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Phosphorus (P) recovery was carried out through struvite precipitation from urines. Human urine, however, contains not only high nutrients for plants, such as P and nitrogen, but also pharmaceuticals and hormones. In this work, effects of magnesium (Mg) dose (in terms of Mg:P ratio) on P recovery efficiency and pharmaceutical amounts contained in struvite were investigated. Batch-scale experiments of synthetic and human urines revealed that struvite precipitation formed more X-shaped crystals with an increased molar ratio of Mg:P, while the amount of pharmaceuticals (tetracycline, demeclocycline, and oxytetracycline) in struvite decreased with an increased molar ratio of Mg:P. The lowest pharmaceutical amounts in struvite were found at the Mg:P ratio of 2:1 from both samples. Moreover, the maximum P recovery efficiency, quantity and purity of struvite were found in the range of 1.21 to 2:1. It indicated that the molar ratio of Mg:P has a significant impact on struvite precipitation in terms of pharmaceutical amounts in struvite; morphology, quantity and purity of struvite; and P recovery.

A rapid and sensitive ultra-HPLC/MSIMS (UHPLC/MSIMS) assay method for the simultaneous determination in human urine of 22 drugs belonging to different pharmaceutical groups was developed. The drugs were extracted from urine samples and then separated on a Zorbax Rapid Resolution High Definition SB-C18 column. The mobile phase consisted of methanol and water containing formic acid with gradient elution. The chromatographic separation time was 7 min. The MSIMS detector, equipped with an electrospray ionization source, was set up in both positive and negative modes. The lower LOQs for the drugs in this method were between 0.05 and 0.60 ng/mL. Calibration curves in human urine were generated in the range of 0.05-600 ng/mL. Method validation parameters such as intraday and interday precision, accuracy, extraction recovery, stability, selectivity, dilution integrity, and carryover effect for all the compounds were within the acceptable ranges. This simple and fast method was applied successfully to study the pharmacokinetics of four selected drugs in human urine collected from patients. This UHPLC/MS/MS method offers an attractive way forward for the development of a routine rapid analysis for selected substances, particularly given the growing amount of new information about drug properties.

The purpose of this study is to conduct a retrospective data analysis for inter-laboratory cross-validation studies to set a reasonable and practical acceptance criterion based on a number of cross-validation results. From the results of cross-validation studies for 16 compounds and their metabolites, analytical bias and variation were evaluated. The accuracy of cross-validation samples was compared with that of quality control (QC) samples with statistical comparison of the analytical variation. An acceptance criterion was derived with a confidential interval approach. As the results, while a larger bias was observed for the cross-validation samples, the bias was not fully caused by analytical variation or bias attributable to the analytical methods. The direction of the deviation between the cross-validation samples and QC samples was random and not concentration-dependent, suggesting that inter-laboratory variability such as preparation errors could be a source of bias. A derived acceptance criterion corresponds to one prescribed in the Guideline on bioanalytical method validation from the Ministry of Health, Labour and Welfare in Japan and is a little wider than one in the European Medical Agency. In conclusion, thorough retrospective data analysis revealed potential causes of larger analytical bias in inter-laboratory cross-validation studies. A derived acceptance criterion would be practical and reasonable for the inter-laboratory cross-validation study.

In addition to having selective potency against the molecular target(s), a compound must be able to reach its intended site of action in vivo in sufficient quantity and for the appropriate duration of time to exert a biological effect. The fate of a compound after in vivo administration depends upon its absorption, distribution, metabolism, and excretion (ADME), as well as its target residence time. The concentration of the compound in the blood, plasma, and other tissues represents the sum of all of these processes. Described in this unit are protocols for administering a compound by various routes to rats and for collecting the appropriate samples to determine the pharmacokinetics profile. The basic terms used in pharmacokinetics studies are defined, and representative examples are given to illustrate important variables to consider., (Copyright © 2014 John Wiley & Sons, Inc.)

In recent years, drug-facilitated crime (DFC) has become an increasing problem. A minimum list of 80 analytes to be monitored in such cases has been proposed by the Society of Forensic Toxicologists (SOFT) including the recommended minimum performance limits (RMPL). In the present study, two liquid chromatography-tandem mass spectrometry-based screening procedures, one in positive (method I) and one in negative (method II) electrospray ionization mode were developed and validated. Gradient elution was performed on a ZORBAX Eclipse XDB-C18 column after protein precipitation of the urine samples. Detection was carried out in the scheduled multiple reaction monitoring (MRM) mode monitoring two transitions per compound. A total of 100 analytes (91 basic in method I and nine acidic in method II) could be identified using the described procedure. No interferences were observed in 30 tested blank urine samples. The RMPLs were achieved for all analytes and ranged from 1 ng/mL for fentanyl to 10 μg/mL for γ-hydroxybutyrate (GHB). Matrix effects (ME) were evaluated using the same 30 urine samples and ranged from -90 % for tetrazepam to >6,000 % for the 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH). The relative standard deviations of ME were below 25 % for the vast majority of analytes. Results for urine specimens from nine authentic DFC cases were always negative with exception of drugs prescribed to the victims. Reanalysis with the developed procedure of 24 urine samples, with a positive screening result during routine clinical toxicology analysis, confirmed the routine findings. In an excretion study after a single oral doxylamine dose (30 mg), the parent drug and its nor metabolite could be detected in urine specimens from a young female volunteer for 10 days. The developed procedure allows a selective and sensitive screening of urine samples for almost all recommended analytes relevant in DFC cases.

Amphetamines, cannabinoids, cocaine, opiates, methadone, and benzodiazepines in authentic hair samples with drug concentrations around the medical and psychological assessment (MPA) guidelines cut-offs were screened by LUCIO-direct ELISA kits. Following confirmation of all positive and a significant number of negatively screened samples with gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods accredited for forensic purposes. Receiver operating characteristics (ROC) were plotted and the area under the curve (AUC) and overall misclassification rate (OMR) were calculated and compared to those obtained for the same drug classes in urine. While fulfilling the validation criteria of the German forensic guidelines, for almost all screening tests in hair and urine the AUC were greater than 0.8, indicating good to excellent performance. Moreover the AUC calculated for the detection of drugs in hair did not differ significantly to the AUC calculated for the detection of the same drug classes in urine, thus showing a comparable screening performance to the well accepted, previously published application of the same ELISAs for the detection of drugs at unconventionally low cut-offs in urine. For the first time, the validation of the immunoassay tests for the complete 6-drug panel MPA profile in hair and urine using a large population of authentic hair and urine samples with drug concentrations around MPA cut-offs, lower than conventional clinical or workplace drug testing guidelines cut-offs as well as those suggested by the Society of hair testing (SoHT) is presented., (Copyright © 2014 John Wiley & Sons, Ltd.)

