ESTIMATION OF HERBICIDE 2,4-D IN COMMERCIAL PREPARATIONS USING SPECTROPHOTOMETRY.

ESTIMATION  OF  HERBICIDE   2,4-D  IN   COMMERCIAL  PREPARATIONS  USING SPECTROPHOTOMETRY.

Arun G. Bhoi

Arun’s Institute of  Forensic Sciences, Research and Education, Pune -411 028 (India).

Email : arun.bhoi@gmail.com

(e-J. Foren. Crime Inv. 2011, 4, 1. Art. 1, 11^th Nov. 2011)

ABSTRACT

A spectrophotometric method is explained for the quantitative determination of 2,4-D (2,4-Dichlorophenoxyacetic acid) present in the commercial preparations. 2,4-D was quantified at λ-max 287 nm and linear function of concentration was observed in the range of 8 μg/ml to 20 μg/ml. The correlation coefficient was found to be o.9985. The presence of  2,4- D was confirmed by TLC using solvent system methyl alcohol : ammonia (100 :1.5 v/v) and Dragendorff’s reagent. It showed brown spot at R_f – value o.65.

KEY WORDS  

2,4-D,  herbicide, weedicide, spectrophotometric determination, TLC, Draggendorff’s reagent.

INTRODUCTION

2,4- Dichlorophenoxyacetic acid and its sodium salts, as also, amine salts are commonly used as weedicides. These are, also, preferred as growth regulators, powerful and systemic herbicides. 2,4-D acid and its salts are selective herbicides used to control broad leaf weeds in farms of orchids, tea, sugarcane because of their total water solubility and systemic post emergent property.  Its poisoning aspects are studied by Fianagan et. al. (1)   Rivers et. al. studied the gas chromatographic detection of 2,4-D (2). Curry (3) studied the poisoning aspects through autopsy. In this work 2,4-D acid is detected using thin-layer chromatography and it is estimated by application of spectrophotometrytechnique.

EXPERIMENTAL : 

Reagents 

All reagents and solvents used were of analytical reagent grade. Distilled water and 95% ethyl alcohol were used throughout the experiment, wherever necessary.

Standard 2,4-D solutions:

The Commercial Grade 2,4-D preparation was procured from the market was used for the experimental studies. Standard solutions containing 8, 10, 12, 14, 16, 18 and 20 µg of 2,4-D per milliliter were prepared using requisite quantities of 2,4-D in 95% ethyl alcohol, in separate measuring tubes.

PROCEDURE 

Thin Layer Chromatography 

           The glass plates (20 cm x 20 cm) were coated with slurry of silica Gel G with water (1:2) to a thickness of 0.25 mm. The plates were air dried at room temperature and then activated at 105⁰ C – 110⁰ C at least for half an hour. Std. Microgram quantities of 2,4 – D solution  were   spotted on the activated TLC plates. The plates were developed in a chamber (saturated) containing solvent system  methyl alcohol : ammonia (100 :1.5 v/v). After the solvent front had travelled 10 cm distance from the original spotting line, the plates were taken out from the chamber and dried in air. The spots were visualized by spraying the  TLC plate with Draggendorff’s reagent.

 Spectrophotometric  Determination:

            2,4-D was quantitatively determined using spectrophotometric device Specord 200 – 1.8 – 0502. The absorbances of every standard 2,4-D solutions were measured at  λ-max  : 287 ± 2 nm   in the UV range 200 – 340 nm using pair of matching quartz cells. 95% ethyl alcohol was used as a reference material. The respective absorbances are tabulated as below in Table – 1.

Table – 1: Table showing concentrations of  standard 2,4-D solutions and its respective absorbances at λ-max  : 287 ± 2 nm.

Sr. NO.	Conc.  of  2,4-D μg / ml	Absorbance at λ-max  : 287 ± 2 nm
1	8	0.0296
2	10	0.0419
3	12	0.0634
4	14	0.0823
5	16	0.1025
6	18	0.1163
7	20	0.1365

 RESULTS & DISCUSSION :

The TLC plates spotted with standard 2,4-D, eluted using above mentioned solvent system were air dried and sprayed with routinely used Draggendorff’s reagent. It showed the presence of  2,4-D as a brown spot at R_f  – value 0. 65.   

The absorbances of standard 2,4-D solutions measured at λ-max 287±2 nm with corresponding concentrations of 2,4-D are recorded in Table – 1.  The calibration curve of these absorbances versus respective concentrations of 2,4-D procured is as shown below in Fig. 1.

Figure – 1 : Calibration curve for the determination of 2,4-D, Conc. of std. 2,4-D (μg/ml) vs.  Absorbance measured at   λ-max  287±2 nm

Beer’s law is obeyed good in the range of 8 μg to 20 μg per ml.  The calibration equation found is “ Y = 0.009x – 0.047” and “correlation coefficient = 0.998”. Detection Limit  was < 1 μg/ml. Diluents in the commercial preparations did not interfere in the measurements. This method can also be applied to the forensic toxicological analyses, successfully.

 Acknowledgements

The authors thanks are due to the  Director, Directorate of Forensic Science Laboratories, Mumbai, M. S.(India) and Chitra S. Kamat, Regional Forensic Science Laboratory, Nashik, M. S., (India).

REFERENCES

1) Fianagan, R.J., Meredith, T.J., Ruprah, M.,Onyon, L.J. and Liddie,   The lancet, 1990, Feb 24, 454-458.

2) Rivers, J.B., Yauger, Jr. W.L and Klemaner, H.W.;  J. of chromatography, 1970, 50, 334-337.

3) Curry, A.S.; British Medical Journal, 1962, 10,687-688.

