Technical Note ] Importance of Vacutainer Selection in Forensic ...

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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 4~ until analysis which was carried out within two weeks. Immunological screening was performed using the automated AxSym analyzer (Abbott, Wiesbaden, Germany) with Abbott fluor- escence-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 ini- tially 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 GC-MS was performed. Immunological screening for drugs of abuse in serum/plasma with Abbott FPIA For protein separation, 400 tJL of serum/plasma was added dropwise to 400 IJL of acetone. The solution was vortex mixed and centrifuged for 10 min at 1900 xg. The supernatant was analyzed by the Abbott FPIA tests. According to the results of a previous study (9), the following cutoffvalues 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 non- drug 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 4~ to allow the development of hemolysis, after which it was also centrifuged for serum/plasma separation. From each of these 24 serum/plasma samples, 5001JL was added dropwise to 500 IJL of acetone. After vortex mixing, the samples were centrifuged for 10 min at 1900 xg. To 500 tJL of the supernatant 25 IJL of a solution containing amphetamine (1000 ng), benzoylecgonine (1000 ng), morphine (100 ng), and 11-nor-Ag- tetrahydrocannabinol-9-carboxylic acid (THCCOOH) (40 rig) in acetone was added. These preparations were analyzed in the Abbott AxSym analyzer. The Student's t-test was applied to test for significant differences. Determination of basic analytes in serum/plasma by GC-MS Serum samples (1 mL) were diluted with 4 mL of 0.1M phosphate buffer (pH 6.0), and 100 IJL of an internal standard solution (1 ng/lJL of amphetamine-d5, 3,4-methylenedioxymetham- phetamine-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- 340 Journal of Analytical Toxicology, Vol. 25, July/August 2001 propanol/concentrated aqueous ammonium hydroxide (80:20:2, v/v/v) at I mL/min. The extracts were evaporated, the dry residue was derivatized with 40 IJL MBTFA followed by derivatization with 30 IJL MTBSTFA, and 1 tJL of this solution was analyzed by GC-MS. GC conditions were as follows: injection port tempera- ture 260~ held at 60~ for 2 min, increased to 170~ at 20~ then to 310~ at 12~ and held for 5 min. Quantitation of amphetamine, 3,4-methylenedioxymetham- phetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethamphetamine (MDEA), morphine, 6- monoacetylmorphine, 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. Determination of cannabinoids in serum/plasma by GC-MS For the determination of the cannabinoids A9-tetrahydrocannabinol (THC), 11-hydroxy-A94etrahydrocannabinol (THC- OH), and THCCOOH, 1 mL of serum/plasma was mixed with 4 mL deionized water and 50 IJL of internal standard solution (0.5 ng/IJL of THC-d3 and THC-OH-d3 and 2 ng/IJL of THCCOOH-d3 in methanol), vortex mixed, and centrifuged for 10 min at 1900 xg. Manual SPE was performed using 3-mL Bakerbond C18 500-rag cartridges (Baker, Griesheim, Germany) conditioned with 3 mL methanol and 2 x 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 x 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 IJL isooctane, and 1 IJL was analyzed by GC-MS. GC conditions were as follows: injection port tempera- ture 250~ held at 100~ for 2 rain, increased to 310~ at 30~ and held for 6 rain. Quantitation ofTHC, 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 TWenty milliliters of pooled blank serum (pH 7.5) was spiked with cocaine to obtain 1 tJg/mL and incubated in a 25~ water bath for 80 h. At selected times, 1-mL aliquots were analyzed for COC, EME, and BZE by GC-MS. Study on the stability of benzoylecgonine in unpreserved serum Ten milliliters of pooled blank serum (pH 7.5) was spiked with BZE to obtain 1 IJg/mL and incubated in a 25~ water bath. At 0, 15, 23, and 38 h, two 1-mL aliquots were analyzed for BZE by GC-MS. 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
Journal of Analytical Toxicology, Vol. 25, July/August 2001 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 25~ water bath for 250 h, and at selected times, 1-mL aliquots were analyzed for THCCOOH by GC-MS. 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 compo- nents 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 18o Cocaine 350 ~. 160 "st" ~" 300 140 E ~120 + ~ 250 8 1oo 8 200 2000 ,~1500 1000 c 0 0 500 + ,- + ++O ~_ 0 29 pairs of samples 45 pairs of samples Figure 1. Concentrations of cocaine, benzoylecgonine, ecgoninemethylester and THCCOOH measured with GC-MS in serum from normal Vacutainers (O, ascending order) and in the corresponding plasma from Vacutainers containing fluoride/oxalate (+). 150 1 Ecgonine methylestsr := 80 + ~ 150 O + r 60 40 20 a,,~ 0 ........ --[- + _t_t _[_ .st. _t_.t_ .'~..'~..'~.~.'~.C'~"i.'~..']..'].~.']..'~.~.']. ~ =.- 0 0 + 100 ooOO+ + 50 ~_ .,~. ~i~+ +++ ++ 0 19 pairs of samples 20 pairs of samples 7 3500 3000 ,.~2500 Benzoylecgonine + ~ 2501 THCCOOH 200 0 i'! ~2- s- o ,i I_ c l- G) c o 0 0 =, xO \ 'x%\% 1~176 1 5o - . . . . . 20 40 60 8 Incubation time in serum at 25~ (h) Figure 2. Degradation of cocaine (I), 1000 ng/mL spiked) to benzoylecgonine (O), and ecgonine methylester (A) in serum at 25~ The sum of the molar concentrations of cocaine, benzoylecgonine, and ecgonine methylester at each time is given (9 together with the result of an exponential regres- sion of these data (dashed line). in a 25~ water bath for 250 h, and at selected times, 1-mL aliquots were analyzed for THCCOOH by GC-MS. 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 volo umes of the plasma samples were higher because o 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 cannabi- noid 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). 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 coef- ficients 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, indi- cating 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 GC-MS No differences could be observed during extraction and GC-MS analysis between the two sample 341
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