An Automated Multicycle Immunoaffinity Enrichment Approach Developed for Sensitive Mouse IgG1 Antibody Drug Analysis in Mouse Plasma Using LC/MS/MS.

In the immuno-oncology field, surrogate mouse monoclonal antibodies are often preferred in establishing proper PK/PD/efficacy correlations as well as supporting anticipated mouse to human translation. Thus, a highly sensitive and specific bioanalytical method is needed in quantifying those surrogate mouse antibodies after dosing in mice. Unfortunately, when specific reagents, such as recombinant target antigen and anti-idiotypic antibody, are not available, measuring mouse surrogate antibody drugs in mice is very challenging for ligand binding assay (LBA) due to the severe cross reactivity potential. Different from LBA, if at least one unique surrogate peptide can be identified from the surrogate antibody sequence, the immunoaffinity enrichment based LC/MS/MS assay may be able to differentiate the analyte response from the high endogenous immunoglobulin background and provide adequate sensitivity. Herein, a new automated multicycle immunoaffinity enrichment method was recently developed to extract a surrogate mouse IgG1 (mIgG1) antibody drug from mouse plasma using a commercially available antimouse IgG1 secondary antibody. In the assay, reuse of the capture antibody up to six times mostly resolved the binding capacity issue caused by the abundant endogenous mIgG1 and made the immunoaffinity enrichment step more cost-effective. Combined with a unique surrogate peptide identified from the antibody, the LC/MS/MS assay achieved a limit of quantitation of 5 ng/mL with satisfactory assay precision, accuracy, and dynamic range. The successful implementation of this novel approach in discovery pharmacokinetic (PK) studies eliminates the dependence on specially generated immunoaffinity capturing reagents.

Magnetic Solid-Phase Extraction of Drugs and Pesticides from Human Plasma Using COOH-mMWCNTs.

Carbon nanotubes (CNTs) are useful for extracting chemical compounds due to their properties, such as surface area and the potential for chemical modification. Especially the formation of CNTs with carboxylic acid functional group makes them disperse in water-based samples and have strong interaction forces with cationizable analytes. Based on these features, carboxylic acid functionalized multi-walled CNTs (COOH-MWCNTs) have been used as extraction sorbents. CNT can also be gathered using an external magnet by forming complex with iron oxide (Fe3O4) magnetic nanoparticles (MNPs). In this study, COOH-MWCNTs with MNPs were subjected to magnetic solid-phase extraction (mSPE) in order to extract the targeted substances such as diphenhydramine, doxylamine, tramadol, escitalopram, zolpidem, diphenamid, paclobutrazol, hexaconazole, cyproconazole and mepronil from human plasma samples. The following five factors were optimized: (i) the ratio of COOH-MWCNTs to MNPs as a sorbent from 1:1 to 1:4; (ii) sorbent amount starting from 12.5 to 75%; (iii) sample pH tested pH 2 to pH 10 with 1 N hydrochloride and 1 N sodium hydroxide; (iv) agitating time from 0 to 4 min and (v) elution solvent. Limit of detection of 10 targeted substances in human plasma were in the range of 0.1-0.4 mg/L. The recovery of targeted substances (except diphenamid) in human plasma was 73.06-110.28% for intra-day and 83.00-107.70% for inter-day and the precision (relative standard deviation, %) in human plasma was 0.3-13.3% for intra-day and 2.9-15.6% for inter-day. The method was applied to nine authentic biological samples from overdose patients in the emergency room of Chungnam National University Hospital. The performance of mSPE was compared with the liquid-liquid extraction method using ethyl acetate. The results showed that the newly developed method in this study can be used for screening analysis in forensic and clinical toxicology.

Perspectives on Nanodelivery to the Brain: Prerequisites for Successful Brain Treatment.