BLOOD ALCOHOL CONCENTRATION (BAC), DATA ANALYSIS AND BEHAVIORIAL STUDIES.

BLOOD ALCOHOL CONCENTRATION (BAC), DATA ANALYSIS AND BEHAVIORIAL STUDIES.  

Arun G. Bhoi

Arun’s Institute of  Forensic Sciences, Research and Education, Pune  – 411028, State of Maharashtra, (India).

Email : arun.bhoi@gmail.com

(e-J. Foren. Crime Inv. 2011, 3, 1. Art. 1, 19^th Sept.. 2011)

ABSTRACT

In the State of Maharashtra, India, the cases of alcohol / illicit liquor consumption, its preparation, possession, storage  and transportation are  controlled and governed by Bombay Prohibition Act – 1949. The review of 382 cases registered under Bombay Prohibition Act – 1949 has been taken for the presence of ethyl alcohol. These cases were analysed for blood alcohol concentration (BAC) in an academic year. The BAC was estimated using  Modified Diffusion Oxidation Method, routinely used in  Forensic Laboratories. Further, BAC has been correlated with the Medical Officer’s observations like, smelling of alcohol, impairment of speech and walking as also, the position of pupils.

KEY WORDS

Blood alcohol, ethyl alcohol, BAC, Bombay Prohibition Act, Motor Vehicle Act, Modified Diffusion Oxidation Method.

INTRODUCTION

There is a big rise in the cases of driving vehicles in drunken condition. To prohibit this tendency Government is taking very harsh steps. Mobile units are set up by Traffic Police to check up, whether the person driving the vehicle has consumed the alcohol or not?  The Breath Analyser is used for this purpose. It measures the percentage of the ethyl alcohol present in the breath of the person in question. Thus trying to maintain the road safety and to avoid the uncalled accidents.  Some people also, consume alcohol at public places and create nuisance. The consumed and the absorbed alcohol shows its presence in body fluids such as blood, urine, synovial fluids, likewise breath. In India the alcohol consumption cases are  controlled  by Motor Vehicle Act – 1988, National Highways Act – 1956, National Highways Traffic Safety Act, Rules of the Road Regulations 1989, Delhi Traffic Rules, Indian Penal Code – 1980. Maharashtra Police Department, also, registers such cases under Bombay Prohibition Act – 1949, Amendment dated  31-12-2007 and Maharashtra Motor Vehicles Rules – 1989.

To register the alcohol consumption cases in the Court of Law, blood samples of the person in question are to be  got examined from Forensic Laboratories. Jain and Carvey have taken review of chemical and IR methods (1), gas chromatography (2) for the analysis of alcohol. Yamamoto and Veda (3) have studied breath alcohol analysis for the estimation of blood alcohol concentration (BAC). Meson and  Dubowski (4) have elaborated uses and methods of breath alcohol analysis.

EXPERIMENTAL

Equipments

Vacuum pump, round bottom titration flasks, three way glass tube valve, carbonate bulbs, micro titration unit.

Reagents

1. Potassium dichromate solution N/20 (i.e. 0.05N)
2. Sodium thiosulphate solution N/20 (i.e. 0.05N)
3. Conc. Sulphuric acid
4. Granular Sodium carbonate.
5. Starch
6. Morpholline solution
7. Nitroprusside solution

All chemicals used were of Analytical Reagent Grade. Double glass distilled water was used as and when required.

Procedure

             Blood (5 ml) samples of the people suspected to be under influence of alcohol were collected and preserved in glass phials by Medical Officer as and when they were produced before him for examination. The preservative used was 5 mg of sodium fluoride with 15 mg of potassium oxalate as an anti-coagulant (5) for every 5 ml of venous blood. The cases were referred for the detection and determination ethyl alcohol (BAC) over a span of a year. The micro-chemical test, nitroprusside – morpholine reagent test was carried on the blood samples to judge the presence of ethyl alcohol, therein. The ethyl alcohol vapours produce blue colour with this reagent. Then the ethyl alcohol in blood was quantitatively determined using modified diffusion oxidation method (6, 7) which is routinely used for the determination of blood alcohol concentration (BAC) in forensic laboratories.

RESULTS AND DISCUSSION

            In a year 382 cases were analysed for the presence of ethyl alcohol. The modified diffusion oxidation method was used for this analysis. Of the  382 cases analysed 307 cases found positive for blood alcohol concentration of which 285 cases contained BAC more than 50 mg per 100 ml.  From 285 cases, 22 cases showed BAC below 50mg/100ml of which only one case contained BAC in the range of 30 to 50mg%.  From the received cases 74 cases did not show presence of ethyl alcohol in the blood. One case was found unfit for analysis as the blood was got coagulated. The  analysed 382 cases are distributed over the range of  BAC starting from ‘00’ mg% to 300 mg%  as in TABLE -1. It is graphically represented in Figure -1.

TABLE – 1 : BAC mg% and corresponding number of cases examined.

BAC mg%	No. of Cases	BAC mg%	No. of Cases	BAC mg%	No. of Cases
00	74	110	26	210	8
20	1	120	24	220	4
30	15	130	9	230	7
40	6	140	23	240	4
50	0	150	19	250	4
60	37	160	15	260	3
70	34	170	2	270	2
80	16	180	13	280	2
90	17	190	4	290	0
100	17	200	7	300	0

Figure – 1: Graph Showing Number of Cases Examined Vs Corresponding BAC% (Estimated). (Data utilized from Table – 1.)

From 285 BAC positive cases only 122 cases were accompanied with the duly filled in M. O.s observation Form-A and Form-B. As per M. O.s observations these 122 cases are tabulated below as in TABLE – 2. Groups I, II, III and IV are categorized for the effects smelling of alcohol, smell with slurred speech, staggered walk and blurred – dilated pupils, respectively.

