Sensitive Head-Space Gas Chromatographic Method for the Determination of Ethanol Utilizing Capillary Blood Samples Paul K. Wilkinson, John G. Wagner,‘ and Allen J. Sedman College of Pharmacy and Upjohn Center for Clinical Pharmacology, University Hospital, University of Michigan, Ann Arbor, Mich. 48 104
A head-space analysis method utilizing 20-50 HI blood samples and enabling the determlnation of ethanol concentrations over a range of 0.0030-1.2 mg/ml with an average precision of f 4 . 6 % is descrlbed. This assay displays several Improvements over methods described formerly. These improvements are: increased sensitivity produced by equilibration of blood samples at 60 ‘C, which Increased the partition coefflcient (air/blood) of ethanol approximately 4-fold over that at 30 ‘C; improved preclslon (average 4.6%) due to the use of a water-jacketed, gas-tight syringe malntalned at a constant temperature; use of sodium nltrite, which prevented ethanol oxidation and acetaldehyde accumulation observed at elevated temperatures, and resulted in more complete “salting out” of ethanol from blood solutions; and utilization of sodium fluoride which effectively halted spurious ethanol levels caused by the growth of micro-organisms in stored blood.
The need for a sensitive gas chromatographic method to determine blood ethanol levels was demonstrated by Wagner and Pate1 ( I ) . Their results indicated that blood ethanol concentrations as low as 0.005 mg/ml must be measured to define adequately the time course of blood ethanol in man, including the marked curvature a t the tail end ( 1 ) . Forney ( 2 ) reported that during the absorption-distribution phase, following the oral administration of ethanol (or the distribution phase following intravenous administration), the “concentration of alcohol in arterial blood may be 50 to 100 percent higher than it (is) in venous blood”, and, “during this time, blood from an arm vein will not truly reflect the concentration of alcohol in the brain where its effects are produced”. Therefore, the collection of capillary blood samples, which more closely approximate the ethanol concentration in arterial blood (3) is indicated. Another advantage of sampling capillary blood is that it permits the collection of the large number of samples required to define adequately the total time course of blood ethanol. Numerous venipunctures would be painful to human subjects. The collection of capillary blood samples avoids this problem and, in addition, its withdrawal does not require highly qualified personnel ( 3 ) . Investigators ( 4 ) have expressed the concern that capillary blood may become diluted by fluids located near the capillaries when fingertip samples are collected. Forney et al. ( 4 ) have shown that this hemodilution phenomena is of no importance. Hemoglobin determinations made on capillary and venous bloods, drawn simultaneously, showed no significant differences. Therefore, fingertip blood samples would accurately reflect conditions prevailing in the capillaries. Jain and Cravey (5-8) have extensively reviewed the chemical, infrared, and gas chromatographic methods used for the analysis of ethanol. Hancock et al. (9) compared To whom reprint requests should be directed: Upjohn Center for Clinical Pharmacology, University of Michigan Medical Center, Ann Arbor, Mich. 48104. 1506
ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
blood ethanol concentrations measured by gas-liquid chromatography (GLC), chemical titration, and enzymatic oxidation. They concluded that the latter two methods were not as specific for ethanol as the GLC technique. The review by Jain and Cravey (6) indicated that direct injection and head-space gas chromatographic techniques would be best suited for measuring capillary blood concentrations of 5-10 wg/ml. Several authors (1, I O , 11 ) have discussed the analysis of ethanol by the direct injection of whole blood, serum, plasma, or urine into the gas chromatograph. Use of these techniques, however, has been limited because of the fear that the direct injection of biological samples into the chromatograph would spoil the column, give additional interfering peaks, or plug up the syringe. Jain (10) described a direct injection method which purportedly avoided all of these problems. However, Perez et al. (12) reported that “unless the glass-wool plug a t the injection port is changed daily, non-chromatographable materials in blood accumulate, which interfere with analyses on subsequent days. To avoid this difficulty, the column (must be) removed a t the end of each day and the glass-wool plug at the injection port . . . replaced”. The experiences of this laboratory were similar to those of Perez et al. Consistent results could be obtained only if the glass-wool plug was changed after every 15-20 direct injections of whole blood. Numerous authors have described head-space gas chromatographic methods (6, 13-18) for the analysis of ethanol. Use of these techniques offers distinct advantages over direct injection methods. Most important is prevention of contamination of the column. However, most of the headspace procedures equilibrate blood ethanol samples a t room temperature (25 “C) and, therefore, are useful only at ethanol concentrations in excess of 0.1 mg/ml. Ozeris et al. (15) and Bassette et al. (14) reported a method capable of measuring aqueous ethanol concentration of 0.1 pg/ml utilizing an equilibration temperature of 60 “C. Although the procedure of Glendening and Harvey (13) required large samples (1-2 ml), their work indicated that smaller volumes could probably be used with only moderate loss in sensitivity. Several authors have reported problems associated with the development of such a technique. Glendening et d. (13) reported that the use of elevated temperatures (40-50 “C) gave more erratic results than when samples were equilibrated at slightly above ambient temperatures. In addition, work by Brown et al. ( 1 8 ) and Smalldon and Brown (19) indicated that ethanol in blood was quickly oxidized to acetaldehyde at 60 “C by an oxyhemoglobin-mediated mechanism. Multiple injections of head-space samples from a single blood sample, resulting in long equilibration at elevated temperatures, should be avoided.
