Application of immunocapture and nanoflow LC-MS/MS to overcome the barriers to the quantitation of oxytocin in plasma of women in third stage labour.

AIMS: Postpartum haemorrhage (PPH) is the leading cause of maternal mortality worldwide. To prevent PPH, the WHO recommends administration of oxytocin (OT) immediately after birth, i.e. during the third stage of labour (TSL). Previous studies demonstrate that methods to quantify OT in biological matrices, e.g. enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) lack the specificity and/or sensitivity to accurately quantify OT in plasma from women administered OT during TSL. This is due to increased metabolic clearance of OT in late-stage pregnancy and at the time of childbirth, resulting in extremely low OT plasma concentrations. This study describes the development of an ultra-sensitive bioanalytical method that overcomes the issues previously reported and enables accurate pharmacokinetic analyses of exogenously administered OT in TSL. METHODS: A selective and sensitive assay to quantify OT in TSL plasma was developed. Immunoprecipitation (IP) was applied to selectively extract OT from the TSL plasma, thereby generating clean extracts compatible with nanoflow LC (nLC). nLC-MS/MS was chosen for its high sensitivity and ability to differentiate between OT and potentially co-captured OT-like immunoreactive products. RESULTS: The presented methodology is accurate and precise, with a good linear fit between 100-10 000 fg mL-1 OT. TSL plasma samples from a clinical phase 1 study (NCT02999100) were analysed successfully, enabling OT quantification down to 100 fg mL-1. CONCLUSIONS: The presented IP-nLC-MS/MS method succeeded in overcoming the sensitivity challenge related to the assay of OT in TSL plasma and thereby revealing the PK profiles of OT in TSL plasma clinical study samples.

IP-LC-MSMS Enables Identification of Three Tau O-GlcNAcylation Sites as O-GlcNAcase Inhibition Pharmacodynamic Readout in Transgenic Mice Overexpressing Human Tau.

O-ß-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates tau phosphorylation and aggregation: the pharmacological increase of tau O-GlcNAcylation upon treatment with inhibitors of O-GlcNAc hydrolase (OGA) constitutes a potential strategy to tackle neurodegenerative diseases. Analysis of tau O-GlcNAcylation could potentially be used as a pharmacodynamic biomarker both in preclinical and clinical studies. The goal of the current study was to confirm tau O-GlcNAcylation at S400 as a pharmacodynamic readout of OGA inhibition in P301S transgenic mice overexpressing human tau and treated with the OGA inhibitor Thiamet G and to explore if additional O-GlcNAcylation sites on tau could be identified. As a first step, an immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC-MS) methodology was developed to monitor changes in O-GlcNAcylation around S400 of tau in mouse brain homogenate (BH) extracts. Second, additional O-GlcNAc sites were identified in in-house produced recombinant O-GlcNAcylated human tau at relatively high concentrations, thereby facilitating collection of informative LC-MS data for identification of low-concentration O-GlcNAc-tryptic tau peptides in human transgenic mouse BH extracts. This strategy enabled, for the first time, identification of three low abundant N-terminal and mid-domain O-GlcNAc sites of tau (at S208, S191, and S184 or S185) in human transgenic mouse BH. Data are openly available at data.mendeley.com (doi: 10.17632/jp57yk9469.1; doi: 10.17632/8n5j45dnd8.1; doi: 10.17632/h5vdrx4n3d.1).

Therapeutic siRNA Loaded to RISC as Single and Double Strands Requires an Appropriate Quantitative Assay for RISC PK Assessment.

