RESUMO
Permeability is a key factor driving the absorption of orally administered drugs. In early discovery, the efficient evaluation of permeability, particularly for compounds violating Lipinski's Rule of 5, remains challenging. Addressing this, we established a high-throughput method to measure the experimental polar surface area (HT-EPSA) as an in vitro surrogate to measure permeability. Compared to earlier methods, HT-EPSA significantly reduces data acquisition time with enhanced sensitivity, selectivity, and data quality. In the effort of translating EPSA to human in vitro and in vivo passive permeability, we demonstrated the application of EPSA for predicting Caco-2 cell and human intestinal permeability, showing improvements over topological polar surface area and the parallel artificial membrane permeability assay for rank-ordering permeability in a proteolysis targeting chimera case study. The HT-EPSA method is expected to be highly beneficial in guiding early stage compound rank-ordering, faster decision-making, and in predicting in vitro and/or in vivo human intestinal permeability.
Assuntos
Ensaios de Triagem em Larga Escala , Permeabilidade , Espectrometria de Massas em Tandem , Humanos , Células CACO-2 , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas em Tandem/métodos , Absorção Intestinal , Permeabilidade da Membrana Celular , AnimaisRESUMO
In contrast to quantification of biotherapeutics, endogenous protein biomarker and target quantification using LC-MS based targeted proteomics can require a much more stringent and time-consuming tryptic signature peptide selection for each specific application. While some general criteria exist, there are no tools currently available in the public domain to predict the ionization efficiency for a given signature peptide candidate. Lack of knowledge of the ionization efficiencies forces investigators to choose peptides blindly, thus hindering method development for low abundant protein quantification. Here, the authors propose a tryptic signature peptide selection workflow to achieve a more efficient method development and to improve success rates in signature peptide selection for low abundant endogenous target and protein biomarker quantification.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Cromatografia Líquida , Fluxo de Trabalho , Peptídeos , BiomarcadoresRESUMO
While bioanalytical outsourcing is widely adopted in the pharmaceutical industry, AbbVie is one of the few large biopharmaceutical companies having an internal bioanalytical unit to support nearly all its drug metabolism and pharmacokinetic studies. This article highlights our experience and perspective in building an integrated and centralized laboratory to provide early discovery and preclinical-stage bioanalytical support with high operational efficiency, cost-effectiveness and data integrity. The advantages of in-house nonregulated bioanalytical support include better control of data quality, faster turnaround times, real-time knowledge sharing and troubleshooting, and lower near- and long-term costs. The success of an in-house model depends upon a comprehensively optimized and streamlined workflow, fueled by continuous improvements and implementation of innovative technologies.
Assuntos
Laboratórios , Serviços Terceirizados , Automação , Tecnologia , Indústria FarmacêuticaRESUMO
RATIONALE: The in-sample calibration curve (ISCC) approach of quantification utilizes the response of isotopologue ions from spiked-in stable isotope labeled internal standard (SIL-IS) to build a standard curve. The quantitative analysis of the study sample is achieved based on the response of selected monoisotopic analyte ion against the calibration curve. Although this methodology has been demonstrated to be feasible by unit and high-resolution mass spectrometers, quantitation on high-resolution mass spectrometer with product ions has not been tested. We tested the feasibility of this approach using product ions on an high-resolution mass spectrometer equipped with an Orbitrap detector. METHODS: Using a proteomics workflow for sample preparation, two surrogate peptides were quantified from a complex matrix of protein digest from human peripheral blood mononuclear cells (hPBMCs). SIL-IS was spiked in at different levels to construct calibration curves in a traditional manner. ISCCs were prepared using extracted ion chromatograms from isotopically resolved mass spectra and compared with traditional calibration curves. RESULTS: A linear response was observed with ISCC approach for at least two to three orders of magnitude in MS1 as well as targeted MS2 (tMS2). From protein digests, isobaric interferences were observed for endogenous peptides on the MS1 level; this was circumvented with product-ion-based quantitation where for one peptide, %CV for endogenous levels was more than 20% with ISCC but higher with the traditional calibration curve approach. For the second peptide, endogenous levels could not be determined in the traditional approach as calibrant levels did not bracket the lower end, and with the ISCC approach, isotopologues at abundances lower than the endogenous level allowed for quantitative assessments. CONCLUSIONS: ISCC demonstrated improved precision across surrogate peptides from endogenous protein digests. In samples where endogenous analyte concentrations were low in abundance, ISCC rescued what would have been a non-reportable result in a traditional bioanalytical assay as calibrant levels were not prepared at adequately low levels to bracket unknowns. ISCC using high-resolution mass spectrometer is feasible and ideal compared to unit resolution mass spectrometers. High-resolution mass spectrometer allows for isotopic resolution for analytes with > + 2 charge state and provides flexibility in quantification using multiple product ions. ISCC using high-resolution mass spectrometer allows for simultaneous assaying of low abundance isotopologues, the signal acquisition of which is not constrained by limits in data acquisition or calibrant preparation as with other approaches but rather limited by platform sensitivity. In contrast to unit resolution mass spectrometers, these features offered by high-resolution mass spectrometer could be especially useful for the drug discovery assay support where there is less lead time for assay development than for the assays to support the drug development studies.
