Journal of Analytical Toxicology, Vol. 31, October 2007
Vitreous Fluid Quantification of Opiates, Cocaine, and Benzoylecgonine: Comparison of Calibration Curves in Both Blood and Vitreous Matrices with Corresponding Concentrations in Blood Heather M. Antonides*, Elizabeth R. Kiely, and Laureen J. Marinetti
Montgomery County Coroner's Office, Dayton, Ohio 45402
Abstract I Vitreous fluid specimensare often used in the Montgomery County Coroner's Office as a second matrix confirmation for both cocaineand opiate analyses. In this manuscript, calibration curves constructedfor both vitreous and blood were used to quantify vitreous specimensto evaluate if any matrix effects occur when measuringvitreous specimensusinga calibration curve in blood. Casesthat screened positive by ELISAfor cocaine metabolite and opiates were confirmed by solid-phase extraction. Gas chromatography with massspectral detection in the positiveelectron impact mode was used for the detection and quantificationof oxycodone, free morphine, codeine, 6-monoacetylmorphine, hydrocodone, cocaine, and benzoylecgonine.For interpretive purposes,no significant matrix effects were found in concentrationsof vitreous specimens quantified with a calibration curve constructedin a blood matrix. After determiningthat vitreous fluid can be accurately measured with blood calibrators, a comparison was made between blood and vitreousconcentrations for the above analytes. Concentration differences between blood and vitreous specimensfor each drug are evaluatedwith selected case histories included.
Introduction
Vitreous fluid has proven to be the specimen of choice for the Montgomery County Coroner's Office(MCCO)as a second matrix for confirmation of positive opiate or cocaine metabolite immunoassay screens. The eye is composed of 80% vitreous humor, which is 99% water with the remaining 1% made up of sugars, salts, phagocytes, and a network of collagen fibers (1). Diffusion has been cited as the method of transport from systemic circulation to the vitreous humor. Esterases are lacking from vitreous humor, which could add to the sta* Author to whom correspondenceshould be addressed:E-mail:
[email protected].
bility of opiates and cocaine in this matrix. Vitreous fluid specimens are preferred in this laboratory because of an apparent delay in distribution, and therefore peak concentration, of 6monoacetylmorphine (6-MAM) and also for its cleanliness as compared to other matrices, such as liver or urine. When available, vitreous humor is collected with every case during autopsy for toxicological analysis. Matrix effects for alternative matrices are not assessed at many laboratories because of heavy case loads and large batch runs. Only one set of matrix calibrators are run routinely; for this reason only blood calibrators are used. If possible, matrix matched calibrators are recommended by the American Board of Forensic Toxicologyand the American Academy of Forensic Science Laboratory Guidelines when dealing with vitreous specimens (2). The question then arose as to whether there was a significant matrix effect if quantification of vitreous fluid was performed using calibrators prepared in a blood matrix. Therefore, a comparison was made between the measurement of vitreous fluid using a calibration curve prepared from blood matrix and a calibration curve prepared from vitreous matrix. Opiate and cocaine metabolite immunoassay kits for vitreous specimens were evaluated using both blood and vitreous low positive, positive, and negative controls. Screening resuits for all vitreous specimens and contro]s were consistent with the corresponding blood data. Vitreous specimens that screened positive for either opiates or cocaine metabolite by immunoassay based on the positive control were then confirmed with solid-phase extraction (SPE). The extracted specimens were derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) + 1% TMCS and then analyzed by gas chromatography-mass spectrometry (GC-MS) in the positive electron impact mode using selected ion monitoring (SIM) for opiates and the SCANmode for cocaine analysis. Linear regression graphs were used to evaluate blood and vitreous calibrator responses over several batch runs. Fifteen cases were analyzed for both opiates and cocaine for the purpose of comparing vitreous specimens quantified by both blood
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and vitreous calibration curves. For interpretive purposes, the variability in concentration for the analytes was not significant. In addition, 40 cocaine cases and 52 opiate cases were analyzed by comparing the blood and vitreous concentrations in an attempt to establish a correlation between values. When available, case histories were reviewed to provide further insight into any correlations.
