Elimination and phase II metabolism of ethanol in camels after intravenous administration

The Veterinary Journal 189 (2011) 95–99

Contents lists available at ScienceDirect

The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl

Elimination and phase II metabolism of ethanol in camels after intravenous administration Ibrahim A. Wasfi *, Asmaa M. Kamel, Hanan M. Saeed, Nasreen A.A. Saleh, S. Wajid, Nawal A. Al Katheeri, B.A. Agha Camel Racing Laboratory, Forensic Science Laboratory, PO Box 253, Abu Dhabi, United Arab Emirates

a r t i c l e

i n f o

Article history: Accepted 23 June 2010

Keywords: Ethanol Ethyl glucuronide Camel Racing

a b s t r a c t Ethanol elimination was studied in camels (n = 8) after a single bolus intravenous dose of 0.1 g/kg bodyweight (BW). Blood samples were then collected at set intervals. Ethanol and ethyl glucuronide (EtG) in blood were analysed by validated static headspace gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry (LC–MS) methods, respectively. Blood-ethanol concentration–time profiles were plotted for each camel and these were evaluated. A simple linear regression model was fitted to the selected data points and the slope of the fitted line was used to estimate the elimination rate, the distribution factor and turnover rate, which were 5.15 mg/dL blood/h, 0.55 L/kg and 0.028 g/h/kg, respectively. Blood EtG concentration–time profiles were also plotted for each camel. The elimination half-life of EtG, estimated by linear regression (using the values obtained after ethanol was completely eliminated) was 2.18 h. The theoretical initial blood concentration of EtG (C0), obtained by extrapolation to time zero was 23.4 lg/dL. The results will be useful in monitoring alcohol doping in camels using either parent drug or metabolite. Ó 2010 Elsevier Ltd. All rights reserved.

Introduction The pharmacokinetics of ethanol have been studied extensively in humans since 1930 (Holford, 1987; Kalant, 1996). The absorption of alcohol taken orally is rapid since it is highly lipid soluble, and although the process commences in the buccal cavity and the stomach most absorption takes place from the small intestine. After absorption, alcohol is rapidly distributed throughout the body water and is not selectively stored in any tissues. Widmark (1932) is credited with establishing the basic principles governing the uptake and distribution of ethanol in the human body and his work established a quantitative relationship between a person’s blood-ethanol concentration (BEC) and the amount of ethanol absorbed and distributed in all body fluids and tissues (Kalant, 1996). The classical approach to ethanol elimination kinetics is that of a zero-order process, presuming that ethanol is eliminated at a constant, concentration independent rate (b60) due to the saturation of the oxidative enzymes at low BEC. However, recent literature has shown that BECs are not linear over time and that ethanol elimination rates are concentration dependent (Holford, 1987). In humans, 95–98% of ingested ethanol is oxidised (phase I) and the remainder (2–5%) is excreted unchanged in breath, urine and sweat. The oxidation of ethanol occurs primarily in the liver, first * Corresponding author. Tel.: +971 2 4033098; fax: +971 2 4463470. E-mail address: iawasfi@yahoo.com (I.A. Wasfi). 1090-0233/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2010.06.013

