Elucidating fentanyls differentiation from morphines in chemical and biological samples with surface-enhanced Raman spectroscopy.

Fentanyl and morphine are opioid drugs as well as new psychoactive substances. Even originally introduced as efficient anesthetic drugs to relieve moderate-to-severe pain in clinic, the overdose of new synthetic opioids is currently a serious public health problem in numerous countries worldwide. The entire category of fentanyls has been included in the regulatory list in several countries. There is a great and urgent demand to rapidly recognize fentanyls and morphines in various samples. Here, we report an on-site surface-enhanced Raman spectroscopic method to classify fentanyls from morphines by the Raman spectroscopic signature of the molecular scaffold structure, with an assistance of principle component analysis algorithm. Moreover, by simple but fine-tuning approach of inorganic salt-induced aggregation of gold nanoparticles substrate, we achieved a selective detection of 10 ng/mL fentanyl from 2000-fold of heroin, the most common coexisting substance in chemical samples. Good differentiation of 50 ng/mL fentanyl from 10 000-fold morphine as a main metabolite of heroin in urine samples was also possible after a feasible pretreatment by StageTip procedures. Depending on different structures, the detection sensitivity of five fentanyls ranged from 50 to 2000 ng/mL.

Fingerprinting of morphine using chromatographic purity profiling and multivariate data analysis.

Chromatographic purity profiling (CPP) is the common name of a group of analytical and chemometric applications for detection, identification and quantitative determination of related substances and other impurities in active pharmaceutical ingredients (APIs) and finished dosage forms (FDFs). CPP is used for fingerprinting and discriminating between samples, thus representing a core activity in modern drug analysis. The worldwide demand for morphine and its congeners is tremendous and depends entirely on the supply of natural opiates. The aim of this research was to develop a methodology that enables identification of a source of morphine, thus revealing falsification of the substance. The characteristic and reproducible features of impurity profiles for 28 samples of morphine (6 morphine sulfate, 9 morphine hydrochloride and 13 morphine base) were captured by a new LC/MS method for impurity profiling of morphine. The impurity profile encompasses the related substances specified in relevant Ph.Eur. monographs, as well as the other morphinane like impurities, including the naturally occurring co-extracted alkaloids. Different pattern recognition techniques (unsupervised and supervised) were used to reveal the differentiation features of the morphine fingerprints for classification and authentication purposes. The results described in this research open the possibility of using the chromatographic purity profile combined with multivariate data analysis for fingerprinting of morphine samples.

Development, optimization, and validation of a novel extraction procedure for the removal of opiates from human hair's surface.

Room temperature ionic liquids (ILs) have proved to be efficient extraction media for several systems, and their ability to capture volatile compounds from the atmosphere is well established. We report herein a contactless extraction procedure for the removal of opiate drugs from the surface of human hair. The compounds were chosen as a model drug, particularly due to their low volatility. Equal amounts of IL and hair (about 100 mg) were introduced in a customized Y-shaped vial, and the process occurred simply by heating. After testing several ILs, some of them (e.g. 1-methyl-3-ethanol-imidazolium tetrafluoroborate, phenyl-trimethyl-ammonium triflate or bis(dimethyl) diheptylguanidinium iodide) showed extraction efficiencies higher than 80% for the two studied compounds, morphine and 6-monoacetylmorphine. Using the design of experiments (DOE) approach as an optimization tool, and bearing in mind the hygroscopic properties of the ILs (in particular, 1-methyl-3-ethanol-imidazolium tetrafluoroborate), the process was optimized concerning the following variables: temperature (50-120 ºC), extraction time (8-24 h), IL amount (50-200 mg) and water content of the IL (0.01-60%). This study not only provided the optimum conditions for the process (120 ºC, 16 h, 100 mg of IL containing 40% of water), but has also showed that the water content of the IL represents the variable with the most significant effect on the extraction efficiency. Finally, we validated our method through the comparison of the results obtained by treating hair samples with the described procedure to those obtained using a standard washing method and criteria for positivity.

Chromatographic resolution of closely related species: drug metabolites and analogs.

