Gas Chromatographic Ethanol Determination in Bhunimbadi Kwath

JY Nehete, VV Shewale, VN Deshmukh and MR Narkhede.

Department of Pharmacognosy M.G.V’s Pharmacy College Panchavati, Nashik-422 003 M.S. (India)

*Corresponding Author E-mail: jynehete@yahoo.com

ABSTRACT

Ayurvedic formulations, has to check for self generated as well as added alcohol concentration within specified limits. In present study Gas chromatographic method developed for determination of Bhunimbadi Kwath self generated alcohol concentration. In polyherbal formulation alcohol percentage determined using calibration curve. Bhunimbadi Kwath alcohol concentration was found to be 5.04% v/v, which was within prescribed limit. The method has been applied successfully for the assay of ethanol in Bhunimbadi Kwath.

KEYWORDS: Polyherbal formulation, Gas chromatography, Bhunimbadi Kwath.

INTRODUCTION:

Ayurvedic preparation, Bhunimbadi Kwath, prepared by boiling coarse medicinal plants on slow flame so as the water remains ½, ¼ or 1/8th. It is then filtered, the liquid so obtained is "Kwath" i.e. decoction. Freshly prepared Kwath is generally difficult to preserve for longer duration. Hence, to have optimum use of Kwath, it undergoes the process of Arishta which generates alcohol by fermentation. This facilitates extraction of active principles contained in drugs.

Ethanol is often considered to be the most used and abused chemical substance. As a result, measurement of ethanol is one of the most frequently performed toxicological tests. Ethanol analysis is important for both clinical and forensic purposes. Several body fluids, such as venous blood, capillary blood, serum or plasma, urine, vitreous humor, tear fluid, cerebrospinal fluid (lumbar fluid), saliva, sweat, and breath, are suitable for determination of ethanol in living subjects¹. Methods for the measurement of ethanol range from nonspecific and semi quantitative techniques such as Gap Assessment method reported by Wu AHB.²Enzymatic Ethanol Method reported by Yost DA et al and Cary PL et al.^3,4Electrochemical/Infrared Detection reported by Currier GW et al⁵and Jones AW, Pounder DJ^6.Ethanol in toiletries, official drug preparations¹andwine⁷ determine by Gas chromatography. Recently, Wadher et al, reported GC and mass spectrometric methods for the determination of amount of alcohol in OTC products and methanol¹.

Ayurvedic formulations containing self generated alcohol are needed to be checked for their alcohol concentration as per label claim. For Bhunimbadi Kwath label claim is NMT 12 %.

Alcohol Concentration exceeding the prescribed limit in polyherbal formulation leads to alcohol abuse. In present research article gas chromatographic method for determination of alcohol in marketed ayurvedic formulation is discussed.

MATERIALS AND METHODS:

Drugs and Chemicals:

HPLC grade ethanol, GR grade NaOH and phenolphthalein.

Formulation:

Marketed ayurvedic formulation Bhunimbadi Kwath was procured from Ayurved Seva Sangh, Nashik.

Experimental:

Standard preparation:

A range of standard solutions of ethanol were prepared containing 2, 4, 6, 8, 10, 12% v/v of ethanol using ethanol (99.9%) and HPLC grade water.

Sample preparation:

For the assay, 25 ml formulation was taken in distillation flask, 5 ml 0.1 N NaOH solution, 10 mg phenolphthalein powder and 150 ml HPLC grade water were added to it. Distillate was collected after heating resulting mixture at 110^οC and diluted 1:10 with HPLC grade water.

Instrumental specifications:

Model- GC 8610(Chemito)

Detector- dual flame ionization

Stationary phase-2 mm (i. d.) steel column packed with carbowax 20

Carrier gas- Nitrogen,

Flow rate-1 kg/cm²/min.

Temperature- column 90⁰C injector and detector 110⁰C

Sample applicator -10μl Hamilton syringe

Gradual increase in temperature at rate of 3⁰C/min from 90⁰C to 110⁰C was done.

Chromatographic Analysis:

Ethanol calibration curve plotted in range (2-12% v/v). Sample solution (1μl) injected to record chromatogram. In polyherbal formulation alcohol concentration was calculated from calibration curve.

RESULT AND DISCUSSION:

For the quantitative determination of ethanol, optimum conditions with maximum selectivity were established by number of preliminary experiments. Optimum conditions to resolve peak were fixed by varying one parameter at a time keeping other parameters constant. Stationary phase carbowax 20 M was found to be ideal column for efficient separation of the component with good peak shape. The effects of nitrogen flow rate on chromatogram were recorded; flow rate of 1 kg/cm²/min was selected because of its ideal retention time, sharp peak and less time for analysis. The injector and detector temperature were maintained at 110⁰ through out the analysis for sharp peaks and ideal chromatographic behavior.

The retention time of ethanol was found to be 1.25. Calibration curve plotted was found to be linear over the concentration range of 2-12 % v/v. The data were analyzed by linear regression method. The calibration graph is described by the line equation y = m(x) + c. Linear regression equation y = 552247(x) and slope 113169, with correlation coefficient ‘r’ was found to be 0.997. From the line equation it was found that percentage of alcohol concentration in polyherbal formulation was 5.04 %. From the concentration of alcohol obtained in polyherbal formulation Bhunimbadi Kwath it was found that it satisfies the prescribed limit for alcohol in ayurvedic formulation. Thus above mentioned polyherbal formulation may not be abused for alcohol and safe for its claimed biological activity.

Analyzing five replicates of fixed amount of ethanol checked precision and accuracy of the proposed method. The precision of the method was calculated in terms of the relative standard deviation (1.28% ) indicated high precision and accuracy of the proposed method. In order to study selectivity of the method, the interference of commonly associated excipients in the determination of commonly associated excipients in the determination of ethanol was carried out. It was observed that none of the excipients interfered in the determination as evident from the similar retention time of ethanol.

ACKNOWLEDGEMENT:

We are thankful to Prof. S.S. Kale H.O.D., Pharmacognosy Department, and Prin. V. M. Aurangabadkar, M. G. V’s Pharmacy College, Panchavati, Nashik, for providing us facilities for this research.

REFERENCES:

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2) Wu AHB, Broussard LA, Hoffman RS. National academy of clinical biochemistry laboratory medicine practice guidelines: recommendation for the use of laboratory tests to support the impaired and overdosed patient from the emergency department. Clin Chem.2003; 49:357–379.

3) Yost DA, Boehnlein L, Schaffer M. A novel assay to determine ethanol in whole blood on the Abbott TDX. Clin Chem. 1984; 30:1029 A.

4) Cary PL, Whitter PD, Johnson CA. Abbott radiative energy attenuation method for quantifying ethanol evaluated and compared with gas-liquid chromatography and the Du Pont aca. Clin Chem.1984; 30:1867–1870.

5) Currier GW, Trenton AJ, Walsh PG. Innovations: emergency psychiatry: relative accuracy of breath and serum alcohol readings in the psychiatric emergency service. Psychiatr Serv. 2006; 57:34–36.

6) Jones AW, Pounder DJ. Measuring blood alcohol concentrations for clinical and forensic purposes. In: Drug Abuse Handbook. Karch SB, ed. Boca Raton, FL: CRC Press, 1998.

7) Stackler and Christensen. Quantitative Determination of Ethanol in Wine by Gas chromatography. Am. J. Enol. Vitic.1974; 25: 202-207.

Received on 25.05.2009 Modified on 21.07.2009

Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; Page 876-877