to acetaldehyde and then to acetate, which is further oxidised into carbon dioxide and water mainly in muscle tissues (Jones, 2000). A small amount (<0.1%) of the ethanol ingested becomes conjugated with glucuronic acid and sulfate to form ethyl glucuronide (EtG) (Schmitt et al., 1995; Dahl et al., 2002) and ethyl sulfate (EtS) (Helander and Beck, 2004), respectively. These phase II reactions are catalysed by UDP-glucuronosyltransferase (Foti and Fisher, 2005) and sulfotransferase (Schneider and Glatt, 2004). After alcohol ingestion, EtG and EtS can be detected for considerably longer time than ethanol itself (Schmitt et al., 1995; Dahl et al., 2002). For this reason, urine and blood testing for these minor ethanol metabolites has gained acceptance as a sensitive method to confirm recent alcohol intake. The presence of EtG and EtS provides a strong indication of recent drinking, even if ethanol is no longer detectable (Helander, 2003). In racehorses, ethanol is reported to be used illegally to calm nervous horses, especially during movement to and loading into the starting gate (You et al., 2007). Racehorse practices tend to be adopted in camel racing, so ethanol use in camels can therefore be expected. Because it is quickly absorbed from the gastrointestinal tract without requiring digestion ethanol can be available as a quick source of energy. Furthermore, blood ethanol is not usually screened for in post-race samples, which may encourage camel trainers to use small amounts of ethanol as a doping agent. The objective of the present study was to evaluate the elimination rate of a small dose of ethanol using the Widmark equation

96

I.A. Wasfi et al. / The Veterinary Journal 189 (2011) 95–99

and to develop a sensitive liquid chromatography–mass spectrometry (LC–MS) method for the analysis of EtG and EtS in camel blood as an effective way of controlling illegal ethanol use in camel racing. Materials and methods Animals Eight clinically healthy male camels (Camelus dromedarius), 6–9 year old and ranging in bodyweight (BW) from 400 to 500 kg were used in this study. The camels were out of training and kept in open pens. They were fed good quality hay and lucerne (alfalfa) once daily, with water allowed ad libitum. No camel had received any drug for at least 1 month. Treatment Ethanol (0.1 g/kg) was given as a 50% (w/v) solution in normal saline and administered intravenously (IV) over 5 min into one jugular vein. Venous blood samples were drawn from the opposite jugular vein at 0, 5, 10, 15, 30, 45 min and 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6 and 12 h timed from the start of the infusion of the ethanol. The blood concentration of ethanol was measured on the same day of collection. Determination of blood-ethanol concentration Blood-ethanol concentrations were determined in duplicate using a validated headspace gas chromatography–mass spectrometry method (Wasfi et al., 2004). Determination of blood EtG and EtS EtG and pentadeuterated EtG (Medichem) and ethyl sulfate sodium salt (ABCR) were obtained commercially. All solvents and chemicals were of analytical grade or HPLC grade. The LC–MS/MS analysis was performed using a Thermo Finnigan TSQ Quantum Discovery mass spectrometer equipped with a Surveyor Autosampler and a MS Pump system (Thermo). Solid-phase extraction (SPE) was carried out using a manual manifold system (Jones). A Hypercarb high performance liquid chromatography (HPLC) column (50 mm  2.1 mm internal diameter, 3 mm; Thermo) equipped with a pre-column of the same phase was used for the analyses. The oven temperature was kept at 25 °C. The mobile phase comprised 2 mM ammonium formate (pH 3.0) as solvent A and acetonitrile with 0.1% formic acid as solvent B. The separation was achieved by isocratic elution (20% solvent B) at 0.2 mL/min at t = 0 min until t = 2.5 min. At t = 2.6 the flow was increased to 0.4 mL/min until t = 9.0 min and at t = 9.1 min the flow was returned to 0.2 mL/min until t = 10.1 min. Injection volume was 5 lL each. There was no carry-over effect by using this mobile phase program. The atmospheric pressure ionisation (API) source was operated in negative electrospray ionisation (ESI) mode. A capillary temperature at 300 °C was employed. The nitrogen sheath and auxiliary gas flow rates were set at 40 and 12 arbitrary TSQ quantum units, respectively. Detection of the drugs was performed in the MRM mode with a single time segment. The peak widths for the selection of the precursor and the corresponding product ions in Q1 and Q3 were both set at 0.7 amu (FWHM). The scan width for the selected product ions was set at 0.5 amu and the scan time at 0.04 s per transition. Argon was used in the collision cell and was set at 1.5 mTorr for all experiments. Mass transitions of m/z 221 > 75 and 221 > 85 for EtG and m/z 125 > 80 and 125 > 97 for EtS and 226 > 75 and 226 > 85 for EtG-D5 as internal standard (IS) were used to selectively monitor precursor ions and corresponding product ions. The collision-induced-dissociation (CID) energies ranged from 17 to 40 eV. These parameters were optimized while 1 lg/mL standard analytes were infused into the mobile phase at 5 lL/min. Data processing was performed using the Finnigan Xcalibur Version 1.3 software. Quantitative results were obtained by peak-area calculations. Limit of detection (LOD) and limit of quantification (LOQ) were 0.01 and 0.05 lg/mL, respectively. The values were calculated as a mean of background noise + 3 standard deviations (SD) and +10 SD, respectively. Day to day variations (n = 6) for 1 and 5 lg/dL were 4.7% and 5.27%, respectively. Intra-day variations (n = 6) for 1 and 5 lg/dL were 4.4% and 5.17%, respectively. The EtG calibration curve (1, 2, 5, 10, 15 and 20 lg/dL) was linear (r2 = 0.997). Extraction recovery was investigated by analysing blood samples (n = 3) spiked with EtG (at 2.5 and 5 lg/dL) before sample extraction. The IS was added to each sample after extraction. The peak area ratios of the recovered targets to the IS from the samples spiked with analytes before extraction were compared with the same amount of absolute analytes and IS added to the extracts of blank blood samples. Recovery was 55 ± 5% and 63 ± 6% for 2.5 and 5 lg/dL, respectively. The analytical specificity for EtG was tested by blank blood untreated camel samples (n = 20) containing various drugs (antidepressants, antipsychotics, sedatives, hypnotics, analgesics, etc.). No interfering substances were detected. A postcolumn infusion system was used for the evaluation of possible ion suppression.

