FDA Bioanalytical Method Validation Research Articles

B-115 Software Data Processing and Review LC-MS/MS Workflow Improvements for Clinical Research

Abstract Background In the field of quantitative LC-MS/MS, the need for efficient, accurate and user-friendly data review software is paramount, with the data generated in the clinical laboratory meeting local guidelines, as well as rigorous method performance characteristics defined by the FDA Bioanalytical Method Validation and CLSI C62-Ed2 guidelines. The quantitative LC-MS/MS workflow is dependent on interfacing to LMS/LIMS for sample traceability, batch analysis using matrix-matched external calibrators and quality controls for quantification and acceptance, and stable-isotope labeled internal standards to compensate for sample extraction, recovery, matrix effects and analytical variability. The demands on the laboratory to run multiplexed analyte panels and analyze a greater number of samples produces large amounts of analytical data which must still go through batch and quality review before release from the laboratory to the clinicians. The Waters Quan Review Software significantly reduces the burdensome aspects of batch quality control by introducing a series of innovative features. These include improvements in workflow, a modernized interface, and the ability to flag exceptions based on acceptance criteria. Methods Samples for three multiplexed LC-MS/MS panels with previously defined analytical method performance characteristics were acquired using Waters IVD Systems controlled by MassLynx software. The data was processed, quantified, and reviewed using both the existing TargetLynx XS IVD Application Manager and the new Quan Review Software. The three quantitative LC-MS/MS methods were the analysis of four immunosuppressants drugs in whole blood, a twelve steroid panel in plasma and a urine dilute and shoot method for a drugs of abuse panel containing greater than 80 analytes. Each method used matrix matched calibrators and QCs for quantification and batch control with performance characteristics including calibration linearity, carryover, precision and accuracy. Results The analytical performance data generated using the new MS Quan data review software met the same performance criteria as the TargetLynx XS data. For immunosuppressants, the % difference of the mean repeatability and total precision (%CV) for the individual analyte QCs processed using both software was within ±1.3% and ±4.4% respectively. The mean % bias of External Quality Assurance Samples (n=40) for each immunosuppressant processed using both software was within ±7.9%. The modernized, task-oriented accelerated workflows and focused review by exception reduced review time by up to 50%. Conclusions The new Quan Review Software generated results equivalent to the existing TargetLynx XS Application Manager. The software’s enhanced workflow capabilities streamlined the review process in a new user-friendly interface which reduced the training burden enabling scientists to generate high-quality data quickly and easily. The exception focused review functionality allowed tailored rule sets which reduced the time spent reviewing data by up to 50%. Whilst the batch-level review and task-oriented workflow improved batch acceptance and simplified the review of complicated datasets into easy tasks. By integrating these features, Data Review software offers a comprehensive solution that not only improves the efficiency and accuracy of batch quality control but also contributes to the overall reliability and integrity of quantitative LC-MS/MS analysis. For Research Use Only. Not for Use in Diagnostic Procedures.

Pharmacokinetic Properties of Baitouweng Decoction in Bama Miniature Pigs: Implications for Clinical Application in Humans.

Traditional Chinese medicine (TCM) serves as a significant adjunct to chemical treatment for chronic diseases. For instance, the administration of Baitouweng decoction (BTWD) has proven effective in the treatment of ulcerative colitis. However, the limited understanding of its pharmacokinetics (PK) has impeded its widespread use. Chinese Bama miniature pigs possess anatomical and physiological similarities to the human body, making them a valuable model for investigating PK properties. Consequently, the identification of PK properties in Bama miniature pigs can provide valuable insights for guiding the clinical application of BTWD in humans. To facilitate this research, a rapid and sensitive UPLC-MS/MS method has been developed for the simultaneous quantification of eleven active ingredients of BTWD in plasma. Chromatographic separation was conducted using an Acquity UPLC HSS T3 C18 column and a gradient mobile phase comprising acetonitrile and water (containing 0.1% acetic acid). The methodology was validated in accordance with the FDA Bioanalytical Method Validation Guidance for Industry. The lower limit of quantitation fell within the range of 0.60-2.01 ng/mL. Pharmacokinetic studies indicated that coptisine chloride, berberine, columbamine, phellodendrine, and obacunone exhibited low Cmax, while fraxetin, esculin, fraxin, and pulchinenoside B4 were rapidly absorbed and eliminated from the plasma. These findings have implications for the development of effective components in BTWD and the adjustment of clinical dosage regimens.

