Benzodiazepines are psychoactive compounds having a structure which is combination of benzene and diazepine ring. The whole group of such compounds is called “Benzos” [1,2].

Benzos are frequently prescribed to treat certain ailments. Due to sedative properties, they have a high potential for abuse [3,4] especially when used with other depressants such alcohol and opiates [5,6]. Commonly prescribed benzodiazepines are Xanax (alprazolam), Librium (chlordiazepoxide), Valium (diazepam) and Ativan (lorazepam) (Figure 1).

Figure 1: Benzos.

There are few methods for the colorimetric identification of benzodiazepines in the forensic drug testing and toxicological laboratories. Two types of tests are performed for the identification of drug namely preliminary test and confirmatory test [7-9]. In case of diazepam which is abundantly abused among others benzos not many color test are available in literature. However, for diazepam and flunitrazepam, two different color reactions are available [10,11]. No single color test is available for the benzos as a whole. The object of this preliminary study was to find a single color test for benzos which could give some indication of the benzodiazepine group. Zimmermann’s Reaction was applied for identification of benzos but the test does not give clear indication due to double shade (reddish purple) [12- 14]. Herein a new presumptive color test for the identification of benzodiazepines has been developed. This test is rapid, easy to perform, economical and has reasonable sensitivity towards tested benzos. Another advantage of this test is that it can be used for quantitation of benzos along with its presumptive identification.

Materials

• Cobalt thiocyanate, Chlorpheniramine maleate and Diazepam were imported from Sigma-Aldrich, spruce street St. Louis.

• Analytical grade solvents Hydrochloric acid 37%, n-Hexane 99%, chloroform 99.8%, toluene 99.6%, acetone, isopropanol as well as analytical grade chemicals Sodium hydroxide, Diphenhydramine, Quinine Sulfate, Ditheoximide, Vanillin, Caffeine, Sodium hydrogen tartrate, Calcium carbonate, Hydroxylamine, were purchased from Acros organics new jersey USA.

• Deionized water was used for reagent preparations. Absolute Ethanol was purchased from Merck Darmstadt Germany.

• Sodium Chloride, Sulfamic Acid, Starch, Lactose, and analytical grade methanol were bought from Fischer Scientific Bioreagents Fair Lawn New Jersey USA.

• Oxytetracycline, meloxicam and albendazole were taken as generous gift from Venus pharmaceuticals Lahore.

• Tablets, capsules and injections were acquired from different pharmacies (Table 1).

Table 1: Compound obtained from Pharmacies, Brand Used and Manufacturer.

Instrumentations

• TurboVap Model#10430 (Harris oaks Blvd Suite C Chariotte NC 28269 USA) was used for solvent drying.

• Fisher digital vortex mixer model# 945416 was used for sample mixing.

• Eppendorf centrifuge model 5810 was used for phase separation.

• Gas Chromatograph-Mass Spectrometry (GC model # 7890AMass Spectrometer model #5975C with triple axis detector Agilent technologies).

• Fourier Transform Infrared spectrophotometer (Thermo-Fischer scientific Model # Nicolet is 10) was used for confirmation of extracted samples.

• UV-Visible spectrophotometer Evolution 300 was used for the quantitation of Diazepam.

• Analytical balance (Mettler Toledo PL 303) was used for the weighing purposes

Reagent preparation: 2% cobalt thiocyanate solution was prepared by adding 2 grams of cobalt thiocyanate powder in 100 ml Deionized water.

Extraction procedure: Extraction procedure for various types of dosage forms i.e. tablets, capsules or injectable is given in Table 2.

Table 2: List of Drugs and their Extraction procedures applied.

Color test procedure: Two glass culture tubes were taken and labelled as “Control” and “Sample”. In the control tube, one drop of Conc. HCl was added followed by the addition of two drops of Cobalt thiocyanate reagent whereas in the sample tube, a pinch of test powder was placed in sample tube and added one drop of Conc. HCl followed by the addition of two drops of Cobalt thiocyanate reagent. Any change or transition of color and its shifting to suitable organic solvent was noted.

