Original Article

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

Abid Kamal, Kamal Y. T., Sayeed Ahmad, F. J. Ahmad, Kishwar Saleem1

Bioactive Natural ABSTRACT Products Laboratory, Objective: A simple, sensitive, and specific thin layer chromatography (TLC) densitometry method has Department of been developed for the simultaneous quantification of strychnine and brucine in the seeds of Strychnos nux- Pharmacognosy, Faculty of Pharmacy, Jamia vomica. Materials and Methods: The method involved simultaneous estimation of strychnine and brucine Hamdard, Hamdard after resolving it by high performance TLC (HPTLC) on silica gel plate with –methanol–formic acid Nagar, 1Department of (8.5:1.5:0.4 v/v/v) as the mobile phase. Results: The method was validated as per the ICH guidelines for Chemistry, Jamia Millia precision (interday, intraday, intersystem), robustness, accuracy, limit of detection, and limit of quantitation. Islamia, Jamia Nagar, The relationship between the concentration of standard solutions and the peak response was linear within the New Delhi, India 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 Address for correspondence: brucine, respectively. Accuracy of the method was checked by recovery studies conducted at three different Dr. Sayeed Ahmad, E- mail: sahmad_jh@yahoo. concentration levels and the average percentage recovery was found to be 100.75% for strychnine and 100.52% co.in 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. Received : 11‑04‑11 Review completed : 11-4-2011 Accepted : 23‑05‑11 KEY WORDS: HPTLC, method development, strychnine, brucine, validation

trychnos nux-vomica Linn., commonly known as kuchla glucoside-loganin, 7-O-acetyl loganic acid,[4] caffeotannic acid, S belongs to the family Loganiaceae, is a medium-sized tree and a trace of copper. Its alcoholic seed extract showed good lipid distributed widely in India in the deciduous forest of the eastern peroxidation effect in rat liver.[5] Crude extract of S. nux-vomica and southern parts of the country.[1] Kuchla fruit is used as has been reported to exhibit an inhibitory effect on the reverse appetizer, tonic, astringent to bowels, and antipyretic and useful transcriptase of RNA tumor virus (I), protein kinase, and HIV-1 in the treatment of hiccups, leukoderma, blood disorders, piles, protease.[6-8] Recent research has shown that excitatory effect of ulcers, pneumonia, hemoptysis, occipital headache, cold and strychnine on the central nervous system results from its ability to [9] cough, anemia, jaundice, itching, ear troubles, renal colic, and antagonize the effect of synaptic inhibition. Brucine and brucine urinary infection.[2,3] Some of the major chemical constituents of N- oxide has been reported for its and anti-inflammatory properties.[10] The methods so far reported for the analysis of S. nux-vomica include strychnine [Figure 1a], brucine strychnine and brucine include their estimation using circular [Figure 1b], brucine-n-oxide, and also traces of strychnicine, a chromatography,[11] nonaqueous capillary electrophoresis,[12-16] [17] [18] Access this article online UV spectrophotometry, thin layer chromatography (TLC), [18] [19] Quick Response Code: column liquid chromatography, capillary zone electrophoresis, Website: and voltametry[20] showed low resolution owing to poor www.jpbsonline.org reproducibility. Others have been working on the separation of bioactive components of plants using chromatographic methods. DOI: In this respect, Petruczynik et al. have developed a method 10.4103/0975-7406.94814 for the separation of plant alkaloids on a silica gel plate.[21] Shalaby and Khalil further modified this technique using an RP

How to cite this article: Kamal A, Kamal YT, Ahmad S, Ahmad FJ, Saleem K. Simultaneous HPTLC determination of strychnine and brucine in strychnos nux- vomica seed. J Pharm Bioall Sci 2012;4:134-9.

