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Acetaldehyde accumulation during headspace gas-chromatographic determination of ethanol
Forensic Science International,
21
20 (1982) 21 - 25
ACETALDEHYDE ACCUMULATION DURING HEADSPACE CHROMATOGRAPHIC DETERMINATION OF ETHANOL
GAS-
M. CHIAROTTI and N. De GIOVANNI Zstituto di Medicina Legale e delle Assicurazioni, Rome (Italy)
Universitci Cattolica de1 Sacro Cuore,
(Received June 6, 1981; in revised form September 25, 1981; accepted October 12,198l)
Summary The acetaldehyde accumulation in blood during the equilibration time of samples processed by gas-liquid chromatographic headspace technique is reported. The quantitative loss of blood ethanol concentration is recorded at various temperatures and times of the preheating process. When the equilibration process is performed at 40 “C, minimal errors, due to ethanol oxidation, are observed.
Introduction Today, the quantitative analysis of ethanol in blood is mainly performed by headspace gas-chromatographic technique [ 1 - 41. This method, which is very rapid, specific and sensitive, is also employed routinely by ‘automatic headspace sampler’ devices [ 51. Headspace GLC technique has also been successfully employed for the determination of ethanol in capillary blood samples [6, 71. The choice of
this biological fluid is advisable because the ethanol concentration blood more closely approximates to that in arterial blood [8] .
in capillary
However it has been noted that acetaldehyde is produced in blood samples during the preheating process of the headspace analysis [9]. As this phenomenon. could be a serious limitation on the use of this method for forensic toxicology purposes, experiments have been carried out to evaluate the importance of the acetaldehyde production and to eliminate this drawback.
Materials and methods Blood blood bank Blood by adding
samples were taken from a pool of human blood provided by the of Policlinico Gemelli of Rome. alcohol concentrations (BAC) of 0.05% and 0.1% were prepared to the blood known amounts of 10% wt/vol ethanol solution as
0379-0738/82/0000-0000/$02.75
@ Elsevier Sequoia/Printed in The Netherlands
Fig. 1. Graphical representation preheating times at 60 “C.
of BAC decrease and acetaldehyde accumulation
during
suggested by Karnitis and Porter [lo]. The internal standard used was npropanol (0.05% aqueous solution for 0.05% BAC analysis and 0.1% for 0.1% BAC). Forty microliters of samples were placed in 5 ml glass vials with an equal volume of internal standard. The vials were immediately sealed with a flange-type rubber stopper and an aluminium seal, using a manual crimper, and were then placed in an automatic headspace sampler (Carlo Erba HS Sampler 250). The analyses were performed while maintaining the waterjacket temperature at 40, 50 and 60 OC, taking 1 ml headspace from eight different samples for each BAC every ten minutes for two hours of equilibration time. The vapor phase was introduced into a Carlo Erba Fractovap
23
2300 gas-liquid chromatography apparatus fitted with hydrogen flame ionization detector. A U-shaped glass column, 2 m X 4 mm, packed with styrenedivinylbenzene polymer (Poropak Q) was used. The analytical conditions were: column temperature, 160 “C; detector temperature, 200 “C; carrier gas (nitrogen) flow 30 ml/min. The concentration of ethanol was determined by the peak height ratio method.
Results and discussion The results are reported in Fig. 1 and Tables 1 and 2. Acetaldehyde is detectable in blood samples free of ethanol but the concentration is veryr low; it increases for 40 minutes during the preheating and then remains constant; it is probably the acetaldehyde normally present in red cells [ 1 l] . In addition to this basic amount, increased quantities of acetaldehyde have been observed in blood samples with BAC of 0.05% and 0.1%. At 40 “C acetaldehyde production was not significant and it was comparable with that observed in ethanol free samples. Under these conditions no ethanol decrease was noted either. When the equilibration was performed over 40 “C, the acetaldehyde accumulation was ,associated with a progressive lowering of ethanol concentration by oxyhemoglobin-mediated mechanism as referred to by Smalldon and Brown [ 121. When the equilibration was carried out beyond 50 ‘C!, we observed a significant increase in the acetaldehyde peak which was proportional to the preheating time. To evaluate the significance of the decrease in ethanol, the Student’s ttest was applied to the mean values obtained after 50, 90 and 120 minutes of equilibration and those obtained after 10 minutes. The BAC (0.05%) decrease is appreciable at 90 minutes, both at 50 and 60 “C, (p < 10% and p = 15% respectively) and after 120 minutes becomes more evident (p < 0.5% and p = 0.5%) (Table 1). This is in agreement with the data of Wilkinson et al. [9], who also reported the accumulation of acetaldehyde in samples heated at 60 “C. However no data are reported about the accumulation of acetaldehyde during the preheating time. Brown et al. [13] also reported acetaldehyde production in blood maintained at 62 ‘C, but there are no data for temperatures between 37 “C and 62 “C. The use of headspace automatic samplers can be the cause of serious errors if the heating temperature is 50 “C or higher, because the time elapsed between the first and the last sample could be two hours or more. The BAC failure at 60 “C could be very important as shown in Table 2: e.g., an error of 20% of BAC was observed in samples of 0.05% concentration maintained for 120 minutes at this temperature. In conclusion, preheating at 45 “C or lower, is advisable when an automatic headspace sampler is used, and when capillary blood samples are used.