Materialsand Methods Reagents and supplies Potassium phosphate (monobasic and dibasic), ammonium hydroxide, glacial acetic acid, hydrochloric acid, sodium acetate, zinc sulfate, and all American Chemical Society (ACS)and high-performance liquid chromatography (HPLC)-grade solvents were purchased from Fisher Scientific (Pittsburgh, PA). Selectra-Sil, BSTFAwith 1% TMCS,and ZSDAU020Clean Screen extraction columns were purchased from United Chemical Technologies (UCT)Worldwide Monitoring (Bristol, PA). ColorpHast pH-indicator strips, pH 4.0-7.0 and pH 0-14, and all glassware were purchased from Fisher Scientific. Standards and controls Morphine, codeine, 6-MAM, hydrocodone, oxycodone, hydromorphone, hydromorphone-d3, morphine-d3, codeine-d3, hydrocodone-d3,oxycodone-d3,benzoylecgonine (BE), and BEd8 were purchased from Cerilliant (Round Rock, TX) at a concentration of 1 mg/mL in methanol. Cocaine, ecgonine methyl ester, cocaethylene, and cocaine-d3were also purchased from Cerilliant at a concentration of 1 mg/mL in acetonitrile. Opiate working standards were diluted with methanol. Cocaine and benzoylecgonine working standards were diluted with acetonitrile. Oxycodone Plus Blood Control was purchased from Utak Corporation (Valencia, CA) and contained oxycodone, morphine, hydromorphone, hydrocodone, 6-MAM, and codeine, all at a concentration of 1 IJg/mL. Cocaine Blood Control was purchased from Quality Assurance Service (Augusta, GA) and contained cocaine and cocaethylene at a concentration of 0.5 1Jg/mL and benzoylecgonine and ecgonine methyl ester at a concentration of 0.8 IJg/mL. Instrumentation A Tecan Miniprep 75 Automated Sample Prep instrument was used with the Immunalysis ELISA kits. Two GC-MS instruments were used: an Agilent 6890N GC with a 5973N MS and an Agilent 6890N GC with a 5973N MS with an inert source. Extraction and chromatographicconditions Drug-free blood matrix was donated by the Dayton Community Blood Center and Tissue Bank from expired lots of whole blood. All negative control blood was determined to have no detectable analytes before use in cocaine and opiate confirmation analyses. Synthetic blood purchased from Immunalysis was used as a blank matrix for all immunoassay 470
testing. Vitreous specimens from cases that were determined to have no detectable amount of cocaine, cocaine metabolites, or opiates and were free of blood contamination were combined for the blank vitreous matrix. During routine autopsies, blood samples were collected in grey-top tubes containing sodium fluoride and potassium oxalate. Vitreous samples were collected in red-top tubes which contain no preservatives. All samples were stored at 4~ until analysis. Vitreous specimens were screened for opiates and cocaine metabolite by immunoassay kits used with the Tecan Miniprep 75 automated system. Both blood and vitreous positive controls were prepared by spiking blank synthetic blood and vitreous matrices with morphine and BE for final concentrations of 0.05 IJg/mL and 0.10 IJg/mL, respectively. A low positive control was used for opiates with a final concentration of 0.025 IJg/mL of morphine. All results greater than the positive control were confirmed for BE for both blood and vitreous specimens. In order to detect positive oxycodone cases, results greater than the opiate low positive control were confirmed for opiates for both blood and vitreous specimens. For opiate quantification, 50 IJL of 10 IJg/mLmorphine-d3, codeine-d3, hydrocodone-d3, and oxycodone-d3internal standard was added to 2 mL of all specimens. Specimens were sonicated for 15 rain; 14 mL of 0.1M, pH 6.0 phosphate buffer was added, and specimens were rotated