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ORIGINAL PAPER

Microwave-assisted extraction: a simpler and fastermethod for the determination of ethyl glucuronidein hair by gas chromatographymass spectrometry

Ivn lvarez & Ana Mara Bermejo &Mara Jess Tabernero & Purificacin Fernndez &Pamela Cabarcos & Patricia Lpez

Received: 2 October 2008 /Revised: 6 November 2008 /Accepted: 25 November 2008# Springer-Verlag 2008

Abstract Alcohol is the most frequently abused addictivesubstance that causes serious social problems throughout theworld; thus, alcoholism is of particular interest in clinical andforensic medicine. Alcohol biomarkers are physiologicalindicators of alcohol exposure or ingestion and may reflectthe presence of an alcohol use disorder. The glucuronideconjugation is a minor pathway of ethanol metabolism. Ethylglucuronide (EtG) is a marker of recent alcohol consumptionthat detects alcohol use reliably over a definite time period. The

present paper describes a new method for the determination ofEtG in hair. It is based both in the microwave-assistedextraction (MAE), to extract the analyte from hair samples,

and gas chromatographymass spectrometry (GC-MS), toidentify and quantify the EtG in selected ion monitoring (SIM)mode. The method was applied to 15 hair samples fromoccasional alcohol users, obtaining positive results in all cases.It was fully validated, including a linear range (0.310 ng/mg)and the main precision parameters. In summary, the use ofmicrowave-assisted extraction turned out to be a substantiallysimpler, faster, and a more sensitive procedure than any otherconventional sample preparations.

Keywords Ethyl glucuronide . Hair. Microwave-assistedextraction . Gas chromatographymass spectrometry

Introduction

Alcohol is a depressant drug that has been produced anddrunkin Europe for thousands of years [1]. It has traditionally beenconsidered a serious public health problem because ofseveral physical, mental and social problems associated withits abuse, as it exerts an enormous toll on the lives andcommunities of many nations [2]. In fact, the Spanish 2005

National Report, presented to the European MonitoringCentre for Drugs and Drug Addiction, has shown thatalcohol is by far the psychoactive substance of abuse mostwidely consumed by the Spanish population [3].

Detection of ethanol in body fluids is only possible during arelatively short time after alcohol consumption. About9095% of alcohol is eliminated by oxidation mainly in theliver, whereas biotransformation of ethanol to ethyl glucuro-nide (ethyl--D-6 glucuronic acid; EtG), via conjugation withactivated glucuronic acid, represents only 0.020.06% ofcomplete alcohol elimination [4, 5]. Although the percentageof alcohol metabolized by this later pathway is small, itrepresents a useful tool as EtG becomes detectable up to4 days after complete elimination of alcohol from the body.With its specific time frame of detection, intermediate

between short- and long-term markers, and a high sensitivity

and specificity (i.e., EtG is not detectable unless alcohol has been consumed), EtG is a promising marker of alcoholconsumption in general and a marker for relapse detection[6, 7]. Its detection in hair is more and more studied duringthe last years for the purpose of alcohol abuse monitoring, in

both clinical and forensic toxicology [8]. It was determinedby GC-MS-EI, by GC-MS/NCI, and by LC-MS/MS [4].

Generally, some extraction methods have been used inforensic toxicology, such as liquidliquid extraction, solid

phase extraction or solid phase microextraction and so on

Anal Bioanal ChemDOI 10.1007/s00216-008-2546-2

I. lvarez : A. M. Bermejo (*) : M. J. Tabernero : P. Fernndez :

P. Cabarcos : P. LpezInstitute of Legal Medicine, Forensic Toxicology Service,Faculty of Medicine,C/ San Francisco s/n,15782 Santiago de Compostela, Spaine-mail: [email protected]