EtG standards (0.5 and 1 lg/mL) were infused post-column using a syringe pump directly into a T-connector and mixed with mobile phase at 10 lL/min. Extracts from blood bank, were injected on to the LC-column and the EtG channel was monitored. The matrix effect on the ionisation of EtG was also evaluated by comparing the signals from plasma extracts spiked with 2.5 and 5.0 lg/mL EtG with those from a similar EtG standard concentration in the mobile phase. The matrix effect was expressed as percentage of the mean deviation of the absolute peak area measured in blank plasma from the peak area measured in mobile phase. Ion suppression was estimated to be about 20%. To an aliquot of 200 lL of whole blood was added 50 lL of IS solution (1 lg/mL) and 1.2 mL of cold methanol. The samples were immediately agitated for 1 min and thereafter subjected to a temperature of 20 °C for 20 min, followed by centrifugation at 15,000g for 10 min. An aliquot (750 lL) from the methanol layer was transferred to a 5 mL glass tube and evaporated to dryness at 60 °C under nitrogen. The residue was dissolved in 1 mL 1% formic acid in water. The residue was extracted by solid-phase extraction (CCETG203, 200 mg/3 mL, United Chemical Technologies) previously conditioned with 2 mL of 1% formic acid in water. After loading the sample, the column was washed with 1.4 mL of water and the column was dried under full vacuum for 10 min. Elution of EtG was performed with 2 mL of 1% formic acid in methanol. The solvent was dried under nitrogen at 60 °C and the residue was dissolved in 100 lL 1% formic acid in methanol; 5 lL was then injected into the LC–MS/MS system. Pharmacokinetic analysis Blood-ethanol concentration–time profiles were plotted for each camel and these were evaluated using the Widmark method, which assumes zero-order elimination kinetics. The mean of each duplicate BEC measurement was taken as the BEC at that time point. The estimated elimination rate was calculated for each camel from the BEC data by visually selecting a number of data points where the curve was approximately linear (3–6 points). A simple linear regression model was fitted to the selected data points and the slope of the fitted line was used to estimate the elimination rate (ER). The distribution factor (the Widmark factor; r0) was calculated from the amount of ethanol injected (A), BW (kg) and C0 (y-intercept, g/L) as r0 = A/ (BW  C0). The x-intercept of the regression line estimates the time necessary to eliminate all the ethanol (h0) from the body and the ratio dose/h0 is therefore a measure of the turnover rate of ethanol from the whole body in units of g/kg/h. The curvilinear part of the elimination phase appearing at very low BECs is not considered when calculating the x-intercept (h0) in this way. Blood EtG concentration– time profiles were plotted for each camel. The EtG elimination half-life was estimated by linear regression using the values obtained after ethanol was completely eliminated. The theoretical initial blood concentration of EtG (C0), was obtained by extrapolation to time zero. Data are expressed as means ± SD. Post-race plasma samples were screened for ethanol and for EtG by the methods described.