A high-throughput UPLC-MS-MS Bio-analytical method for the analysis of veterinary pharmaceutical residues in Chicken Tissues, Application of efficient-valid-green (EVG) Framework as a Competence Tool

Antibacterial medications are receiving the most attention due to hypersensitivity reactions and the emergence of bacterial mutants resistant to antibiotics. Treating Animals with uncontrolled amounts of antibiotics will extend beyond their lives and affect humans. This study aims to determine the concentration of the residues of sulfadimidine, sulfaquinoxaline, diaveridine, and vitamin K3 in the tissues of poultry (muscles and liver) after treatment with the combined veterinary formulation. A UPLC-MS-MS method was developed using Poroshell 120 ECC18 and a mobile phase composed of acetonitrile and distilled water, containing 0.1 % formic acid, in the ratio of (85:15 v/v) at a flow rate of 0.6 mL/min. Sample extraction solvent was optimized using response surface methodology (RSM) to be acetonitrile: methanol in the ratio (49.8: 50.2 v/v), and the method was validated according to the FDA bioanalytical method validation protocol over the range (50–1000 µg/Kg) for sulfaquinoxaline and (50–750 µg/Kg) for the other 3 drugs. The greenness of the sample preparation and analytical method was assessed by applying Analytical Eco-scale (AES) and AGREE coupled with AGREEprep. The Competence of the study was evaluated via the EVG framework known as Efficiency, validation, and greenness, to achieve a balance point represented by a radar chart. The method was applied to decide the time required for poultry products to be safe for human use after administration of the studied drugs. It was found that, after the administration of the last dose, minimally 7 days are required till the levels of the drugs drop to the maximum residue limit determined by the FDA/WHO in animal tissues.

A simple and green capillary electrophoresis-mass spectrometry method for therapeutic drug monitoring of colistin in clinical plasma samples

Colistin and other polymyxin antibiotics have become increasingly used in clinical settings as a result of treating multidrug-resistant infections in critically ill patients. The highly variable pharmacokinetics of colistin in these patients is accompanied by a high risk of toxicity or underdosing. An effective tool that allows rational optimization of the drug dosage regimen is therapeutic drug monitoring. Therefore, there is a need to dispose with appropriate, sensitive, and accurate analytical methods. Here, a simple, specific, and accurate on-line capillary electrophoresis – tandem mass spectrometry method was developed and applied for the first time to determine colistin in human plasma. Protein precipitation using acidified acetonitrile was the solitary procedure used to achieve sample pretreatment. A bare fused silica capillary was employed for the separation process, and the background electrolyte used was 50 mM formic acid (pH 2.54). The FDA's bioanalytical method validation guidelines were followed in the validation of the proposed method. For colistin A and colistin B, favorable performance and validation parameters were obtained (such as linearity, limit of detection, lower limit of quantitation, intra-day and inter-day precision, accuracy, and stability).The validated method was then effectively used to analyze real clinical samples taken from patients who were in critical condition. Our newly developed method is comparable with previously published liquid chromatography approaches and has the potential to be applied in the therapeutic monitoring of colistin in routine clinical laboratories. Moreover, according to the greenness assessment, the developed capillary electrophoresis – mass spectrometry method represents a very interesting green and sustainable tool in the field of bioanalysis.

HPLC for simultaneous quantification of free mannose and glucose concentrations in serum: use in detection of ovarian cancer.

