Journal of Analytical Toxicology, Vol.

25, July/August 2001

Technical Note

Importance of Vacutainer Selection in Forensic Toxicological Analysis of Drugs of Abuse

Stefan W. Toennes* and Gerold F. Kauert
Institute of Forensic Toxicology, University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt/Main, Germany

Abstract
The enzymatic degradation of cocaine in blood samples, even during transport to a forensic laboratory, is a common problem in toxicological analysis. This can be avoided by the use of bloodsampling devices such as gray-top Vacutainers containing the cholinesterase inhibitor sodium fluoride. In the present study, which included 147 authentic cases, blood samples were collected into two different tubes, one containing fluoride/oxalate and one without stabilizing agents. In all cases, both samples were analyzed for drugs of abuse using Abbott FPIA immunoassays after precipitation and gas chromatographymass spectrometry (GCMS) for quantitative analysis. The cannabinoid immunoassay showed markedly lower values in the fluoridecontaining samples; this was investigated further and could be explained by hemolysis of these samples. In addition, the concentrations of 11-nor-9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) were lower in these samples. A stability study with the THCCOOH acyl glucuronide showed that it is unstable in unpreserved serum, which could explain our observation. GCMS quantitative data for amphetamine and derivatives, opiates, 9-tetrahydrocannabinol, and 11-hydroxy-9tetrahydrocannabinol were essentially identical; however, they also differed substantially for cocaine, cocaethylene, ecgonine methylester, and benzoylecgonine. Unexpectedly, the concentrations of benzoylecgonine in unpreserved serum were almost half as high as in the fluoride-containing samples.

In the present study, immunochemical and gas chromatographicmass spectrometric (GCMS) analysis data of drugs of abuse from blood samples collected in forensic cases of suspected driving under the influence of drugs were compared. Blood samples were taken from the subjects using two Vacutainer tubes, one free of additives and one containing fluoride/oxalate as stabilizing agents (gray-top Vacutainer). This study design allowed the direct comparison of the analytical results from the two types of blood sampling tubes using authentic material. New information on the stability of certain metabolites could be obtained.

Experimental
Chemicals, reference standards, and apparatus

Introduction
The rapid enzymatic degradation of cocaine in blood samples even during transport to a laboratory is a well-known problem in forensic toxicology. Through the use of blood samples collected in gray-top Vacutainer tubes (Becton Dickinson), which contain the anticoagulant potassium oxalate and the cholinesterase inhibitor sodium fluoride, enzymatic cocaine catabolism can be inhibited, at least for certain period of time (13). Concentrations of other drugs of abuse are reported to be unaffected during storage (48). However, these studies were carried out mostly with reference substances only or without controls in unstabilized material.
* Author to whom correspondence should be addressed. E-mail: toennes@em.uni-frankfurt.de.

The reference standards and their corresponding deuterated internal standards were purchased from Radian (Promochem, Wesel, Germany), except for (l)-9-carboxy-11-nor-9-tetrahydrocannabinol glucuronide (THCCOOH-glucuronide) 10 g/mL solution in methanol which was from Alltech (Deerfield, IL). The derivatization reagents N-methyl-N-(tert-butyldimethyl-silyl)trifluoroacetamide (MTBSTFA), N-methyl-bis(trifluoroacetamide) (MBTFA), and methyl iodide were from Sigma (Munich, Germany). All other reagents and organic solvents were of analytical grade and from Merck (Darmstadt, Germany). GCMS analyses were performed on a Hewlett-Packard (Waldbronn, Germany) GCMS (HP 5890 series II GC, HP 6890 ALS, HP 5972 MSD) with an HP-5 MS capillary column (30 m 0.25-mm i.d., 0.25-m film thickness). The carrier gas was helium with a flow rate of 1.0 mL/min. The MS conditions were 280C transfer line temperature and 70 eV ionization energy. Data analysis was performed on a Windows computer with HP ChemStation software (Rev. C.03.00).
Samples and study design