EXPERIMENTAL Assay Procedure, Preparation of Biological Sample. Equal volumes (20-50 ~ 1of) an internal standard solution and of a blood ethanol sample, rapidly collected with a calibrated glass disposable microsampling pipet (Corning Glass Works, Corning, N.Y.), were placed in a 0.4-ml polypropylene test tube (Eppendorf, Scientific
Products, Romulus, Mich.). The tube was capped with the attached stopper, and the solution vortexed for 10-20 seconds. One half of the resultant mixture was transferred to a 6-ml amber glass serum vial, which was subsequently sealed with a flange-type, uncoated rubber stopper (S-63 86 White, West Company, Phoenixville, Pa.) and an aluminum seal using a manual crimper. The flange-type rubber stoppers were pretreated by boiling in distilled water for 30 minutes followed by thorough rinsing with deionized water. Without this pretreatment, a peak appears in the ethanol region of the chromatogram. The vials were placed in a constant temperature water bath maintained a t 60 "C and allowed to equilibrate for 3 minutes prior to injection of the head-space sample into the gas chromatograph. An experiment in which the equilibration time was altered is described later. Ethanol Reference Standards. Outdated transfusion blood containing citrate phosphate dextrose solution USP as an anticoagulant, was used for all studies unless otherwise stated. A working standard solution of 20 mg ethanol/ml was prepared by transferring 1.25 ml of absolute alcohol (USP) to a 50-ml glassstoppered vial. The solution was diluted to volume with deionized water. Ethanol concentrations of 0.0030 mg/ml to 1.2 mg/ml were prepared by serial dilution of the aqueous working standard solution with outdated transfusion blood. Ethanol standard solutions were freshly prepared for each phase of the study, and were assayed in the same manner as biological samples. Internal Standard Solutions. One aqueous internal standard solution contained 1 mg n-propanollml, 40 units sodium heparin/ ml, 6.9 mg (100mM) sodium nitrite/ml, and 40 mg sodium fluoride/ml. Another aqueous internal standard solution containing only 1 mg n-propanol/ml, 40 units sodium heparin/ml, and 40 mg sodium fluoride/ml was also prepared. Gas Chromatography. The assay was performed on a Varian Series 2100 GLC (Varian Aerograph, Walnut Creek, Calif.) fitted with hydrogen flame ionization detectors and a Model 20 dual pen recorder. Two 6-foot, 3.5-mm i.d. U-shaped glass columns were packed with 80-100 mesh styrene divinyl benzene polymer (Porapak Q, Waters Associates, Framingham, Mass.) and the packed columns were conditioned a t 250 "C for 12 hours. The operating conditions were: column temperature, 150 "C; injector temperature, 165 "C; detector temperature, 200 "C; nitrogen (carrier gas) flow, 30 cm3/min; air flow, 225 cm3/min; and hydrogen flow, 30 cm3/min. The columns were routinely purged a t 150 "C when not in use. Preparation of Head-Space Sample. The needle of a 2-cm3 gastight syringe (Precision Sampling Corporation, Baton Rouge, La.), housed in a constant temperature water-jacket maintained a t 67 "C, was inserted through the rubber stopper of the 6-ml amber glass serum vial into the vapor phase, making sure not to touch the liquid. The plunger was pumped 5 times and was returned to 0.1 ml above the intended sample size. The syringe was withdrawn from the vial and the vial then removed from the hot water bath. T h e sample size (0.30-0.75 ml) was adjusted and the head-space sample injected into the column with a rapid, smooth motion. At least two determinations were made for unknown blood ethanol samples. The exact size of the head-space sample was not critical as the response was the ratio of peak areas for ethanolln-propanol. Each determination, including equilibration time, required 15 minutes. Investigation of Variables. A study utilizing a completely crossed randomized block design with 3 replicates was undertaken to determine the effects of four main factors and to assess the feasibility of multiple head-space injections from a single sample