Interpretation of Results

Urine

Urine is currently the necessary specimen in US federal workplace drug-testing programs. Under the federal program, a government-certified laboratory assays a urine specimen for benzoylecgonine by immunoassay using a 300ng/mL cutoff concentration. Confirmation is performed by GC-MS using a 160ng/mL cutoff concentration. Only benzoylecgonine may be assayed in US workplace urine drug-testing programs.

Urine workplace drug testing, regardless of the analytes assayed, is appropriate only to determine exposure to cocaine. Quantitative benzoylecgonine concentrations especially if corrected for creatinine, in sequential urine specimens, may be functional in substance abuse treatment programs to detect a relapse during a period of abstinence, but single specimens collected in a forensic setting cannot be likewise interpreted because only an accidental urine specimen is characteristically collected. Generally, cocaine and metabolite concentrations measured in urine cannot be correlated with time of drug use or degree of impairment. In typical cases, urinary benzoylecgonine concentrations may be detectable from 2 to 4 days after cocaine use, depending on dose, frequency of use, urinary pH, and clearance.

Urine specimens were gathered for 3 days after 6 subjects were administered a single dose of cocaine by intravenous (25 mg), intranasal (32 mg), and smoked (42 mg) routes of administration. The mean percentage of the total dose recovered over 3 days was 45, and 25%, correspondingly. Cocaine concentrations declined to below detectable limits (1 ng/mL) within 24 h, whereas benzoylecgonine and ecgonine methyl ester were frequently detectable in low concentrations (26ng/mL) through 72 h. The average utmost concentration and peak time for cocaine and the major urinary metabolites are summarized in Tables 12-1 and 12-2. Minor urinary metabolites (norcocaine, benzoylnorecgonine, m- and p-hydroxycocaine, m- and p-hydro-benzoyl-ecgonine) accounted for 2% or less of the total dose administered and were below limits of detection within 48 h. After smoked cocaine, very small amounts of methylecgonidine were measured in the urine.

The exclusion half-lives for cocaine and metabolites in urine have been determined through the consolidation of several published studies. Based on those data, the half-lives were 1.5 h for cocaine, 7.5 h for benzoylecgonine, and 3.6 h for ecgonine methyl ester, though there was considerable inter-individual variation. In a study simulating chronic cocaine use, a longer terminal excretion curve was observed. After 6 subjects received repeated doses of oral cocaine, a biphasic urinary excretion pattern was observed in 5 subjects. The mean initial elimination rates were 4.1 h for cocaine, 7.2 h for benzoylecgonine, and 5.6 h for ecgonine methyl ester. The mean terminal half-lives were 22.8 h for benzoylecgonine (range 15.0-34.5) and 32.8 h (range 22.3-52.4) for ecgonine methyl ester. A terminal excretion curve was only observed in 3 of the subjects for cocaine with a mean half-life of 19.0 h (range 13.5-27.4). Prolonged positive immunoassay results from 5 to 10 days have been reported after compulsive cocaine use with continuous positive results for up to 16 days, consistent with a longer terminal half-life after chronic, heavy cocaine use. Increased plasma protein binding for benzoylecgonine compared to the negligible plasma protein binding of cocaine may also explain extended detection times seen in the urine of chronic high-dose cocaine users.

Stability in Urine

Benzoylecgonine even though relatively stable in urine at frozen temperatures, is vulnerable to hydrolysis in alkaline conditions and at higher temperatures. In a study in which benzoylecgonine was fortified into urine stored at room and refrigerated temperatures, adjusted to pH 5, 7, or 9, the loss of benzoylecgonine was clearly related to temperature and pH. Re analysis of 61 urine specimens for benzoylecgonine after storage at -15 °C from 1 to 8 months (mean 2.3 months) demonstrated an average decrease of 19% with a standard deviation of 28%. Specimens with a pH of 5.3 to 7.9 showed minimal loss, while those with a pH >8.0 showed a loss of >50% of the original benzoylecgonine concentration. One specimen that did not contain any remaining benzoylecgonine had a pH of 8.9. A few specimens tested higher than the original result, possibly due to improper sample mixing or hydrolysis of cocaine to benzoylecgonine

