ethanol estimation from marketed cough syrup preparation using gc

ESTIMATION OF ETHANOL CONTENT IN MARKETED COUGH PREPARATION USING PACKED COULUMN IN GC : 

1 1 ESTIMATION OF ETHANOL CONTENT IN MARKETED COUGH PREPARATION USING PACKED COULUMN IN GC Author: Samir M. Shah M.Pharm Pharmaceutics

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2 Gas Chromatography is a separation method in which the components of a sample partition between two phases: one of these phases is a stationary bed with a large surface area, and the other is a gas which percolates through the stationary bed. The components of the sample (called solutes or analytes) separate from one another based on their relative vapor pressures and affinities for the stationary bed. Hydroalcoholic diluting agents are suitable for drugs soluble in either water or diluted alcohol. The most important in this group are the elixirs. These solutions contain approximately 25% alcohol. Alcohol content: up to 23% Use: A pleasantly flavored vehicle. The chief objection to its extensive use is the high alcohol content (about 22%), which at times may counteract the effect of other medicines. Introduction1,2

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3 Critical Step for GC Method Development3 Gather information from the literature for the Physiochemical properties of the analyte (From Literature, Internet, etc.) Determine solubility/miscibility profile Determine the B.P. Select chromatography method (Based on solubility study, Retention of the compounds, Carrier gas etc.) Perform Initial run for GC conditions Select simple or gradient mode Perform trials to select the optimum conditions for separation (By changing Solvent, Injection volume, Column, Flow Rate, Oven temperature, Detector temperature, Column temperature etc) Perform Linearity and Accuracy to challenge the method Define System Suitability Parameters Summarize Methodology Prepare Development Report Validate the Method

OBJECTIVE : 

4 4 OBJECTIVE Ethanol is generally used as a vehicle for many marketed cough syrup preparations which also contain codeine and these cough syrup are generally prescribed with paracetamol. The CNS-depressant effects opoid analgesics and ethanol may be additive. The combination of these agents may result in additive CNS-depression and impairment of judgement, thinking and psychomotor skills. In more severe cases, respiratory depression, hypotension, profound sedation and coma can occur. Death has been reported in overdosage. Therefore, ethanol should be used within certain limits in cough syrups. Hence, the aim of present work was to estimate the content of ethanol in marketed cough syrup preparation using packed column in GC

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5 LITERATURE REVIEW4,5 Pienta N., Jorgenson J., describe an experiment that teaches students liquid-liquid extraction and gas-liquid chromatography. This experiment shows that the combination of liquid-liquid extraction and gas chromatographic analysis of the resulting solutions quite fruitful. This method for the determination of ethanol in complex aqueous solutions is a simpler and more pragmatic one than many of those reported in the literature. Zilly M., Langmann P., developed a very sensitive and specific gas chromatographic method for ethanol determination in human urine samples. The non-invasive method was performed without any pretreatment and carried out on a Stabilwax capillary column, 30m × 0.53mm × 1.0µm film thickness. Helium was used as carrier gas with a constant inlet pressure of 27.72 kPa (0.277 bar) and a flame ionization detector (FID). Quantification was performed with the use of acetonitrile as an internal standard (IS). The calibration curve was linear throughout the concentration range from 0.5 to 500 mg/l. A clear chromatographic separation of ethanol from methanol, acetone, 1-propanol and 2-propanol was achieved.

EXPERIMENTAL WORK : 

6 6 EXPERIMENTAL WORK Extraction of ethanol from cough syrup using toluene. To extract ethanol from cough syrup toluene was selected as it is more miscible with ethanol. The cough syrup and toluene are immiscible, so both were added in volumetric flask and stoppered. This stoppered volumetric flask was kept in sunlight (oven can be used at 40 ºC) for 1 hour, which helps ethanol and toluene to evaporate in the atmosphere of volumetric flask as both are volatile. The stoppered volumetric flask was then kept in freeze for 15 min, for condensation of vapor. This process ends with ethanol being transferred from cough syrup to toluene. Toluene being lighter than cough syrup remains above it in volumetric flask. Toluene is decanted in another volumetric flask which has ethanol dissolved in it. This solution was injected for determination of ethanol content in GC using packed column.

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7 GC parameters for Ethanol-Toluene after method development. Retention Time (RT) of ethanol : 0.79 min Retention Time (RT) of toluene : 1.16 min Oven Temperature : 150 ºC Detector Temperature : 150 ºC Column Temperature : 150 ºC Flow rate of carrier gas (nitrogen) : 20 ml/min Range : 20 Attenuation : (-) 3 Establish linearity, accuracy, precision and specificity for ethanol-toluene in GC using packed column. Estimation of total amount of ethanol in one bottle of marketed preparation.