60°C
5ooc
0) b) c) d) e)
t
;;;;,e)
peak
t
peak
z:cY
t
"r:;:$
peak
t
peak 8,Zos)
0.0070
-
0.0325
-
-
0.000v
-
0.0000
50'
-
0.0000
-
0.0000
90'
-
0.0000
-
0.0000
120'
-
0.1428
-
0.0145
50'
-
0.1428
-
0.1450
90'
-
0.1428
-
0.1450
120'
Blood ssr~iples with O.OO'j. BAC Equilibration time
10'
-
O.OUOL
-
o.ooco
10'
o.oo$ BAc_ Equilibrstlon time
Ethanol/n-Propanol Mean of 8 determinations (from 8 different vials) Standard error Student's t Acetaldehyde/n-hopanol
Temperature
6OoC
5ooc
Temperature
response
0.7292
to.0424
0.9422
1.0068
0.6683 +0.0355
1.6096
10'
+0.0179
0.0444
ml:;;;
50'
-2.7615
0.1011 +0.0096
O.ObOO*)
0.0145 +0.0028
90'
-6.5818
0.1983 20.0149
-1.2344
_ 0.0242 +0.0048
-7.9604
0.2246 +0.0137
-1.7423
_ 0.0412 +0.0100
120'
2.9087
0.7955 $0341
3.1413
0.7769 +0.0105
120'
Blood samples with 0.05$ BAC Equilibration time
ACIYCALDEHYDEACCUMULATION
0.9680 +0.0180
0.0600*)
+0.0174
O' 0.7701 + 0.0272
9
0.05$ BAC Equilibration time
ETBANOL CONCENTRAl'ION
Effect of equilibration time at 50 “C!and 60 “C on gas-liquid chromatography
TABLE 1
0.0323 +0.0107 _
o.oocJo +0.0060
10'
0.0234
+0.0368
0.7762
-0.4059
0.8409 _ ?? 0.0199
50'
0.5229
+0.0098
0.7691
1.3322
0.7855 to.0146
g0'
120'
3.3258
+0.0145
0.7104
1.1329
+0.0123
0.7928
-1.7625
0.0505 +0.0116
0.0184 ?? 0.0037
50'
-3.6834
0.0748 +0.0095
-0.8503
-4.3326
0.0930 +0.0117
-2.6178
+0.0~23 _
_ +0.0045
120' 0.03oc
0.0234
90'
Blood samples with 0.10% BAC Equilibration time
+0.0123
0.7773
0.8271 e.0275
10'
0.14 BAC Equilibration time
25 TABLE
2
BAG decrease after equilibration of blood samples at 50 “C and 60
Blood alcohol concentration
Equilibration time (minutes)
Blood alcohol concentration (%)
(%)
60
10 50 90 120
0.100 0.099 0.098 0.091
0.050 0.048 0.044 0.040
50
10 50 90 120
0.100 0.098 0.095 0.096
0.050 0.049 0.047 0.046
Temperature (“C)
‘C.
