GAS CHROMATOGRAPHY USING AN INTERNAL STANDARD FOR THE ESTIMATION OF ETHER AND HALOTHANE LEVELS IN BLOOD

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Brit. J. Anaesth. (966), 8, 9 GAS CHROMATOGRAPHY USING AN INTERNAL STANDARD FOR THE ESTIMATION OF ETHER AND HALOTHANE LEVELS IN BLOOD BY BERNARD WOLFSON, HAROLD E. CICCARELLI AND EPHRAIM S.

1 Brit. J. Anaesth. (966), 8, 9 GAS CHROMATOGRAPHY USING AN INTERNAL STANDARD FOR THE ESTIMATION OF ETHER AND HALOTHANE LEVELS IN BLOOD BY BERNARD WOLFSON, HAROLD E. CICCARELLI AND EPHRAIM S. SIKER Department of Anesthesiology, Mercy Hospital, Pittsburgh, Pennsylvania, U.S.A. SUMMARY A method has been described for the estimation of blood levels of ether and halothane by gas chromatography using carbon tetrachloride as a solvent and chloroform as an internal standard. Reference has been made to the previous description of a similar method for the estimation of blood methoxyflurane levels and some technical improvements have been suggested. Extraction of both agents from blood was virtually complete and results were highly reproducible. Gas chromatography holds an established place in chemical analysis and numerous chromatographic techniques are available for the measurement of blood levels of anaesthetic agents. The authors recently described an extraction technique for the measurement of methoxyflurane blood levels incorporating the use of an internal standard (Wolfson, Ciccarelli and Siker, 966). The latter is a substance introduced in identical amounts into both the extracting and the calibrating or s. When a sample is injected into the chroma tograph, peaks are obtained from the agent to be analyzed and from the internal standard and measurements made of the ratio of their heights. Comparison between the ratios obtained from the unknown solution and the yield the concentration of the agent. The main advantage of the technique is that it obviates the necessity for exact knowledge of the size of the sample injected into the chromatograph. (For a more detailed description of the internal standard technique readers are referred to the previous paper.) A similar technique has now been tested for the analysis of ether and halothane. ETHER An extraction technique using carbon tetrachloride as the solvent has been described previously (Baker and King, 96). In the present technique carbon tetrachloride was used as the solvent and chloroform as the internal standard. The extracting solution was made by adding 00 microlitres of chloroform to ml of carbon tetrachloride. (The chloroform need not be measured accurately.) To prepare a solution of standard concentration, a calibrated -ml flask was filled to the calibration mark with extracting solution and then closed with a right-fitting rubber stopper. The flask was then weighed on an accurate electronic balance; microlitres of ether were injected through the rubber cap from a -microlitre syringe and the flask reweighed. Where there was a discrepancy between the actual weight gain and that calculated from volume and specific gravity the actual weight gain was accepted as being more accurate. After thorough mixing, ml of this solution was diluted to ml with the original extracting solution in another calibrated flask and the flask closed tightly with a glass stopper. This dilution technique allowed relatively large, accurately measurable quantities to be used, without undue wastage of tie extracting solution. Further, by varying the dilution factor, calibrating solutions of almost any desired concentration can be produced.* Figure shows a chromatogram from a standard solution containing 9 mg ether per cent (approximately (J per cent). (At least three injections were always made and the average of the three peak height ratios used for purposes of calculation.) * If x rng ether are added to ml of solvent and ml of this mixture is subsequently diluted to y ml, then the final concentration equals (x)/y mg ether per cent.

2 9 BRITISH JOURNAL OF ANAESTHESIA A ETHER A ETHER n A B. B - B -OS O.9I 0.9 B AVERAGE RATIO OF PEAK HEIGHTS I.O FIG. of approximately 0. /4 of standard ether solution (9 mg ether per cent in carbon tetrachloride with chloroform as an internal standard). To extract the ether from blood, equal quantities (I to ml) of extracting solution and blood were mixed on a mechanical rocker and then centrifuged.* Centrifuging was continued until adequate separation of the cellular debris from the clear ether-containing layer was obtained (approximately minutes). As with the AVERAGE RATIO OF PEAK HEIGHTS 0.9 FIG. of approximately 0. ^ of an extraction of a prepared blood ether solution (40 /4=8.4 mg per cent). Figure shows a chromatogram obtained from the injection of approximately 0. JJ. of an extraction of a prepared blood solution containing 8.4 mg ether per cent (40 pj per cent). Comparison of the ratios of peak heights from the standard solution with the ratio of peak heights of the extract according to the formula: ether = Ratio obtained from sample Ratio obtained from at least three injections from this layer were made for each estimation and the average of the three ratios was used. Where low quantities of agent are anticipated, ml of blood may be mixed with ml of extracting solution and vice versa for high quantities. The calculated value is then divided or multiplied by two. This technique prevents too great a difference between the peak heights of the agent and the internal standard, and improves the accuracy. shows the blood to contain (0.9/.0) x 9 = 7.4 mg ether per cent. This represents a recovery rate of 96. per cent. Table I shows the degree of extraction of ether obtained from a series of s of ether in blood. It can be seen that deviations from per cent are well within the range of experimental error. This is higher than the 9 per cent extraction rate reported by Baker and

