Green HPLC-DAD and HPTLC Methods for Quantitative Determination of Binary Mixture of Pregabalin and Amitriptyline Used for Neuropathic Pain Management.

First analytical methods were herein developed for determination of pregabalin (PGB) and amitriptyline (AMT) as an active binary mixture used for management of neuropathic pain whether in pure forms or in human biological fluids (plasma/urine). First method is green high-performance liquid chromatography-diode array detector (HPLC-DAD) after derivatization of PGB with ninhydrin (NIN) on a reversed-phase C18 column using a mobile phase consisting of ethanol:water (97:3%, v/v) pumped isocratically at 0.8 mL/min; AMT were scanned at 215 nm, whereas PGB-NIN was scanned at 580 nm. Second method is High-performance thin-layer chromatography (HPTLC), where PGB and AMT were separated on silica gel HPTLC F254 plates, using ethanol:ethyl acetate:acetone:ammonia solution (8:2:1:0.05, by volume) as a developing system. AMT peaks were scanned at 220 nm, whereas PGB peaks were visualized by spraying 3% (w/v) ethanolic NIN solution and scanning at 550 nm. Linear calibration curves were obtained for human plasma and urine spiked with PGB and AMT over the ranges of 5-100 µg/mL and 0.2-2.5 µg/band for PGB, and 1-100 µg/mL and 0.1-2.0 µg/band for AMT for HPLC-DAD and HPTLC methods, respectively. The suggested methods were validated according to Food and Drug Administration guidelines for bioanalytical methods validation and they can be applied for routine therapeutic drug monitoring for the concerned drugs.

Urine Drug Tests: Ordering and Interpreting Results.

Urine drug testing is an essential component of monitoring patients who are receiving long-term opioid therapy, and it has been suggested for patients receiving long-term benzodiazepine or stimulant therapy. Family physicians should be familiar with the characteristics and capabilities of screening and confirmatory drug tests. Immunoassays are qualitative tests used for initial screening of urine samples. They can give false-positive and false-negative results, so all results are considered presumptive until confirmatory testing is performed. Immunoassays for opioids may not detect commonly prescribed semisynthetic and synthetic opioids such as methadone and fentanyl; similarly, immunoassays for benzodiazepines may not detect alprazolam or clonazepam. Immunoassays can cross-react with other medications and give false-positive results, which have important implications for a patient's pain treatment plan. False-negative results can cause missed opportunities to detect misuse. Urine samples can be adulterated with other substances to mask positive results on urine drug testing. Family physicians must be familiar with these substances, the methods to detect them, and their effects on urine drug testing.

Determination of fluvoxamine maleate in human urine and human serum using alkaline KMnO4 -rhodamine B chemiluminescence.

The flow-injection chemiluminescence (FI-CL) behavior of a gold nanocluster (Au NC)-enhanced rhodamine B-KMnO4 system was studied under alkaline conditions for the first time. In the present study, the as-prepared bovine serum albumin-stabilized Au NCs showed excellent stability and reproducibility. The addition of trace levels of fluvoxamine maleate (Flu) led to an obvious decline in CL intensity in the rhodamine B-KMnO4 -Au NCs system, which could be used for quantitative detection of Flu. Under optimized conditions, the proposed CL system exhibited a favorable analytical performance for Flu determination in the range 2 to 100 µg ml-1 . The detection limit for Flu measurement was 0.021 µg ml-1 . Moreover, this newly developed system revealed outstanding selectivity for Flu detection when compared with a multitude of other species, such as the usual ions, uric acid and a section of hydroxy compounds. Additionally, CL spectra, UV-visible spectroscopes and fluorescence spectra were measured in order to determine the possible reaction mechanism. This approach could be used to detect Flu in human urine and human serum samples with the desired recoveries and could have promising application under physiological conditions.

Analytical methodologies for the determination of benzodiazepines in biological samples.

