EVALUATION OF ALCOLMETER EBA; RESULTS FROM IN-VITRO AND IN-VIVO EXPERIMENTS.A.W. Jones and K.. JossonDepartment of Alcohol Toxicology, National Laboratory of Forensic Chemistry and Departments of Clinical Pharmacology and Internal Medicine, University Hospital, Linkping, Sweden.Summary. We evaluated the precision and accuracy of Alcolmeter eba, a quantitative evidential breath-alcohol analyzer. The Alcolmeter device was calibrated to read directly in terms of BAC with a 2300:1 blood/breath ratio. The Alcolmeter response was linearly related to the concentration of alcohol in air-vapor standards generated in-vitro from 0 to 220 mg/dl BAC equivalent (r —0.99). The SD increased with the concentration of alcohol and the average coefficient of variation (CV) was 1.2%. The recovery of alcohol ranged from 97- 104% of target concentrations. The precision (CV) of breath-alcohol analysis in-vivo was 2.4%. Venous BAC was on average 6% higher than Alcolmeter response and the 95% confidence limits ranged from 12% low to 27% high.IntroductionThis paper reports experiments to test the precision and accuracy of Alcolmeter eba, a microprocessor controlled evidential breath-alcohol analyzer. The alcohol contained in a specimen of breath is oxidized with an electrochemical (fuel cell) sensor. This way of analyzing alcohol offers inherent selectivity of response because neither ketones, such as acetone, nor volatile hydrocarbons, such as toluene, are oxidized at the detector. These volatile agents are potential interfering substances with some currently available breath-test instruments.Our testing protocol comprised two main phases: (1) Experiments in-vitro to establish precision and accuracy of Alcolmeter response when analyzing air-alcohol-vapor standards generated from a breath simulator device. (2) Experiments in-vivo with healthy volunteers given known amounts of alcohol under controlled laboratory conditions. Venous whole blood and end-expired breath were obtained for analysis during the absorption, distribution and elimination stages of ethanol in the body.Materials and MethodsThe Alcolmeter instrument evaluated in this work was made available to us by Lion Laboratories Ltd, Barry, Wales, UK. The instrument is controlled by a microprocessor. A typewriter keyboard is available for entering data and options exist for computer down-loading of results. Alcolmeter eba is equipped with a dry-alcohol-in-gas standard (Nalco) within the unit. In the present work the Nalco gas contained a concentration of ethanol equivalent to breath from a person with a BAC of 80 mg/dl. The following sequence of tests are made during normal use of the instrument; air-blank, calibration with Nalco alcohol standard, air-blank, first subject test, air-blank, second subject test, air- blank, and finally a repeat test with the Nalco standard.In-vitro experimentsIn-vitro tests were made with an alcoholic breath simulator device maintained at 34 °C equilibrium (Dubowski 1979). The simulator was charged with aqueous