In cooperation with the highway police (Hessen, Germany), a study was performed using 147 cases of suspected driving under drug influence. In each case, blood sampling was carried out using two 10-mL Vacutainer tubes (Becton Dickinson, Heidelberg, Germany): one contained 25 mg sodium fluoride and 20 mg potassium oxalate as stabilizing reagents (equipped with gray rubber tops), and the other type was without additives (red rubber tops). In all cases urine samples were also available. Upon

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arrival in the laboratory, the Vacutainer tubes were centrifuged for separation of serum (Vacutainer tubes with coagulated blood, normal samples) or plasma (from blood in Vacutainer tubes containing oxalate/fluoride additive, fluoride samples) and stored at 4C until analysis which was carried out within two weeks. Immunological screening was performed using the automated AxSym analyzer (Abbott, Wiesbaden, Germany) with Abbott fluorescence-polarization immunoassays (FPIA) Amphetamine/ Methamphetamine II, Opiates U, Cannabinoids U, and Cocaine Metabolite U; cutoff levels in urine were 300 ng/mL, 200 ng/mL, 25 ng/mL, and 200 ng/mL, respectively. Urine samples were initially analyzed immunologically, and if a positive result was obtained, the serum and plasma samples were tested using the same immunoassays. If at least one of the two samples tested positive, quantitative analysis with GCMS was performed.
Immunological screening for drugs of abuse in serum/plasma with Abbott FPIA

propanol/concentrated aqueous ammonium hydroxide (80:20:2, v/v/v) at 1 mL/min. The extracts were evaporated, the dry residue was derivatized with 40 L MBTFA followed by derivatization with 30 L MTBSTFA, and 1 L of this solution was analyzed by GCMS. GC conditions were as follows: injection port temperature 260C, held at 60C for 2 min, increased to 170C at 20C/min, then to 310C at 12C/min, and held for 5 min. Quantitation of amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethamphetamine (MDEA), morphine, 6monoacetylmorphine, codeine, dihydrocodeine, cocaine (COC), cocaethylene, and ecgonine methylester (EME) as TFA derivatives and benzoylecgonine (BZE) as tBDMS derivative was performed in single ion monitoring (SIM) mode using internal standards. For the determination of the cannabinoids 9-tetrahydrocannabinol (THC), 11-hydroxy-9-tetrahydrocannabinol (THCOH), and THCCOOH, 1 mL of serum/plasma was mixed with 4 mL deionized water and 50 L of internal standard solution (0.5 ng/L of THC-d3 and THC-OH-d3 and 2 ng/L of THCCOOH-d3 in methanol), vortex mixed, and centrifuged for 10 min at 1900 g. Manual SPE was performed using 3-mL Bakerbond C18 500-mg cartridges (Baker, Griesheim, Germany) conditioned with 3 mL methanol and 2 3 mL water on a vacuum manifold. The samples were slowly applied onto the columns (0.5 mL/min). The columns were washed with 1 mL water, 1 mL 0.25M acetic acid, and 1 mL water, and vacuum was applied for 5 min. The analytes were eluted with 3 0.5 mL acetone, and the eluate was evaporated to dryness. The residue was derivatized using methyl iodide, re-extracted with isooctane, and evaporated again. The residue was dissolved in 50 L isooctane, and 1 L was analyzed by GCMS. GC conditions were as follows: injection port temperature 250C, held at 100C for 2 min, increased to 310C at 30C/min, and held for 6 min. Quantitation of THC, THC-OH, and THCCOOH as methyl derivatives was performed in the SIM mode using internal standards.
Study on the stability of cocaine in unpreserved serum Determination of cannabinoids in serum/plasma by GCMS

For protein separation, 400 L of serum/plasma was added dropwise to 400 L of acetone. The solution was vortex mixed and centrifuged for 10 min at 1900 g. The supernatant was analyzed by the Abbott FPIA tests. According to the results of a previous study (9), the following cutoff values were used: 40 ng/mL for Amphetamine/ Methamphetamine II, 40 ng/mL for Opiates U, 20 ng/mL for Cannabinoids U, and 20 ng/mL for Cocaine Metabolite U.
Influence of the fluoride/oxalate additives on the FPIA results

