FLUORESCENCE POLARIZATION IMMUNOASSAY OF VITREOUS HUMOR TO DETECT DRUG USE

In recent years the number of postmortem cases in which cocaine involvement is suspected has increased considerably. This has created a need for a rapid screening procedure for the postmortem samples. The present study examined the usefulness of vitreous humor specimens, analyzed by a rapid immunological assay, as a possible alternative to the more conventional and relatively slow gas chromatography/mass spectrometry of blood samples to screen for antemortem cocaine use. Medical examiner cases occurring over a six month period were reviewed and forty-ei~ht cases which were determined to be drug related or drug induced deaths were analyzed. Postmortem concentrations of cocaine and its hydrolysis product, benzoylecgonine, recovered from blood, were compared to the concentrations detected in vitreous humor. These specimens were screened via a rapid fluorescence polarization immunoassay (FPIA) and also analyzed by gas chromatography/ mass spectrometry (GC/MS) for a quantitation of cocaine and its primary hydrolysis product, benzoylecgonine. An assessment of FPIA to screen postmortem vitreous humor samples was carried out. The sensitivity of this method was 0.03 mcg/ml. The vitreous humor cocaine metabolite levels by FPIA and GC/MS demonstrated a correlation coeificient of 0.7. Blood and vitreous humor benzoylecgonine levels from the same postmortem case analyzed by GC/MS showed a correlation coef-

iii ACKNOWLEDGEMENTS The author first wishes to dedicate this thesis to the memory of Dr. David R. DeFanti. Having been the major professor and advisor to the author from 1986 until his death in February~ 1991, Dr. DeFanti gave invaluable assistance throughout the author's years of graduate study. For being a mentor, and for his concern and patience, the author expresses her sincere appreciation. The author wishes to thank her co-workers at the Forensic Toxicology Laboratory for their advice and moral support in this investigative study.
iv The author especially wishes to express her gratefulness to her husband and daughters for their understanding and love throughout this thesis research and her whole graduate career.
The author wishes to thank Abbott Laboratories for generously supplying all immunoassay reagents needed for this study.
The author would like to thank the Society of Forensic  Another postmortem fluid which is readily available but is often ignored is vitreous humor. In comparison with blood and urine, it is not as susceptible to major postmortem changes. The vitreous is compartmentalized and isolated, which makes it a suitable toxicological material, even after embalming or putrefaction, when blood may be unavailable (Coe, and Sherman, 1970 rapidly cocaine and/or metabolites diffuse into vitreous humor specimens (Jones and Pounder, 1987). And lastly, this research further demonstrated that FPIA screening of vitreous humor specimens is valid in forensic toxicology. 3.

