Tackling new psychoactive substances through metabolomics: UHPLC-HRMS study on natural and synthetic opioids in male and female murine models.

Novel psychoactive substances (NPS) represent a broad class of drugs new to the illicit market that often allow passing drug-screening tests. They are characterized by a variety of structures, rapid transience on the drug scene and mostly unknown metabolic profiles, thus creating an ever-changing scenario with evolving analytical targets. The present study aims at developing an indirect screening strategy for NPS monitoring, and specifically for new synthetic opioids (NSOs), based on assessing changes in endogenous urinary metabolite levels as a consequence of the systemic response following their intake. The experimental design involved in-vivo mice models: 16 animals of both sex received a single administration of morphine or fentanyl. Urine was collected before and after administration at different time points; the samples were then analysed with an untargeted metabolomics LC-HRMS workflow. According to our results, the intake of opioids resulted in an elevated energy demand, that was more pronounced on male animals, as evidenced by the increase in medium and long chain acylcarnitines levels. It was also shown that opioid administration disrupted the pathways related to catecholamines biosynthesis. The observed alterations were common to both morphine and fentanyl: this evidence indicate that they are not related to the chemical structure of the drug, but rather on the drug class. The proposed strategy may reinforce existing NPS screening approaches, by identifying indirect markers of drug assumption.

Unveiling Morphine: A Rapid and Selective Fluorescence Sensor for Forensic and Medical Analysis.

Opioid use, particularly morphine, is linked to CNS-related disorders, comorbidities, and premature death. Morphine, a widely abused opioid, poses a significant global health threat and serves as a key metabolite in various opioids. Here, we present a turn-off fluorescent sensor capable of detecting morphine with exceptional sensitivity and speed in various samples. The fluorescent sensor was developed through the dimerization process of 7-methoxy-1-tetralone and subsequent demethylation to produce the final product. Despite morphine possessing inherent fluorophoric properties and emitting light in an approximately similar wavelength as the sensor's fluorescent blue light, the introduction of the target molecule (morphine) in the presence of the sensor caused a reduction in the sensor's fluorescence intensity, which is attributable to the formation of the sensor-morphine complex. By utilizing this fluorescence quenching sensor, the chemo-selective detection of morphine becomes highly feasible, encompassing a linear range from 0.008 to 40 ppm with an impressive limit of detection of 8 ppb. Consequently, this molecular probe demonstrates a successful application in determining trace amounts of morphine within urine, yielding satisfactory analytical results. The study also explores the effect of several variables on the sensor's response and optimizes the detection of morphine in urine using a response surface methodology with a central composite design.

Urine and hair drug test results associated with daily consumption of codeine-predominant poppy seed food products.

This study examined the urine and hair opiate profiles associated with the daily consumption of presumptive codeine-predominant poppy seed food products. Ten participants consumed one of five food products at breakfast for 10 consecutive days. Baseline urine and hair samples were collected on Day 1. The urine samples were collected 4, 8 and 12 h following poppy seed consumption on Days 1 and 10, and the first morning void urine samples were collected on Days 2-10. A second hair specimen was collected on Day 20 ± 2. Urine drug test results: Three of the food products were associated with opiate-negative urine drug test results at all time points at a 300 ng/mL cut-off. Two of the food products were associated with opiate-positive drug test results at all non-baseline time points at a 300 ng/mL cut-off. Of these, all samples (n = 60) were codeine-positive, and 27 (45%) were morphine-positive. Codeine concentrations exceeded morphine concentrations in every sample and always by multiples. Thirty-nine of the 60 samples (65%) were codeine-positive at a 2,000 ng/mL cut-off, while none of these samples were morphine-positive at this cut-off. None of the 60 samples reached an opiate threshold of 15,000 ng/mL, although one participant produced a maximum codeine concentration of 13,161 ng/mL (13,854 ng/mg creatinine). There was no clear trend toward increasing urinary opiate concentrations over the course of the study. Hair drug test results: The hair samples of two participants produced quantifiable codeine (41 pg/mg and 51 pg/mg), but no sample reached a common reporting threshold of 200 pg/mg for codeine or morphine.

