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Direct vapor generation Fourier transform infrared spectrometric determination of ethanol in blood
ANALYTICA CHIMICA ACTA
ELSEVIER Analytica
Chimica
Acta 336 (1996) 123-I 29
Direct vapor generation Fourier transform infrared spectrometric determination of ethanol in blood Amparo Pkez-Ponce, Universiry of Valencia, Department
Salvador Garrigues, of Analytical
Miguel de la Guardia*
Chemistry, 50 DI: Moliner St., 46100
Received 3 June 1996; accepted
9 August
Burjassot,
Valencia, Spain
1996
Abstract A new procedure is proposed for a direct determination of ethanol in plasma and whole blood. The method is based on the injection of a discrete sample volume of 10~1 into an electrically heated Pyrex glass reactor in which, at a temperature of 90°C the ethanol is volatilized and introduced by means of a N2 carrier flow inside a long-path infrared gas cell and the corresponding flow analysis recording registered as a function of time. The measurement of the area of the flow injection recording, obtained from the absorbance of the transient signal in the range 1150-950 cm-’ , allows the direct quantification of ethanol upto 2g 1-l. with a limit of detection of O.O2Ogl-’ and coefficient of variation between 0.3 and 1.9% for three replicate analyses of the same sample. The analysis frequency of the method is 40 hh’, and it can be applied to a single drop of
finger blood. Keywords;
Infrared spectrometry;
Fourier transform;
Flow injection;
Vapor generation;
1. Introduction Fourier transform infrared (FTIR) sectrometry is a rapid analytical technique that provides very interesting qualitative information [l-3] and, as has been clearly established [4-81, is a very useful tool for accurate quantitative analysis without requiring complex sample preparation procedures. The FTIR quantitative analysis can be carried out on solid, liquid and gaseous samples, the determinations in gaseous or vapor phase being highly sensitive due to the transparency of gases, low background values and possibilities offered by the use of multi-
* Corresponding
author. Fax: (+34) 6 386 43 22.
OOO3-2670/96/$15.00 @> 1996 Elsevier Science B.V. All rights resen fed PII SOOO3-2670(96)00386-8
Ethanol determination;
Blood
ple-pass cells [9,10]; the only drawback is the fact’ that the gaseous state is not so frequent in real samples to be analyzed. Our research group has developed a simple system for the generation of vapor phases from liquid samples which can be adapted to any FTIR spectrometer equipped with a gas cell. This system permits us to obtain transient FTIR signals in the gas phase from samples injected directly through a septum into a heated Pyrex glass reactor, the vapor phase generated inside the reactor being transported away by means an inert carrier gas flow. Our early applications of this system were focused on the analysis of real samples including the determination of ethanol in chloroform (where it is added as a protective agent to avoid phosgene
124
A. Phrez-Ponce et al./Analytica Chimica Acta 336 (1996) 123-129
formation) [ 111, the direct determination of benzene, toluene and methyl t-butyl ether in gasolines [ 121 and the determination of ethanol in alcoholic beverages [13]. This last application suggested the tremendous possibility of this system to develop a simple procedure for the determination of ethanol in complex matrices based on the selective vaporization of ethanol. So, in this paper, the aforementioned methodology has been employed for direct blood analysis. The determination of ethanol in blood is very important, especially in establishing responsibilities in traffic accidents. The upper permissible concentration levels of ethanol in blood for drivers depends on different national laws; for example, in Spain the upper limit is 0.8 g of ethanol in 11. The determination of ethanol in blood is usually carried out, after previous treatment, by headspace gas chromatography using a mass-spectrometric detector [14] or a flame ionization detector [ 151. This paper describes the development a simple method for FTIR spectrometric determination of ethanol in blood by direct injection of samples in a volatilizer and measurement in the vapor phase, in order to avoid the interference of the sample matrix components. Furthermore, the measurements were made at 1050 cm-’ to increase the selectivity, this corresponds to the C-O stretching vibration in ethanol and has been demonstrated to be useful for breath analysis [ 151.
2. Experimental
2.1. Instruments A Magna 550 FTIR spectrometer (Nicolet, Madison, WI), equipped with a temperature-stabilized DTGS detector, a long-lasting Ever-G10 source and a KBr beamsplitter was employed for spectral measurements with a nominal resolution of 8cm-’ using a Wilmad (Buena, USA) ultramini longpath cell, Model 3.2, with a volume of lOOm1 and a permanently aligned multiple band-pass of 3.2m, equipped with a ZnSe window. OMNIC software was used to control the instrument, for data acquisition and also for processing the analytical results.
