Drugs In Biological Samples Research Articles

Electrochemical Sensors Based on Molecularly Imprinted Polymers for Pharmaceuticals Analysis

<P>Background: The electrochemical sensing of drugs in pharmaceutical formulations and biological matrices using molecular-imprinting polymer (MIP) as a recognition element combined with different electrochemical signal transduction has been widely developed. The MIP electrochemical sensors based on nanomaterials such as graphene, carbon nanotubes, nanoparticles, as well as other electrode modifiers incorporated into the MIPs to enhance the performance of the sensor, have been discussed. The recent advances in enantioselective sensing using MIP-based electrochemical sensors have been described. </P><P> Methods: The molecular imprinting has more than six decades of history. MIPs were introduced in electrochemistry only in the 1990s by Mosbach and coworkers. This review covers recent literature published a few years ago. The future outlook for sensing, miniaturization and development of portable devices for multi-analyte detection of the target analytes was also given. </P><P> Results: The growing pharmaceutical interest in molecularly imprinted polymers is probably a direct consequence of its major advantages over other analytical techniques, namely, increased selectivity and sensitivity of the method. Due to the complexity of biological samples and the trace levels of drugs in biological samples, molecularly imprinted polymers have been used to improve the response signal, increase the sensitivity, and decrease the detection limit of the sensors. The emergence of nanomaterials opened a new horizon in designing integrated electrochemical systems. The success of obtaining a high-performance electrochemical sensor based on MIPs lies in the kind of material that builds up the detection platform. </P><P> Conclusion: The novel approaches to produce MIP materials, combined with electrochemical transduction to develop sensors for screening different pharmaceutically active compounds have been overviewed. MIPs may appear indispensable for sensing in harsh conditions, or sensing that requires longterm stability unachievable by biological receptors. The electrochemical sensors provide several benefits including low costs, shortening analysis time, simple design; portability; miniaturization, easy-touse, can be tailored using a simple procedure for particular applications. The performance of sensor can be improved by incorporating some conductive nanomaterials as AuNPs, CNTs, graphene, nanowires and magnetic nanoparticles in the polymeric matrix of MIP-based sensors. The application of new electrochemical sensing scaffolds based on novel multifunctional-MIPs is expected to be widely developed and used in the future.</P>

Development and validation of a multiplexed drug level assay in support of combination biologics therapy clinical studies

Clinical development of biotherapeutics for combination therapy requires monitoring the concentrations of both drugs in biological samples. Traditionally, two assays are required to measure drug levels one at a time, which poses challenges in sample management, data reporting, and cost. The Meso Scale Discovery (MSD®) U-PLEX™ platform provides a simple and flexible way to create custom multiplex ligand binding assays (LBAs). We developed and fully validated a two-plex assay on the U-PLEX platform where two therapeutic monoclonal antibodies (mAbs) in Merck’s pipeline, which we call MK-A and MK-B in this manuscript, can be measured simultaneously in one sample. Our results demonstrated that the multiplexed pharmacokinetic (PK) assay has performances, including accuracy, precision, and cross-reactivity, that meet requirements in regulatory guidance. Furthermore, results of MK-A from the multiplex assay are comparable to results from a previously validated MK-A single-plex assay with 80% of samples tested in both assays having concentration differences < 30% relative to the mean of the two measurements. The multiplex assay was used to support a phase I MK-A/MK-B combination therapy clinical study and generated results consistent with historical MK-A monotherapy PK data. The ability to measure both biotherapeutics in a multiplexed assay is beneficial in that it improves consistency and efficiency while reduces sample volume and cost. With the number of combination therapies increasing in development, multiplexed assays can potentially have wide applications in biologics bioanalysis.

Recent advances in microextraction procedures for determination of amphetamines in biological samples.

Amphetamine and its related derivatives have stimulant and hallucinogenic properties. Illegal use of these drugs is an increasing global problem resulting in significant public health and legal problems. Deaths have been reported after intake of these drugs due to overdose. It is important to determine the type and concentration of illicit drugs in biological samples. These compounds are found in complex matrices at low concentration levels. The microextraction techniques are dominant sample preparation procedure and they are widely accepted as the most labor-intensive part of the bioanalytical process. For this purpose, a survey of recent published advances in microextraction procedures for quantification of amphetamines in biological samples found in the different databases from 2008 to date will be conducted.

