DETECTION OF ACETYL SALISYLIC ACID, CAFFEINE AND PARACETAMOL IN CLANDESTINE MEDICINAL PREPARATION USING LIQUID CHROMATOGRAPHIC TECHNIQUES Shilpa

Sharma, Prakash Tiwari, Sandeep Kumar Mourya* and Dr. Devasish Bose Department of Criminology and Forensic Science, Dr. Harisingh Gour Central University, Sagar (M.P.), India *Corresponding author: email- sandeepmourya07@rediffmail.com ABSTRACT In a survey conducted in the Sagar region of Madhya Pradesh, some surprising data was collected that clandestine medicinal preparations in the form of Pudia (Sachet) are widely used by the lower society people especially bidi workers, labourers and villagers as pudias are the most trustworthy medicines for quick relief from minor ailments like: headache, bodyache, fever, common cold and flu. These peoples tend to consume any medicines/drugs in the name of ayurveda. Thus, fake and unregistered medical practitioners have started their business of selling clandestine medicinal preparations under different names such as mathura ki pudia and are playing with the lives of these innocent people by claiming that their preparations are safer, which owes its effect completely to ayurvedic components. Such types of preparations are widely available without any medicinal prescription and are illegal because its dose, composition, manufacture and expiry date is not mentioned. Pudia is shown to have analgesic, antipyretic and tranquilizing effect, and has the potential of causing dependency both physiological and psychological in a long run. Therefore commonly used medicines in minor ailments, containing acetyl salicylic acid, paracetamol (acetaminophen) and caffeine were selected for their presence in the clandestine medicinal preparations (Pudia). Aspirin (acetyl salicylic acid), Paracetamol (acetaminophen) and Caffeine were screened in the Pudia by thin layer chromatography (TLC) on the basis of Rf values, treatment with a visualizing agent which develops colour and then comparing it with the standards. Further the confirmation and analytical separation was done using high performance liquid chromatography, Screened compounds by the TLC were confirmed and identified qualitatively by comparing the chromatograms of clandestine medicinal preparation (Pudia) with the chromatograms of standards of selected compounds. The inference drawn was that the pudia, an illegal clandestine preparation is allopathic in nature constituting acetyl salicylic acid, caffeine and paracetamol in varying concentration in a powder base which may be harmful for human consumption. KEYWORDS Clandestine medicinal preparations, Acetyl salicylic acid, Caffeine, Paracetamol, Thin Layer Chromatography, High Performance Liquid Chromatography.

INTRODUCTION In India, majority of the population is of the villagers who are poor, belong to lower strata and are illiterate and thus tend to consume any medicines/drugs in the name of ayurveda. Thus, illegal clandestine preparations under the brand name mathura ki pudia is by being consumed by these innocent people. Pudias are widely available without any medical prescription. Pudia is widely abused by the lower society people especially servants, as pudias give quick relief from minor ailments like: headache, fever, common cold and flu. Thus shown to have analgesic, antipyretic and tranquilizing effect. Therefore such components namely: aspirin, paracetamol and saridon were selected for study. Aspirin or acetyl salicylic acid is an analgesic used for minor aches as in headache ,migraine and myocardial infarction. Is a non-steroidal anti inflammatory medication, antiplatelet and antipyretic. Paracetamol or acetaminophen is an analgesic(pain reliever),antipyretic(fever reducer) acting on the central and peripheral nervous as nonopioid analgesic. Caffeine is an alkaloid acting as a central nervous system stimulant . A legal psychoactive drug which reduces physical fatique and restore alertness. Numerous analytical techniques have been reported for the analysis of analgesics in various combinations in pharmaceuticals such as: titrimetry, voltametry, flourimetry, colorimetry, UV spectrophotometry, gas chromatography, thin layer chromatography and high performance liquid chromatography. Thin layer chromatography is a simple, cost effective, reliable technique while high performance liquid chromatography is is a rapid, sensitive, highly specialized, accurate and reproducible quality control method for determination of analgesics. Thus, as forensic science students the objective through this work is to determine the composition of the illegal clandestine preparation pudia whether ayurvedic or allopathic using analytical chromatographic techniques thin layer chromatography and high performance liquid chromatography and also to validate its nature whether it is fit for human consumption or not. Thin layer chromatography was used to determine the colour and Rf values of the resoluted spots of the components of pudia and standard. High performance liquid chromatography to confirm the results given by thin layer chromatography by comparison of the chromatograms of the pudia and standard to identify the components of the illegal clandestine preparation pudia ascertaining its genuineness of consumption by humans.