for 5 rain. After centrifugation, specimens were applied to SPE columns preconditioned with methanol and phosphate buffer. The SPE columns were washed using 0.01M acetic acid and 1:1 acetone/chloroform; specimens were eluted with 4% ammonium hydroxide in ethyl acetate. Eluents were evaporated to dryness and reconstituted with 50 IJL BSTFA + 1% TMCS and 50 IJL ethyl acetate for derivatization at 75~ for 45 rain (3-5). Specimens were analyzed for opiates using an Agilent GC-MS with DB-5 MS capillary column (30 m • 0.25-ram i.d., 0.25-1Jm film thickness). The temperature program was 80~ ramped to 170~ at 40~ then ramped to 290~ at 10~ and held for 3.75 rain. The injection volume was 2.0 IJL in splitless mode with an inlet temperature of 250~ The detector temperature was maintained at 310~ The following ions were monitored for the opiate analysis: 399.2, 6-MAM; 429.3, morphine; 432.35, morphine-d3; 371.2, hydrocodone and codeine; 374.2, hydrocodone-d3 and codeine-d3;459.3 and 444.2, oxycodone; and 462.3, oxycodone-d3.Morphine-d3 was used to quantify 6-MAM, and the 444.2 ion was used as the qualifier for oxycodone in order to calculate ion ratios. All other analytes were confirmed by detection in at least two matrices and by a positive immunoassay result. Hydromorphone can also be quantified by this method; however, no positive cases were detected. Quantification for all opiates was based on a four-point calibration curve using the following concentrations: 0.02, 0.07, 0.33, and 0.50 IJg/mL.Blood and vitreous calibrators were run in duplicate only during the evaluation for matrix effects. Both vitreous and blood calibrators and blanks were run with each batch of specimens along with high and low blood controls. Only blood controls were analyzed because of the lack of a second source for the analytes to spike into a blank vitreous specimen. The purchased blood control was within + 20% or
Journal of Analytical Toxicology,Vol. 31, October 2007
cluded with each batch of cases. Again, only the purchased • 2 standard deviations (SD) for both vitreous and blood calibrators. Between-run CV results were 12%, 6-MAM; 11%, free blood control was run for quality control. The control was in the acceptable range using both the vitreous and blood calimorphine; 9%, codeine; 6%, hydrocodone;and 5%, oxycodone brators. The between-run CVvalues for cocaine low and high for the low control and 11%, 6%, 7%, 11%, and 4% for the high control, respectively.Within-run CVresults were 10%, 6controls were 6.5% and 7.4%, respectively.Cocainewithin-run MAM;2%, free morphine; 3%, codeine; 3%, hydrocodone; and CVvalues were 2.1% and 5.2% for the low and high controls, 3%, oxycodonefor the low control and 11%, 1%, 1%, 1%, and respectively. BE CV between-run values were 11% and 8% for the low and high controls, respectively. The within-run CV 2% for the high control, respectively. Both the limit of detecvalues for BE were 2.7% and 3.6% for the low and high contion (LOD)and quantification (LOQ) for this method were detrols, respectively. Both the LOD and LOQ were based on the termined by the low calibrator. The upper limit of linearity was determined by a linearity study to be 1.0 lJg/mL for all analytes. Morphine For cocaine analysis, 50 IJL of both 10 3 Blood Callb. IJg/mL cocaine-d3 and 20 lag/mL of benv = 5.5788x + 0.0036 2.5 R2 = 0"9997 / ~ zoylecgonine-d8 internal standard was added to 2 mL of the specimen. Blood and vitreous ; 2 specimens treated with 4 mL 5% zinc sulfate "= 1.5 precipitating solution in distilled v~ Vitreous Calilx water/methanol (7:3) were vortex mixed and ~ 1 v = s.o4o6x + o.o4~4 centrifuged before applying to the SPE 0.5 R== 1 columns. Columns were preconditioned with o methanol, distilled water, and sodium acetate 0.6 0 0.1 0.2 0.3 0.4 0.5 Concentration (pg/mL) buffer. After the specimens were added to the SPE columns, the columns were washed with [ 9 Vitreous Calib. 