Original Article Simultaneous HPTLC determination of strychnine 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 chloroform–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 analgesic and anti-inflammatory properties.[10] The methods so far reported for the analysis of S. nux-vomica include alkaloids 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. 134 Journal of Pharmacy and Bioallied Sciences April-June 2012 Vol 4 Issue 2 Kamal, et al.: Simultaneous quantification of strychnine and brucine × 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.