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[9, 10, 11]. But some need lots of time, some are expensiveand some require large amounts of organic solvents. Simple,rapid and less labor-intensive extraction techniques areneeded in forensic toxicology. The growing interest inobtaining increasingly better results in this context has ledto the development of microwave-assisted extraction(MAE), which has been widely used for the extraction of

organic pollutants from sediments, soil, water, and othertypes of materials. By contrast, MAE has scarcely been usedto extract drugs of abuse from different materials such asserum, urine, tablets, or coca leaves [12, 13]. Commercialmicrowave equipments with security systems and closedvessels have made MAE an analytical technique of interestsince these instruments enable simultaneous extractions ofanalytes at high pressure and temperature. The MAEtechnique requires less solvent consumption and shorterextraction times, while the extraction yields of the analyteare equivalent to or even higher than those obtained withconventional methods. The MAE has been applied suc-

cessfully to the extraction of ethyl glucuronide from urine by our investigative group, and this method has beenincorporated to our laboratory routine [14]. Because ofthese issues, this work proposes a new, fast, sensitive, andreliable method to detect ethyl glucuronide in human hairsamples by using MAE and GC-MS.

Experimental

Materials and methods

Chemical reagents and standards

Ethyl glucuronide and ethyl glucuronide-d5 were purchasedfrom Medichem (Stuttgart, Germany). Methanol gradientgrade, chloroform, pyridine and N,O-(bistrimethylsilyl)trifluoroacetamide (BSTFA) were obtained from Merck(Darmstadt, Germany).

Preparation of solutions

Stock standard solutions of EtG and EtG-d5 at a concentra-tion of 1 mg/mL in methanol were prepared, and from

these, working solutions were obtained by dilution withmethanol. Samples were stored at 4 C when not in use.

Distilled water was processed through a Milli-Q watersystem (Millipore, Bedford, MA, USA).

Instrumentation

The microwave extractor system was an ETHOS PLUSMPR300/12S (Milestone, Agrigento, Italy) equipped witha solvent detector. The microwave was able to extract 12

samples simultaneously in PTFE -lined extraction closedvessels under the same conditions (temperature and

pressure), with simultaneous magnetic stirring of thesample and solvent inside. An in-board control systemwas installed for monitoring and controlling pressure andconditions inside the extraction vessels. This oven allows amaximum of 1,000 W and the power changes in order to

reach and maintain the temperature selected.Chromatographic analyses for EtG were performed using

an electron impact ionization gas chromatograph model6890 from Hewlett-Packard (Little Falls, DF, USA)interfaced to a mass selective detector (MSD) model 5973inert from Agilent Technologies (Las Rozas, Spain).

Analytical conditions

Chromatographic elution was performed using the previously published fully validated method described above [14].Initially neat standards of EtG and EtG-d5 (2 l of a

0.01 mg/mL solution) were injected in a mixture afterderivatization and analyzed using the full scan mode of theGC-MS. Quantifier and qualifier ions used for each analytewere selected based on their abundance and m/z values.Because of their reproducibility and lack of interference,high mass ions were selected when possible. The ionsselected were as follows: m/z 160, 261, 405 (EtG); m/z 165,266, 410 (EtG-d5), using the underlined ones for quantita-tion. Upon selection of unique ions, the MS was run inselected ion monitoring (SIM) mode.

Extraction procedure and derivatization

Hair samples obtained from subjects who did not drink anyalcohol were used to carry out the calibration curves. Hairsamples were submitted to an initial procedure of decontam-ination by washing twice in 5 mL of a 0.1% solution of Tween80 for 10 min, and rinsing twice with 5 mL of distilled water toeliminate external contamination, and the last wash analyzedto exclude possible interferences, e.g., of any cosmetic

product. After drying, each sample was cut in 1-mm segmentsand then 100 mg of hair were weighted.

Thirty microliters of the deuterated internal standard(10 g/ml) were added to 100 mg of hair. The internal

standard was used to eliminate injection error while main-taining a constant area ratio for concentration quantitation.The sample was mixed with 8 mL ofn-hexan/water (1:1 v/v)and placed in the vessel of the microwave oven forextraction at 110 C for 11 min. After the extraction, thevessel contents were centrifuged at 4,000 rpm for 5 min. Theaqueous layer was removed by evaporation to dryness undera nitrogen stream in a thermostatic bath at 50 C.

Derivatization was performed using the previouslypublished method described above [14].

