Drugs In Biological Samples Research Articles

A novel colorimetric biosensor based on non-aggregated Au@Ag core–shell nanoparticles for methamphetamine and cocaine detection

We report a novel colorimetric biosensor based on non-aggregation Au@Ag core-shell nanoparticles to detect methamphetamine and cocaine. The biosensor consisted of a reporter probe (RP) that is a specific single-stranded DNA (ssDNA) sequence coated on Au@Ag nanoparticles, a capture probe (CP) conjugated with magnetic beads, and an illicit drug-binding DNA aptamer (Apt). Au@Ag nanoparticles were synthesized by seed growth and characterized by scanning electron microscope (SEM), high-resolution transmission electron microscopy (HR-TEM), and UV–vis spectra. Methamphetamine (METH) was used as an example to evaluate the feasibility of the biosensor and to optimize the detection conditions. We demonstrated that this sensing platform was able to detect as low as 0.1nM (14.9ngL−1) METH with a negligible interference from other common illicit drugs. Various concentrations of METH were spiked into urines, and the biosensor yielded recoveries more than 83.1%. In addition, the biosensor also showed a high sensitivity to detect cocaine. These results demonstrated that our colorimetric sensor holds promise to be implemented as a visual sensing platform to detect multiple illicit drugs in biological samples and environmental matrices.

Isoconversional thermal and pyrolytic GC–MS analysis of street samples of hashish

Thermal decomposition of hashish, a widely used illicit drug, was studied by isoconversional and pyrolytic GC–MS analysis. An accurate value of activation energy was determined by the B-1.92 method, which was used to determine degradation mechanism, impact of destruction by incineration of the drug on environment. The thermograms exhibited a weight loss of 11–19% from ambient to 463K due to moisture content. Major degradation occurred in the range 463–643K. The average activation energy was found to be ∼121kJmol−1 that suggested rapid volatilization of the material in the temperature range. The variation of activation energy with conversion suggested a multistep degradation of the samples. The mechanism of degradation was found to be a second order decomposition representing an exothermic chemical change. The residue at 1073K was analyzed by SEM-EDS and found to contain C, O, P, S, K, Mg, Ca, Al and Si to various extents; C and O were present in substantial amounts. Pyrolytic GC–MS analysis identified cannabidiol, limonene, β-caryophyllene and pyrolytic products of acetaminophen and dehydrophenacetin as the major decomposition products. This study shows that the incineration process does not completely destroy the drug, rather it throws large quantities of drug-related toxic substances into the environment. The pyrolysis GC–MS showed that the characteristic peak due to cannabidiol can be used as a signature of the drug in biological samples.

Development of a Nafion/MWCNT-SPCE-Based Portable Sensor for the Voltammetric Analysis of the Anti-Tuberculosis Drug Ethambutol.

Herein we describe the development, characterization and application of an electrochemical sensor based on the use of Nafion/MWCNT-modified screen-printed carbon electrodes (SPCEs) for the voltammetric detection of the anti-tuberculosis (anti-TB) drug ethambutol (ETB). The electrochemical behaviour of the drug at the surface of the developed Nafion/MWCNT-SPCEs was studied through cyclic voltammetry (CV) and square wave voltammetry (SWV) techniques. Electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM) were employed to characterize the modified surface of the electrodes. Results showed that, compared to both unmodified and MWCNTs-modified SPCEs, negatively charged Nafion/MWCNT-SPCEs remarkably enhanced the electrochemical sensitivity and selectivity for ETB due to the synergistic effect of the electrostatic interaction between cationic ETB molecules and negatively charged Nafion polymer and the inherent electrocatalytic properties of both MWCNTs and Nafion. Nafion/MWCNT-SPCEs provided excellent biocompatibility, good electrical conductivity, low electrochemical interferences and a high signal-to-noise ratio, providing excellent performance towards ETB quantification in microvolumes of human urine and human blood serum samples. The outcomes of this paper confirm that the Nafion/MWCNT-SPCE-based device could be a potential candidate for the development of a low-cost, yet reliable and efficient electrochemical portable sensor for the low-level detection of this antimycobacterial drug in biological samples.

Automated hollow-fiber liquid-phase microextraction followed by liquid chromatography with mass spectrometry for the determination of benzodiazepine drugs in biological samples.