The latter is an imperative issue because the detection of benzoylecgonine or ecgonine methyl ester in urine as a result of hydrolysis of exogenously added cocaine could be raised as a defense when these metabolites are detected under regulated testing. When cocaine (1 mg/L) was added to buffers (pH 5, 7.4, and 10) and stored at 4 °C and 25 °C, the rate of cocaine hydrolysis to benzoylecgonine increased with temperature and pH. Ecgonine methyl ester was tested for but not detected as a hydrolysis product of cocaine in buffers, suggesting its presence could be indicative of endogenous cocaine use. However, another study showed that when high concentrations of cocaine (155 mg/L) are fortified into negative urine specimen adjusted to alkaline pH, significant hydrolysis to both benzoylecgonine and ecgonine methyl ester occurs despite the fact that the esterases that hydrolyze cocaine are not present in urine. Expressed as a percentage of the initial amount of cocaine added, the detected amounts of benzoylecgonine and ecgonine methyl ester were 34% and 36%, respectively, in urine adjusted to pH 8.0 and stored at room temperature (25 °C) for 3 days. Comparable data for urine adjusted to pH 9.0 were 43% for both benzoylecgonine and ecgonine methyl ester. No appreciable cocaine loss was observed at pH 5.0 or 7.0. For this reason, some investigators suggest the measurement of the para- and meta-hydroxybenzoylecgonine when it is alleged that benzoylecgonine and ecgonine methyl ester are present because of in vitro adulteration.

Like benzoylecgonine ecgonine methyl ester constancy in urine has been shown to be subject to alkaline hydrolysis. At pH 3-5 ecgonine methyl ester has been shown to be stable for up to 3 years, but at pH 9 no ecgonine methyl ester remained after 30 days when stored at 4-5 °C. Very quick hydrolysis of ecgonine methyl ester in buffers occurs at higher pH values, but these are not likely to be found physiologically. Ecgonine methyl ester has been shown to be stable for at least 6 months under frozen (-20 °C) and refrigerated (4 °C) conditions in investigation urine. Other factors, such as microbial growth or in vitro adulteration with alkaline materials, such as bleach, also affect the stability of cocaine and its metabolites in urine.

Passive Exposure and ingestion

Often, issues are raised about unknowing exposure or ingestion of cocaine. This is particularly true in workplace drug testing and childhood or infant contact, although only the former will be discussed here.

Passive Exposure to Crack Smoke

Even though passive exposure to crack cocaine vapors poses a realistic threat in pediatric cases, studies performed in the adult population do not support this. A possible reason for these differences has been suggested. Crack smoke has been characterized as particulate matter rather than vaporized cocaine. The authors conclude that, unlike smoke, particulate cocaine will not remain suspended in the air and settle quicker, resulting in significant exposure in pediatric cases from oral ingestion via hand-to mouth activity of a child crawling on the floor in an environment of heavy crack use.

There have been 2 studies that purposely examined the issue of passive exposure to free-base cocaine in adults. The first study was limited to a single 7 kg adult male exposed to the volatilization (235-250 °C) of 200 mg cocaine free-base in a 1.2 x 1.4 x 2.4 m (4,050 L) closed room for 30 min. Urine specimens taken 5 min before exposure and at 6, 12, 18, and 24 h after exposure yielded a peak benzoylecgonine concentration of 14 ng/mL at 18 h by GC-MS. RIA screening results indicated <50 ng/mL in all 4 post-exposure samples and <25 ng/mL in 3 samples. The second, more comprehensive study was performed using 6 males exposed to 100 mg and 200 mg cocaine free-base (200 °C) for I h in a 2.1 x 2.5 x 2.4 m room (12,600 L). Urine specimens were collected periodically up to 34.7 h after exposure. Benzoylecgonine concentrations ranged from 22 to 123ng/mL. The peak excretion time occurred at 5 h after passive exposure. No behavioral or physiological effects (pulse, respiration, blood pressure) were observed after exposure. A slight elevation in heart rate was noted for the passive exposure session with 100 mg free-base cocaine, but was not significant (P < 0.05). No specimen tested positive above the 300 ng/mL immunoassay cutoff. The amount of cocaine inhaled by the subjects based on room air measurements was 0.25 mg. For comparison, the same subjects were also given a 1-mg IV injection of cocaine hydrochloride. Four of the six subjects screened positive (300 ng/mL by Emit) following the injection indicating that the minimum amount of cocaine in these subjects necessary to produce a positive result was approximately 1 mg. It was concluded that passive exposure conditions that would result in absorption of 1 mg of cocaine or more could result in the production of positive specimens based on the US federal workplace cutoff of 300 ng/mL benzoylecgonine. However, such conditions are not likely.

