1. INTRODUCTION:

Clonazepam, 5-(2-chlorophenyl)- 1,3-dihydro- 7-nitro-2H -1,4-benzodiazepin-2-one, is a benzodiazepine derivative (Fig 1) with anticonvulsant, muscle relaxant, and anxiolytic properties¹ and easily available in the market at low price. It is a long-acting benzodiazepine which is commonly administered for anxiety / panic disorders and sometimes for epilepsy^2-4. Clonazepam is a nitrobenzodiazepine as it is a chlorinated derivative of nitrazepam and its pharmacological properties as with other benzodiazepines are the enhancement of the neurotransmitter GABA via modulation of the GABA_A receptor⁵. This drug is a common drug of abuse due to its sedative action on the brain and central nervous system^6-9. The specific drug is forensically important as a number of cases pertaining to the abusal of Clonazepam is encountered very often in the Forensic Laboratories.

Several methods has been reported for the quantitative and qualitative analysis of clonazepam in different matrices like tablets^10,15, in blood¹¹ serum¹², in plasma¹³ , in hair¹⁴and in urine¹⁵ using different chromatographic techniques. However no work has so far been carried out by researchers for the analysis of clonazepam in chocolates as a stupefying agent.

The present work describes a method for the identification of Clonazepam using GC-MS followed by its quantification using HPLC in pure standards as well as in actual sample as per the International Conference on Harmonisation (ICH) guidelines¹⁶. The sample was received in our Laboratory as a case exhibit seeking the identification and quantification of any sedative present. The developed method will be highly significant as the studied drug is forensically important and encountered most often.

2. EXPERIMENTAL:

2.1 Chemicals and reagents:

The standard reference material of Clonazepam was provided by M/s Intas Pharmaceuticals Pvt. Ltd, Ahmedabad, India. The solvents, Methanol, Acetonitrile, Chloroform, Water (all HPLC grade) were procured from M/s Qualigens fine Chemicals Pvt Ltd, India. Orthophosphoric acid, potassum dihydrogen phosphate and anhydrous sodium sulphate (all AR grade) were procured from M/s Merck Specialities Pvt. Ltd, India.

Fig 1: Structure of Clonazepam

2.2 Instrumentation:

The GC-MS analysis was performed on a Perkin Elmer Clarus 600S GC-MS system which is controlled by Turbomass 5.3.0 Software. The HPLC analysis was carried out on a Shimadzu HPLC 10 AT VP system with a PDA detector controlled by Class VP-10 software.

2.3 Preparation of sample and standard stock solution:

Sample preparation:

The chocolate sample is accurately weighed, 3.225g and put in about 20 ml of distilled water and kept overnight for homogenisation. The sample was filtered after 24 hours and the filtrate was transferred to a separating funnel, adjusted the pH - 8 to 9 by addition of 0.1M NaOH followed by extraction with chloroform three times. Each time the organic layer was collected by filtering through anhydrous sodium sulphate and made a final volume of 10 ml. 5 ml of this filtrate was kept for GC-MS analysis separately while the rest 5 ml was kept for HPLC analysis.

Standard stock solution:

10 mg of standard reference material of clonazepam was accurately weighed and dissolved in the above said mobile phase solvent in a 10 ml standard flask which is used as the stock solution of concentration, 1mg/ml.

2.4 GC-MS analysis:

The standard reference material of clonazepam was accurately weighed in a standard flask and made up with chloroform so that the final concentration is 500µg/ml. 2 µl standard solution was injected to the GC-MS system with the following conditions to obtain the chromatogram. The sample residue after solvent extraction was reconstituted with 250µl of chloroform and 2 µl of it was also injected to the GC-MS system where injector temp was set to 260⁰C with split flow of 20 ml/min, inline and source temp was about 200ºC, with carrier gas (Helium) flow of 1 ml/min. Oven temp was set initially 180ºC without any hold then it increases to 230ºC with a rate of 25ºC/min then it was increases to 270ºC with a rate of 10ºC, and finally temp was increases to 325 ºC with a rate of 25ºCand was held for 2 minutes with a total run time of 10.20 minutes. The GC column used for the analysis was PE Elite- 5MS column with dimensions 15mx0.25mmx0.1µm.

The detection carried out by mass spectrometer with EI (electron Impact) ionisation and was monitored by both SIM (Selected Ion Monitoring) mode with 10 prominent m/z (mass/charge) values of Clonazepam (280,314,315,286,288,234,240,316,287,317) and full scan mode with mass range of 51 to 330. A solvent delay of 2.5 minutes was given during the run to avoid the solvent.