Nanocarriers (NCs) are promising tools to improve drug delivery across the blood-brain barrier (BBB) for more effective treatment of brain disorders, although there is a scarcity of clinical translation of brain-directed NCs. In order to drive the development of brain-oriented NCs toward clinical success, it is essential to understand the prerequisites for nanodelivery to be successful in brain treatment. In this Perspective, we present how pharmacokinetic/pharmacodynamic (PK/PD), formulation and nanotoxicity factors impact the therapeutic success of brain-specific nanodelivery. Properties including high loading efficiency, slow in vivo drug release, long systemic circulation, an increase in unbound brain-to-plasma concentration/exposure ratio (Kp,uu,brain), high drug potency, and minimal nanotoxicity are prerequisites that should preferably be combined to maximize the therapeutic potential of a brain-targeted NC. The PK of brain-directed NCs needs to be evaluated in a more therapeutically relevant manner, focusing on the released, unbound drug. It is more crucial to increase the Kp,uu,brain than to improve the ability of the NC to cross the BBB in its intact form. Brain-targeted NCs, which are mostly developed for treating brain tumors, including metastases, should aim to enhance drug delivery not just to tumor regions with disrupted BBB, but equally important to regions with intact BBB where the drugs themselves have problems reaching. This article provides critical insights into how a brain-targeted nanoformulation needs to be designed and optimized to achieve therapeutic success in the brain.

Pyrolyzed cotton balls for protein removal: Analysis of pharmaceuticals in serum by capillary electrophoresis.

Because of the inherent affinity of proteins for bare, fused silica capillaries, the analysis of protein-containing samples has proven a challenging task for capillary electrophoresis. The adsorption of proteins to the capillary walls effectively changes the zeta potential and thus affects the electro-osmotic flow leading to significant shifts in migration time, peak broadening, and poor reproducibility. While there are several well-known methods to remove proteins from samples prior to the analysis (including precipitation) or to prevent their adsorption to the capillary (semi-permanent coatings), those approaches are often expensive, time consuming, or simply unreliable. Aiming to address these needs, this manuscript reports on the use of pyrolyzed cotton balls, as a simple and widely accessible hydrophobic material to remove proteins from serum samples. The material retains enough flexibility so it can be placed directly into the sample vials and has enough capacity to capture more than 75% of the proteins in the sample (1% dilution of 1 mL of serum). The advantages of the material are demonstrated by performing the analysis of five representative drugs (in serum) by capillary electrophoresis obtaining a change in migration time of only 5 ± 1%, after 10 consecutive runs.

Profiling of Drug-Metabolizing Enzymes and Transporters in Human Tissue Biopsy Samples: A Review of the Literature.

Within the drug pharmacokinetics (PK)-absorption, distribution, metabolism, and excretion (ADME) research community, investigators regularly generate in vitro data sets using appropriately vendor-sourced and processed human tissue. Such data enable drug screening, the generation of kinetic parameters, extrapolation of in vitro to in vivo, as well as the modeling and simulation of drug PK. Although there are large numbers of manuscripts describing studies with deceased organ donor tissue, relatively few investigators have published studies utilizing living donor tissue biopsy samples. After a review of the available literature, it was possible to find publications describing the use of tissue biopsy samples to determine enzyme inhibition ex vivo, the study of genotype-phenotype associations, the evaluation of tissue expression profiling following an inducer, and assessment of correlations between tissue expression profiles and in vivo-derived trait measures (e.g., biomarker plasma levels and probe drug PK). Some reports described multiple single-tissue biopsies, whereas others described single multiple-organ biopsies. It is concluded that biopsy-derived data can support modeling exercises (as input data and when validating models) and enable the assessment of organ-specific changes in enzyme and transporter profiles resulting from drug interactions, disease (e.g., metabolic disease, fibrosis, inflammation, cancer, infection), age, pregnancy, organ impairment, and genotype. With the emergence of multiorgan axes (e.g., microbiome-gut-liver-kidney) and interest in remote sensing (interorgan communication), it is envisioned that there will be increased demand for single- and multiorgan tissue biopsy data to support hypothesis testing and PK-ADME model building. SIGNIFICANCE STATEMENT: Based on a review of the literature, it is apparent that profiling of human tissue biopsy samples is useful in support of pharmacokinetics (PK)-absorption, distribution, metabolism, and excretion (ADME)-related studies. With conventional tissue biopsy as precedent, it is envisioned that researchers will turn to less invasive "liquid biopsy" methods in support of ADME-related studies (e.g., profiling of plasma-derived tissue-specific nanovesicles). Generation of such multiorgan liquid biopsy data in larger numbers of subjects and at multiple study time points will provide a rich data set for modeling purposes.