Schizophrenia: recent advances in LC-MS/MS methods to determine antipsychotic drugs in biological samples.

Schizophrenia is one of the most debilitating and costly illnesses worldwide. First-generation antipsychotics such as chlorpromazine and haloperidol succeeded in controlling the positive symptoms of schizophrenia, but had significant extrapyramidal effects that led to the search for new agents and the release of second-generation (or atypical) antipsychotics. These drugs had a lower risk of adverse motor symptoms. Therapeutic drug monitoring has become a useful tool to optimize schizophrenia treatment and HPLC-MS/MS has been considered the primary technique to monitor antipsychotics. This review comprises three sections: schizophrenia pathophysiology and treatment; recent advances in LC-MS/MS methods designed to measure levels of antipsychotics and their metabolites in plasma samples (selectivity, matrix effect and sensitivity); and the importance of therapeutic drug monitoring.

A new epirubicin biosensor based on amplifying DNA interactions with polypyrrole and nitrogen-doped reduced graphene: Experimental and docking theoretical investigations

Epirubicin is an effective anticancer drug, but naturally causes several side effects. Hence the determination of this drug in biological samples, plays a key role in evaluating its effects and side effects. In this research, a novel label-free DNA-based biosensor was fabricated for the analysis of epirubicin in biological samples. The sensor was fabricated through modifying a pencil graphite electrode with polypyrrole, nitrogen doped reduced graphene (for improving the electrical conductivity) and salmon sperm ds-DNA (PP/NrG/ds-DNA/PGE) and the changes in the electrode signal were used for the determination of epirubicin. The PP/NrG/ds-DNA/PGE showed a high sensitivity for epirubicin in the concentration range of 0.004–55.0 μM and had a detection limit of 1.0 nM. For further evaluations, the interactions between ds-DNA bases and epirubicin were investigated through a theoretical docking study and the obtained data confirmed the intercalation of epirubicin in the minor grooves of ds-DNA with guanine bases. The PP/NrG/ds-DNA/PGE was used for the analysis of epirubicin in injection and urine samples.

Electrochemical Determination of Sunitinib in Biological Samples Using Polyacrylonitrile Nanofibers/Nickel-Zinc-Ferrite Nanocomposite/Carbon Paste Electrode

This article presents the novel modifier; polyacrylonitrile nanofibers/Ni0.5Zn0.5Fe2O4 nanoparticles nanoparticles as a novel modifier, which has been synthesized using an electrospinning method. This nanocomposite has been used to construct an electrochemical sensor for highly sensitive determination of sunitinib (STB) drug in biological samples. The synthesized nanocomposite was fully characterized using methods such as Fourier transform infrared spectroscopy, scanning electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. For the electrochemical determination of STB, the differential pulse voltammetry signal of the drug electrooxidation on carbon paste electrode modified with the newly developed nanocomposite was investigated. Preliminary experiments showed that the nanocomposite had an electrocatalytic effect on the electrooxidation of STB and a significant improvement in the anodic current of the drug was obtained at +0.66 V. Under the optimum conditions, the analytical figures of merit were extracted and the method was fully validated. A relatively wide dynamic linear range of 0.018–18.77 μ mol L−1 with the detection limit of 0.9 n mol L−1 was obtained. Furthermore, the method was applied for the determination of STB in biological fluid samples such as human serum samples with acceptable recoveries (97–102), satisfactory precision (2–4%RSD) and minimal matrix effects.

