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Background and Objectives: Clearances and the area under the plasma concentration–time curve extrapolated to infinity (AUC0–∞) of intravenous (IV) and oral midazolam and alfentanil are probes for hepatic and first-pass cytochrome P450 3A (CYP3A) activity, drug interactions, and phenotyping. Single-time plasma concentrations are also used as a proxy for clearance and AUC0–∞. Pupil diameter change is a noninvasive surrogate for plasma alfentanil. An ideal probe should have minimal intrasubject (interday) variability. Despite their widespread use, the intrasubject variability of CYP3A probes is not well characterized. This investigation determined the intrasubject (interday) variability of midazolam and alfentanil metrics of hepatic and first-pass CYP3A. Methods: Twelve volunteers were studied in a four-period protocol, with each period identical and separated by approximately 2 weeks. In each period, participants received 1 mg IV midazolam then 15 μg/kg IV alfentanil 1 h later. The next day, they received 3 mg oral midazolam then 60 μg/kg oral alfentanil. Plasma drug concentrations were determined by liquid chromatography-mass spectrometry (LCMS). Dark-adapted pupil diameters were measured coincident with blood sampling. Plasma concentrations and pupil effects (miosis) were analyzed using noncompartmental methods. The results were the coefficient of variation (%CV, mean ± SD) across four sessions in 12 participants. Results: For IV midazolam: AUC0–∞, clearance, and 5 h concentration, the %CVs were 12 ± 3, 12 ± 3, and 18 ± 8. For IV alfentanil AUC0–∞, clearance, 2 h concentration, and area under the effect curve from time zero to infinity (AUEC0–∞), the %CVs were 16 ± 5, 15 ± 4, 22 ± 7, and 50 ± 28. For oral midazolam AUC0–∞, clearance, and 5 h concentration, %CVs were 19 ± 5, 18 ± 4, and 28 ± 11. For oral alfentanil: AUC0–∞, clearance, 4 h concentration, and AUEC0–∞, %CVs were 20 ± 4, 21 ± 4, 42 ± 26, and 37 ± 14. Conclusions: Midazolam and alfentanil had comparable intrasubject variabilities of clearance and AUC0–∞. Single-time point metrics had greater intrasubject variability than AUC0–∞ and clearance. Miosis was a surrogate for alfentanil concentrations and provided real-time results, but intrasubject variability was greater than that of clearances and AUC0–∞. [ABSTRACT FROM AUTHOR]

Objective: People regularly contact emergency medicine services concerned that they have been exposed to drink spiking, i.e., exposure to drugs without their knowledge or permission. We identified drugs in blood and urine samples from patients suspecting exposure to drink spiking, with special consideration for drugs not reported taken by the patient (unreported drugs). Methods: From September 2018 to May 2019, we collected blood and urine samples from patients 16 years or older presenting at an emergency clinic in Oslo, Norway, within 48 hours of suspected exposure to drink spiking. We also collected information on ethanol ingestion and drugs taken. Blood samples were analyzed for 20 classical recreational drugs using ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) and an automated enzymatic method for ethanol. Urine samples were analyzed using immunoassay methods and a specific gas chromatography mass spectrometry (GCMS) method for gammahydroxybutyrate (GHB). Results: From 100 included patients (median age 24 years, 62 females), we collected 100 blood samples and 72 urine samples. Median time since exposure was 5 hours. Unreported drugs were found in 15 patients. Unreported drugs in the blood samples were clonazepam in 3, methylenedioxymethamphetamine (MDMA) in 3, amphetamine in 2, tetrahydrocannabinol (THC) in 2, tramadol in 1, cocaine in 1, and methamphetamine in 1. Unreported drugs in the urine samples were cocaine in 5, amphetamine in 4, ecstasy in 3, and cannabis in 2. Ethanol was found in 69 patients, all reporting ethanol ingestion. Median blood ethanol concentration was higher in patients with no unreported drugs detected, 1.00‰ (interquartile range (IQR) 0–1.52) vs. 0‰ (IQR 0–0.46) (p<0.001). GHB was not detected. Conclusion: Unreported drugs, possibly used for drink spiking, were found in 15% of patients. Blood ethanol concentration was higher when no unreported drugs were found. GHB was not detected in any patient. [ABSTRACT FROM AUTHOR]

Currently, tandem mass spectrometry-based newborn screening (NBS), which examines targeted biomarkers, is the first approach used for the early detection of maple syrup urine disease (MSUD) in newborns, followed by confirmatory genetic mutation tests. However, these diagnostic approaches have limitations, demanding the development of additional tools for the diagnosis/screening of MUSD. Recently, untargeted metabolomics has been used to explore metabolic profiling and discover the potential biomarkers/pathways of inherited metabolic diseases. Thus, we aimed to discover a distinctive metabolic profile and biomarkers/pathways for MSUD newborns using untargeted metabolomics. Herein, untargeted metabolomics was used to analyze dried blood spot (DBS) samples from 22 MSUD and 22 healthy control newborns. Our data identified 210 altered endogenous metabolites in MSUD newborns and new potential MSUD biomarkers, particularly L-alloisoleucine, methionine, and lysoPI. In addition, the most impacted pathways in MSUD newborns were the ascorbate and aldarate pathways and pentose and glucuronate interconversions, suggesting that oxidative and detoxification events may occur in early life. Our approach leads to the identification of new potential biomarkers/pathways that could be used for the early diagnosis/screening of MSUD newborns but require further validation studies. Our untargeted metabolomics findings have undoubtedly added new insights to our understanding of the pathogenicity of MSUD, which helps us select the appropriate early treatments for better health outcomes. [ABSTRACT FROM AUTHOR]

The pharmacokinetic properties of dihydroquercetin (DHQ) were studied after single and repeated (for 3 days) administration to rats in the form of a starch suspension at a dose of 25 mg/kg. Blood samples were collected using a permanent catheter in the jugular vein in 2, 5, 10, 20, and 30 min and in 1, 2, 4, and 6 h after administration. Before the repeated administration (5 min), blood sample was collected to assess the concentration of DHQ at the zero time point. Quantitative analysis was carried out by HPLC-tandem mass spectrometry. DHQ was very quickly absorbed by the gastrointestinal tract and quickly eliminated from the body. Repeated administration of DHQ did not lead to its accumulation in the body but had an effect on the enzymatic system with a subsequent increase in DHQ exposure (accumulation factor >1 by AUC after repeated administration). [ABSTRACT FROM AUTHOR]

Saxitoxins (STXs) are potent neurotoxins produced by marine dinoflagellates or freshwater cyanobacteria known to cause acute and eventually fatal human intoxications, which are classified as paralytic shellfish poisonings (PSPs). Rapid analysis of STXs in blood plasma can be used for a timely diagnosis and confirmation of PSPs. We developed a fast and simple method of STX extraction based on plasma sample acidification and precipitation by acetonitrile, followed by quantification using liquid chromatography-tandem mass spectrometry (LC–MS-MS). Our approach provides the results ≤30 min, with a limit of detection of 2.8 ng/mL and a lower limit of quantification of 5.0 ng/mL. Within-run and between-run precision experiments showed good reproducibility with ≤15% values. Standard curves for calibration were linear with correlation coefficients ≥0.98 across the assay calibration range (5–200 ng/mL). In an interlaboratory analytical exercise, the method was found to be 100% accurate in determining the presence or absence of STX in human plasma specimens, with recovery values of 86–99%. This simple method for STX determination in animal or human plasma can quickly and reliably diagnose STX exposures and confirm suspected PSP cases to facilitate patient treatment or expedite necessary public health or security actions. [ABSTRACT FROM AUTHOR]

Dispersive solid phase extraction/back–extraction of Ibuprofen using multi-walled carbon nanotubes as sorbent was studied. The effects of some factors on Ibuprofen extraction were investigated. pH of IP solution and temperature were more effective on IP adsorption than other investigated parameters. The obtained preconcentration factor was 11.43. A linear range of 10.31 – 206.28 mg/L (R2 = 0.997) was obtained for the HPLC method. The detection limit and relative standard deviation were 4.93 mg/l and 2.27%, respectively. Extraction and HPLC/UV determination of Ibuprofen in the human blood serum samples was carried out efficiently using the suggested procedure. The obtained results showed satisfactory selectivity and efficiency for the extraction of Ibuprofen molecules from the matrix of the Human blood serum samples. The suggested method is simple, efficient, fast, without usage of organic solvents, and low cost for separation, extraction, and determination of Ibuprofen molecules. [ABSTRACT FROM AUTHOR]