vial. The factors and their levels were as follows: Time of storage, Initial, 1 week and 2 weeks; temperature of storage, 4 "C and -17 "C; nitrite concentration, Nil and 100 mM; ethanol concentration, 0.0060, 0.020, 0.060, 0.20, and 0.60 mg/ml; replicate injections per vial; l s t , 2nd, and 3rd. Separate experiments were performed to determine the optimum equilibration time and the variation of response with blood type and the normal range of hematocrit. Study Conditions. Effects of Extended Storage. Blood ethanol standards were prepared and mixed separately with equal volumes of each internal standard solution. Forty-microliter aliquots of the resultant mixtures were transferred to 6-ml amber glass serum vials and the vials were sealed. Each vial was appropriately labeled and placed in a freezer unit (-17 "C) or in a refrigerator (4 "C). The refrigerated samples were assayed immediately and a t 1-week intervals for an additional 2 weeks. The frozen samples were stored overnight and assayed, and then a t weekly intervals for 2 additional weeks. At each storage time, 3 vials with 3 replicate injections
C
2
4
6
RETEN'CN
8 -1ME
' IN
0
'
2
Nl\J'tS
Figure 1. Typical chromatographic tracing 40-pl mixture (blood, internal standard solution with sodium nitrite), 0.3 ml head-space gas injected. Sample 0.60 mg ethanol/mL Sensitivity, ethanol, n-propanol. lo-'* A/mV X 64. Peaks (A, El) delayed injection peaks, (C) ethanol, (D) npropanol
per vial were assayed for each combination of factors. "Frozen with nitrite" samples were also assayed a t the end of 4 weeks. Effects of Equilibration Time. A blood ethanol solution having an ethanol concentration of 0.060 mg/ml was prepared. Aliquots of this solution were mixed separately with equal volumes of each internal standard solution. Sample vials were prepared and placed in a constant temperature water bath for varying time periods (1-45 min). Three separate vials were assayed for ethanol content a t each equilibration time. Acetaldehyde response was also determined for all samples not containing sodium nitrite and incubated for 45 minutes. In a separate experiment, a blood ethanol solution of 0.20 mg/ml was prepared. An aliquot of this solution was mixed with an equal volume of internal standard solution containing no sodium nitrite and 40-p1 aliquots were transferred to 6-ml serum vials. These vials were placed in a 60 "C water bath for 45 minutes and the acetaldehyde and ethanol responses quantitated. Effects of Hematocrit a n d Blood Type. The hematocrit of a sample of freshly drawn whole blood was determined (50%) and a 5-ml portion centrifuged to obtain the plasma. A sample having a hematocrit of 42% was prepared by mixing 0.95 ml of plasma with another 5 ml of whole blood. The normal range of hematocrit for adult males is 42-50%. Blood ethanol solutions of 0.13 mg/ml were prepared by dilution of an aqueous ethanol standard with the 42% and 50% hematocrit blood samples. In a separate experiment blood samples of all four blood types (A, B, AB, and 0 ) were used to prepare blood ethanol solutions of 0.13 mg/ml. Analysis of Unknown Blood Ethanol Samples. An ethanol calibration curve was prepared by utilizing blood ethanol standard solutions of 0.0060, 0.020, 0.060, 0.20, 0.60, and 1.0,mg ethanol/ml. Three "unknown" blood ethanol samples (0.0083, 0.040, and 0.28 mg/ml) were prepared by a coworker and the code not broken until after the samples were assayed. Ethanol Blood Level Curue. To demonstrate the utility of the assay method, an adult male volunteer was administered 15 ml of 95% ethanol diluted to 150 ml with unsweetened orange juice. The subject had fasted from 1O:OO p.m. the evening before the study until 3 hours post dosing. Whole capillary blood samples (50 pl) were collected a t 0, 4, 8, 12, 16, 20, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, and 180 minutes. The samples were mixed with an equal volume of internal standard solution containing sodium nitrite, transferred to 6-ml serum vials, and stored in a freezer a t -17 "C until they were assayed.