TABLE-2: Groupwise distribution of 122 cases observed and opined by M. O. for after consumption effect.

BAC Range mg/100ml	Smelling Alcohol Group – I	Smell with Slurred Speech Group – II	Walking  Staggered Group – III	Pupils Blurred Dilated Group – IV
50 – 60	7	4	0	0
61-70	5	7	1	1
71-80	4	6	0	0
81-90	4	0	0	2
91-100	3	3	0	1
101-110	2	6	0	3
111-120	1	6	0	1
121-130	1	2	2	1
131-140	1	1	1	8
141-150	1	1	2	1
151-160	1	4	0	5
161-170	0	0	0	0
171-180	0	3	0	3
181-190	1	0	1	0
191-200	1	0	0	2
201-210	1	0	1	2
211-220	0	0	0	1
221-230	0	0	0	1
231-240	0	0	1	0
241-250	0	1	0	0
251-260	0	0	0	3
261-270	0	0	0	0
271-280	0	0	0	1

It is considered that if BAC is above 50 mg% w/v then the person under observation always smells of alcohol and he is under influence of alcohol. For our studies (Table-2, Group-I) it applies nicely. The person having BAC level below this may or may not smell of the alcohol and may not be under the influence of alcohol. However, the fellow may start smelling for BAC as low as 30mg%, depending on age, sex, addiction, quantity consumed and time lapsed after consumption. The approximate range of BAC, 100mg% to 180 mg/100 ml of blood shows changes in speech, it is slurred and he speaks irrelevantly.  As tabulated above (Group-II) and as per our studies, impairment of speech is observed starting from 60mg% to 180mg%.  BAC in the approx. range 180 to 280 mg% w/v brings the person in confused condition and he finds difficulty in walking. He starts showing signs of impairment. Our study reflects this range as 130 mg% to 240mg% (Table-2, Group-III). Person with BAC above 280 mg% w/v (approx.) has pupils dilated, fails to respond properly and leads to Comma. Our data range (Group – IV) reflects that BAC 90mg% and above starts showing effect on vision. Medical Officer have observed dilated pupils for this range.   With BAC above 350 mg% w/v, the person marches towards death and it is certain.

CONCLUSION

The BAC data obtained reveals that the opinion and observations made by the Medical Officer (M. O.) about the drunkards at the time of collection of blood samples for BAC are deviated by about 25% for Group-I. This group counts for smelling of the alcohol and it relates with the quantity of alcohol consumed and delay of time after consumption. The person may smell of alcohol irrespective the quantity of alcohol got absorbed in the blood. Even if small dose of alcohol is taken and the person is examined immediately, he may smell of alcohol. The observations made by M. O. stand almost correct for Group – II. The number of cases observed for Group – III are less. Some cases from Group – IV should have reflected in this group. However, 75% observations from Group – IV come true for the considerations made above. It means, if higher dose of alcohol is consumed by a person, the BAC will be higher and gets in for Group – IV. It is likely that higher percentage of BAC (Group-IV) shows all the signs of influence of alcohol meant for  lower concentrations.

In many cases police have given the history of consumption of alcohol, however, M. O.s did not mention their observations about consumption after examining the person in question. Our studies also, come across no consumption or no history cases (about 15%) which showed the presence of alcohol distributed on the wide range of BAC i.e. from 60mg% to 250mg%. This is to be understood that no BAC or Breath Analysis is 100% accurate. It is a rough estimation. Furthermore, no demarcation line for BAC will be drawn while relating it with the physical activities. Studies explored, herein also, pinpoints that M. O.s should be more careful while examining the persons who are  in drunken condition and recording their physique.

ACKNOWLEDGEMENT

            Author’s thanks are due to Director, Directorate of Forensic Science Laboratory, State of Maharashtra, India.

REFERENCES

1. Jain N. C. and  Cravey R. H., J. Chromatogr. Sci., May 1972, 10 (5), p- 257-262.
2. Jain N. C. and  Cravey R. H., J. Chromatogr. Sci., May 1972, 10 (5), p- 262-267.
3. Yamamoto K and  Veda A., J. Forensic Sci. July, 1972, 1(2), p- 207-224.
4. Meson M. F. and Dubowski K. M., J. Forensic Sci., Jan. 1976, 21(1), p- 9-41.
5. Maharashtra Civil Medical Code ( Supplement – Chapter-13 ), 1988, p-12.
6. Mahal H. S., Anal. Chem., 1959, 31, p-1908.
7. Modi, A Text Book of Medical Jurisprudence and Toxicology, 1977, p-655.

USE OF SIMPLIFIED TLC FOR THE DETECTION OF OXAZEPAM AND DIAZEPAM TRANQUILIZERS IN EATABLES

USE  OF  SIMPLIFIED  TLC  FOR  THE  DETECTION  OF  OXAZEPAM  AND  DIAZEPAM  TRANQUILIZERS  IN EATABLES

Arun G.  Bhoi

 Arun’s Institute of Forensic Sciences, Research and Education, Kale Padal, Pune-411 028 (India)

 Email : arun.bhoi@gmail.com

C. S. KAMAT

 Regional Forensic Science Laboratory, Nasik (India)

 

( e-J. Foren. Crime Inv. 2011, 2, 1,  Art. 1, 9^th June 2011)

 

 ABSTRACT

               Benzodiazepines like oxazepam, diazepam are malafidely used by the opportunist thieves to steal the belongings of the pilgrims and/or travelers by mixing it homogeneously with eatables. The method of extracting these drugs from ammonical mass of eatables using chloroform is described, here in, with the aid of easy solvent system and iodine-potassium iodide (alc.) and copper sulphate (aq.) solutions. Oxazepam, diazepam, lorazepam, nitrazepam give brown spots at respective R_f-values with this reagent. The detection limit is 1μg.