To update traditional "wet" matrices to dried blood spot (DBS) sampling, based on the liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) technique, and develop a method for simultaneous analyzing caffeine and its three primary metabolites (theobromine, paraxanthine, and theophylline), supporting routine therapeutic drug monitoring (TDM) for preterm infants. DBS samples were prepared by a two-step quantitative sampling method, i.e., volumetric sampling of a quantitative 10 μL volume of peripheral blood and an 8 mm diameter whole punch extraction by a methanol/water (80/20, v/v) mixture containing 125 mM formic acid. Four paired stable isotope labeled internal standards and a collision energy defect strategy were applied for the method optimization. The method was fully validated following international guidelines and industrial recommendations on DBS analysis. Cross validation with previously developed plasma method was also proceeded. The validated method was then implemented on the TDM for preterm infants. The two-step quantitative sampling strategy and a high recovery extraction method were developed and optimized. The method validation results were all within the acceptable criteria. Satisfactory parallelism, concordance, and correlation were observed between DBS and plasma concentrations of the four analytes. The method was applied to provide routine TDM services to 20 preterm infants. A versatile LC-MS/MS platform for simultaneous monitoring caffeine and its three primary metabolites was developed, fully validated, and successfully applied into the routine clinical TDM practices. Sampling method switching from "wet" matrices to "dry" DBS will facilitate and support the precision dosing of caffeine for preterm infants. [ABSTRACT FROM AUTHOR]

This perspective explores the feasibility of smart sampling with dried blood spots for the determination of proteins and peptides from human biomatrices using liquid chromatography coupled to mass spectrometry for clinical purposes. The focus is on innovative approaches to transform filter paper from a mere sample carrier to an active element in sample preparation, with the aim of reducing the need for extensive and intensive sample preparation in the conventional sense. Specifically, we discuss the use of modified cellulose to integrate sample preparation at an early stage of sample handling. The use of paper immobilized with either trypsin or monoclonal antibodies for protein digestion and affinity clean‐up is discussed as a potential benefit of starting sample preparation instantly at the moment of sampling to optimize time efficiency and enable faster analysis, diagnosis, and follow‐up of patients. [ABSTRACT FROM AUTHOR]

Aims: Measuring venous plasma paracetamol concentrations is time‐ and resource‐consuming. We aimed to validate a novel electrochemical point‐of‐care (POC) assay for rapid paracetamol concentration determinations. Methods: Twelve healthy volunteers received 1 g oral paracetamol, and its concentrations were analysed 10 times over 12 h for capillary whole blood (POC), venous plasma (high‐performance liquid chromatography tandem mass spectrometry (HPLC‐MS/MS)), and dried capillary blood (HPLC‐MS/MS). Results: At concentrations >30 μM, POC showed upward biases of 20% (95% limits of agreement [LOA] −22 to 62) and 7% (95% LOA −23 to 38) compared with venous plasma and capillary blood HPLC‐MS/MS, respectively. There were no significant differences between mean concentrations for the paracetamol elimination phase. Conclusions: Upward biases in POC compared with venous plasma HPLC‐MS/MS were likely due to higher paracetamol concentrations in capillary blood than in venous plasma and to faulty individual sensors. The novel POC method is a promising tool for paracetamol concentration analysis. [ABSTRACT FROM AUTHOR]

Due to the high prevalence of cannabinoids in forensic toxicology analysis, it is crucial to have an efficient method that allows the use of a small sample amount and that requires a minimal sample preparation for the determination and quantification of low concentrations. A simple, highly selective and high throughput liquid chromatography–tandem mass spectrometry methodology (LC–MS-MS-MS3) was developed for the determination and quantification of ∆9-tetrahydrocannabinol (THC), 11-hydroxy-∆9- tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THC-COOH) in blood samples. Chromatographic analysis of THC, THC-OH and THC-COOH and their deuterated internal standards was preceded by protein precipitation (PPT) of 0.1 mL of blood samples with acetonitrile. Chromatographic separation was achieved by use of an Acquity UPLC® HHS T3 (100 mm × 2.1 mm i.d. 1.8 μm) reversed-phase column, using a gradient elution of 2 mM aqueous ammonium formate, 0.1% formic acid and methanol at a flow rate of 0.4 mL/min, with a run time of 10 min. For the MS-MS-MS3 analysis, a SCIEX QTRAP® 6500+ triple quadrupole linear ion trap mass spectrometer was used via electrospray ionization (ESI), operated in multiple reaction monitoring (MRM) and linear ion trap mode (MS3). The method was validated in accordance with internationally accepted criteria and guidelines, and proved to be selective and linear between 0.5 and 100 ng/mL (r2 > 0.995). The lower limits of quantification (LLOQ) corresponded to the lowest concentrations used for the calibration curves. The coefficients of variation obtained for accuracy and precision were <15%. The mean recoveries were between 88.0% and 117.2% for the studied concentration levels (1 ng/mL, 5 ng/mL and 50 ng/mL). No significant interfering compounds, matrix effects or carryover were observed. The validated method provides a sensitive, efficient and robust procedure for the quantification of cannabinoids in blood, using LC–MS-MS-MS3 and a sample volume of 0.1 mL. This work is also a proof of concept for using LC–MS3 technique to determine drugs in biological samples. [ABSTRACT FROM AUTHOR]

Therapeutic drug monitoring (TDM) is a specialized area of laboratory medicine which involves the measurement of drug concentrations in biological fluids with the aim of optimizing efficacy and reducing side effects, possibly modifying the drug dose to keep the plasma concentration within the therapeutic range. Plasma and/or whole blood, usually obtained by venipuncture, are the "gold standard" matrices for TDM. Microsampling, commonly used for newborn screening, could also be a convenient alternative to traditional sampling techniques for pharmacokinetics (PK) studies and TDM, helping to overcome practical problems and offering less invasive options to patients. Although technical limitations have hampered the use of microsampling in these fields, innovative techniques such as 3-D dried blood spheroids, volumetric absorptive microsampling (VAMS), dried plasma spots (DPS), and various microfluidic devices (MDS) can now offer reliable alternatives to traditional samples. The application of microsampling in routine clinical pharmacology is also hampered by the need for instrumentation capable of quantifying analytes in small volumes with sufficient sensitivity. The combination of microsampling with high-sensitivity analytical techniques, such as liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), is particularly effective in ensuring high accuracy and sensitivity from very small sample volumes. This manuscript provides a critical review of the currently available microsampling devices for both whole blood and other biological fluids, such as plasma, urine, breast milk, and saliva. The purpose is to provide useful information in the scientific community to laboratory personnel, clinicians, and researchers interested in implementing the use of microsampling in their routine clinical practice. [ABSTRACT FROM AUTHOR]

Direct infusion of lipid extracts into the ion source of a mass spectrometer is a well-established method for lipid analysis. In most cases, nanofluidic devices are used for sample introduction. However, flow injection analysis (FIA) based on sample infusion from a chromatographic pump can offer a simple alternative to shotgun-based approaches. Here, we describe important modification of a method based on FIA and tandem mass spectrometry (MS/MS). We focus on minimizing contamination of the FIA/MS both to render the lipidomic platform more robust and to increase its capacity and applicability for long-sequence measurements required in clinical applications. Robust validation of the developed method confirms its suitability for lipid quantitation in human plasma analysis. Measurements of standard human plasma reference material (NIST SRM 1950) and a set of plasma samples collected from kidney cancer patients and from healthy volunteers yielded highly similar results between FIA-MS/MS and ultra-high-performance supercritical fluid chromatography (UHPSFC)/MS, thereby demonstrating that all modifications have practically no effect on the statistical output. Newly modified FIA-MS/MS allows for the quantitation of 141 lipid species in plasma (11 major lipid classes) within 5.7 min. Finally, we tested the method in a clinical laboratory of the General University Hospital in Prague. In the clinical setting, the method capacity reached 257 samples/day. We also show similar performance of the classification models trained based on the results obtained in clinical settings and the analytical laboratory at the University of Pardubice. Together, these findings demonstrate the high potential of the modified FIA-MS/MS for application in clinical laboratories to measure plasma and serum lipid profiles. [ABSTRACT FROM AUTHOR]

Quick, easy, cheap, effective, rugged, and safe extraction strategies are becoming increasingly adopted in various analytical fields to determine drugs in biological specimens. In the present study, we developed two fully automated quick, easy, cheap, effective, rugged, and safe extraction methods based on acetonitrile salting‐out assisted liquid‐liquid extraction (method 1) and acetonitrile salting‐out assisted liquid‐liquid extraction followed by dispersive solid‐phase extraction (method 2) using a commercially available automated liquid‐liquid extraction system. We applied these methods to the extraction of 14 psychotropic drugs (11 benzodiazepines and carbamazepine, quetiapine, and zolpidem) from whole blood samples. Both methods prior to liquid chromatography–tandem mass spectrometry analysis exhibited high linearity of calibration curves (correlation coefficients, > 0.9997), ppt level detection sensitivities, and satisfactory precisions (< 8.6% relative standard deviation), accuracies (within ± 16% relative error), and matrix effects (81–111%). Method 1 provided higher recovery rates (80–91%) than method 2 (72–86%), whereas method 2 provided higher detection sensitivities (limits of detection, 0.003–0.094 ng/mL) than method 1 (0.025–0.47 ng/mL) owing to the effectiveness of its dispersive solid‐phase extraction cleanup step. These fully automated extraction methods realize reliable, labor‐saving, user‐friendly, and hygienic extraction of target analytes from whole blood samples. [ABSTRACT FROM AUTHOR]