The manifold employed for the vapor generation FTIR measurements [ 1l-131 was a single-channel assembly with a nitrogen carrier, which includes a volatilization reactor of 2.5 ml internal volume with inlet and outlet. Samples are injected inside the reactor through a septum using Hamilton (Reno, NY) gas-tight syringes, Model 1701, of different volumes with a removable needle (0.26mm i.d.) having a bevelled point with a 17” bevel, style 1. The temperature of the reactor was measured by means of a thermocouple and operated using an electrically controlled heater, Model AK0 (Barcelona). The reactor is connected with the IR gas cell using Viton (Isoversinic) tubes of 4 mm i.d.. 2.2. Chemicals Analytical-reagent grade absolute ethanol (99.5%) from Panreac (Barcelona), was employed for the preparation of standards and nitrogen gas (C-45), from Carburos Metalicos (Barcelona) was employed as the carrier gas. 2.3. General procedure 10 ul of untreated blood or plasma sample was injected into the open 2.5 ml reactor, preheated at 90°C and the vapor phase generated was transported inside the gas cell of the FTIR spectrometer using a carrier nitrogen flow of 400mlmin-i. In some experiments, a glass-bead packed reactor of 5.5 ml internal volume and 8.5 ml dead volume was employed in order to retain the partially decomposed matrix and to avoid clogging of the small reactor (see Fig. 1 for details). The Gram-Schmidt plot, which is a representation of the infrard (IR) intensity arriving at the detector as a function of time, is obtained for the transient signals generated after the injection of samples. Each GramSchmidt point corresponds to a FAIR spectrum in which an appropriate spectral range corresponding to absorption by ethanol can be selected. With the absorbance data found in the 1150-950 cm-’ range, an appropriate chemigram corresponding to the ethanol signal can be constructed. The chemigram provides a flow-injection (FI) recording which corresponds to the absorbance of ethanol, in the selected wave-number range. The area of the peaks
A. P&ez-Ponce
et al./Analytica
125
Chimica Acta 336 (1996) 123-129
a
r( Septum couple Electically controled heater Flow controled
;In
carrier .
-1
Reactor
1
1 Waste
Fig. I. Manifold
employed
for vapor generation
FHR spectrometry.
were employed as the quantitative analytical variable, taking into consideration a baseline correction established between the two troughs found before and after sample injection, in order to minimize the noise. Data obtained for samples were interpolated on the corresponding calibration graph established for aqueous standard solutions of ethanol, injected in the same way as the samples.
3. Results and discussion 3. I. Vapor-phase FTIR spectra generated from aqueous standards and samples
The injection of 10 11 of an aqueous standard of 1.2gl ’ ethanol in a carrier flow of 400 ml min-’ N2 provides a Gram-Schmidt recording of approximately two and a half minutes, the absorbance being is predominately that of the generated water vapor (see FIIR spectra found after 1.206 and 2.004 min in
Inset (a) glass-bead
packed reactor, and (b) open reactor.
Fig. 2). However, 0.724min after injection the spectrum of the vapor phase corresponds to a mixture of ethanol and water, as can be seen in Fig. 2. Considering the spectral range between 115s 950 cm-‘, which includes the main characteristic bands of ethanol (C-O), the chemigram depicted in Fig. 2 was obtained. It shows that after 0.724 min, the FI recording for ethanol reaches the maximum value and in 1.206 min has returned to the baseline. Thus, fast and easily measurable results can be found from the treatment of the output corresponding to a small wave-number range. To establish the baseline, the average value of the noise found before and after the peak can be employed (see Fig. 2). The injection of a sample of plasma, containing 1.2 g ll’ ethanol, provides Gram-Schmidt and chemigram recordings comparable to those found for aqueous standards. Fig. 3 depicts, as an example, the spectra obtained on the maximum of the corresponding outputs for two samples, one with and another without ethanol. The band of ethanol at 1050cmP’
126
A. P&z-Ponce
et al./Analytica
Chimica Acta 336 (1996) 123-129
chemigram \
‘I
”
baselin6’
\--
F-c;u
\ r \’
Time (minutes) Fig. 2. Gram-Schmidt recording (from 2000 to 500 cm-‘) and chemigram (from 1150 to 950 cm-‘) provided by the injection of 10 ul of an aqueous standard of ethanol and the spectra obtained in the vapor phase at different time values: (A) 0.724min. (B) 1.206min, and (C) 2.CMMmin. Experimental conditions: ethanol concentration 1.2 g ll’, reactor temperature 90°C carrier flow 400 ml mini’ and resolution 8 cm-‘.
contaminated by water. Thus, it can be concluded that both real samples and aqueous standards of ethanol can be injected directly into the reactor and the corresponding absorbance signals treated in the 1150-950 cm-’ range providing a sample throughout frequency of the order of 40 hh’ without matrix effects from proteins (which remain inside the reactor without being volatilized) and water (which is evolved more slowly). For quantitative analysis the area values provide better sensitivity and reproducibility than the use of peak height values. Fig. 3. Vapor phase FAIR spectra of plasma samples. (A) spectrum of a plasma without ethanol and (B) spectrum of a plasma sample containing 1.2 g 1-l ethanol. Inset: amplification of the 1200-8OCJcm~’ range. Experimental conditions: injection volume 10 ~1, reactor temperature 90°C carrier flow 400 ml mini’ and resolution 8 cm-‘.
can clearly be identified because it is far from the water absorption range and these signals are obtained for periods
3.2. Effect of experimental parameters Carrier-gas flow rate, reactor temperature and injection volume are the main parameters which influence the size and shape of the peaks obtained for ethanol by the injection of plasma samples. An flow rate dramatically increase of N2 carrier decreases the sensitivity of the vapor generation
A. Pe’rez-Ponce et al./Analytica
04 Carrie4r”
flow
(ml
12bo
6
“2k’)
Fig. 4. Effect of the N2 carrier flow on the chemigram areas. Experimental conditions: ethanol concentration 0.8 g I-‘, injection volume 10 ~1, reactor temperature 90°C and resolution 8cm-‘.