In recent years, therapeutic siRNA projects are booming in the biotech and pharmaceutical industries. As these drugs act by silencing the target gene expression, a critical step is the binding of antisense strands of siRNA to RNA-induced silencing complex (RISC) and then degrading their target mRNA. However, data that we recently obtained suggest that double-stranded siRNA can also load to RISC. This brings a new understanding of the mechanism of RISC loading which may have a potential impact on how quantification of RISC loaded siRNA should be performed. By combining RNA immune precipitation and probe-based hybridization LC-fluorescence approach, we have developed a novel assay that can accurately quantify the RISC-bound antisense strand, irrespective of which form (double-stranded or single-stranded) is loaded on RISC. In addition, this novel assay can discriminate between the 5'-phosphorylated antisense (5'p-AS) and the nonphosphorylated forms, therefore specifically quantifying the RISC bound 5'p-AS. In comparison, stem-loop qPCR assay does not provide discrimination and accurate quantification when the oligonucleotide analyte exists as a mixture of double and single-stranded forms. Taking together, RISC loading assay with probe-hybridization LC-fluorescence technique would be a more accurate and specific quantitative approach for RISC-associated pharmacokinetic assessment.

Absolute quantitation of binding antibodies from clinical samples.

The quantitation of antibody responses is a critical requirement for the successful development of vaccines and therapeutics that often relies on the use of standardized reference materials to determine relative quantities within biological samples. The validity of comparing responses across assays using arbitrarily defined reference values is therefore limited. We developed a generalizable method known as MASCALE (Mass Spectrometry Enabled Conversion to Absolute Levels of ELISA Antibodies) for absolute quantitation of antibodies by calibrating ELISA reference sera using mass spectrometry. Levels of proteotypic peptides served as a proxy for human IgG, allowing the conversion of responses from arbitrary values to absolute amounts. Applications include comparison of binding assays at two separate laboratories and evaluation of cross-clade magnitude-breadth responses induced by an investigational HIV-1 vaccine regimen. MASCALE addresses current challenges in the interpretation of immune responses in clinical trials and expands current options available to make suitable comparisons across different settings.

Pharmacokinetics and pharmacodynamics of TMC207 and its N-desmethyl metabolite in a murine model of tuberculosis.

TMC207 is a first-in-class diarylquinoline with a new mode of action against mycobacteria targeting the ATP synthase. It is metabolized to an active derivative, N-desmethyl TMC207, and both compounds are eliminated with long terminal half-lives (50 to 60 h in mice) reflecting slow release from tissues such as lung and spleen. In vitro, TMC207 is 5-fold more potent against Mycobacterium tuberculosis than N-desmethyl TMC207, and the effects of the two compounds are additive. The pharmacokinetic and pharmacodynamic (PK-PD) response was investigated in the murine model of tuberculosis (TB) infection following oral administration of different doses of TMC207 or N-desmethyl TMC207 at 5 days per week for 4 weeks starting the day after intravenous infection with M. tuberculosis and following administration of different doses of TMC207 at various dosing frequencies for 6 weeks starting 2 weeks after infection. Upon administration of N-desmethyl TMC207, maximum plasma concentration (C(max)), area under the plasma concentration-time curve from time zero to 168 h postdose (AUC(168h)), and minimum plasma concentration (C(min)) were approximately dose proportional between 8 and 64 mg/kg, and the lung CFU counts were strongly correlated with these pharmacokinetic parameters using an inhibitory sigmoid maximum effect (E(max)) model. Administration of the highest dose (64 mg/kg) produced a 4.0-log(10) reduction of the bacillary load at an average exposure (average concentration [C(avg)] or AUC(168h) divided by 168) of 2.7 µg/ml. Upon administration of the highest dose of TMC207 (50 mg/kg) 5 days per week for 4 weeks, the total reduction of the bacillary load was 4.7 log(10). TMC207 was estimated to contribute to a 1.8-log(10) reduction and its corresponding exposure (C(avg)) was 0.5 µg/ml. Optimal bactericidal activity with N-desmethyl TMC207 was reached at a high exposure compared to that achieved in humans, suggesting a minor contribution of the metabolite to the overall bactericidal activity in TB-infected patients treated with TMC207. Following administration of TMC207 at a total weekly dose of 15, 30, or 60 mg/kg fractionated for either 5 days per week, twice weekly, or once weekly, the bactericidal activity was correlated to the total weekly dose and was not influenced by the frequency of administration. Exposures (AUC(168h)) to TMC207 and N-desmethyl TMC207 mirrored this dose response, indicating that the bactericidal activity of TMC207 is concentration dependent and that AUC is the main PK-PD driver on which dose optimization should be based for dosing frequencies up to once weekly. The PK-PD profile supports intermittent administration of TMC207, in agreement with its slow release from tissues.