In this study, we investigate the separation of a variety of mixtures of drugs, metabolites, and related analogs including representatives of the carbamazepine, methylated xanthine, steroid hormone, nicotine, and morphine families using several automated chromatographic method development screening systems including ultra high performance liquid chromatography, core-shell HPLC, achiral supercritical fluid chromatography (SFC), and chiral SFC. Of the 138 column and mobile phase combinations examined for each mixture, a few chromatographic conditions afford the best overall performance, with a single achiral SFC method (4.6 × 250 mm, 3.0 µm GreenSep Ethyl Pyridine, 25 mM isobutylamine in methanol/CO2) affording good separation for all samples. Four of these mixtures were also resolved by achiral SFC on the Luna HILIC and chiral SFC Chiralpak IB columns using methanol or ethanol with 25 mM isobutylamine as polar modifiers. Modifications of standard chromatography screening conditions afforded fast separation methods (from 1 to 5 min) for baseline resolution of all components of each of these challenging sets of closely related compounds.

Matrix solid phase dispersion assisted enzymatic hydrolysis as a novel approach for cocaine and opiates isolation from human hair.

The possibility of assisting enzymatic hydrolysis (EH) procedures by sample disruption mechanisms inherent to matrix solid phase dispersion (MSPD) has been explored in the current study. EH of hair specimens from poly-drug abusers was assisted by dispersing/blending the sample (0.05 g) with alumina (2.25 g) before loading the dissolved enzyme (6 mL of 1 mg mL(-1) Pronase E in 1.4 M/1.4 M Tris/HCl, pH 7.3) through the hair-alumina solid phase packaged inside a disposable MSPD syringe. The MSPD-EH method was developed, and it proved to offer quantitative results when isolating cocaine, benzoylecgonine (BZE), codeine, morphine and 6-monoacethylmorphine (6-MAM) from human hair samples. The procedure allows an on column clean-up/pre-concentration procedure of the isolated targets by attaching a previously conditioned Oasis HLB cartridge to the end of the MSPD syringe. The EH procedure of human hair with Pronase E can therefore be shortened to approximately 30 min. Within this time, sample blending/dispersion, MSPD syringe package, elution (EH when dissolved Pronase E is passing through the sample-dispersant bed), and extract clean-up and target pre-concentration stages are achieved. Gas chromatography-mass spectrometry (GC-MS) was used for determining each target after elution from the Oasis HLB cartridges with 2 mL of 2% (v/v) acetic acid in methanol, concentration by N2 stream evaporation, and dried extract derivatization with N-methyl-tert-butylsilyltrifluoroacetamide (BSTFA) and chlorotrimethylsilane (TMCS). The method was validated according to the guidance for bioanalytical method validation of the US Department of Health and Human Services, Food and Drug Administration. The simplicity of the proposed approach makes it a useful procedure for screening/quantifying drugs of abuse in hair specimens from poly-drug abusers.

Chromatographic determination of drugs of abuse in vitreous humor using solid-phase extraction.

A simple method is presented for the simultaneous determination of morphine, 6-acetylmorphine, codeine, cocaine, benzoylecgonine, cocaethylene, methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in vitreous humor by high-performance liquid chromatography with photodiode array detector after solid-phase extraction with Oasis® HLB cartridges and dichloromethane as eluent. The chromatographic process was carried out using an XTerra® RP8 column (250 × 4.6 mm i.d., 5 µm particle size) and a mobile phase composed of acetonitrile and pH 6.5 phosphate buffer in gradient mode. A linear response from the detector was obtained within the concentration range of 0.1-4 µg ml(-1) , with correlation coefficients higher than 0.99. The limits of detection were lower than 30 ng ml(-1) for all the drugs studied, the coefficients of variation fluctuated between 0.1 and 12.4%, and the average recoveries were higher than 78% for all the drugs except for EDDP, with a value of 66.4%. Finally, the proposed method was applied to 15 vitreous humor samples coming from individuals who had died from opiate and/or cocaine overdose, showing consumption of cocaine in 14 cases, methadone in five cases and heroin in three cases. Average concentrations of 0.30 µg ml(-1) for morphine, 0.24 µg ml(-1) for 6-acetylmorphine, 0.10 µg ml(-1) for codeine, 0.81 µg ml(-1) for cocaine, 1.26 µg ml(-1) for benzoylecgonine, 0.15 µg ml(-1) for cocaethylene, 0.11 µg ml(-1) for methadone and 0.68 µg ml(-1) for EDDP were obtained.

In-line solid-phase extraction-capillary electrophoresis coupled with mass spectrometry for determination of drugs of abuse in human urine.

