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Examination of postmortem fluids and tissues for the presence of methylecgonidine, ecgonidine, cocaine, and benzoylecgonine using solid-phase extraction and gas chromatography-mass spectrometry.

Typically, biological tissues have been analyzed for cocaine (COC See chip on chip. )[1] and/or COC metabolites to detect COC use, but defining the route of administration is difficult based on this information alone. Additional useful information may be obtained from the detection of methyl ecgonidine (MED), which has been reported as a pyrolysis py·rol·y·sis
n.
Decomposition or transformation of a chemical compound caused by heat.


pyrolysis (pīrol´isis),
n
 product from COC smoking (1-3). In actuality, the smoking of crack COC is greatly affected by individual technique, and the amount of COC inhaled is dependent on several factors (1, 4-6). The temperature in the pipe or smoking device has a significant impact on the amount of COC inhaled and on the amount of MED produced. At temperatures of 255-420[degrees]C, the amount of COC converted to MED is 50-80% (4) and at a temperature of 650[degrees]C, as much as 89% of COC is converted to MED (1, 4). These temperatures are not unrealistic because the glowing end of a cigarette can easily reach temperatures of ~800[degrees]C.

On the basis of this information, it would be reasonable to expect individuals smoking crack COC to inhale significant amounts of MED. As a result, the detection of MED would be a valuable marker to aid in identifying smoking as a route of administration and in estimating COC concentrations at the time of death.

There are additional considerations in the analysis of MED. Once in the body, the concentration of MED might be expected to be lower because of conversion to ecgonidine (ED). The hydrolysis hydrolysis (hīdrŏl`ĭsĭs), chemical reaction of a compound with water, usually resulting in the formation of one or more new compounds.  of MED to ED has been reported, and ED has been detected in human urine (7, 8). Urine specimens that had tested positive for benzoylecgonine (BZ) in the military drug-testing program were tested for MED and ED. In 22 of the 23 specimens tested, ED was detected, and the concentrations were at least an order of magnitude A change in quantity or volume as measured by the decimal point. For example, from tens to hundreds is one order of magnitude. Tens to thousands is two orders of magnitude; tens to millions is three orders of magnitude, etc.  greater than those of MED. These results suggest the value of quantifying both MED and ED to identify COC smoking as the route of administration.

Although MED and/or ED have been examined in various tissues and fluids (2, 3, 7-13), MED has not been examined in liver and brain tissue, and ED has not been examined in liver, brain, and blood specimens. We examined the available specimens from 15 postmortem postmortem /post·mor·tem/ (post-mort´im) performed or occurring after death.

post·mor·tem
adj.
Relating to or occurring during the period after death.

n.
See autopsy.
 cases for the presence of MED, ED, COC, and BZ.

Materials and Methods

CHEMICALS, REAGENTS, AND SUPPLIES

N-desmethyl-N-[[sup.2.H.sub.3]]methylcocaine ([d.sub.3] COC), BZ, N-des-methyl-N-[[sup.2.H.sub.3]]methylbenzoylecgonine ([d.sub.3]-BZ), N-desmethyl-N-[[sup.2.H.sub.3]]methylecgonine ([d.sub.3]-EC), ecgonine, MED (anhydroecgonine methyl ester), and ED (anhydroecgonine) were purchased from Radian International. COC (free base) was purchased from Sigma Chemical, and dimethylformamide (DMF (Distribution Media Format) A floppy disk format from Microsoft that was used to distribute its software. DMF floppies compressed more data (1.7MB) onto the 3.5" diskette, and the files could not be copied with normal DOS and Windows commands. A DMF utility had to be used. ), DMF dipropyl acetal acetal /ac·e·tal/ (as´e-t'l)
1. any of a class of organic compounds formed by combination of an aldehyde molecule and two alcohol molecules.

2.
 (DMF-DPA), and DMF dimethyl di·meth·yl  
n.
An organic compound, especially ethane, containing two methyl groups.
 acetal were purchased form Aldrich Chemical. Solid-phase extraction (SPE SPE - Software Practice and Experience ) columns containing silica-based [C.sub.8] and S[O.sub.3]H (200 mg) and a glass extraction chamber were purchased from United Chemical Technologies. Reacti-vials, bis(trimethylsilyl)trifluoroacetamide (BSTFA BSTFA N,o-Bis (Trimethylsilyl) trifluoroacetamide (derivatization reagent) ), and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA MTBSTFA N-Methyl-N- (Tert-Butyldimethylsilyl)trifluoroacetamide (derivatization reagent) ) containing 1% tert-butyldimethylchlorosilane (TBDMCS) were purchased from Pierce. All solvents and reagents were analytical or HPLC HPLC high-performance liquid chromatography.

HPLC

high performance liquid chromatography.

HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed
 grade. Acetone acetone (ăs`ĭtōn), dimethyl ketone (dīmĕth`əl kē`tōn), or 2-propanone (prō`pənōn), CH3COCH3  was dried over molecular sieves (3A, 8-12 mesh) for at least 24 h.

SPECIMENS

Specimens from 15 postmortem cases were obtained from the Postmortem/ Human Performance Laboratory, Division of Forensic Toxicology, Armed Forces Medical Examiners' Office, Armed Forces Institute of Pathology. The research protocol was approved by the Armed Forces Institute of Pathology Institutional Review Board. Specimens reported to be positive for COC/BZ had been screened positive by immunoassay Immunoassay

An assay that quantifies antigen or antibody by immunochemical means. The antigen can be a relatively simple substance such as a drug, or a complex one such as a protein or a virus.
, confirmed positive by gas chromatography-mass spectrometry (GC-MS), and stored frozen at -18[degrees]C for 6 months after testing was completed. Before use in this study, specimen identity was removed in preparation for disposal. Complete information concerning the specific source for each fluid and tissue was not available for any case. A significant impact on results was unlikely for the liver specimens because drug distribution in liver tissue would be expected to be uniform as a result of normal high hepatic profusion and metabolic activity. Brain tissue may have been obtained from various portions of the brain because there is no standardized procedure concerning the source of brain tissue. This most likely did not have a significant impact on our results, based on the reported uniform distribution of COC in postmortem brain tissue (14). Urine was obtained directly from the bladder. In a few cases, blood specimens were obtained directly from the heart or from an intravenous catheter.

INSTRUMENT

The Hewlett-Packard GC-MS system consisted of an HP 5890 Series II Plus gas chromatograph and a Model 5972 quadrupole A quadrupole is one of a sequence of configurations of electric charge or gravitational mass that can exist in ideal form, but it is usually just part of a multipole expansion of a more complex structure reflecting various orders of complexity.  mass-selective detector. An HP 18593B auto-injector was used to inject the samples into the GC-MS instrument.

SYNTHESIS OF METHYL N-DESMETHYL-N-[[sup.2.H.sub.3]]METHYLECGONIDINE ([d.sub.3]-MED)

The starting compound, N-desmethyl-N-[[sup.2.H.sub.3]]methylecgonidine ([d.sub.3]-ED) was prepared from 200 [micro]g of [d.sub.3] EC by the procedure published previously (7). DMF dimethyl acetal (50 [micro]L) was added to the compound in a Reactivial. The vial was tightly capped, vortex-mixed, and heated at 50[degrees]C for 5 h. The solution was cooled to room temperature. The [d.sub.3]-MED, without further purification, was quantitatively transferred to a 50-mL volumetric flask and diluted to the mark with acetonitrile acetonitrile /ac·e·to·ni·trile/ (as?e-to-ni´tril) a colorless liquid with an etherlike odor used as an extractant, solvent, and intermediate; ingestion or inhalation yields cyanide as a metabolic product. . When compared against a known amount of nondeuterated MED, the concentration of [d.sub.3]-MED was found to be 2.08 mg/L. On the basis of the molar concentration compared to the starting material, [d.sub.3]-ED, the yield was 84%.

PREPARATION OF STOCK SOLUTIONS

Solutions of COC, [d.sub.3] COC, MED, and [d.sub.3]-MED at appropriate concentrations were prepared in acetonitrile. Hydroxy hy·drox·y  
adj.
Containing the hydroxyl group.



[From hydroxyl.]


hydroxy  

Containing the hydroxyl group (OH).

Adj. 1.
 solvents were not used to avoid hydrolysis of methyl esters. Solutions of BZ, [d.sub.3]-BZ, ED, and [d.sub.3]-ED were prepared in methanol.

SAMPLE PREPARATION FOR COC, BZ, MED, AND ED DETERMINATION

Liver and brain tissue samples. The flowchart for the extraction is shown in Fig. 1. For each specimen and control, 1 g of tissue (negative tissue for control) was weighed into flat-bottomed plastic tubes and stored frozen. Controls were prepared at concentrations of 100, 50, and 20 ng/g of tissue by adding COC (100 [micro]L of 1.0, 0.5, or 0.2 mg/L), BZ (100 [micro]L of 1.0, 0.5, or 0.2 mg/L), MED (100 [micro]L of 1.0, 0.5, or 0.2 mg/L), and ED (100 [micro]L of 1.0, 0.5, or 0.2 mg/L) into three empty glass centrifuge centrifuge (sĕn`trəfyj), device using centrifugal force to separate two or more substances of different density, e.g., two liquids or a liquid and a solid.  tubes. Internal standards (100 [micro]L of 0.5 mg/L [d.sub.3] COC, 100 [micro]L of 0.5 mg/L [d.sub.3]-BZ, 50 [micro]L of 0.68 mg/L [d.sub.3]-MED, and 100 [micro]L of 0.5 mg/L [d.sub.3]-ED) were added to the control tubes and to empty glass centrifuge tubes that were later used for specimen analysis.