FTIR spectrometric determination of ethanol, as can be seen in Fig. 4, which shows that an increase of the carrier flow rate sharply decreases the sensitivity. Carrier flow rates <400mlmin-’ are the most appropriate to obtain sensitive and reproducible measurements. However, it must be taken into account that the use of slow carrier-gas flows involves an increase of the volatilization time, thus reducing the sampling throughput. Throughout this study, a flow rate of 400ml min-’ was chosen as a compromise between sensitivity and speed of analysis. Although the boiling temperature of ethanol is 78.17”C, sensitive measurements can be obtained between 60 and 110°C. Table 1 summarizes the data obtained for ethanol at different temperatures. The use of higher temperatures increases the sampling frequency from 20hP’ at 60°C to 40 hh’ at 110°C.
Table 1 Effect of volatilization
(“C)
Coefficient variation of (%)
Sample throughput
60 70 80 90 100 110
5.250.1 6.1f0.5 5.9f0.3 6.5&O. 6.0f0.2 6.3fO.l
2.5 8.2 5.1 1.2 3.5 1.9
20 24 27 34 34 40
‘Mean&d.
(n=4)
127
However, the sensitivity remains constant in all the temperature ranges assayed, of the order of 6 area units gg ’ 1; the repeatability of the area values in general was better for temperatures higher than 80°C as compared to temperatures below the boiling point of ethanol (see Table 1). A value of 90°C has been selected from these experiments to obtain the best sensitivity and repeatability. An increase of the injected sample volume obviously increases the back area for a fixed ethanol concentration, thus improving the sensitivity of the analytical determinations. Fig. 5 shows the effect of injection volume, from 2.5-17.5 ul and it can be seen that there is a good linear regression between area values and injection volume (r=O.993). The graph indicates that the results are not affected by contamination, which could affect the blank value, probably because samples are injected directly without adding any reagent. Besides, this system is free from memory effects. The good calibration linearity shows that the heating conditions are appropriate for obtaining complete volatilization of both small and large sample volumes, thus offering exciting possibilities for improving the sensitivity by means of the use of large injection volumes. However, on comparing the sample throughput values found for different injection volumes, it can be seen that it is reduced from 60 hh’ for 2.5 ul to 40 hh’ for 17.5 ~1. A value of
14 ,
I
temuerature
Sensitivity (area units g-11)”
Temperature
Chimica Acta 336 (1996) 123-129
I
(hh’)
2y// 0
I,1
0
,,.,li,
5
1,/I,,,
10
15
20
Injection volume @I) Fig. 5. Effect of the injection volume on chemigram area of a solution of 0.8 g 1. ’ ethanol. Experimental conditions: temperature 90°C carrier flow 402 ml min-’ and resolution 8 cm -‘,
A. Pkrez-Ponce et al./Analytica
128
Chimica Acta 336 (1996) 123-129
10 ~1 (10 pl syringe) was chosen to reach a compromise between sensitivity and speed.
3.4. Analytical features of vapor generation FTIR spectrometry for ethanol determination in blood
3.3. Effect of the matrix The vapor-phase generation avoids matrix and spectral interferences because, working at a relativly low temperature, only ethanol and water are volatilized and transported by the carrier flow through the measurement cell. The matrix remains and decomposes inside the reactor and must be eliminated by cleaning the system after approximately every ten injections. To avoid this problem, additional experiments were carried out using another reactor with a high internal volume, which is occupied almost entirely by glass beads. In such a reactor, the residue remains between the beads and does not obstruct subsequent injections. So, a long series of samples can be injected without cleaning the reactor. However, results obtained at 90°C and 300mlmin-’ N2 carrier flow (see Table 2) indicate that the use of the packed reactor gives greater sensitivity than the unpacked reactor, but the limit of detection and sampling frequency are better with the unpacked reactor. Thus, it may be preferable to use the simple open reactor and to clean the system after a series of injections. Table 2 Comparison spectrometry
of reactors
employed
for vapour
generation
FIIR
(hh’)
Reactor
Sensitivity (area units gg’ 1)”
LOD (gll’)h
Sample throughput
Packed Unpacked
1.1210.06 0.87?~0.10
0.15 0.04
20 34
“Meants.d. (n=3). ‘LOD, limit of detection
(k=3) for a probability
Table 3 Figures of merit of the vapor-generation Matrix
Water Plasma Water
level of 99.86%.
