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Measurement of Anesthetics in Blood Using a Conventional Infrared Clinical Gas Analyzer

Section Editor(s): Feldman, Jeffrey M.Peyton, Philip J. MD, MBBS, FANZCA*; Chong, Michael MBBS, FANZCA*; Stuart-Andrews, Christopher MEng; Robinson, Gavin J. B. MBBS, FANZCA; Pierce, Robert MBBS, MD§; Thompson, Bruce R. PhD

doi: 10.1213/01.ane.0000278126.94161.33
Technology, Computing, and Simulation: Research Report

BACKGROUND: Measurement of the partial pressure of volatile anesthetics in blood is usually done using a “headspace equilibration” method with gas chromatography. However, it is not often performed in clinical studies because of the technical, equipment, and logistic requirements. To improve the accessibility of this measurement, we tested the use of a common infrared clinical gas analyzer, the Datex-Ohmeda Capnomac, for this purpose.

METHODS: After characterization of the linearity of the device in measuring the volatile anesthetic concentration in the presence of nitrous oxide, carbon dioxide, and water vapor, blood was tonometered with known concentrations of sevoflurane (actual value between 0.5% and 5.0%) in oxygen and oxygen/nitrous oxide mixtures, as well as mixtures of isoflurane and desflurane in oxygen.

RESULTS: Mean bias (standard deviation) overall for sevoflurane in oxygen relative to the tonometered reference partial pressure was −4.5 (4.8%) of the actual concentration. This was not altered significantly by measurement in 40% oxygen/60% nitrous oxide. For isoflurane and desflurane it was −3.9 (3.3%) and −4.6 (3.8%), respectively, of the actual concentration.

CONCLUSIONS: The accuracy and precision of measurement of volatile anesthetic gas partial pressures in blood by a double headspace equilibration technique, using a clinical infrared gas analyzer, were comparable to that achieved by previous studies using gas chromatography.

IMPLICATIONS: Measurement of anesthetic gas tension in blood using a sidestream gas analyzer is comparable in accuracy and precision to analysis by gas chromatography. This simplified approach facilitates direct studies of anesthetic vapor delivery to blood, instead of relying upon measuring exhaled gas concentrations.

From the *Department of Anaesthesia, Austin Hospital, and University of Melbourne, Melbourne; †Department of Electrical and Computer Systems Engineering, Monash University; ‡Department of Anaesthesia and Perioperative Medicine, The Alfred; §Institute for Breathing and Sleep, Austin Hospital and University of Melbourne; and ∥Department of Allergy, Immunology and Respiratory Medicine, The Alfred Hospital and Monash University, Melbourne, Australia.

Accepted for publication May 9, 2007.

Address correspondence and reprint requests to A. Philip Peyton, MD, MBBS, FANZCA, Department of Anaesthesia, Austin Hospital, Heidelberg 3084, Melbourne, Australia. Address e-mail to

Measurement of the partial pressure of volatile anesthetics administered to patients is important for the conduct of clinical studies regarding the behavior and effects of inhaled anesthesia. Measurement of an anesthetic's partial pressure in inspired and end-expired alveolar gas is most commonly used as a surrogate for this purpose because of its noninvasiveness, and because it can be performed using rapid gas analyzers routinely used for patient monitoring during the conduct of anesthesia.

However, end-expired volatile anesthetic partial pressures differ widely from those in arterial blood (1,2). Arterial levels more accurately reflect the partial pressure perfusing the brain and other organs. Although more accurate, measurement of the partial pressures of anesthetics in the blood is not commonly done.

