anshu R, Neena S, Sayyada K, Ajay K, Rawat S. TLC determination of strychnine and brucine of Strychnos nuxvomica in ayurveda and homeopathy drugs. J. Chromatography, 2008; 67, 607-613.

6. Wan-Sheng Chen, Li-Li Liu, Xiang Li, Jie-Wei Li, Song-Gang Ji, Guo-qing Zhang Yi-Feng Chai. Separation and determination of strychnine and brucine in Strychnos nux-vomica L. and its preparation by capillary zone electrophoresis. J. of Biomed. Chromato. 2000; 90, 477-482

7. Synder KL, Krikland JJ, Glajch JL, Practical HPLC Method Development, 2^nd Edn, Wiley-Interscience Publication, USA. 1983; 1-10.

8. ICH Harmonized Tripartite Guidelines, Validation of Analytical Procedures: Text and Methodology, Q2R (1).

9. Validation analytical procedures and methodology, ICH Haromonizies Tripati Guidelines. 1996; 1-8.

10. David MB, Validating Chromatographic Methods - A Practical Guide, Wiley-Interscience Publication, John Wiley and Sons, New York. 1997; 9-47.

Received on 24.03.2010 Modified on 27.04.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 720-723