2021 White Paper on Recent Issues in Bioanalysis: Mass Spec of Proteins, Extracellular Vesicles, CRISPR, Chiral Assays, Oligos; Nanomedicines Bioanalysis; ICH M10 Section 7.1; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A - Recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC & Part 1B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

Evaluation of micropillar array columns for chromatographic separation of phosphorothioated oligonucleotides and their diastereomers.

Chromatographic analysis of therapeutic oligonucleotides is challenging due to the presence of closely related impurities, degradants or metabolites and due to the presence of phosphorothioate bonds, which introduce chiral centers. In the present study, ion pair reversed phase chromatography of oligonucleotides on micropillar array columns was investigated. Two commonly used mobile phase conditions were included. With 16.3 mM triethylamine and 400 mM hexafluoroisopropanol, the separation of 16-mer oligonucleotides differing in the number and positions of phosphorothioate linkages as well as some n-1 and n-2 truncations demonstrated complete suppression of diastereoselectivity. Although the positional phosphorothioate isomers evaluated could not be resolved, an increase in phosphorothioate bonds resulted in more retention. A therapeutic 19-mer RNA sequence with 2'-fluor and 2'-O-methyl modifications showed partial separation of some very close impurities. When using 15 mM triethyl ammonium acetate in the mobile phase, diastereomer selectivity was clearly observed for all analytes. The best result was obtained for the 19-mer RNA therapeutic mimic with four phosphorothioate bonds, since all 16 theoretical diastereomers were clearly observed under the conditions tested. A limited benchmark exercise demonstrated the improved capability of the new micropillar array columns. Therefore, these columns can be positioned as a valuable alternative when challenging oligonucleotide separations are expected.

Development of immunoprecipitation - two-dimensional liquid chromatography - mass spectrometry methodology as biomarker read-out to quantify phosphorylated tau in cerebrospinal fluid from Alzheimer disease patients.

In Alzheimer's disease (AD) brain, one of the histopathological hallmarks is the neurofibrillary tangles consisting of aggregated and hyperphosphorylated tau. Currently many tau binding antibodies are under development to target the extracellular species responsible for the spreading of the disease in the brain. As such, an in-house developed antibody JNJ-63733657 with picomolar affinity towards tau phosphorylated at both T212 and T217 (further named p217+tau) was recently tested in phase I clinical trial NCT03375697. Following multiple dose administration in healthy subjects and subjects with AD, there were dose dependant reductions in free p217+tau fragments in cerebrospinal fluid (CSF) following antibody administration, as measured with a novel single molecule ELISA assay (Simoa PT3 x PT82 assay), demonstrating epitope engagement of the therapeutic antibody [Galpern, Haeverans, Janssens, Triana-Baltzer, Kolb, Li, Nandy, Mercken, Van Kolen, Sun, Van Nueten, 2020]. Total p217+tau levels also were reduced in CSF as measured with the Simoa PT3 x PT82 assay. In this study we developed an orthogonal immunoprecipitation - liquid chromatography - triple quadrupole mass spectrometry (IP-LC-TQMS) assay to verify the observed reductions in total p217+ tau levels. In this assay, an excess of JNJ-63733657 is added to the clinical CSF to ensure all p217+tau is bound by the antibody instead of having a pool of bound and unbound antigen and to immunoprecipitate all p217+tau, which is followed by on-bead digestion with trypsin to release surrogate peptides. Tryptic peptides with missed cleavages were monitored when phosphorylation occurred close to the cleavage site as this induced miscleavages. Compared with acidified mobile phases typically used for peptide analysis, reversed phase LC with mobile phase at basic pH resulted in sharper peaks and improved selectivity and sensitivity for the target peptides. With this setup a diphospho-tau tryptic peptide SRTPSLPTPPTREPK*2 could be measured with pT217 accounting for at least one of the phospho-sites. This is the first time that the presence of a diphopsho-tau peptide is reported to be present in human CSF. A two-dimensional LC-TQMS method was developed to remove matrix interferences. Selective trapping of diphospho-peptides via a metal oxide chromatography mechanism was achieved in a first dimension with a conventional reversed phase stationary phase and acidified mobile phase. Subsequent elution at basic pH enabled detection of low picomolar p217+tau levels in human CSF (lower limit of quantification: 2 pM), resulting in an approximate 5-fold increase in sensitivity. This enabled the quantification of total p217+tau in CSF leading to the confirmation that in addition to reductions in free p217+tau levels total p217+tau levels were also reduced following administration of the tau mAb JNJ-63733657, correlating with the previous measurement with the PT3 x PT82 Simoa assay. An orthogonal sample clean-up using offline TiO2/ZrO2 combined with 1DLC-TQMS was developed to confirm the presence of mono-ptau (pT217) tryptic peptides in CSF.