Simple Sonochemical Synthesis of Cupric Oxide Sphere Decorated Reduced Graphene Oxide Composite for the Electrochemical Detection of Flutamide Drug in Biological Samples

Reduced graphene oxide/cupric oxide (RGO/CuO) composites were developed by a simple, eco-friendly sonochemical method. This system has been explored as a potential electrocatalyst toward the electrochemical detection of flutamide. The prepared RGO/CuO composite was confirmed by scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction, and Raman spectra studies. In addition, the electrochemical properties of the prepared electrode were investigated by electrochemical impedance spectroscopy, cyclic voltammetry, and chronoamperometry techniques. The RGO/CuO composite modified glassy carbon electrode (GCE) exhibits high electrocatalytic activity toward the detection of flutamide. The RGO/CuO/GCE modified electrode also detects flutamide with a low detection limit (0.001 μM), wide linear range (0.005 to 71.32 μM) and acceptable sensitivity (5.54 μA μM−1cm−2). A fabricated sensor showed good selectivity, repeatability, reproducibility and long-term operational stability. Appreciable recoveries were achieved for the detection of flutamide in water and in biological samples, demonstrating the practical feasibility of the RGO/CuO/GCE modified electrode.

Polyethylenimine@Fe3O4@carbon nanotubes nanocomposite as a modifier in glassy carbon electrode for sensitive determination of ciprofloxacin in biological samples

This paper reports on the synthesis of a novel nanocomposite of multi-wall carbon nanotubes (CNTs), magnetite nanoparticle (Fe3O4), and polyethylenimine (PEI) to construct an electrochemical sensor for highly sensitive determination of ciprofloxacin (CIP) drug in biological samples and pharmaceutical formulations. The nanocomposite contains many amine functional groups provided by PEI conductive polymer. Due to the high conductivity of CNTs and moderate conductivity of the polymer along with rich active sites of amine functional groups, the PEI@Fe3O4@CNTs nanocomposite displayed excellent electro-catalytic effect on the electro-oxidation of CIP. The nanocomposite was fully characterized using Fourier transform infrared spectroscopy, field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, and transmission electron microscopy. For the electrochemical determination of CIP, the differential pulse voltammetry signal of the drug electro-oxidation on a glassy carbon electrode modified with the newly developed nanocomposite was investigated. Under the optimized experimental conditions, the electrochemical responses of the modified electrode were proportional to the drug concentrations in the range of 0.03–70.0μmolL−1 with a limit of detection 3.0nmolL−1. Furthermore, the method was applied for the determination of CIP in the drug tablets, urine, and serum samples with acceptable recoveries (97–108) and satisfactory precisions (1–3%RSD).

Rapid, Validated UPLC-MS/MS Method for Determination of Glibenclamide in Rat Plasma.

Quick and specific bioanalytical methods are required for analyzing drugs in biological samples. A simple, quick, sensitive, and specific UPLC-MS/MS method was developed and validated for glibenclamide determination in plasma samples. The plasma samples were processed by protein precipitation technique. Glimepiride was used as internal standard (IS). Glibenclamide and glimepiride were eluted on C18 column (Acquity UPLC®BEH). Mobile phase consisting of acetonitrile (0.1% formic acid) and water (0.1% formic acid) was pumped in binary gradient mode at flow rate of 150 μL/min. Glibenclamide and IS elution time was about 1.0 min, and total run time was 2.0 min. The mass spectrometer (triple-quadrupole) was operated in positive electrospray ionization mode. Sodium adducts [M + Na]+ of glibenclamide and IS were monitored in MRM mode. A linear calibration curve was obtained in the range of 10-1280 ng/mL, with regression equation Y = 0.0076 X – 0.0165 and linear regression coefficient r2 = 0.999. Lower limit of quantitation was 10 ng/mL. Accuracy of the method at LQC, MQC, and HQC was 109.7% (± 6.7), 93.6% (± 0.4), and 99.3% (± 1.9), respectively. The coefficient of variation for precision at all QC concentrations was less than 6%. Recovery at LLQC, MQC, and HQC was 104.2% (± 4.9), 100.6% (± 0.9), and 102.9% (± 5.8), respectively. The method was successfully implemented for pharmacokinetic investigations (in-house data).

Electrochemically controlled fiber-in-tube solid-phase microextraction method for the determination of trace amounts of antipsychotic drugs in biological samples.

