GC-MS determination of heroin metabolites in meconium: evaluation of four solid-phase extraction cartridges

A procedure for extraction of heroin and metabolites for gas chromatography-mass spectrometry (GC-MS) analysis of meconium specimens that would allow detection of these analytes at low levels was needed. Solid-phase extraction (SPE) cartridges were therefore evaluated for their effectiveness in sample preparation. Four different types of commercially available extraction cartridges were used. Heroin, 6-monoacetylmorphine (6-MAM), morphine, and codeine were extracted from meconium samples using these SPE cartridges and then simultaneously analyzed using GC-MS. In each case, the extraction efficiency, linearity range, limit of detection (LOD), limit of quantitation (LOQ), between-run precision, and within-run precision were determined. Although satisfactory results were obtained with the four different types of SPE cartridges, best overall performance was observed using Clean Screen columns following the procedures outlined here. LODs as low as 20 ng/g for codeine, 10 ng/g for morphine, and 2.5 ng/g for 6-MAM were obtained, and LOQs as low as 20 ng/g for codeine, 10 ng/g for morphine, and 5 ng/g for 6-MAM were obtained. In all cases linearities were observed (r = > 0.99) for codeine, morphine, and 6-MAM over a wide concentration range (100-2000, 100-2000, and 5-100, respectively). At 50 ng/g codeine and morphine and 10 ng/g 6-MAM, the precision of analysis using these cartridges showed coefficients of variation ranging from 4.75% to 15.5%.     

Postmortem distribution of heroin metabolites in femoral blood, liver, cerebrospinal fluid, and vitreous humor

The presence of 6-monoacetylmorphine (6-MAM) is often used to distinguish between heroin (diacetylmorphine) and morphine exposures. 6-MAM, however, is rapidly metabolized to morphine and may not be present in detectable quantities in blood following heroin exposure. Recent studies have shown that 6-MAM may persist in cerebrospinal fluid (CSF) and this specimen may be preferable for establishing heroin exposure. This study reports postmortem distribution of 6-MAM, unconjugated morphine, and codeine in different tissues from 25 deceased individuals. In all cases, 6-MAM was detected in vitreous humor, and in CSF in 16 of the 25 cases (64%). When 6-MAM was detected in blood (13 of 25 cases), the level of 6-MAM in vitreous humor and CSF was higher than in blood, with a mean concentration ratio of 11.3 (range: 1.7-27) for vitreous humor and 6.6 (range: 2.6-17.3) for CSF. 6-MAM was not detected in liver in any of the cases examined. Free morphine levels were highest in liver, followed by blood, CSF, and vitreous humor. The concentration ratios (mean +/- standard deviation) for free morphine in vitreous humor, CSF, and liver to that in blood were 0.36 +/- 0.18, 0.64 +/- 0.27, and 2.99 +/- 2.12, respectively. The liver/blood ratio was consistent with previously reported values for morphine in heart and femoral blood. Codeine levels following heroin overdose were consistently low relative to the morphine concentration. For blood, liver, and CSF, the ratio of codeine to morphine was essentially the same (0.06), whereas the vitreous codeine/morphine concentration ratio was slightly higher (0.19). These results characterize the distribution of heroin metabolites in postmortem tissues. Vitreous humor appears to be a useful specimen for determining 6-MAM and establishing the morphine was derived from heroin.     

Interpretation of the presence of 6-monoacetylmorphine in the absence of morphine-3-glucuronide in urine samples: evidence of heroin abuse