KEY WORDS : Oxazepam, diazepam, lorazepam, nitrazepam, tranquilizer, sweets, extraction, tlc, iodine, copper sulphate.

INTRODUCTION

             In an outskirt areas of the holy places the complaints of thefts are raised by the pilgrims. Also, the police receive complaints of lifting the belongings of the people while on journey. To succeed in stealing the money and valuables, a specific and wise modus operandi is exercised by the thieves. They distribute, among the devotees / travelers, sweets, cream biscuits, eatables such as pedhas, burfi, kachori etc. which are premixed with benzodiazepines the sleep inducing drugs, under the farce of “Mahaprasada of the Lord”. The benzodiazepines produce sedation and unconsciousness in 5-10 min.(1). The tranquilizers such as oxazepam and diazepam are preferred for this purpose. Sometimes these have been mixed with drinks like tea, coffee, soft drinks and served to devotees/ pilgrims. As they chew – swallow the eatables, slowly and slowly, enter in the state of drowsiness. Taking this as the best occasion their valuable belongings are stolen by the opportunist thieves. Many methods and techniques have already been reported for the detection and estimation of the tranquilizers (2,3,4). However, a simple technique is described here for the extraction of oxazepam and diazepam tranquilizers from the eatables and their detection on Silica Gel G coated TLC plates using iodine-potassium iodide (alc.) and copper sulphate (aq.) solutions, as spray reagents.

EXPERIMENTAL

Equipments:

1. Standard thin-layer chromatographic equipment
2. Filtration assembly
3. Separating funnels (500 ml cap.)

Reagents :

 (A) Iodine – Potassium iodide alcoholic reagent:

10 g of Iodine and 10 g potassium iodide dissolved in 100 ml of  95 % ethanol.

(B) 10 % Copper sulphate aq. solution : 10 g of copper suphate was dissolved in distilled water and was made up to 100 ml.

All reagents and solvents used were of analytical reagent grade.

Standard Solutions :

              Standard solutions of  oxazepam, diazepam, lorazepam and nitrazedpam drugs procured from market were prepared by dissolving weight equivalent to 10 mg of respective active ingredients in 10 ml of 95% ethyl alcohol separately.

Preparation of Synthetic samples :

Powdered masses of tablets/capsules constiturting 10 mg of oxazepam diazepam were homogeneously mixed with 10 gm of sweets (pedhas) separately, as also, with 10 g of spicy/chili powder preparations, taken in separate big sized, wide mouth test tubes.

Procedure:

Extraction :

25 ml of diluted ammonia solution (1 part 25 % ammonia + 1 part distilled water) was added to all the synthetic samples and were allowed to stand for at least three hours with intermittent shaking and swirling and then filtered through the glass wool, separately. The residues left on the glass wool were washed at least thrice with 10 ml of dilute ammonia (5%), every time. The filtrates of two different synthetic samples were transferred to separate separating funnels, then mixed and extracted with 50 ml of solvent chloroform. The extraction process was repeated thrice, so as to achieve the maximum recovery of the tranquilizers of interest. The solvent from extracts was allowed to get evaporated and the concentrated aliquots (10 ml) were used for further analytical purposes.

Thin-layer Chromatography :

             The glass plates (20 cm x 20 cm) were coated with slurry of silica Gel G with water (1:2) to a thickness of 0.25 mm. The plates were air dried at room temperature and then activated at 105⁰ C – 110⁰ C at least for half an hour. Aliquots of each of the synthetic samples were spotted on the activated TLC plate ( equivalent to 5μg). The reference tranquilizers oxazepam and diazepam along with additional control samples of lorazepam and nitrazepam (hyphotic), 5 μg  each were also spotted on TLC plate. The plates were developed in a pre-saturated chamber containing solvent system Benzene, Acetonitrile , Methanol (8:1:1 v/v). After the solvent front had travelled 10 cm distance from the original spotting line, the plate was taken out from the chamber and dried in air. The spots were visualized on TLC by spraying with (A) Iodine- Potassium iodide reagent fllowed by (B) 10 % copper sulphate aq. solution.

RESULTS AND DISCUSSION

             The brown spots were observed at specific R_f-values for oxazepam, diazepam, lorazepam and nitrazepam, immediately after spraying the above reagents. The corresponding R_f x 100 values are cited in the Table – 1 given below. The reagents has a sensitivity to detect the oxazepam and diazepam to the level of 1 μg,  where as routinely used Dragendorff’s reagent does not responds to this limit.

 TABLE- 1 :    Showing R_f x 100 values of the tranquilizers / hypnotics studied .

Sr. No.	Drug	Rf x 100 value
1	Oxazepam	40
2	Diazepam	85
3	Lorazepam	45 (*)
4	Nitrazepam	65 (*)

(*) Synthetic samples are not studied being its non usability for the purpose mentioned above .

Furthermore, no interferences were noticed either by additives or constituting substances from the eatables. Though this method has also been proved helpful to detect other tranquilizers/hypnotics such as lorazepam, nitrazepam, no synthetic samples of these were prepared with eatables for the detailed study looking at their remote possibility of use for the purpose mentioned, herein. The spray reagent described here can also be used for the detection of tranquilizers in the biological materials referred to Forensic Laboratory.

ACKNOWLEDGEMENT

The authors are grateful to Director, Directorate of Forensic Science Laboratories, M.S., Bombay-400 098.