9 Blood Calib. ] distilled water, 0.1M hydrochloric acid, and Figure 1. Comparisonof vitreous and blood calibration curves for morphine. methanol. The specimens were eluted with methylene chloride/2-propanol/ammonium hydroxide (78:20:2), evaporated to dryness, Oxyoodone and reconstituted with BSTFA + 1% TMCS Blood CaUb. for derivatization at 70~ for 20 rain (3,7). 2.5 y = 4.5157x + 0.0451 / The specimens were injected onto the hgilent ~ 2 R2= 0 ~ ~ ~'~ GC-MS with a DB-5MScapillary column (30 ! 1.5 m x 0.25-ram i.d., 0.25-1Jm film thickness). " Vitreous Calib. The temperature program was 80~ ramped 1 y = 4.5K~ + 0.fl242 to 170~ at 40~ held for 2.0 rain, then 0.5 f R 2 0.9995 ramped to 290~ at 10~ and held for o 2.0 min. The injection volume was 1.0 IJL in 0.6 0 0.1 0.2 0.3 0.4 0.5 Concentration (pg/mL) splitless mode with an injector temperature of 250~ The detector temperature was main[ 9 Vitreous Ca,b. - Blood Calib. ] tained at 310~ In full scan mode, the folFigure2. Comparisonof vitreous and blood calibration curvesfor oxycodone. lowing ions were monitored for the cocaine analysis (asterisks denote quantifying ions): 303", 198, 182 for cocaine; 361', 240, 256 for Codeine BE; 306 for cocaine-d3; and 369 for BE-ds. Area ratios were calculated for cocaine and 3 Blood Calib. v = 4.884~ + 0.0019 BE using the qualifying ions. Ecgonine 2.5 =
methyl ester was also detected but not quan-
tiffed. Cocaethylenecan also be quantified by this method; however,no positive cases were detected. Cocaine analysis calibration concentrations were 0.05, 0.1, 0.5, and 2.5 IJg/mLand 0.1, 0.2, 1.0, and 5.0 IJg/mL for cocaine and BE, respectively. Blood and vitreous calibrators were run in duplicate only during the evaluation for matrix effects. Positive low and high blood controls, vitreous and blood blanks, and both matrix calibrators were in-
.~
R 2 = 0.9997
2
.~ 1.5 ~ 1 ~ 0.5
Vitreous C.alib. v = 4A19~: + 0.028 R 2 = 0.9997
o
o
0.1
0.2
0.3
0.4
0.5
0.6
Concentration (pg/mL)
I 9 Vitreous Calib. 9 Blood Calib. ] Figure 3. Comparisonof vitreous and blood calibration curves for codeine.
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Journal of Analytical Toxicology,Vol. 31, October 2007
codone was greater than 0.10 t~g/mL, immunoassay results for oxycodone screened positive in both blood and vitreous positive specimens based on the ELISA opiate positive control. In cases where the oxycodone concentration was less than 0.10 IJg/mL, all specimens screened positive by immunoassay Results and Discussion based on the ELISA low positive opiate control. This could result from the lower cross reactivity for oxycodone using the Immunoassay results for all opiates, except oxycodone, were opiate ELISA kit. Approximately 0.120 IJg/mL of oxycodone is consistent with confirmation results for both blood and vitlisted by the package insert as equivalent to 0.025 IJg/mL of reous controls and cases. When the concentration of oxymorphine, the target analyte. Therefore, the low positive control was used to determine positive opiate cases. For cocaine immunoassay results, 100% 6 -MAM of cases and controls screened positive in both 6 B l o o d Calib. matrices. As BE is the target analyte for this 5 .v = 9.7428x + 0.0267 immunoassay kit, these results were expected, R z = 0.9989 and BE was confirmed in all specimens where 4 a positive immunoassay result occurred. 