Liquid chromatographic/electrospray ionization tandem mass spectrometric analysis for the quantification of buprenorphine, norbuprenorphine, buprenorphine-3-beta-D-glucuronide and norbuprenorphine-3-beta-D-glucuronide in human plasma

A liquid chromatographic/electrospray ionization tandem mass spectrometric method for the quantification of the synthetic opiate buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine-3-beta-D-glucuronide (BUP-3-G) and norbuprenorphine-3-beta-D-glucuronide (NBUP-3-G) in human plasma was developed and validated. Identification and quantification were based on the following transitions: m/z 468 to 396 and 414 for BUP, m/z 414 to 326 and 340 for NBUP, m/z 644 to 468 for BUP-3-G and m/z 590 to 414 for NBUP-3-G. Calibration by linear regression analysis utilized deuteratated internal standards and a weighting factor of 1/x. The method was accurate and precise across a linear dynamic range of 0.6-50.0 ng ml(-1). Pretreatment of plasma samples using solid-phase extraction was sufficient to limit matrix suppression to <30% for all four analytes. The method proved to be suitable for the quantification of BUP and the related metabolites in plasma samples collected from BUP-maintained study participants.     

High-performance liquid chromatographic determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine in biological samples using multi-wavelength forward optical detection.

An isocratic high-performance liquid chromatographic method has been developed for the determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine in plasma, urine and cerebrospinal fluid. The use of an efficient solid-phase extraction procedure together with a forward optical scanning detector allows a detection limit of 500 pg/ml. The method was evaluated by examination of biological samples taken from newborn infants following the intravenous administration of morphine sulfate.     

Simultaneous determination of morphine and its glucuronides in rat hair and rat plasma by reversed-phase liquid chromatography with electrospray ionization mass spectrometry

The simultaneous determination of morphine and the glucuronide metabolites [morphine-3-beta-D-glucuronide (M3G) and morphine-6-beta-D-glucuronide (M6G)] in rat hair and rat plasma was carried out using reversed-phase high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS). The chromatographic separation of the analytes was achieved using a semi-micro-HPLC column (3 microm particle size; 100 x 2.0 mm id) by gradient elution with 50 mM ammonium acetate and acetonitrile as eluents. After separation, morphine and the glucuronides were determined by selected ion monitoring (SIM) of ESI-MS using the quasi-molecular ions [M + H]+ at m/z = 286 and 462, respectively. The calibration curves were linear between the concentration of the analytes and the deuterium-labelled morphine (M-d3) selected as internal standard. The method was applied for the determination of the incorporation of morphine and the glucuronides into the hair shafts and hair roots of Dark Agouti rats after single intraperitoneal administration of morphine hydrochloride. Plasma concentrations of morphine and glucuronides were simultaneously determined after administration. Morphine and M3G were detected in the hair shafts and the hair roots. The concentrations of M3G in the hair root were lower than those of morphine in all sampling periods. In contrast, M3G concentrations in plasma were relatively higher at each sampling time. Small quantities of M6G were also identified in the plasma up to 4 h after administration. The concentration difference between the hair root and plasma seems to be due to the incorporation ratio of morphine and glucuronide into hair. As M3G was also identified in the hair shaft 1 week after administration, the incorporation of glucuronide metabolites into hair is obvious. This is the first report of the identification of morphine glucuronide in hair samples without the use of acid hydrolysis or enzyme digestion.     

Certification of morphine-3-β-D-glucuronide in a human urine standard reference material

 The U.S. National Institute of Standards and Technology (NIST) has developed and certified a standard reference material, SRM 2382, for use in testing for bias in the determination of morphine present as a glucuronide in human urine. This SRM consists of three levels of morphine-3-β-D-glucuronide (M-3-G) in lyophilized urine. Two independent hydrolysis procedures, enzymatic and acidic, for quantitative release of morphine were investigated. The results of hydrolysis efficiency from these two procedures agreed well. These two hydrolysis procedures along with two analytical methods developed previously for measurement of free morphine [1] were used to certify M-3-G in SRM 2382. Enzymatic hydrolysis was used with the GC/MS method and acidic hydrolysis was used with LC/MS. The results from these two pairs of methods were in good agreement, and were statistically combined to yield certified values of 209±20, 437±21, and 853±39 ng/mL for morphine, as the free base. Round-robin studies on this material among ten military laboratories demonstrated the suitability of the SRM for its intended purpose. Received: 6 February 1996/Accepted: 29 April 1996     