2.5 HPLC analysis:

A Phenomenex Gemini C18 HPLC column with dimensions (150mmx4.6mmx5µ) protected with a Phenomenex guard cartridge was used throughout the analysis. The mobile phase consist of Acetonitrile: 40mM potassium dihydrogen phosphate (6:4, v/v ) (pH adjusted to 3.9 by addition of orthophosphoric acid and sonicated for half an hour to remove the dissolve gases) with a flow rate of 1 ml/min. Analytes were scanned from 200-370 nm range using Photo diode array detector (PDA). Total run time was about 5 minutes and about 10 µl sample was injected into the system.

2.6 Method validation studies:

Linearity and range:

Twelve different concentrations of the standard clonazepam ranging from 0.1ug/ml to 1000 ug/ml were prepared and the chromatographic run with the above mentioned conditions for the same samples was carried out. 10 µl of each of the concentrations was injected to the HPLC system for the calibration studies. The chromatogram was examined and the peak areas of the corresponding concentrations are noted at a wavelength of 214nm. A calibration curve of peak area against concentration is drawn to study the linearity and range of the developed method. The linear regression coefficient also is calculated from the calibration curve.

Precision:

The precision of the method developed was examined by checking the repeatability of retention time and peak area of three different concentrations 250, 100 and 50µg/ml with two replicates of each of the concentrations. The intra and inter day precision was studied for the same concentrations and expressed in terms of percentage relative standard deviation.

Robustness:

The robustness of the HPLC method was checked by making slight variations in pH and composition of the mobile phase and carrying out the analysis for a single concentration 100ug/ml. The slight variations made for the analysis were

· pH of mobile phase: 3.95, 3.9, 3.85 with constant mobile phase ratio 6:4

· Mobile phase composition ratio: 5.9:4.1, 6:4 and 6.1:3.9 with constant pH at 3.9

The Rt value and the peak area was noted for each of the analysis and the percentage relative standard deviation was calculated to study the robustness of the method.

Detection limits (LOD/LOQ):

The standard Clonazepam was prepared in different lower concentrations coming down upto 0.1ug/ml and carried out the analysis to determine the limit of detection (LOD) and the limit of quantification (LOQ). Blank mobile phase was also injected and the signal to noise ratio was determined.

Specificity:

The specificity of the method was determined by comparing the analytical results given by the standard clonazepam and the chocolate samples. The Rt values and the UV spectra of the standard and that of the samples are compared to obtain the specificity. The peak purity of the standard was assessed by comparing the spectra at three different levels, at peak start, at peak apex and at peak end of the corresponding chromatographic peak.

Quantification of Clonazepam from chocolate:

The residue obtained by evaporation of 5 ml chocolate filtrate is reconstituted with 1 ml of mobile phase and taken for HPLC analysis. 10 ul of the sample was injected during the chromatographic run. The chromatogram obtained for the sample was integrated at a wavelength of 214 nm for quantification. The calibration curve drawn with the external standard was used for estimating the concentration of clonazepam from the chocolate sample.

3. RESULTS AND DISCUSSION:

3.1GC-MS Analysis:

The GC-MS chromatograms obtained for the standard clonazepam as well as the sample in both SIM and full scan modes are given in the figure-2. Clonazepam eluted from the column at retention time (Rt): 4.00 minutes. Clonazepam was detected in chocolate sample by comparing the Rt of standard clonazepam and also comparing the mass spectra (Fig 3) obtained for standard clonazepam with respects to sample peak at Rt 4.00 minutes. Under given conditions of GC the clonazepam was well separated from the other constituents present in the chocolate sample.

Fig 2: GC-MS chromatograms of standard clonazepam and chocolate extract in Full scan and SIM modes.

Fig 3: Mass Spectra of Clonazepam

3.2 HPLC analysis:

The HPLC analysis was carried out as discussed above and the chromatogram obtained is given in figure-4. The retention time was found to be 2.848 minutes. The measurement of clonazepam was recorded at 309 nm, which is based upon the UV spectra obtained in vivo (Fig 5). The purity of the chromatographic peak is determined by the taking the spectra at different points of the peak (from peak start to peak end) and overlaying the different spectra obtained.