Longitudinal exposure of English primary care patients to pharmacogenomic drugs: An analysis to inform design of pre-emptive pharmacogenomic testing.

AIMS: To investigate the longitudinal exposure of English primary care patients to pharmacogenomic drugs to inform design of pre-emptive testing. METHODS: Sixty-three drugs were identified with dosing guidelines based on variants of 19 pharmacogenes in the Pharmacogenomics Knowledgebase on 01 September 2018. Prescribing of these pharmacogenomic drugs between 1993 and 2017 was summarised for a sample of 648 141 English patients aged 50-99 years on 01 January 2013, registered with Clinical Practice Research Datalink practices during 2011-12. Exposure of patients to pharmacogenomic drugs retrospectively (2, 10, 20 y) and prospectively (5 y) was described. RESULTS: During 2011-12, 58% of patients were prescribed at least 1 pharmacogenomic drug, increasing to 80% over the previous 20 years. Multiple exposure was common, with 47% patients prescribed ≥2 pharmacogenomic drugs and 7% prescribed ≥5 pharmacogenomic drugs over the next 5 years. The likelihood of exposure to pharmacogenomic drugs increased with age, with 89% patients ≥70 years prescribed at least 1 pharmacogenomic drug over the previous 20 years. Even among those aged 50-59 years, 71% were prescribed at least 1 pharmacogenomic drug over the previous 20 years. The pharmacogenomic drugs prescribed to the most patients were for pain relief, gastroprotection, psychiatric and cardiovascular conditions. Three pharmacogenes (CYP2D6, CYP2C19 and SLCO1B1) accounted for >95% pharmacogenomic drugs prescribed. CONCLUSIONS: In primary care patients, exposure to pharmacogenomic drugs is extremely common, multiplicitous and has commenced by relatively early adulthood. A small number of pharmacogenes account for the majority of drugs prescribed. These findings could inform design of pre-emptive pharmacogenomic testing for implementation in primary care.

A fast and simple approach for the quantification of 40 illicit drugs, medicines, and pesticides in blood and urine samples by UHPLC-MS/MS.

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.

Development of "Quick-DB forensic": A total workflow from QuEChERS-dSPE method to GC-MS/MS quantification of forensically relevant drugs and pesticides in whole blood.

Trends in forensic toxicology show the advancement of rapid and sensitive analytical methods for qualitative and quantitative analysis of drugs of abuse. However, forensic toxicologists are continuously faced with the challenges of identifying and quantifying drug blood concentration while simultaneously struggling with manpower shortage. In view of developing a simple and productive toxicological analysis method encompassing total workflow from sample preparation to quantitative analysis, here we describe a simple, robust, and sensitive method for the simultaneous determination and quantification of 63 forensically relevant drugs and pesticides in human whole blood. The method is based on sample preparation by a modified QuEChERS extraction and dispersive solid-phase extraction (dSPE) clean-up followed by gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis. Limits of detection of the target analytes in whole blood ranged in the few ng/mL-order levels. Intra- and inter-day validation result ranges were 0-24% for accuracy (% error) and 0.8-26% for precision (%RSD). Recovery rates ranged from 66% to 84% for barbiturates, 36% to 110% for benzodiazepines, 41% to 86% for tri/tetracyclic antidepressants, 15% to 81% for drugs of abuse, 28% to 44% for phenethylamines, and 25% to 118% for pesticides. The validated results were used to develop a user-friendly, systematic, and quantitative toxicological GC/MS/MS system and software "Quick-DB Forensic".