A procedure based on solid-phase extraction (SPE) followed by ultra-high-performance liquid chromatography (UHPLC) with UV detection has been developed for the analysis of multiple drugs in human urine. The compounds evaluated were aliskiren, prasugrel, rivaroxaban, prednisolone, propranolol, ketoprofen, nifedipine, naproxen, terbinafine, ibuprofen, diclofenac, sildenafil and acenocoumarol. Seventeen different solid phase extraction (SPE) cartridges were tested to evaluate their applicability for the isolation of drugs from human urine. Comparison were recovery of different drugs and reproducibility. The samples were analyzed by UHPLC using a Poroshell 120 EC-C18 column and acetonitrile -0.05% TFA in water as the mobile phase under gradient elution conditions. SPE combined with UHPLC-UV allowed the determination of drugs over a linear range of 0.01-30.0μg/mL, with limits of detection at 0.003-0.217μg/mL and precision of 0.8-7.1%. Phenyl (C6H5) sorbent was found to provide the most effective clean-up, removing the greatest amount of interfering substance and simultaneously ensuring analyte recoveries higher than 85.5% with relative standard deviations (RSD) <10%. The method was applied with good accuracy and precision in the determination of drugs in human urine obtained from patients treated with selected drugs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Although chemical derivatization for signal enhancement in drug testing is most often associated with gas chromatography, it also has the potential to improve the detection of analytes poorly ionized by atmospheric pressure ionization techniques, such as electrospray ionization used in liquid chromatography-mass spectrometry. A number of acidic compounds, namely drug glucuronides (e.g. conjugates of temazepam, oxazepam, lorazepam, morphine, testosterone, epitestosterone, 5-α-dihydrotestosterone, androsterone, p-nitrophenol, and paracetamol) were successfully derivatized with tris(trimethoxyphenyl) phosphoniumpropylamine to introduce a quaternary cation functionality to the analytes. Benzodiazepine glucuronides were more specifically investigated, and following positive mode electrospray ionization mass spectrometry, average improvements to peak areas as a result of derivatization were 67-, 6-, and 7- fold for temazepam, oxazepam, and lorazepam glucuronides. Average improvements to the signal-to-noise ratios for temazepam, oxazepam, and lorazepam glucuronides were 1336-, 371- and 217-fold, respectively. The values obtained for the derivatized conjugate were also typically higher than those for the underivatized parent drug. Urine containing benzodiazepine glucuronides was also successfully derivatized. The data indicates potential for the use of charge derivatization to improve the detection of molecules with acidic functionalities by liquid chromatography-mass spectrometry (LC-MS) techniques in certain scenarios., (Copyright © 2014 John Wiley & Sons, Ltd.)

This contribution describes properties and utilization of free liquid membranes (FLMs) in micro-electromembrane extraction (μ-EME) of analytes from samples with complex matrices. An FLM was formed as a plug of a selected organic solvent, 1-ethyl-2-nitrobenezene (ENB) or 2-nitrophenyloctyl ether, in a narrow bore polymeric tubing and was sandwiched between a plug of aqueous donor and aqueous acceptor solution. The FLM acted as a phase interface that enabled selective transfer of analytes from donor into acceptor solution. Acceptor solution after μ-EME was analysed by capillary electrophoresis (CE). Fundamental characteristics of FLMs were depicted and discussed by presenting experimental data on their performance for various basic operational parameters, such as composition and volume of donor/acceptor solution, applied extraction voltage, thickness of FLM and extraction time. Positively charged basic drugs (nortriptyline, haloperidol and loperamide) and their solutions in water, urine and blood serum served as model samples. It was shown that FLMs may offer fast, efficient and selective pretreatment of crude biological samples providing that basic operational parameters of μ-EME are set properly. At optimised conditions, basic drugs in 1.5μL of a biological sample were transferred across 1.5μL of FLM (ENB) into 1.5μL of acceptor solution in about 5min at an extraction voltage of 100V. Repeatability values of μ-EMEs and CE-UV analyses of the three basic drugs were better than 7.7% for peak areas, recoveries ranged between 19 and 52% and linear relationship was obtained for analytical signal vs. concentration in 1-50mgL(-1) range (r(2) better than 0.996). Limits of detection, defined as 3×S/N, were below 1mgL(-1) for all examined matrices., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Recent analyses demonstrated that metabolites are unlikely to contribute significantly to clinical inhibition of cytochrome P450 (P450)-mediated drug metabolism, and that only ∼2% of this type of drug interaction could not be predicted from the parent drug alone. Due to generally increased polarity and decreased permeability, metabolites are less likely to interact with P450s, but their disposition is instead more likely to involve transporters. This commentary presents case studies illustrating the potential importance of transporters as determinants of metabolite disposition, and as sites of drug interactions, which may alter drug efficacy and safety. Many of these examples are hydrophilic phase II conjugates involved in enterohepatic cycling, where modulation of transporter-dependent disposition may alter pharmacokinetics/pharmacodynamics. The case studies suggest that characterization of metabolite disposition, toxicology, and pharmacology should not focus solely on metabolites with appreciable systemic exposure, but should take into consideration major excretory metabolites. A more thorough understanding of metabolite (phase I and II; circulating and excreted) transport properties during drug development may provide an improved understanding of complex drug-drug interactions (DDIs) that can alter drug and/or metabolite systemic and intracellular exposure. Knowledge and capability gaps remain in clinical translation of in vitro and animal data regarding metabolite disposition. To this end, useful experimental and modeling approaches are highlighted. Application of these tools may lead to a better understanding of metabolite victim and perpetrator DDI potential, and ultimately the establishment of approaches for prediction of pharmacodynamic and toxicodynamic consequences of metabolite transport modulation.

Little research using dried blood spot samples to assess adherence to medication has been reported. The World Health Organisation estimates that only half of the patients in the developed world take their medication as prescribed. Additional costs to the healthcare provider include wasted medicines, avoidable additional hospital visits and non-optimum patient care. There is little evidence of information concerning medication adherence being made available to inform clinical decision making. In this article we explore the potential of the dried blood spot sample collection methodology as a means of identifying medication adherence to facilitate medicines optimization for a range of disparate diseases. Furthermore, the opportunity to personalize healthcare for different patients by assessing the clinically necessary therapeutic level of the relevant drugs is highlighted.

