SIMULTANEOUS ESTIMATION OF TURPENTINE OIL, CHLORBUTOL, PARA-DICHLOROBENZENE AND BENZOCAINE BY CAPILLARY GAS CHROMATOGRAPHY

Headnote

ABSTRACT

This paper describes a new, simple, precise and accurate GC method for simultaneous estimation of Turpentine oil, Chlorbutol, Para- dichlorobenzene and Benzocaine in formulation with flame ionization detection (GC-FID). A baseline separation of active drugs and internal standard was achieved in 12min. using BP1 (100% Dimethyl polysiloxane) capillary column. During analysis the oven temperature was programmed. Validation was carried out in compliance with the International Conference on Harmonization guidelines. The proposed method was validated for linearity, LOD, LOQ, accuracy, precision. It can be conveniently adopted for routine quality control analysis.

Keywords: Capillary Gas Chromatography; Turpentine oil; Chlorbutol; Para- dichlorobenzene; Benzocaine.

INTRODUCTION

Turpentine Oil is obtained by distillation from the oleoresin from various species of Pinus and rectified.1 Turpentine is composed primarily of monoterpene hydrocarbons, the most prevalent of which are the pinenes, camphene, and 3-carene. It shows the antibacterial activity.2 Chlorbutol is 1, 1, 1-trichloro-2- methylpropan-2-ol.hemihydrate having analgesic as well as local anaesthetic activity.3 Para-dichlorobenzene is 1, 4- dichlorobenzene and can be used as insecticidal fumigant.4 Benzocaine is ethyl p-aminobenzoate used as local anesthetic.5 The structures of Chlorbutol, Paradichlorobenzene, Benzocaine shown in Figure 1a), 1b) and 1c) respectively. Literature survey revealed that even though there is method for the determination of these compounds by gas chromatography it is vague.6 Therefore a GC method was developed for determination of Turpentine oil, Chlorbutol, Para- dichlorobenzene and Benzocaine from their dosage form.

MATERIALS AND METHODS

Chemicals and Reagents

Soliwax ear drop manufactured by NuLife Pharmaceuticals, pune, India was procured from the market. Soliwax ear drop is a combination of Turpentine oil (15%w/v), Chlorbutol (5%w/v), Para-dichlorobenzene (2%w/v) and Benzocaine (2.7%w/v). Methanol was procured from RUNA Chemicals Pvt. Ltd. All dilutions were performed in standard volumetric flasks.

Instrumentation

The analysis was performed by using the analytical balance Shimadzu, the GC used is of Thermo Scientific Trace GC 800 system with FID detector. Column used in GC is a capillary column SGE forte BP-1, 30m × 0.32mm, 0.25µm.

Method Development

A suitable GC method has been developed for the analysis of Turpentine oil, Chlorbutol, Para- dichlorobenzene and Benzocaine in their dosage form different columns were tried. Chromatographic separation was performed on Thermo Scientific Trace GC 800 system with Flame Ionization Detector. Chromatograms and data were recorded by means of Iris 32 software. BP-1 (100% Dimethyl polysiloxane) capillary column showed satisfied results for analysis. The system was run at a flow rate of 2 mL/min, 0.2 µL of sample was injected in the chromatographic system. Nitrogen was used as a carrier gas. Oven temperature was kept at 80°C for 5 min. then increased at a rate of 20°C/min. to 200°C and held at 200°C. Injector temperature and detector temperature were kept at 250°C. The split ratio was kept at 1:10.

Preparation of Standard Stock Solutions: Standard stock solution of concentration 10000 µg/mL of Turpentine oil, 3000 µg/mL of Chlorbutol, 1000 µg/mL of Para- dichlorobenzene and 2000 µg/mL of Benzocaine was prepared using 0.3% camphor (prepared in methanol used for internal standard). From the standard stock solution, the mixed standard solutions were prepared using methanol to contain 6480 µg/mL of Turpentine oil, 2160 µg/mL of Chlorbutol, 864 µg/mL of Para- dichlorobenzene and 1164 µg/mL of Benzocaine.

Sample Preparation: For analysis of the ear drop formulation accurately weighed 1 gm sample in 25 mL 0.3% camphor solution, close and clamp the volumetric flask with the help of stopper and shake the flask with wrist shaker for 45 min. filter the contents through anhydrous sodium sulphate using Whatmann filter paper No.1, From this sample 0.2 µL was injected and analyzed by GC-FID for the concentrations of Turpentine oil, Chlorbutol, Para- dichlorobenzene and Benzocaine.

Method Validation

The method validation was carried out as per ICH guidelines.7 Various method validation parameters were performed.

System Suitability Test: System suitability tests are used to verify that the reproducibility of the equipment is adequate for the analysis to be carried out. System suitability tests were performed as per the USP 31 to confirm the suitability and reproducibility of the system. Various parameters such as tailing factor and resolution between the peaks were obtained.

LOD and LOQ: Sensitivity was determined by establishing the limit of detection (LOD) and limit of quantification (LOQ) at signal-to-noise ratio of 3:1 and 10:1 respectively.

Linearity: Linearity was evaluated by analysis of working standard solutions of Turpentine oil, Chlorbutol, Para- dichlorobenzene and Benzocaine of six different concentrations. The range of linearity was from 40 - 90 µg/mL for Turpentine oil, 10 - 15 µg/mL for Chlorbutol, 5 - 10 µg/mL for Para- dichlorobenzene and 6 - 11 µg/mL for Benzocaine. The peak area ratio and concentration of each drug was subjected to regression analysis to calculate the calibration equations and correlation coefficients.

