chromatographic determination of drugs of abuse in vitreous humor using solid-phase extraction

Research Article

Received: 22 November 2011, Revised: 22 December 2011, Accepted: 22 December 2011 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jat.2722

Chromatographic determinationof drugs of abusein vitreous humor using solid-phase extractionPurificación Fernández,a* Santiago Seoane,a Cristina Vázquez,a

María Jesús Tabernero,a Antonia M. Carrob and Rosa A. Lorenzob

ABSTRACT: A simple method is presented for the simultaneous determination of morphine, 6-acetylmorphine, codeine, co-caine, benzoylecgonine, cocaethylene, methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in vitreoushumor by high-performance liquid chromatography with photodiode array detector after solid-phase extraction with OasisW

HLB cartridges and dichloromethane as eluent. The chromatographic process was carried out using an XTerraW RP8 column(250� 4.6mm i.d., 5mm particle size) and a mobile phase composed of acetonitrile and pH 6.5 phosphate buffer in gradientmode. A linear response from the detector was obtained within the concentration range of 0.1–4mg ml�1, with correlationcoefficients higher than 0.99. The limits of detection were lower than 30ng ml�1 for all the drugs studied, the coefficientsof variation fluctuated between 0.1 and 12.4%, and the average recoveries were higher than 78% for all the drugs exceptfor EDDP, with a value of 66.4%. Finally, the proposed method was applied to 15 vitreous humor samples coming fromindividuals who had died from opiate and/or cocaine overdose, showing consumption of cocaine in 14 cases, methadone in fivecases and heroin in three cases. Average concentrations of 0.30mg ml�1 for morphine, 0.24mg ml�1 for 6-acetylmorphine,0.10mg ml�1 for codeine, 0.81mg ml�1 for cocaine, 1.26mg ml�1 for benzoylecgonine, 0.15mg ml�1 for cocaethylene,0.11mg ml�1 for methadone and 0.68mg ml�1 for EDDP were obtained. Copyright © 2012 John Wiley & Sons, Ltd.

Keywords: drugs of abuse; vitreous humor; HPLC-PDA; SPE

* Correspondence to: Purificación Fernández, Institute of Forensic Sciences,Forensic Toxicology Service, Faculty of Medicine, 15782-Santiago de Compostela,Spain.E-mail: [email protected]

a Institute of Forensic Sciences, Forensic Toxicology Service, Faculty of Medicine,15782-Santiago de Compostela, Spain

b Department of Analytical Chemistry, Faculty of Chemistry, 15782-Santiago deCompostela, Spain

INTRODUCTIONThe consumption of drugs of abuse in Spain continues to behigh, with cocaine consumption being higher than that of her-oin in recent years. In burnt or embalmed bodies, as well aswhen decomposition processes or hemorrhagic shocks havetaken place, it is not possible to obtain blood or urine specimens;thus, vitreous humor plays an important role in forensic toxicol-ogy in these cases. Vitreous humor is a clean fluid with a lowercontent of protein than urine, which can be easily obtained bydirect suction during the autopsy. As it is protected by the osse-ous structure around the eyeball, its postmortem contaminationis difficult. Also, its limited irrigation preserves vitreous humorfrom blood infection. These features make vitreous humor a veryuseful specimen for the detection of drugs of abuse, when bloodor urine specimens are not available (Drummer, 2007; Fernándezet al., 2006). It is well known that drugs of abuse and theirmetabolites enter vitreous humor as a result of passive diffusionfrom blood, with significant concentrations of drugs that are notstrongly bound to proteins being found (Ziminski et al., 1984;Cox et al., 2007).

For the diagnosis of opiates and/or cocaine overdose, forensicsurgeons base their analysis fundamentally on the results oflaboratory tests, which must be rapid, sensitive and specific, inorder to guarantee the correct determination of drugs in differ-ent biological fluids. This justifies the continuous developmentof new analytical methods that allow analysts to distinguishbetween the drugs and their metabolites. However, postmortemlevels of drugs in vitreous humor are only sporadically reported,probably owing to the difficult interpretation of results. There-fore, much effort has been dedicated to determining therelationship between the levels of the drugs in vitreous humor

J. Appl. Toxicol. 2012 Copyright © 2012 John

and those observed in blood, but a common conclusion hasnot yet been reached. Wyman and Bultman (2004) confirmedthe suitability of vitreous humor for the analysis of drugs ofabuse, it being possible to discern whether morphine originatedfrom heroin consumption or codeine metabolism; if the co-deine/morphine ratio is much lower than 1, it can be stated thatmorphine comes from heroin consumption. Others authors havefound different ratios between blood and vitreous humor levels.With regard to other alternative specimens, Moolchan et al.