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography for analysis of morphine and its four metabolites in human urine

A cation-selective exhaustive injection and sweeping micellar EKC (CSEI-Sweep-MEKC) was established to analyze morphine and its four metabolites, including codeine, normorphine (NM), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G). After SPE, the urine samples were analyzed by this CE method. The phosphate buffer (75 mM, pH 2.5) containing 30% methanol was first filled into an uncoated fused-silica capillary (40 cm, 50 microm id), then a high-conductivity buffer (120 mM phosphate, 10.3 kPa for 99.9 s) followed. The pretreated urine sample was loaded by electrokinetic injection (10 kV, 600 s). The stacking and separation were performed by using phosphate buffer (25 mM, pH 2.5) containing 22% methanol and 100 mM SDS at -20 kV, and detected at 200 nm. During method validation, calibration plots were linear (r > or = 0.998) over a range of 30-3000 ng/mL for morphine, NM, and codeine, 100-2000 ng/mL for M6G, and 80-3200 ng/mL for M3G. The LODs (S/N = 5, sampling 600 s at 10 kV) were 10 ng/mL for morphine, NM, and codeine, 35 ng/mL for M6G, and 25 ng/mL for M3G. This stacking CE method could increase 2500-fold sensitivity of codeine, when comparing with CZE. Five addicts' urine specimens were analyzed. Their results were compared with those of LC-MS-MS, and showed good coincidence. This method could be feasible for monitoring morphine and its metabolites in forensic interest and pharmacokinetic investigations.     

On-line desalting and determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in microdialysis and plasma samples using column switching and liquid chromatography/tandem mass spectrometry

A sensitive and reproducible method for the determination of morphine and the metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) was developed. The method was validated for perfusion fluid used in microdialysis as well as for sheep and human plasma. A C18 guard column was used to desalt the samples before analytical separation on a ZIC HILIC (hydrophilic interaction chromatography) column and detection with tandem mass spectrometry (MS/MS). The mobile phases were 0.05% trifluoroacetic acid (TFA) for desalting and acetonitrile/5 mM ammonium acetate (70:30) for separation. Microdialysis samples (5 microL) were directly injected onto the system. The lower limits of quantification (LLOQ) for morphine, M3G and M6G were 0.50, 0.22 and 0.55 ng/mL, respectively, and the method was linear from LLOQ to 200 ng/mL. For plasma, a volume of 100 microL was precipitated with acetonitrile containing internal standards (deuterated morphine and metabolites). The supernatant was evaporated and reconstituted in 0.05% TFA before the desalting process. The LLOQs for sheep plasma were 2.0 and 3.1 ng/mL and the ranges were 2.0-2000 and 3.1-3100 ng/mL for morphine and M3G, respectively. For human plasma, the LLOQs were 0.78, 1.49 and 0.53 ng/mL and the ranges were 0.78-500, 1.49-1000 and 0.53-500 ng/mL for morphine, M3G and M6G, respectively.     

Characteristics of the gastrointestinal absorption of morphine in rats

The absorption characteristics of morphine were investigated by using rat gastrointestine. Absorption and transport experiments were carried out by the in situ loop and the in vitro everted sac methods, respectively. Brush border membrane vesicles (BBMVs) were used for uptake experiments. Morphine and its metabolites, morphine-3-glucuronide (M-3-G), and morphine-6-glucuronide (M-6-G), in biological samples were simultaneously determined by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection and electrochemical detection. In the in situ loop method, morphine was well absorbed in the order of jejunal site greater than duodenal site greater than ileal site greater than middle intestinal site greater than rectal site, but it was poorly absorbed from the stomach. In each of the everted duodenal and jejunal sacs, 2,4-dinitrophenol, a metabolic inhibitor, inhibited the transport of morphine from the mucosal side to the serosal side. Further, HgCl2 pretreatment reduced the absorption of morphine from the duodenal and the jejunal loops. The initial uptake of morphine by BBMVs was stimulated in the presence of an H+ gradient (inner pH 7.5 and outer pH 5.0) and an overshoot phenomenon was observed. The initial uptake showed concentration dependence, i.e., it was saturable. Results obtained in this study indicate that carrier-mediated transport stimulated by the H+ gradient is partly involved in the duodeno-jejunal absorption of morphine, although morphine is passively absorbed from other sites.     