Strong π-Metal Interaction Enables Liquid Interfacial Nanoarray-Molecule Co-assembly for Raman Sensing of Ultratrace Fentanyl Doped in Heroin, Ketamine, Morphine, and Real Urine.

Toward the challenge on reliable determination of trace fentanyl to avoid opioid overdose death in drug crisis, here we realize rapid and direct detection of trace fentanyl in real human urine without pretreatment by a portable surface enhanced Raman spectroscopy (SERS) strategy on liquid/liquid interfacial (LLI) plasmonic arrays. It was observed that fentanyl could interact with the gold nanoparticles (GNPs) surface, facilitate the LLI self-assembly, and consequently amplify the detection sensitivity with a limit of detection (LOD) as low as 1 ng/mL in aqueous solution and 50 ng/mL spiked in urine. Furthermore, we achieve multiplex blind sample recognition and classification of ultratrace fentanyl doped in other illegal drugs, which has extremely low LODs at mass concentrations of 0.02% (2 ng in 10 µg of heroin), 0.02% (2 ng in 10 µg of ketamine), and 0.1% (10 ng in 10 µg of morphine). A logic circuit of the AND gate was constructed for automatic recognition of illegal drugs with or without fentanyl doping. The data-driven analog soft independent modeling model could quickly distinguish fentanyl-doped samples from illegal drugs with 100% specificity. Molecular dynamics (MD) simulation elucidates the underlying molecular mechanism of nanoarray-molecule co-assembly through strong π-metal interactions and the differences in the SERS signal of various drug molecules. It paves a rapid identification, quantification, and classification strategy for trace fentanyl analysis, indicating broad application prospects in response to the opioid epidemic crisis.

Poppy Seed Consumption May Be Associated with Codeine-Only Urine Drug Test Results.

Consumption of poppy seed-containing food products can result in opiate-positive urine drug test results and may pose challenges in distinguishing poppy seed consumption from opiate administration. In this context, guidance has suggested that codeine concentrations exceeding 300 ng/mL coupled with morphine-to-codeine ratios <2 are indicative of codeine consumption and, therefore, exclude poppy seed consumption as a legitimate explanation for the test result. In recent years, we performed independent medical examinations of three individuals who produced codeine-positive/morphine-negative (300 ng/mL) forensic urine drug test results but denied codeine administration, attributing their test results to the consumption of specific poppy seed-containing food products. In the present study, 11 participants consumed one of the 10 unique poppy seed-containing food products, including the three implicated food products. Six of 33 non-baseline urine samples (18%)-representing three food products-were positive for codeine and negative for morphine at 300 ng/mL cut-offs (and therefore featured morphine-to-codeine ratios <2). This study adds to a small literature indicating that consumption of poppy seed-containing food products cannot reliably be distinguished from codeine administration based on previously published urinary opiate concentrations and ratios. An important caveat is that in none of these cases did maximum urinary codeine concentrations exceed 1,300 µg/g creatinine.

Urine thebaine determination by liquid chromatography tandem mass spectrometry after poppy seed consumption.

Laboratories are challenged to distinguish whether a positive urine morphine result is due to heroin use or possible poppy seed consumption. Thebaine is an opium alkaloid that has been shown to be present in the urine of individuals who have consumed poppy seeds, as well as those who have used opium. It is not present in heroin. We present a sensitive, specific liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for thebaine. We show that thebaine is detectable after consumption of two different poppy seed-containing products for up to 72 h in urine. We discuss limitations of the assay and suggest how the test might best be used.

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.

A hierarchical 3D camellia-like molybdenum tungsten disulfide architectures for the determination of morphine and tramadol.