REFERENCES 

 (1) Tripathi K. D., Essentials of Medical Pharmacology, Third Edition, Jaypee Brothers, Medical Publishers Pvt. Ltd., New Delhi, India, 1994, p-3,  .

(2) E.G.C. Clarke, Isolation And Identification of Drugs in Pharmaceuticals, body fluids and post mortem material, First Edition 1969, The Pharmaceutical Press, LONDON, Reprint – 1974, p – 294, 443, 458.

(3) Fartushnyi, A.F., Muzhanovskii, Sukhin, Sedov, Kvasov,E.B. Farm. Zh. (Kiev), Sep-Oct, 1986(5),47-50 (Ukrain).

(4)  Yan, E.; Jin, X. (  Yiyao Gongye, 1986,17 (5), 21-23 (ch).

Spectrophotometric Determination of Pyridine in Denatured Spirits

Arun G. Bhoi

Arun’s Institute of  Forensic Sciences, Research and Education, Pune  – 411028, State of Maharashtra, (India).

Email : arun.bhoi@gmail.com

(e-J. Foren. Crime Inv. 2011, 1, 1. Art. 3, 26^th Jan. 2011)

ABSTRACT

The rectified spirit (ethyl alcohol) is intentionally denatured using denaturants like caoutchoucine, pyridine, crystal violet, denatonium saccharide, some aldehydes and petroleum hydrocarbons for specific use or to avoid its misuse. A spectroscopic method is reported here for the determination of pyridine in denatured spirit samples (DNS). The spirit samples containing pyridine are allowed to react with tri-chloroacetic acid in the presence of sodium hydroxide at 85˚c temperature for 75 minutes. The absorbance of the crimson colour produced is measured at λ-max 362 ± 1 nm. Bear’s law is found in the range of 275μg to 1472 μg. The other denaturants present in the denatured spirit samples do not interfere.

KEY WORDS : pyridine, denatured spirit, denaturant, sodium hydroxide, tri-chloroacetic acid.

INTRODUCTION

Various miscible denaturants such as acetaldehyde, acetone, caoutchoucine, formaldehyde, acetaldehyde, crotonaldehyde, ethyl acetate, ether, methanol, chloroform, pyridine, petroleum hydrocarbon solvent etc. are added to rectified spirit to make the later unfit for human consumption. Usually 0.5% (v/v) of pyridine bases are added to rectified spirit to make it non-potable. Many of the methods like gas chromatography [1,2], spectrophotometry [3-6], titremetry [7] and thin-layer chromatography [8] are reported in the literature for determination of pyridine.

The spectrophotometric method using tri-chloroacetic acid in alkaline condition is described here for the determination of pyridine.

EXPERIMENTAL

Equipments :

A Shimadzu  double beam ultra violet / visible spectrophotometer and a matching pair of quartz cells were used for the absorbance measurements.

Reagents :

All reagents used were of Analytical Reagent grade. Glass distilled water was used throughout the experiment. 95 % ethyl alcohol was used as and when required.

1. Sodium hydroxide solution (10%) : 10 g of sodium hydroxide palettes were dissolved in small quantity of distilled water and made up to 100 ml with the same.
2. Tri-chloroacetic acid solution (1%) : 1 g of tri-chloroacetic acid was dissolved in distilled water and made up to 100 ml.
3. Standard Pyridine solution (1 %)  : Pyridine ( 1 ml ) was dissolved in 95% of ethyl alcohol and was made up to 100 ml.

Procedure :

Exact quantities ( ml ) of standard pyridine solution such as 0.3, 0.6, 0.9, 1.2 and 1.5 ml equivalent to 294, 588, 883, 1177 and 1472  μg  respectively, were taken in separate reaction/test tubes. Distilled water was added to each of these to make the total volume 3 ml. For blank 3 ml of distilled water was in one additional test tube. Then 2 ml of 10 % sodium hydroxide solution and 5 ml of 1 % tri-chloroacetic acid solution was added to each of these reaction tubes. The water bath was adjusted to 85˚c approx. and all the above reaction tubes were kept in the water bath for 75 min. The temperature of water bath was regulated to 85˚c during the reaction time. Afterwards the tubes were taken out and allowed to stand for half an hour. Then the absorbances of the crimson color developed were measured at λ-max 362 ± 1 nm using blank reagent as reference with the help of double beam Shimadzu ultraviolet – visible spectrophotometer and matching pair of quartz cells.

Figure 1 : Showing absorbance spectra of colour developed for Pyridine  Standard 1 to Standard  5    (λ-max 362±1 nm).

TABLE 1 : Showing Concentrations of Standard Pyridine /10ml and its corresponding Absorbances

StandardNo.	Concentration of Std. Pyridine per 10 ml		Absorbance (Av. of two)At λ-max 362 ±1 nm
	ml of 1% Std. Pyridine	Equivalent μg of Pyridine
1	0.30	294	0.266
2	0.60	588	0.670
3	0.90	833	1.099
4	1.20	1177	1.594
5	1.50	1472	1.960

RESULTS AND DISCUSSION

Fig. 1 shows the absorbance spectra of the crimson color developed. The absorbances with corresponding concentrations of pyridine are recorded in Table – 1.  The calibration curve of absorbance versus respective concentration of pyridine is shown in Fig. 2.  Beer’s law is obeyed good in the range of 275 μg to 1472 μg per 10 ml. The calibration equation for this is “ Y = 0.001x – 0.158” and “correlation coefficient = 0.997”. Other denaturants such as acetone, aldehydes, methanol, chloroform, pyridine,  ethylacetate, ether etc. did not interfere in the determination of the pyridine. Thus, this method is very much useful for the determination of pyridine in spirit (DNS). The requisite quantities of the DNS samples can be taken directly for the estimation of pyridine along with standards for colour development. By measuring absorbance of the colour developed at λ-max 362±1 nm, the corresponding concentration of pyridine can be found from calibration curve. Thus the method reported here can be used preferentially for the determination of pyridine in DNS and does not require any pre-treatment of the sample.