Unknowing Ingestion of Cocaine in Adults

Another issue often raised is that cocaine ingestion was unknowing through the addition of cocaine to a beverage. Most studies performed on cocaine intake in a beverage did not involve fortified beverages, but rather beverages that naturally contained cocaine. Specifically, in the 1980s it was determined that Health Inca Tea, which was sold in health food stores in the United States, contained trace amounts of cocaine. The FDA has since banned the imports of any tea containing residual cocaine, yet several studies performed with Health Inca Tea and other teas imported from South American countries clearly show that even very low amounts of cocaine in a beverage can result in a positive urine drug test.

Health Inca Tea has been reported to contain an average of 4.8 mg of cocaine/bag and between 1.87 and 2.15 mg of cocaine/cup of prepared tea. In 4 subjects ingesting 1 cup of Health Inca Tea containing 1.87 mg of cocaine, peak urinary benzoylecgonine concentration ranged from 1,400 to 2,800ng/mL 4 to 11 h after ingestion. Positive immunoassay results determined by FPIA were obtained for 21 to 26 h post tea ingestion. Total benzoylecgonine excretion in 36 h ranged from 1.05 to 1.45 mg, representing 59 to 90% of the ingested cocaine dose. In another study where 1 subject drank a cup of tea containing 2.15 mg of cocaine, the peak urinary benzoylecgonine concentration of 1,274 ng/mL occurred 2 h after tea ingestion. Total benzoylecgonine excreted in 29 h was 0.82 mg, or 38.1% of the ingested dose. Benzoylecgonine concentrations as determined by GC-MS exceeded 300ng/mL for 17.5 h after ingestion and 150 ng/mL for 22 h after ingestion. In a similar study, Peruvian and Bolivian coca tea was found to contain 5.11 mg and 4.86 mg of cocaine, respectively, per 1 ounce tea bag and 4.14 mg and 4.29 mg of cocaine, respectively, as per cup of prepared tea. Mean ecgonine methyl ester concentrations in prepared tea were 1.16 mg and 1.81 mg, respectively, for Peruvian and Bolivian teas. Benzoylecgonine concentrations were much lower. After consumption of a cup of Peruvian tea by 1 individual, peak urine concentrations were 3,368 ng/mL for benzoylecgonine at 10 h, 2,520 ng/mL for ecgonine methyl ester at 10 h, and 196 ng/mL for cocaine at 5 h. Consumption of Bolivian coca tea resulted in peak concentrations of 4,150 ng/mL for benzoylecgonine, 2,314 ng/mL for ecgonine methyl ester, and 587 ng/mL for cocaine, all at 3.5 h. Total benzoylecgonine excretion in 48 h was 3.11 mg and 2.69 mg after consumption of Peruvian and Bolivian coca tea, respectively, and benzoylecgonine concentrations remained above 300 ng/mL for at least 20 h. No pharmacological effects were reported in any of the studies where coca teas were ingested.

In a study where 6 ounces of cola fortified with 25 mg cocaine hydrochloride was consumed by a 165-lb male, peak urinary concentrations of cocaine 269ng/mL and benzoylecgonine (7,940 ng/mL) were obtained at 1 and 12 h, respectively. Urine benzoylecgonine concentrations remained in excess of 300ng/mL for 48 h. The subject reported a very slight local anesthetic effect on the lips and tongue when consuming the beverage, but no unusual taste. About 1 h after ingestion, the subject noted some dryness of the mouth, lightheadedness, and a slight headache, which lasted about 1.5 h. However, the physiological effects described should be considered in the context of a single subject study without placebo.

These studies demonstrate that very small amounts of cocaine, <5.0mg or about 1/5 of a typical intranasal dose (or line), when added to or contained in a beverage, are sufficient to produce a positive drug test in an unsuspecting subject for cocaine metabolites in urine. In such cases, the detection of unique cocaine metabolites, such as methylecgonine or ecgonine, produced after smoking crack cocaine, may prove to be useful in refuting alleged oral ingestion of cocaine after a positive drug test for benzoylecgonine.

