ABSTRACT
The 10th Global CRO Council (GCC) Closed Forum was held in Orlando, FL, USA on 18 April 2016. In attendance were decision makers from international CRO member companies offering bioanalytical services. The objective of this meeting was for GCC members to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at this closed forum included reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, biomarker assay validation, processed batch acceptance criteria, electronic laboratory notebooks and data integrity, Health Canada's Notice regarding replicates in matrix stability evaluations, critical reagents and regulatory approaches to counteract fraud. In order to obtain the pharma perspectives on some of these topics, the first joint CRO-Pharma Scientific Interchange Meeting was held on 12 November 2016, in Denver, Colorado, USA. The five topics discussed at this Interchange meeting were reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, processed batch acceptance criteria and electronic laboratory notebooks and data integrity. The conclusions from the discussions of these topics at both meetings are included in this report.
Subject(s)
Biomarkers/analysis , Chemistry Techniques, Analytical/standards , Data Collection/standards , Guidelines as Topic , Pharmaceutical Preparations/analysis , Drug Stability , Government Regulation , Humans , Research ReportABSTRACT
The 9th GCCClosed Forum was held just prior to the 2015 Workshop on Recent Issues in Bioanalysis (WRIB) in Miami, FL, USA on 13 April 2015. In attendance were 58 senior-level participants, from eight countries, representing 38 CRO companies offering bioanalytical services. The objective of this meeting was for CRO bioanalytical representatives to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues selected at this year's closed forum include CAPA, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, and ELNs. A summary of the industry's best practices and the conclusions from the discussion of these topics is included in this meeting report.
Subject(s)
Biomarkers/analysis , Biosimilar Pharmaceuticals/analysis , Drug Evaluation, Preclinical/methods , Biomarkers/blood , Electronic Health Records , Laboratories , Societies, Medical , Validation Studies as TopicABSTRACT
Asunaprevir (BMS-650032) is a selective hepatitis C virus (HCV) NS3 protease inhibitor with potent activity against HCV genotypes 1, 4, 5 and 6. It has been developed in conjunction with direct-acting antiviral agents, in interferon- and ribavirin-free regimen, to improve existing therapies for HCV infection. To support the pharmacokinetic analyses in asunaprevir clinical studies, we have developed and validated a highly sensitive and robust LC-MS/MS method to quantify asunaprevir in human EDTA plasma with an LLOQ of 0.05ng/mL, which was a 20-fold sensitivity improvement over a previously reported assay for asunaprevir. A deuterated labeled [D9]-asunaprevir was used as the internal standard (IS). The analyte and the IS were extracted using a semi-automated liquid-liquid extraction (LLE) at pH 7 with methyl-t-butyl ether (MTBE) in a 96-well plate containing 10µL of 10% CHAPS as the surfactant to prevent non-specific binding issue. Chromatographic separation was achieved on a Genesis C8 column (2.1×50mm, 4µm) with a gradient elution using 0.1% formic acid in water as mobile phase A and a mixture of methanol: acetone: formic acid (95:5:0.1; v/v/v) as the mobile phase B. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 748â648 for asunaprevir and m/z 757â649 for [D9]-asunaprevir,and a collision energy of 30 electron Volts (eV). The assay was validated over a standard curve range from 0.05 to 50ng/mL for asunaprevir in human plasma. The intra- and inter assay precisions were within 7.1% CV, and the % deviation was within 5.5% of their nominal concentrations. This assay has been successfully applied to multiple clinical studies with excellent assay ruggedness and reproducibility.