AKNOWLEDGEMENT

Author’s thanks are due to Director, Directorate of Forensic Science Laboratories, State of Maharashtra, India.

REFERENCES

1. Habboush, A. E., Farroha, S. M. and Al-Bayat, R. I.; Chromatographia., 1978, 11, 662.
2. Iskenderov, R. A., Ali-Zade, N. I., Bairamov, F. G. and Nagiev, T. M.; Zh. Anal. Khim., 1988, 43, 181 (Russ.).
3. Zhadanov, B. V., Dobrakova, G. M. and Adamova, G. M.; Ref. Zh. Khim., 1977, 20, 19 GD.
4. Rawat, J. P. and Bhattacharjee Priti; Indian J. Chem., Sect. A, 1976, 14, 544.
5. Amlathe, S., Upadhyay, S. and Gupta, V. K.; Microchem. J., 1988, 37(2), 225.
6. Ramchandran, K. N. and Gupta, V. K.; Microchem. J., 1991, 44 (3), 272.
7. Indian Standard, Specification for Ordinary Spirit (Revised), Indian Standards Institution, New Delhi, 18:324, 1959, p.26.
8. Land, E. and Lang, H. Z.; Analyt. Chem., 1976, 278, 368.

Figure 2 : Calibration curve for the determination of pyridine, Conc. of std. pyridine (μg/10ml) vs.  Absorbance measured at       λ-max 362±1 nm

Simultaneous Detection of Cardiovascular Drugs Clopidogrel and Aspirin in Combined Formulation by HPTLC

Arun G. Bhoi

Arun’s Institute of  Forensic Sciences, Research and Education, Pune -411 028 (India).

Email : arun.bhoi@gmail.com

(e-J. Foren. Crime Inv. 2011, 1, 1. Art. 2, 5^th Jan. 2011)

ABSTRACT

Clopidogrel as clopidogrel bisulphate and aspirin drugs are prescribed in the combined dosage form on the cardiovascular disease. Both of these drugs are analysed using HPTLC (high performance thin layer chromatography). HPTLC glass plates (10 cm x 10 cm) pre-coated with silica gel 60F₂₅₄ were used as stationary phase. Mobile solvent system was hexane : acetone (8:2 v/v) and UV detection carried at 254 nm. Clopidogrel and aspirin were detected at R_f-values 0.60 ± 0.02 and 0.25  ±0.02, respectively. The LOD for these drugs was found to be 100 ng per spot.

KEY WORDS : clopidogrel, aspirin, detection, cardiovascular, HPTLC, UV.

 

INTRODUCTION

Clopidogrel as clopidogrel bisulphate (Methyl-2-chlorophenyl-(4,5,6,7 tetrahydrothieno [3,2-c] pyridine – 5 yl) acetate bisulphate) and aspirin (acetyl salicylic acid, 2-acetoxy benzoic acid) are routinely used in the treatment of cardiovascular diseases. Aspirin works as anti-platelet, vasodilatory drug and also as weaker analgesic, anti-pyretic and anti-inflammatory [1]. Himani et. al. have studied susceptibility of clopidogrel bisulphate to acids, alkali, oxidation and heat using HPTLC [2]. Mitacos, A. and Panderi, I. have validated clopidogrel in pharmaceutical preparations using LC [3]. Aspirin in the presence of other drugs has been estimated using RP-HPLC [4,5,6], Rapid HPLC [7], HPTLC [8] and spectrophotometry [9]. Determination of clopidogrel has been reported in the literature using HPLC [10] and spectrophotometry [11]. Studies of clopidogrel bisulphate in combined form have been explored using HPLC [12,13,14,15]. Shrivastav et. al. [16] have validated clopidogrel bisulphate and aspirin drugs using RP-HPLC. Thus, heavy use and demand for cardiovascular drugs clopidogrel bisulphate and aspirin made it necessary to find easy method for its detection. HPTLC technique is explained here for the simultaneous detection of  this duo using simple, specific and speedy line up.

EXPERIMENTAL

Equipments:

The following material / equipment was used for the experiment.

1. Merck, HPTLC glass plates pre-coated with silica gel 60 F 254 ( 10 cm x 10 cm).
2. Camag Development Chamber ( small size, twin type ).
3. Camag UV Viewing Chamber equipped with 254 nm and 366 nm wavelength exposure facility.

High Performance Thin-layer Chromatography (HPTLC):

Reagents: All reagents and solvents used were of analytical reagent grade.

95% ethyl alcohol and glass distilled water was used wherever required.

Standard Solutions:

Clopitab A 150 capsules, each  containing clopidogrel bisulphate powder equivalent to 75 mg of clopidogrel and aspirin in tablet form, two tablets of 75 mg each (i.e.150 mg of aspirin), marketed by LUPIN LTD., MUMBAI, INDIA, were used for studies.

1. Clopidogrel Standard Solution ( 1mg/ml) : Clopidogrel bisulphate powder equivalent to 10 mg of clopidogrel was weighed from powder contents of capsule (Clopitab A 150), dissolved in small quantity of 95% of ethyl alcohol and made up to 10 ml with the 95% ethyl alcohol.
2. Aspirin Standard Solution ( 1mg/ml) : Two tablets of aspirin were separated out carefully, from the contents of  Clopitab A 150 capsule, finely powdered and quantity equivalent to 10 mg of aspirin was weighed, dissolved in small quantity of 95% ethyl alcohol which was further made up to 10 ml using the same.