Results The pharmacokinetic parameters (means ± SD) of ethanol in camels after injection of an IV dose of 0.1 g/kg BW are shown in Table 1. The ethanol elimination rate in camels of 5.15 ± 1.01 mg/dL/h

Table 1 Pharmacokinetic parameters of ethanol in camels (n = 8) after injection of an intravenous dose of 0.1 g/kg BW. Measurement Ethanol Maximum blood-ethanol concentration (mg/dL) C0 (mg/dL) b60 (mg/dL blood/h) Distribution factor (L/kg) Distribution volume (L) Turnover rate (g/h/kg) Ethyl glucuronide Elimination half-life (h) The theoretical initial blood concentration (lg/dL)

Mean

SD

Range

29.8

6.62

21.3–40.5

18.5 5.15 0.55 274.3 0.028

3.2 1.01 0.09 47.1 0.004

13.7–24.6 3.29–5.94 0.41–0.73 205–365 0.023–0.033

2.18 23.4

0.09 4.36

2.0–2.34 14.9–29.4

The maximum blood-ethanol concentration was calculated 5 min after the IV dose. C0, blood-ethanol concentration at time zero; b60, zero-order elimination rate constant (the slope of the regression equation). The distribution factor was calculated from the ethanol dose injected, BW and C0. The distribution volume was calculated by multiplying the distribution factor by the BW.

I.A. Wasfi et al. / The Veterinary Journal 189 (2011) 95–99

Concentration (mg/dL)

30

Camel a Camel b Camel c

20

Camel d Camel e Camel f

10

Camel g Camel h

0 0

1

2

4

3

5

Time(h) Fig. 1. Blood-ethanol concentration–time profile in eight camels after an IV dose of 0.1 g ethanol/kg BW. The rate of disappearance of ethanol from blood was 5.15 mg/ dL/h and was derived according to Widmark (1932) (i.e., the slope of the linear elimination phase) where 3–7 measurements of the blood-ethanol concentration were included in calculating the slope of the linear elimination phase.

(C0) was 18.5 ± 3.2 mg/dL. The linear part of the individual concentration–time profiles of ethanol in blood from which the ethanol parameters were estimated are shown in Fig. 1. The mean concentration–time profiles of ethanol and EtG in blood are shown in Fig. 2. The multiple reaction monitoring chromatograms of EtG and EtS (10 lg/dL) and EtG-D5 (25 lg/dL) in control camel blood are shown in Fig. 3. The blood-ethanol concentration 3.5 h after ethanol injection was below the LOQ of the method (1 mg/dL), while EtG was above the LOQ of the method at 6 h and was easily quantified. The mean EtG concentration (3.5 lg/dL) at 12 h was slightly below the LOQ of 5 lg/dL. A larger sample volume of 1 mL blood (rather than 0.2 mL) may have allowed quantification of EtG for a longer period of time. The EtS concentration in camel blood following ethanol injection was low and erratic and was therefore not included in the study. In post-race plasma samples, the concentrations of ethanol and of EtG were below the LOQ of 1 mg/dL and below those of 0.05 lg/mL for ethanol and EtG, respectively. Discussion