Clinical Investigation on Endogenous Biomarkers to Predict Strong OAT-Mediated Drug-Drug Interactions.

BACKGROUND: Endogenous biomarkers are promising tools to assess transporter-mediated drug-drug interactions early in humans. METHODS: We evaluated on a common and validated in vitro system the selectivity of 4-pyridoxic acid (PDA), homovanillic acid (HVA), glycochenodeoxycholate-3-sulphate (GCDCA-S) and taurine towards different renal transporters, including multidrug resistance-associated protein, and assessed the in vivo biomarker sensitivity towards the strong organic anion transporter (OAT) inhibitor probenecid at 500 mg every 6 h to reach close to complete OAT inhibition. RESULTS: PDA and HVA were substrates of the OAT1/2/3, OAT4 (PDA only) and multidrug resistance-associated protein 4; GCDCA-S was more selective, having affinity only towards OAT3 and multidrug resistance-associated protein 2. Taurine was not a substrate of any of the investigated transporters under the in vitro conditions tested. Plasma exposure of PDA and HVA significantly increased and the renal clearance of GCDCA-S, PDA and HVA decreased; the magnitude of these changes was comparable to those of known clinical OAT probe substrates. PDA and GCDCA-S were the most promising endogenous biomarkers of the OAT pathway activity: PDA plasma exposure was the most sensitive to probenecid inhibition, and, in contrast, GCDCA-S was the most sensitive OAT biomarker based on renal clearance, with higher selectivity towards the OAT3 transporter. CONCLUSIONS: The current findings illustrate a clear benefit of measuring PDA plasma exposure during phase I studies when a clinical drug candidate is suspected to be an OAT inhibitor based on in vitro data. Subsequently, combined monitoring of PDA and GCDCA-S in both urine and plasma is recommended to tease out the involvement of OAT1/3 in the inhibition interaction. CLINICAL TRIAL REGISTRATION: EudraCT number: 2016-003923-49.

Multimodal biomarker discovery for active Onchocerca volvulus infection.

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated.

2020 White Paper on Recent Issues in Bioanalysis: BMV of Hybrid Assays, Acoustic MS, HRMS, Data Integrity, Endogenous Compounds, Microsampling and Microbiome (Part 1 - Recommendations on Industry/Regulators Consensus on BMV of Biotherapeutics by LCMS, Advanced Application in Hybrid Assays, Regulatory Challenges in Mass Spec, Innovation in Small Molecules, Peptides and Oligos).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by Mass Spectrometry (hybrid assays, LCMS and HRMS) were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 1) Hybrid Assays, Innovation in Small Molecules, & Regulated Bioanalysis. Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation), Part 2B (Regulatory Input) and Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 5, and 6 (2021), respectively.

Capillary microsampling in clinical studies: opportunities and challenges in two case studies.