[FIGURE 1 OMITTED]

Freshly prepared NaF (200 [micro]L; 10 g/L) was added to the tissues in the plastic tubes. The tissues were thawed, homogenized with 1 mL of 0.1 mol/L phosphate buffer (pH 6.0), and poured into the corresponding glass tubes containing drugs and internal standards for controls and internal standards only for specimens. Two milliliters of 0.1 mol/L phosphate buffer (pH 6.0) was then added to the residual tissue and homogenized again. The resulting homogenate homogenate /ho·mog·e·nate/ (ho-moj´in-at) material obtained by homogenization.

homogenate

material obtained by homogenization.
 was combined with the initial homogenate.

After homogenization homogenization (həmŏj'ənəzā`shən), process in which a mixture is made uniform throughout. Generally this procedure involves reducing the size of the particles of one component of the mixture and dispersing them evenly , the tubes were capped and placed in an ice bath. The homogenates were vortex-mixed for 10-15 s and centrifuged for 60 min at 3834g at 7-10[degrees]C. A set of SPE columns was placed in the extraction chamber and conditioned with 3 mL each of methanol, water, and 0.1 mol/L phosphate buffer (pH 6.0), in that sequence, under slightly reduced pressure.

A set of glass centrifuge tubes was placed in the chamber to collect the next elution elution /elu·tion/ (e-loo´shun) in chemistry, separation of material by washing; the process of pulverizing substances and mixing them with water in order to separate the heavier constituents, which settle out in solution, from the  fraction (containing ED). The supernatants from the tissue homogenates were poured onto the columns and allowed to elute e·lute  
tr.v. e·lut·ed, e·lut·ing, e·lutes
To extract (one material) from another, usually by means of a solvent.



[From Latin
 by gravity or under slightly reduced pressure. Deionized water (1 mL) was added to the columns, and that fraction was also collected. The tubes from the chamber were then removed and set aside for extraction of ED (Fig. 1). The SPE columns were washed with 2 mL of deionized water, 3 mL of 0.1 mol/L HCl, and 3 mL of isopropanol isopropanol, isopropyl alcohol, or 2-propanol (ī'səprō`pənōl, ī'səprō`pĭl), (CH3)2CHOH, a colorless liquid that is miscible with water. , and then dried for 5 min under reduced pressure. The MED, COC, and BZ were then eluted with 3 mL of a mixture (9:1:0.2 by volume) of dichloromethane-methanol-aqueous N[H.sub.3] (14.8 mol/L).

The solutions were evaporated to dryness under nitrogen at room temperature to minimize loss of MED. The extracts were dissolved in 50 [micro]L of dry acetone, transferred into autosampler vials, and tested for MED and COC by two separate GC-MS procedures. After the MED and COC tests, the samples were tested for BZ as the propyl propyl /pro·pyl/ (pro´pil) the univalent radical CH3CH2CH2—, from propane.

pro·pyl
n.
A univalent organic radical, CH3CH2CH2, derived from propane.
 derivative, using another GC-MS procedure.

To extract ED from the eluates, the pH of the solutions was adjusted to 2.0 [+ or -] 0.2 with 0.5 mol/L HCl. Methylene chloride (1 mL) was added to each tube and vortex-mixed. The solutions were centrifuged at 2177g for 3 min. The clear upper aqueous layers were then poured onto a second set of SPE columns preconditioned with 3 mL of methanol, 3 mL of deionized water, and 1 mL of 0.01 mol/L HCl. The solutions were allowed to elute by gravity flow or under slightly reduced pressure. The columns were washed with 1 mL of 0.1 mol/L HCl and 3 mL of methanol, and dried under reduced pressure for 5 min. The ED was eluted with 3 mL of a mixture (4:6:0.25 by volume) of methanol-isopropanol-aqueous N[H.sub.3] (14.8 mol/L). The solutions were evaporated under nitrogen at 50[degrees]C. To remove the white residue from ED, 2 mL of dry acetone was added. The solutions were vortex-mixed and centrifuged. The acetone solutions were separated and evaporated to dryness. The liver specimens were tested for ED as the tert-butyldimethylsilyl derivative by a GC-MS method, and the brain specimens were tested for ED as the trimethylsilyl derivative by GC-MS.