I. lvarez et al.


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Fig. 1 a Chromatogram for ablank hair sample. b Chromato-gram for a hair sample spikedwith EtG (1 ng/mg). c Chro-matogram of a real sample (EtG:0.53 ng/mg)

Microwave-assisted extraction: a simpler and faster method for the determination of ethyl glucuronide in hair


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Validation of the method

The analytical validation of the method was performed byestablishing selectivity, linearity, limits of detection andquantitation, intra- and inter-day precision and accuracy, andrecovery [10, 11].

Selectivity of the method was demonstrated by analyzingten hair samples from different people who had notconsumed ethanol. Samples were extracted and analyzedfor assessment of potential interferences from endogenoussubstances. The apparent response at the retention times ofthe analytes under investigation was compared with the

response of analytes at the limit of quantitation.Standard calibration curves were obtained as previously

described using drug-free control hair spiked with standardsolution to obtain the concentration range of 0.310 ng/mgfor EtG. Quantitation was based on target peak area ratio ofEtG (m/z 261) to its internal standard (m/z 266).

Sensitivity of the method was determined by calculationof the limit of detection (LOD) and the lower limit ofquantitation (LLOQ). LOD was determined by an empiricalmethod that consists of analyzing a series of hair samplescontaining decreasing amounts of the analytes. LOD wasthe lowest concentration that presented a signal-to-noise

ratio higher than 3 for at least three diagnostic ions for eachsubstance. The LLOQ is the lowest concentration of analytethat can be determined quantitatively with appropriate

precision and accuracy.Precision and accuracy were determined by inter- and

intra-day assays. Inter-day precision and accuracy wereevaluated by six determinations per concentration in differ-

ent days. Intra-day precision and accuracy were determinedat three concentrations, 0.3, 3, and 10 ng/mg, by preparingand analyzing on the same day five replicates for each level.

Precision, expressed as the coefficient of variation (CV)of the measured values, calculated as (standard deviation/mean)100, is expected to be less than 15% at allconcentrations, except for the LLOQ for which 20% isacceptable. In the same way, accuracy was evaluated usingthe mean relative error (MRE), which had to be less than15% of the theoretical values at each concentration levelexcept for the LLOQ, for which 20% is acceptable [15].

Recovery or extraction efficiency (%) for the analyte was

determined at low and high concentration levels. Calculationswere performed by comparing the areas of the peaks afterextraction of samples with the internal standard and the EtG,with those obtained containing only the internal standard andsubsequently spiked with the drug at the same concentration.

Results and discussion

Sample preparation is one of the most important steps in themajority of analytical procedures to determine constituentsin samples with complex matrices. An ideal sample

preparation technique should be simple, inexpensive,efficient, selective, and compatible with various analyticaltechniques. It should give as high a recovery as possible,use the minimum amount of solvent, and be environmen-tally friendly. Microwave-assisted extraction has risenrapidly in the last decade, and for most applications it has

proven to be effective in all the aspects mentioned abovecompared to traditional extraction procedures.

It is well known that ethylglucuronide can accumulate inhair, where it can be detected following alcohol consumption.Furthermore, hair is easy to obtain through non-invasive

procedures. Some studies have demonstrated that EtG is

highly specific and sensitive as an alcohol marker [16].

Table 1 Limit of detection, lower limit of quantitation and calibration results of EtG

LOD (ng/mg) LLOQ (ng/mg) Linearity Slope standard error Intercept standard error R

0.1 0.3 y=0.2466x+0.1061 0.00459 0.02450 0.990

Table 2 Precision and accuracy obtained for the EtG in hair

Concentrationadded (ng/mg)

Intra-day study (n=5) Inter-day study (n=6)

C.V. (%) Relative meanerror (%)

C.V. (%) Relative meanerror (%)

0.3 6.76 5.35 9.32 4.770.5 7.95 9.641 4.36 13.433 3.81 7.71 5.11 5.005 6.12 0.788 5.59 1.8810 2.17 3.59 5.90 0.69

Table 3 Recoveries of EtG in hair (n=5)

Concentration (ng/mg) Mean Recovery (%) CV (%)

10 86.54 2.8770 88.45 11.86

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In the consulted literature, it has been possible to findsome papers in which EtG was extracted from hair. In mostof them, ultrasonication for 2 or 3 h is required and a solid

phase extraction procedure of approximately 45 min isneeded. Some authors even describe a previous step ofincubation at 2530 C during 5 and 12 h [2, 4, 1621].This kind of extraction is more laborious than the onedescribed in our paper, with a higher time and solventconsumption.