RESULTS AND DISCUSSION A typical c h r o m a t o g r a m is shown i n Figure 1. Acetaldehyde, if observed, would a p p e a r between peaks B a n d C, a p p r o x i m a t e l y 2 m i n u t e s a f t e r the first solvent (air) peak, peak A. N - P r o p a n o l was selected for t h e i n t e r n a l s t a n d a r d since i t is a s u b s t a n c e n o t normally f o u n d i n h u m a n blood and its r e t e n t i o n t i m e is different f r o m t h a t of ethanol or isopropyl alcohol (IPA). IPA (retention t i m e = 7 minutes) is the bactericidal disinfectant often used prior to blood withdrawal a n d , therefore, t e n d s to c o n t a m i n a t e t h e capillary samples. ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
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There was not any significant difference in response for any storage time with “frozen with nitrite” samples. The random differences observed were attributed to day to day variability in instrument setting. “Refrigerated with nitrite” samples were characterized by an increase in response with storage time. The presence of sodium nitrite generally resulted in higher GLC response ratios. This probably can be attributed to a “salting out” effect produced by the sodium nitrite. Sodium nitrite reduced the variability (as measured by deviations of the extreme value from the mean) of replicate determinations a t the lower ethanol concentrations measured (Table IV). The variability a t higher ethanol concentrations was unchanged by the presence of sodium nitrite. The reduction in variability a t low ethanol concentrations became evident after long periods of storage. Samples containing internal standard solution with sodium nitrite can be stored in a freezer for a t least four weeks without deleterious effects. A 3-minute equilibration time (total elapsed time of 9 minutes for 3 repeat determinations) appears to be optimal. The effects of sodium nitrite are demonstrated in Table V. With sodium nitrite, equilibration is reached in 3 minutes and maintained for 45 minutes; while, without sodium nitrite, there appears to be a steady fall off of response with time following 15 minutes of equilibration a t 60 “C. Sodium nitrite prevents the oxyhemoglobin-mediated oxidation of ethanol to acetaldehyde (18, 19). Previous authors (18, 1 9 ) have reported that this oxidation of ethanol to acetaldehyde has a zero order dependence with respect to ethanol. The following results, seen at low ethanol concentrations, did not support the former reports. Blood samples containing 0.20 and 0.060 mg/ml of ethanol were analyzed (using internal standard solution without sodium nitrite) following a 45-minute incubation a t 60 “C and the acetaldehyde response was quantitated (Table VI). A zero order
Table I. A Typical Calibration Curve” Ethanol concentration Calcdb
Obsd‘
Relative error,
0.0030 0.0060 0.020 0.060 0.20 0.60
0.0032 0.0057 0.019 0.063 0.20 0.61 1.2
-6.7
1.2
ou
+5.0 15.0 -5.0 0.0 -1.7
0.0
Least squares parabola: In ( R ) = -0.151 + 1.16 In ( C ) + 0.0347 [In (C)I2 b B y dilution. mg/ml ethanol in blood. C B y chromatographic analysis, mean of 2 replicate injections. a
Calibration data are given in Table I. The responses were calculated as the ratios of peak areas of ethanol to n-propanol, with peak area determined as the product of peak height times peak width a t half-height. The data were fitted to the least squares parabola (Table I): In ( R ) = a0
+ a1 in (C) + a2 [In (C)Iz
(1)
where C = ethanol concentration (mg/ml) and R = response. This approach was used for the following reasons: the responses over the large range of ethanol concentrations were not linear; and the equal weights used in linear least squares estimation resulted in large deviations a t the lower concentrations. The In-ln weighting and the use of a parabola eliminated these problems. Unknown blood ethanol concentrations were determined by use of a quadratic solution program using an electronic calculator. F o u r Main Factors. Analysis of the data for the four main factors (storage time, storage temperature, presence of sodium nitrite, and ethanol concentration) and replicate injections from the same vial gave the following results (see Tables I1 and 111). Table 11. Results of Extended Storage at 4 “C
*
ResponseQ) a t storage t i m e
l-week
2-v;eeh
sodium nitrite
sodium nitrite
Initial sodium nitrite‘ Ethanol concn, m g / m l
with
without
with
0.0060
0.003 07 0.00335 0.020 0.0110 0.0116 0.060 0.0375 0.03 54 0.20 0.130 0.140 0.60 0.434 0.420 a Ratio of ethanol peak area to n-propanol peak area. Mean = nil.