Benzodiazepine drugs belong to important and most widely used medicaments. They demonstrate such therapeutic properties as anxiolytic, sedative, somnifacient, anticonvulsant, diastolic and muscle relaxant effects. However, despite the fact that benzodiazepines possess high therapeutic index and are considered to be relatively safe, their use can be dangerous when: (1) co-administered with alcohol, (2) co-administered with other medicaments like sedatives, antidepressants, neuroleptics or morphine like substances, (3) driving under their influence, (4) using benzodiazepines non-therapeutically as drugs of abuse or in drug-facilitated crimes. For these reasons benzodiazepines are still studied and determined in a variety of biological materials. In this article, sample preparation techniques which have been applied in analysis of benzodiazepine drugs in biological samples have been reviewed and presented. The next part of the article is focused on a review of analytical methods which have been employed for pharmacological, toxicological or forensic study of this group of drugs in the biological matrices. The review was preceded by a description of the physicochemical properties of the selected benzodiazepines and two, very often coexisting in the same analyzed samples, sedative-hypnotic drugs.

Pregabalin Abuse amongst Opioid Substitution Treatment Patients.

Pregabalin (Lyrica®) is used in treating epilepsy, nerve pain and anxiety. Pregabalin was initially thought to have a low misuse potential however there are emerging reports of Pregabalin being abused. A study was commenced at the National Drug Treatment Centre's (NDTC) Drug Analysis Laboratory to determine the level of usage of Pregabalin within the addiction services population in Ireland. A total of 498 urine samples representing samples from 440 individual opioid substitution patients, initially screened by immunoassay for drugs of abuse, were subjected to further analysis for Pregabalin by Liquid Chromatography/Mass Spectrometry (LC/MS). Of 440 patients tested, 39 tested positive for Pregabalin (9.2%). Only 10 patients from this group were prescribed this drug to our knowledge thus giving an estimated rate of misuse of 7.0%. Other drugs detected in the Pregabalin positive patients were Opiates (31.8%), Cocaine (11.4%), Benzodiazepines (79.5%) and Cannabis (77.8%). Our study confirms that Pregabalin abuse is taking place amongst the addiction services population. We believe that misuse of this prescription drug is a serious emerging issue which should be monitored carefully.

Out of the frying pan, into the fire: a case of heat shock and its fatal complications.

Exertional heat stroke incidence is on the rise and has become the third leading cause of death in high school athletes. It is entirely preventable, yet this is a case of a 15-year-old, 97-kg male football player who presented unresponsive and hyperthermic after an August football practice. His blood pressure was 80/30, and his pulse was 180. He had a rectal temperature of 107.3°F, and upon entering the emergency department, he was rapidly cooled in 40 minutes. As he progressed, he developed metabolic acidosis, elevated liver enzymes, a prolapsed mitral valve with elevated troponin levels, and worsening hypotension even with extracorporeal membrane oxygenation support. After 3 days in the hospital, this young man was pronounced dead as a result of complications from exertional heat stroke. We address not only the complications of his hospital course relative to his positive blood cultures but also the complications that can result from attention-deficit/hyperactivity disorder medication our patient was taking. As the population of young adults becomes more obese and more highly medicated for attention-deficit/hyperactivity disorder, we sought out these growing trends in correlation with the increase in incidence of heat-related illness. We also address the predisposing factors that make young high school athletes more likely to experience heat illness and propose further steps to educate this susceptible population.

Optimization of dispersive liquid-liquid microextraction with central composite design for preconcentration of chlordiazepoxide drug and its determination by HPLC-UV.