Subject(s)
Antiviral Agents/blood , Chromatography, High Pressure Liquid/methods , Hepacivirus/enzymology , Isoquinolines/blood , Sulfonamides/blood , Tandem Mass Spectrometry/methods , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Calibration , Humans , Isoquinolines/chemistry , Isoquinolines/pharmacokinetics , Limit of Detection , Liquid-Liquid Extraction , Molecular Structure , Reference Standards , Reproducibility of Results , Sulfonamides/chemistry , Sulfonamides/pharmacokineticsABSTRACT
Dual or triple combination regimens of novel hepatitis C direct-acting antivirals (DAA, daclatasvir, asunaprevir, or beclabuvir) provide high sustained virological response rates and reduced frequency of resistance compared to clinical monotherapy. To support pharmacokinetic (PK) assessments in clinical studies, a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitation of daclatasvir, asunaprevir, beclabuvir (BMS-791325) and its active metabolite (BMS-794712) in human plasma was developed and validated. Human plasma samples were extracted with methyl-t-butyl ether followed by an LC-MS/MS analysis, which was conducted in a multiple reaction monitoring (MRM) mode. The lower limits of quantitation (LLOQ) were 1 ng/mL for daclatasvir, asunaprevir, and BMS-794712, and 2 ng/mL for beclabuvir. Intra-run precision (≤4.5% CV), inter-run precision (≤2.9% CV), and accuracy (±5.3% deviation) based on different concentration levels (low, geometric mean, mid and high) of the quality control samples (QCs) provided evidence of the methods accuracy and precision. Selectivity and matrix effect on LC-MS/MS detection, stability in plasma, and potential interference of coadministered drugs (ribavirin and interferon) were all evaluated and the results were acceptable. Method reproducibility was demonstrated by the reanalysis of a portion of study samples. The cross-validation results for QCs demonstrated the equivalency between this method and two single-analyte methods which were previously validated for quantitation of daclatasvir in human plasma. This approach of using a multiplexed LC-MS/MS method for the simultaneous quantitation of three DAAs is time- and cost-effective, and can maintain good data quality in sample analysis.
Subject(s)
Antiviral Agents/chemistry , Benzazepines/chemistry , Imidazoles/blood , Indoles/chemistry , Isoquinolines/chemistry , Plasma/chemistry , Sulfonamides/chemistry , Antiviral Agents/blood , Antiviral Agents/pharmacology , Benzazepines/blood , Carbamates , Chromatography, Liquid/methods , Hepacivirus/drug effects , Humans , Imidazoles/chemistry , Indoles/blood , Interferons/blood , Interferons/chemistry , Isoquinolines/blood , Pyrrolidines , Reproducibility of Results , Ribavirin/blood , Ribavirin/chemistry , Sulfonamides/blood , Tandem Mass Spectrometry/methods , Valine/analogs & derivativesABSTRACT
BMS-791325 is a novel hepatitis C NS5B inhibitor which is currently in clinical development. To support pharmacokinetic (PK) assessments, sensitive, accurate, precise, and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed and validated for the quantitation of BMS-791325 and its active N-demethyl metabolite (BMS-794712) in human plasma and urine. Plasma and urine samples were extracted with methyl-t-butyl ether followed by an LC-MS/MS analysis which was conducted in a multiple reaction monitoring (MRM) mode for the simultaneous detection of the two analytes in human plasma (0.1-50 ng/mL) and in human urine (5-2500 ng/mL). Intra-run precision (3.0% R.S.D.), inter-run precision (5.3% R.S.D.), and accuracy (±4.7% deviation) from plasma and urine quality control samples provide evidence of the methods accuracy and precision. Selectivity, stability in matrices, extraction recovery, matrix effect on LC-MS detection, and interference of coadministered drugs (famotidine and ritonavir) were all acceptable. Reproducibility of the plasma method was demonstrated by reanalysis of a portion of study samples. The results of cross-validations demonstrated the equivalency of two methods validated in two labs. The plasma method was applied to the analysis of several thousand clinical study samples for PK evaluations of the drug in normal healthy subjects and in patients. The urine method was used in the first in human study to evaluate renal clearance and urinary recovery.
Subject(s)
Antiviral Agents/blood , Antiviral Agents/urine , Benzazepines/blood , Benzazepines/urine , Indoles/blood , Indoles/urine , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Benzazepines/metabolism , Benzazepines/pharmacology , Chromatography, Liquid/methods , Hepacivirus/drug effects , Humans , Indoles/metabolism , Indoles/pharmacology , Reproducibility of Results , Sensitivity and Specificity , Tandem Mass Spectrometry/methodsABSTRACT
This paper represents the consensus views of a cross-section of companies and organizations from the USA and Canada regarding the validation and application of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for bioanalysis of protein biotherapeutics in regulated studies. It was prepared under the auspices of the AAPS Bioanalytical Focus Group's Protein LC-MS Bioanalysis Subteam and is intended to serve as a guide to drive harmonization of best practices within the bioanalytical community and provide regulators with an overview of current industry thinking on applying LC-MS/MS technology for protein bioanalysis. For simplicity, the scope was limited to the most common current approach in which the protein is indirectly quantified using LC-MS/MS measurement of one or more of its surrogate peptide(s) produced by proteolytic digestion. Within this context, we considered a range of sample preparation approaches from simple in-matrix protein denaturation and digestion to complex procedures involving affinity capture enrichment. Consideration was given to the method validation experiments normally associated with traditional LC-MS/MS and ligand-binding assays. Our collective experience, thus far, is that LC-MS/MS methods for protein bioanalysis require different development and validation considerations than those used for small molecules. The method development and validation plans need to be tailored to the particular assay format being established, taking into account a number of important factors: the intended use of the assay, the test species or study population, the characteristics of the protein biotherapeutic and its similarity to endogenous proteins, potential interferences, as well as the nature, quality, and availability of reference and internal standard materials.