Objectives: Conduction of pharmacokinetic (PK) study in pediatric patients is challenging due to blood sampling limits. The dried blood spots (DBS) method represents a potential matrix for microsampling in support of PK studies in children. Herein, we used the Capitainer® qDBS device to develop a DBS method that can collect an exact 10 µL volume of blood on a paper card. This DBS method was developed to simultaneously quantify the concentrations of eight antibiotics, including sulbactam, tazobactam, ampicillin, meropenem, cefotaxime, cefoperazone, piperacillin, and metronidazole using ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). Methods: The prepared DBS samples were extracted in methanol containing acetaminophen as the internal standard at 20 °C on a block bath shaker at 500 rpm for 30 min. The extracted antibiotics were eluted on an Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) using gradient elution with a total chromatographic run time of 6.5 min. The precursor and product ions of the analytes were detected by use of the multiple reaction monitoring (MRM) mode. Results: No interfering peaks at the respective retention times of the analytes were observed in DBS samples. The lower limits of quantification (LLOQ) for the antibiotics were between 0.25 and 2.0 μg/mL, and satisfactory accuracies (intra/inter-assay bias −16.7 to +13.6%) and precisions (intra/inter-assay coefficient of variations 1.5–15.6%) were obtained for the analytes. As a proof of concept, the method was applied to DBS samples obtained from neonatal patients treated with ampicillin and piperacillin/sulbactam. Conclusions: The DBS method is simple and robust, and it can be used in children with limited blood sampling. [ABSTRACT FROM AUTHOR]

Endogenous sex hormones and DNA methylation both play important roles in various diseases. However, their interplay is largely unknown. A deeper understanding of their interrelationships could provide new insights into the pathology of disease development. We, therefore, investigated associations between circulating sex hormones, sex hormone binding globulin (SHBG), and DNA methylation in blood, using samples from 77 men (65 with repeated samples), from the population-based Northern Sweden Health and Disease Study (NSHDS). DNA methylation was measured in buffy coat using the Infinium Methylation EPIC BeadChip (Illumina). Sex hormone (oestradiol, oestrone, testosterone, androstenedione, dehydroepiandrosterone, and progesterone) and SHBG concentrations were measured in plasma using a high-performance liquid chromatography tandem mass spectrometry (LC/MS-MS) method and an enzyme-linked immunoassay, respectively. Associations between sex hormones, SHBG, and DNA methylation were estimated using both linear regression and mixed-effects models. Additionally, we used the comb-p method to identify differentially methylated regions based on nearby P values. We identified one novel CpG site (cg14319657), at which DNA methylation was associated with dehydroepiandrosterone, surpassing a genome-wide significance level. In addition, more than 40 differentially methylated regions were associated with levels of sex hormones and SHBG and several of these mapped to genes involved in hormone-related diseases. Our findings support a relationship between circulating sex hormones and DNA methylation and suggest that further investigation is warranted, both for validation, further exploration and to gain a deeper understanding of the mechanisms and potential consequences for health and disease. [ABSTRACT FROM AUTHOR]

Benzodiazepines (BZDs) and Z-drugs are among the most commonly prescribed pharmaceuticals in the world and are considered standard care for various mental illnesses and for the treatment of sleeping and anxiety disorders, alcohol withdrawal, muscle spasms and epilepsy. Some BZDs are not allowed as pharmaceuticals in many countries, and they are used as designer benzodiazepines (DBZDs). All these compounds are typically screened in routine toxicological analyses for forensic purposes. Knowledge of time-dependent decreases in drug concentrations during storage or transport of samples is of considerable significance and allows forensic toxicologists to achieve reliable data, proper interpretation and high-quality results. The aim of this study was to evaluate changes in the amounts of selected BZDs, DBZDs and Z-drugs in blood samples stored at various temperatures. The study involved BZDs (19), DBZDs (3) and Z-drugs (2) spiked into blank blood. Subsequently, the blood samples were stored at various temperatures (room temperature, 4°C, −20°C and −80°C) for up to 6 months. Analyses were performed at 1- to 2-week intervals using liquid chromatography–tandem mass spectrometry. The stability of compounds was evaluated under four temperature conditions over a 6-month period. Some BZDs were stable at all temperatures tested (e.g. diazepam, oxazepam, nordazepam and prazepam) with a degradation rate of only 0–10%. The highest instability was observed for analyte samples kept at room temperature, and the losses in content for some compounds, e.g. lorazepam and chlordiazepoxide, were almost 100%. For other compounds, the stability was clearly different at each tested temperature. To the best of our knowledge, this is one of the first such comprehensive study of the long-term stability of BZDs covering a wide range of different storage temperatures. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths in the world. HCC is often diagnosed late because patients with early-stage cancer have no apparent symptoms. Therefore, it is desirable to find a reliable method for an early diagnosis based on the detection of metabolites – biomarkers, that can be detected in the early stages of the disease. Untargeted metabolomics is often used as a tool to find a suitable biomarker for several diseases. In this work, untargeted metabolomics was performed on blood plasma samples of HCC patients and compared with healthy individuals and patients with liver cirrhosis. A combination of liquid chromatography and high-resolution mass spectrometry was used as an analytical method. More than a thousand peaks were detected in the blood plasma samples, from which mainly amino acids, carboxylic acids, lipids, and their derivatives were evaluated as potential biomarkers. The data obtained were statistically processed using the analysis of variance, correlation analysis, and principal component analysis. • Hepatocellular carcinoma is one of the leading causes of death related to cancer. • The metabolism of cancer cells is different from that of healthy cells. • Metabolomics represents a promising tool for early diagnostics. [ABSTRACT FROM AUTHOR]

• An LC-MS/MS method was optimized for cardiovascular drugs in dried blood spots (DBS). • Drugs in DBS were simply extracted with organic solvents in two-step protein precipitation. • This method was fully validated for linearity, matrix effect, accuracy, and stability. • Drug distribution within spots was analyzed using punched discs. • Feasibility of accuracy-based analysis for cardiovascular drugs in DBS was proposed. Dried Blood Spots (DBS) revolutionize therapeutic drug monitoring using LC-MS for the precise quantification of cardiovascular drugs (CDs), enabling personalized treatment adapted to patient-specific pharmacokinetics with minimal invasiveness. This study aims to achieve simultaneous quantification of eight CDs in DBS, overcoming physicochemical challenges. A two-step protein precipitation method was used for simple and precise sample preparation. The drugs were analyzed using LC-MS/MS in ESI positive-ion mode, showing high sensitivity and linearity, with a correlation coefficient (r2) exceeding 0.999, after being separated on a reversed-phase chromatography by gradient elution of DW-acetonitrile containing 0.1 % formic acid + 2 mM ammonium formate. The validation results indicate good selectivity, with no observed matrix effect and carry-over. The intra- and inter-day accuracy and precision were within 6 % for most drugs, except for digoxin and deslanoside at low therapeutic levels where the variation was within 20 %. Stability tests confirmed suitable DBS handling and storage conditions, indicating drug stability for at least 30 days at room temperature. The analysis of whole spot has demonstrated remarkable precision and reliability in all target drugs. The analysis of 3 mm internal diameter discs, punched in and out of DBS, presumed to contain 3 µL of blood, showed acceptable accuracy for most drugs, with less polar drugs like digoxin and deslanoside showing lower accuracy, indicating a need for further correction due to non-uniform drug distribution. Consequently, the developed LC-MS/MS method enables the quantification of multiple CDs in a single DBS analysis, while suggesting the potential for accuracy-based analysis. [ABSTRACT FROM AUTHOR]

Synthetic drugs for recreational purposes are in constant evolution, and their consumption promotes a significant increase in intoxication cases, resulting in damaging public health. The development of analytical methodologies to confirm the consumption of illicit drugs in biological matrices is required for the control of these substances. This work exploited the development of an extraction method based on homogenous liquid–liquid microextraction with switchable hydrophilicity solvent (SHS) as extraction phase for the determination of the synthetic drugs 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine and N-methoxybenzyl-methoxyphenylethylamine derivates (25B, 25C and 25I) in postmortem blood, followed by liquid chromatography coupled to mass spectrometry in tandem. The optimized sample preparation conditions consisted of using 250 µL of ZnSO4 10% and 50 µL of NaOH 1 mol/L in the protein precipitation step; N,N -dimethylcyclohexylamine was used as SHS, 650 μL of a mixture of SHS:HCl 6 mol/L (1:1 v/v), 500 μL of whole blood, 500 μL of NaOH 10 mol/L and 1 min of extraction time. The proposed method was validated, providing determination coefficients higher than 0.99 for all analytes; limit of detection and limit of quantitation ranged from 0.1 to 10 ng/mL; intra-run precision from 2.16% to 9.19%; inter-run precision from 2.39% to 9.59%; bias from 93.57% to 115.71% and matrix effects from 28.94% to 51.54%. The developed method was successfully applied to four authentic postmortem blood samples from synthetic drugs users, and it was found to be reliable with good selectivity. [ABSTRACT FROM AUTHOR]