A simple, rapid, and sensitive method based on dispersive liquid-liquid microextraction combined with HPLC-UV detection applied for the quantification of chlordiazepoxide in some real samples. The effect of different extraction conditions on the extraction efficiency of the chlordiazepoxide drug was investigated and optimized using central composite design as a conventional efficient tool. Optimum extraction condition values of variables were set as 210 µL chloroform, 1.8 mL methanol, 1.0 min extraction time, 5.0 min centrifugation at 5000 rpm min(-1), neutral pH, 7.0% w/v NaCl. The separation was reached in less than 8.0 min using a C18 column using isocratic binary mobile phase (acetonitrile/water (60:40, v/v)) with flow rate of 1.0 mL min(-1) The linear response (r(2) > 0.998) was achieved in the range of 0.005-10 µg mL(-1) with detection limit 0.0005 µg mL(-1) The applicability of this method for simultaneous extraction and determination of chlordiazepoxide in four different matrices (water, urine, plasma, and chlordiazepoxide tablet) were investigated using standard addition method. Average recoveries at two spiking levels were over the range of 91.3-102.5% with RSD < 5.0% (n = 3). The obtained results show that dispersive liquid-liquid microextraction combined with HPLC-UV is a fast and simple method for the determination of chlordiazepoxide in real samples.

Acute intoxication caused by overdose of flunitrazepam and triazolam: high concentration of metabolites detected at autopsy examination.

A 52-year-old woman was found dead on the floor of the living room on the first floor of a house, which belonged to the man with whom she shared the house. On visiting the site, her clothes were found to be undisturbed. Packages of flunitrazepam (Silece, 2 mg/tablet) and triazolam (Halcion, 0.25 mg/tablet) were found strewn around the victim. Toxicological analysis was performed, and the concentrations of flunitrazepam, triazolam, and their metabolites in the victim's blood and urine were measured by high-performance liquid chromatography coupled with photodiode array and mass spectrometry. A high blood concentration of 7-aminoflunitrazepam was detected (1,270 ng/g), and further metabolites such as 7-acetamidoflunitrazepam, 7-acetamidodesmethylflunitrazepam, and 7-aminodesmethylflunitrazepam were detected in the blood and urine samples. In addition, 4-hydroxytriazolam and α-hydroxytriazolam were detected in her urine at a concentration of 950 and 12,100 ng/mL, respectively.On the basis of the autopsy findings and toxicology results of high concentrations of both flunitrazepam and triazolam derivatives, the cause of death was determined to be acute intoxication from flunitrazepam and triazolam.

Applying cloud point extraction technique for the extraction of oxazepam from human urine as a colour or fluorescent derivative prior to spectroscopic analysis methods.

Two new methods based on cloud point extraction (CPE) technique were developed and optimized for the extraction and preconcentration of oxazepam from human urine, as an azo or fluorescent derivative. The first method is a spectrophotometric one, which is based on the acid hydrolysis of the oxazepam to a benzophenone, diazotization of the benzophenone, and then the coupling with oxine to form an azo dye. The second method is a spectrofluorimetric one, which involves reduction of the target compound using Zn°/HCl at room temperature with the formation of a highly fluorescent derivative. The main factors affecting the chemical reactions and CPE were investigated and optimized systematically. Under optimum experimental conditions, the calibration graphs were linear in the range of 0.1 to 1.5 (0.05 to 2.0) µg/ml with correlation coefficients of 0.9989 (0.9985), for the CPE-spectrophotometric (CPE-spectrofluorimetric) method. The limit of detection was found to be 0.034 (0.018) µg/ml and the relative standard deviation was calculated to be 1.35 (2.52)%. Recoveries in the spiked samples ranged from 87 to 94%. Finally, the proposed methods were applied to the determination of oxazepam in human urine.

[Pharmacokinetics of afobazole metabolite (M-11) in rats].

Pharmacokinetics of compound M-11 (main metabolite of afobazole) after administration via different routes was studied in rats. After oral and intravenous administration, M-11 exhibited weakly pronounced bioconversion with the formation of a few metabolites that could be detected in plasma samples for about 3 hours. The absolute bioavailability of M-11 after oral administration was 68.3%. It was found that M-11 was completely absorbed from gastrointestinal tract of rats and characterized by "the first pass effect", after which approximately 70% of administered dose entered the circulation. The parent substance was determined neither in urine nor in feces.

Metabolites of lorazepam: Relevance of past findings to present day use of LC-MS/MS in analytical toxicology.

