Microfluidic Device for Patient-Centric Multiplexed Assays with Readout in Centralized Laboratories.

Patient-centric sampling strategies, where the patient performs self-sampling and ships the sample to a centralized laboratory for readout, are on the verge of widespread adaptation. However, the key to a successful patient-centric workflow is user-friendliness, with few noncritical user interactions, and simple, ideally biohazard-free shipment. Here, we present a capillary-driven microfluidic device designed to perform the critical biomarker capturing step of a multiplexed immunoassay at the time of sample collection. On-chip sample drying enables biohazard-free shipment and allows us to make use of advanced analytics of specialized laboratories that offer the needed analytical sensitivity, reliability, and affordability. Using C-Reactive Protein, MCP1, S100B, IGFBP1, and IL6 as model blood biomarkers, we demonstrate the multiplexing capability and applicability of the device to a patient-centric workflow. The presented quantification of a biomarker panel opens up new possibilities for e-doctor and e-health applications.

Increasing prevalence of illicit drug use among employees at Swedish workplaces over a 25-year period.

BACKGROUND: Reports indicate that the proportion of adults using drugs of abuse has been increasing in recent years in Europe. Although there are various indicators of increased drug use in Sweden over time, few studies could demonstrate an increase in the proportion of adults using drugs. To investigate changes in drug use prevalence over time, drug testing at the workplace has been used for a 25-year period. METHODS: The urine samples of employees sent by occupational health services from all over Sweden during a 25-year period were analyzed. The analyzing capacity increased over time (from 3411 in 1994 to 60 315 samples analyzed in 2019), and the majority of the samples was analyzed for the following drugs: cannabis (tetrahydrocannabinol), amphetamine, opiates, cocaine, and benzodiazepines. RESULTS: There was an overall increase in the proportion of samples that tested positive for illicit drugs over a 25-year period. This increase seemed to take place step-wise, with phases of linear increases and plateaus that over time became shorter. About 1.3% of samples tested positive for drugs in 1994, whereas 5.6% tested positive in 2019. Since 2007, the rate of positive samples has increased for cannabis and decreased for benzodiazepines. Although the rate of samples tested positive for opiates had remained relatively stable over the last 20 years, this rate had increased for amphetamine and cocaine between 2013 and 2019. CONCLUSION: The results indicate that the use of illicit drugs among employees at Swedish workplaces has increased during a 25-year period.

Peanuts in the air - clinical and experimental studies.

BACKGROUND: Allergic reactions to food allergens usually occur after ingestion. However, fear of reactions to airborne peanut is a common concern for people with peanut allergy. There are no scientific reports on severe reactions with airborne peanut allergen. OBJECTIVE: To investigate the occurrence of allergic reactions in peanut-allergic children undergoing airborne peanut challenge and to determine levels of airborne peanut protein in a separate experimental evaluation. METHODS: Eighty-four children with peanut allergy underwent an airborne peanut challenge, 0.5 m from a bowl of peanuts for 30 min under controlled conditions. In a separate experiment, airborne peanut proteins from roasted and dry-roasted peanuts were collected at varying distances and at varying times with an electret SensAbues filter connected to an air pump. Collected airborne peanut proteins were extracted, dissolved and detected by ELISA. Basophil activation test was used to confirm biological activity. RESULTS: No moderate/severe allergic reactions to airborne peanut allergens were observed. Two children (2%) had mild rhino-conjunctivitis which required no treatment. The IgE-antibodies to peanut or Ara h 2 did not predict a reaction. In the experimental set-up, biological active peanut proteins were detected, in a very low amount, in median 166 ng/ml for dry-roasted and 33 ng/ml for roasted peanuts and decreased dramatically when the collection occurred at a greater distance (0.5-2 m) from the peanut source. Increased exposure time did affect the amount of collected peanut protein at 0 m, and the highest median was obtained after 60 min (p = .012); for time trend p = .0006. CONCLUSIONS AND CLINICAL RELEVANCE: Allergic reactions to airborne peanut proteins are rare and cannot be predicted by high levels of IgE-antibodies to peanut or Ara h 2. Only small amounts of biologically active peanut proteins were detected in the air and seem unlikely to trigger moderate/severe allergic reactions.

