Urinary Excretion of Ecgonine and Five Other Cocaine Metabolites Following Controlled Oral, Intravenous, Intranasal, and Smoked Administration of Cocaine

Abstract

Urinary excretion of ecgonine (EC) was compared to that of cocaine, benzoylecgonine, ecgonine methyl ester and minor metabolites, meta-hydroxybenzoylecgonine, para-hydroxybenzoylecgonine, and norbenzoylecgonine, following controlled administration of oral, intravenous, intranasal, and smoked cocaine. Urine EC concentrations peaked later than all other analytes and had longer detection times than the other minor metabolites. With a 50 ng/mL cutoff concentration and following low doses of 10 to 45 mg cocaine by multiple routes, detection times extended up to 98 h. Maximum concentrations (Cmax) were 6–14 mole % of those for benzoylecgonine, Cmax increased with dose, time to maximum concentration (Tmax) was independent of dose, and route of administration did not have a significant impact on Cmax or Tmax for metabolites. EC is an analyte to consider for identifying cocaine use due to its stability in urine and long detection times.

Introduction

Cocaine was ranked second only to heroin as one of 20 most misused substances causing significant physical and social harm to humans (1). This is one of the reasons for cocaine monitoring in workplace, parole and treatment programs. The most common testing method is detection of benzoylecgonine (BE), a major cocaine metabolite, in urine specimens (2).

BE usually has higher urine concentrations and longer detection times than other cocaine metabolites; however, BE identification of cocaine use has limitations. For example, BE can be produced by hydrolysis of cocaine added directly to urine, amounts increasing with increasing pH, making it difficult to distinguish ingestion from external contamination (3). The cocaine metabolite ecgonine methyl ester (EME) was suggested as an alternative analyte, but Klette et al. (3) demonstrated that EME also could be produced in vitro from cocaine in basic urine specimens. They suggested identifying meta-hydroxy-BE (mOH-BE) or para-hydroxy-BE (pOH-BE) in urine to prove cocaine use. The hydroxy metabolites are more specific for ingestion but have much shorter detection times than BE or EME, regardless of route of administration (4,5). For single doses of less than 50 mg cocaine, mOH-BE and pOH-BE could not be detected beyond 2 days.

Another limitation of BE and EME as cocaine biomarkers is lack of stability in urine. Most urine specimens stored at −20°C or lower had less than 20% BE and EME degradation over one year (6); however, in select specimens, both major metabolite concentrations significantly decreased during frozen storage (6). Some deterioration can be attributed to higher specimen pH, but other specimen-specific causes are not well understood. This is one reason that ecgonine (EC) was suggested as a hydrophilic, stable metabolite for detecting cocaine use (7).

There are few controlled EC pharmacokinetic studies. EC is highly water soluble and usually requires special procedures for gas chromatography–mass spectrometry (GC–MS) analysis (8). It can be formed in urine from BE, EME or the hydroxy metabolites (Figure 1). Following our smoked cocaine administration study, we found that this metabolite peaks later and lasts longer than other minor metabolites (5); following 10 to 40 mg smoked cocaine, EC was detected up to 80 h. The current study examines EC, cocaine, BE, EME, mOH-BE, pOH-BE, and norbenzoylecgonine (NBE) disposition in urine after oral, intravenous (IV), and intranasal (IN) cocaine administration. Results are compared to those published previously for smoked (SM) cocaine.

Figure 1.

Ecgonine (EC) can be formed by demethylation or hydrolysis of cocaine and metabolites.

Materials and Methods

Research protocols

Subjects were healthy adult males who provided written informed consent. The protocol was approved by the Institutional Review Board for the National Institute on Drug Abuse and adhered to Federal guidelines for the protection of human subjects. All participants had a history of cocaine ingestion by the IV and/or SM routes of administration. Participants resided on a secure research unit with 24 h surveillance throughout the study and were monitored to prevent use of unauthorized drugs. Prior to controlled drug administration, subjects excreted self-administered cocaine until negative urine specimens were obtained at a cutoff concentration of 300 ng/mL by enzyme multiplied immunoassay technique (EMIT®, Siemens Healthcare Diagnostics). The period of time from entry on the ward to dosing was more than three days. The first administration for all subjects was placebo.