Assuntos
Leucócitos Mononucleares , Espectrometria de Massas em Tandem , Calibragem , Humanos , Isótopos , Peptídeos , Espectrometria de Massas em Tandem/métodosRESUMO
Background: Currently, no regulatory guidelines are available for parallelism assessment for LC-MS biomarker quantification. Spike recovery, standard addition and dilutional linearity are recommended with no mention of the implications of applying these approaches. Results: Here, using human urine creatinine, the authors compared spike recovery and standard addition in LC-MS biomarker quantification, and evaluated a new hybrid approach: parallelism QCs. The authors drew different conclusions based on which approach was used (<15% cutoff). Conclusion: Current recommended approaches may lead to different conclusions and are not equivalent and interchangeable. The authors recommend that standard addition should be the universal 'go-to' method for LC-MS biomarker parallelism assessment; parallelism QCs, which consider the total concentration as the theoretical value, can be used if the authentic matrix is limited.
Assuntos
Espectrometria de Massas em Tandem , Biomarcadores , Cromatografia Líquida/métodos , Creatinina , Humanos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodosRESUMO
LC/MS quantification of leukotoxin (LTX) and leukotoxin diol (LTXdiol) in plasma has been previously reported, however large sample volumes are required for achieving stated assay Lower Limit of Quantification (LLOQ). Reported here is a fit-for-purpose LC/MS method that reduces plasma volume from 700 to 25 µL and omits pre-concentration steps. These improvements make for a method with increased utility in mouse studies offering limited sample volumes. Additionally, omitting pre-concentration steps streamlines sample processing, which can now be completed in under 10 min. This method can be used to quickly answer if the ratio of LTX to LTXdiol changes with the dose of the therapeutic drug so this could be used as a potential biomarker for correlating PK/PD effects. No extensive assay characterization was performed before application to an exploratory in-life study. Basal levels of LTX and LTXdiol in plasma were quantified by LC-MRM across 10 individual mice, and the average signal-to-noise was 36 for LTX and 3039 for LTXdiol, with CVs of 29.4% and 15.2%, respectively. Addition of LTX and LTXdiol reference standard at 5, 25, and 75 ng/mL into pooled mouse plasma was quantifiable within 30% relative error using a surrogate matrix calibration curve ranging from 0.8 to 200 ng/mL. The average ratio of LTX to LTXdiol across the 10 mice was 0.32, consistent with previous reports. Finally, the method was applied to a mouse PK/PD study to monitor LTX/LTXdiol kinetics after a single oral dose of a soluble epoxide hydrolase inhibitor. The mean plasma ratio of LTX to LTXdiol increased up to 10-fold by 3 h post-dose followed by a decrease to near pre-dose levels by 24 h, consistent with transient inhibition of sEH-mediated conversion of LTX to LTXdiol. The method improvements described here will make subsequent quantification of LTX and LTXdiol in mouse studies significantly easier.