The NAD(+) depending enzyme SIRT1 regulates the mitochondrial biogenesis, fat and glucose metabolism through catalyzing the deacetylation of several metabolism-related protein-substrates. Recently, synthetic activators of SIRT1 referred to as STACs (Sirtuin activating compounds, e.g. SRT2104) were identified and tested in clinical studies for the treatment of aging-related diseases such as type 2 diabetes, Alzheimer's and obesity. Although the mechanism of SIRT1 activation by small molecules has caused considerable controversy, STACs demonstrated a significant performance enhancement in mice experiments including an improvement of endurance, muscle strength, and locomotor behavior. Due to their potential to increase exercise tolerance in healthy individuals, SIRT1 activators are currently being monitored by anti-doping authorities. In the present study, the in vivo metabolic clearance of three SIRT1 activators was investigated in rats by the collection of urine, DBS (dried blood spots) and plasma samples following a single oral administration. The resulting metabolic products were studied by positive electrospray ionization - (tandem) mass spectrometry and confirmed by the comparison with in vitro generated metabolites using human and rat liver microsomal preparations. Subsequently, a screening procedure for five SIRT1 activators and the metabolite M1-SRT1720 in DBS specimens was developed. Liquid-liquid-extraction and liquid chromatography/tandem mass spectrometry was employed based on diagnostic ion transitions recorded in multiple reaction monitoring mode and two deuterated internal standards namely d8-SRT1720 and d8-M1-SRT1720 were utilized. The doping control assay was characterized with regard to specificity, limit of detection (10-50ng/ml), recovery (65-83%) and imprecision (7-20%) and ion suppression/enhancement effects (<10%), demonstrating its fitness-for-purpose for sports drug testing applications., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A "cocktail" approach, which involves simultaneous administration of multiple CYP-specific probes, concurrently detects the activity of multiple CYP enzymes. We developed and validated a rapid and selective LC-MS/MS method for determining the plasma concentrations of 5 CYP probe drugs and metabolites (caffeine/paraxanthine, CYP1A2 substrate; losartan/losartan carboxylic acid (E3174), CYP2C9 substrate; omeprazole/5-hydroxyomeprazole, CYP2C19 substrate; dextromethorphan/dextrorphan, CYP2D6 substrate; and midazolam/1'-hydroxymidazolam, CYP3A4 substrate) by single-step extraction, followed by a single LC-MS/MS run. An Ostro™ 96-well plate was used for extraction of CYP substrates and metabolites from human plasma and urine. Following optimization of the chromatographic conditions, all the peaks were well separated, and retention times ranged between 4.4 and 11.7 min. The total run time for a single injection was within 13 min. The accuracy and precision values suggested that the assay had high accuracy and reliability in plasma and urine samples. No significant matrix interference was observed. To demonstrate the efficacy of this method, plasma and urine concentrations of 5 CYP probe substrates and their metabolites were determined after simultaneous oral administration of 5 drugs to 4 healthy volunteers. All the substrates and metabolites were detected over an 8 h period, and the plasma concentrations of each substrate at 8 h after administration were above the lower limit of quantification. Urine concentrations of drugs and their metabolic ratio were evaluated after the administration. In conclusion, the advantage of our cocktail approach is that it enables in vivo assessment of the activity of various drug-metabolizing enzymes in a single assay.

The kidney is the most important organ for the excretion of pharmaceuticals and their metabolites. Among the complex structures of the kidney, the proximal tubule and renal interstitium are major targets of nephrotoxins. Despite its importance, there are only a few in silico models for predicting human nephrotoxicity for drug candidates. Here, we present quantitative structure-activity relationship (QSAR) models for three common patterns of drug-induced kidney injury, i.e., tubular necrosis, interstitial nephritis, and tubulo-interstitial nephritis. A support vector machine (SVM) was used to build the binary classification models of nephrotoxin versus non-nephrotoxin with eight fingerprint descriptors. To build the models, we constructed two types of data sets, i.e., parent compounds of pharmaceuticals (251 nephrotoxins and 387 non-nephrotoxins) and their major urinary metabolites (307 nephrotoxins and 233 non-nephrotoxins). Information on the nephrotoxicity of the pharmaceuticals was taken from clinical trial and postmarketing safety data. Though the mechanisms of nephrotoxicity are very complex, by using the metabolite information, the predictive accuracies of the best models for each type of kidney injury were better than 83% for external validation sets. Software to predict nephrotoxicity is freely available from our Web site at http://bmdrc.org/DemoDownload .

Equine forensic science can now detect concentrations down to 25 femtograms/mL (parts per quadrillion, ppq) or less in blood and urine. As such, horsemen are increasingly at risk of inadvertent 'positives' due to therapeutic medication 'overages' or trace identifications of dietary or environmental substances. Reviewed here are the factors which determine detection times and 'withdrawal times' for substances administered to horses. Withdrawal times are affected by many factors, including dose, formulation, route and frequency of administration, bioavailability, plasma half-life, sensitivity of the analytical process, the testing matrix (plasma, urine, or other), and the environmental presence and/or persistence of administered substances. Of these factors only dose is known precisely. For any given administration, horse-to-horse differences in the volumes of distribution, systemic clearance, and terminal plasma elimination half-life of substances are major and totally uncontrollable factors driving horse-to-horse variability in withdrawal times. A further complication is that chemically stable medications administered to horses and eliminated in the urine inevitably become part of the environment of the horse. The presence of these substances in the equine environment is increasingly giving rise to trace identifications long after nominal administration of these substances has ceased. Because of the unknown and uncontrollable horse-to-horse variability in medication pharmacokinetics, any therapeutic medication administration to a horse by definition includes the possibility of an inadvertent medication overage. As such, the caveat that there are no guarantees in life most assuredly applies to advisories concerning equine therapeutic medication withdrawal times., (Copyright © 2013. Published by Elsevier Ltd.)

Biological fluids such as urine, saliva and whole blood were analyzed for contents of drugs by a new combination of desorption electrospray ionization mass spectrometry (DESI-MS) and thin liquid membrane extraction (TLME). Analytes from the sample were extracted into a thin liquid membrane of hexadecane deposited on a porous Teflon membrane, from which they were subsequently analyzed directly by DESI. The total analysis time was 15 minutes for analysis of several samples with a potential analysis time of less than a minute per sample. Thanks to the pre-concentration and sample clean-up built into the method, methadone was detected in urine in full-scan mode with an LOD of 4 ng mL(-1), while amitriptyline, nortriptyline and pethidine showed LODs of 17 ng mL(-1). Quantification was possible for several basic drugs using one common internal standard, providing relative accuracies in the range of 10-30%. A reliability test was performed on 20 samples with methadone, amitriptyline, nortriptyline and pethidine in urine, showing that none of the samples having concentrations above the LOD were missed and no false positives were found. Diphenhydramine and one of its metabolites were detected in authentic samples of urine and saliva, and methadone was detected from a whole-blood sample spiked to a concentration of 100 ng mL(-1). The method has several advantages, such as extremely low price in consumables, the possibility of fast analysis of very crude biofluids such as whole blood and the potential for a very high sample throughput.