II. LITERATURE REVIEW
Vitreous humor is the fluid material which fills the posterior cavity of the eye. It is present at a volume of 2 to 2.5 milliliters in each eye (Logan and Stafford, 1989).
The vitreous (hyaloid body) occupies about 80% of the volume of the ocular globe. It provides structural support and maintains intraocular pressure • Chemically, the vitreous humor is a mucopolysaccharide gel composed of hyaluronic acid and collagen, and it has a water content of between 98 and 99.7% (Table 1). This table shows the relative simplicity and stability of vitreous humor.
Vitreous humor is a clean bilogical fluid containing less protein than urine, and it can be collected with ease. In the forensic domain, the primary use of vitreous humor to date has been to aid in the postmortem diagnosis of disease, the analysis of electrolyte composition and enzymatic activity to determine "time-since-death" determinations (Devgun and Dunbar, 1986).
Vitreous is a partitioned fluid; but the amount of water movement is very high (T 1/2 = 10-15 minutes) • This tends to make one expect that drug levels might be expected to show good correlation with corresponding blood levels. This is true of ethanol determinations and vitreous humor samples which are often analyzed postmortem for alcohol concentration.
The mean ratio of vitreous humor to blood ethanol concentration is 1.17 (Caplan and Levine, 1990). The use of vitreous humor for toxicological analysis was first published involving ethanol determinations (Sturner and Cournbis, 1966) .
In this publication the vitreous humor ethanol levels were compared to blood ethanol levels, obtained simultaneously at autopsy. There have been various reports subsequent to this, verifying the usefulness and accuracy of this approach (Coe and Sherman, 1970, Felby and Olsen, 1969, Ziminski et.al., 1984.
In the literature, various drug distributions studies do mention vitreous drug levels, but little is said about the relation of these levels to blood levels or their ultimate interpretation.
A study on alcohol levels illustrated that when patients have long survival times, they reveal blood/ vitreous values approaching unity (Sturner and Garriot, 1975).
There is speculation that the ratios reached at equilibrium depend on the drug's lipid solubility, plasma protein binding, and solubility in vitreous humor.
The substances that have been studied include such drugs as barbiturates, meprobamate, salicylates, ethchlorvynol, digoxin, quinine, and lithium (Felby and Olsen, 1969). Other studies have also appeared that relate levels of such drugs as barbiturates, methadone and morphine in vitreous humor to other tissues and fluids (Ziminski et. al., 1984). Quantitative postmortem determinations of vitreous drug levels have been reported rather sporadically in the literature.
The physical properties of vitreous humor make it a very good sample material for initial drug screening in postmortem cases.
Active transport of some compounds into the eye has been observed in some cases. This has been observed in some tests conducted on rabbit eyes with delta-9-tetrahydrocannabinol transport (Sorensen, 1971 In the human body, the drug, cocaine, is rapidly metabolized (T 1/2 = o. 7 to 1. 5 hours) to ecgonine methyl ester, ecgonine and other fragments by serum cholinesterase and liver esterases (Baselt and Cravey, 1989). Cocaine is broken down to benzoylecgonine by chemical hydrolysis ( Figure   1). These processes probably continue postmortem in the blood, liver and in drawn blood "in vitro". Enzymatic breakdown can be inhibited by any number of enzyme inhibitors, including fluoride, organophosphate pesticides, physostigrnine, and heavy metals. Hydrolysis can also be slowed by refrigeration or freezing and by decreasing the pH of the sample below neutral. Refrigeration retards both processes, while freezing blood samples containing sodium fluoride can preserve the cocaine for extended periods of time.
One would expect cocaine in the blood to hydrolyze extensively between the time of death and the time when the autopsy blood specimens are taken. In one review of the cocaine literature, it was evident that death from cocaine is rapid and unpredictable (Smart and Anglin, 1987) . None of the papers examined were able to illustrate how to translate postmortem values into actual doses taken. Many of the authors state that following an overdose of cocaine, death is astonishingly rapid (Welti and Fishbain, 1985, Welti and Wright, 1979, Mittleman and Welti, 1984. Thus, it may not yet be possible to state the lethal dose of cocaine with any degree of certainty. It has been suggested that brain tissue may be the better sample for cocaine determination rather than postmortem blood or liver tissue (Sphiehler and Reed, 1985). Cocaine is known to enter and leave the brain rapidly. Some researchers feel that there is an active transport of cocaine into the brain tissue, rather than a passive diffusion. At peak plasma concentrations, the brain cocaine concentration is just over four times the plasma or serum concentration. This brain/blood ratio is the most frequent median found for lO.
cocaine in cocaine overdose fatalities. As blood cocaine concentrations fall slightly more rapidly than brain tissue concentrations, the brain/blood ratio increases. This increase reaches a peak of approximately 10 (range 8 to 12) between one and two hours after cocaine administration. These authors recommend brain tissue for cocaine determinations over postmortem blood or liver tissue. Brain is a better matrix than blood for cocaine analysis because the parent drug is more stable in the brain's lipid-rich environment. However, the technical difficulties in getting the tissue prepared for ana lysis may outweigh the accuracy of the brain tissue for cocaine concentration.
The examination of three human brains in the study by Browne, illustrated that cocaine and benzoylecgonine appear to be distributed throughout the different regions of the organ (Browne et al., 1991). This was in agreement with the findings of an earlier study by Spiehler and Reed (Spiehler and Reed, 1985). The concentrations of cocaine found in the brain were generally four to eight times greater than that found in blood. The addition of this study to the archives further suggests that brain tissue is a better sample than either blood or liver for cocaine determinations. However, working with brain tissue is difficult, as stated earlier.
The presence in the brain of large concentrations of fats and other endogenous materials, make use of this specimen difficult. This material necessitates the use of lengthy and laborious extraction procedures.