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solutions of ethanol at the following target concentrations; 50 mg/dl, 100 mg/dl, 150 mg/dl, 200 mg/dl and 250 mg/dl. The exact concentrations of these standards were checked by gas chromatography against those available from Merck Ltd., Damstadt, FRG. The simulator charge was prepared from absolute ethanol by first weighing an exact quantity to make a 10% w/v stock solution and then volumetric dilutions from this to the desired concentration. These solutions where equilibrated with air when the operator blew into the simulator for about30 seconds to produce a continuous stream of air-alcohol vapor. This was passed directly into the Alcolmeter eba through the breath-inlet tube and analyzed. With liquid simulator charges of 50, 100, 150, 200 and 250 mg/dl, the simulated breath corresponds to BAC of 44, 88, 132, 176, and 220 mg/dl respectively when the blood/breath ratio of alcohol is 2300:1. At least ten determinations were made at each concentration of ethanol in the simulator.In-vivo experimentsThe in-vivo experiments involved tests with human volunteers after they had consumed 0.8 g/kg ethanol as a 20-30% v/v solution in orange juice. This cocktail was finished in 30 min. At the end of drinking, each subject washed out his mouth with warm water, before providing the first specimen of breath for analysis with Alcolmeter eba. Accordingly, some but not all of these 5 min breath specimens were contaminated with residual alcohol in the mouth. At 10, 20, 30, 45, 60, 90, 120, 150, 180, 240, 300 and 360 min intervals timed from start of drinking, specimens of venous whole blood were drawn through an indwelling catheter. The concentration of alcohol in blood was determined by headspace gas chromatography as described in detail elsewhere (Jones and Schuberth 1989).Evaluation of resultsThe blood-alcohol and breath-alcohol measurements were plotted against time after drinking. The absorption and elimination phases were identified and the blood/breath comparisons were made for each stage of alcohol metabolism. Mean, standard deviation (SD) and coefficient of variation (CV) were calculated by ordinary statistical methods. The precision of alcohol analysis in blood and breath was assessed from the variance of differences between duplicate determinations. The relationship between blood and breath alcohol was determined by least squares regression analysis after converting the readings into logarithms. We also plotted the differences (log BAC - log BrAC) against the mean value on the log scale. The antilogarithm of the mean log difference gives the average concentration ratio of blood alcohol to breath alcohol for this particular instrument and group of subjects. Confidence limits for the ratio and the percentage of overestimates and underestimates of BAC are derived from the variance in terms of logarithms (Bland & Altman 1986). Quality control charts were used to plot the differences between duplicate breaths over time.ResultsThe result of analyzing the Nalco alcohol-gas standard after testing each subject was almost always less than the target calibration value. In 178 tests, the mean drop was -1.03 mg/dl (SD 0.806) and the range was -3 to +2 mg/dl. This decrease was statistically significant; Student's t = 17, degree of freedom 178. The second analysis ranged from 2.5% above to 3.8% below the target value. Table 1 shows the precision and accuracy (recovery) of Alcolmeter eba response

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under in-vitro conditions. The recovery data establishes the accuracy of Alcolmeter eba for analyzing alcohol and results ranged from 97% to 104% of the target concentrations.Figure 1 shows the time-course of venous blood-alcohol concentration compared with the Alcolmeter response for 4 volunteer subjects. The alcohol concentration profiles in blood and breath are in good general agreement but show small systematic differences depending on the phase of metabolism. The SD of analyzing alcohol in breath with Alcolmeter eba was 2.4 mg/dl and this corresponds to a CV of 3.5% because the mean BAC equivalent was 68 mg/dl, N=72. Figure 2 illustrates a quality control chart used to monitor the differences between duplicate determinations over time. Note the occurrence of several outlying values when breath was analyzed within 5 min of drinking.The quantitative relationship between Alcolmeter response and venous BAC is plotted as a scatter diagram in figure 3. The raw data were transformed into logarithms to make the variance around the regression line more or less the same over the range of BAC studied. The correlation coefficient 0.98 indicates a significant linear relationship between blood and breath alcohol concentration. Figure 4 shows differences between the logarithms of BAC and BrAC plotted against mean concentration of alcohol in blood and breath also in logs. The antilogarithm of the mean log difference is a ratio, being 1.06. This implies that BAC is on the average 6% higher than Alcolmeter response. The average blood/breath ratio of alcohol in this series of tests is therefore 2438:1 (1.06 x 2300). The broken horizontal lines in figure 4 show the mean difference and its 95% confidence limits in terms of logarithms. On transformation into the original scale of measurements, the lower and upper confidence limits were 0.88 and 1.27. This suggests that Alcolmeter reads from 12% higher than BAC to 27% lower than BAC. The 95% range of apparent blood/breath ratios with Alcolmeter eba was 2024:1 to 2921:1.DiscussionBlood-alcohol and breath-alcohol concentrations are both equally valid as objective tests of alcohol load in the body. Because of the practical advantages of breath testing compared with sample blood for later analysis at a laboratory, several countries in Europe have created statutory limits of breath-alcohol concentration. This paper gives examples of laboratory procedures involving in-vitro and in-vivo techniques that might prove useful when called upon to evaluate the performance of breath-alcohol instruments.The main purpose of evaluations under field conditions is to get feed-back from the police about ease of handling the instrument. The frequency of breakdowns, and percentage of aborted tests are useful information to document. With the statutes written in terms of BrAC, the agreement between BAC and BrAC under field conditions has academic interest only. Indeed, a comparison of BAC and BrAC under field conditions is hampered by the fact that adjustments are necessary to compensate for metabolism of alcohol between the times of sampling blood and breath. Whether the BAC profile is rising or falling at the time of analysis and the rate of disappearance of alcohol from blood is never known in any individual case.Controlled laboratory studies allow an assessment of precision and accuracy in relation to the coexisting blood-alcohol concentration. The specificity of the