Background: Abnormal levels of monosaccharides in blood have been linked to tumorigenesis. In this study, a novel high-performance liquid chromatography (HPLC) method was established for the simultaneous determination of free mannose and glucose in the serum. Methods: The serum was directly derivatized by 1-phenyl-3-methyl-5-pyrazolone under alkaline conditions using L-rhamnose as an internal standard. The chromatographic separation was then performed on a Poroshell EC-C18 chromatographic column (4.6 × 100 mm, particle size 2.7μm, Agilent) with gradient elution using NH4Ac-HAc and acetonitrile as the mobile phases. The method was thereafter validated according to international guidelines. The serum samples obtained from 200 healthy individuals and 200 ovarian cancer (OC) patients were analyzed for free mannose and glucose. Results: The method was found to be reproducible for quantification within 20min and included online sample purification. The method displayed excellent linearity in the concentration range (for mannose: 0.5-500μg/mL; glucose: 0.5-1500μg/mL). The precision, recovery, and stability met the FDA bioanalytical method validation acceptance criteria. Overall, the measurement of glucose content by HPLC correlated well with the different enzymatic methods. Ovarian cancer mannose levels in the serum were significantly higher in the advanced stage (61.22μmol/L, p < 0.0001) than those in healthy volunteers and early-stage patients (44.51μmol/L versus 50.09μmol/L, p < 0.0001). The AUC for the ratio of serum free glucose to mannose (G/M) was 0.98 (p < 0.0001), with a sensitivity of 91.46% and a specificity of 98.50%, which served as a biomarker for OC diagnosis. Conclusion: We report a simple, repeatable, and attractive analytical method by HPLC, which can be used for quantitative estimation of free mannose and glucose simultaneously in human serum. Our results indicate that the serum level of mannose could be used as a potential biomarker of ovarian cancer.

The solvent- and surface-dependent adsorption of the lipopeptide antibiotic daptomycin: The general necessity of adsorption tests

The impact of poor or non-reproducible analyte recoveries due to non-specific drug adsorption on various analytical assays is often underestimated. Even internationally approved guidelines for pharmaceutical analysis such as the EMA guideline on bioanalytical method validation, the ICH guideline M10 on bioanalytical method validation and study sample analysis or the FDA bioanalytical method validation guidance do not adequately encourage more detailed investigations. Furthermore, other areas of research in which the concentration of active pharmaceutical compounds plays a crucial role, for example screening for minimal inhibitory concentrations of bacterial isolates, are potentially affected as well. The aim of this study was to demonstrate the general necessity of drug adsorption tests, using the lipopeptide antibiotic daptomycin as an example. A wide range of typical materials used in processing samples in pharmaceutical and biological analysis, as well as various solvents and biological matrices were included in the experiments. A fully validated LC-MS/MS method was applied for the determination of daptomycin concentrations, which were subsequently used to calculate the recovery. Recovery results (n = 3) ranged from 0.00% to 102.12% with a maximum relative standard deviation of 12.78%. These findings demonstrate that recovery can vary greatly depending on the solvent and the contact material, indicating the need to be optimized and, if applicable, validated. Hence, high reproducibility can only be achieved if all materials (and their manufacturers) used in a method are specified, not just those used in steps considered critical.

A Fast LC-MS/MS Methodology for Estimating Savolitinib in Human Liver Microsomes: Assessment of Metabolic Stability Using In Vitro Metabolic Incubation and In Silico Software Analysis

Savolitinib (Orpathys®), was developed by (HUTCHMED (Shanghai, China) and, AstraZeneca (Gaithersburg, Maryland, USA), is an inhibitor of the c-Met receptor tyrosine kinase that is orally bioavailable. It was designed for the treatment of pillary and clear-cell renal-cell carcinoma (RCC), colorectal cancer, gastric cancer, and metastatic non-small-cell lung cancer (NSCLC). The current work aimed to develop a rapid, specific, green, and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) methodology for estimating savolitinib (SVB) in human liver microsomes (HLMs) with application to an in vitro metabolic stability assessment of SVB in HLMs. The validation steps of the current LC-MS/MS methodology in the HLMs were carried out following US FDA bioanalytical method validation guidelines including sensitivity, selectivity, linearity, accuracy, stability, precision, extraction recovery, and matrix effect. SVB and olmutinib (OLM) were chromatographically separated on an Eclipse Plus C8 column using an isocratic mobile phase. SVB parent ions were generated using the positive mode of an electrospray ionization (ESI) source. SVB daughter ions were detected and quantified using the multiple reaction monitoring (MRM) mode of a triple quadrupole mass analyser. The constructed SVB calibration curve showed linearity over the range from 1 to 3000 ng/mL. The interday and intraday accuracy and precision of the developed LC-MS/MS analytical methodology were −6.67%–4.11% and −0.51%–8.75%, respectively. A lower limit of quantification (LLOQ) of 0.87 ng/mL confirmed the sensitivity of the established method. Furthermore, the eco-scale methodology using the in silico AGREE software was used for the greenness assessment of the current LC-MS/MS method, and the outcomes showed that the established method was very eco-friendly. The intrinsic clearance (Clint) and in vitro half-life (t1/2) of SVB were 33.05 mL/min/kg and 24.54 min, respectively. SVB exhibited a moderate extraction ratio. The current study is the first to establish and validate LC-MS/MS for estimating SVB and assessing the metabolic stability of SVB.