Assuntos
Cromatografia Líquida/métodos , Exotoxinas/sangue , Ácidos Esteáricos/sangue , Espectrometria de Massas em Tandem/métodos , Animais , Biomarcadores/sangue , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos TestesRESUMO
Background: To support the clinical studies of cabiralizumab, an immunogenicity assay for detecting anti-cabiralizumab antibodies is required. Results: Strategies were developed to overcome two major bioanalytical challenges: poor drug tolerance of the anti-drug antibodies assay and very low cut point observed in the screening and confirmatory assays. By using acid dissociation (400 mM glycine solution at pH 2.0), drug tolerance of 200 µg/ml drug was achieved for both the screening and confirmatory assays. Effects of biological matrix (disease state vs normal serum) and assay conditions (capture/detector reagent concentration, minimum required dilution, acid pretreatment) on assay cut points were systematically evaluated. Conclusion: A bridging immunogenicity assay for detecting anti-cabiralizumab antibodies in human serum has been successfully developed, validated and applied to clinical studies.
Assuntos
Anticorpos Monoclonais Humanizados/sangue , Bioensaio , Tolerância a Medicamentos , HumanosRESUMO
The microsampling workshop generated recommendations pertaining to blood sampling site (venous blood versus capillary blood), when to conduct a bridging study, statistical approaches to establish correlation/concordance and deciding on sample size, opportunities and challenges with patient-centric sampling, and how microsampling technology can enrich clinical drug development. Overall, the goal was to provide clarity and recommendations and enable the broader adoption of microsampling supporting patients' needs, convenience, and the transformation from clinic-centric to patient-centric drug development. The need and adoption of away-from-clinic sampling techniques has become critical to maintain patient safety during the current COVID-19 pandemic.
Assuntos
Coleta de Amostras Sanguíneas , Assistência Centrada no Paciente , Desenvolvimento de Medicamentos , HumanosRESUMO
Glucuronides, especially acyl glucuronides, were often found to be unstable in vitro and in vivo. Acyl glucuronide metabolites can convert back to the parent drugs at physiological pH through hydrolysis. Glucuronides can also undergo in-source fragmentation during MS ionization to form the same ions as those of the parent compounds, which could cause interference to the analysis of the parent compounds. All of these may cause significant challenges in developing LC-MS/MS bioanalytical assays of labile glucuronides or parent compounds in the presence of glucuronide metabolites. In this manuscript, we will discuss these challenges and summarize recommended strategies and practices for fast and efficient method development. Critical considerations in assay development will also be discussed.
Assuntos
Bioensaio , Glucuronídeos/análise , Glucuronídeos/metabolismo , Cromatografia Líquida , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Espectrometria de Massas em TandemRESUMO
Acoustic liquid handlers deliver small volumes (nL-µL) of multiple fluid types with accuracy and dynamic viscosity profiling. They are widely used in the pharmaceutical industry with applications extending from high-throughput screening in compound management to gene expression sequencing, genomic and epigenetic assays, and cell-based assays. The capability of the Echo to transfer small volumes of multiple types of fluids could benefit bioanalysis assays by minimization of sample volume and by simplifying dilution procedures by direct dilution. In this study, we evaluated the Labcyte Echo 525 liquid handler for its ability to deliver small volumes of sample preparations in biological matrix (plasma and serum) and to assess the feasibility of integration of the Echo with three types of bioanalytical assay platforms: microplate enzyme-linked immunosorbent assay, Gyrolab immunoassay, and liquid chromatography with tandem mass spectrometry. The results demonstrated acceptable consistency of dispensed plasma samples from multiple lots and species by the Echo. Equivalent assay performance demonstrated between the Echo and manual liquid procedures indicated great potential for the integration of the Echo with the bioanalytical assay, which allows the successful implementation of microsampling strategies in drug discovery and development.
Assuntos
Acústica , Ensaios de Triagem em Larga Escala/métodos , Animais , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Padrões de Referência , Espectrometria de Massas em TandemRESUMO
Bioanalytical methods evolve throughout clinical development timelines, resulting in the need for establishing equivalency or correlation between different methods to enable comparison of data across different studies. This is accomplished by the conduct of cross validations and correlative studies to compare and describe the relationship. The incurred sample reanalysis acceptance criterion seems to be adopted universally for cross validations and correlative studies; however, this does not identify any trends or biases between the two methods (datasets) being compared. Presented here are graphing approaches suitable for comparing two methods and describing equivalence or correlation. This article aims to generate awareness on graphing techniques that can be adopted during cross validations and correlative studies.