Two blood samples were obtained from each of six healthy nondrug users using the two different Vacutainer types (gray top and red top). Each blood sample was split: one part was immediately centrifuged for serum/plasma separation, and the other part was stored for two days at 4C to allow the development of hemolysis, after which it was also centrifuged for serum/plasma separation. From each of these 24 serum/plasma samples, 500 L was added dropwise to 500 L of acetone. After vortex mixing, the samples were centrifuged for 10 min at 1900 g. To 500 L of the supernatant 25 L of a solution containing amphetamine (1000 ng), benzoylecgonine (1000 ng), morphine (100 ng), and 11-nor-9tetrahydrocannabinol-9-carboxylic acid (THCCOOH) (40 ng) in acetone was added. These preparations were analyzed in the Abbott AxSym analyzer. The Students t-test was applied to test for significant differences.
Determination of basic analytes in serum/plasma by GCMS

Twenty milliliters of pooled blank serum (pH 7.5) was spiked with cocaine to obtain 1 g/mL and incubated in a 25C water bath for 80 h. At selected times, 1-mL aliquots were analyzed for COC, EME, and BZE by GCMS.
Study on the stability of benzoylecgonine in unpreserved serum

Serum samples (1 mL) were diluted with 4 mL of 0.1M phosphate buffer (pH 6.0), and 100 L of an internal standard solution (1 ng/L of amphetamine-d5, 3,4-methylenedioxymethamphetamine-d5, morphine-d3, codeine-d3, cocaine-d3, benzoylecgonine-d3, and ecgonine methylester-d3 in acetonitrile) was added. The samples were vortex mixed. The diluted samples were extracted using 3-mL Bond Elut Certify HF 300-mg solid-phase extraction (SPE) cartridges (Varian, Darmstadt, Germany) with the extraction robot RapidTrace (Zymark, Idstein, Germany). The extraction protocol was as follows: conditioning with 2 mL methanol and 3 mL phosphate buffer, application of the sample onto the column at 1 mL/min, rinsing with 2 mL 0.1M acetic acid and 3 mL methanol at 1.5 mL/min, and elution of the analytes with 3 mL of freshly prepared solution of methylene chloride/2-

Ten milliliters of pooled blank serum (pH 7.5) was spiked with BZE to obtain 1 g/mL and incubated in a 25C water bath. At 0, 15, 23, and 38 h, two 1-mL aliquots were analyzed for BZE by GCMS.
Study on the stability of THCCOOH-glucuronide in unpreserved serum and in fluoride/oxalate containing plasma

Blood from a healthy non-drug user was collected in vacutainers with and without fluoride/oxalate and centrifuged for serum/plasma separation. Aliquots of 5 mL of the unpreserved serum and of the fluoride/oxalate plasma were each spiked with a solution of THCCOOH-glucuronide to contain 37.8, 75.6, or

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151.2 ng THCCOOH-glucuronide per milliliter of serum/plasma (equivalent to 25, 50, or 100 ng/mL THCCOOH). The spiked samples were incubated in a 25C water bath for 250 h, and at selected times, 1-mL aliquots were analyzed for THCCOOH by GCMS. The residue of the unpreserved blood sample, which contained the erythrocytes, was washed three times with aqueous 0.9% sodium chloride solution to completely remove serum components and was reconstituted with the sodium chloride solution to the original volume of the blood sample. A 10-mL aliquot was spiked with THCCOOH-glucuronide to contain 37.8 ng/mL (equivalent to 25 ng/mL THCCOOH). The sample was incubated

in a 25C water bath for 250 h, and at selected times, 1-mL aliquots were analyzed for THCCOOH by GCMS.