An Albumin Sandwich Enhances in Vivo Circulation and Stability of Metabolically Labile Peptides.

The effectiveness of numerous molecular drugs is hampered by their poor pharmacokinetics. Different from previous approaches with limited effectiveness, most recently, emerging high-affinity albumin binding moieties (ABMs) for in vivo hitchhiking of endogenous albumin opens up an avenue to chaperone small molecules for long-acting therapeutics. Although several FDA-approved fatty acids have shown prolonged residence and therapeutic effect, an easily synthesized, water-soluble, and high-efficiency ABM with versatile drug loading ability is urgently needed to improve the therapeutic efficacy of short-lived constructs. We herein identified an ideal bivalent Evans blue derivative, denoted as N(tEB)2, as a smart ABM-delivery platform to chaperone short-lived molecules, through both computational modeling screening and efficient synthetic schemes. The optimal N(tEB)2 could reversibly link two molecules of albumin through its two binding heads with a preferable spacer, resulting in significantly extended circulation half-life of a preloaded cargo and water-soluble. Notably, this in situ dimerization of albumin was able to sandwich peptide therapeutics to protect them from proteolysis. As an application, we conjugated N(tEB)2 with exendin-4 for long-acting glucose control in a diabetic mouse model, and it was superior to both previously tested NtEB-exendin-4 (Abextide) and the newly FDA-approved semaglutide, which has been arguably the best commercial weekly formula so far. Hence, this novel albumin binder has excellent clinical potential for next-generation biomimetic drug delivery systems.

Assessment of Drug-Drug Interactions between Taspoglutide, a Glucagon-Like Peptide-1 Agonist, and Drugs Commonly Used in Type 2 Diabetes Mellitus: Results of Five Phase I Trials.

BACKGROUND AND OBJECTIVE: Taspoglutide, a glucagon-like peptide-1 agonist, like native glucagon-like peptide-1, delays gastric emptying time and prolongs intestinal transit time, which may alter the pharmacokinetics of concomitantly administered oral drugs. The effect of taspoglutide on the pharmacokinetics of five oral drugs commonly used in patients with type 2 diabetes mellitus was assessed in healthy subjects. METHODS: Five clinical pharmacology studies evaluated the potential drug-drug interaction between multiple subcutaneous taspoglutide doses and a single dose of lisinopril, warfarin, and simvastatin and multiple doses of digoxin and an oral contraceptive containing ethinylestradiol and levonorgestrel. The extent of interaction was quantified using geometric mean ratios and 90% confidence intervals for the maximum plasma concentration and area under the plasma concentration-time curve. In addition to pharmacokinetics, pharmacodynamic effects were assessed for warfarin and the oral contraceptive. RESULTS: Among the tested drugs, the effect of taspoglutide on the pharmacokinetics of simvastatin was most pronounced, on the day of taspoglutide administration, the average exposure to simvastatin was decreased by - 26% and - 58% for the area under the plasma concentration-time curve and maximum plasma concentration, respectively, accompanied by an increase in average exposure to its active metabolite, simvastatin ß-hydroxy acid (+ 74% and + 23% for area under the plasma concentration-time curve and maximum plasma concentration, respectively). Although statistically significant changes in exposure were observed for other test drugs, the 90% confidence intervals for the geometric mean ratio for maximum plasma concentration and area under the plasma concentration-time curve were within the 0.7-1.3 interval. No clinically relevant changes on coagulation (for warfarin) and ovulation-suppressing activity (for the oral contraceptive) were apparent. CONCLUSION: Overall, multiple doses of taspoglutide did not result in changes in the pharmacokinetics of digoxin, an oral contraceptive containing ethinylestradiol and levonorgestrel, lisinopril, warfarin, and simvastatin that would be considered of clinical relevance. Therefore, no dose adjustments are warranted upon co-administration.