During pregnancy, a drug's pharmacokinetics may be altered and hence anticipation of potential systemic exposure changes is highly desirable. Physiologically based pharmacokinetics (PBPK) models have recently been used to influence clinical trial design or to facilitate regulatory interactions. Ideally, whole-body PBPK models can be used to predict a drug's systemic exposure in pregnant women based on major physiological changes which can impact drug clearance (i.e., in the kidney and liver) and distribution (i.e., adipose and fetoplacental unit). We described a simple and readily implementable multitissue/organ whole-body PBPK model with key pregnancy-related physiological parameters to characterize the PK of reference drugs (metformin, digoxin, midazolam, and emtricitabine) in pregnant women compared with the PK in nonpregnant or postpartum (PP) women. Physiological data related to changes in maternal body weight, tissue volume, cardiac output, renal function, blood flows, and cytochrome P450 activity were collected from the literature and incorporated into the structural PBPK model that describes HV or PP women PK data. Subsequently, the changes in exposure (area under the curve (AUC) and maximum concentration (C max)) in pregnant women were simulated. Model-simulated PK profiles were overall in agreement with observed data. The prediction fold error for C max and AUC ratio (pregnant vs. nonpregnant) was less than 1.3-fold, indicating that the pregnant PBPK model is useful. The utilization of this simplified model in drug development may aid in designing clinical studies to identify potential exposure changes in pregnant women a priori for compounds which are mainly eliminated renally or metabolized by CYP3A4.

Achieving sufficient selectivity in bioanalysis is critical to ensure accurate quantitation of drugs and metabolites in biological matrices. Matrix effects most classically refer to modification of ionization efficiency of an analyte in the presence of matrix components. However, nonanalyte or matrix components present in samples can adversely impact the performance of a bioanalytical method and are broadly considered as matrix effects. For the current manuscript, we expand the scope to include matrix elements that contribute to isobaric interference and measurement bias. These three categories of matrix effects are illustrated with real examples encountered. The causes, symptoms, and suggested strategies and resolutions for each form of matrix effects are discussed. Each case is presented in the format of situation/action/result to facilitate reading.

The purpose of this study was to clarify the relationship between the physicochemical properties of drugs and their urinary excretion mechanisms. Three hundred twenty-five drugs were classified into the reabsorption, intermediate, and secretion types based on their ratio of renal clearance to protein-unbound fraction glomerular filtration rate. Fifty percent of ionized and neutral drugs were the secretion and reabsorption types, respectively. The mean molecular weight of the neutral drugs was slightly smaller than those of the ionized drugs (296 vs. 330-368 g/mol). The reabsorption-type anionic drugs were characterized by their low molecular weights (mean value 269 g/mol) and the logarithmic measure of the acid dissociation constants (pKa s) greater than 4.5, whereas the secretion-type anionic drugs all had pKa s below 4.5. Cationic drugs with pKa s lower than 8.0 tended to be the reabsorption type. Some cationic drugs were classified as the secretion type, despite their high molecular weights (734-811 g/mol) and high log P values (3.1-5.3). The organic anion transporter (OAT)1 and OAT3 substrates were all secretion-type drugs. The same trend was observed for the substrates of organic cation transporter 2, multidrug and toxin extrusion, multidrug resistance-associated protein 4, and multidrug resistance 1/breast cancer resistance protein, but substantial fractions of the substrates were categorized as the intermediate or reabsorption types (9%-38%). This work provides a clue to the renal elimination mechanism of new chemical entities during drug development., (Copyright © 2013 Wiley Periodicals, Inc.)

Electromembrane extraction (EME) is a novel sample preparation technique in pharmaceutical, chemical, clinical and environmental analysis. This technique uses electromigration across artificial liquid membranes for selective extraction of analytes and sample enrichment from complex matrices. This review focuses on the setup, general procedure and parameters affecting the extraction efficiency of EME. An overview of innovations in EME (on-chip EME, low voltage EME, drop-to-drop EME, pulsed EME and EME followed by low-density solvent based ultrasound-assisted emulsification microextraction) is also presented in this article and attention is focused on the use of EME for pharmaceutical, chemical, clinical and environmental analysis.

This White Paper is focused on the technical aspects regarding quantifying pharmaceutically derived inorganic elements in biomatrices in support of GLP nonclinical and clinical studies using inductively coupled plasma (ICP) techniques. For decades ICP has been used in support of Environmental Protection Agency analyses and has more recently been applied for use in the pharmaceutical industry. Current bioanalytical method validation and sample analysis regulatory guidance applies to chromatographic platforms used for analysis of large- and small-molecule PK and TK assessments; however, it is not directly applicable to all aspects of various ICP techniques. Increasingly, quadrupole and high-resolution ICP-MS methods of analysis are being used to quantify inorganic elements contained in pharmaceutical compounds and biomatrices. Many elements occur endogenously in biomatrices, affecting quantification of blanks, standard curve samples, QC samples, and the selection of appropriate levels for the LLOQ.

An extensive search of published research and review articles indicates that enhancing the sensitivity and resolution of capillary electrophoresis (CE) is a very active area of interest. This review focuses on developments and applications in this field over several years, especially from 2009-present. It first reviews developments in the fields of online sample preconcentration and highly sensitive detection for sensitivity enhancement. The online sample preconcentration techniques cover all methods, from electrophoretic preconcentration, extraction-based preconcentration, micelle-based preconcentration and hybrid preconcentration to nanoparticle-based preconcentration. Attention is also given to multiple dimension separations, additives in buffer solution and capillary inner surface modifications that have been used to enhance the resolution and separation efficiency of complex samples in CE. The additives include nanoparticles, chiral selectors and other buffer additives that have been added to the buffer solution.

In medical practices, the determination of enantiomeric ratio of the chiral drugs is very important for their activities, bioavailabilities and biodegradation. Only homochiral medication is safe for humans. The chiral analysis in biological samples is the first and most important step. The present article describes the technical strategies of the enantiomeric resolution of racemic drugs in biological samples. Attempts have been made to describe sample preparation by solid-phase extraction and enantiomeric resolution by chiral high-performance liquid chromatography. Various chiral stationary phases used in chiral separations of racemic drugs have been described. Efforts are also made to discuss the chiral recognition mechanism and future perspectives of chiral analyses in biological samples.

Benzodiazepines have become commonly prescribed medicines worldwide in the therapy of anxiety, sleep disorders and convulsive attacks because they are relatively safe, with mild side effects. The availability of rapid, sensitive and selective analytical methods is essential for the determination of these drugs in clinical and forensic cases. Benzodiazepines are usually present at trace levels (μg/mL or ng/mL) in a complex biological matrix, and the potentially interfering compounds need to be removed before analysis. Therefore, a sample preparation technique is often mandatory, both to extract the drugs of interest from the matrices and to increase their concentration. An extended and comprehensive review is presented herein, focusing on bio-sample preparation (pretreatment, extraction and derivatization) and gas chromatographic methods applied for the quantification of 1,4-benzodiazepines.