3 ESTIMATION OF ETHER AND HALOTHANE LEVELS IN BLOOD 9 TABLE I Percentage recovery of ether from s of ether in blood. ml) Sample number Recovery rate 97 smaller quantities of halothane anticipated in the blood samples to be analyzed, the extracting solution contained only 6 j of chloroform and die calibrating solution only jj (9. mg) of halothane per cent. Preparation and storage of solutions, extraction of halothane from blood samples and mediod of calculation of results were the same as for ether. A HALOTHANE TABLE II Effect of duration of mixing on degree of recovery of ether from s of ether in blood. C" Percentage recovery Duration of mixing (min) 0 0 Blood A (^ per cent) 4 4 Blood B ( /d per cent) King (96). The only major difference in the technique reported here was the use of a hydrogen flame detector as opposed to the thermal conductivity detector used by Baker and King, and it may be that the greater sensitivity of the former type of detector is responsible for the apparent increase in extraction rate. Table II shows the effect of duration of mixing on degree of extraction. From these figures it appears that minutes is sufficient time for complete extraction to occur. HALOTHANE An extraction technique for halothane using heptane as the solvent has been reported (Buder and Freeman, 96; Rudedge et al., 96). A disadvantage of this technique was the interval necessary between injections because of the length of time taken for the heptane to clear the column. A technique similar to that described above for ether using carbon tetrachloride as a solvent was tried and proved satisfactory with a much smaller delay between injections. Because of the B' ST B" AVERAGE RATIO OF PEAK HEIGHTS -.4 FIG. of approximately 0. /A. of standard halothane solution (9. mg halothane per crnt in carbon tetrachloride with chloroform as an internal standard). Figures and 4 show a chromatogram from a containing 9. mg ( \>X) halothane per cent and an extract from a prepared blood sample also containing 9. mg halothane per cent. A similar calculation to that used for ether shows the blood to contain (./.4) x 9.=9. mg halothane per cent. This represents a recovery rate of. per cent. It can be seen from the chromatograms that succeeding peaks do not always take off from, or return to, the original baseline. Where this occurs, a new line is drawn through the bases of

4 94 BRITISH JOURNAL OF ANAESTHESIA A HALOTHANE occurred from day to day, and indeed within the same day, with both ether and halothane; some of these variations are seen in table V. This emphasizes the need for the frequent use of a during a series of analyses. AVERAGE RATIO OF PEAK HEI0HT6 I 88 FIG. 4 of approximately 0. /< of an extraction of a prepared blood halothane solution ( ^=9. mg per cent). the peaks to be measured. It will often be found that this new baseline forms part of a previous peak. This may be seen in figure, where the second ether peak began before the first carbon tetrachloride peak had returned to the baseline. A rising baseline produces the effect seen with the last set of peaks in figure 4. The reason for this rise may not always be apparent but it sometimes represents a recovery phase from a previous negative deflection. Peak height is the distance, measured along a line perpendicular to the original baseline, from the apex of the peak to the new baseline (see figure 4). Table III shows the amount of halothane recovered from a series of prepared blood samples and it can be seen that this again is per cent for all practical purposes. Comparable figures using heptane are "in excess of 9 per cent" (Butler and Freeman, 96) and "97.9 ±.9 per cent" (Rutledge et al., 96). Table IV shows the effect of duration of mixing on degree of extraction and it would appear that minutes is sufficient time for complete extraction. As with methoxyflurane, variations in the ratio of the peak heights of the same s TABLE Percentage recovery of halothane from s of halothane in blood (ji\ per cent) Sample number Recovery rate (%) TABLE Effect of duration of mixing on degree of recovery of halothane from s of halothane in blood. Duration of mixing (min) 0 III IV Blood A (/il per cent) Percentage recovery Blood B ( i>\ per cent) TABLE V Daily variations in the ratio of peak heights of standard solutions of ether and halothane (solutions prepared on first day). Day Standard ether solution* Ratio of peak heights Standard halothane solution! * 9 mg ether per cent in carbon tetrachloride with chloroform as an internal standard. t 9. mg halothane per cent in carbon tetrachloride with chloroform as an internal standard.