FTIR spectrometric
determination
Table 3 summarizes the main features of the methodology developed for the determination of ethanol using vapor generation-FfIR spectrometry. In this table, calibration lines obtained with aqueous standards, and standards prepared with a plasma matrix, are reported. As can be seen, linear calibration plots found for plasma and aqueous solutions, under the same conditions, are comparable, and provide the same intercept and slope values and, as is evident from Table 1, the sensitivity can be increased by increasing the temperature from 80 to 90°C. The calibration lines have been determinated for the O-2 g 1-l concentration range in order to define an appropriate calibration for the quantitative analysis of samples corresponding to persons whose level of ethanol in blood could be around the maximum allowed for driving, which in Spain corresponds to 0.8 g lP ’ . So, under the recommended conditions, quantitative information about the ethanol level of drivers blood could be obtained in less than 2 min and requires only a few 10 ul of untreated blood. The limit of detection has been established from the repeatability of five measurements of a blank solution (water or plasma without ethanol). The limit of detection obtained, under the most sensitive conditions (90”(Z), corresponds to 0.020 g l-‘, indicating that the methodology developed could be applied to a wide range of ethanol concentrations in human blood, including persons with a level well below 0.8 g 1-l.
of ethanol
Flow carrier (ml min-‘)
Temperature
Calibration equation
r
(“C)
397 397 394
80 80 90
A=0.23+0.745C A=0.22+0.741 C A=0.13+0.905C
0.98 0.99 0.995
LOD (g 1-l)
0.16 0.22 0.020
Afs,
,
0.872f0.003 0.931&0.018 0.926ztO.010
Coefficient of variation
(%)
0.3 1.9 1.1
Calibration line: A, area; C, concentration of ethanol in g I-‘; r, regression coefficient (n=5); LOD, limit of detection (k3) for a probability level 99.86%; coefficient of variation for 4 analysis of a sample with a concentration of O.Sgl-’ of ethanol; A& ~,, area and the corresponding standard deviation of 4 independent measurements carried out on IO ul injections of a sample containing 0.8 g I-’ ethanol.
A. Pbez-Ponce
et al./Analytica
Table 4 Results obtained for the determination of ethanol in spiked samples by vapor-generation FTIR spectrometry Ethanol (gl
Sample
added&s,+, Plasma
0.40010.003 0.800*0.009 0.96OxtO.011
Blood
0.400*0.003 0.80010.009 0.960*0.011
‘)
Error (%)
a
found&. 0.41 f0.03 0.82*0.03 0.96&0.08 1.18+10.06 0.41 lto.03 0.78ztO.02 0.92ztO.02 1.12ztO.09
1.200*0.012
1.200+0.012 a Standard
deviation
3.5. Analysis
for n-1
,’ 2.5 2.5 0 -2.4 2.5 -2 4.2 -6.7
Chimica Acta 336 (1996) 123-129
129
allows direct analysis without any sample pretreatment and requires only an extremely small amount of sample which can be obtained from a finger puncture. The 0.02 g 1. ’ limit of detection found for small injection volumes, fast analysis of samples and avoidance of interference from low volatility sampIe components are the main characteristics of the simple procedure developed, which could also be applied for the direct determination of other easily volatile compounds in complex matrices.
Acknowledgements
degrees of freedom (n=3).
ofreal samples
To test the applicability of the procedure developed, a series of samples of human plasma and blood, spiked with ethanol at known concentrations, was analyzed, using in all cases aqueous solutions of ethanol as standards. Table 4 summarizes the results found over a concentration range 0.4-1.2 g I-‘. Quantitative recoveries were found in all cases, with relative errors lower than 2.5%. Errors of 4.6 and 6.7% were obtained only for whole blood analysis at concentration levels clearly higher than those allowed by law, thus ensuring certainty in the characterization of the samples. The analysis of a real unspiked blood sample was also carried out both on a small drop of blood from a finger puncture and 2ml of venous blood. Results found by vapor generation FTIR spectrometric determination of ethanol in these samples were 0.14+0.05g1-’ and 0.16*0.06 g 1-l (mean&s.d., n=5), respectively. This shows that the method could be applied with little injury to persons owing to the extremely small amount of sample required, althought the accuracy of the measurements was not tested.
4. Conclusions The method developed alternative to conventional ing ethanol in human blood these procedures, the vapor
is a rapid procedures or plasma. generation
and accurate for determinIn contrast to FTIR method
The authors acknowledge the financial support by the Conselleria de Educaci6n y Ciencia de la Generalitat Valenciana (project GV 1021/93) and the Spanish DGICYT (project PB 92-0870). A.P.P. acknowledges the grant of the Conselleria de Educaci6n y Ciencia de la G.V.
