Comparative Snake Venom Analysis for Facilitating Wildlife Forensics: A Pilot Study.

Confirm and authentic identification of species is required for the implementation of wildlife laws in cases of illegal trafficking of snake venoms. Illegally trafficked snake venom might be misidentified with other drugs of abuse, and sometimes, the species of venom-yielding snake cannot be verified. Snake venoms from medically important snake species, Naja naja and Daboia russelii, were procured from Irula Snake Catcher's Society, Tamil Nadu, India. Comparative analyses of both venoms were carried out using SDS-PAGE, LC-MS/MS, ICP-MS, and mtDNA analysis. The protein concentration of Naja naja and Daboia russelii venoms was 76.1% and 83.9%, respectively. SDS analysis showed a distinct banding pattern of both venoms. LC-MS/MS results showed proteins and toxins from 12 to 14 protein families in Naja naja and Daboia russelii venoms. Elemental analysis using ICP-MS showed a different profile of some elements in both venoms. mtDNA analysis of venoms using universal primers against Cyt b gene showed homology with sequence of Naja naja and Daboia russelii genes. The study proposed a template of various conventional and advanced molecular and instrumental techniques with their pros and cons. The template can be used by forensic science laboratories for detection, screening, and confirmatory analysis of suspected venoms of snakes. Clubbing of various techniques can be used to confirm the identification of species of snake from which the alleged venom was milked. The results can be helpful in framing charge-sheets against accused of illegal venom trafficking and can also be used to verify the purity and quality of commercially available snake venoms.

First-hand knowledge about snakes and snake-bite management: an urgent need.

Snake-bite is a well-known but fairly ignored medical problem in India. Lack of precise first aid knowledge for snake-bite is a substantial reason for its severe fatality in human beings. The present study is comprised of a pilot survey that assesses and evaluates the knowledge of people of different occupations (teachers, students, farmers, medical residents, and miscellaneous) about snakes and snake-bite management. The pilot survey was conducted through a well-structured open-ended questionnaire about experiences with snakes and snake-bites and first aid measures for accidental snake-bites. Proper knowledge of snakes and snake-bite management was either diminutive or absent in the majority of the subjects, especially amongst teachers. Even the medical professionals were not well acquainted with knowledge about snakes and snake-bite management. Only 13% knew about 'big four', 18% knew 'dry bite', and 21% of subjects knew about anti-snake venom (ASV) used in India. 39% of subjects knew about the whereabouts of traditional healer. Only 12% of subjects, mostly medical residents, knew of any bedside test for diagnosis of snake-bite, and 11% of respondents also knew of LD50 of Indian cobra. A well-timed first aid treatment is always decisive in the management of life-threatening snake-bite cases but the present survey has found that most of the study groups had inadequate and little misleading fundamental knowledge comprising regional snakes, first aid measures for accidental snake-bite, and welfare schemes for snake-bite victims. Therefore, the present study proposes to conduct more such appraisals and strengthening of education curricula on snake-bite that would surely inculcate an adequate level of primary skill in ignorant societies.

Quantitation of Indian krait (Bungarus caeruleus) venom in human specimens of forensic origin by indirect competitive inhibition enzyme-linked immunosorbent assay.

An indirect competitive inhibition enzyme-linked immunosorbent assay was reported to detect krait venom in human specimens of forensic origin. Polyclonal anti-krait venom antibodies were characterized by indirect antibody capture assay. The calibration plot was constructed based on linear regression analysis (y = 72.85 - 12.29x, r(2) = 0.98) with concentration ranges from 0.013 to 1000 ng/well of krait venom with a limit of detection of 0.2 ng/mL in the assay system. The IC50 (inhibitory concentration at 50% displacement) value of krait venom was observed to be 70 ng. Spiking studies indicated recoveries of 95-100% and 94-100% when various concentrations of krait venom were spiked to rat tissues (skin, liver, and kidneys) and pooled human serum, respectively. Polyclonal anti-krait venom antibodies showed no cross-reactivity with cobra and viper venom when tested in the assay system. The coefficient of variation of various concentrations of working range in intra-assay (n = 6) was <5%, whereas in interassay (n = 6) it was observed to be < or 7%. Further, the method was used to quantitate krait venom in human autopsy and biopsy specimens of forensic origin. Concentration of krait venom was found to be in the range of 4-172 ng/100 mg skin or skin scrapings and 64-378 ng/mL blood or serum. The methodology may find application in forensic laboratories to assess the cause of death in the cases of krait-bite victims.