Urine and blood samples. An initial 3-mL aliquot aliquot (al-ee-kwoh) adj. a definite fractional share, usually applied when dividing and distributing a dead person's estate or trust assets. (See: share)  of each urine specimen was prepared as published previously (7). The procedure used for preparing blood specimens was the same as for the liver and brain tissues with the following exceptions. The initial 1-mL aliquots of blood were diluted with 4 mL of 0.1 mol/L phosphate buffer (pH 6.0). No methylene chloride wash was used in preparing the ED-containing fractions for the SPE steps.

DERIVATIZATION

Propylation of BZ at the GC injection port for specimen analysis. After COC and MED analysis in acetone solution, DMF-DPA (20 [micro]L) was added to the autosampler vials. The contents were mixed, and the vials were recapped. Analysis was performed for propyl-BZ as reported previously (7).

Silylation of ED by MTBSTFA for specimen analysis. The extracts from liver and urine containing ED were dissolved in MTBSTFA containing 1% TBDMCS and prepared as described previously (7).

Silylation of ED by BSTFA for specimen analysis. The extracts from brain and blood containing ED were dissolved in 50 [micro]L of BSTFA and heated in closed tubes at 70[degrees]C for 15 min. The samples were centrifuged immediately at 7-10[degrees]C for 2-3 min at 1700g and transferred to autosampler vials for GC-MS analysis.

GC-MS ANALYSIS

Samples were introduced in 1- to 4-[micro]L volumes using an autoinjector. The GC analysis was performed using a DB-5MS capillary column [5:95 phenyl-methylsiloxane; 15 m x 0.25 mm (i.d.); J & W Scientific] or a ZB-5 [5% phenyl phenyl (fĕn`əl), C6H5, organic free radical or alkyl group derived from benzene by removing one hydrogen atom.  polysiloxane; 15 m x 0.25 mm (i.d.); Phenomenex] at 10 psi constant pressure helium flow. The mass-selective detector was operated in the electron ionization mode at 70 eV with a source temperature of 200-250[degrees]C. The electron multiplier voltage of the detector was set at 200-700 V above autotune, and the dwell time for each ion monitored was 50 ms.

GC-MS conditions for MED. The analysis was performed at injector and transfer line temperatures of 140 and 280[degrees]C, respectively. The oven temperature started at 90[degrees]C (held for 1 min) and increased to 140[degrees]C at 20[degrees]C/min (held for 2 min). The GC was started in splitless mode, and injector port purge was turned on after 0.3 min. The monitored ions were m/z 181, 166, and 152 for MED and m/z 184 and 155 for the [d.sub.33]-MED. Ions m/z 152 and 155 were used for quantification. Because COC, BZ, and MED were extracted together, injector temperatures were kept [less than or equal to] 140[degrees]C to avoid possible formation of MED from any COC present in the sample. After each injection, 2-3 [micro]L of acetone was injected as a solvent blank.

GC-MS conditions for COC. The analysis was performed at injector and transfer line temperatures of 280 and 270[degrees]C, respectively. The oven temperature started at 170[degrees]C (held for 0.5 min) and increased to 270[degrees]C at 30[degrees]C/min (held for 2.7 min). The GC was started in splitless mode, and injector port purge was turned on after 0.3 min. The monitored ions were m/z 303, 272, and 182 for COC and m/z 306 and 185 for the [d.sub.3] COC. Ions m/z 303 and 306 were used for quantification. After each injection, 3 [micro]L of acetone was injected as a solvent blank.

GC-MS conditions for propyl BZ. The analysis was performed at injector and transfer line temperatures of 280 and 270[degrees]C, respectively. The oven temperature started at 170[degrees]C (held for 0.5 min) and increased to 270[degrees]C at 30[degrees]C/min (held for 2.7 min). The GC was started in splitless mode, and injector port purge was turned on after 0.3 min. The monitored ions were m/z 331, 272, and 210 for BZ and m/z 334 and 213 for [d.sub.3]-BZ. Ions m/z 331 and 334 were used for quantification. After each injection, 3 [micro]L of MTBSTFA containing 1% TBDMCS was injected as a solvent blank.

GC-MS conditions for tert-butyldimethylsilyl ED. The analysis was performed at injector and transfer line temperatures of 250 and 270[degrees]C, respectively. The oven temperature started at 135[degrees]C (held for 0.5 min), increased to 175[degrees]C at 10[degrees]C/min (held for 0.5 min), and increased to 275[degrees]C at 30[degrees]C/min (held for 1 min). The GC was started in splitless mode, and injector port purge was turned on after 0.2 min. The monitored ions were m/z 281, 252, and 224 for ED and m/z 284 and 227 for the [d.sub.3]-ED. Ions m/z 224 and 227 were used for quantification. After each injection, 3 [micro]L of MTBSTFA containing 1% TBDMCS was injected as a solvent blank.