In this study, a novel 'fiber-in-tube' configuration was applied to electrochemically controlled fiber-in-tube solid-phase microextraction of antipsychotic drugs (perphenazine and chlorpromazine) from biological samples. To prepare an electrochemically controlled fiber-in-tube solid-phase microextraction column, first eight stainless-steel wires were placed into the stainless-steel column. Then, a nanostructured Cu-Cr-Al ternary layered double hydroxide/polythiophene coating was prepared on the inner surface of the stainless-steel tube and on the surfaces of the stainless-steel wires by a facile in situ electrodeposition method. The nanostructured coating exhibited enhanced long lifetime, good mechanical stability, high porosity, and large specific surface area compared with polythiophene and Cu-Cr-Al layered double hydroxide coatings. Under the optimal conditions, the limits of detection were in the range of 0.07-0.8 μg/L. This method showed good linearity for perphenazine and chlorpromazine in the ranges of 0.3-300 and 0.2-300 μg/L, respectively, with coefficients of determination more than 0.9982. The inter- and intra-assay precisions (RSD%, n=3) were in the ranges of 3.0-5.1 and 2.5-4.5% at three concentration levels of 5, 25 and 50 μg/L, respectively. Finally, the method was applied for the analysis of the drugs in human urine and plasma samples.

Microparticles with size/charge selectivity and pH response for SERS monitoring of 6-thioguanine in blood serum

Surface-enhanced Raman scattering (SERS) spectroscopy is an ultrasensitive analytical technique with high specificity, making it an ideal candidate for therapeutic drug monitoring. However, SERS measurement of drugs in biological samples is inevitably affected by the proteins adsorbed nonspecifically on metal nanoparticles, so the drugs usually require separation before SERS detection. In this work, semipermeable alginate microparticles containing gold nanoparticles were fabricated via a centrifugal microfluidic chip, and served as a reliable substrate for SERS detection of 6-thioguanine in serum without protein removal. In comparison with the conventional microfluidic methods using oil for droplet generation, this oil-free method can fabricate more clean and versatile microparticles. The microparticles were characterized by size/charge selectivity and pH response, which effectively improved anti-interference ability and SERS sensitivity for 6-thioguanine detection in serum. The microparticles were further endowed with magnetic property and SERS activity simultaneously by forming Janus structures embedded with magnetic nanoparticles and gold nanoparticles separately in independent hemispheres, which was convenient for manipulation in SERS detection. More broadly, the microparticles are potentially applicable for SERS detection of other small molecules in complex real samples.

Determination of benzodiazepines in pericardial fluid by gas chromatography–mass spectrometry

In Forensic Toxicology it is sometimes impossible to obtain a valid blood sample to perform toxicological analysis due to several factors like advanced state of decomposition, severe burns, bleed to death…. Pericardial Fluid has already been studied during the last years and has been proposed as a valid specimen for toxicological tests.Over the years, the consumption of benzodiazepines spread among the drug dependent population and became noticeable in drug facilitated assault cases and road accidents. Improvement of the analytical methodology required for detecting the presence of these drugs in biological samples is of great importance for forensic toxicology, in order to correctly diagnose an exposure or a poisoning.In this study, 9 benzodiazepines (diazepam, nordiazepam, midazolam, bromazepam, oxazepam, temazepam, lorazepam, clonazepam and alprazolam) have been determined in pericardial fluid. For this purpose a solid phase extraction (SPE) was carried out using Bond Elut Certify cartridges. After the derivatization of six of the nine benzodiazepines, gas chromatography coupled to a selective mass detector was used as the technique for the separation of the analytes.The method developed was fully validated for the 9 analytes and was applied to real samples of pericardial fluid received at the Forensic Toxicology Service of the University of Santiago de Compostela. Finally, they were compared with blood results looking for the existence of a possible correlation between both biological samples.