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instrument for ethanol and the response to interfering substances is another important property that can be tested in-vitro. If specimens of breath are not stored for later analysis at a laboratory, the presence of interfering substances cannot be excluded when non-specific detectors are used.The precision of analyzing alcohol in breath shows a tendency to increase at

higher concentrations of alcohol. The SD was maximum at 30-60 min after drinking compared with late into the post-absorptive phase. The Alcolmeter eba is the latest in a range of fuel cell instruments intended for evidential purposes. Besides its microprocessor control, the instrument includes an important new feature. The built-in alcohol gas standard is used to generate a response factor which is then used to calculate the concentration of alcohol in a subject's breath. Immediately after testing a subject, the alcohol standard is reanalysed and its concentration calculated with the same response factor. This offers a check on the stability of single point calibration during the time the suspect DUI offender is tested. This modification has effectively eliminated the observed drop in response of Alcolmeter fuel cell instruments when many repeated tests are made at short intervals (Jones, 1985). The linearity of the response is also improved. A documented proof of correct calibration of evidential breath-alcohol analyzers is necessary for legal purposes. Duplicate analysis on separate breaths bracketed by tests with an alcohol gas standard has become a recommended procedure in some juristictions. The choice between wet simulator alcohol standards and dry alcohol-in-gas mixtures is open to discussion.The venous BAC was on average 6% higher than Alcolmeter response despite the use of a 2300:1 blood:breath calibration factor. In my earlier studies (Jones, 1985), capillary BAC was compared with Alcolmeter response. We recently showed that venous BAC was higher than capillary BAC during the post-absorptive phase of ethanol metabolism suggesting that venous BAC/BrAC is higher than capillary BAC/BrAC (Jones et al. 1989). The average venous BAC/BrAC ratio was 2438:1 (range 2024 to 2921). The blood/breath ratios were higher when the BAC was low. The lowest ratios were observed shortly after drinking when the alcohol profiles were rising and before complete dissipation of mouth alcohol. References.Bland, J.M. and Altman, D.G. (1986). Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1, 307-310. Dubowski, K.M. (1979). Breath alcohol simulators: theoretical basis and actual performance. Journal of Analytical Toxicology 3; 177-182.Jones, A.W. (1985). Electrochemical measurements of breath- alcohol concentra­tion: precision and accuracy in relation to blood levels. Clinica Chimica Acta 146: 175-183.Jones, A.W., Jnsson, K.and Jorfeldt, L. (1989). Differences between capillary and venous blood-alcohol concentrations as a function of time after drinking, with emphasis on sampling variations in left vs right arm. Clinical Chemistry 35; 400-404.Jones, A.W. and Schuburth, J. (1989). Computer-aided headspace gas chromatography applied to blood-alcohol analysis: Importance of on-line process control. Journal of Forensic Sciences 34; 1116-1127.

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Table 1. Relationship between the precision of analysis of air-alcohol vapor standards generated by breath simulator.

BAC equivalent* Alcolmeter response, mg/dl Recovery %mg/dl Mean SD CV% Range Mean Range44 44.6 0.83 1.86 43-46 101 98-10588 88.3 1.01 1.14 86-90 100 98-102132 131.7 1.49 1.13 130-134 99 98-102176 174.8 1.89 1.08 172-178 99 98-101220 216.8 1.87 0.87 214-219 98 97-100

* blood/breath response factor 2300:1Fig 1. Time course of alcohol concentration in venous blood compared with

Alcolmeter eba response. Results for 4 different subjects representative of the group. The blood/breath ratio was 2300:1.

Fig 2. Quality control chart of differences between duplicate determinations with Alcolmeter eba. The broken horizontal lines are 95% and 99% upper limits for differences. Note the outlying values associated with a mouth alcohol effect.

Fig 3. Blood and breath alcohol scatter plot after logarithm transformation. N = number of blood/breath correlates, r = correlation coefficient.

Fig 4. Differences between log blood-alcohol and log breath-alcohol concentrations plotted against the mean concentration on the logarithm scale. The broken horizontal lines show mean difference and the 95% confidence limits.

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