Assuntos
Bioensaio/métodos , HumanosRESUMO
Microsampling techniques enable the minimization of blood collection volume from animals and subsequent handling of the blood samples or their derived plasma or serum samples. This offers advantages over conventional large-volume sampling, such as eliminating the need for satellite animals and improving animal welfare aspects, and providing the opportunity for additional assessments in small animals where blood volume constraints limit endpoints. This study evaluated the feasibility of implementation of capillary microsampling (CMS) in a single-dose study in mice with the ultimate goal of enabling its use in toxicology studies. The focus was on the impact of microsampling on toxicokinetic assessment and on the subsequent hematology assessment in the same animal. A seventy (70)-µL blood collection via CMS from the tail vein had a minimal effect on the hematology parameters of mice (strain C57BL/6) in samples taken within 24 h of blood collection. TK parameters were similar in plasma samples collected via CMS and cardiac puncture sampling. A bioanalytical assay was developed which enabled the quantification of concentration of both the parent drug and a metabolite using only 5-µL plasma sample per analysis. Incurred sample reanalysis (ISR), unexpected event investigation, and re-assay were successfully performed on the limited samples (≤ 20 µL) collected from CMS. The results of this study confirmed the feasibility of implementing CMS in regulated mouse toxicity studies and demonstrated that it is possible to eliminate or reduce satellite animals.
Assuntos
Coleta de Amostras Sanguíneas , Eritrócitos/efeitos dos fármacos , Testes Hematológicos , Cauda/irrigação sanguínea , Testes de Toxicidade , Ureia/análogos & derivados , Valina/análogos & derivados , Administração Oral , Animais , Contagem de Eritrócitos , Eritrócitos/metabolismo , Estudos de Viabilidade , Hematócrito , Hemoglobinas/metabolismo , Camundongos Endogâmicos C57BL , Toxicocinética , Ureia/administração & dosagem , Ureia/sangue , Ureia/toxicidade , Valina/administração & dosagem , Valina/sangue , Valina/toxicidade , Fluxo de TrabalhoRESUMO
Traditionally, for a liquid chromatography tandem mass spectrometry (LC-MS/MS) bioanalytical assay, an external calibration curve is required to achieve accurate quantitation of an analyte. Recently, a novel in-sample calibration curves (ISCC) methodology that can achieve quick and accurate LC-MS/MS bioanalysis without the use of an external calibration curve was reported. The ISCC methodology utilizes the presence of multiple naturally occurring isotopologues of a stable isotopically labeled analyte to construct an in-sample calibration curve for the quantification. This methodology has great potential in many applications, for example biomarker measurement, quantitative proteomics and clinical diagnosis. Here, we assessed the feasibility of applying this ISCC-LC-MS/MS methodology in regulated bioanalysis using BMS-984478, a drug candidate, as the model compound. We also proposed method validation procedures/processes for this new approach for industry peers' consideration and feedback. A LC-MS/MS method using the ISCC strategy was successfully developed and validated for the quantitative analysis of BMS-984478 in human plasma over the range of 1.33-993.42â¯ng/mL. The validated ISCC-LC-MS/MS method was compared with a previously validated method using the conventional external calibration curve approach, and the two methods showed equivalent performance. Critical considerations and practical approaches in method development, validation and sample analysis were also discussed. Our work demonstrated that the ISCC-LC-MS/MS methodology is a promising approach for regulated LC-MS/MS bioanalysis. ISCC-LC-MS/MS methodology has its unique advantages and has great potential to be widely applied for various quantitative applications, and may even change the landscape of quantitative analysis.