(2000) and Jenkins et al. (1995) studied the levels in plasmaand oral fluid without being able to establish a correlationbetween one and the other. Similar conclusions were drawn bySchramm et al. (1993) when comparing blood, urine and oralfluid levels. Other authors, like Toennes et al. (2005) and Specklet al. (1999), stated that similar levels to those found in bloodwere also found in oral fluid. Cone (1998) went even further withhis statement, confirming this correlation when the route ofadministration is intravenous; however, this balance betweenlevels takes more time to occur when the route of administrationis different. In the case of vitreous humor, these studies aremore unusual, and the positive results obtained should be

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P. Fernández et al.

useful to confirm the presence of drugs of abuse, especiallywhen 6-monoacetylmorphine is found (confirming heroin con-sumption), owing to its longer presence in vitreous humor(Antonides et al., 2007), although these results must be inter-preted with care when used to evaluate blood levels at thetime of death.

Toxicological analyses are based on sample preparation, whichis a very important stage in the analytical process. Sensitivity,selectivity and sample clean-up can be enhanced by commonlyemployed techniques, such as solid-phase extraction, which isused to target particular compounds and remove excessiveimpurities, thus helping to reduce the time involved and thesolvent volume used (Thurman and Mills, 1998).

In this work, a solid-phase extraction (SPE) procedure wasoptimized using OasisW HLB cartridges for the determination ofmorphine (MRP), codeine (COD), 6-monoacetylmorphine (6AM),methadone (MTD), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrro-lidine (EDDP), cocaine (COC), benzoylecgonine (BZE) andcocaethylene (CCE) in vitreous humor by high-performance liq-uid chromatography coupled with a photodiode array detector(HPLC-PDA). Finally, this methodology was applied to the quan-titative analyses of real samples sent to our laboratory in theUniversity of Santiago de Compostela for their toxicologicalinterpretation.

MATERIAL AND METHODS

Chemicals and Reagents

Drug standards (cocaine, benzoylecgonine, cocaethylene,morphine, codeine, 6AM, methadone and EDDP) were suppliedby Cerilliant (Round Rock, TX, USA) and all reagents (sodiumhydroxide, ammonium hydroxide, potassium hydroxide, hydro-chloric acid, potassium dihydrogen phosphate, phosphoric acid,boric acid, acetonitrile, methanol and dichloromethane) werepurchased from Merck (Darmstadt, Germany). OasisW HLBcartridges were obtained from WatersW. A 0.02 M phosphatebuffer pH 6.5 was prepared by dissolving 2.72 g of potassium

Table 1. Regression analysis in vitreous humor (range 0.1–4mg m

Retention time

MRP 7.74

COD 12.48

6AM 16.02

BZE 14.50

COC 22.38

CCE 23.28

EDDP 24.30

MTD 24.93

MRP, morphine; COD, codeine; 6AM, 6-monoacetylmorphine; BZ2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; MTD, methado

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dihydrogen phosphate in 1 l of Milli-Q water and adjusting thepH with phosphoric acid. A borate buffer (pH 9) was preparedby dissolving 12.37 g of boric acid in 100ml of OHNa 1 M andmaking up to 1 l with distilled water, then mixing 83.5ml of thissolution with 16.5ml of HCl 0.1 M.

Standard solutions containing 1mg ml�1 of each drug inmethanol (MRP, COD, BZE, MTD, EDDP) or acetonitrile (6AM,COC, CCE) were prepared. Calibration curves were constructedby using working-strength solutions, containing 0.1, 0.2, 0.4,1.0, 2.0 and 4.0 mg ml�1 of each drug in vitreous humor, thatwere made from the respective standards.