Simultaneous determination of candesartan and hydrochlorothiazide in human plasma by LC-MS/MS

A simple, sensitive, rapid and highly efficient LC-MS/MS method was developed for the determination of Candesartan and Hydrochlorothiazide simultaneously in human plasma. The method employed Zorbax eclipse C18 (150 X 4.6 mm, 5µ) column using acetate buffer: acetonitrile (25:75%, v/v) as the mobile phase. The mobile phase flow rate is 1 mL/min which was delivered into the mass spectrometer electron spray ionization chamber. The Liquid/liquid extraction procedure was used in the method for the extraction of analytes. The chromatograph was attached to a negative ion mode tandem mass spectrometer and the method was validated for all the parameters as per the guidelines of US-FDA. The ions were detected in multiple reaction monitoring mode and the transitions are m/z 439.00®309.10 and 295.80®268.80 for candesartan and hydrochlorothiazide respectively. Isotopic standards were used as internal standards for effective recovery of the analytes. The drugs were analyzed over a calibration range of 1.027-302.047 ng/mL for candesartan and 1.044-306.945 ng/mL for hydrochlorothiazide respectively with regression coefficient greater than 0.99. The mean extraction recoveries are 96.95±5.61 and 100.55±4.82 for candesartan and hydrochlorothiazide respectively. The precision and accuracy values for all the studies were within the range of ≤15% and 85-115%. The performed stability studies indicate that the developed method is stable in plasma for 15 h at room temperature (bench top); 52 h (in injector); for 112 days at -70 oC for long term stability; five successive freeze and thaw cycles. The developed method could be successfully employed for the determination of selected drugs in biological samples.

Greening Reversed-Phase Liquid Chromatography Methods Using Alternative Solvents for Pharmaceutical Analysis.

The greening of analytical methods has gained increasing interest in the field of pharmaceutical analysis to reduce environmental impacts and improve the health safety of analysts. Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most widely used analytical technique involved in pharmaceutical drug development and manufacturing, such as the quality control of bulk drugs and pharmaceutical formulations, as well as the analysis of drugs in biological samples. However, RP-HPLC methods commonly use large amounts of organic solvents and generate high quantities of waste to be disposed, leading to some issues in terms of ecological impact and operator safety. In this context, greening HPLC methods is becoming highly desirable. One strategy to reduce the impact of hazardous solvents is to replace classically used organic solvents (i.e., acetonitrile and methanol) with greener ones. So far, ethanol has been the most often used alternative organic solvent. Others strategies have followed, such as the use of totally aqueous mobile phases, micellar liquid chromatography, and ionic liquids. These approaches have been well developed, as they do not require equipment investments and are rather economical. This review describes and critically discusses the recent advances in greening RP-HPLC methods dedicated to pharmaceutical analysis based on the use of alternative solvents.

Development of electrochemically controlled packed-in-tube solid phase microextraction method for sensitive analysis of acidic drugs in biological samples

In the present research, for the first time, a novel “packed-in-tube” configuration has been applied to electrochemically controlled in-tube solid phase microextraction, followed by high performance liquid chromatography. In order to prepare a mini packed column, small beads of stainless steel were first placed into the stainless steel column. Then, a nanostructured polypyrrole film was prepared on the internal surface of a stainless steel tube and the surface of stainless steel particles through a facile in-situ electrodeposition method. Filling the column with tiny particles of stainless steel effectively reduces the dead volume of the extraction tube and increases the extraction phase volume. The column was used for separation and preconcentration of diclofenac and mefenamic acid as model analytes from biological samples. Several important factors affecting extraction efficiency, such as extraction and desorption times, flow rates of the sample solution and eluent, and extraction and desorption voltages were investigated and optimized. This method showed good linearity for the drugs in the range of 0.3–200.0 μg L−1, 1.1–200.0 μg L−1, and 1.8–200.0 μg L−1 with coefficients of determination better than 0.9986, 0.9973, and 0.9973 in water, urine, and plasma samples, respectively. Intra- and inter-assay precisions (RSD%, n = 3) were in the range of 2.6–4.8% and 2.9–5.1, respectively, at three concentration levels of 10, 25, and 75 µg L−1. In addition, the limits of detection were in the range of 0.02–0.04 μg L−1. The validated method was successfully applied to the analysis of diclofenac and mefenamic acid in some biological samples. Finally, it is concluded that this method can be a general and reliable alternative to the analysis of ionic compounds in biological matrices.