In this study, two-phase hollow-fiber liquid-phase microextraction and three-phase hollow-fiber liquid-phase microextraction based on two immiscible organic solvents were compared for extraction of oxazepam and Lorazepam. Separations were performed on a liquid chromatography with mass spectrometry instrument. Under optimal conditions, three-phase hollow-fiber liquid-phase microextraction based on two immiscible organic solvents has a better extraction efficiency. In a urine sample, for three-phase hollow fiber liquid-phase microextraction based on two immiscible organic solvents, the calibration curves were found to be linear in the range of 0.6-200 and 0.9-200 μg L(-1) and the limits of detection were 0.2 and 0.3 μg L(-1) for oxazepam and lorazepam, respectively. For two-phase hollow fiber liquid-phase microextraction, the calibration curves were found to be linear in the range of 1-200 and 1.5-200 μg L(-1) and the limits of detection were 0.3 and 0.5 μg L(-1) for oxazepam and lorazepam, respectively. In a urine sample, for three-phase hollow-fiber-based liquid-phase microextraction based on two immiscible organic solvents, relative standard deviations in the range of 4.2-4.5% and preconcentration factors in the range of 70-180 were obtained for oxazepam and lorazepam, respectively. Also for the two-phase hollow-fiber liquid-phase microextraction, preconcentration factors in the range of 101-257 were obtained for oxazepam and lorazepam, respectively.

Dispersion of hydrophobic magnetic nanoparticles using ultarsonic-assisted in combination with coacervative microextraction for the simultaneous preconcentration and determination of tricyclic antidepressant drugs in biological fluids

A two-step sample preparation technique based on dispersive micro solid-phase extraction combined with coacervative microextraction is presented for preconcentration and determination of tricyclic antidepressant drugs in biological samples. An important feature of the method is the application of hydrophobic magnetic nanoparticles, which in combination with coacervative microextraction method enables development of rapid and efficient extraction procedure in order to achievement of a high extraction efficiency. Simultaneous optimization by experimental design lead to improvement of method with low cost which supply useful information about interaction among variables. Under the optimized conditions, a linear range of 5–1000ngmL−1 with detection limits from 0.51 to 1.4ngmL−1 were obtained for target analytes. The method was successfully used for the determination of analytes in biological fluids (plasma and urine) with relative recoveries in the range of 89–105% (RSDs<3.5%).

Simultaneous determination of cyclic antidepressants and their related drugs and the estimation of new metabolites in human whole blood and urine by MALDI-QTOF-mass spectrometry

A rapid and sensitive method using matrix-assisted laser desorption ionization (MALDI)-quadrupole time-of-flight (QTOF)-mass spectrometry (MS) was developed for the analysis of six tricyclic antidepressants (ADs) and their related drugs, such as amitriptyline, carbamazepine, clomipramine, imipramine, nortriptyline, quetiapine, and two tetracyclic ADs, mianserin and mirtazapine, because these eight drugs are commonly observed medicines in poisoning cases in Japan. In the present MALDI-MS method, the limits of detection were 0.3–2 ng/mL. The present method provided a simple and high throughput simultaneous analysis of these drugs. This is the first report dealing with quantitation of ADs and their related drugs in biological samples by MALDI-MS. In addition, we applied the MALDI-MS to two authentic cases, and three new metabolites of amitriptyline and three new metabolites of quetiapine could be detected in blood and urine samples in the two cases when regioisomers were counted as one metabolite.

LC-MS/MSによる生体試料中の薬物濃度測定

LC-MS/MSによる生体試料中の薬物濃度測定

Surfactant assisted pulsed two-phase electromembrane extraction followed by GC analysis for quantification of basic drugs in biological samples

In this work, a simple and efficient surfactant assisted pulsed two-phase electromembrane extraction (SA-PEME) procedure combined with gas chromatography (GC) has been developed for the determination of alfentanil, sufentanil and methadone in various samples. It has been found that the addition of anionic surfactant causes the accumulation of the cationic analytes at the SLM/solution interface resulting in an easier transfer of the analytes into the organic phase. The method was accomplished with 1-octanol as the acceptor phase and supported liquid membrane (SLM) by means of an 80V pulsed electrical driving force and the extraction time of 20min. The model analytes were extracted from 3.0mL sample solution (pH 4.0) containing 0.02% w/v surfactant (sodium dodecyl sulfate). The duty cycle of 92% and frequency of 0.357Hz gave the best performance. Extraction recoveries in the range of 70.5–95.2% and satisfactory repeatability (7.6<RSD<13.7) were obtained. The limits of detection were 1.5, 1.5 and 0.6ngmL−1 for sufentanil, alfentanil and methadone, respectively. The method offers an acceptable linearity (2.0–1000.0ngmL−1) for each analyte with coefficient of determination higher than 0.998. Furthermore, the figures of merit of SA-PEME were compared with the results from the conventional two-phase EME which confirm the advantages of the proposed technique. The method was applied for the determination and quantification of the model compounds in the wastewater, plasma, breast milk and urine samples.