Group 1 consisted of six males (5 African American (AA), 1 Caucasian (C); ages 27–39 years; subjects A–F) who ingested a single oral dose of 22.4 mg cocaine hydrochloride and one or more IV cocaine hydrochloride doses, 0 (n = 1), 11.2 (n = 4), 22.4 (n = 4), and 44.8 mg (n = 1). These same participants also smoked three different doses of cocaine base with a controlled smoking device [only smoked data previously reported (5)]. There were 2 to 14 days between doses. Urine specimens were collected prior to and for 2 to 7 days after each dose and stored at −20°C or lower for 12.6 to 12.8 years before analysis.

In Group 2, there were six males (2 AA, 4 C; ages 30–44 years; subjects G–L) who received 32 mg IN cocaine hydrochloride and in a separate session, 25 mg IV cocaine hydrochloride. These participants also smoked 42 mg cocaine base in a glass pipe [only smoked data previously published (5)]. There were at least 3 days between different doses. Urine specimens collected for 3 days were stored at −20°C or lower for 12.7 to 13.2 years before analysis.

Forty-six quality control samples were prepared in urine, contained all analytes except EC in concentrations covering the full analytical range of the assay, and were kept frozen with urine specimens collected from both clinical studies. Fifteen drug-free urine samples also were included in this group for quality control. Quality control samples were prepared at the National Institute on Drug Abuse (Baltimore, MD), submitted with specimens for blind analysis to the Armed Forces Institute of Pathology (Rockville, MD), and stored at −20°C or colder for the last 5 years prior to analysis.

GC–MS analysis

GC–MS analysis was performed by a validated published method (8). Cutoff concentrations for cocaine and major metabolites BE and EME were 10, 20, and 10 ng/mL, respectively, and 25 ng/mL for minor metabolites, mOH-BE, pOH-BE, and NBE. The cutoff concentration for EC, also a minor metabolite, was 50 ng/mL. Each analytical assay contained negative quality control samples and samples fortified at or above cutoff concentrations; acceptance criteria for these quality control samples were acceptable chromatography, quantification within ±20% of target, and two ion ratios within ±20% of target. In addition, two urine control samples were included in each assay with high concentrations of cocaine, BE, EME, and EC; control 1 contained 1500, 8000, 7500, and 1500 ng/mL and control 2 contained 3000, 16,000, 15,000, and 3000 ng/mL of these analytes, respectively.

Results

Excretion profiles for EC following each route of cocaine administration are shown in Figure 2. Subjects received a single dose by the intranasal and oral routes, four different doses intravenously, and four different doses by smoking. Mean urine Cmax for EC was 6–14 mole % of mean BE Cmax, for all routes of administration (Table I). Mean Cmax were similar for minor metabolites, mOH-BE, pOH-BE, and NBE, and increased with dose. BE and EME had the highest peak concentrations, consistent with previous reports (4). Cocaine Cmax were significantly lower following the oral route compared to comparable IV and SM doses (all t > 3.5, p < 0.01), but did not significantly differ between IV, IN, and SM routes (all t < 1.7, p > 0.05). Peak concentrations for all metabolites were independent of route of administration.

Figure 2.

Urine excretion profiles for ecgonine (EC) following four routes of cocaine administration. Cocaine doses (number of subjects) were intranasal 32 mg (n = 6); intravenous 11.2, 22.4, 25, 44.8 mg (n = 12); oral 22.4 mg (n = 6); and smoking 10, 20, 40, 42 mg (n = 12).

Table I.