Techniques for measuring volatile anesthetic partial pressures in the blood are well described (3–5), and most commonly involve equilibration of blood with “headspace” gas in a sealed container, such as a glass syringe. Calculation of the initial partial pressure from that at the end of an equilibration requires that the partition coefficient of the gas be known. Techniques using two (or more) headspace equilibrations allow both partial pressure and partition coefficient of a gas in a blood sample to be simultaneously determined (4). Published studies have used gas chromatography to measure partial pressure after each equilibration, which allows the various gas species present in the blood to be distinguished from each other. The gas chromatograph is a generic measurement device that requires prior calibration against known concentrations of the specific gases to be measured, and in the anesthetic setting this is commonly done using a clinical anesthetic gas analyzer (5). Although gas chromatography represents the accepted standard for this purpose, and allows measurement using small volumes of blood and headspace gas, it requires specialized equipment which is not available in many clinical centers. This limitation likely contributes to the lack of clinical studies in which arterial partial pressures are directly measured.

To improve the accessibility of this measurement, we tested in vitro the use of a widely available clinical infrared gas analyzer, instead of gas chromatography, to directly determine the partial pressure in tonometered blood samples of the volatile anesthetics (sevoflurane, desflurane, or isoflurane) currently used in clinical practice. The method was designed to be practical, requiring no more than 10 mL of blood for accurate and precise measurement. The accuracy of the analyzer in measuring anesthetic vapor concentration in the presence of other gases which absorb infrared radiation (carbon dioxide, CO2; nitrous oxide, N2O; and water vapor) was also characterized.

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Characterization of Analyzer's Accuracy

The gas analyzer chosen was a Datex-Ohmeda Capnomac Ultima (Datex-Ohmeda, Helsinki, Finland). After calibration according to the manufacturer's recommendations, using a cylinder of proprietary calibration gas, the absolute accuracy and linearity of the analyzer in measurement of volatile anesthetic concentration were confirmed using a volumetric technique similar to that used by previous authors (6). This involved delivery of a precisely measured volume of liquid anesthetic, drawn up using a micropipette (or 0.5-mL microsyringe for larger volumes), into the base of a long-necked glass flask of known volume, which was immediately sealed with a gas-tight nonrubber stopper. After evaporation of the volatile liquid, full mixing within the container was ensured by agitation with a 50-mL glass syringe via a three-way stopcock. After thorough mixing, excess pressure in the flask was released via the stopcock and the contents of the flask were sampled for several seconds by the analyzer via standard sidestream sampling tubing at a rate of approximately 200 mL/min.

All data generated by the analyzer during sampling were downloaded in real-time at a sampling rate of 250 samples/s as analog output from the device's analog/serial port, via a 12-bit analog–digital converter card (USB 6009, National Instruments, Austin, TX) to a notebook computer (Powerbook G4, Apple Corp., Cupertino, CA). The resolution of the data obtained was 0.025% for O2 and N2O and 0.0025% for volatile anesthetics and CO2. The data were displayed on the computer using Labview 7.0 (National Instruments), indicating the presence of a flat concentration plateau, which confirmed full mixing within the flask. Averaging of 75 data points from this plateau allowed a high precision concentration measurement to be made. It was found that this method gave a coefficient of variation for the absolute concentration of sevoflurane of <1.0%. A minimum of four independent measurements was made on each occasion, permitting the sem concentration to be determined to within ±1.0% (of the actual concentration).

The absolute accuracy of the analyzer was determined by delivery of 0.2 and 0.4 mL of liquid sevoflurane from a microsyringe into the flask filled with pure O2. At 20°C, liquid to vapor volume ratio for sevoflurane is 1:181 (7), producing expected concentrations of 3.36% and 6.51% respectively, after adjustment for the added volume produced by the evaporation of the liquid, and for measured room temperature and ambient pressure.

The Capnomac uses paramagnetic measurement of O2, and near infrared absorption spectroscopy at different wavelengths to measure CO2, N2O, and the volatile anesthetics. Because of the concern that overlap of infrared absorption spectra of these gases, or related phenomena such as collision broadening, might impair its accuracy where combinations of gases are present in a sample (8), measurement of volatile anesthetic (sevoflurane, desflurane, and isoflurane) concentration in the presence of the other gases which exhibit infrared absorption was done as described above. As a control measurement, the flask was prefilled with 100% O2 before delivery of the liquid anesthetic. The measurement was repeated, prefilling the glass flask with gas mixtures containing incremental concentrations of CO2 or N2O instead. The CO2 concentration was varied from 0% to 5%, and N2O concentration from 0% to 100% with O2 as the balance gas. In each case, mean volatile anesthetic concentration measurement was determined as described above, and compared with that measured in 100% O2.

