Plant Extract and Herbal Products as Potential Source of Sorbent for Analytical Purpose: An Experimental Study of Morphine and Codeine Determination Using HPLC and LC-MSMS.

Solid-phase microextraction (SPME) is an analytical method for microextraction of analytes, in which the analytes bind to the sorbent on the surface of the SPME fiber. Many types of chemical agents are used as sorbent; however, many of these sorbents cause secondary contamination or are not cost-effective. Here, aqueous extract of Ferula gummosa was evaluated as potential source of sorbent for simultaneous microextraction of morphine and codeine. For this purpose, multiwalled carbon nanotubes were carboxylated with H2SO4/HNO3 (3:1) and then functionalized with aqueous extract of F. gummosa. Functionalization was confirmed by Fourier transform infrared and Raman spectroscopy measurements as well as scanning electron microscopy analysis. Porous polypropylene hollow fibers were filled with the functionalized carbon nanotubes (CNTs) and used for analyte extraction in urine sample at 40°C and pH 6 for 2 min. Reversed-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the fiber could preconcentrate 1 ng/mL of morphine and 0.75 ng/mL codeine in urine sample and was successfully used for 30 times with no significant loss in the extraction efficiency. Limit of detection (LOD) and limit of quantification (LOQ) for morphine were 1 and 3.3 ng/mL, respectively. LOD and LOQ for codeine were determined 0.75 and 2.47 ng/mL, respectively. Recovery of the fiber was 80% and 93% for morphine and codeine, respectively. SPME fiber using extract of F. gummosa plant was used for the detection of a small amount of morphine in urine sample. Therefore, plants can be considered as abundant and cheap sources of sorbent for various analytical purposes.

[Simultaneous determination of four opioids in urine by solid-phase extraction and derivatization coupled with gas chromatography-mass spectrometry].

Police officers currently use the colloidal gold rapid testing method to detect heroin in the urine of drug abusers, but the results are often rendered erroneous due to the presence of antitussive drugs, which contain opioids. The traditional manual liquid-liquid extraction method for urine testing has low efficiency and poor sensitivity, and hence, it fails to meet the requirements of the public security department to crack down on drug abusers. Therefore, to avoid punishment, most rapid-test-positive people make false claims about intaking cough suppressants. It is imperative to establish a highly efficient automatic method for the simultaneous determination of multiple opioids in urine, to rule out the use of heroin. A method based on solid-phase extraction and derivatization coupled with gas chromatography-mass spectrometry (GC-MS) has been developed for the simultaneous detection of morphine, O6-acetylmorphine, codeine, and acetyl codeine in urine. Since these four opioids exists as cations in acidic aqueous solution, the urine samples collected from dead bodies or drug addicts were adjusted to pH 6 by using phosphate buffer, enriched, and purified by MCX-SPE columns. Then, morphine, O6-acetylmorphine, and codeine were derivatized by N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) for GC-MS testing. The effects of sample loading and elution flow rate, percentage of formic acid in the wash solvent (methanol), percentage of ammonia in the eluent (methanol), volume of the wash solvent, and drying time of the cartridge on the extraction efficiency were investigated in detail. The best results were obtained under the following conditions:sample loading and elution flow rate, 1.0 mL/min; volume fraction of formic acid in the wash solvent, 3%; volume fraction of ammonia in the eluent solvent, 5%; volume of 3% (v/v) formic acid in methanol (eluent), 1 mL; and drying time of the cartridge, 1 min. The GC-MS results showed good linearity in the range of 0.02-0.8 µg/mL with correlation coefficients (r2) ≥ 0.998. The limits of detection (LODs) and limits of quantification (LOQs) were 0.0016-0.0039 µg/mL and 0.0054-0.0128 µg/mL, respectively. The recoveries of the target analytes were between 93.0% and 110.3% at spiked levels of 0.02, 0.1, and 0.2 µg/mL. As opposed to similar reported methods, our method showed high sensitivity and recovery; furthermore, the matrix interference was eliminated, and the chromatographic peaks of the analytes were completely separated from the impurity peaks at the level of 0.2 µg/mL. The automatic solid-phase extraction equipment is convenient to operate and allows one to process samples in batches. The conditions for solid-phase extraction can be precisely controlled, and the detection accuracy is greatly improved. In addition, a large number of sample tests can be performed by a few experimenters. Hence, this method facilitates simple and rapid forensic toxicology testing and drug abuse monitoring on a large scale.