Bioactive electrode of dispersed graphene oxide in polyaniline composite was electrochemically fabricated onto indium tin oxide substrate for pharmaceutical application. Formations of nanocomposite graphene-polyaniline matrix with diameter 67.99 nm were observed with the use of scanning electron microscope and high resolution transmission electron microscope. Electrochemical interfacial properties and immobilization of enzyme onto the graphene-polyaniline electrode have been evaluated and confirmed with the use of Fourier transform infrared spectroscopic, cyclic voltammetry and electrochemical impedance spectroscopic techniques. The graphene-polyaniline-horseradish peroxidase biosensor was further used for sensing artesunate a potent antimalarial drug. The biosensor shows linearity of 0.05-0.40 ng mL(-1) of artesunate with sensitivity of 0.15 µA ng mL(-1). The procedure was applied to the assay of the drug in dosage form, human serum, plasma and urine without matrix interference. The limits of detection for parenteral artesunate, human urine, human serum and human plasma were 0.012 ng mL(-1), 0.013 ng mL(-1), 0.014 ng mL(-1) and 0.014 ng mL(-1) respectively. The mean percentage recoveries obtained were in the range from 98.23% to 100.3% for parental drug, urine, serum and plasma samples. The resultant precision and accuracy as evidenced have shown a promising selectivity in their application., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) method was developed for simultaneous enantioseparation of three imidazole drugs namely tioconazole, isoconazole and fenticonazole. Three easily available and inexpensive cyclodextrins namely 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD) and heptakis(2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD) were evaluated to discriminate the six stereoisomers of the drugs. However, none of the three CDs gave a complete enantioseparation of the drugs. Effective enantioseparation of tioconazole, isoconazole and fenticonazole was achieved using a combination of 35 mM HP-γ-CD and 10 mM DM-β-CD as chiral selectors. The best separation using both HP-γ-CD and DM-β-CD (35 mM:10 mM) as chiral selectors were accomplished in background electrolyte (BGE) containing 35 mM phosphate buffer (pH 7.0), 50 mM sodium dodecyl sulfate (SDS) and 15% (v/v) acetonitrile at 27 kV and 30 °C with all peaks resolved in less than 15 min with resolutions, Rs 1.90-27.22 and peak efficiencies, N > 180 000. The developed method was linear over the concentration range of 25-200 mg l(-1) (r(2) > 0.998) and the detection limits (S/N = 3) of the three imidazole drugs were found to be 2.7-7.7 mg l(-1). The CD-MEKC method was successfully applied to the determination of the three imidazole drugs in spiked human urine sample and commercial cream formulation of tioconazole and isoconazole with good recovery (93.6-106.2%) and good RSDs ranging from 2.30-6.8%., (© 2013 Wiley Periodicals, Inc.)

The performance of the previously validated LUCIO(®)-Direct-enzyme linked immunosorbent assay (direct ELISA) screening tests according to forensic guidelines is compared to that of cloned enzyme donor immunoassays (CEDIA) test for drugs of abuse in urine as defined in the new re-licensing German medical and psychological assessment (MPA) guidelines. The MPA screening cut-offs correspond to 10 ng/ml 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), 50 ng/ml amphetamine and designer amphetamines, 25 ng/ml morphine, codeine and dihydrocodeine, 30 ng/ml benzoylecgonine, 50 ng/ml methadone metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and metabolites of diazepam, oxazepam, bromazepam, alprazolam, flunitrazepam and lorazepam at 50 ng/ml. Average relative sensitivities and relative specificities were 99.7 % and 98.4 % for direct ELISA and 66 % and 91.4 % for CEDIA, respectively., (Copyright © 2013 John Wiley & Sons, Ltd.)

We describe a sensitive, comprehensive and fast screening method based on liquid chromatography-high resolution mass spectrometry for the detection of a large number of analytes in sports samples. UHPLC coupled to high resolution mass spectrometry with polarity switching capability is applied for the rapid screening of a large number of analytes in human urine samples. Full scan data are acquired alternating both positive and negative ionisation. Collision-induced dissociation with positive ionisation is also performed to produce fragment ions to improve selectivity for some analytes. Data are reviewed as extracted ion chromatograms based on narrow mass/charge windows (±5ppm). A simple sample preparation method was developed, using direct enzymatic hydrolysis of glucuronide conjugates, followed by solid phase extraction with mixed mode ion-exchange cartridges. Within a 10min run time (including re-equilibration) the method presented allows for the analysis of a large number of analytes from most of the classes in the World Anti-Doping Agency (WADA) Prohibited List, including anabolic agents, β2-agonists, hormone antagonists and modulators, diuretics, stimulants, narcotics, glucocorticoids and β-blockers, and does so while meeting the WADA sensitivity requirements. The high throughput of the method and the fast sample pre-treatment reduces analysis cost and increases productivity. The method presented has been used for the analysis of over 5000 samples in about one month and proved to be reliable., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In the kidney, human organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) are the major transporters for the secretion of cationic drugs into the urine. In the human kidney, OCT2 mediates the uptake of drugs from the blood at the basolateral membrane of tubular epithelial cells, and MATE1 and MATE2-K secrete drugs from cells into the lumen of proximal tubules. However, the expression of these transporters depends on the species of the animal. In the rodent kidney, OCT1 and OCT2 are expressed at the basolateral membrane, and MATE1 localizes at the brush-border membrane. Together, these transporters recognize various compounds and have overlapping, but somewhat different, substrate specificities. OCTs and MATEs can transport important drugs, such as metformin and cisplatin. Therefore, functional variation in OCTs and MATEs, including genetic polymorphisms or inter-individual variation, may seriously affect the pharmacokinetics and/or pharmacodynamics of cationic drugs. In this review, we summarize the recent findings and clinical importance of these transporters.

Introduction: Identifying the difficulties of the patient towards following his medication regimen remains complex for the healthcare provider. This can be explained by the multidimensional character of medication adherence and, actually, the evaluation of this phenomenon. The objective of this work was to review the various methods to measure medication adherence., Methods: We performed a search on PubMed completed by a manual one., Results: Two types of measure are described. The "direct" methods are based on the measurement of the level of medicine or metabolite in blood or urine, measurement of biologic markers in blood or measurement of physiologic or clinical markers. The "indirect" methods are represented by the analysis of the administrative databases (prescription, rate of prescription refills); pill counts; electronic medication monitors; the self-reported measures by the patient or his close relations (questionnaires, diaries, interviews); the opinion of the healthcare provider., Discussion: None of these tools supplants the others, each having limits either of feasibility, or reliability. In the end, it is the crossing of the information stemming from these various equipments that allows an idea on the adherence behavior of the patient and especially, dimensions on which he is most in trouble., Conclusion: The identification of these difficulties can allow the healthcare provider to develop behavioral and organizational skills tailored to the patient follow-up., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Understanding which physicochemical properties, or property distributions, are favorable for successful design and development of drugs, nutritional supplements, cosmetics, and agrochemicals is of great importance. In this study we have analyzed molecules from three distinct chemical spaces (i) approved drugs, (ii) human metabolites, and (iii) traditional Chinese medicine (TCM) to investigate four aspects determining the disposition of small organic molecules. First, we examined the physicochemical properties of these three classes of molecules and identified characteristic features resulting from their distinctive biological functions. For example, human metabolites and TCM molecules can be larger and more hydrophobic than drugs, which makes them less likely to cross membranes. We then quantified the shifts in physicochemical property space induced by metabolism from a holistic perspective by analyzing a data set of several thousand experimentally observed metabolic trees. Results show how the metabolic system aims to retain nutrients/micronutrients while facilitating a rapid elimination of xenobiotics. In the third part we compared these global shifts with the contributions made by individual metabolic reactions. For better resolution, all reactions were classified into phase I and phase II biotransformations. Interestingly, not all metabolic reactions lead to more hydrophilic molecules. We were able to identify biotransformations leading to an increase of logP by more than one log unit, which could be used for the design of drugs with enhanced efficacy. The study closes with the analysis of the physicochemical properties of metabolites found in the bile, faeces, and urine. Metabolites in the bile can be large and are often negatively charged. Molecules with molecular weight >500 Da are rarely found in the urine, and most of these large molecules are charged phase II conjugates.