Vitreous Humor Samples

Drug-free control vitreous humor samples and positive cases forcocaine, heroin and/or methadone were used in this study. Allsamples were obtained by direct puncturing of the eyes,collected in our laboratory and frozen at �20 �C until analysis.Samples were centrifuged for 10min at 14 500 rpm in order toseparate compounds that might be coextracted. All tests wereconducted in accordance with the World Medical Association’sPrinciples for Medical Research Involving Human Subjects andthe Spanish National Law.

Chromatographic Conditions

The analyses were performed on a model 2695 liquid chromato-graph from WatersW (Milford, MA, USA) connected to a model996 photodiode array detector also from WatersW. Data were pro-cessed using the software Empower ProW. Samples were injectedinto an XTerra RP8W stainless steel column (250� 4.6mm id, 5mmparticle size) supplied by WatersW.

In order to optimize peak resolution and hence an efficientseparation of the analytes in a reasonably short time, elutionwas performed with a mobile phase consisting of a mixture ofacetonitrile (solvent A) and 0.02 M phosphate buffer pH 6.5(solvent B) at a flow of 0.8ml min�1, in gradient mode: 0–5min,

l�1)

Parameter� standard error

Slope 81283.0� 1316.03Intercept �78.32� 2474.34Slope 92236.4� 1173.99Intercept �1465.99� 2207.29Slope 95301.5� 787.01Intercept 55.07� 1479.71Slope 730432.0� 13379.9Intercept 43297.6� 25156.3Slope 680031.0� 7429.41Intercept 16660.6� 13968.5Slope 865293.0� 11320.6Intercept �19949.9� 21284.6Slope 9064.58� 173.08Intercept �107.80� 301.72Slope 22130.1� 340.34Intercept 43.64� 593.29

E, benzoylecgonine; COC, cocaine; CCE, cocaethylene; EDDP,ne.

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Chromatographic determination of drugs of abuse in vitreous humor

5% solvent A and 95% solvent B; 7min, 10% solvent A and 90% sol-vent B; 10–16min, 15% solvent A and 85% solvent B; 17min, 30%solvent A and 70% solvent B; 20–25min, 50% solvent A and 50%solvent B; 27min, 80% solvent A and 20% Milli-Q water; 30min,5% solvent A and 95% solvent B. The photodiode-array detectorallowed the wavelength range from 200 to 400nm to be scannedto obtain three-dimensional (wavelength� absorbance� time)chromatograms. The sensitivity of the method was maximized byusing the wavelengths with maximal chromatographic response,namely 233nm for COC, BZE and CCE; 285nm for MRP, COD and6AM; and 292nm for MTD and EDDP.

Solid-phase Extraction

One milliliter of borate buffer pH 9.0 was added to 500ml of thevitreous humor sample containing the drugs and centrifuged for

DCM DCM/ MEOH (1:1) MEOH

1350000

1050000

750000

450000

150000

Pea

k ar

ea

100000

50000

0

Figure 1. Eluent optimization. DCM, dichloromethane; MEOH, metha-nol; MRP, morphine; COD, codeine; 6AM, 6-monoacetylmorphine; BZE,benzoylecgonine; COC, cocaine; CCE, cocaethylene; EDDP, 2-ethyli-dene-1,5-dimethyl-3,3-diphenylpyrrolidine; MTD, methadone.

Table 2. Linearity study of HPLC-PDA method in vitreous humor

Correlation coefficient ANOVA re

F-Ratio

MRP 0.9964 3814.79COD 0.9982 6172.696AM 0.9990 14663.52BZE 0.9954 2980.26COC 0.9983 8378.16CCE 0.9976 5842.33EDDP 0.9955 2742.76MTD 0.9971 4228.13

MRP, morphine; COD, codeine; 6AM, 6-monoacetylmorphine; BZEethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; MTD, methadonewith a photodiode array detector.


10min at 14 000 rpm. The drugs were separated from vitreoushumor by means of OasisW HLB (60mg) cartridges, which werepreviously conditioned with 2ml of methanol and 2ml of Milli-Q water. The sample was passed through the cartridge with aManifold (WatersW) and then the cartridge was washed with2ml of methanol–water (20:80, v/v) and 2ml of ammoniumhydroxide–methanol–water (2:18:80, v/v). After drying undervacuum for 10min, analytes were eluted with 2ml of dichloro-methane. The extract obtained was evaporated under a nitrogenstream at 40 �C and reconstituted in 100ml of the mobile phase,yielding a concentration 5 times higher than that originallypresent in vitreous humor. Analyses were performed by injecting20ml of the final extract into the HPLC-PDA system.