The ethanol evaporation from blood volumes of 40 ~1 at 45 “C, is obviously less than that obtained at 60 “C, but enough to be detected by the usual gasliquid chromatography apparatus. References 1 A. A. Ban’Kowskii, M. N. Sadovnik and V. I. Satanovskaia, Determination of ethanol and acetaldehyde in biological media by a method of gas-liquid chromatography in the gas vapor phase. Lab. Delo., 2 (1980) 89. N. C. Jain and R. H. Cravey, A review of breath alcohol methods. J. Chromatogr. Sci., 12 (1974) 214. N. C. Jain and R. H. Cravey, Analysis of alcohol. II. A review of gas-chromatographic methods. J. Chromatogr. Sci., 10 (1972) 263. B. L. Glendening and R. A. Harvey, A simple method using headspace gas for determination of blood alcohol by gas-chromatography. J. Forensic Sci., 14 (1969) 136. T. Meyer, Determination of alcohol in biological samples by headspace gas chromatography using a glass capillary column. Acta Pharmacol. Toxicol., 43 (1978) 164. M. F. Mason and K. M. Dubowski, Breath alcohol analysis: uses, methods, and some forensic problems - review and opinion. J. Forensic Sci., 21 (1976) 9. M. F. Mason and K. M. Dubowski, Alcohol, traffic, and chemical testing in the United States: a resume and some remaining problems. Clin. Chem., 20 (1974) 126. R. B. Forney, F. W. Hughes, R. N. Harger and A. B. Richards, Alcohol distribution in the vascular system. Concentration of orally administered alcohol in blood from various points in the vascular system and in the rebreathed air. Quart. J. Stud. Ale., 25 (1964) 205. 9 P. K. Wilkinson, J. G. Wagner and A. J. Sedman, Sensitive headspace gas-chromatographic method for the determination of ethanol utilizing capillary blood samples. Anal. Chem., 47 (1975) 1506. 10 L. Karnitis and L. J. Porter, A gaschromatographic method for ethanol determination in vapors of biological fluids. J. Forensic Sci., 17 (1972) 318. 11 H. Matthies, Die Wirkung von Aldehyden auf die Metharmoglobinriickbildung in Erythrocyten. Biochem. Z., 329 (1957) 341. 12 K. W. Smalldon and G. A. Brown, The stability of ethanol in stored blood. Part 11: The mechanism of ethanol oxidation. Anal. Chim. Acta, 66 (1973) 285. 13 G. A. Brown, D. Neylon, W. J. Reynolds and K. W. Smalldon, The stability of ethanol in stored blood. Part I: Important variables and interpretation of results. Anal. Chim. Acta, 66 (1973) 271.
21
20 (1982) 21 - 25
ACETALDEHYDE ACCUMULATION DURING HEADSPACE CHROMATOGRAPHIC DETERMINATION OF ETHANOL
GAS-
M. CHIAROTTI and N. De GIOVANNI Zstituto di Medicina Legale e delle Assicurazioni, Rome (Italy)
Universitci Cattolica de1 Sacro Cuore,
(Received June 6, 1981; in revised form September 25, 1981; accepted October 12,198l)
Summary The acetaldehyde accumulation in blood during the equilibration time of samples processed by gas-liquid chromatographic headspace technique is reported. The quantitative loss of blood ethanol concentration is recorded at various temperatures and times of the preheating process. When the equilibration process is performed at 40 “C, minimal errors, due to ethanol oxidation, are observed.
Introduction Today, the quantitative analysis of ethanol in blood is mainly performed by headspace gas-chromatographic technique [ 1 - 41. This method, which is very rapid, specific and sensitive, is also employed routinely by ‘automatic headspace sampler’ devices [ 51. Headspace GLC technique has also been successfully employed for the determination of ethanol in capillary blood samples [6, 71. The choice of
this biological fluid is advisable because the ethanol concentration blood more closely approximates to that in arterial blood [8] .
in capillary
However it has been noted that acetaldehyde is produced in blood samples during the preheating process of the headspace analysis [9]. As this phenomenon. could be a serious limitation on the use of this method for forensic toxicology purposes, experiments have been carried out to evaluate the importance of the acetaldehyde production and to eliminate this drawback.