3 Calib. Calibration curves for both opiate and co/ v= 8~z__6~,9~21985 ~2 caine analysis were constructed using Microsoft Excel software for both blood and vitreous calibrators by analyzing four different 0 o.s calibrator concentrations over several batch 0 0.1 0.2 0.3 0.4 0.5 runs. A total of five batch runs containing duConcentration (pg/mL) plicate measurements for each calibration I 9 Vitreous Calib. 9 Blood Calib. ] concentration point on the respective curve Figure4, Comparisonof vitreous and blood calibration curvesfor 6-monoacetylmorphine. were analyzed. An average of the relative areas for each calibrator was plotted against the target concentration in Figures 1-6. CorrelaCocaine tion coefficients (R2) were calculated to eval12 uate the linearity of each curve. A correlation Blood Calib. 10 y = 4.3557x * 0.0024 coefficient of 0.99 is commonly accepted. To ==8 ; ~ ~ 8 ~ " further assess the matrix effects for each analyte, vitreous case specimens were calculated 6 using both blood and vitreous calibration 4 Vitreous Calib. curves. Vitreous case concentrations of opi2 v = 4.4908x + 0.0088 ates and cocaine are listed in Tables I and II, R 2 = 0.9999 respectively. Any discrepancies that occurred 0 3 between vitreous values were not more than 0 0.5 1 1.5 2 2.5 Concentration ( l l g / m L ) 20% of their average, which is the acceptable precision range. In the instances where con] 9 Vitreous Calib. 9 Blood Calib. [ centrations were out of the acceptable range, 5. Comparison of vitreous and blood calibration curvesfor cocaine. multiple analyses were limited by the volume of vitreous fluid provided to the toxicology section. Given an increased vitreous specimen Benzoylecgonine volume, multiple analyses could have been 12 performed and these discrepancies would have Blood Calib. ~ . m 10 y = 4.0535x + 0.1968 /~...,,'~ been further evaluated. Overall, the data in R z = 0.9983 / / Tables I and II support the absence of matrix ":e effects when quantifying vitreous specimens "E using a blood or vitreous calibration curve. V i t r e o u s Calib. 4 y = 3.7937x + O.166 Calibration curves for morphine and 2 R2 = 0.9988 oxycodone are shown in Figures 1 and 2, respectively. Minimal matrix effects are 0 3 demonstrated by both analytes over the con0 0.5 1 1.5 2 2.5 Concentration(pg/mL) centration range of the curve as established by both figures and the data comparison of Table I * Vitreous Calib. 9 Blood Calib. I I. Differences in measurement of vitreous fluid Figure6, Comparisonof vitreous and blood calibration curvesfor benzoylecgonine. using the vitreous and blood calibration curves
low calibrator for this assay. The upper limit of linearity was determined by the high calibrator.
~treom
Figure
=8
I
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I
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Journal of Analytical Toxicology, Vol. 31, October 2007
publications where blood and vitreous specimens were anawere negligible when the concentration was compared to the lyzed for cocaine and BE (8,9). Acute intoxications were charnormal variability seen with the positive controls. Therefore, acterized by the concentrations of cocaine and BE being much both oxycodone and morphine vitreous concentrations were higher in blood than in vitreous fluid. comparable using the blood and vitreous calibration curves. Opiate concentrations for both blood and vitreous speciCodeine and 6-MAM show increased matrix effects between mens, along with case histories were also reviewed. However, the two specimen types as demonstrated by Figures 3 and 4. distinguishing acute versus chronic use based on case histories Discrepancies occur both at the low and high end of the curve was not as clear as with