The advent of liquid chromatography-tandem mass spectrometry (LC-MS/MS), with the sensitivity it confers, permits the analysis of both phase I and II drug metabolites that in the past would have been difficult to target using other techniques. These metabolites may have relevance to current analytical toxicology employing LC-MS/MS, and lorazepam was chosen as a model drug for investigation, as only the parent compound has been targeted for screening purposes. Following lorazepam administration (2 mg, p.o.) to 6 volunteers, metabolites were identified in urine by electrospray ionization LC-MS/MS, aided by the use of deuterated analogues generated by microsomal incubation for use as internal chromatographic and mass spectrometric markers. Metabolites present were lorazepam glucuronide, a quinazolinone, a quinazoline carboxylic acid, and two hydroxylorazepam isomers, one of which is novel, having the hydroxyl group located on the fused chlorobenzene ring. The quinazolinone, and particularly the quinazoline carboxylic acid metabolite, provided longer detection windows than lorazepam in urine extracts not subjected to enzymatic hydrolysis, a finding that is highly relevant to toxicology laboratories that omit hydrolysis in order to rapidly reduce the time spent on gas chromatography-mass spectrometry (GC-MS) analysis. With hydrolysis, the longest windows of detection were achieved by monitoring lorazepam, supporting the targeting of the aglycone with free drug for those incorporating hydrolysis in their analytical toxicology procedures.

The trazodone metabolite meta-chlorophenylpiperazine can cause false-positive urine amphetamine immunoassay results.

Amphetamines and methamphetamines are part of an important class of drugs included in most urine drugs of abuse screening panels, and a common assay to detect these drugs is the Amphetamines II immunoassay (Roche Diagnostics). To demonstrate that meta-chlorophenylpiperazine (m-CPP), a trazodone metabolite, cross-reacts in the Amphetamines II assay, we tested reference standards of m-CPP at various concentrations (200 to 20,000 g/L). We also tested real patient urine samples containing m-CPP (detected and quantified by HPLC) with no detectable amphetamine, methamphetamine, or MDMA (demonstrated by GC MS). In both the m-CPP standards and the patient urine samples, we found a strong association between m-CPP concentration and Amphetamines II immunoreactivity (r = 0.990 for the urine samples). Further, we found that patients taking trazodone can produce urine with sufficient m-CPP to result in false-positive Amphetamines II results. At our institution, false-positive amphetamine results occur not infrequently in patients taking trazodone with at least 8 trazodone-associated false-positive results during a single 26-day period. Laboratories should remain cognizant of this interference when interpreting results of this assay.

Imprinted functionalized silica sol-gel for solid-phase extraction of triazolamin.

A triazolam-imprinted silica microsphere was prepared by combining a surface molecular-imprinting technique with the sol-gel process. The results illustrate that the triazolam-imprinted silica microspheres provided using γ-aminopropyltriethoxysilane and phenyltrimethoxysilane as monomers exhibited higher selectivity than those provided from γ-aminopropyltriethoxysilane and methyltriethoxysilane. In addition, the optimum affinity occurred when the molar ratio of γ-aminopropyltriethoxysilane, phenyltrimethoxysilane, and the template molecule was 4.2:4.7:0.6. Retention factor (k) and imprinting factor (IF) of triazolam on the imprinted and non-imprinted silica microsphere columns were characterized using high performance liquid chromatography (HPLC) with different mobile phases including methanol, acetonitrile, and water solutions. The molecular selectivity of the imprinted silica microspheres was also evaluated for triazolam and its analogue compounds in various mobile phases. The better results indicated that k and IF of triazolam on the imprinted silica microsphere column were 2.1 and 35, respectively, when using methanol/water (1/1, v/v) as the mobile phase. Finally, the imprinted silica was applied as a sorbent in solid-phase extraction (SPE), to selectively extract triazolam and its metabolite, α-hydroxytriazolam, from human urine samples. The limits of detection (LOD) for triazolam and α-hydroxytriazolam in urine samples were 30 ± 0.21 ng mL(-1) and 33 ± 0.26 ng mL(-1), respectively.