Synthesis of stable isotope-labeled epothilone D using a degradation-reconstruction approach.

The stabilization of microtubules using epothilones represents a novel mechanism of action to treat Alzheimer's disease. Epothilone D is one such microtubule-stabilizing drug that has been investigated by Bristol-Myers Squibb. An important step in the development process was the synthesis of a stable isotope-labeled analog for use in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. A novel synthetic route to stable isotope-labeled epothilone D is described. The synthetic route was based on a strategy to degrade epothilone B and then use that key intermediate to reconstruct stable isotope-labeled epothilone D. Epothilone B was treated with potassium osmate and sodium periodate. The thiazole moiety in epothilone B was efficiently cleaved to give (1S,3S,7S,10R,11S,12S,16R)-3-acetyl-7,11-dihydroxy-8,8,10,12,16-pentamethyl-4,17-dioxabicyclo[14.1.0]heptadecane-5,9-dione. The epoxide in the macrocyclic ring of that intermediate was cleanly removed by treatment with tungsten hexachloride and n-butyllithium to give the corresponding olefin (4S,7R,8S,9S,16S,Z)-16-acetyl-4,8-dihydroxy-5,5,7,9,13-pentamethyloxacyclohexadec-13-ene-2,6-dione. Bis(triethylsilyl) protection produced (4S,7R,8S,9S,16S,Z)-16-acetyl-5,5,7,9,13-pentamethyl-4,8-bis(triethylsilyloxy)-oxacyclohexadec-13-ene-2,6-dione. This intermediate was coupled to a stable isotope-labeled thiazole using a Wittig reaction as the key step to provide (13)C5, (15)N-labeled epothilone D. In summary, the synthesis was completed in nine total steps, only six of which involved isotopically labeled reagents. A total of 168 mg of (13)C5, (15)N-labeled epothilone D was prepared in an 8% overall yield from (13)C2, (15)N-labeled thioacetamide and (13)C3-labeled ethyl bromopyruvate.

A dispersive liquid-liquid microextraction and chiral separation of carvedilol in human plasma using capillary electrophoresis.

Development of simple, rapid and precise analysis of chiral drugs in biological samples is an important issue. Dispersive liquid-liquid microextraction in combination with CE using field amplified sample injection has been of interest because of its capability to analyze trace amount of drugs. Dispersive liquid-liquid microextraction-CE-field amplified sample injection was employed for chiral separation of carvedilol in human plasma using UV-DAD detector and the developed method has been validated according to US FDA method validation guideline for bioanalysis. The method was linear over a concentration range of 12.5-100 ng/ml for each carvedilol enantiomer (R(2) = 0.998) and the mean recoveries ranged from 91 to 107%. The method was adapted for sensitive, selective and rapid determination of carvedilol enantiomers in human plasma samples.

Characterization of the four designer benzodiazepines clonazolam, deschloroetizolam, flubromazolam, and meclonazepam, and identification of their in vitro metabolites

In 2012, the first designer benzodiazepines were offered in Internet shops as an alternative to prescription-only benzodiazepines. Soon after these compounds were scheduled in different countries, new substances such as clonazolam, deschloroetizolam, flubromazolam, and meclonazepam started to emerge. This article presents the characterization of these four designer benzodiazepines using nuclear magnetic resonance spectroscopy, gas chromatography–electron ionization-mass spectrometry, liquid chromatography–tandem mass spectrometry, liquid chromatography–quadrupole time-of-flight-mass spectrometry, and infrared spectroscopy. The major in vitro phase I metabolites of the substances were investigated using human liver microsomes. At least one monohydroxylated metabolite was identified for each compound. Dihydroxylated metabolites were found for deschloroetizolam and flubromazolam. For clonazolam and meclonazepam, signals at mass-to-charge ratios corresponding to the reduction of the nitro group to an amine were observed. Desalkylations, dehalogenations, or carboxylations were not observed for any of the compounds investigated. Furthermore, for clonazolam and meclonazepam, no metabolites formed by a combination of reduction and mono-/dihydroxylation were detected. This knowledge will help to analyze these drugs in biological samples.