References M. Dien, Introduction Modern Vibrational Spectroscopy, Wiley, New York, 1993. L21 B. Schrader (Ed.), Infrared and Raman Spectroscopy, VCH, Weinheim, 1995. (31 G.A. Eiceman. R.E. Clement and H.H. Hiel. Anal. Chem.. 64 (1994) 70. 141 C.L. Putzig, M.A. Leugers, M.L. Mckelvy, G.E. Mitchell, R.A. Nyquist, R.R. Papenfuss and L. Yurga. Anal. Chem.. 66 ( 1994) 26-66. S. Garrigues and M. de la Guardia. Anal. I51 Y. Daghbouche, Chim. Acta, 314 (1995) 203-212. B.A. Hassan, S. Garrigues and M. de la 161 Z. Bouhsain, Guardia, Quim. Anal., 14 (1995) 96. I71 M. Gallignani, S. Garrigues and M. de la Guardia, Analyst, 119 (1994) 653. 181 S. Garrigues, M. Gallignani and M. de la Guardia, Talanta, 40 (1993) 1799. I91 P.L. Hanst, Fresenius’ J. Anal. Chem., 324 (1986) 579. []Ol T. Janatuinen and E. Byckhng, 1111.Lab., 15(7) (1985) 12. S. Garrigues and M. de la Guardia. Anal. 1111 E. L6pez-Anreus, Chim. Acta, 308 (1995) 28-35. S. Garrigues and M. de la Guardia. Anal. 1121 E. Lbpez-Anreus, Chim. Acta, in press. I131 A. PCrez-Ponce. S. Garrigueq and M. de la Guardia, Analyst, in press. [I41 B.K. Logan, R.G. Gullberg and J.K. Elenbash. J. Forensic Sciences, 34(4) (1994) ~107-1111. I151 CM Bell, S.J. Gutowski, S. Young and D. Wells, J. Anal. Toxicol.. 16 (1992) 166-168.
[]I
ELSEVIER Analytica
Chimica
Acta 336 (1996) 123-I 29
Direct vapor generation Fourier transform infrared spectrometric determination of ethanol in blood Amparo Pkez-Ponce, Universiry of Valencia, Department
Salvador Garrigues, of Analytical
Miguel de la Guardia*
Chemistry, 50 DI: Moliner St., 46100
Received 3 June 1996; accepted
9 August
Burjassot,
Valencia, Spain
1996
Abstract A new procedure is proposed for a direct determination of ethanol in plasma and whole blood. The method is based on the injection of a discrete sample volume of 10~1 into an electrically heated Pyrex glass reactor in which, at a temperature of 90°C the ethanol is volatilized and introduced by means of a N2 carrier flow inside a long-path infrared gas cell and the corresponding flow analysis recording registered as a function of time. The measurement of the area of the flow injection recording, obtained from the absorbance of the transient signal in the range 1150-950 cm-’ , allows the direct quantification of ethanol upto 2g 1-l. with a limit of detection of O.O2Ogl-’ and coefficient of variation between 0.3 and 1.9% for three replicate analyses of the same sample. The analysis frequency of the method is 40 hh’, and it can be applied to a single drop of
finger blood. Keywords;
Infrared spectrometry;
Fourier transform;
Flow injection;
Vapor generation;
1. Introduction Fourier transform infrared (FTIR) sectrometry is a rapid analytical technique that provides very interesting qualitative information [l-3] and, as has been clearly established [4-81, is a very useful tool for accurate quantitative analysis without requiring complex sample preparation procedures. The FTIR quantitative analysis can be carried out on solid, liquid and gaseous samples, the determinations in gaseous or vapor phase being highly sensitive due to the transparency of gases, low background values and possibilities offered by the use of multi-
* Corresponding
author. Fax: (+34) 6 386 43 22.
OOO3-2670/96/$15.00 @> 1996 Elsevier Science B.V. All rights resen fed PII SOOO3-2670(96)00386-8
Ethanol determination;
Blood
ple-pass cells [9,10]; the only drawback is the fact’ that the gaseous state is not so frequent in real samples to be analyzed. Our research group has developed a simple system for the generation of vapor phases from liquid samples which can be adapted to any FTIR spectrometer equipped with a gas cell. This system permits us to obtain transient FTIR signals in the gas phase from samples injected directly through a septum into a heated Pyrex glass reactor, the vapor phase generated inside the reactor being transported away by means an inert carrier gas flow. Our early applications of this system were focused on the analysis of real samples including the determination of ethanol in chloroform (where it is added as a protective agent to avoid phosgene
124
A. Phrez-Ponce et al./Analytica Chimica Acta 336 (1996) 123-129
formation) [ 111, the direct determination of benzene, toluene and methyl t-butyl ether in gasolines [ 121 and the determination of ethanol in alcoholic beverages [13]. This last application suggested the tremendous possibility of this system to develop a simple procedure for the determination of ethanol in complex matrices based on the selective vaporization of ethanol. So, in this paper, the aforementioned methodology has been employed for direct blood analysis. The determination of ethanol in blood is very important, especially in establishing responsibilities in traffic accidents. The upper permissible concentration levels of ethanol in blood for drivers depends on different national laws; for example, in Spain the upper limit is 0.8 g of ethanol in 11. The determination of ethanol in blood is usually carried out, after previous treatment, by headspace gas chromatography using a mass-spectrometric detector [14] or a flame ionization detector [ 151. This paper describes the development a simple method for FTIR spectrometric determination of ethanol in blood by direct injection of samples in a volatilizer and measurement in the vapor phase, in order to avoid the interference of the sample matrix components. Furthermore, the measurements were made at 1050 cm-’ to increase the selectivity, this corresponds to the C-O stretching vibration in ethanol and has been demonstrated to be useful for breath analysis [ 151.