Table 111. Results of Extended Storage a t
without
0.00371 0.0129 0.0415 0.148 0.496 of 9 determinations
with
without
0.00279 0.00438 0.00210 0.0116 0.0136 0.00974 0.0367 0.0408 0.0317 0.132 0.151 0.131 0.489 0.484 0.481 ( 3 replicates from 3 vials). With = 100 mM. without
- 17 “C Responsea at storage t i m e
Initial’
1-week
Ethanol concn, n i g l m l
with
*
2 -1veek‘
sodium nitrite
sodium nitrite without
with
sodium nitrite without
with
without
.%-week= sodiLT. nitrite with
0.0060 0.00350 0.00234 0.00421 0.00173 0.00401 0.00159 0.020 0.0120 0.0101 0.0131 0.00938 0.0124 0.00807 0.060 0.0394 0.0384 0.0426 0.0331 0.0401 0.0277 0.20 0.145 0.138 0.152 0.144 0.146 0.137 0.60 0.501 0.475 0.531 0.506 0.477 0.472 a See Table 11. Mean of 9 determinations ( 3 replicates from 3 vials). Mean of 6 determinations (3 replicates from 2 vials). mM, without = nil.
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ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
0.00369 0.0129 0.0420 0.149 0.484 With = 100
Table IV. Effect of Sodium Nitrite on the Variability of Replicate Ethanola Determinations
Table VIII. Effect of Blood Type on Responsea
Storage t i m e Initial
1-week
2-week
sodium n i t r i t e b
sodium nitrite
sodium nitrite
T e m p , OC
with
without
4 -17
12.1c,d 14.3
with
without
with
9.2 13.1
Response
A
0.0706 0.0756 0.07 14 0.0768 0.0031
B AB 0 Std d e v
without
7.3 37.6 9.3 8.5 58.3 31.2 Sodium nitrite cona Ethanol concentration = 0.0060 mg/ml. centration, with = 100 mM, without = nil. e (Extreme - Mean)/ Mean X 100. disregarding arithmetic sign. Mean of 9 determinations (3 replicates from 3 vials). 5.4 9.8
Blood type
See Table VII. ~~~
Table IX. Accuracy of Head-Space Chromatographic Analysis Concentration response a
Calcd
Re1 error
a
ObsdC
~~
Table V. Effect of 60 "Cof Equilibration Time on GLC Response R e npons e a Equilibration t i m e , minute5
\vith sodium nitrite
v;ithout sodium nitrite
1 3 5 10 15 30 45
0.0394 0.0458 0.0429 0.0440 0.0454 0.0448 0.0479
0.0317 0.0337 0.0344 0.0345 0.0334 0.0326 0.0301
a Ratio of ethanol to n-propanol peak area; mean of 3 determinations (1 replicate from 3 vials); ethanol concentration = 0.060 mg/ml.
Table VI. Acetaldehyde Levels at Two Ethanol Concentrations after Equilibration of Blood Samples at 60 "Cfor 45 Minutes Acetaldeh) de response'
t t h a i o l i o n i n , m o ml
0.060 0.20 a
0.0136 0.0276
Ratio of peak areas, acetaldehydeln-propanol.
