Quantitation of Ethchlorvynol by Gas Chromatography

A specific and convenient method is described for the detection and quantitatlon of ethchlorvynol (Placidyl) by gas chromatography using undecane as the Internal standard. Extraction of a blood sample (containing a mass ratio ethchlorvynol to undecane of 2.5 to 1.0) on 6 consecutive days gave an average mass ratio of 2.62 (SD, 0.16; CV, 6.0%) which was considered adequate day-to-day precision for roufine drug analysis. Introduction Ethchlorvynol (l-chloro-3-ethyl-l-penten-4-yn-3-ol; Placidyl) is a short-term hypnotic for the management of insomnia. Present methods for quantitation of ethchlorvynol have proven in one respect or another to be less than ideal. The spectrophotometric method of Wallace, et al. (1) has been found by this author and others (2) to give falsely high values due to the inclusion of metabolites in the final results, particularly in urine. A revised method by Wallace, et al. (3) reports no interference from metabolites. Another commonly used method is the colorimetric procedure of Frings and Cohen (4) which involves the reaction of ethchlorvynol with diphenylamine in an acidic medium. Inherent to all colorimetric methods, these procedures tend to be nonspecific. Published gas chromatographic methods to date which use internal standards are often lacking in other respects; for example. Evenson and Poquette (5) use the solid support Chromosorb 101 for ethchlorvynol which obviously limits the number of other drugs that can be used with this type of column. Gibson and Wright (2) uses chlorobutanol (1, 1, 1trichloro-2-methyl-2-propanol) as the internal standard which has the disadvantage of having a retention time before ethchlorvynol and thus very low column temperatures are required for the more common GC columns. Chlorobutanol has the advantage of being detected by an. electron-capture detector along with ethchlorvynol, however the dosages of ethchlorvynol are usually so large (up to 1 gram per day) that the high sensitivity of the electron-capture detector would not be necessary and the low linearity range of most electron-capture detectors would make it less convenient than with the flame-ionization detector. Thus I wish to describe a gas chromatographic method utilizing a common column packing material, OV-1, and undecane as the internal standard having a relative retention time of 1.8 compared to ethchlorvynol. Materials and Methods Chemicals and reagents. Ethchlorvynol was obtained from Abbott Laboratories (North Chicago, Illinois 60064). Undecane (highest purity) was obtained from Fisher Scientific. The chloroform was spectrograde qualify from MCB. Phosphate buffer (0.067 M) was prepared by dissolving 17.9g potassium phosphate heptahydrate, dibasic, in 1 liter of water. Ethchlorvynol reference solution was prepared by weighing to 3 decimal places 3 drops of ethchlorvynol into a 50-ml volumetric flask. Ethchlorvynol has a relatively short shelf-life (i.e. less than 1 year). If deterioration of the pure drug is visibly evident to any degree, it should not be used. Refrigerated, dilute solutions in ethanol are stable for at least 6 months. Losses in weighing of the liquid have been shown by others ($) to be less than S0 #g in 30 min. The flask is brought to volume with ethanol to give a typical value of 1.144 mg/ml. Likewise one drop of undecane is accurately weighed and dissolved in ethanol (in this experiment 0.35 mg/ml). From the ethclflorvynol stock solution dilute 1.0, 3.0, and 5.0 ml with drug-free whole blood to 100 ml to give the appropriate serial dilutions of blood standards for quantitation. Procedure. A 2-ml aliquot of specimen (blood, urine, gastric contents, etc.) is added to a 125-ml separatory funnel. To three other appropriately labled separatory funnels add 2.0 ml of the blood standards. To each of the separatory funnels add 100 1 of the internal standard stock solution. Add approximately 40 ml of chlorform (volume is not critical) to each of the separatory funnels and shake the mixture for approximately 3 min. Transfer the chloroform layer into another 12S-ml separatory funnel and wash vigorously with 10-ml of 0.067 M phosphate buffer for 30 sec. Collect the chloroform layer into a S0-ml beaker and evaporate on a steam bath to about 2-3 ml. At this point remove the beaker from the steam bath and at room temperature, under a gentle stream of nitrogen, evaporate the remaining chloroform to about 50/tl . Important: Do not allow the chloroform to go to dryness on a s team bath, etc. E thchlorvynol and undecane are extremely volatile. Inject 1-2~ into the gas chromatograph. Instrumentat ion. A Tracor Model 222 gas chromatograph equipped with a dual flame ionization detector, a 1 MV recorder (Linear Instruments Corp.), a U-shaped column, 6' x 4mm i.d. containing 3% OV-1 on high performance Gas-Chrom W, 80/100 mesh. Nitrogen flow rate was 35 ml/min and instrument temperatures were: column, 100~ detector, 260~ and injection port, 290~ The operating conditions were such that the retention time of ethchlorvynol and undecane were 1.9 min and 3.4 respectively. Results and Discussion Figure 1 is the results obtained when three blood ethchlorvynol standards at concentrations of 12.2, 36.6 and 61 p-g/ml are extracted. The curve was drawn by the method of least squares and t.he correlation coefficient was greater than 0.99. Each point is the average of at least 3 determinations on 3 separate days. Ethchlorvynol is linear up to at least 400Fg/ml which is considerably above the maximum lethal levels usually encountered in blood. Recovery: The slope of the standard curve was compared to the slope of unextracted ethchlorvynol/undecane standards prepared in chloroform. The recovery averaged 63% for all concentrations considered when the blood and/or aqueous standard curve is compared to the "unextracted" standard curve. Evanson and Poquette (5) report 87% recovery from serum and 67% recovery from aqueous solutions. In this study, no difference in recoveries was found between blood and aqueous standards. 164*JULYI AUGUST 1977 JOURNAL OF ANALYTICAL TOXICOLOGY*VOL. 1