GC-MS conditions for trimethylsilyl ED. The analysis was performed at injector and transfer line temperatures of 200 and 220[degrees]C, respectively. The oven temperature started at 111[degrees]C (held for 1 min) and increased to 210[degrees]C at 25[degrees]C/min (held for 2 min). The GC was started in splitless mode, and injector port purge was turned on after 0.2 min. The monitored ions were m/z 239, 224, and 210 for ED and m/z 242 and 213 for the [d.sub.3]-ED. Ions m/z 210 and 213 were used for quantification. After each injection, 3 [micro]L of BSTFA was injected as a solvent blank.

STATISTICAL ANALYSIS

The statistical analysis of the data sets was performed using ANOVA anova

see analysis of variance.

ANOVA Analysis of variance, see there
. To be a significant difference, both sets had to show a difference at P <0.05.

Results and Discussion

MED is a pyrolytic py·rol·y·sis  
n.
Decomposition or transformation of a compound caused by heat.



pyro·lyt
 product of COC. Both MED and ED are also produced as artifacts from COC or COC metabolites during GC-MS analysis (7). Therefore, suitable analytical conditions are necessary to test MED and ED in body fluids and tissues. In the extraction, ED was separated from other drugs by selective adsorption on the SPE columns ([C.sub.8] and S[O.sub.3]H). At pH 6.0 [+ or -] 0.5, most of the COC, BZ, and MED (>90%) were retained on the column, whereas ED, which did not adsorb adsorb /ad·sorb/ (ad-sorb´) to attract and retain other material on the surface; to conduct the process of adsorption.

ad·sorb
v.
To take up by adsorption.
, passed through the column and was collected. ED was then extracted from the solution by adjusting the pH to 2-3 and allowing it to pass through a second column. At this pH, ED was more lipophilic lipophilic,
adj/n the ability to dissolve or attach to lipids.

lipophilic (lipōfil´ik),
adj 1. showing a marked attraction to, or solubility in, lipids.
2.
 and suitable for column adsorption adsorption, adhesion of the molecules of liquids, gases, and dissolved substances to the surfaces of solids, as opposed to absorption, in which the molecules actually enter the absorbing medium (see adhesion and cohesion). . The compound was then eluted with a mixture of polar solvents [methanol-isopropanol-ammonia (4:6:0.25 by volume)]. To elute MED, COC, and BZ from the first column, dichloromethane-methanol-ammonia (9:1:0.2 by volume) was preferred over the generally used dichlorobutane-isopropanol-ammonia (8:2:0.2 by volume) because MED is volatile and easily lost during evaporation of the higher boiling isopropanol.

[FIGURE 2 OMITTED]

Separation of ED from COC and COC metabolites minimized the formation of artifact ED. MED in the extract of COC and BZ was analyzed separately at a GC injection port temperature of [less than or equal to] 140[degrees]C. Higher temperatures were avoided to minimize formation of artifact MED. To ensure that artifacts were not formed during analysis, a control containing COC, BZ, EC, and MEC MEC Ministério da Educação (Ministry of Education)
MEC Ministerio de Educación y Ciencia (Spain: Ministry for Education and Science)
MEC Mountain Equipment Co-Op
 at 1, 5, 5, and 5 mg/L, respectively, was tested. No ED or MED was detected under the extraction and GC-MS conditions described. COC was also injected separately before derivatization of BZ because the alkylating agents contained a small amount of methylating agent as impurity im·pu·ri·ty  
n. pl. im·pu·ri·ties
1. The quality or condition of being impure, especially:
a. Contamination or pollution.

b. Lack of consistency or homogeneity; adulteration.

c.
. In our experiment, DMF-DPA, DMF-diisopropylamide, or DMF-diethylamide with only BZ produced a small amount of COC as byproduct (<1%). Although MED and COC were injected under two different conditions (injection temperatures 140 and 280[degrees]C, respectively), the samples could be analyzed in one injection batch by use of an autoinjector with two GC-MS settings. ED extracted from blood and brain and tested as the tert-butyldimethylsilyl derivative showed chromatographic chro·mat·o·graph  
n.
An instrument that produces a chromatogram.

tr.v. chro·mat·o·graphed, chro·mat·o·graph·ing, chro·mat·o·graphs
To separate and analyze by chromatography.
 background. Analyzing the compound as the trimethylsilyl derivative minimized the background.