RESULTS & DISCUSSION : 

8 8 RESULTS & DISCUSSION Establish linearity, accuracy, precision and specificity for ethanol-toluene in GC using packed column. Linearity: As shown in figure 1. Accuracy: For establishing accuracy the RT and % area of test and standard were compared for 100 ppm solution. RT of ethanol for standard and test was found to be 0.7 min. The % area for standard was 0.30% and for test was 0.29%. The difference should be NMT 0.01%. The % area and RT of test matches with that of standard. Hence, the method was accurate. Fig 1. Linearity curve

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9 Precision: For establishing precision six 100 ppm 0.01μl injections of test were injected and %RSD was calculated. For GC with manual injections the %RSD should be less than 15%. The RSD obtained for ethanol is 10.63 %. Specificity: The RT of ethanol for standard was found to be same i.e. 0.7 min. No peak was found at 0.7 min in blank. Hence, the peak of ethanol is pure and no interference is observed. Fig 2. GC chromatogram for pure ethanol Fig 3. GC chromatogram for pure toluene

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10 Estimation of total amount of ethanol in one bottle of marketed preparation. The % area obtained for ethanol in the marketed preparation is used for obtaining ppm, by using % area of standard 0.01μl injection of 100ppm ethanol solution in toluene. The ppm obtained gives us the amount of ethanol in 1μl; hence we can obtain ethanol content in one bottle (50 ml) of cough syrup. Results of % area of all 3 brands of cough syrup are discussed below: Brand 1: 0.29 % (Brand having labeled 5% v/v ethanol.) Brand 2: 0.02 % Brand 3: 0.77 %

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11 Brand 1: As given in figure 4, the % area for Brand 1 is 0.29 %. And % area for 100 ppm standard solution of ethanol in toluene is 0.30 %. So, by cross multiplication it contains 96.67μl/ml (ppm). In 1 ml ---------------------- 96.67μl Therefore, in 50 ml ---------------------- 4833.33μl = 4.8 ml Hence, Brand 1 contains 4.8 ml ethanol in one marketed cough syrup bottle.. Fig 4. GC chromatogram for brand 1

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12 Brand 2: As given in figure 5, the % area for Brand 2 is 0.02 %. And % area for 100 ppm standard solution of ethanol in toluene is 0.30 %. So, by cross multiplication it contains 6.66μl/ml (ppm). In 1 ml ---------------------- 6.66μl Therefore, in 50 ml ---------------------- 333μl = 0.33ml Hence, Brand 2 contains 0.33 ml ethanol in one marketed cough syrup bottle. Fig 5. GC chromatogram for brand 2

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13 Brand 3: As given in figure 6, the % area for Brand 3 is 0.77 %. And % area for 100 ppm standard solution of ethanol in toluene is 0.30 %. So, by cross multiplication it contains 256.66μl/ml (ppm). In 1 ml ---------------------- 256.66μl Therefore, in 50 ml ---------------------- 12833μl = 12.83 ml Hence, Brand 3 contains 12.83 ml ethanol in one marketed cough syrup bottle. Fig 6. GC chromatogram for brand 3

SUMMARY & CONCLUSION : 

14 14 SUMMARY & CONCLUSION SUMMARY: Marketed cough syrup usually contains ethanol in varying amount. Only some of them label the ethanol content. So, in the present work ethanol content from marketed cough syrup was estimated. For that one marketed cough syrup with labeled ethanol content was selected, in order to know specificity of new extraction procedure which was not previously used. And two other marketed cough syrup with unknown ethanol content was also selected. The ethanol was extracted with toluene using new extraction procedure. Finally, the extracted solution was injected to find out the concentration of ethanol in one marketed bottle of cough syrup. % area was used to find out concentration.

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15 CONCLUSION: Method was developed in GC using packed column for determination of ethanol in toluene. This method was found to be simple, linear and accurate and can be used for routine analysis of ethanol in marketed cough syrup preparation. The ethanol content found in all 3 marketed cough syrup preparations. Brand 1 and Brand 2 cough syrups had ethanol within specified limits. Brand 3 had 24% ethanol which is outside the limit.

KEY REFERENCES : 

16 16 KEY REFERENCES McNAIR M., Miller M.; Basic Gas Chromatography; 1st edition; John wiley & sons; 1998; 3-5, 9, 12, 112. Lippincott Williams & Wilkins; Remington, the science and practice of pharmacy; Wolter kluwer health (India) Pvt. Ltd., New Delhi; 21st edition; Vol 1; 2005; 1070-1071. http://www.chem.agilent.com/en-US/Support/FAQs/GC/Applications/ColumnTroubleshooting/Pages/default.aspx, Accessed on 28/03/10. Pienta N., Jorgenson J., et al; Extraction and Gas Chromatographic Determination of Ethanol in Beverages; The chemical educator; Vol 1; 1996; 1-12. Zilly M., Langmann P., et al; Highly sensitive gas chromatographic determination of ethanol in human urine samples; Journal of chromatography B; Vol 798; 2003; 179-186.

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