Assuntos
Antivirais/isolamento & purificação , Fracionamento Químico/métodos , Espectrometria de Massas em Tandem/métodos , Antivirais/administração & dosagem , Antivirais/sangue , Calibragem , Cromatografia Líquida/métodos , Estudos de Viabilidade , Humanos , Limite de Detecção , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização por Electrospray/normas , Espectrometria de Massas em Tandem/normasRESUMO
ND-L02-s0201/BMS-986263 is a lipid nanoparticle (LNP) drug product containing a heat shock protein 47 (HSP47)-specific small interfering ribonucleic acid (siRNA) and being developed for the treatment of liver and idiopathic pulmonary fibrosis. To address immunogenicity-related issues, we developed a robust, fit-for-purpose (FFP) three-tier electrochemiluminescent (ECL) anti-drug antibody (ADA) assay for the detection of antibodies (Abs) generated to surface-exposed components of BMS-986263. The drug was coated directly on plates, and several Abs specific for polyethylene glycol (PEG) and other surface components were tested for use as positive quality controls (QCs). Following selection of a rabbit monoclonal anti-PEG Ab, the assay was optimized, and various method development challenges specific to the modality and pseudo surrogate rabbit control were addressed. Screening, confirmatory, and titer cut points were validated following a statistical evaluation of 41 individual K2EDTA human plasma samples at a minimum required dilution (MRD) of 100. Assay precision, sensitivity, selectivity, drug tolerance, and hook effect were determined for the rabbit Ab prepared in human K2EDTA plasma matrix. The assay was used to interrogate anti-drug Ab (ADA) responses in normal human subjects who were administered 90 mg of the drug intravenously (IV) once every week for 3 weeks in phase I clinical trials. All pre- and post-dose samples were found to be negative for ADA. Based on these results, we concluded that BMS-986263 is not immunogenic. To the best of our knowledge, this work represents the first ADA method developed and reported for an LNP-based drug product.
Assuntos
Anticorpos/imunologia , Proteínas de Choque Térmico HSP47/administração & dosagem , Nanopartículas , RNA Interferente Pequeno/administração & dosagem , Método Duplo-Cego , Técnicas Eletroquímicas , Proteínas de Choque Térmico HSP47/imunologia , Humanos , Lipídeos/química , Medições Luminescentes , Polietilenoglicóis/metabolismo , RNA Interferente Pequeno/imunologiaRESUMO
Aim: The result of investigation on the procedure of sample handling and bioanalysis of small volume of plasma sample for nonclinical studies stored in 0.5-ml micronic tubes was reported. Results/methodology: Sample integrity of the small volume (25 µl) during long-term storage and the feasibility and data reliability of performing multiple re-assays on the small volume sample using 5 µl aliquot per analysis was evaluated. Conclusion: Integrity was maintained in samples (25 µl) stored for up to 1 month in 0.5-ml micronic tubes at -20°C. A 25 µl sample is sufficient for four-times of re-assays. This evaluation demonstrated the feasibility of this workflow of handling and bioanalysis on small volume plasma sample for GLP studies under the US FDA guidance.
RESUMO
Stable isotope labeled (SIL) compounds have been commonly used as internal standards (IS) to ensure the accuracy and quality of liquid chromatography-mass spectrometry (LC-MS) bioanalytical assays. Recently, the application of SIL drugs and LC-MS assays to microdose absolute bioavailability (BA) studies has gained increasing attention. This approach can provide significant cost and time saving, and higher data quality compared to the accelerator mass spectrometry (AMS)-based method, since it avoids the use of radioactive drug, high-cost AMS instrumentation and complex measurement processes. It also eliminates potential metabolite interference with AMS-based assay. However, one major challenge in the application of this approach is the potential interference between the unlabeled drug, the microdose SIL drug, and the SIL-IS during LC-MS analysis. Here we report a convenient and cost-effective strategy to overcome the interference by monitoring the isotopic ion (instead of the commonly used monoisotopic ion) of the interfered compound in MS analysis. For the BMS-986205 absolute BA case study presented, significant interference was observed from the microdose IV drug [13C7,15N]-BMS-986205 to its SIL-IS, [13C7,15N, D3]-BMS-986205, since the difference of nominal molecular mass between the two compounds is only 3 mu, and there is a Cl atom in the molecules. By applying this strategy (monitoring the 37Cl ion for the analysis of the IS), a 90-fold reduction of interference was achieved, which allowed the use of a synthetically accessible SIL compound and enabled the fast progress of the absolute BA study. This strategy minimizes the number of stable isotope labels used for avoiding interference, which greatly reduces the difficulty in synthesizing the SIL compounds and generates significant time and cost savings. In addition, this strategy can also be used to reduce the MS response of the analyte, therefore, avoiding the detector saturation issue of LC-MS/MS assay for high concentration BMS-986205. A LC-MS/MS assay utilizing this strategy was successfully developed for the simultaneous analysis of BMS-986205 and [13C7, 15N]-BMS-986205 in dog plasma using [13C7,15N, D3]-BMS-986205 as the IS. The assay was successfully applied to a microdose absolute BA study of BMS-986205 in dogs. The assay was also validated in human plasma and used to support a human absolute BA study. The same strategy can also be applied to other compounds, including those not containing Cl or other elements with abundant isotopes, or other applications (e.g. selection of internal standard), and the applications were presented.