2. Experimental
2.1. Instruments A Magna 550 FTIR spectrometer (Nicolet, Madison, WI), equipped with a temperature-stabilized DTGS detector, a long-lasting Ever-G10 source and a KBr beamsplitter was employed for spectral measurements with a nominal resolution of 8cm-’ using a Wilmad (Buena, USA) ultramini longpath cell, Model 3.2, with a volume of lOOm1 and a permanently aligned multiple band-pass of 3.2m, equipped with a ZnSe window. OMNIC software was used to control the instrument, for data acquisition and also for processing the analytical results.
The manifold employed for the vapor generation FTIR measurements [ 1l-131 was a single-channel assembly with a nitrogen carrier, which includes a volatilization reactor of 2.5 ml internal volume with inlet and outlet. Samples are injected inside the reactor through a septum using Hamilton (Reno, NY) gas-tight syringes, Model 1701, of different volumes with a removable needle (0.26mm i.d.) having a bevelled point with a 17” bevel, style 1. The temperature of the reactor was measured by means of a thermocouple and operated using an electrically controlled heater, Model AK0 (Barcelona). The reactor is connected with the IR gas cell using Viton (Isoversinic) tubes of 4 mm i.d.. 2.2. Chemicals Analytical-reagent grade absolute ethanol (99.5%) from Panreac (Barcelona), was employed for the preparation of standards and nitrogen gas (C-45), from Carburos Metalicos (Barcelona) was employed as the carrier gas. 2.3. General procedure 10 ul of untreated blood or plasma sample was injected into the open 2.5 ml reactor, preheated at 90°C and the vapor phase generated was transported inside the gas cell of the FTIR spectrometer using a carrier nitrogen flow of 400mlmin-i. In some experiments, a glass-bead packed reactor of 5.5 ml internal volume and 8.5 ml dead volume was employed in order to retain the partially decomposed matrix and to avoid clogging of the small reactor (see Fig. 1 for details). The Gram-Schmidt plot, which is a representation of the infrard (IR) intensity arriving at the detector as a function of time, is obtained for the transient signals generated after the injection of samples. Each GramSchmidt point corresponds to a FAIR spectrum in which an appropriate spectral range corresponding to absorption by ethanol can be selected. With the absorbance data found in the 1150-950 cm-’ range, an appropriate chemigram corresponding to the ethanol signal can be constructed. The chemigram provides a flow-injection (FI) recording which corresponds to the absorbance of ethanol, in the selected wave-number range. The area of the peaks
A. P&ez-Ponce
et al./Analytica
125
Chimica Acta 336 (1996) 123-129
a
r( Septum couple Electically controled heater Flow controled
;In
carrier .
-1
Reactor
1
1 Waste
Fig. I. Manifold
employed
for vapor generation
FHR spectrometry.
were employed as the quantitative analytical variable, taking into consideration a baseline correction established between the two troughs found before and after sample injection, in order to minimize the noise. Data obtained for samples were interpolated on the corresponding calibration graph established for aqueous standard solutions of ethanol, injected in the same way as the samples.
3. Results and discussion 3. I. Vapor-phase FTIR spectra generated from aqueous standards and samples
The injection of 10 11 of an aqueous standard of 1.2gl ’ ethanol in a carrier flow of 400 ml min-’ N2 provides a Gram-Schmidt recording of approximately two and a half minutes, the absorbance being is predominately that of the generated water vapor (see FIIR spectra found after 1.206 and 2.004 min in
Inset (a) glass-bead
packed reactor, and (b) open reactor.
Fig. 2). However, 0.724min after injection the spectrum of the vapor phase corresponds to a mixture of ethanol and water, as can be seen in Fig. 2. Considering the spectral range between 115s 950 cm-‘, which includes the main characteristic bands of ethanol (C-O), the chemigram depicted in Fig. 2 was obtained. It shows that after 0.724 min, the FI recording for ethanol reaches the maximum value and in 1.206 min has returned to the baseline. Thus, fast and easily measurable results can be found from the treatment of the output corresponding to a small wave-number range. To establish the baseline, the average value of the noise found before and after the peak can be employed (see Fig. 2). The injection of a sample of plasma, containing 1.2 g ll’ ethanol, provides Gram-Schmidt and chemigram recordings comparable to those found for aqueous standards. Fig. 3 depicts, as an example, the spectra obtained on the maximum of the corresponding outputs for two samples, one with and another without ethanol. The band of ethanol at 1050cmP’
126
A. P&z-Ponce
et al./Analytica
Chimica Acta 336 (1996) 123-129
chemigram \
‘I
”
baselin6’
\--
F-c;u
\ r \’
Time (minutes) Fig. 2. Gram-Schmidt recording (from 2000 to 500 cm-‘) and chemigram (from 1150 to 950 cm-‘) provided by the injection of 10 ul of an aqueous standard of ethanol and the spectra obtained in the vapor phase at different time values: (A) 0.724min. (B) 1.206min, and (C) 2.CMMmin. Experimental conditions: ethanol concentration 1.2 g ll’, reactor temperature 90°C carrier flow 400 ml mini’ and resolution 8 cm-‘.