Validation of direct injection electrospray LC-MS/MS for confirmation of opiates in urine drug testing

A method based on the direct injection of diluted urine for the identification and quantification of morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, codeine-6-glucuronide, ethylmorphine, ethylmorphine-6-glucuronide and 6-acetylmorphine (6AM) in human urine by electrospray ionisation liquid chromatography-tandem mass spectrometry was validated for use as a confirmation procedure in urine drug testing. Four deuterium labelled analogues were used as internal standards: morphine-3-glucuronide-D3, morphine-D3, codeine-D3 and 6AM-D3. Twenty microlitre aliquots of urine were mixed with 80 mul of the internal standard solution in autosampler vials and 10 mul was injected. The chromatographic system consisted of a 2.0 x 100 mm C18 column and the gradient elution buffers used acetonitrile and 25 mmol/l formic acid. Two product ions produced from the protonated molecular ions were monitored in the selected reaction monitoring mode. The intra- and inter-assay variability (coefficient of variation) was below 10% at higher levels for all analytes, but at the reporting limits the variation was above 20% for 6AM, morphine-3-glucuronide and codeine-6-glucuronide. Ion suppression occurred early after injection but did not affect the identification and quantification of the analytes in authentic samples. The method was further validated by comparison with a reference gas chromatographic-mass spectrometric method using authentic urine samples. The two methods agreed almost completely (99%) regarding the identified analytes, but for the quantitative results there were slightly lower levels when measuring glucuronides directly as compared to total determination after hydrolysis by gas chromatography-mass spectrometry.We conclude that the presented liquid chromatographic-tandem mass spectrometric method is robust and reliable, and suitable for use as a confirmation method in urine drug testing for opiates     

Simultaneous determination of opiates, methadone, amphetamines, cocaine, and metabolites in human placenta and umbilical cord by LC-MS/MS

LC-MS/MS methods for the quantification of morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, 6-acetylmorphine, cocaine, benzoylecgonine, ecgonine methyl ester, hydroxybenzoylecgonine, cocaethylene, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), methadone, and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in human placenta and umbilical cord were developed and validated. Specimens (1?±?0.02 g) were homogenized with the Ultra-Turrax T8 disperser and centrifuged, and the supernatant was submitted to solid-phase extraction with Oasis MCX cartridges. Chromatographic separation was performed using an Atlantis T3 analytical column (100?×?2.1 mm, 3 μm) and a gradient of 0.1 % formic acid and acetonitrile. Selectivity was verified in 10 different blank specimens. The method was linear from 1–5 to 100–500 ng/g, depending on the analyte. Limits of detection and quantification ranged from 0.5 to 2.5 ng/g and 1 to 5 ng/g, respectively. Method imprecision was ≤15.3 %, except for MDMA at low quality control (18.1 %); accuracy, 87.1 to 114 %; extraction efficiency, 16.3 to 154.0 % (%CV?=?1.8-39.4 %); matrix effect, ?75.7 to 449.9 % (%CV?=?3.5–50 %); and process efficiency, 8.7 to 316.0 %. The method was applied to authentic placenta and umbilical cord specimens from drug-user pregnant women.     

Method for quantification of morphine and its 3- and 6- glucuronides, codeine, codeine glucuronide and 6-monoacetylmorphine in human blood by liquid chromatography-electrospray mass spectrometry for routine analysis in forensic toxicology.

Simultaneous determination of opiates and their glucuronides in body fluids has a great practical interest in the forensic assessment of heroin intoxication. A selective and sensitive method for quantification of morphine and its 3- and 6-glucuronides, codeine, codeine glucuronide and 6-monoacetylmorphine (6-MAM) based on liquid chromatography-electrospray ionisation mass spectrometry is described. The drugs were analysed in human autopsy whole blood after solid-phase extraction on a C8 cartridge. The separation was performed on an ODS column in acetonitrile (analysis time 15 min). For the quantitative analysis, deuterated analogues of each compound were used as internal standards. Selected-ion monitoring was applied where the molecular ion was chosen for quantification. The limits of quantification were 0.5 ng/ml for morphine and 6-MAM and 1 ng/ml for the 6-glucuronide of morphine, codeine-6-glucuronide and codeine and 5 ng/ml for the 3-glucuronide of morphine.     