Measurement of the alcohol biomarker phosphatidylethanol (PEth) in dried blood spots and venous blood-importance of inhibition of post-sampling formation from ethanol.

Phosphatidylethanol (PEth) is a group of phospholipids formed in cell membranes following alcohol consumption by action of the enzyme phospholipase D (PLD). PEth measurement in whole blood samples is established as a specific alcohol biomarker with clinical and forensic applications. However, in blood specimens containing ethanol, formation of PEth may continue after sampling leading to falsely elevated concentrations. This study evaluated the use of dried blood spot (DBS) and microsampling specimens to avoid post-sampling formation of PEth. Filter paper cards and three commercial devices for volumetric microsampling of finger-pricked blood were assessed, using PEth-negative and PEth-positive whole blood fortified with 2 g/L ethanol. PEth (16:0/18:1) was measured by LC-MS/MS. Post-sampling formation of PEth occurred in wet blood and in the volumetric devices, but not filter paper cards, when stored at room temperature for 48 h. Addition of an inhibitor of PLD, sodium metavanadate (NaVO3), eliminated post-sampling formation during storage and drying. In conclusion, the present study confirmed previous observations that PEth can be formed in blood samples after collection, if the specimen contains ethanol. The results further demonstrated that post-sampling formation of PEth from ethanol also occurred with commercial devices for volumetric dried blood microsampling. In order for a PEth result not to be questioned, it is recommended to use a PLD inhibitor, whether venous blood is collected in a vacutainer tube or finger-pricked blood is obtained using devices for dried blood microsampling.

Pharmacokinetics of methylphenidate and ritalinic acid in plasma correlations with exhaled breath and oral fluid in healthy volunteers.

PURPOSE: The primary aim of this study was to explore the potential of alternative sampling matrices for methylphenidate by assessing the correlations between dl-threo-methylphenidate and dl-threo-ritalinic acid concentrations in exhaled breath and oral fluid with those in plasma, in repeated samples collected after a single oral dose of methylphenidate. The secondary aim was to study the enantioselective pharmacokinetics of methylphenidate in plasma, with a focus on interindividual variability in the metabolism of methylphenidate to ritalinic acid. METHODS: Twelve healthy volunteers received a single oral dose of dl-threo-methylphenidate (Ritalin® capsules, 20 mg). Venous blood samples were collected for 24 h, and plasma analyzed for threo-enantiomers of methylphenidate and ritalinic acid with LC-MS/MS. Repeated sampling of exhaled breath, using a particle filter device, and of non-stimulated oral fluid, using a felt pad device, was also performed. Exhaled breath and oral fluid were analyzed with a non-enantioselective LC-MS/MS method for dl-threo-methylphenidate and dl-threo-ritalinic acid. RESULTS: In all subjects, d-threo-methylphenidate was detectable in plasma for at least 15 h after the dose with a biphasic profile. l-threo-Methylphenidate was measurable in only five subjects and in most cases in low concentrations. However, one female subject displayed a biphasic concentration-time profile for l-threo-methylphenidate. This subject also had the highest d-threo-methylphenidate AUC (191 ng*h/mL versus 32-119 ng*h/mL in the other subjects). d-threo-Ritalinic acid concentrations were on average 25-fold higher (range 6-126) than the corresponding d-threo-methylphenidate concentrations. Single-time point plasma concentration ratios between d-threo-ritalinic acid and d-threo-methylphenidate 1.5-12 h after dose correlated highly (r = 0.88-0.98) with the d-threo-ritalinic acid AUC/d-threo-methylphenidate AUC ratio. In eleven subjects, dl-threo-methylphenidate in oral fluid mirrored the biphasic profile of methylphenidate (sum of d- and l-threo-enantiomers) in plasma, but the concentrations in oral fluid were on average 1.8 times higher than in plasma. dl-threo-Methylphenidate was detected in exhaled breath in all subjects, but there was no consistent concentration-time pattern. CONCLUSIONS: In some subjects, the pharmacologically less active l-threo-enantiomer may contribute to the total plasma methylphenidate concentrations. Monitoring methylphenidate concentrations without enantiomeric determination carries the risk of missing such subjects, which might affect how the plasma concentrations of methylphenidate are interpreted and used for clinical decision making. The use of exhaled breath and oral fluid to assess medication adherence to MPH in patients with ADHD warrants further studies.

