Simultaneous HPTLC determination of strychnine and brucine in strychnos nux-vomica seed.

OBJECTIVE: A simple, sensitive, and specific thin layer chromatography (TLC) densitometry method has been developed for the simultaneous quantification of strychnine and brucine in the seeds of Strychnos nux-vomica.
MATERIALS AND METHODS: The method involved simultaneous estimation of strychnine and brucine after resolving it by high performance TLC (HPTLC) on silica gel plate with chloroform-methanol-formic acid (8.5:1.5:0.4 v/v/v) as the mobile phase.
RESULTS: The method was validated as per the ICH guidelines for precision (interday, intraday, intersystem), robustness, accuracy, limit of detection, and limit of quantitation. The relationship between the concentration of standard solutions and the peak response was linear within the concentration range of 50-1000 ng/spot for strychnine and 100-1000 ng/spot for brucine. The method precision was found to be 0.58-2.47 (% relative standard deviation [RSD]) and 0.36-2.22 (% RSD) for strychnine and brucine, respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 100.75% for strychnine and 100.52% for brucine, respectively.
CONCLUSIONS: The HPTLC method for the simultaneous quantification of strychnine and brucine was found to be simple, precise, specific, sensitive, and accurate and can be used for routine analysis and quality control of raw material of S. nux-vomica and several unani and ayurvedic formulations containing this as an ingredient.

Strychnos nux-vomica Linn., commonly known as kuchla belongs to the family Loganiaceae, is a medium-sized tree distributed widely in India in the deciduous forest of the eastern and southern parts of the country.[1] Kuchla fruit is used as appetizer, tonic, astringent to bowels, and antipyretic and useful in the treatment of hiccups, leukoderma, blood disorders, piles, ulcers, pneumonia, hemoptysis, occipital headache, cold and cough, anemia, jaundice, itching, ear troubles, renal colic, and urinary infection.[23] Some of the major chemical constituents of S. nux-vomica include alkaloids strychnine [Figure 1a], brucine [Figure 1b], brucine-n-oxide, and also traces of strychnicine, a glucoside-loganin, 7-O-acetyl loganic acid,[4] caffeotannic acid, and a trace of copper. Its alcoholic seed extract showed good lipid peroxidation effect in rat liver.[5] Crude extract of S. nux-vomica has been reported to exhibit an inhibitory effect on the reverse transcriptase of RNA tumor virus (I), protein kinase, and HIV-1 protease.[6-8] Recent research has shown that excitatory effect of strychnine on the central nervous system results from its ability to antagonize the effect of synaptic inhibition.[9] Brucine and brucine N- oxide has been reported for its analgesic and anti-inflammatory properties.[10] The methods so far reported for the analysis of strychnine and brucine include their estimation using circular chromatography,[11] nonaqueous capillary electrophoresis,[12-16] UV spectrophotometry,[17] thin layer chromatography (TLC),[18] column liquid chromatography,[18] capillary zone electrophoresis,[19] and voltametry[20] showed low resolution owing to poor reproducibility. Others have been working on the separation of bioactive components of plants using chromatographic methods. In this respect, Petruczynik et al. have developed a method for the separation of plant alkaloids on a silica gel plate.[21] Shalaby and Khalil further modified this technique using an RP chromatographic plate with ion separation for the estimation of more alkaloids derived from plant sources.[22] Saqui-Sannes, et al. adapted this method for the estimation of strychnine and crimidine in biological samples as well.[23] They determined the amounts of these alkaloids in dog stomach and serum, which are generally used as dog poisons. All these methods used earlier for the estimation of strychnine and brucine are tedious, lengthy, and less sensitive. With this background, we herein report a novel, very simple, specific, sensitive, very economic, and a laboratory friendly validated high performance thin layer chromatography (HPTLC) method for the simultaneous quantification of these marker compounds in the seeds of S. nux-nomica. The method has been validated as per the ICH guidelines[24] similar to the methods reported by laboratory.[25-30]

Figure 1

Structure of (a) strychnine; (b) brucine

Materials and Methods

Plant materials and chemicals

Standard strychnine (98%) and brucine (98%) were procured from Fluka and Sigma Aldrich, USA, respectively. Dried samples of seeds of S. nux-vomica (Family: Loganiaceae) were procured from a Delhi market, which were further authenticated by a Pharmacognosist and voucher specimens were deposited in depository of Bioactive Natural Product laboratory, Department of Pharmacognosy, Jamia Hamdard. All other chemicals used were of analytical reagent grade.