An integrated microfluidic device for solid-phase extraction and spectrophotometric detection of opium alkaloids in urine samples.

A novel lab-on-chip integrated microfluidic device for solid-phase extraction (SPE) and spectrophotometric detection of morphine (MOR), codeine (COD), and papaverine (PAP) was developed. The extracted analytes were analyzed with a miniature UV-Vis spectrophotometer. The SPE adsorptive phase composed of polyurethane/polyaniline (PU/PANI) nanofibers was fabricated by electrospinning and in situ oxidative polymerization techniques. The sorbent was characterized by Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). The main factors of extraction such as desorption conditions, pH, salt effect, and extraction time were investigated. The partial least square (PLS) regression was applied to improve the quantification of analytes. The linear dynamic ranges (LDRs) for MOR, COD, and PAP were 4-240, 4-210, and 1-150 ng mL-1, respectively. Finally, the proposed method was successfully applied for the determination of MOR, COD, and PAP in human urine samples and the extraction recoveries were obtained in the range of 66.7-85.0% with RSDs < 8.3%.

Correlation between interleukin-6 levels and methadone maintenance therapy outcomes.

BACKGROUND: The outcome of methadone maintenance therapy (MMT) varies in each patient with opioid use disorder (OUD). Opioid abuse activates proinflammatory processes by increasing cytokine production and impairing neurotrophic factor expression, and possibly leads to a vicious cycle that hinders recovery. Therefore, we investigated whether markers of inflammation and neurotrophic expression correlate with the MMT outcomes in OUD patients. METHOD: We investigated OUD patients undergoing MMT and followed them up for 12 weeks. We measured plasma tumor necrosis factor (TNF)-α, C-reactive protein (CRP), interleukin (IL)-6, IL-1ß, transforming growth factor (TGF)-ß1, brain-derived neurotrophic factor (BDNF), urinary morphine tests, and plasma morphine levels at baseline and on weeks 1, 4, 8, and 12 during MMT. Multiple linear regressions and generalized estimating equations (GEEs) were used to examine the correlation between the cytokine and BDNF levels and MMT outcomes. RESULTS: We initially enrolled 104 patients, but only 78 patients completed end-of-study assessments. Plasma levels of CRP, TGF-ß1, and BDNF fell during MMT. Plasma IL-6 levels were significantly associated with plasma morphine levels (P = 0.005) and urinary morphine-positive (+) results (P = 0.04), and significantly associated with poor compliance (P = 0.009) and early dropout from MMT (P = 0.001). However, other cytokine and BDNF levels were not consistently associated with MMT outcomes. CONCLUSION: Higher IL-6 levels were associated with poor MMT outcomes. Additional studies on regulating IL-6 expression to improve treatment outcomes in OUD patients might be warranted.

Stability studies in biological fluids during post-analysis custody. Opiate compounds derived from heroin consumption.

