umn enz3~matic conversion and derivatization with fluorimetric detection. ALCOHOL 11(6) 577-582, 1994.-We quantified ethanol by measurement of the ...
Alcohol, Vol. 11, No. 6, pp. 577-582, 1994 Copyright ©1994ElsevierScienceLtd Printed in the USA.All rights reserved 0741-8329/94 $6.00 + .00
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Quantifying Ethanol by High Performance Liquid Chromatography With Precolumn Enzymatic Conversion and Derivatization With Fluorimetric Detection H U I - M I N C H E N A N D C H A R L E S M. P E T E R S O N 1
S a n s u m Medical Research Foundation, 2219 Bath Street, Santa Barbara, C A 93105 Received 23 N o v e m b e r 1993; A c c e p t e d 8 J u n e 1994 CHEN, H.-M. AND C. M. PETERSON. Quantifying ethanol by high performance liquid chromatography with precolumn enz3~maticconversion and derivatization with fluorimetric detection. ALCOHOL 11(6) 577-582, 1994.-We quantified ethanol by measurement of the subsequent increase in acetaldehyde after reaction with alcohol dehydrogenase and nicotinamide adenine dinucleotide (ADH-NAD) with a fluorimetric HPLC method. Ethanol standards ranging from 0.3 to 200 mg/ dl were investigated and the limit of quantitation of the fluorimetric HPLC method was found to be 6 mg/dl. The accuracy of the HPLC method was assessed by assaying blood samples containing 6-200 mg/dl of ethanol and comparing its results to those of the ADH-NAD enzymatic method (r 2 = 0.993). The coefficients of variation for intraassay (assayed ten times) and interassay (assayed on 7 consecutive days) were 6.7°/0 and 9.3°7o for blood samples containing 50 mg/dl of ethanol and 4.0% and ! 7.9% for blood samples containing 200 mg/dl of ethanol. The blood ethanol concentrations of a volunteer after a pulse of 0.3 g/kg of ethanol determined with the described HPLC method were correlated to the results from the ADH-NAD enzymatic method (r 2 = 0.986). In conclusion, the fluorimetric HPLC method for measurement of ethanol here described is of potential clinical utility. Acetaldehyde
Alcohol dehydrogenase
Ethanol
HPLC
Fluorimetric detection
a fluorimetric HPLC assay for the measurement of whole blood, hemoglobin, plasma and platelet associated acetaldehyde (1,7-15). With its specificity, sensitivity (picomole range) and precision (intra-assay CV < 3.5°70 and inter-assay CV < 15%) (11), we hypothesized that this assay could also be utilized to quantify ethanol by the measurement of the consequent increase in acetaldehyde after reaction with alcohol dehydrogenase and nicotinamide adenine dinucleotide (ADHNAD). The purpose of this study was to investigate the limit of quantitation, accuracy and precision of the coupled enzymatic conversion with fluorimetric HPLC of the produced acetaldehyde for ethanol determination.
BLOOD ethanol determination is one of the most frequently performed assays in clinical and forensic laboratories. The importance of an assay that can accurately and promptly quantify ethanol with acceptable precision cannot be overstated. The preferred methods include: gas chromatography (direct injection and head space), enzymatic oxidation with alcohol dehydrogenase, chemical oxidation with acid dichromate, and osmometry (2,4,5,16,18). Among these, gas chromatography is considered the most favorable method due to its sensitivity, specificity and minimal sample preparation (2). However, it has not been accepted as a routine method for alcohol analysis due to its requirement for special expertise of trained personnel and dedicated instrumentation. In recent years, investigators have attempted to refine gas chromatography to improve its applicability in clinical settings, and also have proposed new experimental approaches (18). High performance liquid chromatography (HPLC) has not been easily applied to the determination of ethanol due to lack of a proper detector (18). In our laboratory we have developed
METHODS
Subjects The study protocol was approved by proper institutional review. All subjects had not consumed ethanol for at least 1 week and consented to participate in the study. Blood was
J To whom requests for reprints should be addressed. 577
578 drawn into vacuum tubes (Becton Dickinson Vacutainer Systems, Rutherford, N J) containing ethylenediaminetetraacetic acid as an anticoagulant.