Toxicological findings: A retrospective overview of medico-legal investigations in Parma (Italy).

The aim of this study was to collect all available data from 2009 to 2016 focusing on the epidemiological, clinical and pharmacological issues only related to acute intoxication fatalities in the Unit of Legal Medicine of the Department of Medicine and Surgery at the University of Parma. All death certificates and autopsy reports were retrieved from the archives and evaluated to identify cases in which only acute intoxication from xenobiotics could be defined as the cause of death, however statistical and descriptive analyses were applied to all the data. A more comprehensive analysis on all causes of death showed that out of 1005 total cases the most common is haemorrhagic shock/traumatic shock (36.5%), followed by cardiogenic shock with 27.4%; asphyxia ranks as the third cause of death (11.8%); concerning encephalic injuries, our data show 10.9% of cases, while acute intoxication by xenobiotics accounts for 5.7%. Data show that the majority of subjects are poly-abuser (75.4%); people not enrolled within a preventive treatment (59.4%) were more likely to commit suicide (28.1%), whereas only 6.2% in the sub-population in treatment (40.6%) committed suicide: therefore, data strongly suggest the evidence that joining a preventive programme can decrease the probability of extreme action. Access to a full case history may indeed save considerable time and expense in carrying out tests, but also valuable targeted samplings. The investigating officer should, therefore, submit as much information as possible about the case, as this may influence the type and extent of analysis undertaken, as well as the interpretation of analytical results.

Development of a high-throughput screening analysis for 288 drugs and poisons in human blood using Orbitrap technology with gas chromatography-high resolution accurate mass spectrometry.

The screening analysis for drugs and poisons always symbolizes the capabilities of a forensic laboratory. Due to the rapid emergence of new compounds in clinical and forensic intoxication cases, sensitive and specific methods are necessary for the screening of wide range of target compounds. A novel high-throughput screening method has been developed for the toxicological analysis of 288 drugs and poisons in human blood using Orbitrap technology with gas chromatography-high resolution mass spectrometry (GC-HRMS). This method allows for the fast detection and identification of high-throughput forensically important drugs and poisons, e.g., drugs of abuse (cocaine, amphetamines, synthetic cannabinoids, opiates, hallucinogen), sedative-hypnotics, antidepressants, non-steroidal anti-inflammatory drugs, pesticides (acaricides, fungicides, insecticides, nematicides), and cardiovascular agents in one single GC-Q Exactive run. After a simple extraction with ethyl ether and buffer, following centrifugation, the supernatant was injected into the system. For detection, spiked blood samples were analyzed by Orbitrap-GC-HRMS using an electrospray ionization in full scan mode with a scan range from 40 to 650 (m/z). The identification of drugs and poisons in the samples was carried out by searching the accurate molecular mass of characteristic fragment ions, ion rations and retention time (RT) against the in-house library that we developed with 70 ev electron energy. The limit of detection (LOD) for most compounds (249 in a total of 288 compounds) was below 100 ng/mL. For selectivity, no substances have been identified in drug-free blood samples from six different sources, and the method was suitable for the recovery and the carryover. The coefficient of variation (CV) of the RTs was below 0.99% in all reproducibility experiments. Mass accuracy was always better than 3 ppm, corresponding to a maximum mass error of 1.04 millimass units (mmu). The developed method was applied to 136 real samples from forensic cases, demonstrating its suitability for the sensitive and fast screening of high-throughput drugs in human blood samples.

Effect of OATP1B1 genotypes on plasma concentrations of endogenous OATP1B1 substrates and drugs, and their association in healthy volunteers.