In Forensic Toxicology, the evidences have to be maintained under custody for, at least, one year. Depending on the conditions and duration of storage, drug concentrations might have changed considerably since the first analysis. The aim of this study is to evaluate in vitro stability of opiate compounds, derived from heroin consumption, 6-acetylmorphine (6-MAM), morphine (MOR) and codeine (COD), in blood and urine, during post-analysis custody. Parameters evaluated were: time of custody, temperature, addition of preservative (blood) and pH (urine). Blood and urine samples were spiked with the three analytes to give a final concentration of 1000 ng/mL. The prepared samples were divided into 2 groups and stored at two temperatures (4 °C and -20 °C). Each one of these groups was subsequently divided in other two groups: with and without preservative (1%NaF) for blood, and pH 4 and 8 in the case of urine. 6-MAM, MOR and COD were analyzed by GCMS after SPE and derivatization with BSTFA. Analyses were performed in triplicate every two weeks for a year. In blood samples 6-MAM is the only compound that degrades. The best storage conditions were at -20 °C with NaF, with 6-MAM recoveries, after one year of custody, of 47.1 ± 1.5%; while in the other conditions 6-MAM disappeared after 215 days (at 4 °C with NaF), 45 days (at -20 °C without NaF) and 15 days (at 4 °C without preservative). COD does not degrade, with recoveries higher than 90%, in all of the conditions. They ranged from 89.7 ± 3.6% in samples maintained at -20 °C without NaF to 95.9 ± 2.0% in those maintained at 4 °C with NaF. MOR recoveries were lower than those of COD. They ranged from 66.9 ± 3.6%, in frozen samples added with NaF, to 78.6 ± 0.5% in refrigerated samples without preservative. In urine samples the three compounds were stable in all the studied conditions, with the exception of 6-MAM in samples at pH 8 and stored at 4 °C. In these conditions, 6-MAM disappeared after 135 days of custody; while recoveries in the other conditions ranged from 93.7 ± 6.4%, at 4 °C and pH 4, to 85.1 ± 2.0% at -20 °C and pH 8. MOR and COD recoveries were similar in the four conditions. In the case of MOR, they ranged from 82.1 ± 1.2% at 4 °C and pH 4 to 89.5 ± 6.0% at -20 °C and pH 8. As far as COD is concerned, recoveries ranged from 111.6 ± 5.8% at 4 °C and pH 8 to 102.6 ± 1.2% at 4 °C and pH 4. In conclusion, the study showed that the most labile opiate compound is 6-MAM. Its stability mainly depends on urine pH or the addition of preservative, in blood samples. The best storage conditions for samples from heroin consumers are in the freezer, at -20 °C. In addition, blood samples must be added with 1%NaF and urine samples must be buffered at pH 4.

Potential application of amino acids in analytical toxicology.

The ability of extraction and preconcentration of small quantities of substances from biological samples is important in analytical sciences, particularly forensic medicine. In the present study, we evaluated the binding potential of amino acids to produce a new solid phase microextraction fiber based on carbon nanotube (CNTs) for extraction and preconcentration of small amount of morphine in urine sample. Raw CNTs were first carboxylated and then functionalized with 3 amino acids including glutamate, arginine, and cysteine. Functionalization was confirmed by FTIR analysis, Raman spectroscopy and SEM imaging. The functionalized CNTs were coated on polypropylene hollow fiber and used for preconcentration. The results of HPLC analysis in isocratic elution mode using acetonitrile-sodium acetate (10:90, v/v; pH 4; 0.01 M) as the mobile phase showed that amino acids are able to adsorb morphine and the prepared fiber could preconcentrate a very low concentration of morphine (0.25 ppb) in urine matrix. In addition, the fiber was successfully used for up to 30 times with no significant loss in the extraction efficiency. Lowest limit of detection (LOD) and limit of quantitation (LOQ) was 0.07 and 0.25, respectively. Also, the lowest and best recovery of the fiber was 87.8% and 139% at LOQ, which belonged to glutamate and arginine, respectively. The fibers based on amino acids can be used for the detection of a small amount of morphine in biological samples, which are not detectable by conventional methods. Simple mechanism of these fibers in preconcentrating morphine makes them a novel candidate for detection of other opiates and drugs of abuses in crime scene investigations and postmortem examinations several days after exposure.

Morphine Concentrations in Human Urine Following Poppy Seed Paste Consumption.

Papaver somniferum (opium poppy) is one of the world's oldest medicinal plants which are widely used for medicinal, nutritive and scientific purposes. Turkey is one of the major legal opium poppy producer countries in the world and the seed paste of the poppies is consumed in great deal, even more than 100g per meal. The main objective of this study is to investigate the influence of poppy seed paste ingestion on urine tests for opiates whether or not could lead to opiate positive urine test results. For this purpose, a variety of poppies were used and the morphine content of white, yellow and blue-black poppies were determined as 1.9, 4.0 and 2.6mg/kg, respectively. 100g of these seed pastes were consumed in the breakfast by ten healthy adults enrolled in the study over three days and urine samples were collected before and after the breakfast. Opiate screening analysis was carried out by enzyme immunoassay method and the results were evaluated by two different cut-off values (300 and 2000ng/mL). Morphine confirmation analysis was made by GC-MS system and the chromatographic method was validated in terms of selectivity, extraction efficiency, linearity (25-2000ng/ml), intra-assay precision, accuracy, limit of detection (LOD) and limit of quantitation (LOQ) (3 and 10ng/ml), carryover, matrix effect, dilution integrity and stability. According to cut-off value 300ng/ml, opiate concentrations were found positive up to 48h. For cut-off value 2000ng/mL; this time was up to 12h in collected urine samples after consumption of three different colored poppy seed pastes. In all urine samples, thebaine was detected while the heroin abuse metabolite 6-acetyl morphine (6-AM) was not. Urine drug testing legislation was revised on 2016 in Turkey and opiate screening cut-off values increased from 300 to 2000ng/mL. Overall results have shown that poppy seed paste as food consumption could lead to opiate positive urine test result even if increased cut off levels are used. It can also be deduced that thebaine can be taken as supportive biomarker for poppy seed paste consumption. Awareness of interpretation of urine test results and defining the procedures especially for forensic drug testing must be done in legal aspect to ensure justice for each individual (workplace, traffic, court etc.).