11.
One paper in the literature demonstrated the usefulness of testing decomposed human remains for cocaine in all cases where the presence is suspected (Manhoff et. al., 1991).
Cocaine is generally quite labile and has a plasma half-life of less than one hour. It is known that in vivo it is rapidly hydrolyzed by .enzymatic and nonenzymatic reactions. Despite this, it has been found that cocaine and its metabolites are frequently detectable in decomposed human remains. Cocaine has been detected even in advanced cases of decomposition and mummification.
Recent data now suggests that blood cocaine concentrations change significantly during the interval between death and autopsy . The change appears to be dependent upon the site from which the blood is sampled. Cocaine concentrations in the heart, aorta, and femoral vein blood increase during the interval between death and autopsy, while the subclavian vein blood decreases . This study emphasizes the difficulty in attempting to estimate the perimortem concentration of cocaine in the blood from any postmortem data. The data implies that cocaine accumulates in some tissues, such as the brain and the liver, and these demonstrate higher cocaine concentrations than in the blood.
In another study where the two eyes were sampled at different postmortem intervals, markedly different cocaine levels were demonstrated (Beno and Kriewall, 1989). In this study also, postmortem release is the only reasonable explanation for this unusual finding. In a case study of 12, cocaine involved in a fatal poisoning, cocaine tissue concentrations and body fluid concentrations were reported in the literature for this case (Pokl±s et. al., 1985). The blood level of cocaine was 1. 8 mcg/ml and vitreous humor level was 2.4 mcg/ml.
for the parent drug The blood/vitreous ratio in this case cocaine was 0.75. There was no benzoylecgonine levels reported in this case report as the procedure used did not analyze for benzoylecgonine. The survival time was known to be less than 2 hours and the drug was intravenously administered.
The incidence of cocaine abuse has arisen dramatically over recent years. This has directly resulted in a major increase in deaths attributed to cocaine overdose as well as to increased findings of cocaine during the screening of biological samples for drugs of abuse. There are numerous methods in the literature describing the analysis of cocaine and its hydrolysis product, benzoylecgonine in body fluids, using high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) (Chinn et. al., 1980, Evans and Morarity, 1980, Griesmer et. al~, 1983, Logan and Stafford, 1989, Tebbett and McCartney, 1988 (Jolley, 1981 andCaplan et. al., 1987}. A derivative of the TDx, the ADx, which is dedicated solely to screening specimens for the major classes of abused drugs, was introduced in 1988. . And its entry into the forensic field has, therefore, been relatively recent (Table 2}.
Immunoassays are based on specific antigen-antibody reactions.
The primary advantage of immunoassay is its The vitreous specimens had been stored at -20° c.
since being received in the laboratory from the medical examiner. There were four negative controls which were included in the study ;there was no cocaine and/or metabolites detected in the bloods or urines of these cases.
The vitreous humor specimens were analyzed by fluorescence polarization immunoassay (FPIA) without any pretreatment, dilution, or centrifugation. The Abbott ADx analyzer 17.
was employed for the FPIA analysis. The calibrators, controls, and reagents were supplied by Abbott Laboratories.
The instrument was operated in accordance with the Abbott operators manual for this instrument. Six calibrators were assayed and a valid calbration curve was derived by averaging the net polarization values ( Figure 2). The assay requires a 50 microliter sample to be run on the assay. The assertion was that the simplistic compositon of vitreous humor specimen would allow for this facilitated screening technique to be utilized.
The sensitivity for the cocaine metabolite (benzoylegonine) assay was lowered to a threshold which allowed for low level positives in vitreous to be identified.
This presumptive screening process has already been mentioned as one which detects the primary metabolite of cocaine, benzoylecgonine, in human urine specimens.
demonstrates selective reactivity and The reagent system high specficity.
Benzoylecgonine has a reactivity of 100%, whereas cocaine, ecgonine methyl ester and ecgonine cross react with the assay at less than 1%. The technique has superior sensitivity which allows for an adjustable minimum allowable threshold. Below this threshold, the safety zone extends to the stated sensitivity of the assay (McCord and Mccutcheon, 1988

19.
In forensic toxicology work, any preliminary or presumptive positive on an initial screening test must be confirmed by a confirmatory test. It is generally agreed in the forensic community that gas chromatography coupled with mass spectrometry (   .