This study aimed to elucidate the impact of OATP1B1 genotype (*1b/*1b, *1b/*15, and *15/*15) on plasma concentrations of endogenous OATP1B1 substrates. Healthy volunteers with OATP1B1 *1b/*1b (n = 10), *1b/*15 (n = 7), or *15/*15 (n = 2) received oral administration of a cocktail of statins (atorvastatin, pitavastatin, rosuvastatin, and fluvastatin). Mean area under the plasma concentration of atorvastatin, pitavastatin, and rosuvastatin in OATP1B1 *15/*15 were 2.2, 1.7 and 1.58-times greater than the corresponding values in OATP1B1 *1b/*1b, respectively, whereas that of fluvastatin was identical to those in other OATP1B1 genotypes. OATP1B1 *15/*15 also showed higher mean plasma concentrations of OATP1B1 endogenous substrates compared with the other OATP1B1 genotypes, such as coproporphyrin I, glycochenodeoxycholate sulfate (GCDCA-S), lithocholate sulfate (LCA-S), glycolithocholate sulfate (GLCA-S) and taurolithocholate sulfate (TLCA-S), but not total or direct bilirubin, chenodeoxycholate-24-glucuronide, or ω-dicarboxylic long-chain fatty acids. Area under the plasma concentration-time curves of plasma coproporphyrin I and GLCA-S discriminated OATP1B1 genotype *15/*15 from the other genotypes. In combination with previously published clinical studies, these results support the notion that coproporphyrin I, and GLCA-S and GCDCA-S could be a surrogate probe for assessing human in vivo OATP1B1 activities.

In-line coupling of supported liquid membrane extraction across nanofibrous membrane to capillary electrophoresis for analysis of basic drugs from undiluted body fluids.

Planar polyamide 6 nanofibrous membrane was for the first time used in direct coupling of supported liquid membrane (SLM) extraction to CE analysis. Disposable microextraction device with the nanofibrous membrane was preassembled and stored for immediate use. The membrane in the device was impregnated with 1 µL of 1-ethyl-2-nitrobenzene and the device was subsequently filled with 10 µL of acceptor solution (10 mM HCl) and 15 µL of donor solution (sample). The device was in-line coupled to CE system for selective extraction and direct injection, separation and quantification of model basic drugs (nortriptyline, haloperidol, loperamide and papaverine) from standard saline solutions (150 mM NaCl) and from undiluted human body fluids (urine and blood plasma). Compared to standard polypropylene supporting material, the nanofibrous membrane demonstrated superior characteristics in terms of lower consumption of organic solvents, constant volumes of operational solutions, full transparency and possibility to preassemble the devices. Extraction parameters were better or comparable for the nanofibrous vs. the polypropylene membrane and the hyphenated SLM-CE method with the nanofibrous membrane was characterized by good repeatability (RSD ≤ 11.3%), linearity (r2 ≥ 0.9953; 0.5-20 mg/L), sensitivity (LOD ≤ 0.4 mg/L) and transfer (27-126%) of the basic drugs.

Bioanalytical workflow for novel scaffold protein-drug conjugates: quantitation of total Centyrin protein, conjugated Centyrin and free payload for Centyrin-drug conjugate in plasma and tissue samples using liquid chromatography-tandem mass spectrometry.

AIM: Alternative scaffold proteins have emerged as novel platforms for development of therapeutic applications. One such application is in protein-drug conjugates (PDCs), which are analogous to antibody-drug conjugates. METHODOLOGY: Liquid chromatography-mass spectrometry methods for quantitation of total protein, conjugate and free payload for a PDC based on Centyrin scaffold were developed. Tryptic peptides generated from a region of the Centyrin that does not contain a conjugation site, and another that has the conjugation site with the linker-payload attached were used as surrogates of the total and conjugated Centyrin, respectively. CONCLUSION: The methods were successfully applied to analysis of samples from mice to quantify the plasma and tissue concentrations. This same workflow can potentially be applied to other PDCs and site-specific antibody-drug conjugates.

Simultaneous determination of drugs and pesticides in postmortem blood using dispersive solid-phase extraction and large volume injection-programmed temperature vaporization-gas chromatography-mass spectrometry.