Results and Discussion

The aim of the present study was to investigate possible differences in results obtained by analyzing blood samples collected in Vacutainer tubes with and without stabilizing additives. Such differences have already been observed in the preanalytical phase. An effect of the sodium fluoride was that all plasma samples became markedly hemolytic, and the volumes of the plasma samples were higher because of the anticoagulant potassium oxalate.
Immunological screening for drugs of abuse in serum/plasma with Abbott FPIA

To some extent, the results of the immunoassays in normal serum and fluoride plasma for amphetamine and derivatives (79 cases), opiates (40 cases), cannabinoids (65 cases), and cocaine (46 cases) showed considerable differences. The values for opiates and amphetamine did not differ markedly in average, but in the case of the assay Cocaine Metabolite U, the values in the fluoride samples were, on average, twice as high as the values in the normal samples. For the cannabinoid assay, the results of the fluoride samples were, on average, only half as high as the values in the normal samples, which caused a higher rate of false negatives (28% vs. 5% in normal samples).
Figure 1. Concentrations of cocaine, benzoylecgonine, ecgoninemethylester and THCCOOH measured with GCMS in serum from normal Vacutainers ( O, ascending order) and in the corresponding plasma from Vacutainers containing fluoride/oxalate (+).

Influence of the additives on FPIA results

Drug-free serum/plasma samples were spiked just before measurement to eliminate any differences due to sample preparation. The assays of all six samples were reproducible with variation coefficients of less than 8%. When serum/plasma was separated immediately after blood sampling, none of these samples was hemolytic, and no significant differences in the FPIA values were observed, indicating that the additives themselves did not affect the measurement. This is in agreement with results obtained by Robinson and Smith (10) for an immunoassay of tricyclic antidepressants. During storage for two days, hemolysis developed in the fluoride/oxalate-containing blood samples. FPIA values in the fluoride/oxalate-containing samples before and after storage did not differ except for the assay Cannabinoids U, where the values obtained in the hemolytic samples were significantly lower by 39% ( p < 0.001). This effect must therefore be attributed to hemolysis.
Quantitative analyses in serum/plasma by GCMS

Figure 2. Degradation of cocaine ( I ), 1000 ng/mL spiked) to benzoylecgonine (N), and ecgonine methylester (L ) in serum at 25C. The sum of the molar concentrations of cocaine, benzoylecgonine, and ecgonine methylester at each time is given O) together with the result of an exponential regression of these data (dashed line).

No differences could be observed during extraction and GCMS analysis between the two sample

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types; GCMS signals of analytes and matrix compounds in SIM mode showed no differences. No differences in the quantitative results could be found for the analytes amphetamine (49 cases), MDMA (23 cases), MDA (16 cases), morphine (14 cases), codeine (13 cases), dihydrocodeine (3 cases), THC (35 cases), and THCOH (32 cases). However, clear differences were observed for cocaine, its metabolites (29 cases), and THCCOOH (45 cases) as shown in Figure 1.
Stability of cocaine and its metabolites in serum/plasma

In 29 cases, benzoylecgonine was determined as evidence of previous cocaine use. Cocaine itself was not found in any of the normal samples, but in 65% of the fluoride samples. Cocaethylene was present in two of the fluoride samples (8%), confirming that fluoride has a stabilizing effect on cocaine and cocaethylene. It was surprising that the concentrations of benzoylecgonine in the fluoride samples were almost twice as high (compare immunological results) and those of ecgonine methylester were almost half when compared to those of the normal samples (Figure 1). In serum without additives, cocaine is rapidly hydrolyzed enzymatically to ecgonine methylester as has been shown in various studies (3) and in the present study (Figure 2). In contrast to Isenschmid et al. (3), it was found that cocaine was also hydrolyzed to benzoylecgonine, but only to a minor extent, which is in accordance with Stewart et al. (11). Ecgonine methylester and benzoylecgonine were also instable and were further hydrolyzed to ecgonine. The overall degradation process could be assessed by adding the molar concentrations of cocaine, ecgonine methylester, and benzoylecgonine and performing an exponential regression analysis which yielded a half-life of 15 h (equation: y = 4.22 e0.0462 x, regression coefficient 0.991, Figure 2). The differences observed between the data obtained by analyzing the normal and the fluoride samples are mainly due to the fluoride-mediated inhibition of the plasma cholinesterase (PChE). Consequently, ecgonine methylester concentrations were lower in the fluoride samples because enzymatic hydrolysis of cocaine to ecgonine methylester was inhibited. The literature reports on the stability of benzoylecgonine are