The linearity, correlation coefficient squared ([r.sup.2]), limit of quantification (LOQ LOQ Limit of Quantitation
LOQ Limit Of Quantification
LOQ Loquitur (Latin: speaks)
LOQ Level of Quantification
LOQ List Of Questions
LOQ Laugh Out Quiet
LOQ Leadership Opinion Questionaire
), and extraction efficiency are summarized in Table 1. In the range of linearity, all compounds showed ion ratios within [+ or -] 20% of the mean values. At least eight concentrations were used in the range of linearity. Only one sample was tested at each concentration. Good correlation [r.sup.2] was observed in the linear range mentioned, where acceptable values were defined as [greater than or equal to] 0.9900. Signal-to-noise ratios in all analyses were >4:1. In this study, LOQ was defined as the concentration at which one of the two ion ratios was outside [+ or -] 20% but within [+ or -] 30% and the concentration was within [+ or -] 20% of expected value. Quantification ions in some compounds showed better chromatographic background, allowing a LOQ below the limit of linearity. This observation was most likely attributable to the variability of the biological matrices among the various tissues and fluids. Specimens were analyzed in several batches, and controls at concentrations of 0, 20, 50, and 100 [micro]L were used in each batch analysis to validate the results. The criteria for batch validation were the same as those used for linearity studies.

Case histories for the specimens were extremely limited, although preliminary causes of death were not attributed to drug overdose. The results from the analyses by drug are summarized in Tables 2 and 3. Fig. 2 shows chromatograms for ED extracted from blood specimen 4 (Fig. 2a) and MED extracted from brain specimen 11 (Fig. 2b), where the concentrations were 25 [micro]g/L and 22 ng/g, respectively. The ranges for MED concentrations in liver, brain, blood, and urine were 0-10 ng/g, 0-92 ng/g, 0-42 [micro]g/L, and 0-2030 [micro]g/L, respectively. The number of samples in which MED was detected was 5 of 15, 7 of 14, 3 of 11, and 10 of 13 in liver, brain, blood, and urine, respectively. Although MED was detected in 5 of 15 liver specimens, the observed concentrations did not exceed 10 ng/g in any specimen. In contrast, urine concentrations as high as 2030 [micro]g/L were observed, and 6 of the 13 urine specimens reached concentrations in excess of 100 [micro]g/L. In general, MED concentrations were much lower in brain than in urine. The range of MED concentrations in the liver specimens was slightly larger than in the brain specimens. Although examining brain for estimating COC concentrations at the time of death may have some advantage because of the free passage of COC across the blood-brain barrier, the characteristics of MED relative to the blood-brain barrier have not been established. The high number of blood samples negative for MED or with low MED concentrations may indicate that detection of MED may be difficult in blood and be of limited usefulness in the estimation of COC concentrations at the time of death. On the basis of these observations along with other concerns for the use of blood in evaluating COC use in postmortem cases (15), other tissues and/or fluids may be more useful in postmortem examinations.

ED concentrations were noticeably higher than MED in liver, blood, and urine. In liver, the median ED concentration was 655 ng/g (range, 90-3274 ng/g) compared with 0 ng/g (range, 0-10 ng/g) for MED. Similar results were observed in blood. The amount of ED was relatively low in brain, and there was no statistical difference compared with MED concentrations in brain tissue. This indicates that ED does not easily pass through the blood-brain barrier, which might be expected based on the hydrophilic hydrophilic /hy·dro·phil·ic/ (-fil´ik) readily absorbing moisture; hygroscopic; having strongly polar groups that readily interact with water.

hy·dro·phil·ic
adj.
 nature of ED, and may also be accompanied by extensive first- or second-pass metabolism of MED in the liver and efficient excretion of ED by the body via the kidneys. This may also indicate that any MED that may enter the brain is converted to ED via enzymatic hydrolysis at a very low rate, which may parallel the observation that cocaine methyl esterase esterase /es·ter·ase/ (es´ter-as) any enzyme which catalyzes the hydrolysis of an ester into its alcohol and acid.

es·ter·ase
n.
Any of various enzymes that catalyze the hydrolysis of an ester.
 activity in brain is only 1-6% compared with liver in rats (16). Although MED was detected in only 3 of 11 blood specimens, ED was detected in all blood specimens. This suggests that MED is extensively converted to ED in the blood as well as in the liver, which is supported by the previously reported hydrolysis of MED to ED in liver homogenates (7) and the enzymatic hydrolysis observed in unpreserved sheep plasma (17).

When the ED concentrations in liver and urine were compared, 6 of 13 specimens contained more ED in liver than in urine. In 8 of 15 liver specimens, the amount of ED actually exceeded the amount of BZ. ED was detected in all 15 cases (50 of 53 total specimens), indicating that smoking was a route of COC administration. Compared with the detection of MED in 12 of 15 cases (25 of 53 total specimens), ED appears to be more easily detected than MED and may be a more useful marker than MED for determination of COC smoking in postmortem cases.

In conclusion, a review of the data reveals that ED concentrations were significantly higher than MED concentrations in liver, blood, and urine. In brain the difference was not significant. Of 53 tissue and fluid specimens collected from 15 postmortem cases, 50 specimens were positive for ED compared with only 25 specimens positive for MED. Only three brain specimens were negative for ED. The presence of ED and MED suggests that smoking was the route of COC administration in all 15 postmortem cases.