A d-SPE protocol followed by gas chromatography-mass spectrometry (GC-MS) analysis using large volume injection-programmed temperature vaporization (LVI-PTV) was optimized for simultaneous quantification of 14 pesticides, drugs of abuse, prescription drugs and metabolites in human postmortem blood without derivatization. The validated method showed good repeatability, linearity, intermediate precision, and recovery. LOQs were 0.02 or 0.03µg/mL. The method showed to be fast and easy-to-implement in a forensic laboratory and was satisfactorily applied for the analysis of 10 postmortem blood real samples. Six samples contained cocaine (0.04-3.13µg/mL), two 3,4-methylenedioxymethamphetamine hydrochloride (MDMA, 0.04-0.09µg/mL) and two carbamazepine (0.08-0.98µg/mL). Other analytes found were carbofuran (27.3µg/mL), the metabolite 7-aminoflunitrazepam (1.12µg/mL), amitriptyline (0.21µg/mL) and diazepam (0.03µg/mL).

Systematic forensic toxicological analysis by liquid-chromatography-quadrupole-time-of-flight mass spectrometry in serum and comparison to gas chromatography-mass spectrometry.

Comprehensive screening procedures for psychoactive agents in body fluids are an essential task in clinical and forensic toxicology. With the continuous emergence and adaption of new psychoactive substances (NPS) keeping a screening method up to date is challenging. To meet these demands, hyphenated high-resolution mass spectrometry has gained interest as extensive and expandable screening approach. Here we present a comprehensive method for systematic toxicological analysis of serum by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) with data independent acquisition. The potential of this method was demonstrated by analysis of 247 authentic serum- and 12 post-mortem femoral blood samples. Thus 950 compounds, comprising 185 different drugs and metabolites could be identified. For the detected substances, including pharmaceutical substances, illicit drugs as well as NPS, serum concentrations were confirmed ranging from traces to toxic values indicating the capability for forensic toxicological requirements. Positive identification of drugs was achieved by accurate mass measurement (±5ppm for [M+H]+; ±10ppm for [M-H]-), retention time (±0.35min), isotopic pattern match (less than 10 m/z RMS [ppm]), isotope match intensity (less than 20% RMS) and the presence of at least two fragment ions. The LC-QTOF-MS procedure was shown to be superior to serum screening by GC-MS, since 240% (335 versus 141) more drugs were identified in serum samples compared to GC-MS.

Electrodialytic extraction of anionic pharmaceutical compounds from a single drop of whole blood using a supported liquid membrane.

A method to introduce target analytes to a chromatograph from a single drop of whole blood was investigated for minimally invasive monitoring of anionic pharmaceuticals. In this work, salicylate and loxoprofen were examined as organic anions. A micro ion extractor (MIE) has been developed for extraction of inorganic trace anions from whole blood, but this device is not suitable for extraction of pharmaceuticals. In the present study, we improved and optimized the MIE device for organic anion extraction. Various supported liquid membranes were evaluated for use as the ion transfer membrane, with each membrane placed between a droplet sample (donor) and an acceptor solution. A supported liquid membrane of porous polypropylene impregnated with 1-butanol was selected. In addition, the methods for electric field creation and electrode contact were examined to improve the characteristics of the MIE device. The current and extraction time were also optimized. With the optimized method, salicylate and loxoprofen were successfully extracted from a single drop of whole blood. Changes in the concentrations of these pharmaceuticals in blood over time were monitored after administration. As only 25µL of whole blood was required for analysis, repeat measurements could be conducted to monitor changes in the concentrations. This MIE will be useful for monitoring pharmaceutical concentrations in blood.

Advances in Studies of P-Glycoprotein and Its Expression Regulators.