Maximum Urine Concentrations (Cmax) of Cocaine and Metabolites Following Oral, Intravenous (IV), Intranasal (IN), and Smoked (SM) Controlled Administration of Cocaine

	Cmax (ng/mL)
Subject	EC*	Cocaine	BE	EME	mOH-BE	pOH-BE	NBE
Oral, 22.4 mg
A	271	492	7202	2970	305	243	956
B	512	57	7282	5942	100	404	157
C	300	594	5930	1587	189	235	156
D	194	135	4128	2136	129	67	27
E	174	193	5473	3790	55	522	298
F	396	470	6837	4903	130	468	617
Mean	308	324	6142	3555	151	323	369
SE	48	83	455	619	32	64	131
IV, 11.2 mg
B	220	115	3602	1621	52	116	51
D	118	966	3074	1572	93	47	25
E	192	1170	2735	1167	38	107	150
F	487	209	4356	2676	137	292	176
Mean	254	615	3442	1759	80	141	101
SE	81	266	353	322	22	53	37
IV, 22.4 mg
A	328	2177	9164	3354	451	279	134
C	408	2216	4218	1603	130	158	54
D	260	1106	4656	2177	151	123	76
F	467	637	8095	4647	169	503	447
Mean	366	1534	6533	2945	225	266	178
SE	45	394	1233	674	76	86	91
IV, 25 mg
G	629	52	6338	1773	189	30	44
H	146	921	6276	2637	52	90	45
I	206	301	6260	1548	601	181	134
J	474	672	12,942	6044	322	616	316
K	589	550	14,738	4132	29	471	491
L	0	2326	2514	1544	0	0	0
Mean	409	804	8178	2946	199	231	172
SE	90	328	1902	739	94	104	79
IV, 44.8 mg
D	388	1576	11,887	4207	369	218	184
IN, 32 mg
G	441	772	5514	1637	80	56	48
H	63	0	100	40	0	0	0
I	353	71	9895	2060	111	273	116
J	552	423	18,528	7739	678	653	455
K	2052	193	15,213	8918	19	379	768
L	133	234	4033	2127	13	34	45
Mean	599	282	8881	3754	150	233	239
SE	300	115	2864	1487	107	104	126
SM, 10 mg
E	0	135	1384	610	0	63	35
F†	228	283	2966	1966	64	185	74
F†	287	1004	4570	3467	98	429	196
Mean	172	474	2973	2014	54	226	102
SE	88	268	920	825	29	108	48
SM, 20 mg
C	1053	977	3822	975	167	89	52
D	114	383	3229	1753	93	66	13
E†	177	1682	3619	1614	57	196	114
E†	337	2032	7861	3695	82	538	333
F	444	1435	9577	6206	157	516	389
Mean	425	1302	5622	2849	111	281	180
SE	167	287	1297	955	22	103	76
SM, 40 mg ‡
A	1425	508	6881	1921	415	224	107
B	904	162	6295	3018	113	252	124
E	600	5921	10,766	4645	129	489	640
F	478	9748	12,844	8969	230	1197	1587
Mean	852	4085	9196	4638	222	540	614
SE	211	2303	1569	1548	69	227	347
SM, 42 mg ‡
G	ND	67	1021	410	36	ND	ND
H	245	521	5094	2052	81	53	ND
I	83	94	2199	569	33	76	39
J	343	1635	8871	4344	387	268	92
K	843	186	22,494	7676	37	564	3592
L	149	178	3502	1751	47	46	29
Mean	333	447	7197	2800	104	201	938
SE	135	247	3256	1133	57	99	885

^*Abbreviations: EC, ecgonine; BE, benzoylecgonine; EME, ecgonine methyl ester; mOH-BE, meta-hydroxy-BE; pOH-BE, para-hydroxy-BE; NBE, nor-BE; and ND, not detected.

^†Subject had two separate sessions at the same dose.

^‡Reprinted from Huestis et al. (5).

Unlike Cmax, urinary Tmax for each analyte was independent of dose (all t < 1.2, p > 0.05, Table II). Cocaine tended to have later mean Tmax following oral and IN ingestion but were generally not statistically different from those following other routes. Tmax for major metabolites BE and EME, and pOH-BE were on average 4–7 h (4,5). mOH-BE and NBE peaked later in most instances, with EC as the latest, usually in 8–13 h.

Table II.