References

(1.) Martin BR, Lue LP, Boni JP. Pyrolysis and volatilization volatilization /vol·a·til·iza·tion/ (vol?ah-til-i-za´shun) conversion into vapor or gas without chemical change.

vol·a·til·i·za·tion
n.
See evaporation.
 of cocaine. J Anal Toxicol 1989;13:158-62.

(2.) Jacob PD, Lewis ER, Elias-Baker BA, Jones RT. A pyrolysis product, anhydroecgonine methyl ester (methylecgonidine), is in the urine of cocaine smokers. J Anal Toxicol 1990;14:353-7.

(3.) Jacob PD, Jones RT, Benowitz NL, Shulgin AT, Lewis ER, Elias-Baker BA. Cocaine smokers excrete excrete /ex·crete/ (eks-kret´) to throw off or eliminate by a normal discharge, such as waste matter.

ex·crete
v.
To eliminate waste material from the body.
 a pyrolysis product, anhydroecgonine methyl ester. J Toxicol Clin Toxicol 1990;28:121-5.

(4.) Nakahara Y, Ishigami A. Inhalation efficiency of free-base free·base or free-base  
v. free·based, free·bas·ing, free·bas·es

v.tr.
1. To purify (cocaine) by dissolving it in a heated solvent and separating and drying the precipitate.

2.
 cocaine by pyrolysis of 'crack' and cocaine hydrochloride hydrochloride /hy·dro·chlo·ride/ (-klor´id) a salt of hydrochloric acid.

hy·dro·chlo·ride
n.
A compound resulting from the reaction of hydrochloric acid with an organic base.
. J Anal Toxicol 1991;15:105-9.

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Decomposition of a chemical compound by reaction with water, such as the dissociation of a dissolved salt or the catalytic conversion of starch to glucose.
 product of methylecgonidine, as an indicator of smoking cocaine. J Mass Spectrom 1999;34:651-60.

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(9.) Cone EJ, Hillsgrove M, Darwin WD. Simultaneous measurement of cocaine, cocaethylene, their metabolites, and "crack" pyrolysis products by gas chromatography-mass spectrometry. Clin Chem 1994;40:1299-305.

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(12.) Kintz P, Cirimele V, Sengler C, Mangin P. Testing human hair and urine for anhydroecgonine methyl ester, a pyrolysis product of cocaine. J Anal Toxicol 1995;19:479-82.

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(14.) Kalasinsky KS, Bosy TZ, Schmunk GA, Ang L, Adams V, Gore SB, et al. Regional distribution of cocaine in postmortem brain of chronic human cocaine users. J Forensic Sci 2000;45:1041-8.

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ERIC T. SHIMOMURA, GWENDOLYN D. HODGE, and BUDDHA D. PAUL *

Division of Forensic Toxicology, Office of the Armed Forces Medical Examiner, Armed Forces Institute of Pathology, Rockville, MD 20850.

The opinions expressed herein are those of the authors and are not to be construed as official or as reflecting the views of the Department of the Army, the Department of the Navy, the Department of the Air Force The executive part of the Department of the Air Force at the seat of government and all field headquarters, forces, Reserve Components, installations, activities, and functions under the control or supervision of the Secretary of the Air Force. Also called DAF. See also Military Department. , or the Department of Defense.

[1] Nonstandard non·stan·dard  
adj.
1. Varying from or not adhering to the standard: nonstandard lengths of board.

2.
 abbreviations: COC, cocaine; MED, methylecgonidine; ED, ecgonidine; BZ, benzoylecgonine; [d.sub.3]-COC, N-desmethyl-N-[[sup.2.H.sub.3]]methylcocaine; [d.sub.3]-BZ, N-desmethyl-N-[[sup.2.H.sub.3]]methylbenzoylecgonine; [d.sub.3]-EC, N-desmethyl-N-[[[sup.2.H.sub.3]]]methylecgonine; DMF, dimethylformamide; DMF-DPA, DMF dipropyl acetal; SPE, solid-phase extraction; BSTFA, bis(trimethylsilyl)trifluoroacetamide; MTBSTFA, N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide; TBDMCS, tert-butyldimethylchlorosilane; GC-MS, gas chromatography mass spectrometry; [d.sub.3]-MED, methyl N-desmethyl-N-[[sup.2.H.sub.3]]methylecgonidine; [d.sub.3]-ED, N-desmethyl-N-[[sup.2.H.sub.3]]methylecgonidine; and LOQ, limit of quantification.

* Address correspondence to this author at: Division of Forensic Toxicology, AFIP AFIP Administración Federal de Ingresos Públicos (Argentina)
AFIP Armed Forces Institute of Pathology (US DoD)
AFIP Armed Forces Institute of Pathology (Rawalpindi, Pakistan) 
 Annex, 1413 Research Blvd., Rockville, MD 20850. Fax 301-319-0628; e-mail paul@afip.osd.mil.