Subject(s)
Chromatography, Liquid/methods , Proteins/analysis , Tandem Mass Spectrometry/methods , Animals , Canada , Humans , United States , Validation Studies as TopicABSTRACT
The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years' editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for small molecule bioanalysis using LCMS. Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' input) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the upcoming issues of Bioanalysis.
Subject(s)
Biological Assay , Chromatography, Liquid/methods , Mass Spectrometry/methodsABSTRACT
The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years' editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for Large molecules bioanalysis using LBA and Immunogenicity. Part 1 (Small molecules bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' Input) were published in the Bioanalysis issues 6(22) and 6(23), respectively.
Subject(s)
Chemistry Techniques, Analytical , Immunity , Antibodies, Neutralizing/immunology , Biotransformation , Humans , Pharmaceutical Preparations/metabolism , Pharmacokinetics , Polyethylene/chemistry , Practice Guidelines as Topic , United States , United States Food and Drug AdministrationABSTRACT
AIM: The determination of drug-protein binding is important in the pharmaceutical development process because of the impact of protein binding on both the pharmacokinetics and pharmacodynamics of drugs. Equilibrium dialysis is the preferred method to measure the free drug fraction because it is considered to be more accurate. The throughput of equilibrium dialysis has recently been improved by implementing a 96-well format plate. Results/methodology: This manuscript illustrates the successful application of a 96-well rapid equilibrium dialysis (RED) device in the determination of atazanavir plasma-protein binding. DISCUSSION/CONCLUSION: This RED method of measuring free fraction was successfully validated and then applied to the analysis of clinical plasma samples taken from HIV-infected pregnant women administered atazanavir. Combined with LC-MS/MS detection, the 96-well format equilibrium dialysis device was suitable for measuring the free and bound concentration of pharmaceutical molecules in a high-throughput mode.
Subject(s)
Anti-HIV Agents/blood , Anti-HIV Agents/metabolism , Blood Chemical Analysis/methods , Oligopeptides/blood , Oligopeptides/metabolism , Pyridines/blood , Pyridines/metabolism , Tandem Mass Spectrometry , Anti-HIV Agents/chemistry , Atazanavir Sulfate , Chromatography, Liquid , Clinical Trials as Topic , Dialysis , Female , Humans , Oligopeptides/chemistry , Pregnancy , Protein Binding , Pyridines/chemistry , Reproducibility of ResultsABSTRACT
The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years' editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' Input. Part 1 (Small molecules bioanalysis using LCMS) was published in the Bioanalysis issue 6(22) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the Bioanalysis issue 6(24).
Subject(s)
Clinical Laboratory Techniques , Analytic Sample Preparation Methods , Chromatography, Liquid , Humans , Mass SpectrometryABSTRACT
The topic of incurred sample stability (ISS) has generated considerable discussion within the bioanalytical community in recent years. The subject was an integral part of the seventh annual Workshop on Recent Issues in Bioanalysis (WRIB) held in Long Beach, CA, USA, in April 2013, and at the Global CRO Council for Bioanalysis (GCC) meeting preceding it. Discussion at both events focused on the use of incurred samples for ISS purposes in light of results from a recent GCC survey completed by member companies. This paper reports the consensus resulting from these discussions and serves as a useful reference for depicting ISS issues and concerns, summarizing the GCC survey results and providing helpful recommendations on ISS in the context of bioanalytical method development and application.
Subject(s)
Clinical Chemistry Tests , Data Collection , Reproducibility of ResultsABSTRACT
Background. Early retirement of teachers due to burnout is frequent in Germany. In this study short- and medium-term effects of AFA breathing therapy were evaluated. Methods. This study was designed as a longitudinal controlled intervention design with four points of measurements: before assessment (T1), after intervention (T2), three months (follow up 1) (T3) after intervention, and six months (follow up 2) after intervention (T4). The intervention lasted a total of 11 weeks (weekly group therapy for eight weeks and three weeks of individual breathing session). The effects of intervention were measured with the questionnaire "work-related behaviour and experience Patterns" (AVEM) at four times. Results. In the intervention group 64 teachers and in the self-selected control group 27 teachers were included. The AVEM scales "subjective significance of work" and "professional ambition" changed over time and within both groups (interaction effect). Significant improvements over the four measurements were observed in the intervention group in two AVEM scales: "emotional distancing" (F = 6.3; P < 0.01) and "balance and mental stability" (F = 4.4; P < 0.02). Conclusions. AFA breathing therapy showed short- and medium-term effects in the intervention group over four points of measurements. It may be assumed that breath therapy supports teachers in resisting occupational demand.