Objective: To test the feasibility of an SC mini‐infusion pump to deliver ceftazidime in dogs and produce plasma concentrations sufficient to reach a therapeutic target for 48 hours. Setting: University research laboratory. Animals: Six healthy Beagle dogs. Interventions: Ceftazidime was administered by 2 routes to 6 healthy Beagle dogs. The first route was an IV bolus injection into a cephalic vein at a dose of 25 mg/kg. Blood samples were collected for 8 hours following injection. The second route was a SC infusion for 48 hours using the RxActuator Mini‐Infuser wearable SC constant rate infusion pump. Blood samples were collected for 58 hours following application of the pump. All plasma samples were analyzed by high‐pressure liquid chromatography and subject to pharmacokinetic analysis. Main Results: After the IV bolus injection, there was rapid distribution and elimination. The elimination half‐life was 0.95 hours, and the clearance was rapid at 0.176 ml/h/kg. After the 48‐hour SC infusion, the half‐life was slightly shorter, and the clearance was higher. The percent bioavailability from the SC infusion was approximately 72%. The SC infusion maintained plasma concentration near our target of 8 μg/ml for most of the dose interval but slightly lower after 24 hours. The concentrations below the target were attributed to slight drug loss, less than 100% bioavailability, and faster clearance from SC administration. Conclusions: This study demonstrated the successful application of the RxActuator Mini‐Infuser wearable SC constant rate infusion pump for delivering an antimicrobial needed for serious, and sometimes resistant, infections in dogs. [ABSTRACT FROM AUTHOR]

Abuse of new psychoactive substances (NPS) has become a health and social issue of global concern. p -Methoxyamphetamine (PMA)/ p -methoxymethamphetamine (PMMA) with fluoro- or chloro-derivatives of amphetamine and methamphetamine were among the most common drugs found in specimens from fatal cases in Taiwan during the January 2011 to December 2018 period. A liquid–liquid extraction sample preparation protocol with highly sensitive ultra-high performance liquid chromatography–tandem mass spectrometry approach was developed for the simultaneous analysis of seven phenethylamine-type drugs—PMA, PMMA, p -methoxyethylamphetamine, 4-fluoroamphetamine (4-FA), 4-fluoromethamphetamine (4-FMA), 4-chloroamphetamine (4-CA) and 4-chloromethamphetamine (4-CMA)—in postmortem blood and urine specimens. Separation by liquid chromatography was performed by Agilent Zorbax SB-Aq column. Tandem mass spectrometry was operated in Agilent Jet Stream Technology electrospray ionization in positive-ion multiple reaction monitoring mode. An analytical methodology was evaluated using drug-free blood and urine after fortification with 100–2,000 ng/mL of the seven target analytes. Average extraction recoveries were >80%; slightly higher ion suppression was observed for PMA and 4-CA; intra-/inter-day precision (% coefficient of variation) and accuracy were in the ranges of 0.52–12.3% and 85–110%, respectively. Limit of detection and lower limit of quantitation for these seven analytes were both in the 0.5–5 ng/mL range. Interference and carryover were not significant. This relatively simple methodology was found effective and reliable for routine identification and quantitation of these seven analytes in postmortem and antemortem blood and urine specimens received in 2018. Analytical data obtained from these actual cases indicated the following: (i) compared to findings reported during the 2007–2011 period, the use of substituted phenethylamine-type drugs decreased in 2018; (ii) ketamine and 7-aminonimetazepam (the main metabolite of nimetazepam) were the most common co-ingested substances in specimens containing PMA/PMMA, 4-FA/4-FMA, or 4-CA/4-CMA; and (iii) in drug fatalities, the concentration of PMA was significantly higher than the concentration of PMMA in both urine and blood, while the reverse was true in urine specimens from antemortem cases. [ABSTRACT FROM AUTHOR]

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Capillary dried blood spot (DBS) analysis coupled with multi-analyte steroid liquid chromatography mass spectrometry (LCMS) is attractive for field studies, home-based self-sampling as well as clinical trials by eliminating costly and laborious sample processing involving venipuncture and frozen storage/shipping while providing multiple steroid measurements from a single small sample. We investigated steroid measurements in DBS samples stored for four years at room temperature prior to analysis compared with the original venipuncture serum samples. Healthy women (n=12) provided paired DBS and blood samples over two weeks run-in before seven days treatment with daily transdermal T gel (12.5 mg) and after the end of treatment on days 0, 1, 2, 4, 7 and 14. Compliance with treatment and sampling was high and no adverse effects were reported. Testosterone (T), androstenedione (A 4), 17 hydroxyprogesterone (17OHP) and progesterone (P 4) were measured in extracted DBS samples as whole blood concentrations with and without adjustment for hematocrit. Using the same LCMS methods, DBS T and A 4 measurements had high correlation with minimal bias from prior serum measurements with DBS T displaying the same pattern as serum, with or without hematocrit adjustment. However, serial whole blood measurements of T without hematocrit adjustment provided the best fitting model compared with serum, urine, or hematocrit-adjusted whole blood T measurements. These finding facilitate and simplify DBS methodology for wider field and home-based self-sampling studies of reproductive steroids indicating the need for hematocrit adjustment may be superfluous. [ABSTRACT FROM AUTHOR]

4F-MDMB-BICA is one of the most dangerous new illicit synthetic cannabinoids (SCs) in 2020. Consumption of 4F-MDMB-BICA has been associated with a number of death cases and related serious adverse health effects in Hungary. Therefore, the use of reliable analytical methods to confirm the intake of 4F-MDMB-BICA is an important issue in forensic practice. Besides the detection of the parent compounds of SCs, the screening of their metabolites provides a reliable confirmation of their consumption, in particular, when the parent compound is under the limit of detection. To the best of our knowledge, this is the first report describing the identification of metabolites of 4F-MDMD-BICA after treatment with pooled human liver microsome (pHLM) and in human urine and blood samples using the combination of data obtained by comprehensive ultra-high performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC–HRMS) and semi-targeted UHPLC–HRMS-MS methods. Finally, our routine UHPLC coupled with triple-quadrupole tandem low-resolution mass spectrometry method for screening urine and blood SCs was improved by adding the parent compound and selected main biomarkers of 4F-MDMD-BICA. From the pHLM assay of 4F-MDMD-BICA, 30 phase I metabolites were characterized and structural information thus obtained provided the basis of further identification of in vivo urine and blood metabolites. Overall, 20 urinary and 13 blood in vivo metabolites of 4F-MDMD-BICA have been identified by the investigation of five authentic urine and two blood samples. The ester hydrolysis metabolite was selected as a reliable primary biomarker in urine and blood. As secondary targets, urinary mono-hydroxylation metabolite and ester hydrolysis + dehydrogenation metabolite in blood were recommended due to their abundance and selectivity. Overall, the main phase I metabolites of 4F-MDMD-BICA were successfully characterized, and our routine analytical method with related sample preparation procedure provided a reliable analytical tool for screening both 4F-MDMD-BICA and its selected metabolites in urine and blood samples. [ABSTRACT FROM AUTHOR]

Food contact materials (FCM) polyethylene terephthalate (PET) and polybutylene terephthalate (PBT) used extensively in food packaging may contain cyclic oligomers which may migrate into food and thus cause toxic effects on human health. A simple, fast, and sensitive ultra-high-performance liquid chromatography method quadrupole time-of-flight mass spectrometer was developed for the analysis of 7 cyclic oligomers in post-mortem blood samples. The targeted analytes were separated on a Waters BEH C18 (150 × 2.1 mm, 1.7 µm) analytical column by gradient elution. Calibration curves were constructed based on standard solutions and blood samples and Student's t-test was applied to evaluate the matrix effect. The LODs ranged from 1.7 to 16.7 μg mL−1, while the method accuracy was assessed by recovery experiments and resulting within the range 84.2–114.6%. Such an analytical method for the determination of PET and PBT cyclic oligomers in biological samples is reported for the first time. The developed methodology allows the determination of these oligomers in blood providing a useful analytical tool to assess the exposure and thus the potential hazard and health risks associated with these non-intentionally added substances (NIAS) from PET and PBT FCM through food consumption. The method was validated and successfully applied to the analysis of 34 post-mortem whole blood samples. Polyethylene terephthalate trimer was detected in four of them, for the first time in literature. [ABSTRACT FROM AUTHOR]

The aim of the study was to compare 3 blood sampling methods, including capillary blood sampling, for determining Tamoxifen (TAM), Z-endoxifen (END), and 4-hydroxytamoxifen (4HT) concentrations. High performance liquid chromatography-mass spectrometry was used to quantify concentrations of TAM, END, and 4HT in plasma, venous blood, and capillary blood samples of 16 participants on TAM therapy for breast cancer. The rhelise kit was used for capillary sampling. Calibration curves using 13C-labeled analogs of TAM, END, and 4HT as internal standards were used for quantifications. A capillary sampling kit was used successfully for all participants. Mean TAM concentrations did not differ significantly in the 3 types of samples. Mean END and 4HT concentrations did differ significantly between capillary and venous blood samples, possibly related to photodegradation in the internal standards prior to use or degradation products with chromatographic retention times similar to the metabolites. TAM, END, and 4HT concentrations were relatively stable when stored for 14 days at 8 °C and 20 °C. Therapeutic drug monitoring of TAM using an innovative kit and capillary blood sampling is feasible. Preliminary data from this study will aid in developing a multicenter, randomized clinical trial of personalized TAM dose monitoring and adjustments, with the goal of enhancing the quality-of-life and outcomes of patients with breast cancer. Clinical Trial Identification: EudraCT No 2017-000641-44. [ABSTRACT FROM AUTHOR]