The possibility of error in volatile anesthetic measurement because of the presence of water vapor was also investigated. This could arise either from direct interference in infrared absorption, or simply from dilution of other gases in the gas mixture because of retention of water vapor in the gas reaching the measurement chamber. Nafion tubing is present internally within the Capnomac and, when fresh, is highly efficient in selectively dehydrating sampled gas before its measurement. Parallel measurements of sevoflurane concentration were made as described above using the standard 2-m length of solid (non-Nafion) sampling tubing and an identical length of fresh Nafion tubing. In this series, the flask was prefilled with O2 fully saturated with water vapor and cooled to room temperature before addition of the liquid sevoflurane.

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Measurement of Volatile Anesthetic Partial Pressure in Blood

Preparation of Reference Blood/Gas Mixtures

With approval from the institutional ethics committee and patient consent, 50 mL of blood was collected from patients undergoing total IV anesthesia with propofol before collection. The blood was heparinized and put into a sealed glass flask placed into an agitated warm water bath set to approximately 37°C. It was then tonometered against a continuous 400 mL/min flow of vehicle gas containing a physiologically relevant concentrations of volatile anesthetic. After a minimum of 2 h of tonometering, and after calibration of the gas analyzer using the proprietary calibration gas and precise measurement of the water bath temperature, the gas in the flask was sampled by the analyzer as described above, to provide the reference volatile anesthetic partial pressure (corrected for the presence of water vapor in the flask). This period of time is several times the required duration for blood–gas equilibration demonstrated by previous authors for gases of this range of solubility (4).

First, tonometered blood mixtures containing sevoflurane in O2 at partial pressures typical of those seen during inhaled anesthesia, (concentrations between 0.5% and 5.0%) were prepared. To test the technique for measurement of the other currently used volatile anesthetics (desflurane and isoflurane), partial pressures approximating 1 minimum alveolar concentration for each anesthetic (concentrations of 5.8% and 1.1% respectively) in O2, were also prepared.

Because an effect of N2O on accuracy of sevoflurane measurement was found during characterization of the analyzer (see below), further tonometered blood mixtures were prepared for partial pressures of sevoflurane in a typical anesthetic mixture of 60% N2O/40% O2 to determine whether the presence of high concentrations of N2O would affect the accuracy of the technique with sevoflurane.

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Double Headspace Equilibration Technique

Blood was then drawn from the flask into each of four gas-tight 20-mL glass syringes with a three-way stopcock attached. Excess blood was expelled, along with any bubbles, from the syringe and stopcock so that precisely 10 mL of blood was contained in the syringe, into which a further 10 mL of air was then drawn up. Each syringe then contained 10 mL of blood with 10 mL of air as the headspace for equilibration. Necessary corrections to these measured volumes were incorporated for the volume of the three-way stopcock (0.2 mL) and of the nozzle of the glass syringe (0.5 mL). The syringes were then placed into the agitated warm water bath for the first equilibration.

After a minimum of 1 h, the contents of the gas headspace in each 20-mL syringe were transferred via the three-way stopcock to a dry 10-mL glass syringe with its own stopcock. (This was done to prevent the possibility of inadvertent suction of blood into the analyzer if the headspace gas was delivered directly from the 20-mL equilibration syringe.) The sidestream gas sampling line of the analyzer was attached to the 10-mL syringe and the contents were delivered to the analyzer although the gas concentration measurements were performed and downloaded to the computer.

After any remaining headspace gas was expelled from the 20-mL syringe, a further 10 mL of air was drawn up into it and the process described was repeated for the second equilibration.