Performance of PEth Compared With Other Alcohol Biomarkers in Subjects Presenting For Occupational and Pre-Employment Medical Examination.

AIMS: To compare the performance of short- and long-term alcohol biomarkers for the evaluation of alcohol drinking in employment-related health controls. METHODS: The 519 blood samples originated from 509 patients (80% men) presenting at occupational health units and medical centers at employment agencies for the evaluation of risky drinking. The laboratory investigation comprised the measurement of phosphatidylethanol (PEth 16:0/18:1), carbohydrate-deficient transferrin (CDT; % disialotransferrin), gamma-glutamyl transferase (GGT), mean corpuscular volume (MCV), ethanol and ethyl glucuronide (EtG). RESULTS: Many samples tested positive for acute (57%) and chronic (69%) alcohol biomarkers. PEth was the single most positive biomarker (64%; cut-off 0.05 µmol/l or 35 µg/l) and the only positive chronic biomarker in 100 cases. The highest PEth concentrations were seen in samples positive for all chronic biomarkers, followed by those also being CDT positive (cut-off 2.0%). All 126 CDT-positive samples were positive for PEth using the lower reporting limit (≥0.05 µmol/l) and for 114 cases (90%) also using the higher limit (≥0.30 µmol/l or 210 µg/l). In the CDT-positive cases, the PEth median concentration was 1.71 µmol/l, compared with 0.45 µmol/l for the CDT-negative cases (P < 0.0001). PEth and CDT values were correlated significantly (r = 0.63, P < 0.0001). Among the EtG-positive cases (≥1.0 ng/ml), 95% were also PEth positive, and all ethanol-positive cases (≥0.10 g/l) were also PEth positive. CONCLUSIONS: For optimal detection of drinking habits, using a combination of short- and long-term alcohol biomarkers provided best information. PEth was the single most positive alcohol biomarker, whereas GGT and MCV offered little additional value over PEth and CDT.

Evaluation of a Volumetric Dried Blood Spot Card Using a Gravimetric Method and a Bioanalytical Method with Capillary Blood from 44 Volunteers.

Dried blood spot (DBS) sampling is a promising method for collection of microliter blood samples. However, hematocrit-related bias in combination with subpunch analysis can result in inaccurate quantification of analytes in DBS samples. In this study we use a microfluidic DBS card, designed to automatically collect fixed volume DBS samples irrespective of the blood hematocrit, to measure caffeine concentration in normal finger prick samples obtained from 44 human individuals. Caffeine levels originating from blood drops of unknown volume collected on the volumetric microfluidic DBS card were compared to volume-controlled pipetted DBS samples from the same finger prick. Hematocrit independence and volumetric sampling performances were also verified on caffeine-spiked blood samples in vitro, using both LC-MS/MS and gravimetric methods, on hematocrits from 26 to 62%. The gravimetric measurements show an excellent metering performance of the microfluidic DBS card, with a mean blood sample volume of 14.25 µL ± 3.0% ( n = 51). A measured mean bias below 2.9% compared to normal hematocrit (47%) demonstrates that there is no significant hematocrit-induced bias. LC-MS/MS measurements confirm low CV and hematocrit independence of the sampling system and exhibit no substantial mean bias compared to pipetted DBS. Tests with 44 individuals demonstrated applicability of the microfluidic DBS card for direct finger prick blood sampling, and measured caffeine concentrations show a good agreement with measurements of pipetted DBS. The presented concept demonstrates a good volumetric performance which can help to improve the accuracy of DBS analysis by analyzing a whole spot, equivalent to a defined volume of liquid blood.

An Autonomous Microfluidic Device for Generating Volume-Defined Dried Plasma Spots.