Development and validation of a novel HPLC-UV method for simultaneous determination of azathioprine metabolites in human red blood cells

A rapid, specific and accurate high-performance liquid chromatography with tunable ultraviolet detection method was developed to simultaneously determine azathioprine metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methyl mercaptopurine riboside (6-MMPr) in human red blood cells. Erythrocyte lysate sample was precipitated by perchloric acid under the protection of dithiothreitol, with 6-TGN and 6-MMPr being acid hydrolyzed to produce 6-thioguanine (6-TG) and 6-methymercaptopurine (6-MMP). A Waters Cortecs C18 column (2.1 × 150 mm, 2.7 μm) was used for chromatographic separation with a water (containing 0.01 mol/L ammonium acetate and 0.2% acetic acid)/methanol linear gradient at a flow rate of 0.45 mL/min in a 5.5 min. UV detection wavelengths were 340 nm for 6-TG, 303 nm for 6-MMP and the IS (5-bromouracil). The calibration curves fitted a least squares model (weighed 1/x2) from 0.15 to 15 μmol/L for 6-TG (r2 = 0.9999) and from 1 to 100 μmol/L for 6-MMP (r2 = 0.9998). This method was validated according to the FDA bioanalytical method validation guidance and ICH M10 bioanalytical method validation and study sample analysis guidance for industry, and successfully utilized in ten IBD patients receiving azathioprine therapy.

A Bioanalytical Method for Quantification of N-nitrosodimethylamine (NDMA) in Human Plasma and Urine with Different Meals and following Administration of Ranitidine

Control of N-nitrosoamine impurities is important for ensuring the safety of drug products. Findings of nitrosamine impurities in some drug products led FDA to develop new guidance providing recommendations for manufacturers towards prevention and detection of nitrosamine impurities in pharmaceutical products. One of these products, ranitidine, also had a published in vivo study, which has since been retracted by its authors, suggesting a potential for in vivo conversion of ranitidine to the probable human carcinogen, N-nitrosodimethylamine (NDMA). FDA subsequently initiated a randomized, double-blind, placebo-controlled, crossover clinical investigation to assess the potential for in vivo conversion of ranitidine to NDMA with different meals. A bioanalytical method toward characterization of NDMA formation was needed as previously published methods did not address potential NDMA formation after biofluid collection. Therefore, a bioanalytical method was developed and validated as per FDA's Bioanalytical Method Validation guidance. An appropriate surrogate matrix for calibration standards and quality control sample preparation for both liquid matrices (human plasma and urine) was optimized to minimize the artifacts of assay measurements and monitor basal NDMA levels. Interconversion potential of ranitidine to NDMA was monitored during method validation by incorporating the appropriate quality control samples. The validated methods for NDMA were linear from 15.6 pg/mL to 2000 pg/mL. Low sample volumes (2 mL for urine and 1 mL for plasma) made this method suitable for clinical study samples and helped to evaluate the influence of ranitidine administration and meal types on urinary excretion of NDMA in human subjects.

Determination of five positive control drugs in hERG external solution (buffer) by LC-MS/MS to support in vitro hERG assay as recommended by ICH S7B

ICH S7B recommends screening for hERG channel block using patch clamp recordings to assess a drug's proarrhythmic risk. Block of the hERG channel has been associated with clinical QTC prolongation as well as the rare, but potentially fatal ventricular tachyarrhythmia Torsade de Pointes (TdP). During recording, drug concentrations perfused to the cells can deviate from nominal concentrations due to molecule-specific properties (such as non-specific binding), thereby introducing error when assessing drug potency. To account for this potential source of error, both the original ICH S7B and the newly released ICH E14/S7B Q&As guidelines call for verifying drug solutions' concentrations. Dofetilide, cisapride, terfenadine, sotalol and E-4031 are hERG blockers commonly used as positive controls to illustrate hERG assay sensitivity. The first four compounds are also clinical drugs associated with high TdP risk; therefore, their safety margins may be useful comparators to better understand an investigational product's TdP risk. Having analytical methods to quantify these five compounds in the hERG external solution that will be used for patch clamp recordings is important from a regulatory science research perspective. However, a literature search revealed no analytical methods or stability information for these molecules in the high salt, serum-free matrix that constitutes the hERG external solution. This study was conducted to develop and validate LC-MS/MS methods to quantify these 5 molecules in hERG external solution. The bioanalytical methods for these positive controls were validated as per the FDA's bioanalytical method validation guidance along with various stabilities.

Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes

A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.

Analysis of 10 urinary BTEX metabolites using LC–MS/MS

Benzene, toluene, ethylbenzene, and xylene (BTEX) are a group of volatile organic compounds that are ubiquitous in the environment due to numerous anthropogenic sources. Exposure to BTEX poses a health hazard by increasing the risk for damage to multiple organs, neurocognitive impairment and birth defects. Urinary BTEX metabolites are useful biomarkers for the evaluation of BTEX exposure, because of the ease of sampling and their longer physiological half-lives compared with parent compounds. A method that utilizes LC-MS/MS was developed and validated for simultaneously monitoring of 10 urinary BTEX metabolites. During the sample preparation an aliquot of urine was diluted with an equal volume of 1% formic acid; internal standard solution was added, and then the sample was centrifuged and analyzed. The analytes were separated on the Kinetex-F5 column by applying a linear gradient, consisting of 0.1% formic acid and methanol. The method was validated according to the FDA Bioanalytical Method Validation Guidance for Industry. The mean method's accuracies of the spiked matrix were 81-122%; the inter-day precision ranged from 4 to 20%; the limits of quantitation were 0.5-2μg/L. The method was used for the evaluation of baseline levels of urinary BTEX metabolites in 87 firefighters.

Instability of Efavirenz Metabolites Identified During Method Development and Validation.

Accurate quantification of efavirenz metabolites in patient samples is required to investigate their potential contribution to efavirenz adverse events. This study aimed to validate a LC-MS/MS method to quantify and investigate the stability of efavirenz and metabolites in human plasma. Compounds were extracted from plasma by supported liquid extraction and resolved on a C18 column. Validation was performed following FDA bioanalytical method validation guidelines. Stability under common conditions of sample pre-treatment and storage were assessed. Efavirenz and 8-hydroxyefavirenz were stable for all conditions tested. 7-Hydroxyefavirenz and 8,14-dihydroxyefavirenz were not stable in plasma at room temperature for 24 h (46%-69% loss), -20°C for 90 days (17%-50% loss), or 60°C for 1 h (90%-95% loss). Efavirenz and 8-hydroxyefavirenz concentrations in HIV/AIDS patient (n=5) plasma prepared from pre-treated (60°C for 1 h) whole blood varied from 517-8564 ng/mL and 131-813 ng/mL, respectively. 7-Hydroxyefavirenz and 8,14-dihydroxyefavirenz concentrations were below validated lower limits of quantification (0.25 and 0.5 ng/mL, respectively), most likely due to sample pre-treatment. This is the first report of 7-hydroxyefavirenz and 8,14-dihydroxyefavirenz instability under conditions commonly used in preparation of samples from HIV/AIDS patients. Alternative biosafety measures to heat pre-treatment must therefore be used for accurate quantification of plasma 7-hydroxyefavirenz and 8,14-dihydroxyefavirenz.

Development and validation of assays for the quantification of β-D-N4-hydroxycytidine in human plasma and β-D-N4-hydroxycytidine-triphosphate in peripheral blood mononuclear cell lysates