A more extraordinary claim for positive urine drug tests for cocaine metabolites is through exposure to semen. This has been addressed in a study in which 5 subjects were given cocaine by intravenous (25 mg), smoked (42 mg), and intranasal (32 mg) routes of administration followed by the collection of semen. Semen samples collected before cocaine use were negative for cocaine and metabolizes, whereas semen samples collected 1 h after cocaine use contained parent drug and metabolite in concentrations ranging from 60 to 80% of plasma concentrations. At 24 h, cocaine and metabolite semen concentrations declined to trace or non-detectable amounts. The highest mean cocaine concentration detected in semen was 0.054 mg/g (after 42 mg smoked and the highest mean benzoylecgonine concentration was 0.081 mg/g (after 25 mg 1V). The total content of cocaine and benzoylecgonine in ejaculate did not exceed 0.001 mg in specimens collected 1 h after cocaine administration. Peak semen concentrations could have been as high as 0.002 mg considering the plasma concentration and the half-life of cocaine. In chronic or heavy cocaine users, administering doses in amounts 100 times those in this study, the total amount present in semen would only be 0.2 mg. As 1 to 2.5 mg of intravenous cocaine would be needed to produce 1 positive urine specimen, passive exposure to cocaine via ejaculate will not produce positive test results.

Dermal Exposure to Cocaine

Concern regarding passive exposure to cocaine in workers who come in frequent contact with the drug has also been raised. The issue of dermal exposure to cocaine has been investigated. The drug dose of cocaine free-base (dissolved in absolute ethanol) applied to the palmer surface of the hand of a volunteer resulted in a peak urine benzoylecgonine concentration of 55ng/mL. The maximum urine benzoylecgonine concentrations after application of 5 mg of cocaine hydrochloride under the same conditions, was 15ng/mL. Although these concentrations are well below US federally mandated drug-testing cutoff concentrations, they are nonetheless detectable. In an additional study, a subject who was a chronic nail biter who immersed 2 one-dollar bills in a container of cocaine paste and did not wash his hands for the day, achieved peak urinary benzoylecgonine concentration close to the 300 ng/mL immunoassay cutoff concentration. However, another subject, who was given the contaminated dollar bills and handled them occasionally throughout the day, only reached a peak urine benzoylecgonine concentration of 72ng/mL. A more complete study using 7 different groups of crime laboratory workers with varying degrees of exposure to cocaine demonstrated that higher concentrations of passive exposure to cocaine occurred in workers using gloves but no facemask than in workers using both. In 1 group, where 2 workers where handling a 50-kilo case of cocaine over a period of 3 h, the worker wearing gloves and a facemask showed urine benzoylecgonine concentrations of 112 ng/mL and 278 ng/mL, respectively, 1 h and 8 h after beginning work; the worker Wearing gloves and no facemask showed urine benzoylecgonine concentrations of 137 ng/mL and 1,570 ng/mL, respectively, for the same collection times. Finally, the issue of passive exposure to cocaine in medical personnel (n = 11) has been studied. When exposed to cocaine hydrochloride by means of aerosol and cutaneous application in a manner similar one that may occur in medical practice, all urine drug-screening tests was negative. In generals all of these studies demonstrated that, with appropriate personal protective equipment, casual exposure to cocaine is not likely to produce a benzoylecgonine concentration greater than the 300 ng/mL US federal workplace drug-testing cutoff concentration. If testing occurs at lower cutoff concentrations, studies suggest that data need to be evaluated carefully.

Other Matrices

Hair

Cocaine and metabolites may be demonstrable in hair for longer periods of time than urine. For this reason, analysis of hair may be useful in determining a history of cocaine use. It takes 4 to 5 days for ingested cocaine to begin to appear in the hair. The ability to detect cocaine in hair is based more on concentration than a pharmacological ‘Time windows’ since as long as the hair is not cut, incorporated drug will remain in it. However its utility is limited to obtaining historical information about drug uses not in determining an acute use such as in post-accident testing. Although appropriate forensic methodologies exist for the analysis of cocaine and metabolites in hair, their use for employment-related drug testing is controversial due to a lack of data required for accurate interpretation of results Many important issues related to hair testing are still being studied, including environmental contamination, washing techniques, hair color bias in hair based on the concentration of certain types of melanin, sex bias, specimen adulteration, quality control procedures, proficiency testing, and the establishment of cutoff concentrations. These issues have been reviewed in several articles and a book, and are beyond the scope of the present discussion. The primary analyte detected in hair after cocaine use is parent cocaine, but to minimize environmental contamination issues and to demonstrate cocaine ingestion, the proposed guidelines for hair testing in US federal workplace drug-testing programs require a benzoylecgonine/cocaine ratio of >0.05 in the confirmatory testing process to report a positive result. Still, some studies suggest that benzoylecgonine arises first and foremost from the hydrolysis of cocaine in hair, not due to biological incorporation. Other probable target analytes in hair to demonstrate in vivo cocaine use include norcocaine and cocaethylene. The confirmatory cutoff concentrations for cocaine analytes in hair are cocaine ≥500 pg/mg, benzoylecgonine ≥50 pg/mg, cocaethylene ≥50 pg/mg, and norcocaine ≥50 pg/mg.