Comparison Between Urinalysis Results and Self-Reported Heroin Use Among Patients Undergoing Methadone Maintenance Treatment in China.

BACKGROUND: The validity and concordance of two main measures of drug use behavior, self-report and urinalysis, has long been discussed. More understanding is needed about the underlying factors associated with discordance between these two methods. OBJECTIVES: Describe the pattern and associated factors of discordance between self-reported heroin use and the urinalysis results of opiate use among methadone maintenance therapy (MMT) patients in China. METHODS: A total of 2,448 MMT patients from 68 clinics in five provinces of China participated in a survey, which collected information on demographics, drug use and MMT-related factors, depressive symptoms, and drug avoidance self-efficacy. The most recent urine morphine test result was obtained from medical records and compared with self-reported heroin use. Participants who had urinalysis within 14 days of the survey were included in the analysis. RESULTS: Among the 1,092 participants, 70 (6.4%) self-reported heroin use and 195 (17.9%) had positive urinalysis results. The over-reporters group had significantly higher education, and the under-reporters had significantly higher level of drug-avoidance self-efficacy and lower level of depressive symptoms. Among the participants who either self-reported heroin use or had positive urinalysis results, being young, having higher education, and having lower level of depressive symptoms were associated with discordance between self-reports and urinalysis results. CONCLUSION: The combination of both measures in assessing drug use behavior seems necessary. The validity of self-report should be considered differently based on demographic and psychosocial characteristics.

Impact of blended treatment literacy and psychoeducation on methadone maintenance treatment outcomes in Yunnan, China.

BACKGROUND: Outcomes of methadone maintenance treatment (MMT) in the management of opioid dependency can be impaired by poor adherence and retention, concomitant drug use, poor adjustment of methadone dosage, and low levels of awareness regarding methadone among drug users, among other factors. This study investigated the effects of intensive blended treatment literacy and psychoeducation on treatment compliance, methadone dose, and heroin use among MMT clients in China. METHODS: A total of 492 MMT clients who tested positive for urine morphine at least once during a 12-week intervention period preceding the study were recruited from 16 MMT clinics. Employing a client-centred approach, a blended treatment literacy and psychoeducation intervention was then implemented between March and June 2014, comprising (1) intensified methadone treatment literacy sessions; (2) participatory goal setting; (3) continuous adherence monitoring and support; and (4) engagement of both peers and doctors in delivering psychoeducation. Wilcoxon signed-rank test was used to compare urine morphine positive rates, daily methadone dosage, and the number of days that clients successfully accessed methadone before and during the intervention. RESULTS: During the intervention, urine morphine positive rates reduced to 27% from 49.3% previously; p < 0.001. In response to client needs, methadone dosages increased among 74% of participants, remained unchanged among 12.0%, and reduced among 13.4% during the intervention. In addition, the average daily methadone dose increased from 63.0 to 72.6 mg; p < 0.001, while the average number of days that clients successfully accessed methadone increased from 69.4 to 73.9 over a period of 12 weeks; p < 0.001. CONCLUSIONS: Blended treatment literacy and psychoeducation delivered by a combination of peers and doctors was associated with reduced heroin use, improved treatment adherence, and higher methadone doses among our sample of MMT clients.

Serum and urine concentrations of morphine and morphine metabolites in patients with advanced cancer receiving continuous intravenous morphine: an observational study.