Method for diazepam quantitation

Standard solution: 10 mg of Diazepam powder standard was dissolved in 10 ml of chloroform for making standard stock solution (1 mg/ml). Aliquot concentrations (250, 300, 350, 400, 450 and 500) μg/ml were taken in labelled glass tube by using micropipette. 500 μl of chloroform was taken in glass tube and marked as blank sample. Two concentration levels of 315 μg/ml and 475 μg/ml were prepared as Qc Low and Qc High respectively. All the tubes were completely dried in air stream using turbo evaporator. Color test was performed in “control tube” and “sample tubes” by adding 0.5 ml of conc. HCl followed by addition of 0.5 ml of Cobalt thiocyanate solution. Vortex it thoroughly then 2 ml chloroform was added for extraction of color in chloroform for quantitation. Chloroform from control tube and each calibrator was pipetted out in other tube. Each tube was extracted with chloroform three time. The tubes were dried on dry bath incubator at 60ºC for solvent evaporation. Finally the tubes were reconstituted with 2 ml chloroform. The standard solutions were run on ultraviolet-visible spectrophotometer for checking of absorbance at 364 nm. Instrumental method parameters for diazepam quantitation are shown in Table 3.

Table 3: Method parameter employed during Diazepam quantitation.

Following parameters are evaluated for validation of abovementioned quantitation method.

• Linearity

• Accuracy and precision

• Limit of detection (LOD)

• Limit of quantitation (LOQ)

• Specificity / Interference

• Solution stability

Procedure for diazepam quantitation from pharmaceutical dosage form

Step-1 (Extraction):

Tablets: One tablet was randomly selected and grounded to fine powder. Powder was shifted in eppendorf centrifuge tubes with the addition of 2 ml Chloroform as extraction medium. Sample mixture was vortexed for 3 minutes. Then sample mixture was centrifuged @4500 rpm. The supernatant was pipetted out in separate glass tube. Each tablet was extracted four times with Chloroform. Supernatant was placed on hot plate for solvent evaporation to get fine diazepam powder.

Injections: One injection was taken and transferred into eppendorf tubes. Chloroform was added in it. After addition of chloroform tablet was vortexed for 3 minutes and then centrifuged @4500 rpm. Chloroform was pipetted out in separate glass tube. Each injection was extracted four times with Chloroform and chloroform was collected in same tube for one injection. After completion of extraction, glass tube with chloroform was placed on hot plate for evaporation of chloroform to get diazepam.

Step-2 (Quantitation): After drying color test is performed by adding 0.5 ml of conc. HCl followed by 0.5 ml of Cobalt thiocyanate solution. Vortex it thoroughly then 2 ml chloroform is added for extraction of color in chloroform for quantitation. Then 400 ul from each tube is taken separately and 2 ml volume was made up by adding 1600 ul of chloroform for making final dilution. The final dilution was checked for their absorbance ultraviolet-visible spectrophotometer at 364 nm. Controls were always run with each batch.

Results

An immediate green color was developed with abovementioned benzodiazepines on addition of cobalt thiocyanate reagent (Figure 2-5). When ammonium thiocyanate was tried instead of cobalt thiocyanate reagent, the green color didn’t appeared. Moreover oxytetracycline and diphenhydramine produced different colors (Figures 5-7). The observed green color in case of benzodiazepines or colors produced by other drugs were checked for their shiftibilty in organic solvents. It was found that green color was completely shiftable in chloroform and methylene chloride in case of diazepam while Green color developed by temazepam was only shiftable in chloroform (Table 4). Color produced by lorazepam and nitrazepam didn’t show any shiftibilty in organic solvents (Figure 4). Colors developed by Meloxicam, naproxin were completely shiftable in methylene chloride only while colors observed in case of bupvacaine and chlordiazepoxide were shiftable in chloroform (Table 4).

Table 4: List of different tested compounds and their reaction colour.

Figure 2: Reaction of diazepam with cobalt thiocyanate and its colour shiftibility in chloroform.

Figure 3: Reaction color of Temazepam with cobalt thiocyanate and its shiftibility in chloroform.

Figure 4: Reaction colour of A (Lorazepam) and B (Nitrazepam) with cobalt thiocyanate and their non-shiftible behavior in chloroform.

Figure 5: Oxytetracycline and diphenhydramine produced different colors.

Figure 6: Calibration curve of diazeam cobalt thiocyanate complex.

Figure 7: FTIR spectra of Color complex and Diazepam.

For study accuracy of diazepam quantitation method three different conc. 250 μg/ml, 300 μg/ml and 400 μg/ml were taken and absorbance value was noted for three days.

Following are the calculations done for accuracy and precision studies.

Limit of detection

The detection limit was determined by the analysis of samples with known concentrations of analyte via visual evaluation and by establishing the minimum level at which the analyte was reliably detected.

It was found that 8 μg/ml is LOD for detection of diazepam with this method (Tables 5-8).

Table 5: Benzodiazepine color and suitable solvent for its shifting.

Table 6: Calibration Curve for Diazepam Quantitation.

Table 7: Linearity Parameters.

Table 8: Calibration Curve for Diazepam Quantitation.