35 30

Concenttration

97

EtG (µg/dL) Ethanol (mg/dL)

25 20 15 10 5 0 0

5

10

15

Fig. 2. Mean concentration–time profiles of ethanol and EtG in blood of eight camels after an IV dose of 0.1 g ethanol/kg BW. Values are presented as means ± standard deviation.

was rather slow; the distribution factor of ethanol in camels was 0.55 ± 0.09 (L/kg) while the maximum blood ethanol attained

The purpose of this study was to investigate alcohol pharmacokinetics in the blood of male camels using the classical Widmark equation. The work defined a relationship between blood-ethanol concentration over time with BW and volume of distribution, and enhanced the understanding of the phase II metabolites of ethanol. To our knowledge, this is the first study which has addressed this issue in camels. The results should be helpful in interpreting positive cases of ethanol in camel racing. A low ethanol dose was used to see whether these would result in prohibited levels of ethanol and its metabolite EtG. The study revealed that camels eliminate ethanol at a slow rate (5.15 mg/dL/h; range 4.45–5.88 mg/dL/h), which is similar to that of horses (6.3 mg/dL/h; Chapman and Rudram, 1978). However, extensive controlled alcohol dosing studies in humans have established an average elimination rate of about 15 mg/dL/h, with a range from 10 to 25 mg/dL blood/h (Jones and Holmgren, 2009), which is much higher than in camels. It should be noted that the elimination rate established in humans was based on a moderate

Fig. 3. Multiple reaction monitoring (MRM) chromatograms of EtG and EtS (10 lg/dL) and EtG-D5 (25 lg/dL) in spiked camel blood for the precursor-to-product-ion transitions of m/z 221 > 75 and 221 > 85 for EtG (B), 125 > 80 and 125 > 97 for EtS (A) and 226 > 75 and 226 > 85 (C) for EtG-D5 as internal standard. The two transitions are summed for each analyse.