contaminated by water. Thus, it can be concluded that both real samples and aqueous standards of ethanol can be injected directly into the reactor and the corresponding absorbance signals treated in the 1150-950 cm-’ range providing a sample throughout frequency of the order of 40 hh’ without matrix effects from proteins (which remain inside the reactor without being volatilized) and water (which is evolved more slowly). For quantitative analysis the area values provide better sensitivity and reproducibility than the use of peak height values. Fig. 3. Vapor phase FAIR spectra of plasma samples. (A) spectrum of a plasma without ethanol and (B) spectrum of a plasma sample containing 1.2 g 1-l ethanol. Inset: amplification of the 1200-8OCJcm~’ range. Experimental conditions: injection volume 10 ~1, reactor temperature 90°C carrier flow 400 ml mini’ and resolution 8 cm-‘.
can clearly be identified because it is far from the water absorption range and these signals are obtained for periods
3.2. Effect of experimental parameters Carrier-gas flow rate, reactor temperature and injection volume are the main parameters which influence the size and shape of the peaks obtained for ethanol by the injection of plasma samples. An flow rate dramatically increase of N2 carrier decreases the sensitivity of the vapor generation
A. Pe’rez-Ponce et al./Analytica
04 Carrie4r”
flow
(ml
12bo
6
“2k’)
Fig. 4. Effect of the N2 carrier flow on the chemigram areas. Experimental conditions: ethanol concentration 0.8 g I-‘, injection volume 10 ~1, reactor temperature 90°C and resolution 8cm-‘.
FTIR spectrometric determination of ethanol, as can be seen in Fig. 4, which shows that an increase of the carrier flow rate sharply decreases the sensitivity. Carrier flow rates <400mlmin-’ are the most appropriate to obtain sensitive and reproducible measurements. However, it must be taken into account that the use of slow carrier-gas flows involves an increase of the volatilization time, thus reducing the sampling throughput. Throughout this study, a flow rate of 400ml min-’ was chosen as a compromise between sensitivity and speed of analysis. Although the boiling temperature of ethanol is 78.17”C, sensitive measurements can be obtained between 60 and 110°C. Table 1 summarizes the data obtained for ethanol at different temperatures. The use of higher temperatures increases the sampling frequency from 20hP’ at 60°C to 40 hh’ at 110°C.
Table 1 Effect of volatilization
(“C)
Coefficient variation of (%)
Sample throughput
60 70 80 90 100 110
5.250.1 6.1f0.5 5.9f0.3 6.5&O. 6.0f0.2 6.3fO.l
2.5 8.2 5.1 1.2 3.5 1.9
20 24 27 34 34 40
‘Mean&d.
(n=4)
127
However, the sensitivity remains constant in all the temperature ranges assayed, of the order of 6 area units gg ’ 1; the repeatability of the area values in general was better for temperatures higher than 80°C as compared to temperatures below the boiling point of ethanol (see Table 1). A value of 90°C has been selected from these experiments to obtain the best sensitivity and repeatability. An increase of the injected sample volume obviously increases the back area for a fixed ethanol concentration, thus improving the sensitivity of the analytical determinations. Fig. 5 shows the effect of injection volume, from 2.5-17.5 ul and it can be seen that there is a good linear regression between area values and injection volume (r=O.993). The graph indicates that the results are not affected by contamination, which could affect the blank value, probably because samples are injected directly without adding any reagent. Besides, this system is free from memory effects. The good calibration linearity shows that the heating conditions are appropriate for obtaining complete volatilization of both small and large sample volumes, thus offering exciting possibilities for improving the sensitivity by means of the use of large injection volumes. However, on comparing the sample throughput values found for different injection volumes, it can be seen that it is reduced from 60 hh’ for 2.5 ul to 40 hh’ for 17.5 ~1. A value of
14 ,
I
temuerature
Sensitivity (area units g-11)”
Temperature
Chimica Acta 336 (1996) 123-129
I
(hh’)
2y// 0
I,1
0
,,.,li,
5
1,/I,,,
10
15
20
Injection volume @I) Fig. 5. Effect of the injection volume on chemigram area of a solution of 0.8 g 1. ’ ethanol. Experimental conditions: temperature 90°C carrier flow 402 ml min-’ and resolution 8 cm -‘,
A. Pkrez-Ponce et al./Analytica
128
Chimica Acta 336 (1996) 123-129
10 ~1 (10 pl syringe) was chosen to reach a compromise between sensitivity and speed.
3.4. Analytical features of vapor generation FTIR spectrometry for ethanol determination in blood
3.3. Effect of the matrix The vapor-phase generation avoids matrix and spectral interferences because, working at a relativly low temperature, only ethanol and water are volatilized and transported by the carrier flow through the measurement cell. The matrix remains and decomposes inside the reactor and must be eliminated by cleaning the system after approximately every ten injections. To avoid this problem, additional experiments were carried out using another reactor with a high internal volume, which is occupied almost entirely by glass beads. In such a reactor, the residue remains between the beads and does not obstruct subsequent injections. So, a long series of samples can be injected without cleaning the reactor. However, results obtained at 90°C and 300mlmin-’ N2 carrier flow (see Table 2) indicate that the use of the packed reactor gives greater sensitivity than the unpacked reactor, but the limit of detection and sampling frequency are better with the unpacked reactor. Thus, it may be preferable to use the simple open reactor and to clean the system after a series of injections. Table 2 Comparison spectrometry
of reactors
employed
for vapour
generation
FIIR
(hh’)
Reactor
Sensitivity (area units gg’ 1)”
LOD (gll’)h
Sample throughput
Packed Unpacked
1.1210.06 0.87?~0.10
0.15 0.04
20 34
“Meants.d. (n=3). ‘LOD, limit of detection
(k=3) for a probability
Table 3 Figures of merit of the vapor-generation Matrix
Water Plasma Water
level of 99.86%.