EXPERIMENTAL/METHODS MATERIALS Analytical grade chemicals namely acetone, methanol, benzene were of qualigens fine chemicals(mumbai.India).Ammonia, glacial acetic acid, chloroform were of BDH-EMerck(mumbai,India), ferric chloride was fron Himedia(mumbai,India),digital balance was used of A & D co.ltd(japan). Hplc grade water, sodium di hydrogen phosphate and acetonitrile procured from qualigens fine chemicals(mumbai,India). Standard samples of pharmaceutical drugs namely: paracip(paracetamol)[1] from cipla(mumbai,India), dispirin(aspirin)[2] from reckitt benckiser(Karnataka,India) and saridon(caffeine)[3] from piramal healthcare(himachal pradesh,India). Clandestine preparation pudia from unregistered medical practitioners.

Pre-coated 0.20 micrometer kiselguar-F260-50x50cm TLC plates and 0.25micronmeter silica gel-G20x20TLC plates from E-merck(mumbai,India). Glass tank was used to develop the tlc plates. Used UV chamber was of ideal scientific concern(kolkata,India). Porcelain pistal mortar was used for grinding the tablets into powder form. Oven for activation of the tlc plates prior to spotting was used. Borosil glass vials and test tubes, microcapillaries were used throughout the experiments. All experiments were performed at ambient temperature.

CHROMOTOGRAPHIC CONDITIONS FOR THIN LAYER CHROMATOGRAPHY Samples (D,P,S) weighing 25mg each were extracted with various extraction solvents namely: 10ml water and 10 ml methanol separately, filtered and stored in glass vials. Solvents like methanol, glacial acetic acid, benzene, chloroform(1:15:100:30v/v);chloroform, acetone(80:20v/v);ethyl acetate, glacial acetic acid(99.5:0.5v/v) absolute methanol(100v/v). UV light and ferric chloride were used to visualize the spots.

THE TECHNIQUE OF THIN LAYER CHROMATOGRAPHY: CHAMBER SATURATION AND DEVELOPMENT Standard samples( D,P,S) and pudias(P1,P2) were applied at the starting line, about 1cm away from one edge of the pre coated TLC plate. Precision micro capillary was used for sample application. Solvent front was kept at 10cms away from the start line. After the application of the sample spot, plate was kept almost vertical in a saturated glass chamber containing the mobile solvent. Plate was then allowed to dry and subjected to detection. Firstly TLC plate was viewed under UV light in UV chamber and then detection was made by visualizing agent. After visualization the spots on the plate were marked with pencil and Rf value of the resolved spots of pudias and standard was calculated by using the above formula:
Rf=distance from base line to centre of the spot/distance from base line to solvent front

CHROMATOGRAPHIC CONDITIONS FOR HPLC A high performance liquid chromatography shimatzu-2006 with an auto sampler was used for analysis using the LC-Sols. software for recording data having a stainless steel coloumn150mm long,4.6mm internal diameter filled with octadecyl silane chemically bonded to porous silica particles of 5micronmeter diameter with mobile phase- 10mM potassasium di hydrogen phosphate buffer at pH6 and acetonitrile(60:40v/v) at ambient temperature flow rate-0.8ml/min sample loop-10 microlitres run time 20 mins UV detection- 207,230 and 254nm

RESULT AND DISCUSSION Optimization of the mobile phase , detection and identification of the spots In order to establish the mobile phase that gives the best result for screening of analgesics, according to elutropic series of solvents, the tried mobile phases were varied from non polar to polar solvents and various combinations tried. When chloroform and acetone was used(80:20/)spots were located at the base line and sample did not move with the solvent. Then with combination of methanol, ammonia(100:1.5v/v), ethyl acetate and glacial acetic acid (99.5:0.5v/v) was applied as mobile phase, tailing was found and spots moved with the solvent. On trying the combination of methanol, glacial acetic acid, benzene, chloroform(1:15:100:30v/v) samples D,P,S,P1,P2 were fully retained on the stationary phase and did not move with the solvent and finally optimum resolution was found and Rf was calculated. When absolute methanol (100v/v)was tried spots were fully retained by the stationary phase and colour of the spots and Rf was calculated.