Saliva

Saliva may be a helpful specimen for the detection of recent cocaine use in clinical studies and workplace testing. It offers the divergent advantage of collection by direct observation without any invasive procedure. Cocaine is the predominant analyte detected in saliva drug test following single doses of cocaine administered via IV (25 mg), smoked (42 mg), and intranasal (32 mg) routes of administration and was detectable in saliva and plasma for 3 to 6 h (arbitrary cutoff = 25 ng/mL). Benzoylecgonine and ecgonine methyl ester were detected, but at consistently low concentrations, peaking several hours after administration. The detection times of cocaine in saliva and plasma paralleled the appearance and disappearance of pharmacologic effects. These findings, as well as an earlier study, suggest that saliva may be a useful option matrix to blood in determining impairment, although more studies are essential as the kinetics of cocaine in saliva appear to be altered after chronic use. Drug concentrations in saliva can also be considerably affected by saliva flow rate, although pH has been shown to be the most important variable in determining saliva concentrations of ionizable drugs. Cocaine (pKa = 8.6) is particularly vulnerable to the effects of pH and salivary flow- as demonstrated by a 6.5-fold higher concentration in saliva that was collected un-stimulated versus stimulated.

For workplace drug-testing purposes, benzoylecgonine remains the target analyte in the proposed revisions to the guidelines for the use of “Oral fluids” saliva and its Components in US workplace drug testing, as it detected more frequently and at higher concentrations than cocaine, particularly after cessation of chronic cocaine use fit, despite studies to the contrary after acute use. Detection times for benzoylecgonine after chronic oral cocaine administration were comparable for saliva and plasma (45 h versus 47 h, cutoff = 10 ng/mL), but far less than urine, which had an average minimum detection time of 165 h, using the same low cutoff concentration. However, cocaine could be detected nearly twice as long in saliva than in plasma (15 h versus 9 h, cutoff = 10 ng/mL).

It is vital to develop standardized compilation protocols for the collection of saliva not only because of the effects of pH and stimulated versus un-stimulated sampling, but because there is a risk for collecting a contaminated specimen from the oral cavity, especially shortly after smoking, intranasal, or oral administration. To pass saliva drug test, saliva/plasma cocaine ratios have been have been shown to be well over 100 shortly after smoking; and over 1,000 shortly after intranasal administration. Contamination of the oral cavity appeared to be cleared 2 h after dosing in these studies. The use of benzoylecgonine as a target analyte in workplace testing should also help manage this issue. However, if cocaine is analyzed in saliva to aid in the determination of impairment, caution must be taken to ensure there is no contamination of the oral cavity.

Sweat

Sweat may be a useful, noninvasive specimen for monitoring drug use and is currently being used in that capability for some parolees. Sweat may be collected using a collection patch for a prearranged period, allowing a continuous period of monitoring or gathering of excreted drug. In sweat, cocaine is excreted chiefly as the parent drug. In patches worn for 7 days, it was possible to demonstrate a single episode of cocaine use. Depending on the device used to collect sweat, any attempt at tampering with the collection device would be evident. Although wearing a sweat patch for monitoring cocaine use may offer a wider detection window than urine, correlation between accumulated sweat cocaine concentrations with injury and time of use are not likely. Because trace amounts of cocaine can be detected in sweat after the IV administration of as little as 1 mg of cocaine, considerable concern has been raised over possible environmental contamination, particularly since it is the parent drug that is present in greatest concentrations in sweat. One study demonstrated that cocaine could he detected in sweat patches worn by drug-free volunteers after the application of cocaine, in its uncharged state, to the outside of the sweat patch membrane. It was also shown that positive sweat patch results could be obtained from patches that were applied to the skin of drug-free volunteers up to 6 days after cocaine was placed onto their slain surface and cleansed with normal hygiene procedures, as well as recommended clearing procedures before the application of me sweat patch.

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