cocaine and BE data. In all cases posfor 6-MAM;however, Table I shows the results were still comitive for 6-MAM, vitreous concentrations were higher than parable. A discrepancy was also seen for codeine at the high end blood concentrations regardless of postmortem intervals. In of the curve, but it was negligible based on the results in Table case histories where heroin was mentioned, postmortem inI. The hydrocodone calibration curve was similar to that of codeine, in that a discrepancy occurred at the higher end of the curve. Data was provided in Table I. Concentration (pg/mL) of Opiates in Vitreous Specimen Cases Table I to illustrate that these differences were insignificant for this analyte as well. Morphine Codeine 6.MAM Hydrocodone Oxycodone Cocaine and BE calibration data are preBid Cal* Vit Cal Bid Cal Vit Cal Bid Cal Vit Cal Bid Cal Vit Cal Bid Cal Vit Cal sented in Figures 5 and 6, respectively. No matrix effects were present for cocaine data 0.02 0.02 0.01 0.03 0.05 0.02 0.02 0.03 0.02 between specimen types and a discrepancy o.o2 0.03 0.02 0.02 0.03 0.03 0.02 0.02 0.04 0.04 0.02 was exhibited by BE only at higher concen0.02 0.09 0.09 0.04 0.05 0.02 0.02 0.04 0.04 0.02 trations, The difference in measurement of 0.03 0.03 0.17 0.12 0.04 0.02 0.03 0.02 0.05 0.05 the vitreous fluid for these analytes was neg0.04 0.03 0.25 0.23 0.08 0.02 0.03 0.03 0.06 0.05 ligible when calculated by either a blood or 0.o4 0.04 0.09 0.05 0.03 0.04 0.19 0.17 vitreous calibration curve, as demonstrated o.04 0.06 0.17 0.07 0.04 0.03 0.19 0.17 in Table II. After the opiate and cocaine o.05 0.05 0.27 0.34 0.05 0,05 0.22 0.16 0.08 0.05 0.05 0.24 0.21 methods for quantification of vitreous fluid 0.07 0.09 0.06 0.06 0.24 0.23 with a blood calibration curve were evaluated o.o7 0.06 0.18 0.14 0.27 0.24 and no significant matrix effects were found, 0.12 0.12 0.41 0.35 results were gathered from cases for both opi0.14 0.10 0.41 0.37 ates (n = 52) and cocaine (n = 40) in blood 0.15 0.15 0.53 0.57 and vitreous fluid. Concentrations of analytes 0.16 0.29 0.60 0.52 0.22 from these cases are listed in Tables IlI and IV. After reviewing several publications re* Abbreviations: Bid Cal, blood calibration curve and Vit Cal, vitreous calibration curve. garding vitreous versus blood specimen concentrations for opiates and cocaine, it was noted that case histories are usually not provided. In this Table II. Concentration (pg/mL) of Cocaine and Cocaine manuscript, individual case histories and concentrations for Metabolite in Vitreous Specimen Cases both specimens were utilized when available to evaluate the resuits with respect to interpretive value. Cocaine Benzoylecgonine Cocaine concentrations were higher in vitreous fluid in 72% Bid Cal Vii Cal Bid Cal Vit Cal of the cases reviewed where cocaine was detected in one or both matrices. After reviewing case histories, blood concentra0.01 0.01 0.30 0.29 tions of cocaine were higher in cases where acute intoxication 0.01 0.01 0.37 0.37 was apparent. Postmortem intervals for these cases were short 0.04 0.05 0.41 0.41 as the deaths were witnessed and reported promptly. For ex0.05 0.06 0.42 0.42 ample, in cases 17 and 27 in Table IV, the decedents had both 0.10 0.11 0.61 0.64 0.21 0.23 0.87 0.94 swallowedcocaine while running from the police. In cases 7 and 0.23 0.25 1.10 1.20 24, the decedents had long histories of drug abuse; however, ac0.26 0.28 1.30 1.40 cording to witnesses, they became unconscious or had seizures 0.29 0.27 1.65 1.38 upon acute ingestion of cocaine. Benzoylecgonine was higher in 0.32 0.29 1.70 1.40 blood 78% more often than in vitreous. In these cases where co0.33 0.31 2.00 2.20 