Materials and methods Blood blood bank Blood by adding
samples were taken from a pool of human blood provided by the of Policlinico Gemelli of Rome. alcohol concentrations (BAC) of 0.05% and 0.1% were prepared to the blood known amounts of 10% wt/vol ethanol solution as
0379-0738/82/0000-0000/$02.75
@ Elsevier Sequoia/Printed in The Netherlands
Fig. 1. Graphical representation preheating times at 60 “C.
of BAC decrease and acetaldehyde accumulation
during
suggested by Karnitis and Porter [lo]. The internal standard used was npropanol (0.05% aqueous solution for 0.05% BAC analysis and 0.1% for 0.1% BAC). Forty microliters of samples were placed in 5 ml glass vials with an equal volume of internal standard. The vials were immediately sealed with a flange-type rubber stopper and an aluminium seal, using a manual crimper, and were then placed in an automatic headspace sampler (Carlo Erba HS Sampler 250). The analyses were performed while maintaining the waterjacket temperature at 40, 50 and 60 OC, taking 1 ml headspace from eight different samples for each BAC every ten minutes for two hours of equilibration time. The vapor phase was introduced into a Carlo Erba Fractovap
23
2300 gas-liquid chromatography apparatus fitted with hydrogen flame ionization detector. A U-shaped glass column, 2 m X 4 mm, packed with styrenedivinylbenzene polymer (Poropak Q) was used. The analytical conditions were: column temperature, 160 “C; detector temperature, 200 “C; carrier gas (nitrogen) flow 30 ml/min. The concentration of ethanol was determined by the peak height ratio method.
Results and discussion The results are reported in Fig. 1 and Tables 1 and 2. Acetaldehyde is detectable in blood samples free of ethanol but the concentration is veryr low; it increases for 40 minutes during the preheating and then remains constant; it is probably the acetaldehyde normally present in red cells [ 1 l] . In addition to this basic amount, increased quantities of acetaldehyde have been observed in blood samples with BAC of 0.05% and 0.1%. At 40 “C acetaldehyde production was not significant and it was comparable with that observed in ethanol free samples. Under these conditions no ethanol decrease was noted either. When the equilibration was performed over 40 “C, the acetaldehyde accumulation was ,associated with a progressive lowering of ethanol concentration by oxyhemoglobin-mediated mechanism as referred to by Smalldon and Brown [ 121. When the equilibration was carried out beyond 50 ‘C!, we observed a significant increase in the acetaldehyde peak which was proportional to the preheating time. To evaluate the significance of the decrease in ethanol, the Student’s ttest was applied to the mean values obtained after 50, 90 and 120 minutes of equilibration and those obtained after 10 minutes. The BAC (0.05%) decrease is appreciable at 90 minutes, both at 50 and 60 “C, (p < 10% and p = 15% respectively) and after 120 minutes becomes more evident (p < 0.5% and p = 0.5%) (Table 1). This is in agreement with the data of Wilkinson et al. [9], who also reported the accumulation of acetaldehyde in samples heated at 60 “C. However no data are reported about the accumulation of acetaldehyde during the preheating time. Brown et al. [13] also reported acetaldehyde production in blood maintained at 62 ‘C, but there are no data for temperatures between 37 “C and 62 “C. The use of headspace automatic samplers can be the cause of serious errors if the heating temperature is 50 “C or higher, because the time elapsed between the first and the last sample could be two hours or more. The BAC failure at 60 “C could be very important as shown in Table 2: e.g., an error of 20% of BAC was observed in samples of 0.05% concentration maintained for 120 minutes at this temperature. In conclusion, preheating at 45 “C or lower, is advisable when an automatic headspace sampler is used, and when capillary blood samples are used.