Assuntos
Acetamidas/análise , Cromatografia Líquida/métodos , Inibidores Enzimáticos/análise , Quinolinas/análise , Espectrometria de Massas em Tandem/métodos , Acetamidas/administração & dosagem , Acetamidas/farmacocinética , Animais , Disponibilidade Biológica , Cromatografia Líquida/economia , Análise Custo-Benefício , Cães , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Humanos , Marcação por Isótopo , Quinolinas/administração & dosagem , Quinolinas/farmacocinética , Espectrometria de Massas em Tandem/economiaAssuntos
Desenvolvimento de Medicamentos/métodos , Lasers , Manejo de Espécimes , Tecnologia Farmacêutica/métodos , Vibração , Drogas em Investigação/farmacocinética , Humanos , Raios Infravermelhos , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Distribuição Tecidual , Pesquisa Translacional BiomédicaRESUMO
Recombinant adeno associated viruses (rAAV) have become an important tool for the delivery of gene therapeutics due to long-standing safety and success in clinical trials. Since humans often become exposed to AAVs and develop anti-AAV antibodies (Abs), a potential impediment to the success of gene therapeutics is neutralization of the viral particle before it has had a chance to bind and enter target cells to release the transgene. Identification of subjects with preexisting Abs having neutralizing potential, and exclusion of such subjects from clinical studies is expected to enhance drug efficacy. In vitro cell-based reporter assays are most often employed to determine the level of neutralizing antibodies in a given population. Such assays measure the ability of the Abs to prevent viral binding and entry into cells by engaging epitopes on the viral capsid involved in host cell receptor binding. In general, cell-based assays are low throughput and labor intensive and may suffer from high variability and low sensitivity issues. In contrast, enzyme-linked immunosorbent assays (ELISAs) are simpler, less variable, and have higher throughput. Demonstrating a correlation between neutralizing Abs assessed by a cell-based assay and total binding Abs measured in an ELISA will enable the use and substitution of the latter for screening and exclusion of subjects. In this work, we describe the development of a highly sensitive, specific, robust, and reproducible chemiluminescent ELISA method for the detection of total anti-AAV9 Abs. Using this method, we analyzed the prevalence of preexisting anti-AAV9 Abs in 100 serum samples from heart disease patients. Analysis of neutralizing Abs in the same samples using an in vitro cell-based assay showed a strong correlation between total anti-AAV9 Abs and neutralizing Abs, indicating the feasibility of using the total Ab ELISA in the future for patient screening and exclusion.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Dependovirus/imunologia , Luciferases de Vaga-Lume/metabolismo , Animais , Biomarcadores/sangue , Linhagem Celular , Cricetinae , Cricetulus , Dependovirus/genética , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Luciferases de Vaga-Lume/genética , Luminescência , Sensibilidade e Especificidade , SorogrupoRESUMO
The 18th Annual Land O'Lakes Bioanalytical Conference, titled 'Cutting-Edge Bioanalytical Technologies and Concepts - Issues, Solutions and Practical Considerations for Applications in Novel and Emerging Modalities', was held 10-13 July 2017 in Madison, WI, USA. This Land O'Lakes Conference is presented each year by the Division of Pharmacy Professional Development within the School of Pharmacy at the University of Wisconsin-Madison (USA). The purpose of this conference is to provide an educational forum to discuss issues and applications associated with the analysis of xenobiotics, metabolites, biologics and biomarkers in biological matrices. The conference is designed to include and encourage an open exchange of scientific and methodological applications for bioanalysis. This report summarized the presentations at the 18th Annual Conference.