Principal opium alkaloids as possible biochemical markers for the source identification of Indian opium.

A total of 124 opium samples originating from different licit opium growing divisions of India were analyzed for their principal alkaloid (thebaine, codeine, morphine, papaverine, and narcotine) content by capillary zone electrophoresis (CZE) without derivatization or purification. Absence of papaverine in Bareilly, Tilhar, and most of the samples originating from Kota is a significant observation in relation to the source of Indian opium. Multiple discriminant analysis was applied to the quantitative principal alkaloid data to determine an optimal classifier in order to evaluate the source of Indian opium. The predictive value based on the discriminant analysis was found to be 85% in relation to the source of opium and the study also revealed that all the principal alkaloids have to be analyzed for source identification of Indian opium. Chemometrics performed with principal alkaloids analytical data was used successfully in discriminating the licit opium growing divisions of India into three major groups, viz., group I, II, and III. The methodology developed may find wide forensic application in identifying the source of licit or illicit opium originating from India, and to differentiate it from opium originating from other opium producing countries.

Determination of diazepam in cream biscuits by liquid chromatography.

An analytical procedure was developed for the detection and quantitation of diazepam in cream biscuits, which were used to commit crime. The method involves the extraction of diazepam with ethanol at room temperature, and the extract is filtered, evaporated to dryness, and redissolved in the mobile phase, methanol-acetonitrile-tetrahydrofuran-water (15 + 55 + 4 + 26, v/v). The separation is achieved on a C18 reversed-phase column with the mobile phase and diode array detection (lambda(max)) at 230 nm. Medazepam is used as the internal standard is for quantification. The calibration plot for the determination of diazepam is based on linear regression analysis (y = 0.6687x + 0.0372; r2 = 0.995). The limit of detection for diazepam in the biscuit samples was estimated as 600 ng/mL. The limit of quantitation for diazepam was estimated as 1.75 microg/mL. The diazepam detected per piece of biscuit was found to be in the range of 0.27-0.45 mg. Pure diazepam was added to biscuit samples at 3 levels (100 and 500 microg/g, and 1 mg/g), and the recoveries were found to be 95%. The mean retention time of diazepam was 2.7 min and that of medazepam (IS) was 4 min. The relative standard deviations of the diazepam level in the biscuit samples were estimated to be 0.4% for retention time and 1.02% for peak area in intraday analysis, whereas the corresponding values were and 0.61 and 2.34% in interday analysis. The method is rapid and reliable for qualitative and quantitative analysis of cream biscuits laced with diazepam, and it can be used by law enforcement laboratories for routine analysis.

Application of capillary zone electrophoresis in the separation and determination of the principal gum opium alkaloids.

We describe the use of capillary zone electrophoresis (CZE) for the qualitative and quantitative determination of major alkaloids (i.e., thebaine, codeine, morphine, papavarine and narcotine) in gum opium involving the analysis of alkaloids without derivatization or purification. Three extractions with 2.5% w/v aqueous acetic acid quantitatively extracted major alkaloids. The separation was carried out by CZE using a 7:3 mixture of methanol and sodium acetate (100 mM, pH 3.1) at a potential of 15 kV, with UV detection at 224 nm. Spiking of pure reference alkaloid standards in the opium extract was used for peak identification. The influences of buffer composition, pH and voltage on the separation of alkaloids were studied. The detection limit of each alkaloid dissolved in methanol was found to be 850 ng/mL (morphine), 450 ng/mL (thebaine), 500 ng/mL (codeine), 550 ng/mL (papaverine), and 500 ng/mL (narcotine) at an injection pressure of 300 mbar (injection volume, 4 nL) with a signal-to-noise ratio of 3:1. The external standard method was used for the quantification of alkaloids. The calibration plot was based on linear regression analysis. The relative standard deviation (RSD) for peak area and migration time was in the range of 1.03-3.56% and 0.34-0.69%, respectively. Percentage compositions (g%) of opium alkaloids in five gum opium samples were found to be in the range of 14.45-15.95 (morphine), 2.0-3.45 (codeine), 1.32-2.73 (thebaine), 0.92-2.37 (papavarine), and 3.85-5.77 (narcotine). The method developed is suitable for the routine analysis of major gum opium alkaloids in samples of forensic importance.