ABSTRACT
The 5th GCC in Barcelona (Spain) and 6th GCC in San Antonio (TX, USA) events provided a unique opportunity for CRO leaders to openly share opinions and perspectives, and to agree upon recommendations on biomarker bioanalytical method validation.
Subject(s)
Biological Assay/standards , Biomarkers/analysis , Calibration , Chromatography, High Pressure Liquid/standards , Contract Services/organization & administration , Humans , Tandem Mass Spectrometry/standards , Validation Studies as TopicSubject(s)
Biomarkers/analysis , Biosimilar Pharmaceuticals/analysis , Pharmaceutical Preparations/analysis , Biosimilar Pharmaceuticals/pharmacokinetics , Biosimilar Pharmaceuticals/standards , Chromatography, High Pressure Liquid/standards , Drug Industry , Mass Spectrometry/standards , Pharmaceutical Preparations/standards , Quality Control , Reference StandardsABSTRACT
BACKGROUND: An absolute bioavailability study that utilized an intravenous [(14)C]microdose was conducted for saxagliptin (Onglyza(®)), a marketed drug product for the treatment of Type 2 diabetes mellitus. Concentrations of [(14)C]saxagliptin were determined by accelerator MS (AMS) after protein precipitation, chromatographic separation by UPLC and analyte fraction collection. A series of investigative experiments were conducted to maximize the release of the drug from high-affinity receptors and nonspecific adsorption, and to determine a suitable quantitation range. RESULTS: A technique-appropriate validation demonstrated the accuracy, precision, specificity, stability and recovery of the AMS methodology across the concentration range of 0.025 to 15.0 dpm/ml (disintegration per minute per milliliter), the equivalent of 1.91-1144 pg/ml. Based on the study sample analysis, the mean absolute bioavailability of saxagliptin was 50% in the eight subjects with a CV of 6.6%. Incurred sample reanalysis data fell well within acceptable limits. CONCLUSION: This study demonstrated that the optimized sample pretreatment and chromatographic separation procedures were critical for the successful implementation of an UPLC plus AMS method for [(14)C]saxagliptin. The use of multiple-point standards are useful, particularly during method development and validation, to evaluate and correct for concentration-dependent recovery, if observed, and to monitor and control process loss and operational variations.
Subject(s)
Adamantane/analogs & derivatives , Carbon Radioisotopes/blood , Dipeptides/blood , Dipeptidyl-Peptidase IV Inhibitors/blood , Mass Spectrometry/methods , Adamantane/administration & dosage , Adamantane/blood , Adamantane/pharmacokinetics , Administration, Oral , Biological Availability , Calibration , Chromatography, High Pressure Liquid/methods , Diabetes Mellitus, Type 2/drug therapy , Dipeptides/administration & dosage , Dipeptides/pharmacokinetics , Dipeptidyl-Peptidase IV Inhibitors/administration & dosage , Dipeptidyl-Peptidase IV Inhibitors/pharmacokinetics , Drug Evaluation/methods , Humans , Injections, Intravenous , Male , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A liquid-liquid extraction (LLE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed and validated for the quantification of BMS-790052 (daclastasvir) in human plasma and urine. The samples were extracted with methyl-t-butyl ether (MTBE) before analyzing by an API 4000 mass spectrometer which was operated in a multiple reaction monitoring (MRM) mode for detection of positively charged ions of BMS-790052 and its internal standard, ¹³C10-BMS-790052. The standard curves ranged from 0.050 to 50.0 ng/mL for BMS-790052 in human plasma, and 1.00-1000 ng/mL in human urine. The intra-assay precision (%CV), based on four levels of analytical QCs (low, geometric mean, mid and high), was within 8.6%; inter-assay precision (%CV) was within 6.7% for both plasma and urine methods, and the mean assay accuracy (%Dev) was within ±3.0% for both plasma and urine methods. The ruggedness of this accurate, precise, and selective LLE-LC-MS/MS method has been demonstrated in the successful analysis of several thousand clinical study samples.