Suspect Screening Using LC-QqTOF Is a Useful Tool for Detecting Drugs in Biological Samples.

High-resolution mass spectrometers (HRMS), including quadrupole time of flight mass analyzers (QqTOF), are becoming more prevalent as screening tools in clinical and forensic toxicology laboratories. Among other advantages, HRMS instruments can collect untargeted, full-scan mass spectra. These datasets can be analyzed retrospectively using a combination of techniques, which can extend the drug detection capabilities. Most laboratories using HRMS in production settings perform untargeted data collection, but analyze data in a targeted manner. To perform targeted analysis, a laboratory must first analyze a reference standard to determine the expected characteristics of a given compound. In an alternate technique known as suspect screening, compounds can be tentatively identified without the use of reference standards. Instead, predicted and/or intrinsic characteristics of a compound, such as the accurate mass, isotope pattern, and product ion spectrum are used to determine its presence in a sample. The fact that reference standards are not required a priori makes this data analysis approach very attractive, especially for the ever-changing landscape of novel psychoactive substances. In this work, we compared the performance of four data analysis workflows (targeted and three suspect screens) for a panel of 170 drugs and metabolites, detected by LC-QqTOF. We found that retention time was not required for drug identification; the suspect screen using accurate mass, isotope pattern, and product ion library matching was able to identify more than 80% of the drugs that were present in human urine samples. We showed that the inclusion of product ion spectral matching produced the largest decrease in false discovery and false negative rates, as compared to suspect screening using mass alone or using just mass and isotope pattern. Our results demonstrate the promise that suspect screening holds for building large, economical drug screens, which may be a key tool to monitor the use of emerging drugs of abuse, including novel psychoactive substances.

Multiplexed therapeutic drug monitoring (TDM) of antiviral drugs by LC–MS/MS

BackgroundTherapeutic drug monitoring (TDM) can be a useful tool in the clinical management of anti-hepatitis C virus (anti-HCV) drugs. Methods for the determination of various types of anti-HCV drugs in biological samples are, therefore, needed for clinical laboratories. ObjectiveIn this work, employing the LC–MS/MS approach, we aimed to develop a multiplexed method for identification of the following anti-HCV drugs: Ribavirin (RBV), Boceprevir (BOC), Telaprevir (TVR), Simeprevir (SIM), Daclatasvir (DAC), Sofosbuvir (SOF) and its metabolite GS 331007 (SOFM) in liquid plasma and in dried plasma spots (DPSs). MethodA single-step extractive-deproteinization was employed for both liquid plasma and DPSs. Reverse-phase liquid chromatography coupled with MRM detection was developed for multiplexed drug detection and quantification. ResultsSensitivities (expressed as LOQ) were 10 (±1.2), 10 (±4.9), 10 (±4.4), 10 (±4.4), 10 (±6.4), 10 (±3.4), 10 (±6.4) ng/ml for RBV, SOFM, SOF, DAC, BOC, TVR, and SIM, respectively; accuracy (expressed as BIAS%) was <10% for all drugs; reproducibility (intra- and inter-day CV%) was <10% for all drugs; dynamic range was 10–10,000 ng/ml for all drugs. ConclusionsA novel, simple, rapid and robust LC–MS/MS multiplex assay for the TDM of various anti-HCV drugs that are currently in the clinic was successfully developed. Application to DPS samples enabled TDM to be used for outpatients as well.

Metabolism of the synthetic cannabinoid 5F-PY-PICA by human and rat hepatocytes and identification of biliary analytical targets by directional efflux in sandwich-cultured rat hepatocytes using UHPLC-HR-MS/MS