Obtaining plasma from a blood sample and preparing it for subsequent analysis is currently a laborious process involving experienced health-care professionals and centrifugation. We circumvent this by utilizing capillary forces and microfluidic engineering to develop an autonomous plasma sampling device that filters and stores an exact amount of plasma as a dried plasma spot (DPS) from a whole blood sample in less than 6 min. We tested 24 prototype devices with whole blood from 10 volunteers, various input volumes (40-80 µL), and different hematocrit levels (39-45%). The resulting mean plasma volume, assessed gravimetrically, was 11.6 µL with a relative standard deviation similar to manual pipetting (3.0% vs 1.4%). LC-MS/MS analysis of caffeine concentrations in the generated DPS (12 duplicates) showed a strong correlation ( R2 = 0.99) to, but no equivalence with, concentrations prepared from corresponding plasma obtained by centrifugation. The presented autonomous DPS device may enable patient-centric plasma sampling through minimally invasive finger-pricking and allow generatation of volume-defined DPS for quantitative blood analysis.

Dose-Response Characteristics of the Alcohol Biomarker Phosphatidylethanol (PEth)-A Study of Outpatients in Treatment for Reduced Drinking.

AIM: Measurement of whole-blood phosphatidylethanol (PEth) offers high sensitivity and specificity as alcohol biomarker. A remaining issue of importance for the routine application is to better establish the relationship between PEth concentration and amount and duration of drinking. METHODS: The study included 36 subjects (32-83 years) voluntarily attending outpatient treatment for reduced drinking. At ~ 3- to 4-week intervals, they provided a diary on their daily alcohol intake and gave blood samples for measurement of PEth and carbohydrate-deficient transferrin (CDT). Whole-blood PEth 16:0/18:1 was measured by liquid chromatography-tandem mass spectrometry and serum CDT (%disialotransferrin) by high-performance liquid chromatography. RESULTS: At start, the self-reported past 2-week alcohol intake ranged 0-1260 (median 330) g ethanol, the PEth 16:0/18:1 concentration ranged 0.05-1.20 (median 0.23) µmol/L, and the CDT value ranged 0.7-13.0% (median 1.5%). At the final sampling after 5-20 (median 12) weeks, neither reported alcohol intake nor PEth and CDT levels differed significantly from the starting values. The PEth concentration showed best association with past 2-week drinking, followed by for intake in the next last week. The changes in PEth concentration vs past 2-week alcohol intake between two successive tests revealed that an increased ethanol intake by ~ 20 g/day elevated the PEth concentration by on average ~ 0.10 µmol/L, and vice versa for decreased drinking. CONCLUSIONS: The PEth concentration correlated well with past weeks alcohol intake, albeit with a large inter-individual scatter. This indicates that it is possible to make only approximate estimates of drinking based on a single PEth value, implying risk for misclassification between moderate and heavy drinking.

Elimination Characteristics of the Alcohol Biomarker Phosphatidylethanol (PEth) in Blood during Alcohol Detoxification.

AIMS: The study documented elimination characteristics of three phosphatidylethanol (PEth) homologs in serially collected blood samples from 47 heavy drinkers during ~2 weeks of alcohol detoxification at hospital. METHODS: Venous whole blood and urine samples were collected every 1-2 days during treatment. Concentrations of PEth, and of urinary ethyl glucuronide (EtG) and ethyl sulfate (EtS) to detect relapse drinking, were measured using liquid chromatography-tandem mass spectrometry. RESULTS: When included in the study, negative or decreasing breath ethanol concentrations demonstrated that the patients were in the elimination phase. The EtG and EtS measurements further confirmed alcohol abstinence during the study, with three exceptions. On admission, all patients tested positive for PEth, the total concentration ranging 0.82-11.7 (mean 6.35, median 5.88) µmol/l. PEth 16:0/18:1, 16:0/18:2 and 16:0/20:4 accounted for on average ~42%, ~26% and ~9%, respectively, of total PEth in these samples. There were good correlations between total PEth and individual homologs (P < 0.0001). There was no significant difference in PEth values between male and female subjects. During abstinence, the elimination half-life values ranged 3.5-9.8 days for total PEth, 3.7-10.4 days for PEth 16:0/18:1, 2.7-8.5 days for PEth 16:0/18:2 and 2.3-8.4 days for PEth 16:0/20:4. CONCLUSIONS: The results demonstrated a very high sensitivity (100%) of PEth as alcohol biomarker for recent heavy drinking, but considerable differences in the elimination rates between individuals and between different PEth forms. This indicates that it is possible to make only approximate estimates of the quantity and recency of alcohol intake based on a single PEth value.