Pharmacokinetics, tissue distribution, and excretion of buagafuran in rats.

The pharmacokinetics, tissue distribution, and excretion of buagafuran (BF, 4-butyl-α-agarofuran), a promising antianxiety drug isolated from Gharu-wood (Aquilaria agallocha Roxb), were investigated in rats. BF plasma concentration was determined in rats after oral and intravenous doses by GC-TOF-MS. BF showed nonlinear pharmacokinetics after oral and intravenous administration of 4, 16, and 64 mg/kg. The AUC(0-∞) and C(max) did not increase proportionally with doses, indicating the saturation in absorption kinetics of BF in rats after oral dosage. BF absorption was extremely poor with an absolute bioavailability below 9.5%. After oral administration of (3)H-BF (4 mg/kg) to rats, radioactivity was well distributed to the tissues examined. The highest radioactivity was found in gastrointestinal tract, followed by liver and kidney. Radioactivity in brain, as a target organ, was about 20-40% of that in plasma at all time points. Total mean percent recovery of radioactive dose was about 80% in rats (51.2% in urine; 28.7% in feces). Bile elimination was also the major excretion route of BF, and 45.4% of the radioactive dose was recovered in bile.

Characterization and evaluation of two-dimensional microfluidic chip-HPLC coupled to tandem mass spectrometry for quantitative analysis of 7-aminoflunitrazepam in human urine.

Microfluidic chip-based high-performance-liquid-chromatography coupled to mass spectrometry (chip-HPLC-MS) has been widely used in proteomic research due to its enhanced sensitivity. We employed a chip-HPLC-MS system for determining small molecules such as drug metabolites in biological fluids. This chip-HPLC-MS system integrates a microfluidic switch, a 2-dimensional column design including an enrichment column (160 nL) for sample pre-concentration and an analytical column for chromatographic separation, as well as a nanospray emitter on a single polyimide chip. In this study, a relatively large sample volume (500 nL) was injected into the enrichment column for pre-concentration and an additional 4 µL of the initial mobile phase was applied to remove un-retained components from the sample matrix prior to chromatographic separation. The 2-dimensional column design provides the advantages of online sample concentration and reducing matrix influence on MS detection. 7-Aminoflunitrazepam (7-aminoFM2), a major metabolite of flunitrazepam (FM2), was determined in urine samples using the integrated chip-HPLC-MS system. The linear range was 0.1-10 ng mL(-1) and the method detection limit (signal-to-noise ratio of 3) was 0.05 ng mL(-1) for 7-aminoFM2. After consecutive liquid-liquid extraction (LLE) and solid-phase extraction (SPE), the chip-HPLC-MS exhibited high correlation between 7-aminoFM2 spiked Milli-Q water and 7-aminoFM2 spiked urine samples. This system also showed good precision (n = 5) and recovery for spiked urine samples at the levels of 0.1, 1.0, and 10 ng mL(-1). Intra-day and inter-day precision were 2.0-7.1% and 4.3-6.0%, respectively. Clinical urine samples were also analyzed by this chip-HPLC-MS system and acceptable relative differences (-1.3 to -13.0%) compared with the results using a GC-MC method were determined. Due to its high sensitivity and ease of operation, the chip-HPLC-MS system can be utilized for the determination of small molecules such as drug metabolites and neurotransmitters in biological fluids for clinical diagnosis.

Gas chromatography-mass spectrometry detection of a norfluoxetine artifact in hydrolyzed urine samples may falsely indicate tranylcypromine ingestion.