Synthesis and bioanalytical evaluation of morphine-3-O-sulfate and morphine-6-O-sulfate in human urine and plasma using LC-MS/MS

The aim of this work was to synthesize morphine‐3‐O‐sulfate and morphine‐6‐O‐sulfate for use as reference substances, and to determine the sulfate conjugates as possible heroin and morphine metabolites in plasma and urine by a validated LC‐MS/MS method. Morphine‐6‐O‐sulfate and morphine‐3‐O‐sulfate were prepared as dihydrates from morphine hydrochloride, in overall yields of 41 and 39% with product purities of >99.5% and >98%, respectively. For bioanalysis, the chromatographic system consisted of a reversed‐phase column and gradient elution. The tandem mass spectrometer was operated in the positive electrospray mode using selected reaction monitoring, of transition m/z 366.15 to 286.40. The measuring range was 5–500 ng/mL for morphine‐3‐O‐sulfate and 4.5–454 ng/mL for morphine‐6‐O‐sulfate in plasma. In urine, the measuring range was 50–5000 ng/mL for morphine‐3‐O‐sulfate and 45.4–4544 ng/mL for morphine‐6‐O‐sulfate. The intra‐assay and total imprecision (coefficient of variation) was below 11% for both analytes in urine and plasma. Quantifiable levels of morphine‐3‐O‐sulfate in authentic urine and plasma samples were found. Only one authentic urine sample contained a detectable level of morphine‐6‐O‐sulfate, while no detectable morphine‐6‐O‐sulfate was found in plasma samples.     

Rapid simultaneous determination of codeine and morphine in plasma using LC‐ESI‐MS/MS: Application to a clinical pharmacokinetic study

A rapid and sensitive high‐performance LC‐MS/MS method was developed and validated for the simultaneous quantification of codeine and its metabolite morphine in human plasma using donepezil as an internal standard (IS). Following a single liquid‐liquid extraction with ethyl acetate, the analytes were separated using an isocratic mobile phase on a C₁₈ column and analyzed by MS/MS in the selected reaction monitoring mode using the respective [M+H]⁺ ions, mass‐to‐charge ratio (m/z) 300/165 for codeine, m/z 286/165 for morphine and m/z 380/91 for IS. The method exhibited a linear dynamic range of 0.2–100/0.5–250 ng/mL for codeine/morphine in human plasma, respectively. The lower LOQs were 0.2 and 0.5 ng/mL for codeine and its metabolite morphine using 0.5 mL of human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated LC‐MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 30 mg codeine phosphate.     

Fast reliable assay for morphine and its metabolites using high-performance liquid chromatography and native fluorescence detection

A method for the fast analysis of morphine (M), normorphine (NM), morphine-3- and -6-glucuronides (M3G and M6G) and codeine (C) is described which has the advantages of sensitivity, speed and specificity. Dihydrocodeine and heroin can also be assayed. The method is based on extraction of the opiates from serum, plasma and cerebrospinal fluid using reversed-phase solid-phase extraction columns, followed by reversed-phase high-performance liquid chromatography with native fluorescence detection. The extraction step provides greater than 95% recovery, and the response of the detection system is linear from 0.5 to beyond 750 ng. The method allows analysis of M, NM, M3G, M6G and C. No other drugs have been found to interfere with the assay. The assay offers a quick, cheap and reliable method of specifically determining morphine and its metabolites, including the potent M6G, from a small sample volume; this will be of advantage to both clinician and basic scientist.     

High-throughput simultaneous analysis of buprenorphine, methadone, cocaine, opiates, nicotine, and metabolites in oral fluid by liquid chromatography tandem mass spectrometry

A method for simultaneous determination of buprenorphine (BUP), norbuprenorphine (NBUP), methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine, benzoylecgonine (BE), ecgonine methyl ester (EME), anhydroecgonine methyl ester (AEME), morphine, codeine, 6-acetylmorphine (6AM), heroin, 6-acetylcodeine (6AC), nicotine, cotinine, and trans-3′-hydroxycotinine (OH-cotinine) by liquid chromatography tandem mass spectrometry in oral fluid (OF) was developed and extensively validated. Acetonitrile (800 μL) and OF (250 μL) were added to a 96-well Isolute-PPT+protein precipitation plate. Reverse-phase separation was achieved in 16 min and quantification was performed by multiple reaction monitoring. The assay was linear from 0.5 or 1 to 500 μg/L. Intraday, interday, and total imprecision were less than 13% (n?=?20), analytical recovery was 92–114% (n?=?20), extraction efficiencies were more than 77% (n?=?5), and process efficiencies were more than 45% (n?=?5). Although ion suppression was detected for EME, cocaine, morphine, 6AC, and heroin (less than 56%) and enhancement was detected for BE and nicotine (less than 316%), deuterated internal standards compensated for these effects. The method was sensitive (limit of detection 0.2–0.8 μg/L) and specific (no interferences) except that 3-hydroxy-4-methoxyamphetamine interfered with AEME. No carryover was detected, and all analytes were stable for 24 h at 22 °C, for 72 h at 4 °C, and after three freeze–thaw cycles, except cocaine, 6AC, and heroin (22–97% loss). The method was applied to 41 OF specimens collected throughout pregnancy with a Salivette® OF collection device from an opioid-dependent BUP-maintained pregnant woman. BUP ranged from 0 to 7,400 μg/L, NBUP from 0 to 71 μg/L, methadone from 0 to 3 μg/L, nicotine from 32 to 5,020 μg/L, cotinine from 125 to 508 μg/L, OH-cotinine from 11 to 51 μg/L, cocaine from 0 to 419 μg/L, BE from 0 to 351 μg/L, EME from 0 to 286 μg/L, AEME from 0 to 7 μg/L, morphine from 0 to 22 μg/L, codeine from 0 to 1 μg/L, 6AM from 0 to 4 μg/L, and heroin from 0 to 2 μg/L. All specimens tested negative for EDDP and 6AC. This method permits a fast and simultaneous quantification of 16 drugs and metabolites in OF, with good selectivity and sensitivity.     