ESTIMATION OF ETHER AND HALOTHANE LEVELS IN BLOOD 9 It should be noted that carbon tetrachloride is extremely volatile and should be kept in tightlystoppered glass bottles or tubes and exposed to the

5 ESTIMATION OF ETHER AND HALOTHANE LEVELS IN BLOOD 9 It should be noted that carbon tetrachloride is extremely volatile and should be kept in tightlystoppered glass bottles or tubes and exposed to the air for as short a time as possible during any mixing procedure. When many estimations are being made, necessitating frequent exposures of the extracting and calibrating solutions to the air, it has become the practice in this laboratory to make fresh solutions at least once a week. This applies not only to ether and halothane but also to methoxyflurane. CHROMATOGRAPHIC DETAILS The apparatus used was the "Hy-Fi" Aerograph Chromatograph supplied by the Wilkins Instrument and Research Company of Walnut Creek, California. The column used was feet x i inch stainless steel packed with per cent SE.0 on 60/80 chromosorb W. Nitrogen with a flow rate of ml/min was the carrier gas. The detector was of the hydrogen flame ionization type and the flow rate of hydrogen was 8 ml/min. The temperature was set at C for both halothane and ether. Range and attenuation was used for halothane and range and attenuation 4 for ether. The size of injection was usually about jj for halothane and fj for ether. REFERENCES Baker, J. M., and King, B. D. (96). Rapid blood ether determinations using gas chromatography. Anesthesiology, 4,. Butler, R. A., and Freeman, J. (96). Gas chromatography as a method for estimating concentrations of volatile anaesthetics in blood. Brit. J. Anaesth., 4, 440. Rutledge, C. O., Seifen, E., Alper, M. H., and Flacke, W. (96). Analysis of halothane in gas and blood by gas chromatography. Anesthesiology, 4, 86. Wolfson, B., Ciccarelli, H. E., and Siker, E. S. (966). Gas chromatography using an internal standard for the estimation of methoxyflurane levels in blood, Brit. J. Anaesth., 8, 9. CHROMATOGRAPHIE GAZEUSE AU MOYEN D'UN STANDARD INTERNE POUR LA DETERMINATION DES TAUX D'ETHER ET D'HALOTHANE DANS LE SANG SOMMAIRE Description d'une mithode pour la determination des taux sanguins d'6ther et d'halothane par chromatographie gazeuse au moyen du t trachlorure de carbone comme solvant et du chloroform comme standard interne. Un rappel a 6t6 fait sur la description pricidente d'une mithode similaire pour la determination des taux sanguins du m6thoxyflurane et quelques ameliorations techniques ont ete suggerees. L'extraction des deux produits du sang a iti virtuellement complete et les resultats 6taient hautement reproduisibles. GAS-CHROMATOGRAPHIE MIT VERWEND- UNG EINES INTERNEN STANDARDWERTES FOR DIE BESTIMMUNG VON ATHER- UND HALOTHANKONZENTRATIONEN IM BLOT ZUSAMMENFASSUNG Es wird eins Methode fur die Bestimmung von Atherund Halothankonzentrationen im Blut beschrieben, wobei fiir die Gas-Chromatographie Tetrachlorkohlenstoff als Losungsmittel und Chloroform als internet Standard verwendet wird. Auf eine friihere Beschreibung einer ahnlichen Methode fur die Bestimmung von Methoxyflurankonzentrationen im Blut wird hingewiesen, und es werden einige technische Verbesserungen vorgeschlagen. Beide Substanzen werden praktisch vollstandig aus dem Blut extrahiert, und die Ergebnisse sind im hohen Mafie reproduzierbar.

CORESTA RECOMMENDED METHOD N 8

CORESTA RECOMMENDED METHOD N 8 DETERMINATION OF WATER IN THE MAINSTREAM SMOKE OF CIGARETTES BY GAS CHROMATOGRAPHIC ANALYSIS (August 1991) 1. FIELD OF APPLICATION The method is applicable to the particulate