Figure 1 shows typical partial pressure wave forms captured by the system from a double headspace equilibration of a reference blood/gas mixture containing 2% sevoflurane in 60% N2O and 40% O2. The values for N2O and O2 have been scaled by 1/10th. For all gases measured, an adequate plateau was achieved indicating that an undiluted bolus of headspace gas was sampled by the analyzer when delivered into the sampling tubing from the 10-mL glass syringe. The notch in this plateau was an artifact because of momentary interruption of gas flow caused by turning of the three-way stopcock. All data from the plateau distal to this point, indicated by the vertical line, were averaged to obtain the equilibration partial pressure of each gas species measured. At this point, the sampling line is open to atmosphere, and the concentration measured is unaffected by artifact caused by resistance to sampling from, for instance, sticking of the plunger of the syringe. The O2 wave form had been deliberately advanced by approximately 1 s, to align it with the other gases, as the response time of the paramagnetic O2 analyzer of the Capnomac was slower than that of the infrared analyzer. The minimum volume of headspace gas required for the measurement was found to be 5–6 mL, as smaller volumes delivered into the gas sampling line did not reliably provide a clear plateau concentration.

Figure 1

Figure 1

Using the data from these two equilibrations, the original partial pressure (Ρ0) of the volatile anesthetic in the blood sample was then calculated, using an adaptation of the technique described by others (4,5). This was done according to mass balance principles as described in the Appendix:

where, P1 and P2 are the measured partial pressures in the syringe after the first and second equilibrations, PB is atmospheric pressure, ΣPgases is the sum of the partial pressures of all the gases measured by the gas analyzer after each equilibration, and PH2O is the known saturated water vapor pressure at the measured temperature.

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Characterization of Analyzer's Performance

Absolute Accuracy and Linearity

Mean (standard deviation) measured concentrations of sevoflurane delivered from the microsyringe into the 1-L flask filled with O2, were 3.33 (0.02%) and 6.45 (0.07%). These underestimated the expected concentrations by 0.03% and 0.06%, respectively. In relative terms, in each case, this represented an underestimate of 0.9% of the actual concentration.

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Accuracy in the Presence of Other Gases

There was a progressive underestimation of sevoflurane concentration with increasing N2O concentration, which was statistically significant (r = −0.81, P < 0.001). The line of best fit followed the following equation

In the presence of 60% N2O, this produced a relative underestimate of sevoflurane of 3% of the actual concentration. This proportional error was maintained across a range of sevoflurane concentrations from 0.5% to 3.5% (P < 0.001). There was no demonstrable error in measurement of isoflurane or desflurane with N2O relative to the measurement made in O2.

Similarly, no significant effect of CO2 on measured concentration of sevoflurane, isoflurane, or desflurane was found. No difference in sevoflurane concentration in O2 saturated with water vapor was demonstrable between samples obtained with standard sampling tubing and Nafion tubing. This confirmed empirically that either the sampled gas was fully dehydrated by the internal Nafion tubing within the device, or that the presence of residual water vapor causes no error in sevoflurane measurement.

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Measurement of Volatile Anesthetic Partial Pressure in Blood

Measurements of sevoflurane partial pressure in tonometered blood at six different reference concentrations are shown in Figure 2. The double headspace equilibration method underestimated the reference partial pressure by 4.5% of the actual concentration (P < 0.001 on the paired t-test). Ninety-five percent confidence limits for the bias at each reference concentration are shown, as well as those for individual measurements, which were overall ±9.6% of the actual concentration.

Figure 2

Figure 2

Figure 3 shows the results for sevoflurane partial pressure measured in the presence of 60% N2O. Correction of both reference concentration and measured partial pressures of sevoflurane was done with each equilibration for the presence of N2O, according to Eq. 2. The mean bias (at three different reference concentrations) was −4.7% of the actual concentration, which was not significantly different from that in the absence of N2O. This confirmed that Eq. 1 compensated accurately for the changes in headspace volume produced by equilibration of high concentrations of N2O in blood (Appendix). It was observed that the headspace volume expanded by 2–3 mL at the end of the first equilibration where N2O was present.