In several cases, fluoxetine, its metabolites, its known artifacts, and supposedly tranylcypromine were detected in urine using the authors' systematic toxicological analysis (STA) procedure based on acid hydrolysis, extraction, and acetylation. As fluoxetine and tranylcypromine are absolutely contraindicated drugs and in none of the cases was tranylcypromine prescribed, the question of whether the detected compound might have been formed by fluoxetine and/or its metabolites arose. Therefore, rat urine taken after dosing with fluoxetine was screened in the same way. In addition, aqueous solutions of fluoxetine, norfluoxetine, tranylcypromine, and a mixture of the latter two drugs were worked-up and analyzed according to the STA and without hydrolysis. In urine specimens obtained from rats dosed with fluoxetine, tranylcypromine was detected as well as in the solution of worked-up norfluoxetine including hydrolysis. Its underlying mass spectrum could be identified by detailed interpretation of the fragmentation patterns as acetylated 3-phenyl-propyl-2-ene-amine. This compound could be postulated as hydrolysis product of norfluoxetine formed by ether cleavage and water elimination. Although this spectrum shows nearly the same fragmentation patterns as that of acetylated tranylcypromine, both compounds could finally be differentiated by their retention indices and by using the positive-ion chemical ionization mode.

Urinary detection times and excretion patterns of flunitrazepam and its metabolites after a single oral dose.

We investigated the excretion profiles of flunitrazepam metabolites in urine after a single dose. Sixteen volunteers received either 0.5 or 2.0 mg flunitrazepam. Urine samples were collected after 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 240, and 336 h. Samples were screened using CEDIA (300 microg/L cutoff) and quantitated using liquid chromatography-tandem mass spectrometry. The cutoff was 0.5 microg/L for flunitrazepam, N-desmethylflunitrazepam, 7-aminoflunitrazepam, 7-aminodesmethylflunitrazepam, 7-acetamidoflunitrazepam, and 7-acetamidodesmethylflunitrazepam. None of the subjects receiving 0.5 mg were screened positive, and only 23 of 102 samples from the subjects given 2.0 mg were positive with CEDIA. The predominant metabolites were 7-aminoflunitrazepam and 7-aminodesmethylflunitrazepam. For all subjects given the low dose, 7-aminoflunitrazepam was detected up to 120 h, and for two subjects for more than 240 h. Seven subjects given the high dose were positive up to 240 h for 7-aminoflunitrazepam. We conclude that the ratio 7-aminodesmethylflunitrazepam to 7-aminoflunitrazepam increased with time, independent of dose, and may be used to estimate the time of intake. For some low-dose subjects, the metabolite concentrations in the early samples were low and a chromatographic method may fail to detect the intake. We think laboratories should consider this when advising police and hospitals about sampling as well as when they set up strategies for analysis.

Excretion of afobazole and its metabolites with urine and feces in rats.

The amount of afobazole and identified metabolites was measured in the urine and feces of rats after intraperitoneal and peroral administration of the drug in a dose of 25 mg/kg. Over 1 day after intraperitoneal or peroral treatment with afobazole, urine and feces contained 0.1% initial compound (from administered dose) and 42.1% metabolites.

Determination of diazepam and its metabolites in human urine by liquid chromatography/tandem mass spectrometry using a hydrophilic polymer column.

Diazepam and its major metabolites, nordazepam, temazepam and oxazepam, in human urine samples, were analyzed by liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using a hydrophilic polymer column (MSpak GF-310 4B), which enables direct injection of crude biological samples. Matrix compounds in urine were eluted first from the column, while the target compounds were retained on the polymer stationary phase. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All compounds showed base-peak ions due to [M+H]+ ions on LC/MS with positive ion electrospray ionization, and product ions were produced from each [M+H]+ ion by LC/MS/MS. Quantification was performed by selected reaction monitoring. All compounds spiked into urine showed method recoveries of 50.1-82.0%. The regression equations for all compounds showed excellent linearity in the range of 0.5-500 ng/mL of urine. The limits of detection and quantification for each compound were 0.1 and 0.5 ng/mL of urine, respectively. The intra- and inter-day coefficients of variation for all compounds in urine were not greater than 9.6%. The data obtained from actual determination of diazepam and its three metabolites, oxazepam, nordazepam and temazepam, in human urine after oral administration of diazepam, are also presented.