High-Yield Passive Plasma Filtration from Human Finger Prick Blood.

Whole-blood microsampling provides many benefits such as remote, patient-centric, and minimally invasive sampling. However, blood plasma, and not whole blood, is the prevailing matrix in clinical laboratory investigations. The challenge with plasma microsampling is to extract plasma volumes large enough to reliably detect low-concentration analytes from a small finger prick sample. Here we introduce a passive plasma filtration device that provides a high extraction yield of 65%, filtering 18 µL of plasma from 50 µL of undiluted human whole blood (hematocrit 45%) within less than 10 min. The enabling design element is a wedge-shaped connection between the blood filter and the hydrophilic bottom surface of a capillary channel. Using finger prick and venous blood samples from more than 10 healthy volunteers, we examined the filtration kinetics of the device over a hematocrit range of 35-55% and showed that 73 ± 8% of the total protein content was successfully recovered after filtration. The presented plasma filtration device tackles a major challenge toward patient-centric blood microsampling by providing high-yield plasma filtration, potentially allowing reliable detection of low-concentration analytes from a blood microsample.

Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics.

Adverse reactions or lack of response to medications are important concerns for drug development programs. However, faithful predictions of drug metabolism and toxicity are difficult because animal models show only limited translatability to humans. Furthermore, current in vitro systems, such as hepatic cell lines or primary human hepatocyte (PHH) 2-dimensional (2D) monolayer cultures, can be used only for acute toxicity tests because of their immature phenotypes and inherent instability. Therefore, the migration to novel phenotypically stable models is of prime importance for the pharmaceutical industry. Novel 3-dimensional (3D) culture systems have been shown to accurately mimic in vivo hepatic phenotypes on transcriptomic and proteomic level, but information about their metabolic stability is lacking. Using a combination of targeted and untargeted high-resolution mass spectrometry, we found that PHHs in 3D spheroid cultures remained metabolically stable for multiple weeks, whereas metabolic patterns of PHHs from the same donors cultured as conventional 2D monolayers rapidly deteriorated. Furthermore, pharmacokinetic differences between donors were maintained in 3D spheroid cultures, enabling studies of interindividual variability in drug metabolism and toxicity. We conclude that the 3D spheroid system is metabolically stable and constitutes a suitable model for in vitro studies of long-term drug metabolism and pharmacokinetics.-Vorrink, S. U., Ullah, S., Schmid, S., Nandania, J., Velagapudi, V., Beck, O., Ingelman-Sundberg, M., Lauschke, V. M. Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics.

Potential of Oral Fluid as a Clinical Specimen for Compliance Monitoring of Psychopharmacotherapy.

BACKGROUND: Oral fluid (OF) is being developed as a specimen for the determination of drug intake as an alternative to serum and plasma. It is generally considered as an attractive specimen due to the noninvasive nature of the sampling procedure and the relation to the free fraction of drug in the blood. These features are of particular value in drug treatment of psychiatric disorders. To establish OF for the purpose of monitoring drug therapy, the relationship between concentrations in OF and serum/plasma must be documented. This study explored one promising sampling device and comprised the following 10 drugs: aripiprazole, citalopram, duloxetine, escitalopram, mirtazapine, pipamperone, pregabalin, promethazine, quetiapine, and venlafaxine. METHODS: For this purpose, 100 paired serum and OF samples were collected from patients undergoing pharmacotherapy and analyzed using a liquid chromatography-tandem mass spectrometry method. A commercial method from Chromsystems for the determination of these drugs in plasma was used and was adapted for OF and ultrafiltrated (Centrifree device) serum. RESULTS: The ratio of each individual pair of samples was used to calculate a mean and SD value between OF and serum free and total concentrations. The OF concentration ratios to serum total fraction differed markedly between substances and differed from 10-fold lower to 8-fold higher. The ratios to serum free fractions were always higher. The relation between the OF and serum concentrations was also evaluated by regression analysis and determination of slopes and correlation coefficients. For all measured relations, there was a statistically significant relation between the OF and serum concentrations. The degree of drug protein binding was in agreement with literature. The aripiprazole, duloxetine, pipamperone, pregabalin, and promethazine concentrations in ultrafiltrated serum were not possible to measure because of low concentrations and nonspecific binding. CONCLUSIONS: Despite a strong statistical correlation between OF and serum concentrations observed for most of the studied substances, it is still evident that OF concentrations cannot simply substitute serum/plasma as therapeutic drug monitoring specimen, but rather be considered as a unique specimen. We believe that OF is a promising matrix especially for compliance testing in psychiatry settings. The Greiner Bio-One device used in this study provides a sampling procedure that offers advantages over the available alternatives.