Fig 4.1: Chromatogram obtained for standard Clonazepam

Fig 4.2: Chromatogram obtained for chocolate sample

Fig 4: Chromatogram showing the clonazepam peak.

Fig 5: UV spectra obtained for Clonazepam

3.3 Method validation studies:

Linearity and range:

The calibration curve (plot of peak area versus concentration) for Clonazepam (Fig 6) was drawn as per the data obtained for various concentrations ranging from 0.1ug/ml to 400 ug/ml, analysed using HPLC method. The linear regression equation obtained from the calibration curve is given as y = 51.405x + 84.924 with linear regression coefficient, R²= 0.9989 which clearly shows the excellent linearity of the developed method. The range of linearity of clonazepam is found to start from 0.1ug/ml onwards below which it is not detectable and upto 400ug/ml above which the linearity decreases. In other words clonazepam is found to be linear in the concentration range of 0.1 to 400ug/ml.

Fig 6: Showing the Calibration curve obtained for clonazepam.

Precision:

The precision of the developed method was examined by carrying out the repeatability of the method with different concentrations both intraday and inter day. The mean peak area as well as retention times recorded each time was taken and the standard deviation and the % RSD calculated. The lower values of %RSD (0.2836 to 3.9% for peak area and 0.5195 to 1.03% with respect to retention time) (Table-1) shows the good precision of the method.

Table1: Data for precision studies with respect to peak area and retention time.

Concentration ug/ml	% R.S.D for peak area		% R.S.D for retention time
Concentration ug/ml	Inter day	Intra day	Inter day	Intra day
50	0.6927	0.2836	1.0312	0.5462
100	3.9016	0.4865	0.5195	0.5233
250	0.9833	0.5779	0.7959	0.5233

Robustness:

The robustness of the method was checked by making slight variations in mobile phase pH and composition. The method was found to be robust as the slight variation in different parameters does not make significant deviation in the values of peak area and retention time for the particular concentration, 100 µg/ml. The lower values (given in the table-2) for %R.S.D makes it clear that the method is robust enough to carry out the analysis of clonazepam.

Table 2: Data for robustness with respect to peak area and retention time.

Parameter	% R.S.D for peak area	% R.S.D for retention time
Mobile phase pH (3.95, 3.9, 3.85)	0.9975	0.8566
Mobile phase ratio (5.9:4.1, 6:4 and 6.1:3.9)	0.7494	1.1343

Detection limits (LOD/LOQ):

The Limit of detection and Limit of quantification for clonazepam using the developed method was determined using the Signal to Noise ratio method. A concentration of 0.1ug/ml was found to be the detection limit and the quantification limit was found to be 0.35ug/ml. The lower value of LOD and LOQ suggests the method is sensitive enough to detect and quantify the analyte in trace amounts.

Quantification of clonazepam from samples and Specificity:

The amount of clonazepam present in the samples was calculated from the calibration curve of standard clonazepam using the linear regression equation obtained. The concentration of clonazepam obtained from the sample is calculated and found to be 246.67ug/ml which is equal to 0.0763ug/mg of the chocolate. The method was found to be highly specific for clonazepam as the chromatographic peak obtained for the samples have identical retention time with the standard. The UV spectra obtained with the samples was perfectly matching with that of the standard with the lambda max 309 nm. The purity of the peak also was checked and found to be excellent. All the results suggest the developed method is highly specific. The results obtained clearly suggest the method is highly sensitive enough to detect the presence of clonazepam in chocolate.

4. CONCLUSIONS:

The analytical method reported in this paper for the estimation of clonazepam from chocolate using HPLC and GC-MS is found to be very sensitive, robust and a precise one which can be routinely used in Forensic Laboratories encountering cases related to the same analyte. The solvent extraction method adopted was found to be good enough to give acceptable results. The same method can be easily applied for the determination of Clonazepam in similar kind of food items.

5. ACKNOWLEDGEMENT:

The authors are highly thankful to the Chief Forensic Scientist, Directorate of Forensic Science, New Delhi for providing the fellowship to carry out the research work. Also the authors are thankful to Shri. A.K. Ganjoo, Director, CFSL Hyderabad for providing the facilities to carry out the research work. Thanks are also due to M/s Intas Pharmaceuticals Pvt. Ltd, Ahmedabad, India for gifting the free reference standard of Clonazepam.

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Received on 01.02.2011 Modified on 28.02.2011

Asian J. Research Chem. 4(5): May, 2011; Page 761-765