60°C
5ooc
0) b) c) d) e)
t
;;;;,e)
peak
t
peak
z:cY
t
"r:;:$
peak
t
peak 8,Zos)
0.0070
-
0.0325
-
-
0.000v
-
0.0000
50'
-
0.0000
-
0.0000
90'
-
0.0000
-
0.0000
120'
-
0.1428
-
0.0145
50'
-
0.1428
-
0.1450
90'
-
0.1428
-
0.1450
120'
Blood ssr~iples with O.OO'j. BAC Equilibration time
10'
-
O.OUOL
-
o.ooco
10'
o.oo$ BAc_ Equilibrstlon time
Ethanol/n-Propanol Mean of 8 determinations (from 8 different vials) Standard error Student's t Acetaldehyde/n-hopanol
Temperature
6OoC
5ooc
Temperature
response
0.7292
to.0424
0.9422
1.0068
0.6683 +0.0355
1.6096
10'
+0.0179
0.0444
ml:;;;
50'
-2.7615
0.1011 +0.0096
O.ObOO*)
0.0145 +0.0028
90'
-6.5818
0.1983 20.0149
-1.2344
_ 0.0242 +0.0048
-7.9604
0.2246 +0.0137
-1.7423
_ 0.0412 +0.0100
120'
2.9087
0.7955 $0341
3.1413
0.7769 +0.0105
120'
Blood samples with 0.05$ BAC Equilibration time
ACIYCALDEHYDEACCUMULATION
0.9680 +0.0180
0.0600*)
+0.0174
O' 0.7701 + 0.0272
9
0.05$ BAC Equilibration time
ETBANOL CONCENTRAl'ION
Effect of equilibration time at 50 “C!and 60 “C on gas-liquid chromatography
TABLE 1
0.0323 +0.0107 _
o.oocJo +0.0060
10'
0.0234
+0.0368
0.7762
-0.4059
0.8409 _ ?? 0.0199
50'
0.5229
+0.0098
0.7691
1.3322
0.7855 to.0146
g0'
120'
3.3258
+0.0145
0.7104
1.1329
+0.0123
0.7928
-1.7625
0.0505 +0.0116
0.0184 ?? 0.0037
50'
-3.6834
0.0748 +0.0095
-0.8503
-4.3326
0.0930 +0.0117
-2.6178
+0.0~23 _
_ +0.0045
120' 0.03oc
0.0234
90'
Blood samples with 0.10% BAC Equilibration time
+0.0123
0.7773
0.8271 e.0275
10'
0.14 BAC Equilibration time
25 TABLE
2
BAG decrease after equilibration of blood samples at 50 “C and 60
Blood alcohol concentration
Equilibration time (minutes)
Blood alcohol concentration (%)
(%)
60
10 50 90 120
0.100 0.099 0.098 0.091
0.050 0.048 0.044 0.040
50
10 50 90 120
0.100 0.098 0.095 0.096
0.050 0.049 0.047 0.046
Temperature (“C)
‘C.
The ethanol evaporation from blood volumes of 40 ~1 at 45 “C, is obviously less than that obtained at 60 “C, but enough to be detected by the usual gasliquid chromatography apparatus. References 1 A. A. Ban’Kowskii, M. N. Sadovnik and V. I. Satanovskaia, Determination of ethanol and acetaldehyde in biological media by a method of gas-liquid chromatography in the gas vapor phase. Lab. Delo., 2 (1980) 89. N. C. Jain and R. H. Cravey, A review of breath alcohol methods. J. Chromatogr. Sci., 12 (1974) 214. N. C. Jain and R. H. Cravey, Analysis of alcohol. II. A review of gas-chromatographic methods. J. Chromatogr. Sci., 10 (1972) 263. B. L. Glendening and R. A. Harvey, A simple method using headspace gas for determination of blood alcohol by gas-chromatography. J. Forensic Sci., 14 (1969) 136. T. Meyer, Determination of alcohol in biological samples by headspace gas chromatography using a glass capillary column. Acta Pharmacol. Toxicol., 43 (1978) 164. M. F. Mason and K. M. Dubowski, Breath alcohol analysis: uses, methods, and some forensic problems - review and opinion. J. Forensic Sci., 21 (1976) 9. M. F. Mason and K. M. Dubowski, Alcohol, traffic, and chemical testing in the United States: a resume and some remaining problems. Clin. Chem., 20 (1974) 126. R. B. Forney, F. W. Hughes, R. N. Harger and A. B. Richards, Alcohol distribution in the vascular system. Concentration of orally administered alcohol in blood from various points in the vascular system and in the rebreathed air. Quart. J. Stud. Ale., 25 (1964) 205. 9 P. K. Wilkinson, J. G. Wagner and A. J. Sedman, Sensitive headspace gas-chromatographic method for the determination of ethanol utilizing capillary blood samples. Anal. Chem., 47 (1975) 1506. 10 L. Karnitis and L. J. Porter, A gaschromatographic method for ethanol determination in vapors of biological fluids. J. Forensic Sci., 17 (1972) 318. 11 H. Matthies, Die Wirkung von Aldehyden auf die Metharmoglobinriickbildung in Erythrocyten. Biochem. Z., 329 (1957) 341. 12 K. W. Smalldon and G. A. Brown, The stability of ethanol in stored blood. Part 11: The mechanism of ethanol oxidation. Anal. Chim. Acta, 66 (1973) 285. 13 G. A. Brown, D. Neylon, W. J. Reynolds and K. W. Smalldon, The stability of ethanol in stored blood. Part I: Important variables and interpretation of results. Anal. Chim. Acta, 66 (1973) 271.