The novel antiviral prodrug molnupiravir is under evaluation for the treatment of SARS-CoV-2. Molnupiravir is converted to β-D-N4-hydroxycytidine (NHC), which is the primary form found in systemic circulation. β-D-N4-hydroxycytidine-triphosphate (NHCtp) is the bioactive anabolite produced intracellularly. Sensitive and accurate bioanalytical methods are required to characterize NHC and NHCtp pharmacokinetics in clinical trials. Human K2EDTA plasma or peripheral blood mononuclear cell (PBMC) lysates were spiked with NHC (plasma) or NHCtp (PBMC), respectively. Following the addition of isotopically-labeled internal standards and sample extraction via protein precipitation or lysate dilution, respectively, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Methods were validated in accordance with FDA Bioanalytical Method Validation recommendations. NHC can be quantified in plasma with a lower limit of quantification (LLOQ) of 1 ng/mL; the primary linearity of the assay is 1–5000 ng/mL. Assay precision and accuracy were ≤ 6.40% and ≤ ± 6.37%, respectively. NHC is unstable in whole blood and has limited stability in plasma at room temperature. The calibration range for NHCtp in PBMC lysates is 1–1500 pmol/sample, and the assay has an LLOQ of 1 pmol/sample. Assay precision and accuracy were ≤ 11.8% and ≤± 11.2%. Ion suppression was observed for both analytes; isotopically-labeled internal standards showed comparable ion suppression, resulting in negligible (<5%) relative matrix effects. Sensitive, specific, and dynamic LC-MS/MS assays have been developed and validated for the quantification of NHC in plasma and NHCtp in PBMC lysates. The described methods are appropriate for use in clinical trials.

Simultaneous quantification of dexamethasone and 6β-hydroxydexamethasone in rabbit plasma, aqueous and vitreous humor, and retina by UHPLC-MS/MS.

Aim: To develop and validate a fit for purpose method for the simultaneous determination of dexamethasone and its major metabolite, 6β-hydroxydexamethasone, in rabbit plasma and ocular matrices to measure the in vivo release and distribution profile of dexamethasone from intravitreal implants. Materials & methods: An UHPLC-MS/MS system was employed to perform the bioanalysis. The method was validated according to the USFDA Bioanalytical Method Validation Guidance for Industry. Results & conclusion: The method was found to be fit-for-purpose for the described biological matrices and had a LLOQ of 0.1ng/ml.

Novel simultaneous method for the determination of avobenzone and oxybenzone in human plasma by UHPLC-MS/MS with phospholipid removal pretreatment: An application to a sunscreen clinical trial

Application of sunscreen is one of many ways to protect skin from the harmful effects of UV radiation. Sunscreen products are widely used and regulated as over-the-counter drug products in the United States. The U.S. Food and Drug Administration recommends an assessment of human systemic absorption of sunscreen active ingredients with a Maximal Usage Trial. The FDA conducted a clinical study to determine the systemic exposure of sunscreen active ingredients present in 4 commercially available sunscreen products of different formulation types under maximal usage conditions. To support this clinical study, a sensitive and specific LC-MS/MS method for the simultaneous determination of the two sunscreens avobenzone and oxybenzone in human plasma was developed. Phospholipid removal 96-well protein precipitation plates were used for sample clean-up and the extracted samples were chromatographed on an Ethylene-Bridged Hybrid (BEH) C18 column in isocratic flow using 10 mM ammonium formate in 0.1% formic acid and methanol (24:76, v/v) as a mobile phase. A triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode was used to acquire data. The method was validated as per current FDA bioanalytical method validation guidance, in the ranges 0.20–12.00 ng/mL for avobenzone and 0.40–300.00 ng/mL for oxybenzone. The validated method was used toanalyzethese active ingredients in human clinical study samples.

Determination of obeticholic acid and its glycine conjugate in human plasma using liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatography/tandem mass spectrometry method was recommended by the authors for the determination orally bioavailable farnesoid X receptor agonist obeticholic acid along with its glycine conjugate glycoobeticholic acid human plasma. Obeticholic acid_d5 and glycoobeticholic acid_d5 were used as internal standards, respectively. After extraction with a mixture of tert-butyl methyl ether and dichloromethane, samples were separated on a phenyl column with a mobile phase of 2mM ammonium acetate with 0.01% formic acid and methanol (15:85, v/v). Analysis was performed on an AB Sciex 4500 triple quadrupole mass spectrometer and data acquisition was performed by multiple reaction monitoring (MRM) in the negative ionization mode. Obeticholic acid and its conjugate were quantified in the linearity range of 0.50-100.00 ng/mL and the correction coefficients were≥0.99 during the validation. Precision and accuracy in different days (inter-day) and single day (intra-day) were meeting the acceptance criteria specified in the recent US FDA bioanalytical method validation guidelines. A variety of stability experiments in neat samples and plasma samples were conducted and the results are meeting the acceptance criteria. A run time of 2.5 min for each sample made it possible to analyze a greater number of samples in a short time, thus increasing productivity. This method could be useful to quantitate obeticholic acid and glycoobeticholic acid in real clinical samples.