Specificity: The specificity of the method towards the drug was established through study of resolution factor of the drug peak from the nearest resolving peak. The peak purity of Turpentine oil, Chlorbutol, Para- dichlorobenzene and Benzocaine were determined by comparing the spectrum at three different regions of the spot i.e. peak start (S), peak apex (M) and peak end (E). Effect of excipients of formulation was studied for whether it interfered with the assay.

Precision: The precision of the method was verified by repeatability and intermediate precision studies. Repeatability studies were performed by analysis of three different concentrations 40, 50, 60 µg/mL for Turpentine oil, Chlorbutol, Para- dichlorobenzene and Benzocaine six times on the same day. Repeating studies on three different days checked the intermediate precision of the method.

Robustness: To evaluate robustness of a GC method, few parameters were deliberately varied. To study the effect of flow rate on system suitability parameters, ± 0.2 units changed. The effect of column temperature was studied at ± 5°C. The injector temperature and detector temperature were kept constant.

Accuracy: Accuracy was determined over the range 80, 100 and 120% of the sample concentration. Calculated amount of Turpentine oil, Chlorbutol, Para-dichlorobenzene and Benzocaine from standard stock solution was added in placebo to attain 80%, 100% and 120% of sample concentration. Each sample was prepared in triplicate at each level.

Stability of Solution: The solution stability of Turpentine oil, Chlorbutol, Para-dichlorobenzene and Benzocaine was carried out by leaving the test solutions of sample in a tightly capped volumetric flask at room temperature for 72 hours. The same sample solutions were assayed for 24 hours interval up to the study period against freshly prepared standard solution.

RESULTS AND DISCUSSION

System Suitability Test

The % RSD values were found to be satisfactory (RSD less than 2.0%). A typical GC chromatogram for simultaneous determination of Turpentine oil, Chlorbutol, Para-dichlorobenzene and Benzocaine from pharmaceutical formulation is shown in Figure 2 and Figure 3. System suitability parameters are mentioned in Table 1.

LOD and LOQ

The LOD and LOQ of Turpentine oil, Chlorbutol, Para-dichlorobenzene and Benzocaine were experimentally determined by six injections of each drug. The LOD of Turpentine oil Chlorbutol, Para-dichlorobenzene and Benzocaine were found to be 20, 2, 1 & 0.05 µg/mL respectively. The LOQ of Turpentine oil, Chlorbutol, Para-dichlorobenzene and Benzocaine were found to be 30, 8, 3 & 0.1 µg/mL respectively.

Linearity

Linearity was evaluated by analysis of working standard solutions of Turpentine oil, Chlorbutol, Para-dichlorobenzene and Benzocaine of six different concentrations. Figure 4, 5, 6 & 7 represents the linearity plots of Turpentine oil, Chlorbutol, Para-dichlorobenzene and Benzocaine respectively. The regression data obtained for the same constituent is represented in Table 2. The range of linearity was from 125 µg/mL to 375 µg/mL for Turpentine oil, 250 µg/mL to 750 µg/mL for Chlorbutol, 125 µg/mL to 375 µg/mL for Para-dichlorobenzene and 250 µg/mL to 750 µg/mL for Benzocaine. The peak area ratio and concentration of each drug was subjected to regression analysis to calculate the calibration equations and correlation coefficients. The result shows that within the concentration range mentioned above, there was an excellent correlation between peak area ratio and concentration.

Specificity

No peak was observed at the retention time of Turpentine oil, Chlorbutol, Para-dichlorobenzene, Benzocaine and camphor in diluents and Placebo chromatogram. Hence the method was specific.

Precision

The repeatability of the retention time was studied by repeatedly injecting mixtures containing 40, 50 and 60 µg/mL of each standard (n = 6). The relative standard deviation (RSD) for the retention time ranged from 0.779% to 1.766%. The reproducibility over different days was carried out by injecting the same standard solution (40, 50 and 60 µg/mL) over 3 days. The RSD values obtained for all standards were below 2.0%, indicating that the retention times were highly reproducible. The results of the repeatability and intermediate precision experiments are shown in Table 3.

Robustness

To evaluate the robustness of the developed GC-FID method, small deliberate variations in the optimized method parameters were done. The effect of change in flow rate and column temperature was studied. The method was found to be unaffected and therefore this method is robust shown in Table 4.

Accuracy

Accuracy was expressed as the percentage of analytes recovered by the assay. Table 5 lists the recoveries of the drug from a series of spiked concentrations. The results indicate that the method is highly accurate for simultaneous determination of Turpentine oil, Chlorbutol, Para-dichlorobenzene and Benzocaine.

Stability of solution

The % assay of Turpentine oil, Chlorbutol, Para-dichlorobenzene and Benzocaine were checked in the test solutions. The % RSD of assay of these drugs during solution stability experiment was within 1.0. No significant changes were observed in the content of Turpentine oil, Chlorbutol, Para-dichlorobenzene and Benzocaine during solution stability experiment. Sample solutions used during the experiment were stable up to the study period of 72 hours. The results are reported in Table 6.

CONCLUSION

The method after being completely validated showed satisfactory data for all the method validation parameters. Method validation study showed that the method is specific, linear, accurate, and easily reproducible; can be used for simultaneous determination of Turpentine oil, Chlorbutol, Para-dichlorobenzene and Benzocaine from pharmaceutical preparations. The method seems to be suitable for quality control in the pharmaceutical industry because of its sensitivity, simplicity and selectivity.

References

REFERENCES

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4. Merck Index No. 3057, 14th edition, published by Merck Research Laboratories Division of MERCK & CO. INC. White house station, NJ, USA2006. 518.

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