Acetaldehyde Assay The fluorimetric acetaldehyde assay was based on the procedure of Peterson and Polizzi (11). Free acetaldehyde or the liberated bound acetaldehyde in the presence of heat, HCI, and NH4 + was reacted with 1,3-cyclohexanedione to form a fluorophore (1,2,3,4,6,7,8,9,10-decahydro-9-methylacridine1,8-dione) that can be separated from other reacted aliphatic aldehydes on a C18 column. The cyclohexanedione (CHD) reagent was prepared as followed: 6.85 g NH4CI, 2.0 ml CHD stock (2.0 molar cyclohexanedione in 50% acetonitrile, -20°C), 5.0 ml ammonium acetate stock (10 g ammonium acetate, 2.0 ml CHD stock, to 40 ml with HPLC-grade H20). To remove contaminants, the reagent was heated in a 50 ml screw cap conical centrifuge tube at 70°C for 1 h, chilled on ice, and passed twice through C-18 Mega Bond Elut cartridges (Analytichem, Harbor City, CA). The cartridges were preconditioned with methanol followed by HPLC-grade HzO. To obtain the standard curve of the acetaldehyde, 200 #1 of CHD reagent was added to 200 #1 of varying concentrations of acetaldehyde (0, 0.25, 0.5, 1, 2, 4, 8, 16 #M) in 1.5-ml screw cap centrifuge tubes (Sarstedt Inc., Princeton, N J) and reacted for 1 h at 70°C. The reaction was stopped by placing samples in ice. Similarly, 200 #1 of blood sample was reacted with 200 /~1 of CHD, chilled and spun to remove precipitate prior to injection into the HPLC. The Gilson ASTED HPLC (Gilson Medical Electronics, Inc., Middleton, WI) was used for the determination of acetaldehyde concentration. In place of a typical sample loop is a mini C18 column (1.6 x 5 ram, the trace enrichment cartridge, Hypersil ODS, 70rag). The 80 #1 crude sample is passed through one side of a dialysis chamber (15 Kd MW cutoff) while 1 ml pure water is drawn by the other and through the trace enrichment cartridge. In the Inject position mobile phase (20°70 acetonitrile in Hz0, 2 ml/min) passes back through the trace enrichment cartridge, and onto the main column (Zorbax RX-C18, 4.6 mm x 15 cm, Mac-Mod Analytical Inc., Chadds Ford, PA). Peaks were detected by measuring fluorescence with a 305-395 nm excitation filter and a 450 +_ 3.5 nm emission filter. Results were determined form the linear regression line of the standards.
CHEN AND PETERSON consequent increase in absorbance at 340 nm is related to the ethanol concentration in the sample. The 200 ttl of sample was first deproteinized with 1.8 ml of 6.25% (w/v) trichloroacetic acid and centrifuged for 5 min at 1000 × g to obtain the supernatant. Two hundred #l supernatant was reacted with 3 ml of ADH-NAD reagent at room temperature for l0 rain following the spectrophotometric measurement of sample vs. blank (0 mg/dl ethanol) NADH absorbance at 340 nm.
Fluorimetric HPLC Method for Ethanol Determination Figure 1 is a flow chart showing the various steps of the fluorimetric HPLC method. Sample was separated into two aliquots. The first aliquot was assayed to determine the background acetaldehyde level. The second aliquot was first reacted with a commercial ADH-NAD assay to convert ethanol to acetaldehyde. The 200 #L of protein free supernatant was reacted with 3 ml ADH-NAD reagent (containing 600 units of alcohol dehydrogenase and 8.4 #moles of NAD, pH 8.8) for 10 min at 25°C. The reaction solution was then diluted 10 times with HPLC grade H20 before mixing with an equal amount of CHD reagent to obtain the optimal pH (pH 5) for the fluorimetric acetaldehyde assay. In addition, 200/A of phosphate buffered saline was also incubated with 3 ml ADHNAD reagent as mentioned to determine the fluorescence background resulting from ADH-NAD reagent. The amount of subsequent increase in acetaldehyde was calculated by subtracting the background acetaldehyde level and the fluorescence background from the acetaldehyde level postreaction with ADH-NAD reagent.
=Blood Sample } I Background Acetaldehyde Level
l Post ADH-NAD Acetaldehyde Levell ....
Background Fluorescence of ADH-NAD
Re~gent
6.25=/0TCA 5 Minutes, 25°C
I Centrifuge t000 x g for 5 Minutes I
Ethanol
~
Precipitates t
Ethanol was obtained as 200 proof ethanol (Quantum Chemical Corporation, Tuscola, IL). Ethanol standards of 0.3, 0.75, 1.5, 3, 6, 12.5, 25, 50, 100, and 200 mg/dl were prepared in phosphate buffered saline (PBS). In addition, blood samples containing 0.3, 0.75, 1.5, 3, 6, 12.5, 25, 50, 100, and 200 mg/dl of ethanol were also prepared by mixing equal volume of blood and ethanol. Similarly, blood sample containing 0 mg/dl of ethanol was obtained by mixing 0.5 ml of PBS with 0.5 ml of blood.