Subject(s)
Chromatography, Liquid/methods , Hepatitis C/blood , Hepatitis C/urine , Imidazoles/blood , Imidazoles/urine , Tandem Mass Spectrometry/methods , Viral Nonstructural Proteins/antagonists & inhibitors , Carbamates , Hepacivirus/drug effects , Hepacivirus/genetics , Hepacivirus/metabolism , Humans , Imidazoles/isolation & purification , Liquid-Liquid Extraction , Pyrrolidines , Sensitivity and Specificity , Valine/analogs & derivativesABSTRACT
The Global CRO Council for Bioanalysis (GCC) was formed in September 2010. Since then, the representatives of the member companies come together periodically to openly discuss bioanalysis and the regulatory challenges unique to the outsourcing industry. The 4th GCC Closed Forum brought together experts from bioanalytical CROs to share and discuss recent issues in regulated bioanalysis, such as the impact of coadministered drugs on stability, some differences between European Medicines Agency and US FDA bioanalytical guidance documents and lessons learned following recent Untitled Letters. Recent 483s and agency findings, as well as issues on method carryover, were also part of the topics discussed.
Subject(s)
Chemistry Techniques, Analytical/standards , Guidelines as Topic , Organizations, Nonprofit/standards , United States Food and Drug Administration/standards , Analytic Sample Preparation Methods , Calibration , Chemistry, Pharmaceutical , Documentation , Drug Combinations , Drug Stability , Europe , Reference Standards , Reproducibility of Results , United StatesABSTRACT
A liquid chromatography and tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine the concentrations of saxagliptin (Onglyza™, BMS-477118) and its major active metabolite, 5-hydroxy saxagliptin to support pharmacokinetic analyses in clinical studies. The dynamic range of the assay was 0.1-50 ng/mL for saxagliptin and 0.2-100 ng/mL for 5-hydroxy saxagliptin. Protein precipitation (PPT) with acetonitrile was used to extract the analytes from plasma matrix before injecting on an Atlantis(®) dC18 column (50 mm × 2.1 mm, 5 µm) for LC-MS/MS analysis. The sample pre-treatment process was carefully controlled to disrupt DPP4-specific binding and non-specific binding observed at lower concentrations. The recoveries for both analytes were >90%. The assay was selective, rugged and reproducible; storage stability of at least 401 days at -20°C was demonstrated. Under these chromatographic conditions, the isomers of saxagliptin and 5-hydroxy saxagliptin were chromatographically separated from saxagliptin and 5-hydroxy saxagliptin. The assay has been used to support multiple clinical studies and regulatory approvals.
Subject(s)
Adamantane/analogs & derivatives , Chromatography, Liquid/methods , Dipeptides/blood , Tandem Mass Spectrometry/methods , Adamantane/blood , Adamantane/chemistry , Adamantane/pharmacokinetics , Dipeptides/chemistry , Dipeptides/pharmacokinetics , Drug Stability , Humans , Reproducibility of Results , Sensitivity and Specificity , StereoisomerismABSTRACT
Dalbavancin is a novel second-generation lipoglycopeptide antibiotic with activity against broad range of Gram-positive pathogens. In order to determine the pharmacokinetics (PK) of dalbavancin in pediatric patients, a new High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) bioanalytical method has been developed for quantification of dalbavancin in plasma and in urine. The plasma method was validated for dalbavancin in the linear range from 0.5 µg/mL to 500 µg/mL using 50 µL of K(2) EDTA plasma. For dalbavancin spiked in urine, non-specific binding (NSB) of the drug to polypropylene (PP) urine collection containers was observed. The loss amounted to about 10% per transfer. After successfully establishing the collection/sampling procedure for urine by addition of Triton X-100 to the collection vessels (with a purpose of preventing NSB), the method was validated for dalbavancin in the range from 0.05 µg/mL to 50 µg/mL, using 100 µL of urine. These methods were used to quantify dalbavancin in plasma and urine of hospitalized children in a pediatric dalbavancin PK study. Eighteen percent of the total number of plasma study samples was reassayed for incurred samples reproducibility (ISR) and all the reassayed dalbavancin concentrations were within the ± 20% limits. For urine, all the collected samples were reassayed for ISR and the original dalbavancin concentration was confirmed within the ± 20% limits for 17 (94%) samples; the one remaining urine sample had its reassayed concentration confirmed within ± 25% of the original result.