BACKGROUND: The feasibility and clinical implication of drug monitoring of morphine, morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G) need further investigation. This study aimed to determine what predicts serum concentrations of morphine in cancer patients receiving continuously intravenous morphine, the relationships between serum concentration of morphine/its metabolites and urinary concentrations, and the relation between morphine concentrations and with clinical outcomes. METHODS: We collected serum and urine samples from 24 patients with advanced cancer undergoing continuously intravenous morphine therapy. Serum samples were obtained at day one. Spot urine samples were collected once daily on three consecutive days. Pain and adverse drug events were assessed using the Korean version of MD Anderson Symptom Inventory. RESULTS: A total of 96 samples (72 urine and 24 serum samples) were collected. Median dose of morphine was 82.0 mg/24 h. In a multivariate analysis, total daily morphine dose was the most significant predictors of both serum and urine concentration of morphine. Morphine, M6G, and M3G in serum and urine were statistical significantly correlated (correlation coefficient = 0.81, 0.44, 0.56; p values < 0.01, 0.03, 0.01, respectively). CONCLUSION: Spot urine concentrations of morphine and its metabolites were highly correlated to those of serum. Total dose of daily morphine was related to both serum and urine concentration of morphine and its metabolites.

Validation of a New Recombinant Antibody Fragment (rFab)-Based Homogeneous Enzyme Immunoassay for the Highly Specific Detection of 6-Acetylmorphine in Urine.

The detection of 6-acetylmorphine (6-AM) in urine by immunoassay methods is challenging due to its short half-life and its similarity in structure to many commonly abused opiates that are often present at very high concentrations in urine. Current 6-AM homogeneous enzyme immunoassays use lyophilized reagents because of the instability of 6-AM in water or lack of the required specificity due to high cross-reactivity with morphine. A new 6-AM rFab-based homogeneous enzyme immunoassay (HEIA) has been developed with highly improved specificity. Using a cutoff concentration of 10 ng/mL, morphine or morphine glucuronides did not produce a positive signal up to 300,000 or 1,000,000 ng/mL, respectively. Assay imprecision (n = 80) was less than 1.5% using four replicates per day for 20 days over the range 0-20 ng/mL. Cross-reactivity with structurally related or non-related compounds was assessed at concentrations up to 1,000,000 ng/mL. Interferences from endogenous compounds at ±25% cutoff were also performed at the concentrations ranging from 100,000 to 500,000 ng/mL. The effect of varied pH values on assay performance at ±25% cutoff was investigated; no false-positive or false-negative results were observed between pH 4 and -11. Based on the analysis of 149 authentic urine samples, the accuracy of the 6-AM HEIA compared with LC-MS-MS was 100%. These results demonstrated that rFab can be suitable for traditional HEIA with desired detection sensitivity and stability.

Illicit Heroin and Methamphetamine Use among Methadone Maintenance Treatment Patients in Dehong Prefecture of Yunnan Province, China.

OBJECTIVE: Methadone maintenance treatment (MMT) was introduced to China in 2004 to reduce the harm of injecting drug users (IDUs). However, little is known about continued drug use, especially methamphetamine (MAMP), among MMT patients. METHODS: A survey was conducted among patients attending five major MMT clinics in Dehong Prefecture in 2014 to investigate the heroin and MAMP use and their associated risk factors. Participants were administered with face-to-face interviews, and urine tests for morphine and MAMP. RESULTS: A total of 2,121 were eligible and participated in the study. Among them, 220 (10.4%) were only positive for morphine, 12.9% were only positive for MAMP, and 196 (9.2%) were positive for both morphine and MAMP. Compared with neither use of heroin nor MAMP during MMT, heroin use (not using MAMP) was associated with ethnicity, shorter duration of MMT, lower dose of methadone, and having had no more than two sex partners in the past year; MAMP use (not using heroin) was associated with ethnicity, longer duration of MMT, higher dose of methadone and being aged <30 years (vs. ≥50 years); use of both heroin and MAMP was associated with being Dai minority (vs. Han), a marital status of divorced or widowed, having used drugs for ≥10 years and shorter duration of MMT. CONCLUSION: These findings indicate the complexity in the treatment of heroin users and underscore the importance in prescribing appropriate methadone dosages in order to reduce both heroin and MAMP use.

Possible mechanism for inhibition of morphine formation from 6-acetylmorphine after intake of street heroin.