In-line solid-phase extraction-capillary electrophoresis coupled with mass spectrometric detection (SPE-CE-MS) has been used for determination of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), codeine (COD), hydrocodeine (HCOD), and 6-acetylmorphine (6AM) in urine. The preconcentration system consists of a small capillary filled with Oasis HLB sorbent and inserted into the inlet section of the electrophoresis capillary. The SPE-CE-MS experimental conditions were optimized as follows: the sample (adjusted to pH 6.0) was loaded at 930 mbar for 60 min, elution was performed with methanol at 50 mbar for 35 s, 60 mmol L(-1) ammonium acetate at pH 3.8 was used as running buffer, the separation voltage was 30 kV, and the sheath liquid at a flow rate of 5.0 µL min(-1) was isopropanol-water 50:50 (v/v) containing 0.5% acetic acid. Analysis of urine samples spiked with the four drugs and diluted 1:1 (v/v) was studied in the linear range 0.08-10 ng mL(-1). Detection limits (LODs) (S/N = 3) were between 0.013 and 0.210 ng mL(-1). Repeatability (expressed as relative standard deviation) was below 7.2%. The method developed enables simple and effective determination of these drugs of abuse in urine samples at the levels encountered in toxicology and doping.

Investigation of in-line solid-phase extraction capillary electrophoresis for the analysis of drugs of abuse and their metabolites in water samples.

In this study, in-line solid-phase extraction (SPE) was used as an enrichment technique in combination with CE for the preconcentration and separation of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine (COC), codeine (COD) and 6-acetylmorphine (6AM). The separation buffer (BGE) used was 80 mM disodium phosphate anhydrous and 6 mM of HCl (final BGE pH of 3). The SPE extractor consists of a small segment of capillary filled with Oasis HLB sorbent and inserted into the inlet section of the electrophoretic capillary. Different parameters affecting preconcentration were evaluated, such as sample pH, the volume of the elution plug and sample injection time. The detection limits (LODs) reached for standard samples by in-line SPE-CE-UV ranged between 50 and 200 ng/L, with sensitivity enhancement factors ranging from 2300 to 5300. Reproducibility values (expressed in terms of relative standard deviation) were below 7.6% for standard samples. This is a simple and an effective method for the determination of the studied drugs of abuse and their metabolites. The applicability of the developed method was demonstrated in tap and river water samples which were directly analyzed without any off-line pretreatment. Analytical parameters were evaluated and LODs were between 70 and 270 ng/L with relative recoveries between 85 and 97%.

Disposable pipette extraction for gas chromatographic determination of codeine, morphine, and 6-monoacetylmorphine in vitreous humor.

The availability of a sensitive and rapid analytical method for the determination of opiates, and other substances of forensic interest, in a variety of biological specimens is of utmost importance to forensic laboratories. Solid-phase extraction is very popular in the pre-treatment of forensic samples. Nevertheless, a new approach, disposable pipette extraction (DPX), is gaining increasing interest in sample preparation. DPX has already been applied to the analysis of drugs of abuse in common biological matrices, such as urine and blood, but has not yet been evaluated on alternative biological samples, such as vitreous humor. The objective of this study was to evaluate the applicability of DPX on the analysis of opiates in vitreous humor. The currently developed method is fast, reliable, and easy to perform. The sensitivity, precision, and accuracy are satisfactory. Recoveries obtained are within the range of 72-91%, whereas the sample volume of vitreous humor required is only 100 µL.

Disposable microfluidic device with ultraviolet detection for highly resolved screening of illicit drugs.

A disposable microfluidic device was constructed by conveniently integrating one poly(methyl methacrylate) board with four reservoirs and one fractured fused-silica capillary with 50 microm i.d. and 7.5 cm total length on a printed circuit board for applying sampling and separation voltages. The disposable microfluidic device combined with a home-made ultraviolet workstation could be conveniently used for efficient screening and quantitative detection of microg mL(-1) illicit drugs. Using eight illicit drugs as models, they could be baseline-separated within 240 s with the separation efficiency up to 600,047 plates m(-1) at the designed device. The novel device and proposed protocol were successfully used to screen illicit drugs in human urine. This work presented a simple and low-cost method to fabricate the microfluidic device and provided a powerful way for sensitive and specific multi-screening of different drugs with high resolution, fast separation and low-cost.

Antifungal activity of the alkaloids from Eschscholzia californica.

The isoquinoline alkaloids hunnemanine and norsanguinarine have been isolated from methanolic extract of the whole plant of Eschscholzia californica. These two alkaloids were checked for their antifungal activity against phytopathogenic fungi Alternaria melongenae, A. brassicola, A. brassicae, Curvularia lunata, C. maculans, Helminthosporium pennisetti, H. oryzae, H. turcicum, Fusarium undum and F. lini. Hunnemanine exhibited 100 % inhibition of spore germination of A. brassicae, H. pennisetti and F. lini at 1000 ppm whereas norsanguinarine exhibited 100 % inhibition of A. brassicicola and C. maculans at this concentration.