The presence of morphine in a urinary sample may be caused not only by intake of heroin but also by intake of poppy-seed-containing food shortly before urine sampling or intake of drugs containing morphine, ethyl morphine, or codeine. To facilitate the interpretation, the heroin-specific metabolite 6-monoacetylmorphine (6-MAM) can be analyzed along with morphine-3-glucuronide (M3G) in an LC-MS verification analysis. In sporadic samples positive in the immunologic opiate screening test, 6-MAM, but not M3G, was found. To systematically analyze the finding all specimens with positive 6-MAM and/or M3G found during a 1-year period were investigated (n = 1923). Of these, 423 were positive for 6-MAM. In 32 (7.6%) of the samples 6-MAM was detected while the M3G concentrations were below cutoff (300 ng/mL) and in some cases even below the limit of detection (15 ng/mL). The 32 samples with this excretion pattern came from 13 different individuals, all but one with previously known heroin abuse. Eleven urine samples, nine containing M3G and 6-MAM and two with only 6-MAM, were also analyzed for the presence of heroin. In six samples, including the two with only 6-MAM, heroin was detected. There are several plausible explanations for these findings. The intake may have taken place shortly before urine sampling. High concentrations of heroin and 6-MAM may inhibit UGT 2B7, the enzyme responsible for glucuronidation of morphine. The hydrolyzation of 6-MAM to morphine may be disturbed by either internal or external causes. To elucidate this, further studies are required. Nevertheless, our finding demonstrates that routine measurement of 6-MAM when verifying opioid-positive immunologic screening results facilitates interpretation of low concentrations of M3G in urine specimens.     

Opiate recidivism in a drug-treatment program: comparison of hair and urine data

The objective of this preliminary study was to determine whether hair can be used as an adjunct specimen for the monitoring of opiate use in a drug-treatment program. Subjects (n = 10) initiating clinical therapy for opiate addiction were monitored for up to 17 weeks with hair and urinalysis. Questionnaires were administered weekly to document hair cuts and chemical treatments. Hair specimens were collected weekly by cutting at the scalp and segmented into 1-cm lengths prior to analysis. Codeine (COD), morphine (MOR), and 6-monoacetylmorphine (6-MAM) concentrations in hair were measured by liquid chromatography-mass spectrometry (LC-MS) [limit of detection (LOD): 20 pg/mg for COD and 6-MAM; 50 pg/mg MOR]. Urine specimens were analyzed by semiquantitative radioimmunoassay (25-ng/mL cutoff) and LC-MS for codeine (COD), morphine (MOR), morphine-3beta-glucuronide (M3G), morphine-6 beta-glucuronide (M6G), and 6-monoacetylmorphine (6-MAM). The LOD and limit of quantitation (LOQ) in urine for COD, M3G, M6G, and 6-MAM were 10 ng/mL and 25 ng/mL for MOR. Interpretation of the segmental hair data in this study was complex and generally was not in agreement with urine data in most cases. Evaluation of hair data suggested that 6 of 10 subjects discontinued opiate use by the end of the study, whereas 3 of 10 appeared to have reduced their use. One subject appeared not to have used opiates throughout the entire study. In contrast, evaluation of urine data suggested that only 4 of 10 subjects significantly reduced use, and 6 of 10 continued drug use on at least an intermittent basis. Urine appeared to be a more sensitive indicator of changes in the pattern(s) of drug use during the course of clinical drug treatment.     

Multiple aspects of hair analysis for opiates: methodology, clinical and workplace populations, codeine, and poppy seed ingestion

Levels of morphine, 6-monoacetylmorphine (MAM) and codeine in hair in both clinical and workplace subjects are presented. Aggressive wash procedures, consisting of 1 isopropanol wash, three 30-min, and two 1-h buffer washes, followed by digestion, extraction and confirmation of digested samples, resulted in values from the cutoff of 2 ng morphine/10 mg hair to greater than 200 ng/10 mg hair. Both morphine and MAM were present above the cutoff in all hair samples from 69 clinical subjects. Only 39 of the 69 heroin-using subjects had urine tests positive for 6-MAM. In a study of morphine in hair following poppy seed consumption, ten subjects ingested 150 g of poppy seed over 3 weeks. Urine samples were collected on the days of poppy seed ingestion and hair samples were taken in the 5th week of the study. The range among the 10 subjects of the highest urine value for each subject was 2929 to 13,827 ng morphine/mL. Hair morphine levels were 0.05-0.48 ng/10 mg hair (average 0.17 ng/10 mg hair). Hair opiate levels of workplace subjects ranged somewhat lower than those of clinical subjects. While all clinical hair samples contained MAM, many workplace samples did not. From workplace samples, a maximum amount of morphine likely to be present from codeine use was 0-3.7% of the codeine in the hair.     