A practical technique was applied to fabricate MoWS2 nanocomposite through a one-pot hydrothermal method for use as the electrocatalyst. The characterization of MoWS2 nanocomposite was investigated by several techniques to identify the size, crystal structure, and elemental composition. MoWS2 nanocomposite exhibited a unique and well-defined hierarchical structure with neatly and densely piled nanopetals acting as the active sites in the electrocatalytic reactions. A carbon screen-printed electrode (CSPE) modified with interesting MoWS2 nanopetals (MoWS2/CSPE) was constructed. Subsequently, the electrochemical oxidation of morphine on fabricated MoWS2/CSPE was studied. Experimental results confirm that under optimized conditions, the maximum oxidation current of morphine occurs at 275 mV in the case of MoWS2/CSPE that is around 100 mV more negative than that observed in the case of the unmodified CSPE and about 2.6 times increase was observed for the oxidation peak current. The analytical approach was obtained by differential pulse voltammetry in accordance with the relationship between the oxidation peak current and the morphine concentration. The oxidation peak currents for morphine were found to vary linearly with its concentrations in the range of 4.8 × 10-8-5.05 × 10-4 M with the detection limit of 1.44 × 10-8 M. Two completely separated signals occured at the potentials of 275 mV and 920 mV for oxidation of morphine and tramadol at the surface of MoWS2/CSPE which are sufficient for determination of morphine in the presence of tramadol. The presence of morphine was also detected in real samples using the introduced approach. Graphical abstract Schematic representation of fabrication of the MoWS2 nanocomposite through a one-pot hydrothermal method for use as the electrocatalyst. A carbon screen-printed electrode was modified with MoWS2 nanocomposite. Subsequently, the electrochemical oxidation of morphine on the fabricated electrode was studied.

A Study of Opiate, Opiate Metabolites and Antihistamines in Urine after Consumption of Cold Syrups by LC-MS/MS.

Studying the origin of opiate and/or opiate metabolites in individual urine specimens after consumption of cold syrups is vital for patients, doctors, and law enforcement. A rapid liquid chromatography-tandem mass spectrometry method using "dilute-and-shoot" analysis without the need for extraction, hydrolysis and/or derivatization has been developed and validated. The approach provides linear ranges of 2.5-1000 ng mL-1 for 6-acetylmorphine, codeine, chlorpheniramine, and carbinoxamine, 2.5-800 ng mL-1 for morphine and morphine-3-ß-d-glucuronide, and 2.5-600 ng mL-1 for morphine-6-ß-d-glucuronide and codeine-6-ß-d-glucuronide, with excellent correlation coefficients (R2 > 0.995) and matrix effects (< 5%). Urine samples collected from the ten participants orally administered cold syrups were analyzed. The results concluded that participants consuming codeine-containing cold syrups did not routinely pass urine tests for opiates, and their morphine-codeine concentration ratios (M/C) were not always < 1. In addition, the distribution map of the clinical total concentration of the sum of morphine and codeine against the antihistamines (chlorpheniramine or carbinoxamine) were plotted for discrimination of people who used cold syrups. The 15 real cases have been studied by using M/C rule, cutoff value, and distribution map, further revealing a potential approach to determine opiate metabolite in urine originating from cold syrups.

[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.

Direct detection of morphine in human urine by surface-ionization mass spectrometry.

Detection and analysis of drugs-of-abuse in biological fluids are the essential tasks for the law enforcement, forensic toxicology, doping research and social health. In this work, we developed a surface-ionization mass spectrometry method for direct detection of trace amount of morphine spiked in blank urine. The mass-spectrometric analysis of spiked samples was carried out without preliminary extraction and chromatographic separation. It was found out that the major fragment ions at m/z 144, 146 could be served as indicator lines of morphine spiked in blank urine. Limit of detection of morphine spiked in blank urine was 100 pg (50 ng/ml), and a linear range of calibration curve was more than two orders of magnitude. The spiked samples were also analyzed by gas chromatography-mass spectrometry without preliminary extraction and derivatization procedures. No morphine was found in the spiked samples. The experimental data show that the high selectivity of the surface ionization can be allowed to direct analysis of morphine spiked in blank urine without its preliminary extraction and chromatographic separation.

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.

Rapid lateral flow assays based on the quantification of magnetic nanoparticle labels for multiplexed immunodetection of small molecules: application to the determination of drugs of abuse.