The combination of dispersive liquid-liquid microextraction (DLLME) with capillary electrophoresis (CE) and a time-of-flight mass spectrometer (TOF-MS) was evaluated for the toxicological screening in urine samples. A methodology based on design of experiments (DOE) was implemented to increase the extraction efficiency. Dichloromethane and isopropanol were selected as the extraction and dispersing solvents, respectively. Seven factors for DLLME were screened with the help of a Plackett-Burmann DOE using two model compounds before fine investigation of the important parameters to maximise the compound extraction. These experiments were performed in the CE-UV configuration to overcome potential MS matrix effects. The performance of the entire procedure was then evaluated using CE-ESI-TOF-MS. With a preconcentration factor of more than 130, the highly sensitive DLLME-CE-ESI-TOF-MS method allowed for the detection of 30 toxicological compounds (i.e., amphetamines and their derivatives, opiates, cocaine and its metabolites and pharmaceuticals) in urine with limits of detection in the sub-ng/mL level and was used to analyse real toxicological samples. The combination of DLLME and CE was particularly attractive because of the small amount of organic solvents required., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The development of comprehensive methods able to tackle with the systematic identification of drug metabolites in an automated fashion is of great interest. In this article, a strategy based on the combined use of two complementary data mining tools is proposed for the screening and systematic detection and identification of urinary drug metabolites by liquid chromatography full-scan high resolution mass spectrometry. The proposed methodology is based on the use of accurate mass extraction of diagnostic ions (compound-dependent information) from in-source CID fragmentation without precursor ion isolation along with the use of automated mass extraction of accurate-mass shifts corresponding to typical biotransformations (non compound-dependent information) that xenobiotics usually undergo when metabolized. The combined strategy was evaluated using LC-TOFMS with a suite of nine sport drugs representative from different classes (propranolol, bumetanide, clenbuterol, ephedrine, finasteride, methoxyphenamine, methylephedrine, salbutamol and terbutaline), after single doses administered to rats. The metabolite identification coverage rate obtained with the systematic method (compared to existing literature) was satisfactory, and provided the identification of several non-previously reported metabolites. In addition, the combined information obtained helps to minimize the number of false positives. As an example, the systematic identification of urinary metabolites of propranolol enabled the identification of up to 24 metabolites, 15 of them non previously described in literature, which is a valuable indicator of the usefulness of the proposed systematic procedure., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A simple sample pretreatment device was developed employing extractions across supported liquid membranes (SLMs) and in-line coupling to a commercial CE instrument. The device consisted of two polypropylene conical units interspaced with a polypropylene planar SLM, which were impregnated with 1-ethyl-2-nitrobenzene. The two units and the SLM were pressed against each other, donor unit was filled with 40 μL of an untreated body fluid and acceptor unit with 40 μL of DI water. The device was then placed into conventional CE vial fitted with a soft spring, which was depressed during injection into CE capillary and ensured that the SLM was not ruptured. Position of separation capillary injection end and high-voltage electrode in the CE instrument was optimized in order to ensure efficient injection of pretreated body fluids. The device can be easily assembled/disassembled and SLMs can be replaced after each extraction thus minimizing sample carry-over, avoiding tedious SLM regeneration, and reducing total pretreatment time and costs. The pretreatment device was examined by direct injection of human urine and serum spiked with nortriptyline, haloperidol, and loperamide. The basic drugs were diffusionaly transported across the SLM within 10 min and were injected into the separation capillary directly from the SLM surface in the acceptor unit, whereas matrix components were retained by the SLM. The in-line SLM-CE method showed good repeatability of peak areas (3.8-11.0%) and migration times (below 1.4%), linear relationship (r(2) = 0.990-0.999), and low LODs (12-100 μg/L)., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The simultaneous extraction of acidic and basic drugs from biological samples is a significant challenge for sample preparation. A novel and efficient method named dual hollow fibre electromembrane extraction combined with CE was applied for the simultaneous extraction and preconcentration of acidic and basic drugs in a single step. Under applied potential of 40 V during the extraction, ibuprofen as an acidic drug and thebaine as a basic drug migrated from a 4 mL aqueous sample solution at neutral pH into 20 μL of each basic (pH 12.5) and acidic (pH 2.0) acceptor phase, respectively; 1-octanol and 2-nitrophenyl octyl ether were immobilised in the pores of anodic and cathodic hollow fibres as supported liquid membranes, respectively. A Box-Behnken design and the response surface methodology were used for the optimisation of different parameters on the extraction efficiency. Under the optimised conditions, the enrichment factors were between 150 and 170 and also the LODs ranged from 3 to 7 ng/mL in different samples. The method was reproducible so that intra- and inter-day RSDs% (n = 5) were less than 5.9%. Finally, the method was successfully applied for the simultaneous extraction and determination of acidic and basic drugs from plasma and urine samples., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Working together to uncover the truth: A molecule-sized diagnostic system combining several recognition elements and four fluorescence-emission channels enabled the identification of a wide range of pharmaceuticals on the basis of distinct photophysical processes. The molecular sensor (see simplified representation; ID = identification) was also used to analyze drug concentrations and combinations in urine samples in a high-throughput manner., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A GC-QqQ-MS method was developed for the detection of over 150 compounds from different classes (steroids, narcotics, stimulants, β-blockers, β-2-agonists and hormone antagonists) in a qualitative way. In the quantitative part, the traditional steroid profile with the most important endogenous steroids is expanded with six minor metabolites, which further improves the detection and identification of endogenous steroid abuse. In addition to these, norandrosterone, salbutamol and the major metabolite of cannabis are also quantified. Methods developed for anti-doping purposes should be subjected to the highest level of quality. Here, the addition of a combination of (deuterated) internal standards allows for an accurate quality control of every single step of the methodology: hydrolysis efficiency, derivatization efficiency and microbiological degradation are monitored in every single sample. Additionally, special attention is paid to the relationships between parameters indicating degradation by micro-organisms and the reliability of the steroid profile. The impact of the degradation is studied by evaluation of the quantities and percentages of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione. The concept of measurement uncertainty was introduced for the evaluation of relative abundances of mass-to-charge ratios and the obtained ranges were compared with the World Anti-Doping Agency regulations on tolerance windows for relative ion intensities. The results indicate that the approaches are similar., (Copyright © 2012 John Wiley & Sons, Ltd.)