Animal experiments have consistently shown that estrogen receptor β (ERβ)-selective ligands have antidepressant and anxiolytic effects. In humans, endogenous ligands for ERβ include 5α-androstane-3β, 17β-diol (3βAdiol) and androstenediol (Δ5-diol). We determined, for the first time, the exact serum levels of 3βAdiol and Δ5-diol in young healthy volunteers using liquid chromatography–tandem mass spectrometry (LC–MS/MS). We investigated the effect of the menstrual cycle on the levels of these steroids in women; then, we performed a gender comparison. Blood samples were collected from 48 subjects: 23 women (mean age = 28.4±7.8 years) and 25 men (mean age = 31.4±7.8 years). We collected the blood samples of women at three time-points in the menstrual cycle: the early follicular phase, ovulatory or mid-cycle phase, and mid-luteal phase. A total of 92 blood samples were analyzed using LC–MS/MS. The levels of two well-studied steroids, namely dehydroepiandrosterone (DHEA) and 17β-estradiol (E2), were simultaneously measured. Depression rating scale (Hamilton Rating Scale for Depression, Beck Depression Inventory-II and Quick Inventory of Depressive Symptomatology) scores were also recorded at the time of blood sampling. Significant differences in the levels of 3βAdiol and E2 and in the depression rating scale scores were observed over the duration of the menstrual cycle of the women. The levels of 3βAdiol and Δ5-diol were significantly lower in women than in men. E2 levels were higher in women than in men, and DHEA levels did not differ significantly between men and women. Further, women had higher scores than men on the Hamilton Rating Scale for Depression. Sex differences in depressive symptoms can be explained by 3βAdiol and Δ5-diol levels, and the effect of the menstrual cycle on mood can be explained by 3βAdiol and E2 levels, not by Δ5-diol level. [ABSTRACT FROM AUTHOR]

Adenosine is a purine ribonucleoside with important roles in various physiological processes. A number of studies have indicated the importance of adenosine in cardiovascular diseases including syncope; however, the accurate determination of adenosine in human blood is challenging due to the molecule's instability. In the present study, we report a simple method for the pre-treatment of blood samples and the development of a fast and efficient hydrophilic interaction chromatographic tandem mass spectrometry method for the analysis of adenosine in patient blood. During collection, samples were mixed directly with a solvent mixture containing 95% acetonitrile and 10 mM ammonium formate in a Vacutainer tube, resulting in successful prevention of adenosine metabolic processes and direct blood sample deproteinization. The method was validated according to bioanalytical industry guidelines and found to be accurate, repeatable, specific and sensitive with LLOQ 0.005 μg/mL, thus allowing its application in the analysis of real clinical samples. [ABSTRACT FROM AUTHOR]

Metomidate and etomidate belong to the non‐barbiturate imidazole family of sedative–hypnotics and elicit little analgesic action when used alone. Metomidate, in particular, has little analgesic activity in humans and is, therefore, used for veterinary purposes. In 2019, a Korean woman in her twenties was found unconscious in a motel bath and eventually died. Etomidate, alprazolam, escitalopram, and metomidate were detected in the postmortem specimens. To our knowledge, this is the first case of human metomidate abuse reported in the Republic of Korea. In this research, a simple and reliable method was developed for the analysis of metomidate and etomidate in human blood samples using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Blood samples were deproteinized with acetonitrile, filtered, and analyzed by LC–MS/MS. Linear calibration curves were obtained with six concentrations ranging from 1 to 50 ng/ml for metomidate and 10 to 500 ng/ml for etomidate. The method was validated by assessing the selectivity, linearity, limit of detection (LOD), limit of quantitation (LOQ), intra‐ and inter‐day precision and accuracy, matrix effect, and stability and successfully applied to the analysis of metomidate and etomidate in human blood samples. In a postmortem case, the concentrations of metomidate and etomidate were found to be 8 and 110 ng/ml in femoral blood and 6 and 210 ng/ml in cardiac blood, respectively. [ABSTRACT FROM AUTHOR]

Autism Spectrum Disorder is a neurodevelopmental disease with an average diagnosis age of over 3 years. Carnitine levels in ASD are important because they show potential mitochondrial dysfunction and abnormal fatty acid metabolism. In this study, in ASD children carnitine levels in dried blood spot samples were evaluated and compared with the control group. Twentythree children diagnosed with ASD in Research and Training Hospital (19 boys, 4 girls) and age and gender matched 24 children without ASD were enrolled in this study. 17 carnitines in dried blood samples were measured with LC-MS/MS. C0, C2, C4-OH, C5, C5-OH, C6, C16, C18 carnitines were lower (p value 0.037, 0.010, 0.005, 0.032, 0.005, 0.003, 0.043, 0.003, respectively) and C18:1 carnitine was higher (p<0.025) in ASD group compared with control group. Comprehensive carnitine levels for ASD are important to establish a treatment protocol for the treatment of ASD behavior and severity. C18:1 carnitine, detected for the first time in the cases with ASD, is important for its high levels and for being a glycine transporter two inhibitor. In ASD cases, the molecular analysis might be suggested for enzymes involved in carnitine metabolism and for glycine transporter 2. [ABSTRACT FROM AUTHOR]

Background: Monocarboxylate transporter 8 (MCT8) deficiency is an X-chromosome-linked neurodevelopmental disorder resulting from impaired thyroid hormone transport across the cell membrane. The diagnosis of MCT8 deficiency is typically delayed owing to the late appearance of signs and symptoms as well as the inability of standard biomarkers of neonatal screening to provide early detection. In this study, we report, for the first time, the ability to detect MCT8 deficiency at birth using dried blood spot (DBS) samples. Methods: We retrospectively measured triiodothyronine (T3), thyroxine (T4), and reverse T3 (rT3) levels in DBS samples obtained at 4–5 days of life from 6 infants with genetically confirmed MCT8 deficiency and from 110 controls. The latter consisted of 58 healthy term neonates obtained at the same time, 16 were stored for more than 1 year before measurement to match samples from the MCT8-deficient infants. Ten DBS samples were collected at day 1 of life and 42 samples were from prematurely born neonates. Measurements were carried out in extract from eight millimeters diameter DBS using liquid chromatography-tandem mass spectrometry. Results: Contrary to characteristic iodothyronine abnormalities of MCT8 deficiency during later life, T3 and T4 values were not discriminatory from those of other study groups. In contrast, rT3 was significantly lower. The T3/rT3 ratio was higher in the DBS samples from the MCT8-deficient infants compared with all other groups with no overlap (p < 0.0001). Conclusions: rT3 and T3/rT3 ratio in DBS samples obtained from neonates can serve as biomarkers to detect MCT8 deficiency at birth. [ABSTRACT FROM AUTHOR]

A sensitive LC-MS/MS method for the simultaneous quantification of the (9 R)- and (9 S)- hexahydrocannabinols (HHCs), and their metabolites, in human urine, oral fluid (OF) and blood samples were developed, validated and used to the biological samples of volunteers. The analytes were extracted from 100 μL human samples. An isocratic elution mode with methanol was used for chromatographic separation of (9 R)- and (9 S)-HHC on an immobilized amylose tris(3-chloro-5-methylphenylcarbamate)-based chiral column Lux i-Amylose-3. The flow-rate of the mobile phase was 0.5 mL/min. An isocratic elution mode of methanol and water (80/20, v/v) was used for chromatographic separation of metabolites of (9 R)- and (9 S)-HHC on a Lux AMP chiral column (with a proprietary chiral selector) at a flow rate of 0.5 mL/min. MS/MS analysis was performed in positive ionization mode for HHC epimers, while in negative ionization mode was used for metabolites of HHCs. The calibration curves for HHCs and their metabolites in human samples ranged from 0.25- 240 ng mL−1 and 1 - 100 ng mL−1, respectively, with determination coefficients (r2) of ≥ 0.99. All analytes were stable at room temperature, 4 °C, in the autosampler (+10 °C) and −20 °C for 24 h, after three freeze/thaw cycles, and when stored at −20 °C up to one week after quality control (QC) sample preparation (concentration differences less than 20% with respect to time zero response), in blood, urine and OF. [Display omitted] • An enantioselective method for the quantification of the (9R) and (9S)-HHC, and their metabolites in real human samples. • The method is simple, fast and fully validated. • Application to real biological fluids for the quantification of parent drug and metabolites. [ABSTRACT FROM AUTHOR]