Time to Maximum Urine Concentration (Tmax) of Cocaine and Metabolites Following Oral, Intravenous (IV), Intranasal (IN), and Smoked (SM) Controlled Administration of Cocaine

	Tmax (ng/mL)
	EC*	Cocaine	BE	EME	mOH-BE	pOH-BE	NBE
Oral, 22.4 mg (n = 6)
Mean	13.3	4.5	6.0	4.5	7.3	6.6	7.5
SE	3.1	0.9	1.0	0.9	1.1	0.9	1.3
IV, 11.2 mg (n = 4)
Mean	9.9	3.8	4.7	4.7	8.2	5.2	5.2
SE	2.0	0.8	1.5	1.5	0.7	1.3	1.3
IV, 22.4 mg (n = 4)
Mean	10.2	2.1	4.6	3.5	10.2	4.6	10.6
SE	1.6	0.5	1.3	0.9	1.6	1.3	5.2
IV, 25 mg (n = 6)
Mean	13.3	3.8	4.2	4.6	5.4	5.0	5.2
SE	4.2	1.0	0.9	1.0	0.8	0.5	1.1
IV, 44.8 mg (n = 1)
	3.0	3.0	3.0	3.0	11.0	3.0	3.0
IN, 32 mg (n = 6)
Mean	12.8	4.8	6.4	4.8	6.1	4.8	5.0
SE	2.6	1.1	1.8	1.2	1.1	1.1	1.3
SM, 10 mg (n = 3)
Mean	10.4	1.8	5.1	5.1	8.9	4.1	9.0
SE	1.6	0.3	2.0	2.0	3.1	1.1	5.7
SM, 20 mg (n = 5)
Mean	8.3	2.8	5.5	4.3	7.6	5.5	6.4
SE	1.1	0.4	1.3	0.8	0.9	1.3	1.1
SM, 40 mg (n = 4) †
Mean	9.3	2.2	6.6	5.6	7.8	4.4	6.0
SE	1.3	0.3	0.9	1.4	0.5	1.5	2.0
SM, 42 mg (n = 6) †
Mean	11.1	2.4	5.6	4.9	6.4	5.2	6.2
SE	4.3	0.3	0.9	0.9	0.9	1.0	2.5

^*Abbreviations: EC, ecgonine; BE, benzoylecgonine; EME, ecgonine methyl ester; mOH-BE, meta-hydroxy-BE; pOH-BE, para-hydroxy-BE; and NBE, nor-BE.

^†Reprinted from Huestis et al. (5).

All metabolites were initially detected in the first or second urine void for most subjects and all routes examined. Last detection times for pOH-BE, mOH-BE, and NBE were usually one day or less, but occasionally extended to 55 h. Last detection of EC following moderate doses exceeded 50 h for most individuals with the latest positive urine specimen excreted at 98 h (Figure 1, Table III). The last detection times for cocaine were usually less than 36 h, with BE and EME indeterminate because they were still positive at the time of last urine collection.

Table III.

Time from Administration of Cocaine by Various Routes to the Last Urine Specimen Containing a Positive Minor Metabolite

	Final Detection Time (h)
	EC* 50 ng/mL cutoff	mOH-BE 25 ng/mL cutoff	pOH-BE 25 ng/mL cutoff	NBE 25 ng/mL cutoff
Oral, 22.4 mg (n = 6)
Mean	50.5	26.2	20.9	25.5
SE	8.6	3.4	3.1	2.1
Min	20.8	20.2	10.8	20.0
Max	78.3	33.6	33.0	33.0
IV, 11.2 mg (n = 4)
Mean	59.3	16.7	9.4	12.6
SE	19.1	5.5	2.2	6.1
Min	21.9	6.6	5.3	5.3
Max	98.4	31.5	15.3	31.0
IV, 22.4 mg (n = 4)
Mean	40.3	28.8	16.1	21.8
SE	2.0	1.8	3.9	3.0
Min	35.8	25.8	9.0	13.5
Max	44.6	32.8	27.4	26.8
IV, 25 mg (n = 6)
Mean	36.1	22.8	13.7	14.9
SE	8.8	4.5	3.9	4.8
Min	ND	4.2	ND	ND
Max	64.1	33.7	23.0	31.2
IV, 44.8 mg (n = 1)
	60.5	43.5	31.3	24.2
IN, 32 mg (n = 6)
Mean	49.6	19.8	14.6	16.7
SE	8.7	6.5	5.3	5.1
Min	13.3	2.3	3.2	3.2
Max	69.0	36.3	34.2	34.2
SM, 10 mg (n = 3) †
Mean	31.2	21.4	19.2	15.0
SE	0.1	9.6	6.0	8.5
Min	ND	ND	10.9	2.2
Max	31.3	31.0	31.0	31.0
SM, 20 mg (n = 5) †
Mean	53.5	26.2	25.0	18.3
SE	11.2	7.8	8.2	4.8
Min	25.4	10.8	10.8	9.5
Max	80.3	55.3	55.3	31.3
SM, 40 mg (n = 4) †
Mean	53.0	26.0	18.5	21.0
SE	9.4	5.3	6.5	4.8
Min	33.3	16.8	6.9	12.9
Max	77.4	37.0	37.0	32.4
SM, 42 mg (n = 6) †
Mean	36.8	15.0	9.5	18.2
SE	7.6	4.2	0.9	0.2
Min	ND	3.9	ND	ND
Max	57.0	28.2	12.8	31.9