FTIR spectrometric
determination
Table 3 summarizes the main features of the methodology developed for the determination of ethanol using vapor generation-FfIR spectrometry. In this table, calibration lines obtained with aqueous standards, and standards prepared with a plasma matrix, are reported. As can be seen, linear calibration plots found for plasma and aqueous solutions, under the same conditions, are comparable, and provide the same intercept and slope values and, as is evident from Table 1, the sensitivity can be increased by increasing the temperature from 80 to 90°C. The calibration lines have been determinated for the O-2 g 1-l concentration range in order to define an appropriate calibration for the quantitative analysis of samples corresponding to persons whose level of ethanol in blood could be around the maximum allowed for driving, which in Spain corresponds to 0.8 g lP ’ . So, under the recommended conditions, quantitative information about the ethanol level of drivers blood could be obtained in less than 2 min and requires only a few 10 ul of untreated blood. The limit of detection has been established from the repeatability of five measurements of a blank solution (water or plasma without ethanol). The limit of detection obtained, under the most sensitive conditions (90”(Z), corresponds to 0.020 g l-‘, indicating that the methodology developed could be applied to a wide range of ethanol concentrations in human blood, including persons with a level well below 0.8 g 1-l.
of ethanol
Flow carrier (ml min-‘)
Temperature
Calibration equation
r
(“C)
397 397 394
80 80 90
A=0.23+0.745C A=0.22+0.741 C A=0.13+0.905C
0.98 0.99 0.995
LOD (g 1-l)
0.16 0.22 0.020
Afs,
,
0.872f0.003 0.931&0.018 0.926ztO.010
Coefficient of variation
(%)
0.3 1.9 1.1
Calibration line: A, area; C, concentration of ethanol in g I-‘; r, regression coefficient (n=5); LOD, limit of detection (k3) for a probability level 99.86%; coefficient of variation for 4 analysis of a sample with a concentration of O.Sgl-’ of ethanol; A& ~,, area and the corresponding standard deviation of 4 independent measurements carried out on IO ul injections of a sample containing 0.8 g I-’ ethanol.
A. Pbez-Ponce
et al./Analytica
Table 4 Results obtained for the determination of ethanol in spiked samples by vapor-generation FTIR spectrometry Ethanol (gl
Sample
added&s,+, Plasma
0.40010.003 0.800*0.009 0.96OxtO.011
Blood
0.400*0.003 0.80010.009 0.960*0.011
‘)
Error (%)
a
found&. 0.41 f0.03 0.82*0.03 0.96&0.08 1.18+10.06 0.41 lto.03 0.78ztO.02 0.92ztO.02 1.12ztO.09
1.200*0.012
1.200+0.012 a Standard
deviation
3.5. Analysis
for n-1
,’ 2.5 2.5 0 -2.4 2.5 -2 4.2 -6.7
Chimica Acta 336 (1996) 123-129
129
allows direct analysis without any sample pretreatment and requires only an extremely small amount of sample which can be obtained from a finger puncture. The 0.02 g 1. ’ limit of detection found for small injection volumes, fast analysis of samples and avoidance of interference from low volatility sampIe components are the main characteristics of the simple procedure developed, which could also be applied for the direct determination of other easily volatile compounds in complex matrices.
Acknowledgements
degrees of freedom (n=3).
ofreal samples
To test the applicability of the procedure developed, a series of samples of human plasma and blood, spiked with ethanol at known concentrations, was analyzed, using in all cases aqueous solutions of ethanol as standards. Table 4 summarizes the results found over a concentration range 0.4-1.2 g I-‘. Quantitative recoveries were found in all cases, with relative errors lower than 2.5%. Errors of 4.6 and 6.7% were obtained only for whole blood analysis at concentration levels clearly higher than those allowed by law, thus ensuring certainty in the characterization of the samples. The analysis of a real unspiked blood sample was also carried out both on a small drop of blood from a finger puncture and 2ml of venous blood. Results found by vapor generation FTIR spectrometric determination of ethanol in these samples were 0.14+0.05g1-’ and 0.16*0.06 g 1-l (mean&s.d., n=5), respectively. This shows that the method could be applied with little injury to persons owing to the extremely small amount of sample required, althought the accuracy of the measurements was not tested.
4. Conclusions The method developed alternative to conventional ing ethanol in human blood these procedures, the vapor
is a rapid procedures or plasma. generation
and accurate for determinIn contrast to FTIR method
The authors acknowledge the financial support by the Conselleria de Educaci6n y Ciencia de la Generalitat Valenciana (project GV 1021/93) and the Spanish DGICYT (project PB 92-0870). A.P.P. acknowledges the grant of the Conselleria de Educaci6n y Ciencia de la G.V.
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