• NUDT15 polymorphisms are associated with leukocytopenia and alopecia. • The assay system for quantifying NUDT15 enzymatic activity using LC-MS/MS was developed and validated. • The NUDT15 activity in cell extracts can be measured using the LC-MS/MS assay system. • Using human blood samples, the LC-MS/MS assay system can detect genotype-dependent differences in the NUDT15 activity. Thiopurine is metabolized to 6-thio-(deoxy) guanosine triphosphate (6-thio-(d) GTP), which is then incorporated into DNA or RNA and causes cytotoxicity. Nudix hydrolase 15 (NUDT15) reduces the cytotoxic effects of thiopurine by converting 6-thio-(d) GTP to 6-thio-(d) guanosine monophosphate (6-thio-(d) GMP). NUDT15 polymorphisms like the Arg139Cys variant are strongly linked to thiopurine-induced severe leukocytopenia and alopecia. Therefore, measurement of NUDT15 enzymatic activity in individual patients can help predict thiopurine tolerability and adjust the dosage. We aimed to develop a quantitative assay for NUDT15 enzymatic activity in human blood samples. Blood samples were collected from donors whose NUDT15 genetic status was determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the 6-thio-GTP metabolic activity in cell extracts. Because 6-thio-guanosine diphosphate (6-thio-GDP) and 6-thio-GMP were generated upon incubation of 6-thio-GTP with human blood cell extracts, the method detecting 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP was validated. All three metabolites were linearly detected, and the lower limit of quantification (LLOQ) of 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 5 μM, 1 μM, and 2 μM, respectively. Matrix effects of human blood cell extracts to detect 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 99.0 %, 100.5 %, and 101.4 %, respectively, relative to the signals in the absence of blood cell extracts. The accuracy and precision of the method and the stability of the samples were also assessed. Using this established method, the genotype-dependent differences in NUDT15 activities were successfully determined using cell extracts derived from human blood cells with NUDT15 wild-type (WT) or Arg139Cys variant and 6-thio-GTP (100 μM) as a substrate (18.1, 14.9, and 6.43 μM/h/106 cells for WT, Arg139Cys heterozygous, and homozygous variant, respectively). We developed a method for quantifying intracellular NUDT15 activity in peripheral blood mononuclear cells (PBMCs), which we defined as the conversion of 6-thio-GTP to 6-thio-GMP. Although PBMCs preparation takes some time, its reproducibility in experiments makes it a promising candidate for clinical application. This method can tell the difference between WT and Arg139Cys homozygous blood samples. Even in patients with WT NUDT15, WT samples showed variations in NUDT15 activity, which may correlate with variations in thiopurine dosage. [ABSTRACT FROM AUTHOR]

Insulin degludec is an ultra-long-acting insulin analogue that is increasingly being used in diabetes due to its favourable efficacy and safety profile. Thus, there is an increasing demand for a reliable and specific analytical method to quantify insulin degludec for research, pharmaceutical industry and clinical applications. We developed and validated an automated, high-throughput method for quantification of insulin degludec in human blood samples across the expected clinical range combining immunopurification with high-resolution mass spectrometry. Validation was performed according to the requirements of the US Food and Drug Administration. The method satisfyingly met the following parameters: lower limit of quantification (120 pM), linearity, accuracy (error < 5%), precision (CV < 7.7%), selectivity, carry-over, recovery (89.7–97.2%), stability and performance in the presence of other insulin analogues. The method was successfully applied to clinical samples of patients treated with insulin degludec showing a good correlation with the administered dose (r2 = 0.78). High usability of the method is supported by the small specimen volume, automated sample processing and short analysis time. In conclusion, this reliable, easy-to-use and specific mass spectrometric insulin degludec assay offers great promise to address the current unmet need for standardized insulin analytics in academic and industrial research. [ABSTRACT FROM AUTHOR]

Psychotropic drugs are regularly present in cases of sudden, unexpected death. Such drugs also tend to express significant postmortem redistribution. To facilitate further investigation of this phenomenon, reliable quantitative methods applicable to multiple biological matrices are needed. We present a validated ultra-performance liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of quetiapine, clozapine and mirtazapine in postmortem whole blood, skeletal muscle, brain tissue and liver tissue using high-performance liquid chromatography–tandem mass spectrometry. Sample preparation was performed using liquid–liquid extraction. The validated ranges were 3.8–1534, 16–1960 and 13–1060 μg/L for quetiapine, clozapine and mirtazapine, respectively. Within-run and between-run accuracy (87.4–122%) and precision (CV 1.5–8.9%), matrix effects (95–101%) and recovery (35.7–92%) were validated at two concentration levels; 5.8 and 1227 μg/L for quetiapine, 25 and 1568 μg/L for clozapine and 20 and 849 μg/L for mirtazapine. Stability in a 10°C environment was assessed for treated samples of brain, liver and muscle tissue, showing deviations in analyte concentrations ranging from −8% to 9% after 3 days. The analyte concentrations in treated samples of whole blood stored at 4°C deviated by <5% after 5 days. The method was applied in three forensic autopsy cases implicating quetiapine, clozapine and mirtazapine, respectively, in supratherapeutic concentrations. [ABSTRACT FROM AUTHOR]

Background: This study investigated the agreement between various dried blood spot (DBS) and venous blood sample measurements of phenylalanine and tyrosine concentrations in Phenylketonuria (PKU) and Tyrosinemia type 1 (TT1) patients.Study Design: Phenylalanine and tyrosine concentrations were studied in 45 PKU/TT1 patients in plasma from venous blood in lithium heparin (LH) and EDTA tubes; venous blood from LH and EDTA tubes on a DBS card; venous blood directly on a DBS card; and capillary blood on a DBS card. Plasma was analyzed with an amino acid analyzer and DBS were analyzed with liquid chromatography-mass spectrometry. Agreement between different methods was assessed using Passing and Bablok fit and Bland Altman analyses.Results: In general, phenylalanine concentrations in LH plasma were comparable to capillary DBS, whereas tyrosine concentrations were slightly higher in LH plasma (constant bias of 6.4 μmol/L). However, in the low phenylalanine range, most samples had higher phenylalanine concentrations in DBS compared to LH plasma. Remarkably, phenylalanine and tyrosine in EDTA plasma were higher compared to all other samples (slopes ranging from 7 to 12%). No differences were observed when comparing capillary DBS to other DBS.Conclusions: Overall agreement between plasma and DBS is good. However, bias is specimen- (LH vs EDTA), and possibly concentration- (low phenylalanine) dependent. Because of the overall good agreement, we recommend the use of a DBS-plasma correction factor for DBS measurement. Each laboratory should determine their own factor dependent on filter card type, extraction and calibration protocols taking the LH plasma values as gold standard. [ABSTRACT FROM AUTHOR]

Electromembrane extraction (EME) in small, stagnant and chip-like devices has the potential for future in-field operation. Literature briefly discuss such systems, but performance suffered from evaporative losses of sample and acceptor. To address this, the current paper reports electromembrane extraction (EME) of five basic drugs (model analytes) from aqueous buffer solutions and whole blood samples under stagnant conditions in a completely closed system. A laboratory-made polyoxymethylene (POM) well plate served as compartment for the sample solution, while a commercially available well filter plate was used to immobilize 2-nitrophenyl octyl ether (NPOE) as supported liquid membrane (SLM) and as closed compartment for the acceptor solution. Major design parameters (sample compartment and electrode geometry) and operational parameters (sample volume, voltage and extraction time) were investigated and optimized. Electrode geometry was not very critical, but extraction efficiency increased with decreasing sample volume. Extraction from 50 μL aqueous buffer solution for 60 min and with a voltage of 75 V was considered exhaustive (sample was depleted), with recoveries ranging between 75% and 87% for loperamide, haloperidol, methadone, nortriptyline, and pethidine (RSD: 2–12%). Extraction from whole blood samples under optimized conditions yielded slightly lower recoveries, ranging between 57 and 96% (RSD: 3–12%). Stagnant EME was evaluated in combination with liquid chromatography-mass spectrometry (LC-MS) as a highly specific instrumental method, and provided evaluation data on methadone from blood samples in accordance with regulatory requirements (LOD: 0.4 ng/mL, LOQ: 1.4 ng/mL, RSD: 6–20%). This work has improved upon the design of stagnant EME, moving it further towards a viable in-field operation device. Image 1 • Electromembrane extraction under totally stagnant conditions. • Extraction from whole blood. • Very simple device with potential for in-field operation. [ABSTRACT FROM AUTHOR]