Heroin is de-acetylated in the body to morphine in two steps. The intermediate 6-acetylmorphine (6-AM) is formed rapidly and is considered important for the pharmacological effect of heroin. In urine drug testing, an atypical pattern of morphine and 6-AM is known to occur in low frequency. The aim of this study was to investigate this atypical pattern in more detail and to identify responsible substances for a possible inhibition of the conversion from 6-AM to morphine. Urine samples were selected from a routine flow of samples sent for drug testing. Out of 695 samples containing morphine and 6-acetylmorphine, 11.5% had the atypical pattern of a 6-AM to morphine ratio above 0.26 as derived from a bimodal frequency distribution. An in vitro study of the conversion of 6-acetylmorphine to morphine in human liver homogenates demonstrated that a number of known carboxylesterase inhibitors were able to inhibit the reaction mimicking the situation in vivo. Compound 3 (3,6-Dimethoxy-4-acetoxy-5-[2-(N-methylacetamido)ethyl]phenanthrene) a substance formed from thebaine during the production of heroin was found to be a strong inhibitor. Liquid chromatography-mass spectrometry was used to identify possible inhibitors present in vivo. This part of the investigation demonstrated that several components may contribute to the effect. It is concluded that inhibition of liver carboxylesterase activity is a possible mechanism causing the atypical pattern and that one candidate compound is the result of the heroin production process. An inhibition of 6-AM metabolism is likely to increase the pharmacological effect of heroin and may be related to a higher risk of lethal toxicity.

Analysis of codeine positivity in urine of pain management patients.

The opioids codeine and morphine have legitimate uses in managing chronic pain conditions, but they are frequently abused. Patients prescribed opioids submit urine samples for medication compliance monitoring, and the interpretation of the results is complex. The purpose of this study was to evaluate the percentage of codeine- and morphine-positive urine drug tests that result from morphine use only, with the positive codeine result arising from low levels of codeine present in pharmaceutical formulations of morphine. This study included 80 urine samples which tested positive for codeine and morphine after pre-analytical hydrolysis and analysis by gas chromatography-mass spectrometry. Quantitative results were correlated with patient prescription information and immunoassay results to classify patients into one of four categories: heroin users (50%), codeine users (34%), codeine and morphine users (5%), and morphine users (11%). The percentage of codeine-positive resulting from morphine use was higher than previous estimates. Urine from patients prescribed morphine only was found to contain codeine at <1% of the morphine concentration, a ratio that was also observed in patients who used heroin. Careful analysis of urine drug testing results, including assessing the ratio of codeine to morphine (C/M), can help providers determine if patients are compliant with their pain management regimens.

Sensitivity of an opiate immunoassay for detecting hydrocodone and hydromorphone in urine from a clinical population: analysis of subthreshold results.

Urine drug testing (UDT) is an emerging standard of care in the evaluation and treatment of chronic non-cancer pain patients with opioid analgesics. UDT may be used both to verify adherence with the opioid analgesic regimen and to monitor abstinence from non-prescribed or illicit controlled substances. In the former scenario, it is vital to determine whether the drug is present in the urine, even at low concentrations, because failure to detect the drug may lead to accusations of opioid abuse or diversion. Opiate immunoassays typically are developed to detect morphine and are most sensitive to morphine and codeine. Although many opiate immunoassays also detect hydrocodone (HC) and/or hydromorphone (HM), sensitivities for these analytes are often much lower, increasing the possibility of negative screening results when the drug is present in the urine. We selected 112 urine specimens from patients who had been prescribed HC or hydromorphone but were presumptive negative by the Roche Online DAT Opiate II™ urine drug screening assay, which is calibrated to 300 ng/mL morphine. Using a GC/MS confirmatory method with a detection limit of 50 ng/mL both for HC and for HM, one or both of these opiates were detected in 81 (72.3%) of the urine specimens. Examination of the raw data from these presumptive negative opiate screens revealed that, in many cases, the turbidity signal was greater than the signal obtained for the negative control, but less than the signal for the 300 ng/mL (morphine) threshold calibrator. A receiver operating characteristic curve generated for the reciprocal of the ratio of turbidity measurements in the patient specimens and negative (drug-free) controls, against the presence or absence of HC and/or HM by confirmatory analyses, produced an area under the curve of 0.910. We conclude that this opiate immunoassay has sufficient sensitivity to detect HC and/or HM in some urine specimens that screen presumptive negative for these commonly prescribed opiates at the established threshold.