Identification and quantitation of phosphatidylethanols in oral fluid by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Phosphatidylethanols (PEth) are formed from phosphatidylcholines and ethanol and are used as a specific and sensitive alcohol biomarker. An analytical method for analysis of PEth in oral fluid based on high-performance liquid chromatography coupled to a quadrupole tandem mass spectrometer (LC-MS/MS) was developed and validated and applied on samples collected from patients undergoing alcohol detoxification. METHODS: A 200-µL aliquot of oral fluid, collected using the QuantisalTM device, was extracted with chloroform/methanol containing internal standard and subjected to LC-MS/MS analysis of three selected PEth forms (16:0/16:0, 16:0/18:1, and 16:0/18:2). Chromatographic separation was achieved on a UPLC BEH phenyl column, using a mobile phase consisting of acetonitrile and water containing 10 mmol/L ammonium hydrogen carbonate with 0.1% ammonia. The MS instrument was operated in negative electrospray ionization and selected reaction monitoring mode. RESULTS: The detection limit for PEth 16:0/16:0, 16:0/18:1, and 16:0/18:2 was ~0.1 ng/mL, and the extraction recoveries at 2.0 ng/mL were in the range of 99%-114%. Method linearity over a concentration range up to 200 ng/mL was ≥0.99. No significant deviation in results was observed in an analyte stability study of two different concentrations at two different temperatures over 3 months. In 35 oral fluid samples collected from patients undergoing alcohol detoxification, the highest concentration was observed for PEth 16:0/18:1 (Detected range, 0.51-55.3 ng/mL; mean, 8.5; median, 3.1). In addition, all three PEth forms were variably identified in a majority (63%) of the oral fluid samples. The PEth 16:0/18:1 values in oral fluid showed a weak positive correlation with the corresponding values in whole blood samples (r=0.50, p=0.026, n=20). CONCLUSIONS: The LC-MS/MS method could reliably detect and quantify PEth in oral fluid samples collected after alcohol exposure. The method was characterized by validation data with satisfactory recovery, sensitivity, accuracy, and imprecision, and applied for analysis of clinical samples. The results suggest that measurement of PEth in oral fluid can be used as a biomarker for alcohol consumption, and as such a non-invasive complement to analysis in blood. However, further studies are required to evaluate the test characteristics (e.g. sensitivity and half-life) in comparison with PEth in blood.

Potential of Mass Spectrometry in Developing Clinical Laboratory Biomarkers of Nonvolatiles in Exhaled Breath.

BACKGROUND: Exhaled breath contains nonvolatile substances that are part of aerosol particles of submicrometer size. These particles are formed and exhaled as a result of normal breathing and contain material from distal airways of the respiratory system. Exhaled breath can be used to monitor biomarkers of both endogenous and exogenous origin and constitutes an attractive specimen for medical investigations. CONTENT: This review summarizes the present status regarding potential biomarkers of nonvolatile compounds in exhaled breath. The field of exhaled breath condensate is briefly reviewed, together with more recent work on more selective collection procedures for exhaled particles. The relation of these particles to the surfactant in the terminal parts of the respiratory system is described. The literature on potential endogenous low molecular weight compounds as well as protein biomarkers is reviewed. The possibility to measure exposure to therapeutic and abused drugs is demonstrated. Finally, the potential future role and importance of mass spectrometry is discussed. SUMMARY: Nonvolatile compounds exit the lung as aerosol particles that can be sampled easily and selectively. The clinical applications of potential biomarkers in exhaled breath comprise diagnosis of disease, monitoring of disease progress, monitoring of drug therapy, and toxicological investigations.

Identification of main human urinary metabolites of the designer nitrobenzodiazepines clonazolam, meclonazepam, and nifoxipam by nano-liquid chromatography-high-resolution mass spectrometry for drug testing purposes.