DETECTION AND IDENTIFICATION Firstly the spots were observed under UV light at 254 nm and 325 nm. Colours were observed against green background. Then solutions like dragondorff, tannic acid and ferric chloride was used for visualization of the spots. Ferric chloride was found to be most sensitive and gave the best result because it has chromophoric group which impart colour on coming in contact with other non-chromophoric compounds. Purple, orange and brown spots were observed for acetyl salicylic acid, caffeine and paracetamol respectively. The purple spots were of acetyl salicylic acid but varied in colour intensity due to different concentration in different samples. P1 showed most intense purple colour proving maximum concentration of aspirin then P2 while in standard P the least. Also orange colour spots of caffeine identified but dull in appearance showing its minute quantities in both pudias P1,P2 and standard D,P,S. Brown colour spots of paracetamol were identified in pudias P1 and P2 and was most intense in standard P and pudia P2. On identifying the coloured spots of standard and pudias under UV and by using ferric chloride Rf was measured and was concluded that: orange spots were observed in P1,P2 and S having Rf- 3.6 showing caffeine is one of the constituent of pudia as Rf of pudias matched with that of the standard. Purple spots were observed in D,P1,P2 having similar Rf- 7 in both pudias and standard indicating acetyl salicylic acid to be a constituent of pudia. Intense brown colour spots were observed in P1,P2 and P having Rf- 4.8 showing paracetamol to be a component of pudia present in high concentration. The above results given by thin layer chromatography for pudias P1,P2 were confirmed analytically by high performance liquid chromatography after performing the qualitative analysis of the standard samples and pudias and measuring the retention time of each component peak versus the retention time of the standard(D,P,S). Then finally the components were identified after comparison of the chromatograms of P1,P2 and D,P,S. Separation and eluting out of the components were completed in less than 20 minutes. Retention time for paracetamol was the least-2.97 mins then caffeine- 4.86 mins and acetyl salicylic acid took the maximum- 7.88 mins to elute out completely as separate peaks.

CONCLUSION The survey analysis for illegal clandestine preparation pudia showed that nearly 95% of illiterate lower strata people consume pudia for minor ailments like: fever, headache and common cold although they have shown to develop dependency when consumed by people on regular basis. This is due to its easy availability, cheap price and well being effect due to its composition constituting of analgesics, antipyretic and stimulant namely: acetyl salicylic acid, paracetamol, caffeine in varying concentration . It components namely acetyl salicylic acid present in enormous concentration is unsafe for ulcer patients causing allergy and destroys stomach lining. Similarly paracetamol present in moderate concentration causes kidney and liver damage and causes dependency when consumed for prolonged period. Caffeine though in minor amount is reported unsafe for people suffering from hypertension. It is thus concluded that pudia an illegal clandestine preparation is allopathic in nature in a powder base. Thus our work of determining the compostion and nature of pudias has been been justified and validated analytically by thin layer chromatography and high performance liquid chromatography. Pudias whose dosage, composition , manufacturing and expiry date is not mentioned has thus proven to be illegal, unsafe and harmful for consumption both by adults and children.

ABBREVIATIONS [1] [2] [3] [4] [5]

D P S P1 P2

Acetyl salicylic acid paracetamol caffeine Pudia1 Pudia2

Structure

Chemical formula and molecular weight

Physical properties
Density-1.40 g/cm Melt.point-135 C (275 F) Boiling point-140 C (284 F) decomposes Solubility in water-3 mg/mL (20 C)

C9H8O4 180.157 g/mol

Density-1.263 g/cm Melt. point-169 C (336 F) Solubility in water12.78 mg/mL (20 C)

C8H9NO2 151.17 g/mol

C8H10N4O2 194.19

Density-1.23 g/cm3, solid Melt point-227228 C, 500-501 K (anhydrous) 234235 C, 507-508 K (monohydrate) Solubility in water-2.17 g/100 mL (25 C)

Figure-1 structure, chemical formula and physical properties of the standard analgesics

P1

P2

FIGURE2:thin layer chromatography of pudias P1,P2 And standards D,P,S

Figure 3: chromatogram analgesics in standard(D,P,S)

Sample(P1,P2) Composition Acetyl salicylic acid paracetamol caffeine

Retention time(mins) 7.88 2.97 3.86

Figure 4: chromatogram for pudias(P1,P2)

ACKNOWLEDGEMENT Shilpa Sharma and Prakash Tiwari thanks deptt. of forensic science, Dr.H.S. Gour Central University, Sagar(M.P.) for providing guidance and facilities for conducting experiments.

REFERENCES 1. Clarkes Analysis of Drugs and Poisons,ed. Moffat Anthony C, Osselton, David M., Widdop Brian, London, UK., 3rd edn., 2004. 2. Meyler's Side Effects of Drugs: The International Encyclopedia of Adverse Drug Rections and Interactions,15th edn.,( 2006): 2555-82. 3. http://en.wikipedia.org/wiki/Aspirin /paracetamol/caffeine 4. M. Levent ALTUN, HPLC Method for the Analysis of Paracetamol, Turk J Chem, 26 (2002) , 521- 528. 5. Franeta JT, Agbaba D, Eric S, Pavkov S, Aleksic M, Vladimirov S, Hplc assay of analgesics in tablets, (2002);57(9):709-13 6. Glenda K. Ferguson, Quantitative HPLC Analysis of an Analgesic/Caffeine Formulation, J. Chem. Educ., (1998), 75 (4): 463- 467