Validation of the Method

The linearity of the proposed method was examined from acalibration graph for each drug in vitreous humor. The curveswere plots of the analyte peak area as a function of theanalyte concentration, using three replicates per concentra-tion level. We constructed regression lines of the typey= ax + b, and linearity was assumed when the slope of thecalibration curve, a, was statistically nonzero, the independentterm, b, was not statistically different from zero, and the corre-lation coefficient was not significantly different from unity(Bressolle et al., 1996).The limit of detection (LOD) was determined in five blank

vitreous humor samples, from the lowest detectable concentra-tion with a signal to noise (S:N) ratio of at least 3, and the lowerlimit of quantification (LLOQ) was taken to be the lowest con-centration in the calibration curves, if the following require-ments are met: the response for the analyte in the LLOQ is atleast ten times higher than the response for the blank; also,the analyte peak must be identifiable and reproducible with aprecision of 20% and an accuracy of �20% (Peters and Maurer,2002).The precision of an analytical method determines the proxim-

ity between the values obtained for a series of samples from thesame stock solution, analyzed under the same conditions, usingfive replicates for each concentration level (low, medium andhigh) the same day (intraday precision or repeatability) or on dif-ferent days (inter-day precision or reproducibility). It is calculated

gression model ANOVA lack of fit

P-Value F-Ratio P-Value

0 0.10 0.98200 1.12 0.37930 1.40 0.26370 1.51 0.23000 0.18 0.94430 1.72 0.17950 1.67 0.19480 1.51 0.2357

, benzoylecgonine; COC, cocaine; CCE, cocaethylene; EDDP, 2-; HPLC-PDA, high-performance liquid chromatography coupled

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from the coefficient of variation (CV), which must not exceed15% (Shah et al., 2000; Peters and Maurer, 2002).

The recovery of an analytical method measures the efficiencyof the extraction procedure. It is calculated by comparing theanalytical response of five blank replicates, containing a knownconcentration of the analyte before the extraction, with theanalytical response of other five replicates similarly spiked, after

Figure 2. Chromatograms obtained at 233nm from (a) a blank vitreous hcase, no. 1 from Table 4. MRP, morphine; COD, codeine; 6AM, 6-monoacEDDP, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; MTD, methadone.

Table 3. Recovery and precision data

Parameter (n=5) 0.1 mg ml�1

MRP % Recovery 91.1% CV intra-day 0.9% CV inter-day 4.6

COD % Recovery 89.8% CV intra-day 4.5% CV inter-day 2.8

6AM % Recovery 95.0% CV intra-day 1.3% CV inter-day 4.9

BZE % Recovery 83.7% CV intra-day 8.8% CV inter-day 10.0

COC % Recovery 98.3% CV intra-day 1.7% CV inter-day 2.5

CCE % Recovery 91.3% CV intra-day 1.5% CV inter-day 5.2

EDDP % Recovery 69.1% CV intra-day 1.5% CV inter-day 1.9

MTD % Recovery 86.2% CV intra-day 9.5% CV inter-day 12.4

MRP, morphine; COD, codeine; 6AM, 6-monoacetylmorphine; BZEethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; MTD, methadone


carrying out the extraction procedure. These last samples wouldgive the analytical response of a hypothetical 100% recovery.The recovery percentage is calculated as follows: A� 100/P,where A is the average value of the replicates spiked beforethe extraction, and P is the average value of the replicates spikedafter the extraction. Recovery values higher than 60% areconsidered adequate.

umor sample (blue) and a standard of 4mg ml�1 (black) and (b) a realetylmorphine; BZE, benzoylecgonine; COC, cocaine; CCE, cocaethylene;

1mg ml�1 4mg ml�1 Average data

82.8 97.8 90.51.6 0.4 1.01.3 0.4 2.1

97.2 96.9 94.61.7 0.7 2.30.9 0.2 1.3

99.1 97.3 97.11.1 0.6 1.01.4 0.4 2.2

93.3 98.6 91.93.2 1.1 4.40.9 0.6 3.9

94.9 97.7 97.00.9 0.1 0.91.1 0.4 1.3

96.1 95.9 94.42.4 0.6 1.50.2 0.8 2.1

62.1 68.1 66.42.9 0.2 1.51.2 0.4 1.2

70.8 78.7 78.61.3 0.7 3.82.9 0.8 5.4

, benzoylecgonine; COC, cocaine; CCE, cocaethylene; EDDP, 2-.