controversial. An enzymatic hydrolysis of benzoylecgonine to ecgonine like cocaine to ecgonine methylester could be expected; however, some authors suggest that benzoylecgonine is stable in unpreserved serum (3,8,12). Stewart et al. (11) found a constant, but slow hydrolysis in unstabilized serum samples that was explained by a low affinity of benzoylecgonine to the enzyme PChE. Giorgi and Meeker (7) and Moody et al. (13) reported a substantial decrease of benzoylecgonine concentrations in blood samples stabilized with fluoride, but Baselt et al. (5) reported that benzoylecgonine was stable in blood for one year in evacuated collection tubes containing 100 mg sodium fluoride and 20 mg potassium oxalate. Therefore, an experiment was performed in order to test the stability of benzoylecgonine in unstabilized serum. A sample containing benzoylecgonine in a concentration of 1000 ng/mL and incubated at 25C exhibited a linear decrease of 13 ng BZE/mL per hour (regression coefficient 0.997). The benzoylecgonine concentration was reduced to almost half of the initial value during the 38 h of incubation. Such a delay may be reached easily when a blood sample is sent by mail. The observed differences of the benzoylecgonine concentrations between the normal samples and the fluoride samples can therefore be explained by the hydrolysis of benzoylecgonine in unpreserved samples.
Stability of THCCOOH-glucuronide in serum/plasma

Figure 3. Percentage of hydrolyzed THCCOOH-glucuronide in serum from normal Vacutainers (O) and in plasma from Vacutainers containing fluoride/oxalate ( I ) at 25C. For each type of material are shown the average and standard deviation from three different concentrations of THCCOOHglucuronide (37.8, 75.6, and 151.2 ng/mL spiked).

The cannabinoid immunoassay is designed to measure THCCOOH. The observation that the immunological values in the fluoride samples were about half as high as in the normal samples could be explained by the finding that hemolysis in the fluoride samples markedly affects FPIA measurement. Surprisingly, the GCMS confirmation analyses showed that, in 90% of the fluoride samples, the THCCOOH concentrations were on average 32% lower than in the normal samples. Previous studies suggested that THC, THC-OH, and THCCOOH are stable in blood and plasma during storage for several months (8,1317). A possible explanation for our finding would be a conjugate of THCCOOH that is unstable, probably susceptible to degradation by serum esterases. Such a metabolite could be the ester glucuronide of THCCOOH (18) (THCCOOH-glucuronide). Therefore, we investigated the stability of the THCCOOH-glucuronide in blood samples with and without stabilizing agents. The THCCOOH-glucuronide was stable under the conditions of the analytical procedure as immediately after spiking of the samples no THCCOOH was detectable. No THCCOOH developed during the incubation of THCCOOH-glucuronide in suspended erythrocytes, indicating that the erythrocytes had no influence on the stability of the glucuronide. During incubation of THCCOOHglucuronide in serum or fluoride/oxalate containing plasma, THCCOOH could be detected in increasing concentrations. The absolute concentrations were dependent on the initial amount of glucuronide but in each group (with or without fluoride/oxalate) the THCCOOH concentration relative to the maximum theoretical yield were reproducible (Figure 3). The concentrations at 139 or 236 h incubation time were significantly lower in the fluoride/oxalate plasma samples than in the unstabilized serum samples ( p < 0.01) proving that the addition of fluoride/oxalate had indeed a stabilizing effect on the THC metabolite THCCOOH-glucuronide. This is probably the explanation for the lower concentrations of THCCOOH in the fluoride samples.

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Conclusions
In authentic cases, two blood samples were collected in tubes with and without the stabilizing agents fluoride and oxalate. Analysis results of drugs of abuse using immunoassays and GCMS differed markedly for THCCOOH and cocaine and its metabolites. The cannabinoid immunoassay was shown to be susceptible to hemolysis that developed in fluoride-containing samples. Furthermore, GCMS quantitative analyses revealed lower concentrations of THCCOOH in fluoride-containing samples, which could be explained by the lower stability of the THCCOOHglucuronide in unpreserved serum. Degradation of cocaine and cocaethylene to ecgonine esters was inhibited in fluoride-containing samples, but the massive enzymatic hydrolysis of benzoylecgonine that also occurred in the unstabilized samples was unexpected.
7. 8.

9. 10. 11. 12. 13.

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