HPTLC instrumentation and experimental conditions

Sample solutions were applied with semiautomatic TLC sampler Linomat V (Camag, Muttenz, Switzerland) controlled by WinCATS software 1.4.4. The plates were developed in 20 × 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland). A TLC scanner III was used for scanning the TLC plates. Precoated silica gel aluminum plates 60F254 (E. Merck, Darmstadt, Germany) with thickness 0.2 mm were used for all determinations. The plates were prewashed with methanol and activated at 60°C for 5 min prior to chromatography. Six different volumes (0.1, 0.2, 0.4, 0.8, 1.0, 2.0 μL) of mixed standard solution (strychnine and brucine) were applied on 20 × 10 cm TLC plate for the preparation of calibration curves of strychnine and brucine. A constant application rate of 150 nL/s was employed with a band width of 7.0 mm. The slit dimension was kept at 6.0 × 0.45 mm and scanning speed of 20 mm/s was employed. Twenty milliliters of mobile phase consisting of chloroform:methanol:formic acid (8.5:1.5:0.4, v/v/v) was used per plate. The optimized chamber saturation time for mobile phase was 15 min at room temperature (25 ± 2°C) at relative humidity of 60 ± 5%. The plates were developed and scanned within 10 min using densito metric scanner III in the absorbance mode at 259 and 306 nm for strychnine and brucine, respectively. The source of radiation was deuterium lamp emitting a continuous radiation between 200–400 nm. The data obtained were analyzed by WinCATS software to get linear regression equation.

Preparation of standard solution

A standard solution containing strychnine and brucine was prepared by dissolving 5 mg each in 10 mL of methanol (500 μg/mL). This stock solution was used to make calibration curves of strychnine and brucine.

Preparation of sample solution

Weighed 50 g of S. nux-vomica seeds and boiled for 2 h in a water bath. The seeds were powdered and mixed with a sufficient quantity of alcoholic KOH and dried in an oven at 100°C. Accurately weighed quantity (10 g) of seed powder was extracted with 200 mL of CHCl3 in Soxhlet apparatus and concentrated to 50 mL. Chloroform extract was shaken with successive three portions of dilute sulfuric acid (50 mL each). Combined the acid extract and filtered, and added excess amount of ammonia to the acid extract to precipitate the alkaloids. The alkaline mixture was successively extracted with chloroform thrice (100 mL each) to ensure complete extraction. The chloroform extract was passed over sodium sulfate and evaporated to dryness on water bath. The residue obtained was reconstituted in 10 mL of methanol and used for quantification.

Method validation

The developed method was validated as per ICH guidelines for precision, robustness, limit of detection (LOD) and limit of quantitation (LOQ), specificity and accuracy.

Linearity

A 6-point calibration curve was constructed by plotting peak area against concentrations. Linearity was evaluated by applying each concentration (50–1000 ng/spot) for strychnine and brucine in triplicates per sample and 6 such samples were evaluated (n=3×6).

Precision

The precision of a method is the extent to which the individual test results of multiple injections of a series of standards agree. System repeatability was determined in 6 replicates of a standard solution at three concentration levels of 100, 200, and 400 ng/spot of strychnine and brucine, respectively. The results of repeatability were expressed in terms of relative standard deviation (% relative standard deviation [RSD]). Intraday precision was done by repeating the same assay 6 times on the same day. Intermediate precision was also assessed by the assay of three; six standard solutions were set on different days (interday precision) and on different system (Intersystem precision). The intraday, interday, and intersystem variations for determination of strychnine and brucine were carried out at three different concentration levels 100, 200, and 400 ng/spot.

Robustness of the method

By introducing small changes in the mobile phase composition, the effects on the results were examined. Mobile phases having different compositions like chloroform: methanol: formic acid (8.7: 1.3: 0.4 v/v/v) and (8.3: 1.7: 0.4 v/v/v) were tried and chromatograms were run. The volume of mobile phase was varied in the range of ± 5%. The plates were pre-washed by methanol and activated at 60 ± 5°C for 5, 10 and 12 min prior to chromatography. Robustness of the method was done at three different concentration levels 400, 600 and 800 ng/spot. Plates were developed in varied volume of mobile phase 8, 10 and 12 mL. Time from spotting to chromatography and chromatography to scanning were also varied and % RSD was determined and found to be less than 2 %.