Table 4. Concentrations in real vitreous humor samples

Case Drug Concentration (mgml�1)

1 Benzoylecgonine 6.84Cocaine 1.95

2 Benzoylecgonine 0.133 Benzoylecgonine 3.844 Morphine 0.15

6-Acetylmorphine 0.24Benzoylecgonine 1.98Cocaine 0.10

5 Benzoylecgonine 0.966 Benzoylecgonine 0.12

Methadone 0.127 Benzoylecgonine 0.63

Methadone 0.108 Benzoylecgonine 0.51

Cocaethylene 0.15EDDP 0.69

9 Benzoylecgonine 0.66Codeine 0.10

10 Morphine 0.45Cocaine 0.37

11 EDDP 0.6612 Benzoylecgonine 0.1013 Benzoylecgonine 0.11

Methadone 0.2114 Benzoylecgonine 0.2415 Benzoylecgonine 0.15

EDDP, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine.

Chromatographic determination of drugs of abuse in vitreous humor

RESULTS AND DISCUSSION

Identification of drugs of abuse was done taking into accounttheir retention times (Table 1) and absorption spectra. Thistwo-fold identification approach resulted in increased specificityas it allowed the detection of potential impurities and/or drugscoextracted from vitreous humor, with retention times similarto those of the target drugs but with markedly different spectra.

Solid-phase extraction is commonly used in analytical laborato-ries as a sample preparation and a cleanup or preconcentrationtechnique in the processing of biological fluids using differentsorbents (Fernández et al., 2006; Li et al., 2006; Wey and Thormann,2001; Aturki et al., 2009). In order to optimize the extraction condi-tions, some previous tests were carried out. OasisW HLB cartridgeswere used owing to their ability to retain different types of drugs,and not only acid or basic substances, for which more specificcartridges are available (OasisW MCX and OasisW WAX).

The first optimization stage consisted in determining theproportion of methanol employed in the first washing step.Percentages of 5, 10, 15, 20, 25 and 30% volume were used,observing the presence of BZE from 25% methanol used, andmorphine from 30% methanol used. Thus, a mixture of 80%water and 20% methanol was used to eliminate the maximumamount of impurities without affecting the retention of targetdrugs of abuse. Afterwards, different portions of NH4OH weretested in the second washing step, from 0.5 to 5% volume, with-out noticeable variation. Thus, a mixture of water–methanol–ammonium hydroxide (80:18:2, v/v) was used to enhance thewashing effect and therefore improve the extraction efficiency.The eluent was also optimized, testing 2ml of dichloromethane,a double elution step with 2ml of dichloromethane and 2ml ofmethanol and 2ml of methanol. The chromatographicresponses obtained in each case are shown in Fig. 1, showingthat dichloromethane is the solvent with the best results regard-ing sample purification and drug recoveries.

The linearity of the method was studied in a concentrationrange 0.1–4mg ml�1, by performing three replicates of eachconcentration level. The data obtained were used to perform re-gression analysis and analysis of variance, showing good results,with coefficients of correlation higher than 0.99 for all the drugsstudied (Tables 1 and 2). Analysis of variance (ANOVA) was per-formed to validate the regression data. When P-values (probabil-ity) are lower than 0.05 (significance level), linear regressionmodels are statistically significant. The lack-of-fit test is designedto determine whether the selected model is adequate todescribe the experimental data obtained, or whether a morecomplicated model should be used. The test is performed bycomparing the variability of the current model residuals withthe variability between observations (area counts) at replicatevalues of the independent variable (known concentration ofthe target drugs in the calibration levels). Because P-values inTable 2 are greater than 0.05, linear regression models areadequate for our data in vitreous humor samples.