Camag, HPTLC glass plate pre-coated   with silica gel 60 F 254 of the size of 10 cm x 10 cm was taken for the experiment. 0.1, 0.2, 0.5, 1.0 and 2.0 μg quantities of each of the clopidogrel and aspirin standard solutions were spotted on the HPTLC plate using micro capillaries. The HPTLC plate was eluted in a previously saturated small sized, twin trough Camag HPTLC chamber using 5 ml quantity of  solvent mixture n-hexane : acetone ( 8 : 2 v/v). The solvent was allowed to travel to a height of     5 cm. Then the HPTLC plate was taken out, air dried and viewed in a UV chamber at 254 nm wavelength.

RESULTS AND DISCUSSION

The developed HPTLC plate when observed in the UV viewing chamber showed the presence of two spots by fluorescence quenching phenomenon. Aspirin showed its presence at Rf-value 0.25±0.02. The another constituent of Clopitab A 150 capsule, clopidogrel was found present at Rf-value 0.60 ±0.02. The solvent system used here for the detection of both the constituents of cardiovascular drug Clopitab A 150 shows very good separation from each other. The spots visualized are neither very close to the spotting line and solvent front nor farther away from these. The detection limit for both of these drugs ca. 100 ng. Further, it takes hardly 20 minutes to to complete the detection process. No clumber some technique is used herein. It (working set) can also, be used like a portable kit. Hence, HPTLC technique described here is a simple, specific and speedy one to detect drugs clopidogrel and aspirin separately or simultaneously in the medicinal formulations or biological samples.

ACKNOWLEDGEMENT

Author’s thanks are due to Director, Directorate of Forensic Science Laboratories, Mumbai, State of Maharashtra, India.

 

REFERENCES

[1] Tripathi K. D., Essentials of Medical Pharmacology, Third Edition, Reprint 1995, Jaypee    Brothers, Medical Publishers (P) Ltd., New Delhi, India.

[2] Himani Agarwal, Niraj Kaul, Paradkar A. R. and Mahadik K. R.;  Talanta, 2003, 61(5), 581-589.

[3] Mitakos, A. and Panderi, I.; J. Pharm. Biomed. Anal., 2002, 28, 431-438.

[4] Gandhimathi, M., Ravi, T. K., Abraham, A and Thomas, R.;  J. Pharm. Biomed. Anal., 2003, 32, 1145-1148.

[5] Shah, D. A., Bhatt, K. K., Mehta, R. S., Shankar, M. B., Baldania, S. L. and Gandhi, T. R.; Ind. J. Pharm. Sci., 2007, 69(4), 571-574.

[6] Shah, D. A., Bhatt, K. K., Mehta, R. S., Shankar, M. B., Baldania, S. L. and Gandhi, T. R.; Ind. J. Pharm. Sci., 2007, 69(4), 546-549.

[7] Sawyer, M. and Kumar, V. J.; J. Chromatogr. Sc., 2003, 4, 393-397.

[8] Bernard Fried and Joseph Sharma; Practical Chromatography, A Multidisciplinary Approach, CRC Press, 1996, p-242.

[9] Sena, M. M. and Poppi, R. J.; J. Pharm. Biomed. Anal., 2004, 34, 27-34.

[10] Juliana Sippel, Leticia L. Stair, El Frides E. S. Schapoval, Martin Steepe.; Journal AOAC International, 2008.

[11] Hala E. Zaa-Zaa, Samath, S. Abbas, M. Abdelkawy and Maha M. Abdelrahman.; Talanta, 2009, 78(3), 874-884.

[12] Pankaj K. Pacchadia, Ashish S. Doshi and Hitendra S. Joshi.; J. AOAC Int., 2009, 92(1), 152-157.

[13] Patel R. B., Shankar M. B., Patel, M. R. and Bhatt K. K.; J. AOAC Int.; 2008, 91(4), 750-755.

[14] Patel, R., Patel, M., Shankar, M. and Geetha, M, AAPS 2006-200789.PDF.

[15] Anadkumar K., Ayappan, Y., Raghu Raman V., Vetrichelvan T., Shankar A.S.K., Nagavalli, D.; Ind. J. Pharma. Sci. 2007, 69(4), 597-599.

[16] Shrivastava P. K., Basaiwal P. K., Jain Deepti and Shrivastava S. K.; Ind. J. Pharma. Sci.; 2008, 70(5), 667-669.

 

 

 

 

 

Identification of Impressions of Metal Marking Punches

Arun G. Bhoi

Arun’s Institute of  Forensic Sciences, Research and Education, Kale Padal, Pune – 411 028 (India).

Email : arun.bhoi@gmail.com

(e-J. Foren. Crime Inv. 2011, 1, 1. Art. 1, 1^st Jan. 2011)

ABSTRACT

In this work physical characteristics studied to know whether engine and chassis numbers present on respective sites of  M/Cars are produced by the use of questioned punching nails or not ? The numbers present on the voucher plates (Questioned) comparatively studied with the test impressions produced by the questioned metal marking punches using microscopy. Imperfections, striations present thereon and its positioning at the ‘Clock-type Grid’ helped to correlate these.

KEY WORDS : punches, impression, digits, engine, chassis, number, clock-type grid, forensic.

INTRODUCTION

The M/Cars and the sets of metal marking punches used for punching digits were examined to know falsification in engine and chassis numbers, if any. The stamped impressions found on the various organs of vehicles such as engine, chassis and voucher plate were comparatively studied stamped impressions produced using questioned punches for their identity, individuality and similarity or dissimilarity. Microscopic examination was preferentially carried for doing this work.