caine was higher in blood than vitreous, BE was significantly 0.53 0.58 2.40 2.60 higher in the blood by as much as 10-fold.The pH of all vitreous 2.60 2.20 specimens was determined before analysis and found to be ap3.90 3.30 proximately 7. Therefore, hydrolysis of cocaine to BE could ac5.20 5.90 count for some of the BE in the vitreous matrix (6,7). Cocaine Abbreviations: Bid Cal, blood calibration curve and Vit Cal, vitreous calibration and BE data suggest that these two analytes have slower districurve. bution into the vitreous. These results were similar to other 9
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Journal of Analytical Toxicology,Vol. 31, October 2007/
sults were inconsistent with the pattern for vitreous and blood quantifications mentioned previously. In case 51, vitreous and blood concentrations were 0.36 pg/mL and 0.47 pg/mL, respectively. The case history stated that the decedent ingested Oxycontin and soon became unresponsive. A family member called medics, and upon their arrival the decedent was alert and stated he had no complaints Table III. Opiate Data for Vitreous and Blood Specimens Using a Blood and felt fine. Approximately 3 h later, the deceCalibration Curve dent was again unresponsive, and medics pronounced him dead 6 h after the initial Vitreous Blood Vitreous Blood ingestion. The time-release preparation of oxyResult Result Result Result (pg/mL) codone (Oxycontin) was the most likely explaCase Anal~e (pg/mL) (pg/mL) Case Analyte (pg/mL) nation of the history and toxicology results in 0.02 this case. Case 52 had an oxycodone blood con25 hydrocodone 0.03 1 6-MAM 0.04 < 0.02 0.15 centration of 0.77 pg/mL and a vitreous conoxycodone 0.24 morphine 0.04 0.16 ND centration of 0.42 pg/mL. In this case, the 26 6-MAM < 0.02 codeine 0.02 0.02 morphine 0.04 2 morphine 0.15 0.15 o.12 decedent had a history of pain medication codeine < 0.02 codeine 0.17 0.06 < o.o2 abuse and seeking prescriptions from multiple
tervals ranged from hours to days. Oxycodone and hydrocodone had higher concentrations in vitreous than blood in 96% and 71% of cases, respectively. After all results were compiled for this manuscript, two additional cases of interest were analyzed. Cases 51 and 52 were positive for oxycodone, but the re-
3 4
5 6 7 8 9 10
11 12
13
14
15 16
17 18 19 20 21 22 23 24
oxycodone 6-MAM morphine codeine
0.53 < 0.02 0.02 < 0.02
hydrocodone 0.02 hydrocodone 0.03 oxycodone < 0.07 hydrocodone 0.18 oxycodone 0.22 morphine 0.12 oxycodone 0.24 hydrocodone 0.05 6-MAM 0.27 morphine 0.22 codeine 0.09 morphine 0,14 hydrocodone 0.03 6-MAM 0.03 morphine 0.04 codeine < 0.02 6-MAM 0.09 morphine 0.07 codeine 0,02 morphine < 0.02 codeine 0,25 hydrocodone 0.04 oxycodone 0.41 6-MAM 0.17 morphine 0.02 codeine ND 6-MAM 0.08 morphine ND morphine 0.16 oxycodone 0.07 morphine 0.03 morphine 0.07 oxycodone 0.04 hydrocodone 0.06 oxycodone 0.6 oxycodone 0.41 oxycodone 0.41
0.43 ND 0.03 < 0.02 < 0.02 0.02 < 0,07 0.15 0.17 0.05 0.17 0.05 0.05 0.76 0.2 0.12 0.02 ND 0.18 < 0.02 < 0.02 0.18 0.02 < 0.02 0,12 < 0.02 0.26 0.02 0.02 0.02 ND 0.10 0.53
27 28 29 30 31
32 33
34 35 36 37
38 39 40
41 42 43 44
0.05
45
0.11 0.19 0,03 0.03 0.28 0.28 0,28
46 47 48 49 50 51 52
474
oxycodone hydrocodone oxycodone oxycodone morphine hydrocodone hydrocodone 6-MAM morphine codeine hydrocodone oxycodone 6-MAM morphine codeine oxycodone hydrocodone oxycodone oxycodone hydrocodone 6-MAM morphine codeine morphine hydrocodone 6-MAM morphine codeine morphine hydrocodone oxycodone oxycodone hydrocodone oxycodone hydrocodone hydrocodone codeine oxycodone hydrocodone hydrocodone oxycodone oxycodone oxycodone