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× 10 cm TLC plate for the preparation of calibration curves of strychnine and brucine. A constant application rate of 150 H O H O nL/s was employed with a band width of 7.0 mm. The slit H H dimension was kept at 6.0 × 0.45 mm and scanning speed of 20 mm/s was employed. Twenty milliliters of mobile phase H H H H consisting of chloroform:methanol:formic acid (8.5:1.5:0.4, v/v/v) was used per plate. The optimized chamber saturation N N O N N O 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. H3CO OCH 3 The data obtained were analyzed by WinCATS software to get Strychnine Brucine a b linear regression equation. Figure 1: Structure of (a) strychnine; (b) brucine Preparation of standard solution chromatographic plate with ion separation for the estimation of more alkaloids derived from plant sources.[22] Saqui-Sannes, A standard solution containing strychnine and brucine was et al. adapted this method for the estimation of strychnine and prepared by dissolving 5 mg each in 10 mL of methanol (500 crimidine in biological samples as well.[23] They determined the µg/mL). This stock solution was used to make calibration curves amounts of these alkaloids in dog stomach and serum, which are of strychnine and brucine. generally used as dog poisons. All these methods used earlier for the estimation of strychnine and brucine are tedious, lengthy, and Preparation of sample solution less sensitive. With this background, we herein report a novel, very simple, specific, sensitive, very economic, and a laboratory Weighed 50 g of S. nux-vomica seeds and boiled for 2 h in a water friendly validated high performance thin layer chromatography bath. The seeds were powdered and mixed with a sufficient (HPTLC) method for the simultaneous quantification of these quantity of alcoholic KOH and dried in an oven at 100°C. marker compounds in the seeds of S. nux-nomica. The method has Accurately weighed quantity (10 g) of seed powder was extracted [24] been validated as per the ICH guidelines similar to the methods with 200 mL of CHCl3 in Soxhlet apparatus and concentrated reported by laboratory.[25-30] to 50 mL. Chloroform extract was shaken with successive three portions of dilute sulfuric acid (50 mL each). Combined the Materials and Methods acid extract and filtered, and added excess amount of ammonia to the acid extract to precipitate the alkaloids. The alkaline Plant materials and chemicals mixture was successively extracted with chloroform thrice (100 mL each) to ensure complete extraction. The chloroform extract Standard strychnine (98%) and brucine (98%) were procured was passed over sodium sulfate and evaporated to dryness on from Fluka and Sigma Aldrich, USA, respectively. Dried samples water bath. The residue obtained was reconstituted in 10 mL of seeds of S. nux-vomica (Family: Loganiaceae) were procured of methanol and used for quantification. from a Delhi market, which were further authenticated by a Pharmacognosist and voucher specimens were deposited in Method validation depository of Bioactive Natural Product laboratory, Department of Pharmacognosy, Jamia Hamdard. All other chemicals used The developed method was validated as per ICH guidelines were of analytical reagent grade. for precision, robustness, limit of detection (LOD) and limit of quantitation (LOQ), specificity and accuracy. HPTLC instrumentation and experimental conditions Linearity Sample solutions were applied with semiautomatic TLC sampler Linomat V (Camag, Muttenz, Switzerland) controlled A 6-point calibration curve was constructed by plotting peak area by WinCATS software 1.4.4. The plates were developed in against concentrations. Linearity was evaluated by applying each 20 × 10 cm twin trough glass chamber (Camag, Muttenz, concentration (50–1000 ng/spot) for strychnine and brucine Switzerland). A TLC scanner III was used for scanning the TLC in triplicates per sample and 6 such samples were evaluated n plates. Precoated silica gel aluminum plates 60F254 (E. Merck, ( =3×6). Darmstadt, Germany) with thickness 0.2 mm were used for all determinations. The plates were prewashed with methanol Precision 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 The precision of a method is the extent to which the individual standard solution (strychnine and brucine) were applied on 20 test results of multiple injections of a series of standards

Journal of Pharmacy and Bioallied Sciences April-June 2012 Vol 4 Issue 2 135   Kamal, et al.: Simultaneous quantification of strychnine and brucine agree. System repeatability was determined in 6 replicates of the drug at different levels in the crude drug. a standard solution at three concentration levels of 100, 200, and 400 ng/spot of strychnine and brucine, respectively. The Results and Discussion results of repeatability were expressed in terms of relative standard deviation (% relative standard deviation [RSD]). Optimization of the solvent system Intraday precision was done by repeating the same assay 6 times on the same day. Intermediate precision was also assessed by For the development of mobile phase, different trials were made the assay of three; six standard solutions were set on different using many solvents in different proportions. When mobile days (interday precision) and on different system (Intersystem phase consisting of chloroform:methanol was used in the ratio of precision). The intraday, interday, and intersystem variations 8:2, v/v two spots were observed at the Rf value of 0.60 and 0.69 for determination of strychnine and brucine were carried out at for strychnine and brucine, respectively. But it was found that three different concentration levels 100, 200, and 400 ng/spot. the resolution between the peaks was poor. In order to improve the resolution between the peaks, a new mobile phase with the Robustness of the method 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 By introducing small changes in the mobile phase composition, in achieving very compact spots at the Rf value of 0.60 and 0.69 the effects on the results were examined. Mobile phases having [Figures 2 and 3] for strychnine and brucine, respectively, with different compositions like chloroform: methanol: formic good resolution of more than one. 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 Figure 2: HPTLC chromatogram of standard strychnine (500 µg/spot) at 259 nm 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 Rf 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 Figure 3: HPTLC chromatogram of standard brucine (500 µg/spot) conducted 6 times. This was done to check the recovery of at 306 nm

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Method validation for three different concentration levels are summarized in Table 2. The low % RSD indicated that the method is precise for the Linearity analysis.

Linearity was found between concentration ranges of 50–1000 Robustness of the method ng/spot for strychnine and 100–1000 ng/spot for brucine with r2 value of 0.9977 and 0.9984, respectively [Table 1]. The effect of deliberate changes in the composition of mobile phase were studied as % RSD and depicted in Table 3. Low % Precision RSD indicates the method is robust.