Assessment of CYP450 genetic variability effect on methadone dose and tolerance.

AIM: Methadone dose is related to treatment success in individuals under methadone maintenance treatment (MMT). We constructed a gene matrix using previously identified genetic polymorphisms in CYP450 and determined their genetic influence on methadone dose or tolerance. MATERIALS & METHODS: The allelic combinations of CYP450 genetic variants (two from CYP2C19, four from CYP2B6 and five from CYP3A4) were analyzed in 366 MMT heroin dependent patients as possible determinants of methadone dose and tolerance using analysis of covariance. RESULTS: Methadone dose (p = 0.007) and tolerance (p = 0.06) were mainly influenced by CYP2C19 gene dose. Moreover, dominant influence of the CYP2C19 gene dose on methadone dose and tolerance was only found among MMT patients with negative urine morphine test results, but not among those with positive results. CONCLUSION: The findings suggest that CYP2C19 gene dose may serve as a potential indicator for assessing methadone dose and tolerance in MMT patients.

Comprehensive automation of the solid phase extraction gas chromatographic mass spectrometric analysis (SPE-GC/MS) of opioids, cocaine, and metabolites from serum and other matrices.

The analysis of opioids, cocaine, and metabolites from blood serum is a routine task in forensic laboratories. Commonly, the employed methods include many manual or partly automated steps like protein precipitation, dilution, solid phase extraction, evaporation, and derivatization preceding a gas chromatography (GC)/mass spectrometry (MS) or liquid chromatography (LC)/MS analysis. In this study, a comprehensively automated method was developed from a validated, partly automated routine method. This was possible by replicating method parameters on the automated system. Only marginal optimization of parameters was necessary. The automation relying on an x-y-z robot after manual protein precipitation includes the solid phase extraction, evaporation of the eluate, derivatization (silylation with N-methyl-N-trimethylsilyltrifluoroacetamide, MSTFA), and injection into a GC/MS. A quantitative analysis of almost 170 authentic serum samples and more than 50 authentic samples of other matrices like urine, different tissues, and heart blood on cocaine, benzoylecgonine, methadone, morphine, codeine, 6-monoacetylmorphine, dihydrocodeine, and 7-aminoflunitrazepam was conducted with both methods proving that the analytical results are equivalent even near the limits of quantification (low ng/ml range). To our best knowledge, this application is the first one reported in the literature employing this sample preparation system.

Metabolic profiling of urine and blood plasma in rat models of drug addiction on the basis of morphine, methamphetamine, and cocaine-induced conditioned place preference.

The metabolic profiles of urine and blood plasma in drug-addicted rat models based on morphine (MOR), methamphetamine (MA), and cocaine (COC)-induced conditioned place preference (CPP) were investigated. Rewarding effects induced by each drug were assessed by use of the CPP model. A mass spectrometry (MS)-based metabolomics approach was applied to urine and plasma of MOR, MA, and COC-addicted rats. In total, 57 metabolites in plasma and 70 metabolites in urine were identified by gas chromatography-MS. The metabolomics approach revealed that amounts of some metabolites, including tricarboxylic acid cycle intermediates, significantly changed in the urine of MOR-addicted rats. This result indicated that disruption of energy metabolism is deeply relevant to MOR addiction. In addition, 3-hydroxybutyric acid, L-tryptophan, cystine, and n-propylamine levels were significantly changed in the plasma of MOR-addicted rats. Lactose, spermidine, and stearic acid levels were significantly changed in the urine of MA-addicted rats. Threonine, cystine, and spermidine levels were significantly increased in the plasma of COC-addicted rats. In conclusion, differences in the metabolic profiles were suggestive of different biological states of MOR, MA, and COC addiction; these may be attributed to the different actions of the drugs on the brain reward circuitry and the resulting adaptation. In addition, the results showed possibility of predict the extent of MOR addiction by metabolic profiling. This is the first study to apply metabolomics to CPP models of drug addiction, and we demonstrated that metabolomics can be a multilateral approach to investigating the mechanism of drug addiction.