Figure 3

Figure 3

The accuracy of the method in measurement of partial pressure of desflurane and isoflurane, the other volatile anesthetics used in current anesthetic practice, is shown in Table 1. The reference concentration of desflurane was 5.8% and for isoflurane 1.1%. Data for sevoflurane at a reference concentration of 2.3% are included for comparison. The gases are ordered according to the measured solubility. The accuracy and precision of the method were consistent regardless of the solubility of the gas. Table 1 also lists the measured blood/gas partition coefficients for these four gases [mean (standard deviation)], corrected to 37°C according to published data describing the effect of temperature on solubility of anesthetics in blood (9,10). These corrected values slightly overestimate values found in standard texts (11). Underestimation of the true partial pressure will lead to this, and correction of measured partial pressure to the reference tonometered partial pressure would bring the calculated partition coefficients into closer agreement with these quoted values.

Table 1

Table 1

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A number of published studies have measured volatile anesthetic partial pressure in blood by multiple headspace equilibration (1,3,5,6,12) and all have used gas chromatography. These were reviewed by Smith et al. (5) and only a few have reported the results of formal validation of their technique using tonometry (5,6). In their own study, Smith et al. reported a mean underestimate for volatile anesthetics of 2.3% of the actual concentration, with 95% confidence limits for an individual measurement of ±8.5% of the actual concentration. For sevoflurane, they found a 4.5% underestimate ±7% (5). Dwyer et al. found a 4% underestimate ±7% against tonometerd isoflurane in blood (6). Our bias and confidence limits for sevoflurane over a wide range of concentrations, were comparable to these, and confirm that measurement of volatile anesthetic partial pressure in blood can be done as well using the commonly available clinical gas analyzer we used.

In these studies, the negative bias relative to the tonometered reference concentration is because of a number of likely sources of error. These include cooling of equilibration syringes immediately after removal from the warm water bath, and inevitable loss of gas to atmosphere when expelling gas and bubbles from the syringe before drawing up headspace gas for each equilibration. Inadequate equilibration time is an unlikely explanation in our study, as the time allowed for the tonometry and for the syringe equilibrations well exceeded the required time (4).

Trapping of equilibrated gas in small bubbles before the second equilibration was identified by Smith et al. (5) as a significant potential source of error with measurement in whole blood, which is prone to foaming. They dealt with this by performing their second equilibration using only a 2 mL fraction of a larger blood sample used for the first equilibration, which allowed blood free of bubbles to be used for the second equilibration. We did not do this, but retained all 10 mL of blood for our second equilibration, with results not significantly different from theirs. We subsequently found that flocculation of blood could be eliminated by addition of a small drop of the antifoaming agent, dimethicone. Use of this agent did not appear to affect the accuracy of our assay, but did make the blood specimens more convenient to work with, although not altering the calculated partition coefficient of the volatile anesthetic in the sample.

Previous commentators have questioned the suitability for research purposes of infrared analyzers for measurement of volatile anesthetic concentrations in the presence of other gases, such as N2O, water vapor, and CO2, which also absorb infrared radiation (8). Careful characterization and calibration of the device is important if these potential sources of error are to be adequately addressed. We used one commonly available clinical gas analyzer for this study, and any other device should undergo similar characterization if it is intended to be used. This principle also applies to use of gas chromatographs which, despite the advantage that they distinguish between different gas species, still require calibration against known standards before use (13). We chose to use simple volumetric measurements as our primary calibration standards for this study. These were accurate, precise and reproducible, and provided a robust validation of the gas analyzer's performance. A direct comparison of results using the gas analyzer and gas chromatography would be of further interest, although it would not improve the reliability of our findings.

Despite the apparently significant error in sevoflurane measurement we demonstrated associated with the presence of high concentrations of N2O, the effect on the accuracy of the double headspace equilibration assay was negligible. It should be noted that the correction to the calculated partial pressure of sevoflurane in the tonometered blood samples, produced by adjustment of sevoflurane headspace concentrations using Eq. 2, was only in the order of 1% (relative). No significant effect was caused by the presence of CO2. In addition, our data also suggested that sampled gas was effectively dehydrated by the internal Nafion tubing within the analyzer, eliminating the presence of water vapor as a source of error in measurement of other gases.