Plasma codeine and morphine concentrations after a single oral dose of codeine phosphate

Plasma concentrations of codeine and its O-demethylated metabolite morphine were determined, by a sensitive and specific high performance liquid chromatography (HPLC) method, following a single oral dose of 60 mg codeine phosphate. Ten healthy volunteers received a single dose of 60 mg codeine phosphate. The plasma concentrations were analyzed for codeine and morphine at the 0.5, 1, 3, and 6 hours postdosing. The mean peak codeine plasma concentrations and tmax (time to reach maximum plasma codeine concentrations) were 88.1 ng/mL and 1.2 hours. Mean maximum concentrations of metabolically produced morphine was 2.7 +/- 0.6 ng/mL. The mean ratio of areas under the plasma concentration-time curves for morphine and codeine was 0.027. Thus, free morphine represented only about 2.7 +/- 1.8% of the free codeine area in each case.     

Morphine and codeine in oral fluid after controlled poppy seed administration

Opiates are an important drug class in drug testing programmes. Ingestion of poppy seeds containing morphine and codeine can yield positive opiate tests and mislead result interpretation in forensic and clinical settings. Multiple publications evaluated urine opiate concentrations following poppy seed ingestion, but only two addressed oral fluid (OF) results; neither provided the ingested morphine and codeine dosage. We administered two 45 g raw poppy seed doses, each containing 15.7 mg morphine and 3.1 mg codeine, 8 h apart to 17 healthy adults. All OF specimens were screened by on‐site OF immunoassay Draeger DrugTest 5000, and confirmed with OF collected with Oral‐Eze® device and quantified by liquid chromatography‐tandem mass spectrometry (1 µg/L morphine and codeine limits of quantification). Specimens (n = 459) were collected before and up to 32 h after the first dose. All specimens screened positive 0.5 h after dosing and remained positive for 0.5–13 h at Draeger 20 µg/L morphine cut‐off. Maximum OF morphine and codeine concentrations (C_max) were 177 and 32.6 µg/L, with times to C_max (T_max) of 0.5–1 h and 0.5–2.5 h post‐dose, respectively. Windows of detection after the second dose extended at least 24 h for morphine and to 18 h for codeine. After both doses, the last morphine positive OF result was 1 h with 40 µg/L 2004 proposed US Substance Abuse and Mental Health Services Administration cut‐off, and 0.5 h with 95 µg/L cut‐off, recently recommended by the Driving under the Influence of Drugs and Medicines project. Positive OF morphine results are possible 0.5–1 h after ingestion of 15.7 mg of morphine in raw poppy seeds, depending on the cut‐off employed. Copyright © 2014 John Wiley & Sons, Ltd.     

HUMAN ERYTHROCYTE BUT NOT BRAIN ACETYLCHOLINESTERASE HYDROLYSES HEROIN TO MORPHINE

1. In human blood, heroin is rapidly hydrolysed by sequential deacylation of two ester bonds to yield first 6-monoacetylmorphine (6-MAM), then morphine. 2. Serum butyrylcholinesterase (BuChE) hydrolyses heroin to 6-MAM with a catalytic efficiency of 4.5/min per mumol/L, but does not proceed to produce morphine. 3. In vitro, human erythrocyte acetylcholinesterase (AChE) hydrolyses heroin to 6-MAM, with a catalytic efficiency of 0.5/min per mumol/L under first-order kinetics. Moreover, erythrocyte AChE, but not BuChE is capable of further hydrolysing 6-MAM to morphine, albeit at a considerably slower rate. 4. Both hydrolysis steps by erythrocyte AChE were totally blocked by the selective AChE inhibitor BW284c51 but were not blocked by the BuChE-specific inhibitor, iso-OMPA (tetraisopropylpyrophosphoramide). 5. The brain synaptic form of AChE, which differs from the erythrocyte enzyme in its C-terminus, was incapable of hydrolysing heroin. 6. Heroin suppressed substrate hydrolysis by antibody-immobilized erythrocyte but not by brain AChE. 7. These findings reveal a new metabolic role for erythrocyte AChE, the biological function of which is as yet unexplained, and demonstrate distinct biochemical properties for the two AChE variants, which were previously considered catalytically indistinguishable.     