The limits of detection achieved were: 29 ng ml�1 for MRP,14 ng ml�1 for COD, 25 ng ml�1 for 6AM, 12 ng ml�1 for COC,10 ng ml�1 for BZE, 28 ng ml�1 for CCE, 24 ng ml�1 for EDDPand 28 ng ml�1 for MTD. The lower limit of quantification wasset at 100 ng ml�1 for all the drugs studied. These results arecomparable to those published by Logan and Stafford (1990)to determine cocaine and BZE in vitreous humor. Other studiesconducted on the same sample type, employing solid-phaseextraction and gas chromatography (Mackey-Bojack et al.,


2000; Fernández et al., 2006), gave values of LOD and LLOQ forcocaine and BZE slightly higher or similar to those presentedin this work. The proposed methods by Pragst et al. (1999) andWyman and Bultman (2004), to determine morphine and meta-bolites using GC-MS, showed results lower than those obtainedin our study. Taking into account the usual levels reported invitreous humor from cocaine users who had died owing tooverdose (Fernández et al., 2006; Furnari et al., 2002), the limitsof detection and quantification achieved can be consideredacceptable in this type of sample.Five blank vitreous humor samples were analyzed to assess

the selectivity of the method. The absence of analytical responseat the retention times of the target drugs demonstrated that nointerference occurred, as shown in Fig. 2(a).Intra- and inter-day precisions were examined at three different

concentration levels (low, medium and high) of drugs of abuse invitreous humor. Both studies gave good results with coefficientsof variation less than 12.5%, especially when determining highconcentrations (Table 3). These values are lower than thoseobtained by Fernández et al. (1994) for COC and BZE in intra- andinter-day analysis. In a later publication, Fernández et al. (2006)reported lower values for cocaine (3.4%) using gas chromatographyas the determination technique. Wyman and Bultman (2004)showed coefficients of variation higher than those presented herefor morphine (7.2%) and codeine (4.4%).The recovery of the method was also studied at the same

three different concentration levels as selected for the test ofprecision (Table 3). The values obtained are higher for the highconcentration (4 mg ml�1) of morphine and BZE, the medium

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concentration (1 mg ml�1) of codeine, 6AM and CCE, and the lowconcentration (0.1mg ml�1) of cocaine, EDDP and methadone.Average recoveries ranged from 66.4% for EDDP to 97.1% for6AM. The efficiency of the extraction procedure for BZE wascomparable to that reported by Fernández et al. (2006), andhigher for BZE and cocaine compared with other studies (Loganand Stafford, 1990; Fernández et al., 1994).

Finally, this method was applied to quantification of drugs ofabuse in 15 vitreous humor samples from opiates and/or cocaineabusers, after applying enzymeinmunoassay and gas chroma-tography–mass spectrometry in urine to demonstrate thepresence of these substances. The results obtained can be seenin Table 4. The highest levels of the analytes under study wereobserved in the first case (Fig. 2b) for BZE (6.84mg ml�1) andcocaine (1.95mg ml�1). BZE was detected in 13 cases, followedby cocaine and methadone found in three cases, and morphineand EDDP detected in two cases. It was also noticed that inseven cases only cocaine and/or its metabolites were detected;in another four cases, cocaine appeared together with metha-done; three cases were associated with cocaine and heroinconsumption, and one case was found to be only related tomethadone. In contrast to other studies (Fernández et al., 2006;Mackey-Bojack et al., 2000), BZE and cocaine were detectedsimultaneously in very few cases of our series (two out of 15individuals). Concentrations found for BZE varied from 0.10 to6.84 mg ml�1, with an average value of 1.20� 1.91 mg ml�1. Mostof these values are within the toxic and even lethal levels for thissubstance as described by Logan and Stafford (1990) andMackey-Bojack et al. (2000).

The results of our study demonstrate the suitability of thevitreous humour as an alternative biological sample for thedetermination of drugs of abuse and the effective use ofSPE-HPLC/PDA method for this purpose.

Acknowledgments

The authors wish to acknowledge funding of this work by TheXunta de Galicia (Project 10PXIB208089PR).

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chromatographic determination of drugs of abuse in vitreous humor using solid-phase extraction

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