98

I.A. Wasfi et al. / The Veterinary Journal 189 (2011) 95–99

ethanol dose of 0.5–0.8 g/kg BW, compared to a low dose of 0.1 g/ kg BW in camels, and the elimination rate of ethanol is dose-related (Lieber, 1997). However, dogs appeared to eliminate ethanol at a rate (15 mg/dL/h) approaching that of humans (Clark et al., 1941) and rats eliminated ethanol even faster (40 and 53 mg/dL/ h for males and females, respectively) after an intra-peritoneal dose of 0.8 g/kg (Crippens et al., 1999). The distribution factor (L/kg) of ethanol in camels was 0.55 ± 0.09, which was lower than reported for men (0.79), women (0.72), male rats (0.72), female rats (0.74), macaques (0.83), rabbits (0.76) and sheep (0.68) (Matsumoto et al., 1999; Crippens et al., 1999; Dettling et al., 2007). The volume of distribution (Vd) of ethanol after an IV dose agreed closely with body water, which was not unexpected for a molecule that is not protein bound and easily crosses through cell membranes. Fillali and Shaw (2004) determined the total body water in young camels using triturated water and reported a value of 0.71 L/kg, which was similar to our findings in the current study (0.41–0.73 L/kg). Matsumoto et al. (1999) developed an interesting allometric model for predicting blood ethanol volume of distribution (Vd = 0.762W0.932) in mammals in a BW range of 0.256 kg (rat) to 80 kg (human). Using this equation to estimate the Vd of ethanol in camels resulted in a value of 0.5 L/kg which was similar to the Vd we calculated experimentally in this study (0.55 ± 0.09 L/kg). The EtG concentration in blood peaked approximately 1.5 h after ethanol administration, and the ratio between ethanol and EtG in blood (ethanol in mg/dL, EtG in lg/dL) was >1 only for the first 0.5 h after ethanol administration and was <1 thereafter. These ratios may be useful to approximate the time of ethanol administration. The method for EtG analysis reported here proved to be specific and sensitive and could detect EtG for up to 12 h after administering this low dose of ethanol, while blood ethanol was below the limit of quantification 3 h after administration. EtG is therefore a useful biomarker for the misuse of ethanol in camel racing. You et al. (2007) successfully used an LC–MS/MS method for the quantification of EtG and EtS in post-race urine samples in horses. The theoretical initial blood concentration of EtG (C0), obtained by using the terminal half-life and extrapolation to time zero, was 23.4 lg/dL. The elimination half-life of EtG in camels is comparable to the value of 2.2 h reported in man (Høiseth et al., 2007). Unlike EtG, EtS in camel blood was present at erratic levels and was therefore not quantified. This finding agreed with previous studies from our laboratory which found that camels had good glucuronidation capacity, but limited sulfation (Al Katheeri et al., 2006; Wasfi et al., 2008). The median elimination half-life of EtG, calculated using the values obtained after ethanol was completely eliminated, was 2.0 h. Endogenous methanol and ethanol concentrations are found in the venous blood in humans. It was assumed that microflora in the gastrointestinal tract is the source of these alcohols (‘autobrewing’), as well as from intermediary metabolism. In ruminants such as camels autobrewing might be more significant due to the fermentation process which takes place in the rumen when compared to monogastric animals. Autobrewing in racing camels might even be more pronounced as they are usually given high sugar and carbohydrate diets which may result in blood ethanol levels that are defensible in court. The camel racing authority in United Arab Emirates adopts a zero medication rule in samples taken after racing. Detection of a prohibited substance and/or its metabolite(s) in those samples is regarded a violation of the rules of camel racing. In screening hundreds of post-race plasma samples we found that the concentrations of ethanol and of EtG were below the LOQ of 1 mg/dL and of 0.05 lg/mL for ethanol and EtG, respectively. Accordingly, if a camel had been administered 0.1 g/kg of ethanol 2–3 h before racing, a prohibited ethanol blood concentration