O-demethylation of codeine to morphine inhibited by low-dose levomepromazine

Purpose  Codeine/paracetamol (C/P) and levomepromazine (L) are frequently co-administered for the treatment of acute back pain, but the efficacy/effectiveness of this combination drug therapy has not been evaluated. The demethylation of codeine to morphine is catalyzed by the polymorphic enzyme cytochrome P450 2D6 (CYP2D6), of which levomepromazine (methotrimeprazine) is a known inhibitor. The aim of this study was to investigate whether low-dose levomepromazine inhibits the formation of morphine from codeine in a patient population of homozygous extensive (EM) and heterozygous extensive (HEM) metabolizers of CYP2D6. Methods  Our patient cohort consisted of 29 patients hospitalized for acute back pain who were randomized to a 24-h treatment with either C/P (60 mg codeine + 1000 mg paracetamol) four times daily or to L+C/P (levomepromazine 5 + 5 + 5 + 10 mg + C/P) four times daily. After zero-urine sampling (baseline), the treatment was started and urine collected for 24 h. Blood samples were later genotyped for the CYP2D6*3, *4, and *6 polymorphisms by the PCR (LightCycler system) and for the *5 polymorphism using long PCR, to identify EM and HEM and to eliminate CYP2D6 poor metabolizers. Urine samples were analyzed using the CEDIA immunoassay and gas chromatography–mass spectrometry after enzymatic hydrolysis of glucuronide conjugates. O-demethylation ratios of codeine were calculated as hydrolyzed (total) concentrations of morphine/morphine + codeine. Results  Twenty-two of the patients fulfilled the inclusion criteria, of whom ten were EM (five C/P and five L+C/P) and twelve were HEM (six C/P and six L+C/P) for functional CYP2D6 alleles. In the EM group, the median O-demethylation ratio was significantly higher (P = 0.016, Mann–Whitney test) after the C/P treatment (0.092, range 0.041–0.096) than after the L+C/P treatment (0.031, range 0.009–0.042). However, there was no significant difference between these two treatments in either the HEM group [n = 12; 0.024 (range 0.011–0.042) vs. 0.026 (range 0.009–0.041), respectively; P = 1.00] or in the combined EM/HEM group [11 C/P + 11 L+C/P; 0.041 (range 0.011–0.096) vs. 0.030 (range 0.009–0.042), respectively; P = 0.122]. Conclusions  Our study revealed significant inhibition in the O-demethylation of codeine to morphine in homozygous EM of CYP2D6 treated with low-dose levomepromazine and codeine/paracetamol, compared to treatment with codeine/paracetamol only. No significant difference could be detected in HEM or in the mixed and heterogenous group of EM/HEM. In patients prescribed this drug combination, the amount of morphine generated by the O-demethylation of codeine may be insufficient for effective pain relief. The therapeutic effect of codeine in the treatment of acute back pain should be assessed with and without levomepromazine.     

Preliminary evaluation of the Abbott TDx for benzoylecgonine and opiate screening in whole blood

A method for the detection of benzoylecgonine (cocaine metabolite) and opiates in whole blood is described. This method employs the Abbott TDx fluorescence polarization immunoassay technique, which was designed for urine analysis. Drug-free whole blood was spiked with varying concentrations of benzoylecgonine, morphine, and codeine. Samples were prepared for analysis by adding 300 microL of 10% trichloroacetic acid to 300 microL of blood. Specimens were vortexed and centrifuged with 50 microL of supernatant required per assay. Precision studies of six replicate samples spiked with benzoylecgonine at 0.5 mg/L gave a within-run CV of 4.7% and a between-run CV of 4.7% with a detection limit of 0.1 mg/L. Within-run CVs for morphine at 0.5 mg/L and 0.1 mg/L were 1.7% and 7.9% respectively. The detection limits for morphine and codeine were 0.05 mg/L. Correlation coefficients for spiked whole blood calibration curves of benzoylecgonine, morphine, and codeine were 0.984, 0.999, and 0.997 respectively. This preliminary evaluation demonstrates a potential application of the TDx fluorescence polarization immunoassay technique to the analysis of drugs in whole blood.     