0.05 < 0.02 0.19 0.06 0.02 < 0.02 0.05 0.03 < 0.02 < 0.02 < 0.02 0.03 0.04 0.05 5.0 < 0.05 1.6
21
0.28 2.3
24
0.05 0.63 < 0.05 2.5 0.4 1.6 ND 1.2 0.46 2.9 0.48 15 0.07 2.3 < 0.05 0.53 < 0.05 1.3 0.28 2.3 0.07 < 0.2 0.38 4 7.9 6.7 1.1 1.2 0.11 0.42 4.7 10.7
25
22
23
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Analyte cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine cocaine benzoylecgonine
Vitreous Blood Result Result (pg/mL) (pg/mt) 0.17 0.56 0.11 0.14 0.33 1.8 0.22 8.23 0.8 2.1 0.41 0.58 5.6 3.9 ND 0.14 ND 3.3 0.09 0.79 0.19 0.27 < 0.05 0.58 < 0.05 0.94 0.49 1.5 0.15 1.3 0.83 2.5 ND 0.34 0.47 2
0.34 1.2 0.12 0.28
0.13 1.42 0.05 0.4 0.09 2.9 0.38 17.4 0.25 4.1 0.75
1 9.7 9.6 ND 0.08 < 0.05 5.5 ND 0.99 0.12 0.49 ND 0.24 ND 0.4 0.2 2.5 0.1 3.1 0.52 4.5 ND 0.3 0.22 2.5 0.07 1.3 ND 0.27
Conclusions Both vitreous and blood controls and specimens from cases produced consistent results in the immunoassay screens for benzoylecgonine and opiates. The data comparing vitreous and blood calibrators demonstrated that matrix effects in these analyses were minimal. Therefore, measuring vitreous specimens with blood calibrators was acceptable. Any variability that occurred did not differ from the normal variability that occurs with positive blood controls that are run with each batch, except for those cases where sufficient amount of vitreous fluid specimen was not available. A relationship between vitreous and blood concentration is not established for most drugs including opiates, cocaine, and cocaine rnetabolite. Data have been provided showing concentrations for both specimens in cases analyzed at MCCO. Oxycodone, hydrocodone, and 6-MAMhad higher concentrations in the vitreous fluid than in blood in 96%, 71%, and 100% of the cases, respectively.Morphine and codeine concentrations had greater variance between blood and vitreous specimens in comparison with other opiates, depending on the source of the drug. For cocaine, concentrations in vitreous specimens were higher than the blood in 72% of the cases analyzed. Blood specimens showed higher concentrations of BE than in the vitreous specimens in 78% of the cases analyzed. Acute overdoses were easier to identify in the cocaine cases based on the information provided in the case history along with the blood and vitreous concentrations. The findings suggested that cocaine and BE distribute more slowly into the vitreous fluid because, in cases of acute overdose, values of each analyte were higher in the blood as compared to the 475
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vitreous specimen. Because of different half-livesand dosing intervals within the opiate class, it was often unclear if exposure was acute, chronic, or acute toxicity in addition to chronic use. However, the data were still useful in identifying the use of heroin because of the prolonged detection of 6-MAMin vitreous specimens. Interestingly, many of the oxycodone cases analyzed had much higher vitreous concentrations than blood concentrations where chronic abuse was noted in the case history. The availability and common use of a time released preparation of oxycodone could be a contributing factor in this observation. Analysis of vitreous specimens offers an extended window of drug detection for those drugs with shorter halflives, specificallycocaine and 6-MAM.In several instances both of these analytes were detected in vitreous and not detected in the blood matrix. Lastly, oxycodone, hydrocodone, 6-MAM,cocaine, and BE were detected in higher concentrations in the majority of the cases reported, suggesting that detection is possible for a longer period in the vitreous sample.
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