This review deals with recent advances in studies on P-glycoprotein (P-gp) and its expression regulators, focusing especially on our own research. Firstly, we describe findings demonstrating that the distribution of P-gp along the small intestine is heterogeneous, which explains why orally administered P-gp substrate drugs often show bimodal changes of plasma concentration. Secondly, we discuss the post-translational regulation of P-gp localization and function by the scaffold proteins ezrin, radixin and moesin (ERM proteins), together with recent reports indicating that tissue-specific differences in regulation by ERM proteins in normal tissues might be retained in corresponding cancerous tissues. Thirdly, we review evidence that P-gp activity is enhanced in the process of epithelial-to-mesenchymal transition (EMT), which is associated with cancer progression, without any increase in expression of P-gp mRNA. Finally, we describe two examples in which P-gp critically influences the brain distribution of drugs, i.e., oseltamivir, where low levels of P-gp associated with early development allow oseltamivir to enter the brain, potentially resulting in neuropsychiatric side effects in children, and cilnidipine, where impairment of P-gp function in ischemia allows cilnidipine to enter the ischemic brain, where it exerts a neuroprotective action.

Assessment of Patient Medication Adherence, Medical Record Accuracy, and Medication Blood Concentrations for Prescription and Over-the-Counter Medications.

Importance: Inaccurate medication records and poor medication adherence result in incomplete knowledge of therapy for patients. Objective: To study accuracy of medical records and patient adherence by measuring blood concentrations of medications. Design, Setting, and Participants: This cross-sectional study validated a serum-based liquid chromatography-tandem mass spectrometry assay to simultaneously quantify 263 medications used for acute and chronic conditions. The assay panel was applied to 3 clinical patient cohorts: residual serum from 1000 randomly selected samples sent for routine clinical chemistry testing between April 8 and October 6, 2015 (residuals cohort), 50 prospectively enrolled patients in a gastroenterology clinic between March 1 and March 15, 2016, who were prescribed more than 5 medications (gastroenterology care cohort), and a convenience cohort of 296 patients with hypertension who sought care in an emergency department (ED care cohort) between July 1, 2012, and April 25, 2013. Integrated data analysis of the cohorts was performed from August 22 to November 29, 2017. Main Outcomes and Measures: Medication serum concentrations, electronic health record medication lists, and predicted drug interactions. Results: Of the 1346 total samples, 1000 came from the residuals cohort (640 women and 360 men; median age, 60 years [interquartile range (IQR), 44-71 years]), 50 from the gastroenterology care cohort (30 women and 20 men; median age, 66 years [IQR, 62-70 years]), and 296 from the ED care cohort (160 women and 136 men; median age, 59 years [IQR, 52-66 years]). Median medication adherence, defined as the subset of detected medications from the prescription record, was 83% (IQR, 50%-100%) in the residuals cohort, 100% (IQR, 84%-100%) in the gastroenterology care cohort, and 78% (IQR, 57%-100%) in the ED care cohort. Patients adherent to 1 medication were more often adherent to other medications. Among patients prescribed 3 medications or more, there were no significant associations between medication adherence and sex or number of prescribed medications, and there was a modest association between adherence and age. By comparing detected vs prescribed medications, we detected a median of 0 (IQR, 0-2) medications per patient that were not listed in the electronic health record in the residuals cohort, 1 (IQR, 0-2) medication per patient that was not listed in the electronic health record in the gastroenterology care cohort, and 1 (IQR, 0-2) medication per patient that was not listed in the electronic health record in the ED care cohort. A total of 435 patients (43.5%) in the residuals cohort had no discrepancy between the electronic health record and detected medication lists, 22 patients (44.0%) in the gastroenterology care cohort had no discrepancy between the electronic health record and detected medication lists, and 41 patients (13.9%) in the ED care cohort had no discrepancy between the electronic health record and detected medication lists. Half of adverse drug reaction alerts occurred among medications detected without prescription. Conclusions and Relevance: Comprehensive medication monitoring offers promise to improve adherence, the accuracy of medical records, and the safety for patients with polypharmacy.