Background: The toxicological analysis of blood and urine samples from victims of alleged sexual assault represents a crucial part of the forensic evidence when this crime is investigated., Material and Methods: We searched a national forensic toxicology database (TOXBASE) to find cases registered as sexual assault, rape, including date-rape that the police had requested the analysis of ethanol and other drugs. Between 2008 and 2010, N = 1460 such cases met this criteria. After immunological screening of urine or blood samples, all positive results were verified by more specific analytical methods, such as gas chromatography-mass spectrometry (GC-MS) for illicit drugs. A large number of prescription drugs and their metabolites were determined by capillary GC with nitrogen-phosphorous (N-P) detector. GC with flame ionization detector (FID) was used to analyze ethanol and gamma-hydroxybutyrate (GHB) in blood at limits of quantitation (LOQ) of 0.1 g/L and 8 mg/L, respectively., Results: The average age (± standard deviation) of all victims was 24 ± 10.3 years and 72% were between 15 and 29 years. Ethanol and other drugs were not detected in 31% of cases (N = 459). Blood-ethanol was positive in N = 658 cases at mean, median and highest concentrations of 1.23 g/L, 1.22 g/L and 4.3 g/L, respectively. Ethanol plus drugs were present in N = 188 cases (13%) and one or more other drugs alone in N = 210 cases (14%). Cannabis (marijuana) and amphetamines were the major illicit drugs, whereas diazepam, alprazolam, zopiclone as well as newer antidepressants were the major prescription drugs identified., Conclusions: The mean age of victims of sexual assault in Sweden, the proportion of drug positive to drug negative cases, the predominance of ethanol positive cases as well as the types of other drugs showed a remarkably good agreement in two studies spanning a period of 8 years.

Capillary electrophoresis with a capacitively contactless conductivity detector (CE-C⁴D) is becoming a significant useful technique for the analysis of analytes in various fields such as pharmaceutical, biomedical, food and environmental. This review is an update describing the recent developments in the application of CE with a C⁴D detector., (Copyright © 2012 John Wiley & Sons, Ltd.)

Electromembrane extraction (EME) at low voltage (0-15 V) of 29 different basic model drug substances was investigated. The drug substances with logP<2.3 were not extracted at voltages less than 15 V. Extraction of drug substances with logP≥2.3 and with two basic groups were also effectively suppressed by the SLM at voltages less than 15 V. Drug substances with logP≥2.3 and with one basic group were all extracted at low voltages and with a strong compound selectivity which appeared to have some influence from the polar surface area of the compound. For this group of substances, recoveries varied between 0 and 23% at 5 V, whereas, recoveries varied between 5.5 and 51% at 15 V. Based on mass transfer differences related to charge, polarity, and polar surface, highly selective extractions of drug substances were demonstrated from human plasma, urine, and breast milk. An initial evaluation at low voltage (5 V) was compared with similar extractions at a more normal voltage level (50 V), and this supported that reliable data can be obtained under these low-voltage (mild) conditions by EME., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In the present work, for the first time a new set-up was presented for simultaneous extraction of acidic and basic drugs using a recent novel electrically-enhanced microextraction technique, termed electromembrane extraction at low voltages followed by high performance liquid chromatography with ultraviolet detection. Nalmefene (NAL) as a basic drug and diclofenac (DIC) as an acidic drug were extracted from 24 mL aqueous sample solutions at neutral pH into 10 μL of each acidified (HCl 50 mM) and basic (NaOH 50 mM) acceptor solution, respectively. Supported liquid membranes including 2-nitrophenyl octyl ether containing 5% di-(2-ethylhexyl) phosphate and 1-octanol were used to ensure efficient extraction of NAL and DIC, respectively. Low voltage of 40 V was applied over the SLMs during 14 min extraction time. The influences of fundamental parameters affecting the transport of target drugs were optimized using experimental design. Under optimal conditions, NAL and DIC were extracted with extraction recoveries of 12.5 and 14.6, respectively, which corresponded to preconcentration factors of 300 and 350, respectively. The proposed technique provided good linearity with correlation coefficient values higher than 0.9956 over a concentration range of 8-500 μg L⁻¹ and 12-500 μg L⁻¹ for NAL and DIC, respectively. Limits of detection and quantifications, and intra-day precisions (n=3) were less than 4 μg L⁻¹, 12 μg L⁻¹, and 10.1%, respectively. Extraction and determination of NAL and DIC in human urine samples were successfully performed. In light of the data obtained in the present work, this new set-up for EME with low voltages has a future potential as a simple, selective, and fast sample preparation technique for simultaneous extraction and determination of acidic and basic drugs in different complicated matrices., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: To further expand the use of fraction collection (FC)-microplate scintillation counting (MSC) in detecting trace amount of radioactivity in absorption, distribution, metabolism and excretion (ADME) studies and improve the resolution of UHPLC-FC-MSC, we report the coupling of UHPLC with MS and faster FC (1.2 s/fraction) followed by MSC using 384-deep-well LumaPlate™ (PerkinElmer, MA, USA) for profiling of radiolabeled metabolites in plasma, urine, bile and feces., Results: Collection of 1.2 s/well clearly improved the resolution of the reconstructed radiochromatograms and, at the same time, provided sufficient detection sensitivity that allowed for more accurate integration of peaks, which is required for radiolabeled ADME studies. The introduction of a reversed gradient as a make-up solvent mixture ensured more uniform drops collected in each well, with resolution maintained throughout the UHPLC run. Less sample injection and more frequent FC resulted in less quenching by matrix and accurate integration of peak., Conclusion: UHPLC-FC-MSC-MS is suitable for metabolite profiling in ADME studies and offers higher resolution, higher sensitivity, shorter LC running time, reduced matrix effect and more environmentally friendly experiments compared with conventional online flow scintillation analysis.