^*Abbreviations: EC, ecgonine; mOH-BE, meta-hydroxy-BE; pOH-BE, para-hydroxy-BE; NBE, nor-BE; and ND, none detected.

^†Reprinted from Huestis et al. (5).

Quality control samples stored frozen were tested with specimens. Ninety percent of these quality control samples containing cocaine, BE, EME, mOH-BE, pOH-BE, and NBE quantified within ±20% of target. No EC was detected in any of these samples. No analytes were detected in any of the drug-free quality control samples.

Discussion

Elimination half lives, expected urine concentrations and detection times for cocaine and several metabolites have been reported (2). The current study focuses on the minor cocaine metabolite EC but also reports expected Cmax, Tmax, and detection times for cocaine, BE, EME, mOH-BE, pOH-BE, and NBE following controlled administration of different doses of cocaine by oral, IV, and IN routes. Results for each analyte are compared to those previously published following SM (5) in order to present a comprehensive analysis of the effects of route of administration and dose on excretion profiles.

Mean urine Cmax for EC was 6–14% that of BE (molar) and occurred 3–9 h later than BE Tmax. The longer time to reach peak concentration was expected because in vivo conversion of BE and EME takes time to occur, just as BE and EME peak later than their parent compound cocaine. However, it is interesting that the peak concentration occurs later than the other products of BE metabolism. This may indicate that the esterase and chemical hydrolysis mechanisms that form EC are slower. EC cutoff concentration was higher than for all other analytes due to its being a zwitterion and water soluble, making isolation more difficult. Despite a high cutoff concentration of 50 ng/mL, detection times for EC averaged 31–60 h following low cocaine doses with longest detection at 98 h. There were instances where detectable EC may have been present after four days. Four days after receiving 44.8 mg cocaine IV, one subject smoked placebo as a part of the smoking protocol published previously (5). Placebo was the first administration after entering the ward for subjects in both study groups. Although most urine specimens collected after placebo dosing had no measurable cocaine or metabolites, there were low concentrations of EC in some of the first specimens collected (data not shown). BE and EME also were present at < 250 ng/mL and the concentrations decreased with time. The best explanation for these low, decreasing concentrations of BE, EME, and EC were that they were residual excretion from the previous study. One subject who received placebo as the first administration had low concentrations of BE, EME, and EC in pre- and post placebo urine specimens. BE was present at 22 ng/mL in the first urine void at 2.25 h post dose but was < 20 ng/mL for subsequent specimens. EME and EC were present for up to 39.6 h at concentration ranges of 10–37 ng EME/mL and 50–153 ng EC/mL. The presence of these metabolites was obviously due to residual excretion from the subjects' preadmission cocaine use and demonstrates that EC can have longer detection times than BE and higher terminal concentrations than EME. The longer detection times were characteristic of EC regardless of the route of administration.