Limit of detection and limit of quantitation

In order to estimate the LOD and LOQ, blank solution (methanol) was spotted 6 times following the same method as explained above. The signal to noise ratio was determined. LOD was considered as 3:1 and LOQ as 10:1. LOD and LOQ were experimentally verified by diluting known concentrations of reference solution until the average responses were approximately 3 or 10 times the standard deviation of the responses for 6 replicate determinations.

Specificity

The specificity of the method was ascertained by analyzing standard drug and sample.

The spots for strychnine and brucine in sample were confirmed by comparing R and spectra of spot with that of standard. The peak purity of strychnine and brucine was assessed by comparing the spectra at three different levels, that is peak start, peak apex, and peak end positions of the spot. Purity of sample spot corresponding to strychnine and brucine was determined by taking the spectra and by comparing it with that of standard.

Recovery studies (accuracy)

The pre-analyzed samples were spiked with 50%, 100% and 150% of the standard solution and the mixtures were reanalyzed by the proposed method. The experiment was conducted 6 times. This was done to check the recovery of the drug at different levels in the crude drug.

Results and Discussion

Optimization of the solvent system

For the development of mobile phase, different trials were made using many solvents in different proportions. When mobile phase consisting of chloroform:methanol was used in the ratio of 8:2, v/v two spots were observed at the R value of 0.60 and 0.69 for strychnine and brucine, respectively. But it was found that the resolution between the peaks was poor. In order to improve the resolution between the peaks, a new mobile phase with the composition of chloroform:methanol and formic acid was used in the ratio of 8.5:1.5:0.4, v/v/v. This new mobile phase helped in achieving very compact spots at the R value of 0.60 and 0.69 [Figures 2 and 3] for strychnine and brucine, respectively, with good resolution of more than one.

Figure 2

HPTLC chromatogram of standard strychnine (500 μg/spot) at 259 nm

Figure 3

HPTLC chromatogram of standard brucine (500 μg/spot) at 306 nm

Linearity was found between concentration ranges of 50–1000 ng/spot for strychnine and 100–1000 ng/spot for brucine with r2 value of 0.9977 and 0.9984, respectively [Table 1].

Table 1

Validation parameters of the proposed HPTLC method for estimation of strychnine and brucine

Precision data on the intraday, interday, and interanalyst variation for three different concentration levels are summarized in Table 2. The low % RSD indicated that the method is precise for the analysis.

Table 2

Intermediate precision data of proposed HPTLC method of (a) strychnine and (b) brucine

The effect of deliberate changes in the composition of mobile phase were studied as % RSD and depicted in Table 3. Low % RSD indicates the method is robust.

Table 3

Robustness data of proposed HPTLC method of (a) strychnine and (b) brucine

LOD and LOQ were calculated using signal to noise ratio method and found to be 16.7 and 45.3 for strychnine and 34.3 and 95.9 for brucine, respectively [Table 1].

The specificity of the newly proposed method was ascertained by superimposing the spectrum of both standard and sample and confirmed for its purity [Figures 4 and 5].

Figure 4

Superimposed UV spectra of strychnine standard with samples showing γ max at 259 nm

Figure 5

Superimposed UV spectra of brucine standard with samples showing γ max 306 nm

The accuracy studies were done for the method as recovery studies and the amount of the drug recovered was calculated. The results of the recovery study were depicted in Table 4.

Table 4

Accuracy as recovery data of proposed HPTLC method of (a) strychnine (b) brucine

Analysis of samples

The samples were spotted in triplicate on TLC plate, strychnine comes at R of 0.60 and brucine at R of 0.69, respectively [Figure 6]. No interference was observed in samples with immediate impurities and resolution between the peaks found good.

Figure 6

HPTLC chromatogram of Strychnos nux-vomica seed showing strychnine and brucine

Conclusion

HPTLC method was developed and validated for the simultaneous determination of strychnine and brucine in S. nux-vomica seed and found to be 0.22% w/v for strychnine and 0.26% w/v for brucine, respectively. The method was found to be simple, rapid, accurate, specific, and robust for the analysis of strychnine and brucine in crude drug, which can be adopted by any laboratory for the quality control of crude drugs and formulations that contains strychnine and brucine as active markers or S. nux-vomica seed as an ingredient.