Limit of quantitation

25 μg/ml is LOQ for quantitation of diazepam by above mentioned method.

Stability study

Stability studies are done in two different ways.

Intraday stability study: In this study standard solution of 300 μg/ml is prepared and run on UV-Visible spectrophotometer and its Absorption is noted. This solution was then transferred to screw capped glass tube and its absorbance is noted after every 3 hour at 364 nm till 12 hours consecutively and following calculation are done to check stability.

Interday stability study: In this study standard solution of 300 μg/ml is prepared and run on UV-Visible spectrophotometer and its Absorption is noted. This solution was then transferred to screw capped glass tube and its absorbance is noted after each day for consecutive 5 days at 364 nm and following calculation are done to check stability (Tables 9-11).

Table 9: Accuracy & Precision Determination for Diazepam-cobalt thiocyanate complex Quantitation.

Table 10: Calculation for limit of detection.

Table 11: Calculation for determining LOQ.

Compounds like Stearic acid, Magnesium stearate, Starch, Lactose, Talc, silica, cellulose, mannitol, caffeine and gelatin was run individually on UV-VIS to check interference at wavelength 364 nm. No compound showed any interference at 364 nm (Tables 12 and 13).

Table 12A: Intraday stability study.

Table 12B: Intraday stability study.

Table 13A: Interday stability study.

Table 13B: Interday stability study.

Reaction of hydrochloric on benzodiazepines resulted in the formation of benzophenone [15] which immediately reacted with added cobalt thiocyanate, producing a green color because thiocyanate share negative charge approximately equally between sulfur and nitrogen. As a consequence act as nucleophiles at either sulfur or nitrogen which describes the mechanistic behavior evolving in this reaction. The complexes of cobalt thiocyanate formed in aqueous media were found to consist of two species-[Co(SCS)]+ and [Co(NCS)6]-4 with the number of possible water molecules in the coordination spheres undetermined. The complex [Co(NCS)]+ is present in aqueous solutions containing an excess of cobalt, while an excess of thiocyanate produces [Co( NCS) a]-2 [16,17]. Previous Studies have shown that many metal thiocyanates can be extracted from aqueous solutions by means of non-polar organic solvents. Absorptancy curves have been run on solutions of cobalt thiocyanate in non-aqueous solvents. Such extractions have been utilized extensively in the isolation of thiocyanate complexes of such metals like iron, uranium, and molybdenum. Moreover, shift of green color to organic solvent can be supported by the observation of Young and Hall [18] where blue color of thiocyante was soluble in ether. Their importance to the present discussion lies in the emphasis they add to the solubility phenomena attributable to the thiocyanate group effect.

FTIR analysis of formed Diazepam-cobalt thiocyanate complex was performed. C=N of diazepam showed a substantial shift from 1607.65 cm^-1 to 1628.43 cm^-1 which confirm the reaction occurred between diazepam and cobalt thiocyante and support proposed complexes structure. Whereas C=O group (1681 cm^-1), C-Cl Stretching (781), Ar. C – H Bending (740 cm^-1), Ar. C - C Stretching 1442.8 do not participate in this chemical reaction.

Probably one molecule of cobalt thiocyanate reacted with 2 molcules of diazepam forming dimer. FTIR complex also showed the appearance of band at 2061.71 cm^-1

Diazepam showed a significant absorption in ultraviolet region after the formation of complexes with cobalt thiocyanate reagent in acidic medium which were shiftable in the organic layer. During study, no interference was observed with pharmaceutical excipients and confirms the sensitivity and specificity of developed method.

Previously reported tests lack their propensity to produce a single convinced color for different benzodiazepines to imagine them as a definite class. An earlier method described using formaldehydesulfuric acid gives yellow colour with bromazepam while it gives orange color on reacting with diazepam, estazolam lorazepam, lormetazolam, nitrazepam. Similarly, method using alkaline DMSO produced different color for different benzodiazepines [10]. Another drawback of that test was its high sensitivity towards moisture. Zimmermann’s reaction was also applied for the identification of benzos but the test does not give clear indication due to double shade (reddish purple color) [12-14]. whereas our described method have a unique feature to produce a single color (green) and it was found much yonder towards such limitations as reagent was prepared using deionized water.

A new presumptive color test for the identification of eight benzodiazepines has been developed which were tested during study. This test is rapid, easy to perform, economical and has reasonable sensitivity towards benzodiazepines. It can be used for the quantitation of benzodiazepines like diazepam using UV-visible spectroscopy. Experimental data showed that detector response showed a linear behavior with increasing analyte concentration which resulted in reliable quantitation limits.