Isolation and identification of metabolites from dihydromyricetin

Dihydromyricetin (DHM) is the major bioactive constituent of Rattan Tea, which is the tender stems and leaves of Ampelopsis grossendentata. Seven metabolites (2-8) of DHM (1) were obtained by the chromatographic method. The metabolites 2-5 were obtained from the urine of rats administered orally with DHM; and metabolites 6-8 were detected in the fecal specimens of rats, which were also produced by human intestinal bacteria (HIB) in vitro, and were separated from the cultured media of HIB containing DHM. Their structures were elucidated as 5,7,3',5'-tetrahydroxyflavanonol (2); 5,7,3',5'-tetrahydroxy-4'-methoxyflavanonol (3); 5,7,4',5'-tetrahydroxy-3'-methoxyflavanonol (4); and dihydromyricetin-O-5-beta-D-glucuronide (5); (2R,3S)-5,7,3',4',5'-pentahydroxyflavanonol (6); 3,4,5,7,3',4',5'-hepthydroxyflavan (7) and 5,7,3',4',5'-pentahydroxyflavanone (8) on the basis of UV, NMR and LC-MS/MS data. These seven metabolites were formed through familiar metabolic reactions. Dihydromyricetin-O-5-beta-D-glucuronide (5) is a new compound. The (13)C-NMR data of (2) and (4) are reported for the first time.     

Simultaneous detection and quantitation of O6-monoacetylmorphine, morphine and codeine in urine by gas chromatography with nitrogen specific and/or flame ionization detection

It is of importance to differentiate heroin intake from the absorption of opiate-containing pharmaceuticals or opiates from other sources. A method for the routine determination of O6-monoacetylmorphine (6-MAM), the specific metabolite of heroin in human urine, by gas chromatography and classical detectors without having recourse to gas chromatography/mass spectrometry-selected ion mode (GC/MS-SIM) is described. With dual detection by nitrogen selective and flame ionization detectors, the limits of detection for 6-MAM were determined to be 2 ng/mL and 4 ng/mL urine for a 10 mL sample. When applied to urines preliminarily screened for opiates, the results appeared consistent in comparison with those obtained by GC/MS-SIM. The method was also developed for the simultaneous quantitative analysis of morphine and codeine. The linearity was tested up to 600 ng/mL for the three compounds of interest 6-MAM, morphine and codeine and their absolute recoveries were 76%, 78%, 75% respectively.     

Direct tandem mass spectrometry for the simultaneous assay of opioids,cocaine and metabolites in dried urine spots

A micro-analytical method based on spotting urine samples (20 μL) onto blood/urine spot collection cards followed by air-drying and extraction (dried urine spot, DUS) was developed and validated for the screening/confirmation assay of morphine, 6-methylacetylmorphine (6-MAM), codeine, cocaine and benzoylecgonine (BZE). Acetonitrile (3 mL) was found to be a useful solvent for target extraction from DUSs under an orbital-horizontal stirring at 180 rpm for 10 min. Determinations were performed by direct electrospray ionization tandem mass spectrometry (ESI-MS/MS) under positive electrospray ionization conditions, and by using multiple reaction monitoring (MRM) with one precursor ion/product ion transition for the identification and quantification (deuterated analogs of each target as internal standards) of each analyte. The limits of detection of the method were 0.26, 0.94, 1.5, 1.1, and 2.0 ng mL⁻¹, for cocaine, BZE, codeine, morphine and 6-MAM, respectively; whereas, relative standard deviations of intra- and inter-day precision were lower than 8 and 11%, respectively, and intra- and inter-day analytical recoveries ranged from 94 ± 4 to 105 ± 3%. The small volume of urine required (20 μL), combined with the simplicity of the analytical technique makes it a useful procedure for screening/quantifying drugs of abuse. The method was successfully applied to the analysis of urine from polydrug abusers.     