A rapid lateral flow immunoassay is presented that uses carboxyl-modified superparamagnetic nanoparticles as labels that can be quantified by highly sensitive multi-channel electronic readers. The approach is generic in that it is likely to be applicable to numerous small molecules. The method permits both single- and multiplex assays at a point-of-need without sample pretreatment. It is user-friendly and offers attractive characteristics demonstrated here for detection of morphine, fentanyl and methamphetamine in urine. The competitive immunoassay uses commercially available reagents that do not require special permissions. After migration of sample, the lateral flow test strips are subjected to an alternating magnetic field at two frequencies. The response from the nanolabels is readout at a combinatorial frequency from the entire volume of a porous immunochromatographic membrane by the magnetic particle quantification technique. Even trace concentrations can be quantified within ≤20 min with the limits of detection (LOD) of 0.20 ng·mL-1, 0.36 ng·mL-1 and 1.30 ng·mL-1 for morphine, fentanyl and methamphetamine, respectively. The second variant presented here features highly sensitive quantification of haptens (LOD for fentanyl - 0.05 ng·mL-1). This is due to high-affinity trapping of magnetic nanolabels in a universal streptavidin-based test strip, which can be also used for detection of virtually any other small molecule. The third variant is of the multiplexed type and intended for rapid and simultaneous detection of the drugs of abuse in human urine with LODs equal to 0.60 ng·mL-1 and 3.0 ng·mL-1 for morphine and methamphetamine, respectively. In addition to the low LODs, the RSDs did not exceed 7%, 9%, and 11% for methamphetamine, morphine and fentanyl, respectively. Graphical abstract Three variants of small molecule detection in competitive format at a point-of-need. Single-plex variants feature antibody and high-affinity streptavidin test lines, while multiplex variant - several antibody test lines. Magnetic nanolabels are quantified from the whole volume of test strip.

Profiling of morphine and codeine in urine after the ingestion of curry containing poppy seed as an evidence for opiates defence in Malaysia.

Consumption of curry containing poppy seeds has raised an issue concerning the opiate content in the urine that might exceed the cut-off value (300ng/mL). The main objective of this study was to examine the morphine and codeine contents in the urine of the consumers after partaking poppy seed-enriched curry in. The volunteers were asked to partake: (a) a single meal and their urines were collected within 24h, or (b) Two meals a day for three consecutive days and their urines were collected within 72h. Two different dosages were also tested in this study: (a) low dosage: 1g/100ml curry (containing 138µg of morphine and 66µg of codeine) and (b) high dosage: 5g/100ml curry (containing 690µg of morphine and 330µg of codeine). The subjects were randomised into the groups using the method of stratified randomization with age and gender groups as covariates. A total of 6 subjects was allocated for each group and placebos were used as control. Results showed that all subjects who consumed low dosage of poppy seeds either in single meal or multiple meals experiment were found negative. However, 1 out of 6high dosage subjects was confirmed positive at a period of 3-6h after the consumption of curry in the single meal study. This outlier maybe due to the lack of water consumption after consuming the curry, thus the low volume of urine was collected and the opiate was concentrated in the urine. On the other hand, 5 out of 6high dosage subjects in the multiple meals experiment were found positive. Majority of these subjects were found positive on the second and third day of the experiment after the second curry meal was consumed. The outlier (negative) in this group might be due to the high consumption of water throughout the experiment and the subject's urine volumes and frequency of urine collection were much higher compared to other subjects. From the result of this study, it can be concluded that partaking high dosages of poppy seed in curry could give a positive response (>300ng/ml+uncertainty of measurement) in the urine, and the water consumption after partaking curry has significant influence for the opiate contents in the urine.

Disposable electrochemical immunosensor array for the multiplexed detection of the drug metabolites morphine, tetrahydrocannabinol and benzoylecgonine.

Heroin, marijuana and cocaine are widely abused drugs. Their use can be readily detected by analyzing urine for the metabolites morphine (MOR), tetrahydrocannabinol (THC) or benzoylecgonine (BZC). A multiplex immunosensor is described here for detection of MOR, THC and BZC using screen printed carbon array electrodes modified with gold nanoparticles. Antibodies against MOR, THC and BZC were immobilized on eight electrodes in a sensor array simultaneously, and a competitive assay was used for the detection. The free analytes in the sample compete with bovine serum albumin-conjugated analytes for the immobilized antibodies on the sensor surface. The array is capable of detecting the three drugs simultaneously within 20-40 min. The method has a high sensitivity, with detection limits as low as 1.2, 7.0, and 8.0 pg.mL-1 for MOR, THC and BZC, respectively. Cross reactivity testing was preformed to monitor any nonspecific binding. The results revealed good selectivity. Urine samples were spiked with the 3 drugs and tested with the multiplexed immunosensor. Recovery percentages ranged between 88 to 115%. Graphical abstract Schematic presentation of the multiplexed immunosensor for drugs of abuse,viz. tetrahydrocannabinol (THC), morphine (MOR), and benzoylecgonine (BZC)) by using an array of modified electrodes.