Received January 10, 2001; accepted March 12, 2001.
Table 1. Linearity, correlation coefficient squared ([r.sup.2]),
LOQ, and extraction efficiency for MED, ED, COC, and BZ
from postmortem specimens.

           Linearity,            LOQ, (a)   Extraction
           [mu] g/L   [r.sup.2]  [mu] g/L   efficiency, %

Liver

MED        6-150      0.9982     5          >90
ED         20-500     0.9989     10         50-60
COC        20-500     0.9966     5          >90
BZ         20-500     0.9991     5          >90

Brain

MED        20-300     0.9994     5          >90
ED         20-500     0.9971     10         50-60
COC        15-2000    0.9992     5          >90
BZ         15-2000    0.9969     5          >90

Blood

MED        20-300     0.9982     5          >90
ED         20-300     0.9989     10         50-60
COC        10-500     0.9966     5          >90
BZ         10-500     0.9991     5          >90

Urine

MED        2-300      0.9965     2          >90
ED         10-300     0.9979     10         50-60
COC        5-500      0.9933     5          >90
BZ         5-500      0.9996     5          >90

(a) When LOQ was below the lowest limit of linearity, one of the ion
ratios was outside [+ or ] 20%, but quantification was within
[+ or -] 20% of the expected value.

Table 2. Drug concentrations in liver and brain from
15 postmortem specimens. (a)

                          Liver, ng/g

Specimen     MED        ED           COC        BZ

1            0          90           0          60
2            0          132          0          68
3            0          221          66         612
4            0          293          0          45
5            0          334          21         149
6            0          428          11         440
7            0          596          57         103
8            0          655          9          821
9            0          1131         21         890
10           0          2582         426        1643
11           5          1406         134        2755
12           6          1644         103        3513
13           8          1950         503        4980
14           8          2134         77         2963
15           10         3274         270        2430
Median       0          655          57         821
Range        0-10       90-3274      0-503      45-4980
Positive     5/15       15/15        12/15      15/15

                          Brain, ng/g

Specimen     MED        ED           COC        BZ

1            0          7            0          3078
2            0          0            0          46
3            92         0            1403       474
4            0          0            0          47
5            0          48           33         61
6            0          12           37         283
7            NA (b)     NA           NA         NA
8            0          22           8          1489
9            0          22           14         290
10           31         52           336        222
11           22         26           863        592
12           17         43           704        5153
13           14         40           546        2707
14           29         17           707        2982
15           19         34           490        574
Median       7          22           187        524
Range        0-92       0-52         0-1403     46-5153
Positive     7/14       11/14        11/14      14/14

(a) Values of 0 indicate quantification below the LOQ.
(b) NA, specimen not available.

Table 3. Drug concentrations in blood and urine
from 15 postmortem specimens. (a)

                      Blood, [mu] g/L

Specimen      MED         ED          COC         BZ

1             0           13          0           30
2             0           36          0           60
3             NA (b)      NA          NA          NA
4             0           25          0           32
5             NA          NA          NA          NA
6             0           60          5           332
7             0           299         30          87
8             0           29          5           458
9             0           119         13          628
10            11          214         35          1543
11            0           314         20          1519
12            NA          NA          NA          NA
13            42          143         12          2071
14            8           773         88          1831
15            NA          NA          NA          NA
Median        0           119         12          458
Range         0-42        13-773      0-88        30-2071
Positive      3/11        11/11       8/11        11/11

                         Urine, [mu] g/L

Specimen      MED         ED          COC         BZ

1             0           126         37          917
2             0           468         155         2084
3             62          109         1918        1736
4             0           146         63          999
5             48          456         1229        6768
6             14          226         185         3948
7             132         307         1119        1705
8             40          296         475         14 748
9             116         1143        1208        19 740
10            NA          NA          NA          NA
11            1299        1826        13 520      42 462
12            2030        7452        20 528      116 430
13            616         1116        28 062      39 624
14            999         2729        4214        107 868
15            NA          NA          NA          NA
Median        62          456         1208        6768
Range         0-2030      109-7452    37-28 062   917-116 430
Positive      10/13       13/13       13/13       13/13

(a) Values of 0 indicate quantification below the LOQ.
(b) NA, specimen not available.
COPYRIGHT 2001 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2001 Gale, Cengage Learning. All rights reserved.

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Title Annotation:Drug Monitoring and Toxicology
Author:Shimomura, Eric T.; Hodge, Gwendolyn D.; Paul, Buddha D.
Publication:Clinical Chemistry
Date:Jun 1, 2001
Words:5343
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