~
Table VII. Effect of Hematocrit on GLC Response I limatoLrit,
3
42 50 Av s t d dev
Response"
Std dc\
0.0990 0.0986
0.0011 0.0010 0.0010
0.00504 0.0144 0.0426 0.159 0.459 0.786
0.0060 0.020 0.060 0.20 0.60 1.0
0.0062 0.01 9 0.058 0.21 0.59 0.95
-3.3 +5.0 +3.3
5.0 -1.7 -5.0
Unknown samples
0.00610 0.0083 0.0076 ~ 8 . 4 0.0269 0.040 0.037 17.5 0.216 0.28 0.29 -3.6 " Ratio of ethanol to n-propanol peak area; mean of 2 determinations (2 replicates from 1 vial). By dilution, mg/ml ethanol in blood. By chromatographic analysis, least squares parabola: In ( R ) = -0.222 + 1.08 In ( C ) + 0.0158 [In ( C ) ] * .
Table X. Capillary Ethanol Concentrations Measured Following the Oral Administration of 15 ml of 95% Ethanol Diluted to 150 ml with Orange Juice to an Adult Male Volunteer Time, hr
Concn, m g l m l
T i m e , hr
Concn, m g l m l
0.0 0.067 0.133 0.200 0.267 0.417 0.500 0.750 1.00
0.0 0.017 0.12 0.21 0.29 0.28 0.26 0.19 0.12
1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00
0.081 0.046 0.032 0.017 0.012 0.0074 0.0030 0.0024
aRatio of ethanol to n-propanol peak area, mean of 3 sample vials, ethanol concentration = 0 13 mg/ml
relationship woiild predict an equal acetaldehyde response for both concentrations, not the twofold increase at 0.20 mg/ml as was observed. There appears to be a higher order dependence on ethanol concentration a t the lower concentration ranges investigated, which may be consistent with an enzymatically controlled mechanism obeying MichaelisMenten kinetics. Using internal standard solution containing sodium nitrite, no such effect was observed as the acetaldehyde peaks were too small to quantitate. Effects of Hematocrit and Blood Type. Hematocrit (42-50% for adult males) and blood type had no apparent effect on the assay results (Tables VI1 and VIII). Analysis of Unknown Blood Ethanol Samples. T o test the accuracy of the method, a single blind study was performed. The values obtained by GLC analysis agreed well with those calculated by dilution (Table IX). Sample Blood Ethanol Concentration Curve. Table X
0
3
2
1
TIME
IN
HOURS
Figure 2. Capillary ethanol concentrations measured following oral administration of 15 ml of 9 5 % ethanol to an adult male volunteer ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
1509
and Figure 2 present a capillary ethanol concentrationtime profile measured following the administration of 15 ml of 95% ethanol to a single human subject. Choice of Reagents. Sodium fluoride was added to the internal standard solution to inhibit ethanol metabolism (18, 1 9 ) as well as ethanol production (20) by the growth of micro-organisms, Inclusion of the anticoagulant, sodium heparin, in the internal standard solution eliminated the need for heparinized capillary tubes. Sodium nitrite (100 mM) effectively halted the oxidation of ethanol and, therefore, prevented the subsequent increase in acetaldehyde observed a t elevated temperatures (19). Blood samples turned brown-green to black in color when mixed with the internal standard solution containing sodium nitrite; an indication that the oxyhemoglobin was, indeed, converted to methemoglobin (19).
SUMMARY The head-space analysis method described is capable of a routine precision of 4.6% (average) over a 400-fold range of concentration. A sensitivity of 3 kg ethanol/ml is easily reached, and even lower sensitivity is feasible with increased head-space sample size. Since endogenous ethanol levels average 1.2-1.5 wg/ml (21, 22), the lower sensitivity reported is sufficient for blood level studies. Equilibration of the blood ethanol samples a t an elevated temperature resulted in an ethanol enriched head-space gas (14, 23). A change of equilibration temperature from 30 to 60 "C increased the apparent ethanol partition coefficient (air/ blood) an estimated 4-fold. The use of a constant temperature water-jacketed, gas-tight syringe prevented condensation (24) and reduced the scatter previously reported (13) a t elevated temperatures. Storage time had little effect on response at the preferred storage temperature (-17 "C). Appropriate standard ethanol solutions should be used whenever samples are to be analyzed. The assay described should also be suitable for deter-
mining the concentration of ethanol in biological fluids other than capillary blood, such as in urine, serum, or plasma.