Preparation and evaluation of solid-phase microextraction fibers based on monolithic molecularly imprinted polymers for selective extraction of diacetylmorphine and analogous compounds.

All of the studies on solid-phase microextraction based on molecularly imprinted polymers up to now have been carried out on the synthesis of the polymer on the surface of the fiber which is brittle and the polymer coating strips during handling. The objective of this study was to develop a method for fabrication of a monolithic and robust solid-phase microextraction fiber on the basis of molecularly imprinted polymer for selective extraction of diacetylmorphine and its structural analogues followed by their GC or GC/MS analysis. A fiber was produced by copolymerization of methacrylic acid-ethylene glycol dimethacrylate imprinted with diacetylmorphine. The effective factors influencing the polymerization have been investigated and are detailed here. Also, the influences of pH, extraction time and temperature on the extraction efficiency of analytes were investigated. The prepared fiber was thermally stable up to 300 degrees C which has vital importance in SPME coupled with GC or GC/MS. The adsorption isotherm modeling was performed by fitting the data of studied compounds to bi-Langmuir isotherm model. The evaluated equilibrium constants for diacetylmorphine were 0.011 and 1824.72 microM(-1), and the number of binding sites was 170.37 and 4.64 nmolg(-1), respectively. This fiber was successfully used for extraction of template molecule from aqueous solution and further analysis with GC or GC/MS. The high extraction efficiency was obtained for diacetylmorphine, 6-monoacetylcodeine, and 6-monoacetylmorphine, yielding the detection limits of 300, 47, and 1 ngmL(-1), respectively.

Four years' experience in external proficiency testing programs for hair testing of drugs of abuse in Italy (HAIRVEQ) and comparison with the Society of Hair Testing program in 2005.

Since 2002, the Istituto Superiore di Sanità, Rome, Italy, in cooperation with Institut Municipal d'Investigaciò Mèdica, Barcelona, Spain, has set up an external proficiency testing program (HAIRVEQ) to evaluate reliability in hair testing for drug abuse by laboratories from the Italian National Health Service. The results obtained in the last 2 rounds (2004-2005) by 26 laboratories and the evolution of the performance in hair testing for drugs of abuse by laboratories that have participated during the whole external proficiency testing program are presented. The 3 hair samples from the last exercise (2005) were also included in the proficiency test organized by the Society of Hair Testing (SoHT) and 17 international laboratories reported results. Samples analyzed in both exercises were real hair samples from drug consumers. In 2004, 2 identical samples were sent containing cocaine and opiates. One sample was a pulverized specimen and the second one was cut in short segments. In 2005, 2 samples, one containing MDMA and another containing cocaine, were included together with one blank sample. In 2004, approximately 42% of HAIRVEQ laboratories reported an erroneous qualitative result. The scatter of quantitative results was high, although no statistical differences, except for codeine, were found between results reported for the hair specimen if pulverized or reduced in short cuts. In 2005, 47 incorrect qualitative results were reported by HAIRVEQ laboratories, whereas only 5 were informed by SoHT laboratories. Concerning quantitative results, the ones from HAIRVEQ laboratories were comparable, although more dispersed, than those reported by SoHT laboratories. The scatter in quantitative results remained quite high and similar to those of the previous years; nonetheless, an improvement in the qualitative performance was observed. Considering the few number of laboratories showing a satisfying performance, guidelines have to be provided focused on method validation and qualitative and quantitative data evaluation.

Saludimerines A and B, novel-type dimeric alkaloids with stereogenic centers and configurationally semistable biaryl axes.

The first biarylic bis-morphinanedienone alkaloids, saludimerines A (3a) and B (3b), isolated from a tree of Croton flavens (Euphorbiaceae) are described. These naturally occurring dimers of the known alkaloid salutaridine are joined together via a rotationally hindered biaryl axis, giving rise to atropo-diastereomers that are configurationally stable at room temperature but slowly interconvert in methanolic solution within several days. Their structures were established by spectroscopic methods and by partial synthesis, which was achieved by a highly atropo-diastereoselective biomimetic oxidative coupling of the monomeric precursor, salutaridine. Their axial configurations were elucidated by circular dichroism (CD) investigations, which succeeded despite the fact that the two atropo-diastereomers exhibit near-identical CD spectra. This remarkable phenomenon was rationalized by quantum chemical CD calculations. The configurational assignment of saludimerines A (3a) as P-axial and B (3b) as M was corroborated by atropisomer-specific NOE interactions between protons of the one molecular half with nuclei in the other.