Analgesic effect and plasma concentrations of codeine and morphine after two dose levels of codeine following oral surgery

Summary A double blind randomised cross over investigation was carried out in 25 male patients undergoing two oral surgical extractions, one for each lower wisdom tooth. The two extractions were performed about 6 weeks apart and were carried out under local anaesthesia. One hour after each extraction the patients randomly received 90 or 45 mg codeine. During the following 5 h the patients rated the intensity of their pain on a visual analogue scale. Blood was simultaneously sampled and assayed for codeine and its metabolite morphine.Mean pain intensity difference was just significantly higher after 90 mg codeine compared to 45 mg. The mean plasma concentrations of codeine and morphine were significantly higher after the 90 mg dose. However, for the two dose levels of codeine there was no obvious relationship between the difference in analgesic effect and the difference in the plasma concentration of codeine or morphine. The plasma concentrations of morphine were 2–3% of those of codeine and the levels were relatively low. Local formation of morphine from codeine within the human brain should therefore be investigated.Four patients were unable to demethylate codeine to a detectable plasma concentration of morphine after 90 mg codeine. In those patients the analgesic effect during the first hours was better after 90 mg codeine than after 45 mg.This suggests some analgesic effect of codeine itself.The work was presented in part at the Fourth World Congress on Pain, Adelaide, Australia, April 1–6, 1990     

Infants and young children metabolise codeine to morphine. A study after single and repeated rectal administration.

1. Codeine was administered rectally to thirteen infants and young children undergoing elective surgery. Nine infants (6-10 months old) received a 4 mg suppository and four children (3-4 years old) an 8 mg suppository. Codeine and its metabolite morphine were measured in plasma by GC/MS. 2. The mean concentrations of codeine at 3, 4 and 5 h after administration were 240, 163 and 123 nmol l-1 in the younger and 309, 251 and 169 nmol l-1 in the older patients. The corresponding concentrations of morphine were 8.3, 7.4 and 4.5 nmol l-1 and 6.8, 5.5 and 2.8 nmol l-1 respectively. One patient in each age group had no detectable amounts of morphine. 3. In the four children, the rectal dose was repeated 6-hourly for four doses. The plasma concentrations of codeine and morphine following the fifth dose were similar to those after the first dose. The mean AUC(0,5 h) of morphine was 1.6% that of codeine. 4. In the infants the mean plasma half-lives of codeine and morphine were 2.6 and 2.5 h. The two infants with the lowest body weights had the longest half-lives. 5. The mean morphine/codeine concentration ratio was 4.3% in the infants and 1.6% in the children, suggesting impaired glucuronidation of morphine in the former group. The hourly concentration ratios were almost identical following the first and fifth dose in the children. 6. We conclude that at the age of 6 months infants are capable of O-demethylating codeine to morphine.     

Testing human hair for drugs of abuse. III. Identification of heroin and 6-acetylmorphine as indicators of heroin use

Hair samples from 20 documented heroin users contained 6-acetylmorphine, a unique metabolite of heroin, in all samples. Heroin was identified in smaller amounts in seven of these samples. The identity of 6-acetylmorphine and heroin was established by comparison of full scan spectra of extracts to standard reference materials. The presence of 6-acetylmorphine generally predominated over heroin, morphine, and codeine. The mean concentrations of analytes were as follows: 6-acetylmorphine, 0.90 ng/mg, N = 20; heroin, 0.17 ng/mg, N = 7; morphine, 0.26 ng/mg, N = 20; codeine, 0.18 ng/mg, N = 15. Analysis of hair samples obtained from 10 drug-free control subjects were negative for 6-acetylmorphine, morphine, and codeine. However, a small interfering peak was observed at the retention time for heroin. Control samples soaked in aqueous solutions of heroin and 6-acetylmorphine were found to be contaminated, even though an initial wash step was included in the analysis. These data suggest that hair analysis for 6-acetylmorphine can be used to differentiate heroin users from other types of opiate exposure (e.g., poppy seed, licit morphine, and codeine); however, environmental contamination can potentially produce false positives during opiate testing.     