Analytical strategies for detecting drugs in biological samples rely on information on metabolism and elimination. 5F-PY-PICA belongs to the group of synthetic cannabinoids that are known to undergo excretion into the bile.The aims of this study were the in vitro identification of metabolites of 5F-PY-PICA and to determine which analytical targets are excreted into the bile and urine.Metabolites identified after incubation of 5F-PY-PICA with pooled human liver microsomes (pHLM), pooled human hepatocytes (pHH), or suspended and sandwich-cultured rat hepatocytes (SCRH). Rat hepatocytes were harvested following a two-step perfusion protocol and the SCRH were prepared between layers of rat-tail collagen. The biliary efflux of 5F-PY-PICA and its metabolites was determined in three-day–cultured SCRH by differential efflux into either standard buffer from intact bile canaliculi or standard buffer without divalent cations, which disrupts the bile canaliculi. The metabolites were identified using liquid chromatography–high resolution mass spectrometry/mass spectrometry (LC-HR-MS/MS).The main metabolites were the COOH-ω-metabolite (M4) in pHH, the defluoro-HO-ω-metabolite (M3) in pHLM, and the COOH-pyrrolidine-metabolite (M6) in rat hepatocytes.Efflux into standard buffer without divalent cations was significantly higher (p<0.050) for 5F-PY-PICA, M4, and the HO-indole-glucuronide-metabolite (M22). M6 did not undergo significant biliary efflux, indicating that basolateral efflux dominates for this metabolite.5F-PY-PICA, M4, and M22 are proposed as analytical targets for bile analysis in forensic screening protocols, whereas M6 should be one of the main urinary targets for 5F-PY-PICA analysis.

Development of an LC-MS/MS method with protein G purification strategy for quantifying bevacizumab in human plasma.

Biopharmaceutical products such as protein drugs and monoclonal antibodies (mAb) are currently of great interest with monoclonal antibody drugs being one of the fastest growing categories of biopharmaceutical products. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has gained high interest for measuring mAb drugs in biological samples in recent years due to its high selectivity. Bevacizumab is a humanized immunoglobulin G (IgG) mAb drug against human vascular endothelial cell growth factor A (VEGF-A). It is used for treating many types of cancers. Recent studies have indicated that clinical outcomes vary among patients treated with bevacizumab and produce various side effects, such as vascular disorders. In this study, we developed an LC-MS/MS method to quantify bevacizumab concentration. We selected readily available and economic materials for sample preparation to facilitate its wider use in clinical fields.-Protein G was used to trap bevacizumab from human plasma. In place of an extended stable isotope-labeled internal standard (SIL-IS), the IgG-based drug-IS tocilizumab was used because of its better calibration performance. The method was validated in terms of its precision, accuracy, linearity, and sensitivity. The accuracies which were expressed as percentage recoveries for three concentration levels were within 92.8±3.2 to 112.7±4.5%. Repeatability and intermediate precision in terms of peak area ratios were lower than 5.2 and 12.9% RSD, respectively. The application to patients' sample measurements revealed a wide individual variability of drug concentrations, and the proposed simple and general method may facilitate personalized medicine for improving therapeutic efficacy and safety. Graphical abstract ᅟ.

High sensitivity and selectivity in quantitative analysis of drugs in biological samples using 4-column multidimensional micro-UHPLC-MS enabling enhanced sample loading capacity

Sensitivity is often a critical parameter in quantitative bioanalyses in drug development. For liquid-chromatography-based methods, sensitivity can be improved by reducing the column diameter, but practical sensitivity gains are limited by the reduced sample loading capacity on small internal diameter (I.D.) columns. We developed a set-up that has overcome these limitations in sample loading capacity. The set-up uses 4 columns with gradually decreasing column diameters along the flow-path (2.1 → 1 → 0.5 → 0.15 mm). Samples are pre-concentrated on-line on a 2.1 mm I.D. trapping column and back flushed to a 1 mm I.D. UHPLC analytical column and separated. The peak(s) of interest are transferred using heartcutting to a second trapping column (0.5 mm I.D.), which is back-flushed to a 0.15 mm I.D. micro-UHPLC analytical column for orthogonal separation. The proof of concept of the set-up was demonstrated by the simultaneous analysis of midazolam and 1′-hydroxy midazolam in plasma by injection of 80 μL of protein precipitated plasma. The 4-column funnel set-up proved to be robust and resulted in a 10–50 times better sensitivity compared to a trap-elute approach and 250–500 fold better compared to direct micro-UHPLC analysis. A lower limit of quantification of 100 fg/mL in plasma was obtained for both probe compounds.