Among the new psychoactive substances (NPS), so-called designer benzodiazepines have become of particular importance over the last 2 years, due to their increasing availability on the internet drug market. Therapeutically used nitrobenzodiazepines such as flunitrazepam are known to be extensively metabolized via N-dealkylation to active metabolites and via nitro reduction to the 7-amino compounds. The aim of the present work was to tentatively identify phase I and II metabolites of the latest members of this class appearing on the NPS market, clonazolam, meclonazepam, and nifoxipam, in human urine samples. Nano-liquid chromatography-high-resolution mass spectrometry was used to provide data about their detectability in urine. Data revealed that clonazolam and meclonazepam were extensively metabolized and mainly excreted as their amino and acetamino metabolites. Nifoxipam was also extensively metabolized, but instead mainly excreted as the acetamino metabolite and a glucuronic acid conjugate of the parent. Based on analysis of human urine samples collected in cases of acute intoxication within the Swedish STRIDA project, and samples submitted for routine drug testing, the most abundant metabolites and good targets for urine drug testing were 7-aminoclonazolam for clonazolam, 7-acetaminomeclonazepam for meclonazepam, and 7-acetaminonifoxipam for nifoxipam.

Measurement of Lung Phosphatidylcholines in Exhaled Breath Particles by a Convenient Collection Procedure.

An analytical method based on high-performance liquid chromatography coupled to quadrupole tandem mass spectrometry was developed for the quantitative determination of four phosphatidylcholines (PCs) in human exhaled breath particles. Analytes were conveniently collected on an electrostatic polymer filter and extracted with methanol prior to analysis. Chromatographic separation was performed on an ultraperformance liquid chromatographic ethylene bridged hybrid phenyl column using a mobile phase consisting of water and methanol containing 4 mM ammonium formate and 0.1% ammonia. The mass spectrometer operated in positive electrospray ionization and selected reaction monitoring mode. Detection limits for PC 16:0/16:0 (dipalmitoylphosphatidylcholine, DPPC), PC 16:0/18:1, PC 16:0/18:2, and PC 18:0/18:2 were <0.01 ng/filter. Method recoveries at concentration levels of 0.1 and 10 ng/filter were 100-110% and 101-121%, respectively. Acceptable precision with coefficients of variation <20% and accuracies of 100% ± 20% were achieved. Identification of the individual PCs was performed on the basis of two product ions with correct ion ratios and chromatographic retention times. The highest amount in exhaled breath was found for DPPC with median concentration 1.14 ng/filter (range 0.6-21 ng/filter), and median molar ratios of DPPC/PC (16:0/18:1) of 1.98 (range 0.48-2.75). A different pattern with lower molar ratio (∼0.15) was found for oral fluid. The most significant element of this study was to use a precolumn in the LC system and to collecting exhaled particles in an electret polymer filter. Due to chromatographic interference by background contamination, an isolator column (PFC kit) was installed in between eluent mixer and injector to reduce contamination. This is the first LC/MS study where the method was successfully applied to analyze PCs in human exhaled breath by using a simple and convenient collection procedure.

The Psychostimulant Khat (Catha edulis) Inhibits CYP2D6 Enzyme Activity in Humans.

The use of khat (Catha edulis) while on medication may alter treatment outcome. In particular, the influence of khat on the metabolic activities of drug-metabolizing enzymes is not known. We performed a comparative 1-way crossover study to evaluate the effect of khat on cytochrome P450 (CYP)2D6 and CYP3A4 enzyme activity. After 1 week of khat abstinence, baseline CYP2D6 and CYP3A4 metabolic activities were determined in 40 Ethiopian male volunteers using 30 mg dextromethorphan (DM) as a probe drug and then repeated after 1 week of daily use of 400 g fresh khat leaves. Urinary concentrations of cathinone and cathine were determined to monitor the subjects' compliance to the study protocol. Genotyping for CYP2D6*3 and CYP2D6*4 was done. Plasma DM, dextrorphan and 3-methoxymorphinan concentrations were quantified. CYP2D6 and CYP3A4 enzyme activities were assessed by comparing plasma log DM/dextrorphan and log DM/methoxymorphinan metabolic ratio (MR) respectively in the presence and absence of khat. Cytochrome 2D6 MR was significantly increased from baseline by concurrent khat use (paired t test, P = 0.003; geometric mean ratio, 1.38; 95% confidence interval [95% CI], 1.12-1.53). Moreover, the inhibition of CYP2D6 activity by khat was more pronounced in CYP2D6*1/*1 compared with CYP2D6*1/*4 genotypes (P = 0.01). A marginal inhibition of CYP3A4 activity in the presence of khat was observed (P = 0.24). The mean percentage increase of CYP2D6 and CYP3A4 MR from baseline by khat use was 46% (95% CI, 20-72) and 31% (95% CI, 8-54), respectively. This is the first report linking khat use with significant inhibition of CYP2D6 metabolic activity in humans.