Lack of effect of paracetamol on the pharmacokinetics and metabolism of codeine in man

Summary Plasma and urine concentrations of codeine and its measurable metabolites were determined by HPLC in six healthy subjects after a single 30 mg oral dose of codeine either alone or after 7 doses of 1 g paracetamol 8 hourly.After codeine alone, the t_1/2 (h), AUC (mol·l^–1·h) and CL_R (ml·min^–1) for codeine were 2.2, 0.81, and 252 respectively. These were not significantly altered by paracetamol: 2.2, 0.84, and 291 respectively.For codeine-6-glucuronide the values were 2.4, 22.0, and 29.7 respectively. These were not significantly different from those after codeine plus paracetamol: 2.4, 21.9, and 39.6. There were no significant differences between the two treatments in the apparent partial clearances (ml·min^–1) of codeine to morphine (88 codeine alone, 70 codeine plus paracetamol), to norcodeine (71 codeine alone, 88 codeine plus paracetamol), and to codeine-6-glucoronide (820 codeine alone, 1022 codeine plus paracetamol).The urinary excretion of codeine-6-glucuronide, morphine, norcodeine, and codeine were not significantly different between the two treatments.     

Fingerprint analysis of illicit heroin samples by gas chromatography

For the fingerprint analysis of illicit heroin samples a gas Chromatographic assay has been developed capable of detecting the main components: acetylcodeine, caffeine, codeine, diamorphine, 6-monoacetylmorphine (6-Mam), morphine and quinine, in one run within 25 min. Diamorphine, morphine, codeine and caffeine can be quantitated directly, but as 6-Mam and acetylcodeine are not fully separated quantitation of the latter two requires acetylation of 6-Mam to diamorphine.25 Samples obtained between 1976 and 1979 have been compared with results of samples found in The Netherlands before 1977.     

Acute effect of glipizide on glucose tolerance in obesity and diabetes mellitus (NIDDM)

Summary The polymorphic cytochrome P-450 DB1 (P-450 IID6) is responsible for the O-demethylation of codeine to morphine by human liver microsomes. The influence of P-450 DB1 variable activity on the bioactivation of codeine in vivo to morphine and on its analgesic effect was investigated in phenotyped healthy volunteers — 7 extensive [EM] and 1 poor [PM] metabolizer of debrisoquine. After pretreatment with oral placebo or quinidine sulphate 50 mg, codeine phosphate 100 mg or placebo were administered orally according to a double-blind randomized crossover design.In EM subjects the plasma morphine C_max was 17.9 nmol/l, whereas virtually no morphine was detectable after quinidine pretreatment (1.5 nmol/l), and in the PM subject (0.60 nmol/l). In EM codeine significantly increased subjective (VAS) and objective (R-III reflex) pain thresholds in response to selective transcutaneous nerve stimulation, whereas no significant analgesia was detected after placebo, or after codeine with quinidine pretreatment, or in the PM. In PM of genetic origin, or due to environmental alteration of the phenotypic expression (i.e. drug interaction), codeine is not activated into morphine and is an inefficient analgesic.Presented in part at the 2nd International Congress on Cancer Pain, New York, N.Y., July 1988, and at the Nineteenth Annual Meeting of the American Society For Clinical Pharmacology and Therapeutics, Nashville, Tennessee, March 1989     

Pharmacokinetics of codeine after single- and multiple-oral-dose administration to normal volunteers

The pharmacokinetics of codeine, codeine glucuronide, morphine, and morphine glucuronide were assessed after single- (60 mg) and multiple-dose (60 mg every six hours for nine doses) oral administration of codeine sulfate to six normal volunteers. Multiple blood and urine samples were collected after administration of the single- and last multiple-oral doses. Drug concentrations were analyzed using radioimmunoassay techniques. No significant alterations in codeine pharmacokinetics were noted after multiple-dose oral administration. However, accumulation of morphine during multiple dosing was significant (AUC24 = 102 +/- 33 ng/mL/hr after single dose versus 212 +/- 118 ng/mL/hr after the last multiple dose). Peak concentration and AUC24 data for morphine glucuronide indicated that significant accumulation of this compound occurs upon multiple-dose administration. These data indicate that morphine and morphine glucuronide serum concentrations are significantly increased during chronic oral codeine therapy and suggest that morphine, and perhaps morphine glucuronide, contribute significantly to the analgesic activity of chronic oral codeine therapy.