High-Precision Quantitation of Biofluid Samples Using Direct Mass Spectrometry Analysis.

The transition of mass spectrometry for clinical analysis is highly desirable, and major progress has been made with direct sampling ionization for operation simplification. High-precision quantitation, however, remains a major challenge in this transition. Herein, a novel method was developed for direct quantitation of biofluid samples, using an extremely simplified procedure for incorporation of internal standards selected against the traditional rules. Slug flow microextraction was used for the development, with conditions predicted by a theoretical model, viz., using internal standards of partition coefficients very different from the analytes and large sample-to-extraction solvent volume ratios. Direct quantitation of drug compounds in urine and blood samples was demonstrated. This development enabled an extremely simplified protocol that is expected to have a significant impact on on-site or clinical analysis.

Development and clinical evaluation of a dried urine spot method for detection of morphine among opioid users.

BACKGROUND: In recent years, drug testing in body fluids has gained popularity for validating self-reported drug use. The storage and transportation of urine specimens is a major concern for remote areas where the facilities for performing drug abuse testing are lacking. AIMS AND OBJECTIVES: The aim of the present study was to develop an efficient method for testing opiate in dried urine spots (DUS) and to evaluate its clinical applicability. MATERIALS AND METHODS: The methodology involved optimization of conditions for extraction, recovery, short-, and long-term stability (room temperature, 4°C,-20°C) for detection of opiate from dried urine spots. Further, the extraction efficiency from dried urine spots was compared with the conventional drug testing methodology. The screening was done by using enzyme-linked immunosorbent assay technique, and confirmation was achieved by gas chromatography equipped with nitrogen phosphorus detector. RESULTS: Deionized water was found to be a suitable extracting solvent compare to bi-carbonate buffer (pH 9.2) and saline. Primary screening was achieved by 2 punches taken from a 20-µl (diameter 1.3 cm) spotted urine samples, whereas confirmation was achieved by 2 complete circles each of 20 µl sample volume. The recovery was found to be 99.41% in water. No sign of significant degradation was seen among all storage conditions. CONCLUSIONS: In the current study, DUS has achieved the same level of precision and reproducibility as that of standard methods used for drug testing in urine. Hence, the DUS sampling appears to have potential to detect opiate among drug users in a clinical setting.

Pharmacokinetics and selected pharmacodynamics of morphine and its active metabolites in horses after intravenous administration of four doses.

The objective of the current study was to describe and characterize the pharmacokinetics and selected pharmacodynamic effects of morphine and its two major metabolites in horses following several doses of morphine. A total of ten horses were administered a single intravenous dose of morphine: 0.05, 0.1, 0.2, or 0.5 mg/kg, or saline control. Blood samples were collected up to 72 hr, analyzed for morphine, and metabolites by LC/MS/MS, and pharmacokinetic parameters were determined. Step count, heart rate and rhythm, gastrointestinal borborygmi, fecal output, packed cell volume, and total protein were also assessed. Morphine-3 glucuronide (M3G) was the predominant metabolite detected, with concentrations exceeding those of morphine-6 glucuronide (M6G) at all time points. Maximal concentrations of M3G and M6G ranged from 55.1 to 504 and 6.2 to 28.4 ng/ml, respectively, across dose groups. The initial assessment of morphine pharmacokinetics was done using noncompartmental analysis (NCA). The volume of distribution at steady-state and systemic clearance ranged from 9.40 to 16.9 L/kg and 23.3 to 32.4 ml min-1  kg-1 , respectively. Adverse effects included signs of decreased gastrointestinal motility and increased central nervous excitation. There was a correlation between increasing doses of morphine, increases in M3G concentrations, and adverse effects. Findings from this study support direct administration of purified M3G and M6G to horses to better characterize the pharmacokinetics of morphine and its metabolites and to assess pharmacodynamic activity of these metabolites.