Nanofiber Packed Solid-Phase Extraction for the Analysis of Plasma, Urine, and Saliva with High-Performance Liquid Chromatography

A convenient and green pre-processing method is presented for determining physiological targets and drugs in biological samples using nanofiber-packed solid-phase extraction instead of a traditional cartridge. Biological samples, including plasma (model target molecule is desloratadine), urine (catecholamines), and saliva (cortisol) were used to characterize sample pre-treatment. Nanofiber-based solid-phase extraction sufficiently isolated the compounds from the complex matrixes. The absolute extraction rates were between 52.3% and 85.7%. Because the nanofiber-packed solid-phase extraction cartridge is a small, lightweight, portable, convenient, and labor-saving device, this pre-processing method has great potential for further application.

Simultaneous derivatization and solid-based disperser liquid-liquid microextraction for extraction and preconcentration of some antidepressants and an antiarrhythmic agent in urine and plasma samples followed by GC-FID.

The present work is based on a one-step method including derivatization and solid-based disperser liquid-liquid microextraction followed by gas chromatography-flame ionization detection (GC-FID) for the determination of four antidepressants (fluoxetine, fluvoxamine, tranylcypromine, and nortriptyline) and an antiarrhythmic agent (mexiletine) in human urine and plasma samples. In this method, a mixture of 1,1,2,2-tetrachloroethane (extraction solvent) and butylchloroformate (derivatizing reagent) is added on a sugar cube (solid disperser) and it is introduced into an aqueous sample containing the analytes and a catalyst, e.g. 3-methylpyridine (picoline). During dissolving the sugar cube by manual shaking, the extractant and derivatization agent are gradually released into the sample as very fine droplets. Then the resulted cloudy solution is centrifuged and the sedimented phase is analyzed by GC-FID. The influence of several variables on the efficiency of derivatization/microextraction procedure such as kind and volume of extraction solvent, type and amount of disperser, amount of derivatization agent, and catalyst volume are optimized. Under the optimum conditions the calibration curves are linear in the range of 8-100,000μgL(-1) (coefficient of determination ≥0.994). The relative standard deviations are obtained in the range of 3.0-6.0% for all compounds. Moreover, the detection limits and enrichment factors of the target analytes are obtained in the ranges 1-15μgL(-1) and 228-268, respectively, for both plasma and urine samples. The relative recoveries obtained for the spiked plasma and urine samples are between 70 and 91%. The results show that the proposed method is simple, reliable, low cost, and applicable to determine trace amounts of the studied drugs in biological samples.

Ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction for the determination of eight drugs in biological samples by gas chromatography-triple quadrupole mass spectrometry

A novel microextraction technique based on ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction (UA-LDS-DLLME) has been developed for the determination of multiple drugs of abuse in biological samples by gas chromatography-triple quadrupole mass spectrometry (GC-QQQ-MS). A total of 100 µL of toluene as extraction solvent was dropped into the sample solution. Then the mixture was sonicated drastically in an ultrasonic bath for 3 min with occasional manual shaking to form a cloudy suspension. After centrifugation at 10,000 r/min for 3 min, the upper layer of low-density extractant was withdrawn and injected into the GC-QQQ-MS for analysis. The parameters affecting extraction efficiency have been investigated and optimized. Under the optimum conditions, good linearities were observed for all analytes with the correlation coefficients ranging from 0. 998 4 to 0. 999 4. The recoveries of 79.3%-100.3% with RSDs < 5.7% were obtained. The LODs (S/N = 3) were in the range from 0.05 to 0.40 µg/L. UA-LDS-DLLME technique has the advantages of less extraction time, suitable for batches of sample pretreatment simultaneously, and higher extraction efficiency. It was successfully applied to the analysis of amphetamines in real human urine samples.

Sensitive magnetic solid-phase microextraction based on oxide multi-walled carbon-nanotubes for the determination of methylamphetamine and ketamine in human urine and blood

A solid-phase microextraction (MSPE) procedure, combined with gas chromatography-mass spectrometry (GC-MS), was developed for the quantification of two illegal basic drugs in biological samples.

Determination of unconjugated non-steroidal anti-inflammatory drugs in biological fluids using air-assisted liquid–liquid microextraction combined with back extraction followed by high performance liquid chromatography

AALLME-HPLC-VWD method for determination of naproxen, diclofenac, and ibuprofen.