Background: Point-of-care tests for toxicological screening of patients for drugs of abuse and therapeutic drugs may be helpful in the emergency department (ED) to assist in a rapid diagnosis., Objectives: In this prospective study, the performance of TesTcard9® (Varian; Middelburg, Netherlands), Syva RapidTest d.a.u. 10® (Dade Behring; Leusden, Netherlands), and Triage TOX Drug Screen® (Biosite; Bunnik, Netherlands), when applied on-site in the ED by physicians and nurses, was evaluated., Methods: Patients in the ED were included in the study when a physician thought the patient could benefit from a toxicological screen. Urine samples were screened utilizing the three point-of-care tests. All three tests simultaneously determined the presence of amphetamines, methamphetamine, opiates, methadone (except for TesTcard9), cocaine, cannabis, barbiturates, benzodiazepines, tricyclic antidepressants, and phencyclidine. The same urine specimen was analyzed in the pharmacy department using Syva EMIT II immunoassay and chromatographic confirmation. The results were compared for agreement., Results: During the 6-month study period, 80 urine samples were screened. In total, 62 (78%) specimens were found positive for at least one drug. Amphetamines (n = 16), cocaine (n = 27), cannabis (n = 25), benzodiazepines (n = 25), and opiates (n = 8) were the most frequently found. The sensitivity and specificity of all three devices were higher than 93% for these compounds, with the exception of the sensitivity for cannabis with the TesTcard9 (88%) and the sensitivity for benzodiazepines with the Syva RapidTest d.a.u. 10 (88%) and TesTcard9 (80%)., Conclusion: In the ED setting, the Triage TOX Drug Screen performed better than the other point-of-care tests, probably due to its more objective reading system and its adequate quality controls., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A method based on in-source collision-induced dissociation (ISCID) liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) and reverse target database search was developed and evaluated for drug screening and confirmation in analytical toxicology context. An established LC-TOFMS screening method, in which identification relies solely on protonated molecule accurate mass measurement, isotopic pattern fit, and retention time (RT), was completed to include 1-3 qualifier ions for each analyte in the database. The qualifier ions for 431 compounds were selected from the experimental ISCID spectra, and their molecular formulae were assigned by applying SmartFormula3D and MSFragmenter software. Three qualifier ions were assigned for 64.5%, two or three for 81.4%, one for 14.8%, and none for 3.7% of the compounds studied. Comparison between ISCID LC-TOFMS and LC-TOFMS with 25 authentic autopsy urine samples showed an improved confidence level with the ISCID method, as isomeric interferences were excluded in most cases. However, some false negative (FN) results were obtained at low concentration levels close to the reporting criteria. The cut-off concentration of the ISCID method was 10-100 ng/mL with 80% of the 49 representative compounds tested, and the level was approximately two times higher than that obtained by LC ion trap MS. The presented method enables simultaneous screening and confirmation whenever at least one qualifier ion is available, as applying an accurate mass precursor ion and one product ion surpasses the standard of four identification points that is required by the current EU protocol.

This contribution presents an experimental approach for improvement of analytical performance of electromembrane extraction (EME), which is based on the use of stabilized constant d.c. electric current. Extractions were performed using a high voltage power supply, which provided stabilized constant d.c. current down to 1μA and facilitated current-controlled transfer of ions of interest from a donor solution through a supported liquid membrane (SLM) into an acceptor solution. Repeatability of the extraction process has significantly improved for EME at constant electric current compared to EME at constant voltage. The improved repeatability of the extraction process was demonstrated on EME-capillary electrophoresis (EME-CE) analyses of selected basic drugs and amino acids in standard solutions and in human urine and serum samples. RSD values of peak areas of the analytes for EME-CE analyses were about two-fold better for EME at constant electric current (2.8-8.9%) compared to EME at constant voltage (3.6-17.8%). Other analytical parameters of the EME-CE methods, such as limits of detection, linear ranges and correlation coefficients were not statistically different for the two EME modes. Moreover, EME at constant electric current did not suffer from SLM instabilities frequently observed for EME at constant voltage., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A sensitive method based on gas chromatography-mass spectrometry was used to determine 22 pharmacologically active substances (frequently used in the treatment of human and animal's diseases) including analgesics, antibacterials, anti-epileptics, antiseptics, β-blockers, hormones, lipid regulators and non-steroidal anti-inflammatories in blood and urine samples. Samples were subjected to continuous solid-phase extraction in a sorbent column (Oasis HLB), and then the target analytes were eluted with ethyl acetate and derivatized in a household microwave oven at 350 W for 3 min. Finally, these products were determined in a gas chromatograph-mass spectrometer equipped with a DB-5 fused silica capillary column. The analyte detection limits thus obtained ranged from 0.2 to 1.3 ng L⁻¹ for urine samples and 0.8-5.6 ng L⁻¹ for blood samples. Recoveries from both blood and urine ranged from 85 to 102%, and within-day and between-day relative standard deviations were all less than 7.5%. The proposed method offers advantages in reduction of the exposure danger to toxic solvents used in conventional sample pretreatment, simplicity of the extraction processes, rapidity, and sensitivity enhancement. The method was successfully used to quantify pharmacologically active substances in human and animal (lamb, veal and pig) blood and urine. The hormones estrone and 17β-estradiol were detected in virtually all samples, and so were other analytes such as acetylsalicylic acid, ibuprofen, ketoprofen and triclosan in human samples, and florfenicol, pyrimethamine and phenylbutazone in animal samples., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A liquid chromatography (LC) retention time prediction software, ACD/ChromGenius, was employed to calculate retention times for structural isomers, which cannot be differentiated by accurate mass measurement techniques alone. For 486 drug compounds included in an in-house database for urine drug screening by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOFMS), a retention time knowledge base was created with the software. ACD/ChromGenius calculated retention times for compounds based on the drawn molecular structure and given chromatographic parameters. The ability of the software for compound identification was evaluated by calculating the retention order of the 118 isomers, in 50 isomer groups of 2-5 compounds each, included in the database. ACD/ChromGenius predicted the correct elution order for 68% (34) of isomer groups. Of the 16 groups for which the isomer elution order was incorrectly calculated, two were diastereomer pairs and thus difficult to distinguish using the software. Correlation between the calculated and experimental retention times in the knowledge base tested was moderate, r(2)=0.8533. The mean and median absolute errors were 1.12 min, and 0.84 min, respectively, and the standard deviation was 1.04 min. The information generated by ACD/ChromGenius, together with other in silico methods employing accurate mass data, makes the identification of substances more reliable. This study demonstrates an approach for tentatively identifying compounds in a large target database without a need for primary reference standards., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Sample preparation is an important and necessary step in a measurement process for isolation and concentration of desired components from complex matrices. It is the most time-consuming and error-prone step in analytical methodology, greatly affecting quality and quantity of analytical data. During the past 15 years, solvent microextraction techniques have been introduced as alternatives to conventional sample preparation methods, such as liquid-liquid extraction and solid-phase extraction. These novel methodologies, which have proved to be extremely simple, low-cost and virtually solvent-free sample-preparation techniques provide a high degree of selectivity, sample cleanup and enrichment. The aim of the present review is to explore recent analytical applications of solvent microextraction techniques for quantification of drugs in biological samples, with particular focus on the methods involving MS as a detection system.