Precision data on the intraday, interday, and interanalyst variation Limit of detection and limit of quantitation

Table 1: Validation parameters of the proposed HPTLC LOD and LOQ were calculated using signal to noise ratio method for estimation of strychnine and brucine method and found to be 16.7 and 45.3 for strychnine and 34.3 Validation parameters Strychnine Brucine and 95.9 for brucine, respectively [Table 1]. Linearity range (ng/spot) 50–1000 100–1000 Correlation coefficient 0.9976 ± 0.0004 0.9983 ± 0.00089 Specificity (r2) Regression equation Y = 610.04 + 9.27*X Y = 636.43 + 8.38*X The specificity of the newly proposed method Limit of detection (ng/ 16.7 34.3 spot) was ascertained by superimposing the spectrum of both Limit of quantification 45.3 95.9 standard and sample and confirmed for its purity [Figures (ng/spot) 4 and 5].

Table 2: Intermediate precision data of proposed HPTLC method of (a) strychnine and (b) brucine Conc. (ng/mL) Interday precision Intraday precision Intersystem precision Mean peak area ± SD (n=6) % RSD Mean peak area ± SD (n=6) % RSD Mean peak area ± SD (n=6) % RSD (a) Strychnine 100 1290.3 ± 9.71 0.75 1292.0 ± 16.8 1.30 1307.3 ± 32.32 2.47 200 2518.3 ± 14.50 0.58 2538.3 ± 22.8 0.90 2550.7 ± 28.31 1.10 400 4430.7 ± 45.62 1.03 4387.3 ± 30.4 0.69 4390.7 ± 32.33 0.73 (b) Brucine 100 1241.3 ± 27.6 2.22 1250.7 ± 5.9 0.47 1254 ± 8.7 0.69 200 2340 ± 19.5 0.83 2345.3 ± 36.2 1.54 2358.3 ± 8.6 0.36 400 4168 ± 17.3 0.41 4165 ± 20.8 0.49 4157.6 ± 28.6 0.68 RSD: Relative standard deviation

Table 3: Robustness data of proposed HPTLC method of (a) strychnine and (b) brucine Mobile phase composition (Chloroform:methanol:formic acid) Mean area ± SD (n=3) % RSD of area (a) Strychnine Actual (v/v/v) Used (v/v/v) Level 8.3:1.7:0.4 −2 2582.0 ± 17.06 0.66 8.5:1.5:0.4 8.5:1.5:0.4 0 2573.7 ± 23.86 0.93 8.7:1.3:0.4 +2 2578.0 ± 19.97 0.77 Wavelength change Actual (nm) Used (nm) Level 259 257 −2 2591.0 ± 71.3 2.8 259 0 2570.3 ± 27.7 1.1 261 +2 2558.0 ± 46.9 1.8 Mobile phase change (Chloroform:methanol:formic acid) Mean area ± SD (n=3) % RSD of area (b) Brucine Actual (v/v/v) Used (v/v/v) Level 8.5:1.5:0.4 8.3:1.7:0.4 −2 2386.7 ± 32.52 1.4 8.5:1.5:0.4 0 2373 ± 42.46 1.8 8.7:1.3:0.4 +2 2376.7 ± 47.09 2.0 Wavelength change Actual (nm) Used (nm) Level 306 304 −2 2358 ± 15.10 0.64 306 0 2355.3 ± 3.51 0.15 308 +2 2383.3 ± 37.11 1.56 RSD: Relative standard deviation

Journal of Pharmacy and Bioallied Sciences April-June 2012 Vol 4 Issue 2 137   Kamal, et al.: Simultaneous quantification of strychnine and brucine

Table 4: Accuracy as recovery data of proposed HPTLC method of (a) strychnine (b) brucine % of standard spiked to the sample Theoretical content (µg/mL) Amount of drug recovered (µg ±SD) (n=6) % of drug recovered % RSD (a) Strychnine 0 220 220.3 ± 35.2 100.1 1.6 50 330 336.5 ± 19.1 102.0 0.6 100 440 454.7 ± 46.1 103.3 1.0 150 550 536.8 ± 30.7 97.6 0.6 (b) Brucine 0 260 252.8 ± 25.2 97.2 1.0 50 390 406.0 ± 75.3 104.1 1.9 100 520 525.9 ± 32.3 101.1 0.6 150 650 648.1 ± 18.0 99.7 0.3 RSD: Relative standard deviation

Figure 4: Superimposed UV spectra of strychnine standard with Figure 5: Superimposed UV spectra of brucine standard with samples samples showing γ max at 259 nm showing γ max 306 nm

comes at Rf of 0.60 and brucine at Rf of 0.69, respectively [Figure 6]. No interference was observed in samples with immediate impurities and resolution between the peaks found good.

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. Figure 6: HPTLC chromatogram of Strychnos nux-vomica seed showing strychnine and brucine References

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