[Alkaloids from a Tibetan medicine Meconopsis quintuplinervia Regel].

AIM: To reinvestigate the chemical constituents of the ethanolic extract of Meconopsis quintuplinervia Regel which is a traditional Tibetan medicine used for treatments of hepatitis, tuberculosis etc.. METHODS: The compounds were enriched by column chromatography techniques over silica gel, macro porous resin and Sephadex LH-20 absorbents, and finally purified by reverse phase preparative HPLC methods with isocratic mobile phase systems of methanol-H2O-acetic acid (500:500:1) and acetonitrile-H2O-acetic acid (200:800:1). Structural determination of the pure compounds were based on extensive analyses of modern spectroscopic methods including IR, MS, HRMS, 1D- and 2D-NMR spectra. RESULTS: Three alkaloids were obtained and their structures were elucidated as norsanguinarine (I), O-methylflavinantine (II) and 6-methoxy-17-methyl-2, 3-[methylenebis (oxy)]-morphin-5-en-7-one (III). CONCLUSION: Norsanguinarine (I) was isolated from genus Meconopsis for the first time, and 6-methoxy-17-methyl-2,3-[methylenebis(oxy)]-morphin-5-en-7-one (III) is a new alkaloid named as meconoquintupline.

New dimeric morphine from opium poppy (Papaver somuniferum) and its physiological function.

Together with bismorphine A (1), a new compound bismorphine B (2) was identified in the wounded capsules of Papaver somniferum. On the basis of analyses of the various spectral data, bismorphine B (2) was determined as a novel morphinan alkaloid, in which two morphine units are coupled through a biphenyl ether bond. When the physiological function of bismorphine B (2) in opium poppy was investigated, it was evident that this alkaloid more effectively cross-links cell wall polysaccharide pectins than bismorphine A (1) and that such cross-linking reaction leads to resistance against hydrolysis by pectinase.

Nitrotyrasacutuminine from Menispermum dauricum.

Nitrotyrasacutuminine, an unusual nitrated morphine-type alkaloid was isolated from the roots of Menispermum dauricum. Its structure was determined by various 2D spectra and chemical methods.

The presence of morphine in ganglionic tissues of Modiolus deminissus: a highly sensitive method of quantitation for morphine and its derivatives.

Morphine and morphine-6-glucuronide, a morphine metabolite, have been identified and quantified in Modiolus deminissus pedal ganglia at a level of 2.41 and 0.95 ng/ganglia, respectively. These opiate alkaloids are normally found at low concentrations in invertebrate and vertebrate tissues, including neural. Given this problem, we also describe a new opiate extraction protocol as well as a high-performance liquid chromatography purification procedure that can separate and quantify morphine and its derivatives at sub-nanogram concentrations. Furthermore, both morphine and morphine-6-glucuronide were identified in this mollusk's pedal ganglia by mass spectrometry analysis.

Separation of morphine and its oxidation products by capillary zone electrophoresis.

Separation of morphine and its oxidation products (10-S-hydroxymorphine, pseudomorphine and morphine N-oxide) by capillary zone electrophoresis in Tris-borate buffer in the presence of cyclodextrins was studied. Pyridoxine was used as an internal standard.

Improved one-step solid-phase extraction method for morphine, morphine-3-glucuronide, and morphine-6-glucuronide from plasma and quantitation using high-performance liquid chromatography with electrochemical detection.

This communication describes an improved one-step solid-phase extraction method for the recovery of morphine (M), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) from human plasma with reduced coextraction of endogenous plasma constituents, compared to that of the authors' previously reported method. The magnitude of the peak caused by endogenous plasma components in the chromatogram that eluted immediately before the retention time of M3G has been reduced (approximately 80%) significantly (p < 0.01) while achieving high extraction efficiencies for the compounds of interest, viz morphine, M6G, and M3G (93.8 +/- 2.5, 91.7 +/- 1.7, and 93.1 +/- 2.2%, respectively). Furthermore, when the improved solid-phase extraction method was used, the extraction cartridge-derived late-eluting peak (retention time 90 to 100 minutes) reported in our previous method, was no longer present in the plasma extracts. Therefore the combined effect of reducing the recovery of the endogenous components of plasma that chromatographed just before the retention time of M3G and the removal of the late-eluting, extraction cartridge-derived peak has resulted in a decrease in the chromatographic run-time to 20 minutes, thereby increasing the sample throughput by up to 100%.