21.
22~ on specific masses, this mode is far more sensitive than total ion scanning. It permits analysis of selected compounds at very low levels in very complex mixtures -mixtures that may be too complex for resolution by gas chromatography alone. The rete~tion times obtained in the total ion chromatogram (TIC) were 9.11/9.12 and 9.66/9.67 min. for cocaine and benzoylecgonine, respectively ( Figure 5). All standards were specialty analytical standards obtained from Radian Corporation. These were pure standards in methanol at a concentration of 10 mcg/ml. The standards included cocaine, trideuterated cocaine (cocaine-D 3 ), benzoylecgonine, and trideuterated benzoyl-ecgonine (benzoylecgonine-D 3 ). These standards were diluted accordingly to allow for a standard curve calibration from 25 ng/ml to 1.0 mcg/ml. There were three levels of control urine specimens run along with the research samples to validate the derived calibration curve.
These controls (CON-DOA) were purchased from Diagnostics Products Corporation (DPC) and were in the following concentrations: 0.18, 0.36, and 1.5 mcg/ml. 24.
adsorbed and distributed into a thin film over the hydrophilic packing. In this manner, background interferences are removed from the analysis.
All standards, controls and samples were analyzed by the same cocaine/benzoylecgonine procedure (Appendix 0) adapted from Isenschmid (Isenschmid et. al., 1988). Into a clean 10 ml test tube, 1 ml of aqueous sample, 2 ml of phosphate buffer
1. In the above equat1on1: In the ebove equeUon11

Cone Std
Area Std tube and evaporated to dryness under a stream of nitrogen.
The dried sample is then reconstituted with 100 ul of chloroform and 3 ul are injected into the GC/MS for mass spectral analysis.
Standard curves for cocaine and benzoylecgonine were established by running series of standards from 25 to 1000 ng/ml (Figures 7,8).
The calibration curve for benzoylecgonine produced a slope of 1. 083, correlation coefficient of 0.999.

zero intercepts and a
The procedure was determined to be reliably linear for concentrations between 0.030 and 1.00 mcg/ml. Quantitations for the analytes were achieved by comparing the abundance of the major ions and retention times of the selected ion · chromatogram of the analytes with the corresponding internal standard (Table 3).     (Table 5). The correlation coefficient for the two assays was 0.701 (Table 6) (Table 7). Specifically, the benzoylecgonine quantitations were compared to calculate the correlation coefficient between the two analyses. The correlation between these two biological specimens for the same analyte was 0.510 (Table 8). The scatter plot and the regression line are shown        This has been demonstrated in the laboratory for other drugs, such as ethanol (Caplan and Levine, 1990 The confirmation analyses provided full concordance data between the two assays. The DPC controls run to validate the GC/MS calibration curve gave accurate quantitations. (Table   10) .   It was thought that the addition of a proteolytic agent would eliminate the viscosity factor and allow for better  (Beno and Kriewall, 1989). This variation could be due to the leaching of the drug from the high concentrations of the drug in the brain tissue.
The history in most of the cases did not afford the survival time information. In the majority of the cases, the survival time was unknown and therefore did not clarify the disparity between the two fluids.
As mentioned in other studies, it had been predicted that when survival times were lengthy, the blood/vitreous ratio of an analyte might approach unity. However, this research did not clarify that assumption.
Since vitreous humor is a partitioned fluid, with a Tl/2 of 10 to 15 minutes, the author anticipated a good correlation with corresponding blood levels. Also, since the molecular weights of benzoylecgonine and cocaine are relatively low, 289 and 303 respectively, these low molecular compounds could diffuse rather easily. However, the ratio varied a great deal between the two biological fluids. There are more factors involved, and the most important probably 47.
being the difference between dosing and the time of death as indicated in the study by Poklis (Poklis et al., 1985). The biggest liability in the screening of vitreous humor is the lack of sufficient sample volume to repeat analyses and generate statistical data on a case by case basis. The low sample volume did not allow for repetitive analyses, which then would allow for CVs to be calculated on each sample. This is not true of blood and urine, which when available, are usually of sufficient volume.
One point for discussion is the validity of testing infants and children by vitreous humor for cocaine exposure in the absence of a urine sample. Lowering the threshold 48.
as was done in this study, to a level of 0.03 mcg/ml would be necessary to detect the low levels of cocaine and metabolites found in infants and children.
In 25 cases reviewed of maternal substance abuse, all showed benzoylecgonine levels in the blood of the fetuses and neonates in the range of 0.12 to 3.2 mcg/ml (Sweeney, 1991). The routine drug screening of infants and children would now appear to be a modern necessity. 49.