EXPERIMENTAL

Physical Examination

The punched numbers – impressions (Questioned) were observed on the engine number site, chassis number site and voucher plate of the M/car vehicle. Being engine as a heavy – voluminous machinery and the chassis panel a long – one piece part of the vehicle, the punched numbers present on these were examined, in situ. Further, tracings of these numbers were taken on butter paper using soft graphite pencil. Supportingly, the numbers present at engine and chassis site were dusted with fine graphite powder and its images were lifted on the one inch broad transparent cellophane tape. Then, simulated (Test ) engine and chassis numbers were produced on 1mm thick  aluminum sheet using questioned metal marking punches. The tracings were taken, as also, the numbers were lifted on the cellophane tape.

Afterwards, the design and the shape of the “Questioned” and “Test” impressions were comparatively studied in situ and making use of tracings and lifted numbers present on cellophane tape. The dimensional parameters such as heights, widths, thicknesses, high-low joints of letters/digits, its spacing, length of script were minutely examined and the identity of the impressions of letters/digits was established. Overlaying of the tracings / lifted numbers of “Questioned” and “Test” letters/digits also helped to correlate these.

Microscopic Examination

Voucher plates were examined by this technique using “Metzer Comparison Microscope”. For this, voucher plates bearing punched Model No., Engine No., Chassis No. and Serial No. were removed from the cars. Further, the simulation impressions of the corresponding letters / digits were produced on 1mm aluminum sheet using the respective metal marking punches. Then, the impressions present on the impressions present on the voucher plate (Questioned) and the simulation impressions (Test) from the aluminum sheet were comparatively studied by keeping these on the moveable mounting stage of the microscope and using the illuminating light assembly. Because of the advantages of the good heights, widths and other characteristics the impressions of the higher case letters viz. “O”, “B” and “H” were selectively chosen for this examination.

Below discussed are some of the features used for correlating stamped impressions present on voucher plate and the test impressions of the higher case letters viz. “O”, “B” and “H”.

1. Individual Characteristics : The higher case letter “O” (Questioned) shows the characteristic curve at position –I.           (Fig. 1 (a)). Similar curve has also been seen on the test impression (Fig. 1 (b)).

Figure 1 : Microscopic View of  :  (a)    Impression of higher case letter “O” from Voucher plate  (b)   Test Impression

2. Imperfections : The imperfection seen as on the impression of higher case letter “O” (Questioned) at position –II, has good identity with that of “Test” impression (Fig. 1 (a) and (b)). This imperfection has resulted by the crack/defect present in the punching nail itself.

3. Irregularities in the Plateau: The higher case letter “B” is chosen for studying the irregularities in the plateau. The misalignment of plateau of terminus and that of upper helices is observed in “Questioned” impression of letter “B” at position “I” (Fig. 2 (a)). Also, at the site “II” (Fig. 2 (a)) the major irregularity (discontinuity) in the vertical leg is prominently noticed. These irregularities are also reproduced in “Test” impressions (Fig. 2 (b)).

Figure 2 : Microscopic View of  :  (a)Impression of higher case letter “B” from voucher plate  (b)Test Impression

4. Identical Striations : On the middle arm of letter “B” the striations are seen sequentially produced (Fig. 3). “Questioned” and “Test” impressions show the linear consistency.  The unevenness in the thickness (shown by the dashed line “——-”) at some of the places of the running boundaries, arms and legs of the letters studied is because of the lack of the application of the uniform forces at the time of producing the respective impressions using punches.

Figure 3 : Microscopic View of leg and arm portion of higher case letter “B” showing striations

(a)Questioned Impression   (b)Test Impression

5. Clock-type Grid : Study of higher case letter “H” based on clock-type grid examination helps to correlate the “Questioned” and “Test” (Simulation) impressions and identifies the instrument / marking punch applied there so.

Figure  4 : Microscopic View of higher case letter “H” with marked ‘Clock – type Grid’ pattern.

(a) Questioned Impression from Voucher plate   (b) Test Impression

In Fig. 4, the ridges at the joint of the horizontal arm with the left leg at “9” – O’ clock position and the line running parallel to to this leg crossing the line of “7” – O’ clock position confirms the identity and the individuality of the stamping device. Furthermore, the plateau defects positioning in between “11” – O’ clock and “12”  – O’  clock, as also, the presence of plateau defect in “6” and “7” – O’ clock position supplements it. The deformities in the plateau or at terminals are usually the after effects of the continual/repeated use of the punches for stamping.

RESULTS AND DISCUSSION

By physical, dimensional and superimposition studies the impressions in  “Questioned” and “Test” (simulation) impressions of the punching nails under examination can be correlated. The parameters explained makes one enable to find the match between the corresponding impressions. These may, also, help to judge dissimilarities, if any, in the impressions to be compared. But these examinations are not full proof to decide the origin of the stamped impressions. However, the proper and careful evaluation of the comparative data and the critical features studied microscopically and use of ‘Type-clock Grid’ made it very much certain to say that the “Questioned” and the corresponding “Test” are derived by application of the same punches from the set. The individual characteristics, defects, irregularities observed in the respective ‘PUNCH’, differentiates / correlates it and its impression/s with the others. Thus by avoiding the possibility of the stamped impression  matching with the impression coming from such other punching nail manufactured from the same ‘HOB’.

ACKNOWLEDGEMENT

Author’s thanks are due to Director, Directorate of Forensic Science Laboratories, Mumbai, State of Maharashtra, India.