In particular, our results support the precision and practicality of using the calculated N2 concentration in the headspace gas, to automatically determine the relative equilibration volumes in the syringe for Eq. 1 (Appendix). This avoided potential errors in volume measurement that could occur where the plunger of the glass syringe did not move freely, and the headspace gas was not at atmospheric pressure. This approach is convenient where room air is used for the equilibration, although any insoluble gas would be suitable. However, it would be unsuitable where gas chromatography was used instead, if N2 was used as carrier gas for the assay, as is commonly done.

The advantage of the clinical gas analyzer as a tool for the technique is its accessibility and ease of use. These devices are commonplace in the operating room environment, familiar to anesthesiologists, and readily available for clinical research purposes. Interfacing with computers is not difficult using the analog signal from the device (14), which provides a precise measurement and recording of gas concentration data. We found the accuracy of the analyzer used in the study excellent when calibrated using the manufacturer's proprietary calibration gas mixture, and the stability of the calibration was well maintained over time. Its on-site availability means that blood samples do not need to be processed distant to the point of contact with the patient, avoiding transportation and time delays and reducing the potential for error in processing the samples using the multiple equilibration method.

The greater accessibility of measurements provided by a clinical gas analyzer may allow more frequent measurements of blood partial pressures in studies investigating the pharmacokinetics of anesthetics. Most published studies use expired alveolar gas concentrations, because of the ease with which these data can be obtained, despite the fact that it is arterial blood partial pressures that are of most direct interest in determining anesthetic effects on body tissues. This is especially relevant during inhaled anesthesia, which substantially widens alveolar-arterial (“A-a”) difference for both respiratory and anesthetic gases (1,2,15,16). The accuracy in studies investigating A-a difference for anesthetic gases, where expired and arterial gas partial pressures are compared, may be assisted when the same device is used for both measurements.

In summary, we investigated the reliability of use of a clinical infrared gas analyzer for the measurement of volatile anesthetic gas partial pressures in blood using a double headspace equilibration technique. Accuracy and precision of measurement were comparable to that achieved by previous studies using gas chromatography.

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Measurement of the partial pressure of volatile anesthetic in the blood is done by equilibration of blood with “headspace” gas in a sealed container such as a glass syringe. Calculation of the initial partial pressure from that at the end of a single equilibration requires that the partition coefficient of the gas be known. Two (or more) sequential headspace equilibrations effectively allow calculation of both the original partial pressure (P0) of the anesthetic in the blood sample, and of the blood/gas partition coefficient of the anesthetic (λ) (assuming from Henry's law that this remains constant for an inert gas as its partial pressure changes).

From mass balance principles,

where VB is the blood volume, P1 is the measured partial pressure of the volatile anesthetic and Vg1 is the volume of the headspace gas in the syringe after the first equilibration.

Similarly, for the second equilibration,

Transposing Eq. A1

where, from Eq. A2

Combining Eqs. A3 and A4

Vg1 and Vg2 are difficult to measure directly in the syringe because of the presence of a meniscus at the blood/headspace interface, and the possibility of changes in pressure within the syringe if the plunger should stick to the barrel during equilibration. The headspace volume cannot be assumed to remain constant, as significant volume changes can occur, especially when high concentrations of N2O are present in the blood sample. However, the volume of gas drawn up into the syringe at the commencement of each equilibration (Vg0) is precisely known, and Vg1 and Vg2 can be calculated with much better precision from the partial pressure changes of N2 in the headspace. N2 has negligible solubility in blood, and its partial pressure is calculated after each equilibration by subtraction of the sum of all the other gas partial pressures (including water vapor pressure) from atmospheric (PB).

where ΣPgases is the sum of the partial pressures of all the gases measured by the gas analyzer after each equilibration. In each case PH2O is determined by the known saturated water vapor pressure at the measured temperature. Because a similar equation applies to the second equilibration

then combining Eqs. A6 and A7 with Eq. A5

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