would be detectable and could be confirmed by the sensitive LC– MS/MS method described. Conclusions This study has defined the zero-order elimination kinetics of ethanol in camels after a low IV dose. This will permit prediction of blood-ethanol concentrations and the amount of ethanol administered from samples collected for doping control. Furthermore, the time course of EtG in blood following ethanol administration has been defined. Simultaneous detection of a doping agent and its metabolite(s) increases the efficiency of anti-doping control analysis. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements This research was supported by Col. Abdul Rahman Al Hammadi, Director of the Forensic Science Laboratory. The authors wish to thank O. Mohamad and A. Idris for technical Assistance. References Al Katheeri, N.A., Wasfi, I., Lambert, A.M., Albo, A.G., Nebbia, C., 2006. In vivo and in vitro metabolism of dexamethasone in the camel. The Veterinary Journal 172, 532–543. Chapman, D.I., Rudram, D.A., 1978. The disposition and excretion of ethanol by the horse. Journal of Veterinary Pharmacology and Therapeutics 1, 293–298. Clark, B.B., Morrissey, R.W., Fazekas, J.F., Welch, C.S., 1941. The role of insulin and the liver in alcohol metabolism. Journal of Studies on Alcohol and Drugs 2, 663– 683. Crippens, D., White, M.L., George, M.A., Jaworski, J.N., Brunner, L.J., Lancaster, F.E., Gonzale, R.A., 1999. Gender differences in blood levels, but not brain levels, of ethanol in rat’s alcoholism. Clinical and Experimental Research 23, 414–420. Dahl, H., Stephanson, N., Beck, O., Helander, A., 2002. Comparison of urinary excretion characteristics of ethanol and ethyl glucuronide. Journal of Analytical Toxicology 26, 201–204. Dettling, A., Fischer, F., Bohler, S., Ulrichs, F., Skopp, G., Graw, M., Haffner, H., 2007. Ethanol elimination rates in men and women in consideration of the calculated liver weight. Alcohol 41, 415–420. Fillali, R.Z., Shaw, R., 2004. Water balance in the camel (Camelus dromedarius). Journal of Camel Science 1, 63–65. Foti, R.S., Fisher, M.B., 2005. Assessment of UDP-glucuronosyltransferase catalyzed formation of ethyl glucuronide in human liver microsomes and recombinant UGTs. Forensic Science International 153, 109–116. Helander, A., 2003. Biological markers in alcoholism. Journal of Neural Transmission Suppl. 66, 15–32. Helander, A., Beck, O., 2004. Mass spectrometric identification of ethyl sulfate as an ethanol metabolite in humans. Clinical Chemistry 5, 936–937. Høiseth, G., Bernard, J.P., Karinen, R., Johnsen, L., Helander, A., Christophersen, A.S., Mørland, J., 2007. A pharmacokinetic study of ethyl glucuronide in blood and urine: applications to forensic toxicology. Forensic Science International 172, 119–124. Holford, N.H.G., 1987. Clinical pharmacokinetics of ethanol. Clinical Pharmacokinetics 13, 273–292. Jones, A.W., 2000. Ethanol metabolism in patients with liver cirrhosis. Journal of Clinical Forensic Medicine 7, 48–51. Jones, A.W., Holmgren, A., 2009. Age and gender differences in blood-alcohol concentration in apprehended drivers in relation to the amounts of alcohol consumed. Forensic Science International 188, 40–45. Kalant, H., 1996. Pharmacokinetics of ethanol: absorption, distribution, and elimination. In: Begleiter, H., Kissin, B. (Eds.), The Pharmacology of Alcohol and Alcohol Dependence. Oxford University Press, New York/Oxford, pp. 15–58. Lieber, C.S., 1997. Ethanol metabolism, cirrhosis and alcoholism. Clinical Chemica Acta 257, 59–84. Matsumoto, H., Minowa, Y., Nishitani, Y., Fukui, Y., 1999. An Allometric Model for predicting blood ethanol elimination in mammals. Biochemical Pharmacology 57, 219–223. Schmitt, G., Aderjan, R., Keller, T., Wu, M., 1995. Ethyl-glucuronide: an unusual ethanol metabolite in humans. Synthesis, analytical data, and determination in serum and urine. Journal of Analytical Toxicology 19, 91–94.

I.A. Wasfi et al. / The Veterinary Journal 189 (2011) 95–99 Schneider, H., Glatt, H., 2004. Sulpho-conjugation of ethanol in humans in vivo and by individual sulphotransferase forms in vitro. Biochemical Journal 1, 543–549. Wasfi, I.A., Al-Awadhi, A.H., Al-Hatali, Z.N., Al-Rayami, F.J., Al Katheeri, N.A., 2004. Rapid and sensitive static headspace gas chromatography–mass spectrometry method for the analysis of ethanol and abused inhalants in blood. Journal of Chromatography B 799, 331–336. Wasfi, I.A., Agha, B., Eltoum, A.M.K., Al Briki, N.A., Wajid, S.A., El Ghazali, M., 2008. The detection and confirmation of formoterol in camel serum and urine by LC–

99

MS/MS. In: Proceedings of the 18th International Conference of Racing Analysts and Veterinarians, Antalya, Turkey, p. 144 (Abstract). Widmark, E.M.P., 1932. Die theoretischen Grundlagen und die praktische Verwendbarkeit der gerichtlich-medizinischen Alkoholbestimmung. Urban and Schwarzenberg, Berlin, 1932, p. 140. You, Y., Uboh, C.E., Soma, L.R., Guan, F., Li, X., Rudy, J.A., Chen, J., 2007. Biomarkers of alcohol abuse in racehorses by liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry 21, 3785–3794.