Cocaine and Metabolites in Blood and Plasma
The ester linkage on the cocaine molecule makes the drug susceptible to chemical and enzymatic hydrolysis. In facts the major metabolites of cocaine, benzoylecgonine (BE) and ecgonine methyl ester (EME), are hydrolytic products of cocaine Conversion to BE occurs chemically, especially under alkaline conditions and enzymatically by a liver methyltransferase; conversion to EME occurs enzymatically by pseudo-cholinesterase in the plasma and by a benzoylesterase in the liver. Several early studies tracked the deterioration of cocaine in blood and plasma Jatlow and Bailey observed that plasma cocaine concentrations decreased by 6.5% after 15 min at room temperature and by 12.5% after 30 min at room temperature. Storage of blood for 24 h at -15 °C or on solid carbon dioxide produced decreases of up to 30 Ye in blood cocaine concentrations.
This latter finding was disputed by Stewart et al., who found no significant cocaine degradation when plasma samples fortified with labeled cocaine were stored for 4 days at -15 °C. It was hypothesized that the findings of Jatlow and Bailey could be explained by the alkaline extraction pH. Baselt studied the stability of cocaine in blood at 2 concentrations, 0.1 and 1.0 mg/l, at 4 °C in the absence and presence of fluoride. At 0.1 mg/L, cocaine disappeared rapidly in non-fluoridated blood and was not detected by day 3; 40 to 60% of the original cocaine was detected in fluoridated blood and plasma after 21 days. In similar pattern was observed at 1 mg/L; 80% of the cocaine originally present was still detected in preserved blood and plasma after 21 days. McCurdy et al. studied the stability of cocaine and BE in 4 types of blood collection tubes at room and refrigerated temperatures over a Today period. BE was stable at room and refrigerated temperatures over the 30-day period in unpreserved blood and in blood preserved with heparin, fluoride, and EDTA.
As measured by radioimmunoassay, blood with a cocaine concentration of 0.2 mg/L was stable at refrigerated temperature for 30 days regardless of the additive present; however, when the blood specimens were stored at room temperature, cocaine was stable only in the fluoridated blood. Long-term stability of BE was explored by Giorgi and Meeker. They found that blood specimens stored in gray-top tubes showed significant losses of BE when stored at ambient temperature for 3 months. Short-term stability of cocaine produced by fluoride was demonstrated by Brogan et al.
When cocaine-spiked blood was added to flasks containing 0.0, 0.25, 0.5, and 1.0% sodium fluoride and stored either at room temperature or at 4 °C, 86 to 91% of the drug originally present was still detected in fluoridated tubes after 48 h at either temperature;. Baselt et al. found that substituting oxalic acid for potassium oxalate enhanced the storage of refrigerated, fluoridated blood containing 0.2 mg/L cocaine. Twenty four percent of the cocaine was detected after 1 year. Further addition of echothiophate iodide, a quaternary amine, reduced the cocaine loss to 60% of the original concentration by 1 year. BE concentration was unaffected under all three storage conditions during the 1-year period. A comprehensive study of the stability of cocaine in blood and buffer was performed by Isenschmid et al. In addition to the measurement of cocaine, BE and EME were also quantitated. The effects of temperature, concentration, pH and preservatives were examined in all possible combinations. The following conclusions were drawn:
· Cocaine hydrolysis is independent of concentration;
· Cocaine hydrolysis in unpreserved blood occurs more rapidly as the temperature and pH of the blood increase and the cocaine is hydrolyzed to EME only;
* Cocaine in blood preserved with fluoride or organophosphates at physiological pH is hydrolyzed to BE only;
· Cocaine preserved with fluoride or organophosphates at pH 5 produces the greatest stability of cocaine in blood .
Another comprehensive study was undertaken by Skopp et al., who looked at the stability of cocaine, BE, EME, and ecgonine preserved with 0.25% potassium fluoride at 4 °C and 20 °C over a 15-day period. At 4 °C and 20 °C, BE and EME: could be detected from cocaine l day after storage. Ecgonine was detectable after 2 days of storage. At 4 °C, approximately 25% of the initial cocaine concentration was still detected at day 14. The relative amount of hydrolytic products was EME > BE > ecgonine. At 20 °C, no cocaine was detected by the end of the study. BE and EME concentrations increased, but decreased after 6 and 7 days of storage, respectively. The concentration of ecgonine increased steadily.