For cocaine and metabolites, Cmax increased linearly with dose following the SM route of administration with a controlled smoking device (5). Tmax tended to be longer following oral and IN routes, but this difference was not statistically significant. Tmax was independent of dose for all analytes. Cocaine Tmax usually occurred at the time of the first urine void. Ranges of mean Tmax for BE, EME, and pOH-BE were 3–7 h, for mOHBE and NBE 5–11 h, and for EC 8–13 h. An exception to these Tmax was the single subject receiving the largest IV dose, whose peak concentrations for most metabolites occurred at 3 h.

Detection times varied with cocaine analyte and cutoff concentration. As reported previously, EME (cutoff = 10 ng/mL) had a longer detection time than BE (cutoff = 20 ng/mL) and both major metabolites had detection times much longer than mOH-BE, pOH-BE, and NBE (5). Detection times for BE and EME, with the low cutoff concentrations applied in this study, could not be accurately determined, since the last specimens were still positive. Detection times reported previously following smoking often exceeded 100 h (5). The aromatic hydroxy metabolites were usually detectable in urine for 1 to 1½ days, with mOH-BE detectable longer than pOH-BE. In cases where donors claim that BE or EME in their urine specimen was due to contamination with cocaine, program managers may request quantification of one of the aromatic hydroxy metabolites as a more specific indicator of cocaine ingestion (3), but these metabolites may not be present in urine specimens collected more than one day after cocaine use, depending upon dose. In the current study, subjects were given low single cocaine doses. Multiple ingestions with higher total doses (500 mg to 2 g per day), more typical for cocaine addicts, extended the detection time for pOH-BE up to 170 h (9).

In a similar manner, longer detection times for EC would be expected following higher and/or multiple cocaine doses. EC also is more stable than BE and EME in stored urine specimens, possibly facilitating identification of cocaine use in unusual specimens with accelerated BE deterioration (7). EC's hydrophilic properties having both hydroxy and carboxy functional groups protect it from oxidation, but also make it more difficult to isolate by solid-phase extraction prior to GC–MS analysis. The polar solvent required to elute the compound from the solid-phase extraction column also eluted low concentration interfering compounds present in the urine matrix (10). This yielded a higher LOQ for EC. With improvements in liquid chromatography–MS–MS techniques, it may be possible to develop simpler and more sensitive EC analyses to extend EC detection times.

It is possible that some EC in urine specimens resulted from in vitro hydrolysis of EME or BE. Hydrolysis of the benzoyl group in BE or the methoxy group in EME produces EC. Since the specimens were stored frozen for an extended period of time, we examined the stability of cocaine, BE, and EME in two ways. First, blind quality control samples prepared in urine and containing these analytes in concentrations that covered the analytical range were stored under the same conditions as the specimens for 5 years. Ninety percent of these samples had quantifications of cocaine, BE, and EME within ±20% of expected values. In addition, aliquots of six specimens were analyzed after storage for five years using a different method (4) and the results compared to current concentrations (Figure 3). Cocaine concentrations were similar with two current results higher than those from the past analyses. BE did decrease in 5 of 6 specimens by 17–29%, and EME concentrations were comparable for 5 of 6 specimens.

Figure 3.

Concentration of cocaine, benzoylecgonine (BE), and ecgonine methyl ester (EME) after storage of aliquots of the same urine specimens at −20°C or lower for 5 years (A) and 12.7–13.2 years (B).

The contribution of EC from in vitro conversion appears to be small. This is also consistent with the observation that EC peaked much later than BE and EME in the time course of drug excretion. Urine specimens with the highest BE and EME concentrations did not have the highest EC concentrations, which one would expect if in vitro formation were extensive.

In conclusion, route of cocaine administration did not have an obvious impact on Cmax and Tmax for cocaine metabolites. Half-lives of cocaine and some metabolites were reported to be dependent on route of administration with half-lives of IN > IV > SM (4). As expected, Cmax increased but Tmax was independent of dose. For doses examined in this study, which were lower than reported street doses, last detection times for minor cocaine metabolites were 1–4 days with mean detection times for EC > NBE > mOH-BE > pOH-BE. Analysis of urinary EC may be an alternative biomarker for identifying cocaine use because of its stability in urine and long detection times, especially if improved analytical techniques are developed with lower detection limits.