LITERATURE CITED J. G. Wagner and J. A. Patel, Res. Commun. Chem. Pathol. Pharmacol., 4, 61 (1972). R. B. Forney. Abstracts of Symposia and Contributed Papers Presented to APhA Academy of Pharmaceutical Sciences at the Meeting of the 118th Annual Meeting of the American Pharmaceutical Association, San Francisco, Calif., March 27-April 2, 1971, Vol. 1 , No. 1 , pp 28-29. R. H. Laessig, Anal. Chem., 40, 2205 (1968). R. B. Forney, F. W. Hughes, R. N. Hager, and A. E. Richards, 0. J. Stud. Alcohol, 25, 205 (1964). N. C. Jain and R. H. Cravey, J. Chromatogr. Sci., 10, 257 (1972). N. C. Jain and R. H. Cravey, J. Chromatogr. Sci., 10, 263 (1972). N. C. Jain and R. H. Cravey, J. Chromatogr. Sci., 12, 214 (1974). R. H. Cravey and N. C. Jain, J. Chromatogr. Sci., 12, 209 (1974). J. A. Hancock, F. L. Mill, and J. R . Miles, Ciin. ToxiCol., 4, 217 (1971). N. C.Jain, Ciin. Chem., 17, 82 (1971). M. K. Roach and P. J. Creaven. Clin. Chim. Acta, 21, 275 (1968). V. J. Perez, T. J. Cicero, and E.A. Bahn, Clin. Chem., 17, 307 (1971). B. L. Glendening and R. A. Harvey, J. Forensic Sci., 14, 136 (1969). R. Bassette. S. Ozeris, and C. H. Whitnah, Anal. Chem., 34, 1540
(1962).
S.Ozeris and R. Bassette, Anal. Chem., 35, 1091 (1963). G. Machata, Clin. Chem. News/., 4, No. 2, Winter, 1972. B. E. Coldwell. G. Solomonraj, H. L. Trenholm, and G. S.Wiberg, Ciin. rowicoi., 4, 99 (1971). G. A. Brown, D. Neylan. W. J. Reynolds, and K. W. Smalldon, Anal. Chim. Acta, 88, 271 (1973). K. W. Smalldon and G. A. Brown, Anal. Chim. Acta, 88, 285 (1973). T. U. Marron and J. J. Hilbe, Proc. Iowa Acad. Sci., 47, 225 (1940). F. Lundquist and H. Wolthers. Acta Pharmacoi. Towicoi., 14, 265 (1958). R. D. Hawkins and H. Kaiant, Pharrnacol. Rev., 24, 67 (1972). R. N. Harger, E. B. Raney. E. G. Bridwell, and M. F. Kitchel, J. Biol. Chem., 183, 197 (1950). D. B. Breimer. H. C. J. Ketelaars, and J. M. VanRossum, J. Chromatogr., 88, 55 (1974).
RECEIVEDfor review January 29, 1975. Accepted May 12, 1975. P.K.W. is a Robert Lincoln McNeil Memorial Fellow, American Foundation for Pharmaceutical Education. Work supported by Grant No. 1R01AA00683-01A1 from the National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health.
Determination of Arsenic and Antimony in Environmental Samples Using Gas Chromatography with a Microwave Emission Spectrometric System Yair Talmi and V. E. Norvelll Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830
The applicability of a gas chromatograph with a microwave emission spectrometric detector (GC-MES) to the determination of As and Sb in environmentally-based samples Is described. The analytical procedure is based on cocrystalllzatlon of As3+ and Sb3+ with thlonalld and reaction of the precipitate with phenylmagneslum bromide (PMB). Following the decomposition of excess PMB, the trlphenyi arsine and stiblne formed are extracted Into ether and separated on a GC column. Atomic emisslon detection of As and Present address, D e p a r t m e n t of Chemistry, U n i v e r s i t y of T e n nessee, Knoxville, Tenn. 37916.
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ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
Sb is then accomplished by a MES detector, attached to the GC column outlet. Various parameters affecting the dlgestlon, cocrystalllzatlon, precipitate recovery, and phenylation are discussed, along with some Instrumental problems such as GC column deterlorizatlon and capillary contamination. The detection limits for As and Sb are 20 and 50 pg, respectively, and the relative sensltlvitles are 50 and 125 ng/l. for water samples and 30 and 75 nglgram for solid samples. Samples analyzed included biological and plant tissues, coal and fly ash, and fresh and salt water. The relative error ranges from 1.7 to lq?h and the relative standard deviation from 2.6 to 7.1%.