Ecgonine was stable at 4 °C and at 20 °C over the 2-week period. At 4 °C, BE was converted to ecgonine by day 3 and the concentration of ecgonine was less than 1096 of the initial BE concentration. At 20 °C~ about 40% of the initial BE concentration had been converted to ecgonine by day 14. At 4 °C, the EME concentration decreased by about 90% by day 14, while at 20 °C no EME was detected by the end of the observation period. The conversion of cocaine to BE, EME, and ecgonine appeared to be stoichiometric at all time intervals at both storage temperatures. Cocaethylene or ethylcocaine is produced in vivo following the simultaneous ingestion of alcohol and cocaine. Cocaethylene was found to break down more slowly in the blood than cocaine.
About 25% of cocaethylene was still detected in blood by the third day when stored at 20 to 25°C. Conversely no cocaine was detected after the first day. Methylecgorudine, or anhydroecgomne methyl ester, is a pyrolysis product of heated cocaine. Scheidweiler et al. added methylecgonidine to sheep plasma with and without sodium fluoride and stored it at -80, 1, 21, and 37 °C for 48 h. Decreased temperature and increased sodium fluoride limited methylecgonidine degradation. In addition, methylecgonidine was more stable at pH 6 than at pH 7.6, even at 37 °C .
Cocaine and Metabolites in Urine
The stability of cocaine and BE in urine specimens has also been investigated by several researchers Baselt added cocaine to urine at pH 5 or pH 8 to produce a concentration of 1 mg/L. The specimens were stored at 4 °C in the absence and in the presence of fluoride. At pH 5, the urine concentration was unchanged after 21 days; at pH 8, cocaine concentrations decreased by 40 to 70%. In each case, the presence of fluoride had no effect on the results.
The stability of BE in urine specimens can have significant forensic implications. Any urine testing positive under the Department of Health and Human Services guidelines must be subsequently frozen for at least 1 year. During that year, the specimen is eligible for retesting upon request. Romberg and Past compiled retest data for 61 urine specimens containing BE. For these specimens, an average decrease of 19% was measured.
However, distribution of the percentage of change suggested a bimodal distribution, with one distribution around 10% and a second distribution around 80%. No explanation for these changes was provided. When Gere et al. reanalyzed 60 urine samples testing positive for BE that had been frozen for 8 to 200 days, decreases greater than 20% were found in 33 of the samples. No correlation was found between the amount lost and time elapsed from the original test, or between the amount lost and the length of time the specimens were kept at room temperature before freezing. Cody and Foltz examined the effects of temperature and pH on BE added to urine specimens.
Overall, decreases in concentration were observed at neutral or alkaline pH and at room temperature. However, exceptions to this overall trend were noted. The authors concluded that factors other than pH and temperature, such as microorganism growth, can also influence BE degradation in urine. The stability of cocaine’s other major metabolite, EME has also been investigated. Vasiliades added EME to drug-free urine with pH adjusted to 3, 5, or 9. The specimens were refrigerated and tested at various times over a 3-year period.
EME remained stable in urine at pH 3 and pH 5 with over 80% of the compound originally present being detected after 3 years. Conversely, the EME stored in pH 9 urine was completely lost within a month. Levine et al. studied the stability of EME in 25 postmortem urine specimens stored under refrigerated or frozen conditions for 6 months. Twenty two of the freezer specimens and 19 of the refrigerated specimens were within 20% of their initial EME concentrations after 6 months. At least 50% of EME was present in all specimens after 8 months.
Cocaine in Oral Fluid
Cone and Menchen studied the stability of cocaine in saliva as a function of temperature, container, and preservative. When the samples were refrigerated, more than 9046 of the cocaine were recovered after 4 days, regardless of the storage container. Only when samples were stored in a tube containing sodium citrate was cocaine found to be stable at 22 °C for 7 days.
The instability of cocaine in untreated blood or plasma is well-documented. Loss of cocame can be nursed by adding pseudocholinesterase inhibitor immediately after collection, reducing the pH to 5, and storing the sample frozen. The stability of cocaine in urine is pH-dependent, similar to cocaine stability observed in aqueous solutions. Benzoylecgonine exhibits greater stability in blood and urine but decreases over time has been observed. EME also displays greater stability in urine, especially at neutral or slightly acidic pH.