Application of drug testing using exhaled breath for compliance monitoring of drug addicts in treatment.

AIM: Exhaled breath has recently been identified as a possible matrix for drug testing. This study explored the potential of this new method for compliance monitoring of patients being treated for dependence disorders. METHODS: Outpatients in treatment programs were recruited for this study. Urine was collected as part of clinical routine and a breath sample was collected in parallel together with a questionnaire about their views of the testing procedure. Urine was analyzed for amphetamines, benzodiazepines, cannabis, cocaine, buprenorphine, methadone and opiates using CEDIA immunochemical screening and mass spectrometry confirmation. The exhaled breath was collected using the SensAbues device and analyzed by mass spectrometry for amphetamine, methamphetamine, diazepam, oxazepam, tetrahydrocannabinol, cocaine, benzoylecgonine, buprenorphine, methadone, morphine, codeine and 6-acetylmorphine. RESULTS: A total of 122 cases with parallel urine and breath samples were collected; 34 of these were negative both in urine and breath. Out of 88 cases with positive urine samples 51 (58%) were also positive in breath. Among the patients on methadone treatment, all were positive for methadone in urine and 83% were positive in breath. Among patients in treatment with buprenorphine, 92% were positive in urine and among those 80% were also positive in breath. The questionnaire response documented that in general, patients accepted drug testing well and that the breath sampling procedure was preferred. CONCLUSION: Compliance testing for the intake of prescribed and unprescribed drugs among patients in treatment for dependence disorders using the exhaled breath sampling technique is a viable method and deserves future attention.

Determination of amphetamine and methylphenidate in exhaled breath of patients undergoing attention-deficit/hyperactivity disorder treatment.

OBJECTIVES: It has been discovered recently that exogenous substances are detectable in exhaled breath after intake. Exhaled breath therefore constitutes a new possible matrix in clinical pharmacology and toxicology. The present work was aimed at exploring this possibility further by a study on patients treated for attention-deficit/hyperactivity disorder with D-amphetamine and methylphenidate. METHODS: Thirteen patients (age range: 32-61 years; 5 women) were included in the study, and breath and urine samples were collected at different times in the dose interval. Analyses of breath and urine samples were done with liquid chromatography-mass spectrometry methods. Urine was examined for amphetamine, methylphenidate, and its metabolite ritalinic acid. RESULTS: Among the 9 patients who received D-amphetamine medication in daily doses of 20-100 mg, amphetamine was detected in all subjects in amounts ranging from 1200 to 30,800 picogram per filter. Among 8 patients receiving methylphenidate medication in daily doses of 80-400 mg, it was detected and quantified in 7 of the cases in amounts ranging from 150 to 10,400 picogram per filter and ritalinic acid was detected and quantified in 3 of the cases ranging from 35 to 360 picogram per filter. In 1 case, methylphenidate was only detectable in breath and urine, whereas ritalinic acid was quantifiable in urine, which could indicate noncompliance, with the 4 hours of dose regimen prescribed. In a number of cases, the sampling was performed 24 hours after the last dose intake. Identification of amphetamine and methylphenidate was based on correct chromatographic retention time and correct product ion ratio with detection performed in selected reaction monitoring mode. CONCLUSIONS: The results confirm that amphetamine is present in exhaled breath after intake and demonstrate for the first time the presence of methylphenidate and ritalinic acid after its intake. This gives further support to the potential use of exhaled breath for detecting drug intake.