Silylation is the most widely used derivatization proce-dure for sample analysis by GC. The popularity of silylationreagents is enhanced by their ease of use and formation of

derivatives. In silylation, an active hydrogen is replaced byan alkylsilyl group such as trimethylsilyl (TMS). Comparedto their parent compounds, silyl derivatives are morevolatile, less polar, and more thermally stable. As a result,GC separation and detection is improved. Silylation reagentsare generally moisture sensitive, requiring them to be sealedto prevent deactivation. Some authors compared severalderivatizing agents for EtG like PFPA (pentafluopropionicanhydride), but in our case, we selected the mixture obtainedwith BSTFA and pyridine, and the silyl derivative obtainedshowed to be stable for more than 4 h [9, 14]. BSTFA attacksthe hydroxyl groups of the ethylglucuronide and the result

is a volatile trimethylsilyl derivate, thus making it easilydetected in the GC-MS.

Figure 1a shows a chromatogram obtained for a blankhair sample, and shows no significant interferences of any

peak appearing at the expected retention time for the analyte(12.80 min), demonstrating a good selectivity of the

proposed method. Figure 1b shows a chromatogram for ahair sample spiked with EtG.

The calibration plot was linear for the EtG over thespecific range (0.310 ng/mg). A simple linear regression

analysis was performed. The LOD, LLOQ, and calibrationresults are detailed in Table 1. The confidence parametersof the validated method (inter- and intra-day precision andaccuracy) for the determination of the studied analyte areshown in Table 2. Precision and accuracy of the analyteunder investigation at reported concentrations satisfactorilymet the international established acceptance criteria [15].

The recoveries obtained are presented in Table 3. Theseresults suggest that at a low analyte concentration, themean recoveries are slightly lower than at a high analyteconcentration.

Stability of the derivatized analyte was studied for a period of 4 h for the highest concentration. A constantbehavior was observed throughout this period.

The analytical procedure proposed for the determinationof ethyl glucuronide in hair showed to be highly precisewith the use of the respective deuterated internal standard.Good sensitivity and linearity were also obtained for theanalyte.

Finally, the developed method was used to analyze 15real hair samples that were received in our laboratory(Forensic Toxicology Service, Institute of Legal Medicine,University of Santiago de Compostela) from people whoacknowledged to be occasional alcohol users (Table 4).

Results show that in all cases analyzed it was possible todetect EtG, confirming its usefulness as a marker of alcoholconsumption and obtaining a wide range of concentrations(0.44 to 4.89 ng/mg). A representative chromatogram of areal sample is presented in Fig. 1c.

Conclusions

The GC-MS method reported in this paper to analyze ethylglucuronide in hair was validated in the range (0.310 ng/mg), according to internationally acceptance criteria and itwas found to be sensitive enough to quantify 0.3 ng/mg.The proposed MAE method is being routinely used for thedetermination of EtG in hair in our laboratory. Microwaveenergy expedites extraction of the analyte while maintain-ing its recovery rates. Because of the technique usesstandard laboratory equipment, it can be an effectivemethod relative to potential alternatives such as liquid

liquid extraction and solid phase extraction. In fact, MAEreduces solvent consumption and extraction time, withrespect to others published methods. MAE is an extraction

procedure that will also be useful when applied to moresensitive instrumentation, such as GC/MS/MS, LC/MS, andall types of analytical techniques. GC-MS was found to bespecific, sensitive, and selective enough for determining thelow analyte concentrations to be expected in hair. So, themethod meets the sensitivity and the selectivity require-ments for clinical and forensic toxicology.

Table 4 Results for the 15 analyzed hair samples

Case EtG (ng/mg)

1 0.442 3.093 1.874 0.795 1.566 2.937 0.838 1.969 4.5910 2.7711 2.3212 3.7213 4.2014 3.5815 0.53

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