Determination of CHLORPYRIFOS by GC/ECD in water and its sorption mechanism study in a RHODIC FERRALSOL.

The validation of chromatographic methods is a costly process, however necessary, especially with regard to the validation of methods that accurately determine concentrations of pesticides in different environmental compartments. This research aimed at the development and validation of a simple and fast method for the determination of chlorpyrifos concentrations in water by means of a gas chromatograph with electron capture detection (GC/ECD), and to investigate chlorpyrifos dynamics of adsorption in a Rhodic Ferralsol in Southern Brazilian conditions. The developed chromatographic method was based in EPA 8141 method. Parameters to be checked for method validation were: Selectivity/specificity, linearity, precision, accuracy, robustness, limit of detection (LOD) and limit of quantitation (LOQ). Were employed the following methodologies for the validation process: ANVISA Resolution 899, DOQ-CGCRE-008 and FDA Bioanalytical Method Validation Guide. Also, through laboratory tests, the sorption dynamics of chlorpyrifos in Rhodic Ferralsol was evaluated. Thus, the soil was contaminated with increasing concentrations of chlorpyrifos, which were subjected to solid-liquid extraction with SPE cartridge Chromabond® C18 ec. The obtained results were submitted to the models of Langmuir, Freundlich, Dubinin-Radushkevich and Sips. By this method, chlorpyrifos peaks are obtained at 16.9min, demonstrating practicality and low cost. This method exhibits precision and sensitivity, with satisfactory LQ and LQ values. The models of Langmuir, Freundlich, Dubinin-Radushkevich and Sips suggest the occurrence of simultaneous adsorption in mono and multilayer of chlorpyrifos in Rhodic Ferralsol colloids, as well as the predominance of a chemical, high energy binding process (irreversible). However, the chemisorption of chlorpyrifos is more related to the good fit found for Dubinin-Radushkevich sorption energy values (9.861 and 11.079 KJ mol-1) and Qm values estimated by Langmuir (485.55 and 389.61μgg-1 for linear and nonlinear model).

The Effects of Special Patient Population Plasma on Pharmacokinetic Quantifications Using LC-MS/MS.

Clinical development of lesinurad, a selective uric acid reabsorption inhibitor, required analysis of lesinurad in plasma from special patient populations. EMA and FDA bioanalytical method validation guidance have recommended studying matrix effects on quantitation if samples from special patient populations are to be analyzed. In addition to lesinurad (plasma protein binding 98.2%), the matrix effects from special population plasma on the quantitation of verapamil (PPB 89.6%), allopurinol and oxypurinol (PPB negligible) were also investigated. The plasma from special population patients had no matrix effects on the three quantification methods with stable isotope labeled internal standard, protein precipitation extraction, and LC-MS/MS detection. The validated lesinurad plasma quantification method was successfully applied for the pharmacokinetic evaluations to support the clinical studies in renal impaired patients. Special population plasma did not affect quantitation of drugs with a wide range of plasma protein binding levels in human plasma. With the confirmation that there is no impact on quantification from the matrix, the bioanalytical method can be used to support the pharmacokinetic evaluations for clinical studies in special populations.

Current Perspectives on Ligand-Binding Assay Practices in the Quantification of Circulating Therapeutic Proteins for Biosimilar Biological Product Development.

Bioanalysis in biosimilar biological product development (BPD) plays a critical role in demonstrating pharmacokinetic (PK) similarity across products. The 2018 FDA Bioanalytical Method Validation guidance for industry provides general principles in the development, validation, and conduct of bioanalytical assays. Given that the PK similarity assessment in BPD programs involves two or more non-identical products, there are additional considerations for bioanalytical methods. Here in, we provide our perspectives on the definition of (1) a single bioanalytical method in the context of BPD in supporting a PK similarity study, (2) bioanalytical method comparability during accuracy and precision experiments to determine the potential bias difference prior to assessing other validation parameters, and (3) bioanalytical method validations that support PK similarity assessments.