5-Fluoro ADB, also known as 5-fluoro MDMB-PINACA, is a potent synthetic cannabinoid that is an agonist to the human cannabinoid CB1 and CB2 receptors. Adverse physiological and psychological effects that have resulted in hospitalization and/or death have been associated with 5-Fluoro ADB use. In addition, analytical confirmation of 5-Fluoro ADB use has been reported in both forensic human performance toxicology and postmortem cases. An analytical method for the identification and quantification of 5-fluoro ADB and the 5-fluoro ADB ester hydrolysis metabolite in human blood samples by liquid chromatography–tandem mass spectrometry was created and validated. The linear range of this assay was determined to be 0.01–10 ng/mL for 5-fluoro ADB and 10–500 ng/mL for the 5-fluoro ADB ester hydrolysis metabolite. The method met both precision and accuracy requirements. Endogenous and exogenous interferences were not observed. Ion suppression exceeding 25% was observed for 5-fluoro ADB. However, additional experiments were performed to ensure that the observed suppression did not affect other method validation parameters such as limit of detection and accuracy. Blood samples from 36 postmortem cases were analyzed utilizing this methodology. The average blood concentration of 5-fluoro ADB was 0.29 ng/mL in central blood specimens and 0.05 ng/mL in peripheral blood specimens. The average blood concentration of the 5-fluoro ADB ester hydrolysis metabolite was 49 ng/mL in central blood specimens and 21 ng/mL in peripheral blood specimens. A serum sample was also analyzed and had a serum concentration of 0.12 ng/mL for 5-fluoro ADB and 42 ng/mL for the 5-fluoro ADB ester hydrolysis metabolite. As the concentration of the 5-fluoro ADB ester hydrolysis metabolite was found at a greater concentration than that of 5-fluoro ADB, this metabolite may be a useful marker to monitor in an attempt to confirm 5-fluoro ADB use in toxicological investigations. [ABSTRACT FROM AUTHOR]

Background: Systemic inflammation is a marker of ill health and has prognostic implications in multiple health settings. Urinary neopterin is an excellent candidate as a nonspecific marker of systemic inflammation. Expression as urinary neopterin-to-creatinine ratio (UNCR) normalizes for urinary hydration status. Major attractions include (a) urine vs blood sampling, (b) integration of inflammation over a longer period compared with serum sampling, and (c) high stability of neopterin and creatinine. Methods: A high-throughput ultraperformance LC-MS method was developed to measure neopterin and creatinine together from the same urine sample. The assay was applied in several clinical scenarios: healthy controls, symptomatic infections, and multiple sclerosis. Area under the curve was compared between weekly and monthly sampling scenarios. Analysis of a single pooled sample was compared with averaging results from analysis of individual samples. Results: The assay has excellent intraassay and interassay precision, linearity of dilution, and spike and recovery. Higher UNCR was demonstrated in female vs male individuals, older age, inflammatory disease (multiple sclerosis), and symptomatic infections. In healthy controls, fluctuations in inflammatory state also occurred in the absence of symptomatic infection or other inflammatory triggers. Analysis of a single pooled sample, made up from weekly urine samples, integrates inflammatory activity over time. Conclusions: UNCR is a useful biomarker of systemic inflammation. The method presented offers simplicity, speed, robustness, reproducibility, efficiency, and proven utility in clinical scenarios. UNCR fluctuations underline the importance of longitudinal monitoring, vs a single time point, to capture a more representative estimate of an individual's inflammatory state over time. [ABSTRACT FROM AUTHOR]

A fast and simple approach to overcome challenges in emergency toxicological analysis, using ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC‐MS/MS) has been developed, for the detection of analytes in blood and urine samples from the following drug classes: analgesics, benzodiazepines, antidepressants, anticonvulsants, drugs of abuse, and pesticides. These substances are relevant in the context of emergency toxicology in Brazil. The sample preparation procedure was relatively easy and fast to perform. The method was fully validated giving limits of in the range of 0.5 and 20 ng mL−1 for blood and urine samples. The intraday and interday precision and accuracy were considered adequate for all analytes once the relative standard deviation (RSD) (%) was lower than 20% for quality control (QC) low and lower than 15% for CQ medium and high. The developed method was successfully applied to 320 real samples collected at the Poison Control Center of São Paulo, and 89.1% have shown to be positive for some of the analytes. This confirms its applicability and importance to emergency toxicological analysis, and it could be very useful in both fields of clinical and forensic toxicology. [ABSTRACT FROM AUTHOR]

Toxic metabolites (N-acetylisoniazid, pyrazinoic acid, and desacetylrifampicin) of first-line anti-tuberculosis drugs (ethambutol, pyrazinamide, rifampicin, and isoniazid) are identified by liquid chromatography–mass spectrometry, which is important in selecting the required dosages of these drugs for the maximum therapeutic effects and the reduction of side effects. Characteristic chromatographic profiles of blood plasma samples from donors with pulmonary tuberculosis ("pathology") and a clinically healthy group ("norm") are obtained and processed by principal component analysis and the k-nearest neighbor method. A perspective of this approach for obtaining of additional diagnostic criteria for pulmonary tuberculosis is shown. [ABSTRACT FROM AUTHOR]

Highlights • Boldenone is used by athletes, fitness trainer and bodybuilder. • An LC–MS/MS assay to quantify boldenone in biological specimens was developed. • This method was applied to postmortem case who injected boldenone in his shoulder. • Endogenous levels of boldenone in blood and urine samples were determined. Abstract Boldenone (BOLD), one of androgenic anabolic steroids (AAS), although banned in humans, is still available illegally. AAS abuse has previously been associated with various cardiovascular adverse events including acute myocardial infarction, arrhythmia, and sudden death. In this study, the concentration of BOLD was determined in postmortem specimens from the corpse of a human male who intentionally injected BOLD undecylenate into his shoulder muscle. In addition, the endogenous levels of BOLD in the blood and urine samples of young human males have been reported. A liquid chromatography-tandem mass spectrometry (LC–MS/MS) method with solid-phase extraction (SPE) was developed and validated for the analysis of BOLD in blood, muscular tissue and urine samples. The validation parameters including linearity, accuracy, precision, matrix effect, and recovery were satisfactory. The concentrations of BOLD in the blood of 20 young human males who didn't take BOLD were under the limit of quantitation (LOQ, 0.5 ng/mL). Additionally, the mean level of BOLD in the urine samples was 3.19 ± 1.65 ng/mL (range: 0.37˜6.02 ng/mL). The concentrations of BOLD in the victim's blood from the femoral vein and heart were 140.44 and 25.74 ng/mL, respectively. On the other hand, those in the muscular tissue from the injection site and the urine sample were 142.3 ng/g and 3474 ng/mL, respectively. [ABSTRACT FROM AUTHOR]

Recently, fentanyl analogs account for significant number of opioid deaths in the United States. Routine forensic analyses are often unable to detect and differentiate these analogs due to low concentrations and presence of structural isomers. A data-independent screening method for 14 fentanyl analogs in whole blood and oral fluid was developed and validated using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Data were acquired using Time of Flight (TOF) and All Ions Fragmentation (AIF) modes. The limits of detection (LOD) in blood were 0.1-1.0 ng/mL and 0.1-1.0 ng/mL in TOF and AIF modes, respectively. In oral fluid, the LODs were 0.25 ng/mL and 0.25-2.5 ng/mL in TOF and AIF modes, respectively. Matrix effects in blood were acceptable for most analytes (1-14.4%), while the nor-metabolites exhibited ion suppression >25%. Matrix effects in oral fluid were -11.7 to 13.3%. Stability was assessed after 24 h in the autosampler (4 °C) and refrigerator (4 °C). Processed blood and oral fluid samples were considered stable with -14.6 to 4.6% and -10.1 to 2.3% bias, respectively. For refrigerated stability, bias was -23.3 to 8.2% (blood) and -20.1 to 20.0% (oral fluid). Remifentanil exhibited >20% loss in both matrices. For proof of applicability, postmortem blood (n = 30) and oral fluid samples (n = 20) were analyzed. As a result, six fentanyl analogs were detected in the blood samples with furanyl fentanyl and 4-ANPP being the most prevalent. No fentanyl analogs were detected in the oral fluid samples. This study presents a validated screening technique for fentanyl analogs in whole blood and oral fluid using LC-QTOF-MS with low limits of detection. [ABSTRACT FROM AUTHOR]

In the field of bioanalysis, dried matrix spot sampling is increasingly receiving interest, as this alternative sampling strategy offers many potential benefits over traditional sampling, including matrix volume-sparing properties. By using a microsampling strategy, e.g., volumetric absorptive microsampling (VAMS), the number of samples that can be collected from a patient can be increased, as a result of the limited sample volume that is required per sample. To date, no VAMS-based methods have been developed for the quantification of analytes in cerebrospinal fluid (CSF). The objective of this study was to develop and validate two LC-MS/MS methods for the quantification of paracetamol in dried blood and dried CSF, with both matrices sampled using VAMS. Both methods were fully validated based on internationally accepted guidelines. Paracetamol was chromatographically separated from its glucuronide and sulfate metabolites and no carry-over or unacceptable interferences were detected. The total precision (%RSD) was below 15% for all QC levels and accuracy (%bias) was below 7% (17% for the LLOQ of aqueous VAMS). The influence of the hematocrit on the recovery of blood VAMS samples appeared to be limited within the hematocrit range of 0.21 to 0.62. The blood VAMS samples were stable for 1 week if stored at 50 °C, and for at least 8 months when stored between − 80 °C and room temperature. The aqueous VAMS samples were stable for at least 9 months when stored between − 80 and 4 °C, and for 1 month when stored at room temperature. Application of the methods on external quality control material and analysis of patient samples demonstrated the validity and utility of the methods and provided a proof of concept for the analysis of CSF microsamples obtained via VAMS devices.ᅟ [ABSTRACT FROM AUTHOR]