Direct determination of codeine, norcodeine, morphine and normorphine with their corresponding O-glucuronide conjugates by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method has been developed for the detection, separation and measurement of codeine and its metabolites norcodeine, morphine and normorphine, with their glucuronide conjugates. The glucuronidase Escherichia coli type VIIA hydrolyses codeine-6-glucuronide completely and is used for the construction of the calibration curves of codeine-6-glucuronide. Enzymic hydrolysis of codeine-6-glucuronide depends on the specific activity of the glucuronidase applied. Examples are shown of a volunteer who is able to form morphine from codeine and one who is unable to do so.     

A sensitive solid-phase fluoroimmunoassay for detection of opiates in urine

An automated flow fluorometer designed for kinetic binding analysis was adapted to develop a solid-phase competitive fluoroimmunoassay for urinalysis of opiates. The solid phase consisted of polymer beads coated with commercial monoclonal antibodies (MAbs) raised against morphine. Fluorescein-conjugated morphine (FL-MOR) was used as the fluorescein-labeled hapten. The dissociation equilibrium constant (K _D ) for the binding of FL-MOR to the anti-MOR MAb was 0.23 nM. The binding of FL-MOR to the anti-MOR MAb reached steady state within minutes and was displaced effectively by morphine and other opiates. Morphine-3-glucuronide (M3G), the major urinary metabolite of heroin and morphine, competed effectively with FL-MOR in a concentration-dependent manner for binding to the antimorphine MAb and was therefore used to construct the calibration curve. The sensitivity of the assay was 0.2 ng/mL for M3G. The assay was effective at concentrations of M3G from 0.2 to 50 ng/mL, with an IC₅₀ of 2 ng/mL. Other opiates and heroin metabolites that showed >50% crossreactivity when present at 1 μg/mL included codeine, morphine-6-glucuronide, and oxycodone. Methadone showed very low crossreactivity (<5%), which is a benefit for testing in patients being treated for opiate addictions. The high sensitivity of the assay and the relatively high cutoff value for positive opiate tests allows very small sample volumes (e.g., in saliva or sweat) to be analyzed. A double-blind comparison using 205 clinical urine samples showed good agreement between this single-step competitive assay and a commercially performed enzyme multiplied immunoassay technique for the detection of opiates and benzoylecgonine (a metabolite of cocaine).     

A validated positive chemical ionization GC/MS method for the identification and quantification of amphetamine, opiates, cocaine, and metabolites in human postmortem brain

A sensitive and specific method for the simultaneous detection and quantification of amphetamine, opiates, and cocaine and metabolites in human postmortem brain was developed and validated. Analytes of interest included amphetamine, morphine, codeine, 6-acetylmorphine, cocaine, benzoylecgonine, ecgonine methyl ester, ecgonine ethyl ester, cocaethylene, and anhydroecgonine methyl ester. The method employed ultrasonic homogenization of brain tissue in pH 4.0 sodium acetate buffer and solid phase extraction. Extracts were derivatized with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide and N,O-bis(trimethylsilyl) trifluoroacetamide. Separation and quantification were accomplished on a bench-top positive chemical ionization capillary gas chromatograph/mass spectrometer with selected ion monitoring. Eight deuterated analogs were used as internal standards. Limits of quantification were 50 ng/g of brain. Calibration curves were linear to 1000 ng/g for anhydroecgonine methyl ester and 6-acetylmorphine, and to 2000 ng/g for all other analytes. Accuracy across the linear range of the assay ranged from 90.2 to 112.2%, and precision, as percent relative standard deviation, was less than 16.6%. Quantification of drug concentrations in brain is a useful research tool in neurobiology and in forensic and postmortem toxicology, identifying the type, relative magnitude, and recency of abused drug exposure. This method will be employed to quantify drug concentrations in human postmortem brain in support of basic and clinical research on the physiologic, biochemical, and behavioral effects of drugs in humans.