Enzymatic Method for Ethanol Determination Ethanol concentration was determined with a commercial ADH-NAD enzymatic assay (Sigma Diagnostics, St. Louis, MO.). Alcohol dehydrogenase (ADH) catalyzed the oxidation of ethanol to acetaldehyde with simultaneous reduction of nicotinamide adenine dinucleotide (NAD) to NADH. The
React with ADH-NAD Reagent 10 Minutes, 25°C
I
|
J I
Dilute 10 X wtth H20
I I
I React with equal volume CHD Reagent 70oC for 1 hour Chill on Ice Centrifuge 10,000 x g for 5 Minutes
I
-
m
~ Prectpitates
HPLC
FIG. 1. Flow chart of the fluorimetric high performance liquid chromatography (HPLC) method for ethanol determination. The total run time as described is approximately 90 rain. However, the l-h incubation with CHD reagent at 70oC could be automated in tandem to reduce the technician time.
ETHANOL BY FLUORIMETRIC HPLC
579
A ccuracy Ethanol standards ranging from 6-200 mg/dl were assayed with the enzymatic method as mentioned to establish the standard curve of ethanol added vs. the NADH absorbance at 340 nm. Likewise, the same range of ethanol standards were also assayed with the HPLC method to establish the standard curve of ethanol added vs. the subsequent increase in acetaldehyde. To assess the accuracy of the HPLC method, blood samples containing known concentrations of ethanol (0, 3, 6, 12.5, 25, 50, 100, and 200 mg/dl) were assayed and the results were compared to those of the ADH-NAD method. The amount of ethanol contained in the sample was calculated from the standard curve. The ethanol concentration of the blood sample was considered zero if the increase in acetaldehyde concentration fell below the range of the standard curve.
Precision Blood samples containing 200 and 5 mg/dl of ethanol were assayed to determine the intra-assay (repeatedly assayed for 10 times) and interassay (assayed on 7 consecutive days) precision of the HPLC method. To avoid the oxidation of ethanol through oxyhemoglobin (1), a blood sample was bubbled with nitrogen before the addition of ethanol and stored at room temperature during the whole experimental period.
Ethanol Dosing Study The subject reported to the laboratory at 0830 having consumed only liquid for 10 hours. Baseline blood samples were drawn 24 h, 30 rain, and 15 minutes before and at time 0. The subject was then instructed to drink 0.3 g/kg ethanol over the next l0 minutes. Blood samples were drawn at 15, 30, 45, 60, and 75 min after time 0. The ethanol concentration in the
blood samples was assayed with both the HPLC method and enzymatic method and the results were calculated from the respective standard curve.
Statistical Analysis The results are reported as the mean of the duplicate measurements. The Student's t-test for paired samples and linear regression statistics utilized Statview for Macintosh (Abacus, Berkeley, CA). RESULTS Ethanol standards ranging from 0.3-200 mg/dl were utilized to assess the limit of quantitation of the fluorimetric HPLC method. The HPLC method was unsuitable for ethanol determination in the range of 0.3-3 mg/dl due to a CV exceeding 20°70. The limit of quantitation was found to be 6 mg/dl ethanol. Figure 2 shows a representative fluorimetric HPLC chromatogram from a blood sample containing 50 mg/ dl ethanol. The standard curves of the HPLC method and the enzymatic method were established. The standard curve of the amount of ethanol added to the samples vs. the increase in acetaldehyde concentration assayed with the HPLC method was linear in the range of 6-200 mg/dl ethanol (Fig. 3A). It can be described with a best fit regression line o f y = 0.143x + 2.029, r 2 = 0.971. The standard curve of the amount of ethanol added to the sample vs. the NADH absorbance at 340 nm was also linear in the range of 6-200 mg/dl ethanol (Fig. 3B). It can be described with a best fit regression line of y = 0.004x - 0.002, r 2 = 1. To assess the accuracy of utilizing the HPLC method for quantifying ethanol, blood samples with known concentrations of ethanol in the range of 6-200 mg/di was assayed with both methods: the results were highly correlated with r ~ =
3
o o c o o o
,T > ~B
2 ¢C
i
0
0.5
r
1
1.5
2
2.5
-
-
F
3
Time (minutes)
FIG. 2. A representative fluorimetric high performance liquid chromatography (HPLC) from a blood sample containing 50 mg/dl ethanol. Peak 1 is the void volume. Peak 2 represents inter alia formaldehyde. Peak 3 represents acetaldehyde.
580
CHEN AND PETERSON 30
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