Application of nonionic surfactant as a new method for the enhancement of electromembrane extraction performance for determination of basic drugs in biological samples

This paper proposes for the first time the use of a nonionic surfactant for the enhancement of electromembrane extraction performance. The presence of nonionic surfactant in donor phase promotes ionic analytes efficient migration through the organic liquid membrane. Surfactant assisted electromembrane extraction coupled with capillary electrophoresis was used for the extraction of basic drugs (methamphetamine, ephedrine and methadone) from biological samples. Parameters that affect the extraction efficiency were investigated and optimized for the proposed method. Optimal extractions were accomplished with 2-nitrophenyl octyl ether as the supported liquid membrane, with 125V as the driving force and 0.2mM Triton X-100 with pH 5.0 in donor and pH 1 in acceptor solutions. Equilibrium extraction conditions were obtained after 20min of operation where the whole assembly agitated at 1000rpm. Under the optimum experimental conditions, good limits of detection (0.90–2.42ngmL−1), linearities (R2>0.9995), and repeatability of extraction (RSDs below 4.4%, n=5) were obtained. Finally, the developed method was applied to drug level monitoring in the biological samples including hair and urine samples and satisfactory results were obtained.

Synthesis of [13C4]-labeled ∆9-tetrahydrocannabinol and 11-nor-9-carboxy-∆9-tetrahydrocannabinol as internal standards for reducing ion suppressing/alteration effects in LC/MS-MS quantification.

(−)-∆9-Tetrahydrocannabinol is the principal psychoactive component of the cannabis plant and also the active ingredient in some prescribed drugs. To detect and control misuse and monitor administration in clinical settings, reference samples of the native drugs and their metabolites are needed. The accuracy of liquid chromatography/mass spectrometric quantification of drugs in biological samples depends among others on ion suppressing/alteration effects. Especially, 13C-labeled drug analogues are useful for minimzing such interferences. Thus, to provide internal standards for more accurate quantification and for identification purpose, synthesis of [13C4]-∆9-tetrahydro-cannabinol and [13C4]-11-nor-9-carboxy-∆9-tetrahydrocannabinol was developed via [13C4]-olivetol. Starting from [13C4]-olivetol the synthesis of [13C4]-11-nor-9-carboxy-∆9-tetrahydrocannabinol was shortened from three to two steps by employing nitromethane as a co-solvent in condensation with (+)-apoverbenone.

Synthesis of carbon-14 and stable isotope labeled Avagacestat: a novel gamma secretase inhibitor for the treatment of Alzheimer's disease.

Bristol-Myers Squibb and others are developing drugs that target novel mechanisms to combat Alzheimer's disease. γ-Secretase inhibitors are one class of potential therapies that have received considerable attention. (R)-2-(4-Chloro-N-(2-fluoro-4-(1,2,4-oxadiazol-3-yl)benzyl)phenylsulfonamido)-5,5,5-trifluoropentanamide (Avagacestat) is a γ-secretase-inhibiting drug that has been investigated by Bristol-Myers Squibb in preclinical and clinical studies. An important step in the development process was the synthesis of a carbon-14-labeled analog for use in a human absorption, distribution, metabolism, and excretion study and a stable isotope labeled analog for use as a standard in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. Carbon-14 labeled Avagacestat was synthesized in seven steps in a 33% overall yield from carbon-14 labeled potassium cyanide. A total of 5.95 mCi was prepared with a specific activity of 0.81 μCi/mg and a radiochemical purity of 99.9%. (13) C6 -Labeled Avagacestat was synthesized in three steps in a 15% overall yield from 4-chloro[(13) C6 ]aniline. A total of 585 mg was prepared with a ultraviolet purity of 99.9%.

Analysis of Local Anesthetics in Biological Samples via Kinetically Calibrated Liquid-Phase Solvent Bar Micro-Extraction Combined with HPLC

In this study, a liquid-phase solvent bar micro-extraction technique was used to investigate both the extraction and back-extraction processes of the target analyte. A novel concentration curve method and a classic time curve method, used under the same experimental conditions, verified the symmetry between the extraction process (target analyte moves from sample matrix to the organic solvent-based extraction phase) and the back-extraction process (target analyte moves from organic solvent to the sample matrix), providing the basis to use the target analyte in the back-extraction process to calibrate its extraction process. A quantitative calibration can be achieved using back extraction on the target analyte from the blank sample matrix in the organic solvent. Information from the process of back extraction of the target analyte, such as the time constant a, can be directly used to calculate the initial concentration of the target analyte in the sample matrix. This new kinetic calibration method employs a liquid-phase solvent bar micro-extraction technique combined with high-performance liquid chromatography with a diode array detector (HPLC-DAD) and was successfully used to analyze three local anesthetics in biological samples; it extends the application of the kinetic calibration to HPLC-DAD and establishes a novel, simple and accurate method to determine the concentration of the free drug in biological samples and its protein-binding ratio.