A method was developed to detect and quantify organophosphate nerve agent (OPNA) metabolites in dried blood samples. Dried blood spots (DBS) and microsampling devices are alternatives to traditional blood draws, allowing for safe handling, extended stability, reduced shipping costs, and potential self-sampling. DBS and microsamplers were evaluated for precision, accuracy, sensitivity, matrix effects, and extraction recovery following collection of whole blood containing five OPNA metabolites. The metabolites of VX, Sarin (GB), Soman (GD), Cyclosarin (GF), and Russian VX (VR) were quantitated from 5.0 to 500 ng mL −1 with precision of ≤16% and accuracy between 93 and 108% for QC samples with controlled volumes. For unknown spot volumes, OPNA metabolite concentrations were normalized to total blood protein to improve interpretation of nerve agent exposures. This study provides data to support the use of DBS and microsamplers to collect critical exposure samples quickly, safely, and efficiently following large-scale chemical exposure events. [ABSTRACT FROM AUTHOR]

Dried blood spot (DBS) sampling has evolved to become the method of choice for collecting samples for newborn screening and therapeutic drug monitoring worldwide. The major advantage of this approach is that it requires only a small amount of blood. In addition, the collection of DBSs on filter paper is simple, sample storage costs are small, and the process deactivates microorganisms and viruses. However, despite these advantages, DBS sampling is seldom used in forensic toxicological analyses. Here, we developed and validated an approach that uses liquid chromatography coupled with electrospray ionization−tandem mass spectrometry for quantifying nine psychotropic drugs (citalopram, duloxetine, mirtazapine, olanzapine, paroxetine, quetiapine, sertraline, zolpidem and zopiclone) in cadaveric DBS samples. Most of them are frequently used by self-harm but are not already targeted by an existing drug screening kit. Our method use only one 3-mm disk excised from each DBS and does not require the troublesome purification process. The linearities of the calibration curves were good in the concentration range of 0.05–1.0 μg/mL. Our method allows for repeatable and accurate quantification with intra- and inter-assay coefficients of variation of below 11.9% and below 12.5%, respectively, for each of the target drugs. In addition, the target drug concentrations in the DBSs remained stable for at least one month when stored at − 80 °C. Compared with our institute's routine method for cadaveric blood sampling, the QuEChERS method, quantifiable concentrations showed a good positive correlation for each of the target drugs. In addition, the concentrations of almost all the target drugs obtained with DBS sampling method were comparable with those obtained with the QuEChERS sampling method. Thus, the present findings extend the possible uses of DBS sampling to the quantification of multiple psychotropic drugs in the field of forensic toxicological testing. • Dried blood spot samples were applied to the forensic toxicological testing. • Nine psychotropic drugs were analyzed by LC/ESI−MS/MS without purification processes. • Target drugs are frequently used by self-harm but are not targeted by an existing drug screening kit. • Developed method required only one 3-mm disk excised from each dried blood spot. • Drugs concentrations obtained by developed method were comparable with those obtained with routine method. [ABSTRACT FROM AUTHOR]

Pharmacokinetic/pharmacodynamic modelling has emerged as a valuable technique for understanding drug exposure and response relationships in drug development. Pharmacokinetic data are often obtained by taking multiple blood samples, which may disturb physiological parameters and complicate study designs. Wearable automatic blood sampling systems can improve this limitation by collecting dried blood samples at programmable time points without disrupting cardiovascular parameters. It is the objective of this study to evaluate the bioanalysis of DBS in comparison to conventional blood sampling techniques and to optimize the recovery of various compounds spiked into canine blood dried on filter paper tape. Incubated blood samples from Beagle dogs were spiked with 16 different compounds and half of the whole blood sample was centrifuged to obtain plasma. After the dried blood sample drops were dried, liquid chromatography-mass spectrometry methods were used to analyze the samples. The study explored different anticoagulants, sample preparation methods and technical approaches to best determine the compound concentrations in dried blood samples. With the two anticoagulants tested and using the optimized sample preparation methods and technical approaches we employed, the bioanalysis of dried blood samples can provide equivalent results to conventional blood sampling techniques. Automated blood sampling systems have the potential to provide increased numbers of blood samples, providing substantially more Pharmacokinetic data within safety pharmacology studies without disrupting physiological parameters. They can provide a viable alternative to traditional methods of obtaining blood for various other types of studies or analyses. [ABSTRACT FROM AUTHOR]

Objective To establish a sensitive method for determination of concentration of lamotrigine (LTG) in rat plasma and to study the pharmacokinetics by LC-MS/MS. Methods 12 rats were divided evenly into model group and shame-operated group. LTG was given as single dose of 10 mg/kg via intragastrical administration. Blood samples were collected from orbital saphenous venous plexus at 5 min,0.25,0.5,1,2,4,6,8,12,24 and 36 h after dosing. The LTG concentrations in rat plasma were assayed by LC-MS/MS. The pharmacokinetic parameters were calculated by DAS software. Results In shame-operated group,cmax (1 382.87±61.17) μg/L,t1/2 (40.43±6.77) h; AUC0-∞(123.45±70.70) mg·h/L. In model group, cmax(1 713.50±65.11) μg/L, t1/2(73.72±17.46) h, AUC0-∞(188.15±76.37) mg·h/L. Conclusion The method is proved to be suitable for the determination of LTG in rat plasma. LTG concentration reached peak value at 2 h in both groups. However, model group had a longer t1/2 and higher concentration than that in shame-operated group, which is a valuable information for further pharmacodynamics study. [ABSTRACT FROM AUTHOR]

Benzodiazepines (BZD) and Z-hypnotics are frequently analyzed in forensic laboratories, and in 2012, the designer benzodiazepines (DBZD) emerged on the illegal drug scene. DBZD represent a particular challenge demanding new analytical methods. In this work, parallel artificial liquid membrane extraction (PALME) is used for sample preparation of DBZD, BZD, and Z-hypnotics in whole blood prior to UHPLC-MS/MS analysis. PALME of BZD, DBZD, and Z-hypnotics was performed from whole blood samples, and the analytes were extracted across a supported liquid membrane (SLM) and into an acceptor solution of dimethyl sulfoxide and 200 mM formic acid (75:25, v/v). The method was validated according to EMA guidelines. The method was linear throughout the calibration range (R2 > 0.99). Intra- and inter-day accuracy and precision, as well as matrix effects, were within the guideline limit of ± 15%. LOD and LLOQ ranged from 0.10 to 5.0 ng mL−1 and 3.2 to 160 ng mL−1, respectively. Extraction recoveries were reproducible and above 52%. The method was specific, and the analytes were stable in the PALME extracts for 4 and 10 days at 10 and − 20 °C. No carry-over was observed within the calibration range. PALME and UHPLC-MS/MS for the determination of DBZD, BZD, and Z-hypnotics in whole blood are a green and low-cost alternative that provides high sample throughput (96-well format), extensive sample clean-up, good sensitivity, and high reproducibility. The presented method is also the first method incorporating analysis of DBZD, BZD, and Z-hypnotics in whole blood in one efficient analysis.Graphical abstract [ABSTRACT FROM AUTHOR]

Toxicokinetics of cyfluthrin after single oral [20 mg/kg body weight (bw)] and intravenous (IV) (3 mg/kg bw) doses were studied in rats. Serial blood samples were obtained after oral and IV administration. Brain tissue samples were also collected after oral administration. Cyfluthrin concentrations in plasma and brain tissues (hypothalamus, striatum, hippocampus and frontal cortex) were quantified using liquid chromatography tandem mass spectrometry (LC/MS). Cyfluthrin disposition was best described by the use of a two-compartment open model. When given orally, plasma kinetics showed an extensive oral absorption of cyfluthrin and a slow elimination. The area under the concentration-time curve [AUC (0–24h) ] and maximal plasma concentration ( Cmax ) were 6.11 ± 1.06 mg h/L and 0.385 ± 0.051 μg/mL, respectively; β phase elimination half-life ( T 1/2 β ) was (17.15 ± 1.67 h). Oral bioavailability was found to be 71.60 ± 12.36%. After oral administration, cyfluthrin was widely distributed to brain tissues. AUC (0–24h) was significant higher in all tested brain tissues than in plasma. The largest discrepancy was found for hypothalamus. AUC (0–24h) , Cmax and T 1/2 β in hypothalamus were 19.36 ± 2.56 mg h/L, 1.21 ± 0.11 μg/g and 22.73 ± 1.60 h, respectively. Assuming the identified toxicokinetics parameters, this study serves to better understand mammalian toxicity of pyrethroid cyfluthrin and to design further studies to characterize its neurotoxicity. [ABSTRACT FROM AUTHOR]