Aim: Capillary microsampling of 15 µl whole blood from fingersticks or heelsticks was used to collect pharmacokinetic (PK) samples from pediatric subjects in two projects. Results: In a mebendazole multisite study in Ethiopia and Rwanda in subjects between 1 and 16 years old, complete PK profiles (7 timepoints) could be obtained, although some of the fingerstick samples were contaminated by the dosing formulation. In a multisite study with a respiratory syncytial virus drug in children between 1 and 24 months old, sparse PK sampling was done (2 samples). All samples were successfully analyzed even though some capillaries were not properly filled. Conclusion: CMS shows potential for PK sampling in pediatrics but may need further optimization.

High-dose etoposide formulations do not saturate intestinal P-glycoprotein: Development, stability, and pharmacokinetics in Sprague-Dawley rats.

It has been suggested that oral absorption of low-permeable P-glycoprotein (P-gp) substrates can be increased through saturation of P-gp. For BCS class IV drug substances, saturating P-gp is challenging due to low aqueous solubility. The present study investigated if the BCS IV drug substance etoposide could be solubilized to a concentration saturating P-gp after oral administration. A formulation consisting of 10% (w/v) of pluronic® F-127 and polyvinylpyrrolidone/vinyl acetate (PVP/VA), and 57% (v/v) ethanol enhanced etoposide's solubility approximately 100 times (16 mg mL-1) compared to its aqueous solubility. In vitro, this formulation was stable upon dilution in simulated intestinal fluid. In male Sprague-Dawley rats, oral administration of increasing solubilized etoposide doses using the formulation matrix increased the AUC0-∞ of etoposide dose-proportionally but resulted in a lower absolute oral bioavailability (F) and rate of absorption as compared to control. At the highest investigated dose (100 mg kg-1), AUC0-∞ and Cmax were significantly increased by 2.9- and 1.4-fold, respectively, compared to control dosed at 20 mg kg-1. A single oral dose of 20 mg kg-1 zosuquidar followed by 20 mg kg-1 oral etoposide increased F 8.6-fold. In conclusion, a stable formulation with improved etoposide solubility was developed, yet the formulation did not result in increased oral bioavailability of etoposide.

2-Methyl-pentanoyl-carnitine (2-MPC): a urine biomarker for patent Ascaris lumbricoides infection.

Infections with intestinal worms, such as Ascaris lumbricoides, affect hundreds of millions of people in all tropical and subtropical regions of the world. Through large-scale deworming programs, World Health Organization aims to reduce moderate-to-heavy intensity infections below 1%. Current diagnosis and monitoring of these control programs are solely based on the detection of worm eggs in stool. Here we describe how metabolome analysis was used to identify the A. lumbricoides-specific urine biomarker 2-methyl pentanoyl carnitine (2-MPC). This biomarker was found to be 85.7% accurate in determining infection and 90.5% accurate in determining a moderate-to-heavy infection. Our results also demonstrate that there is a correlation between 2-MPC levels in urine and A. lumbricoides DNA detected in stool. Furthermore, the levels of 2-MPC in urine were shown to rapidly and strongly decrease upon administration of a standard treatment (single oral dose of 400 mg albendazole). In an Ascaris suum infection model in pigs, it was found that, although 2-MPC levels were much lower compared to humans, there was a significant association between urinary 2-MPC levels and both worm counts (p = 0.023) and the number of eggs per gram (epg) counts (p < 0.001). This report demonstrates that urinary 2-MPC can be considered an A. lumbricoides-specific biomarker that can be used to monitor infection intensity.

LC-MS quantification of oligonucleotides in biological matrices with SPE or hybridization extraction.

Aim: Quantitative LC-MS analysis of oligonucleotides (OGNs) in biological matrices is needed to support candidate selection of new therapeutic OGNs. Methodology & results: A set of 20 single stranded antisense oligonucleotides (ASO) and five siRNAs were extracted from plasma and tissue homogenates. Anion Exchange (AEX) SPE was selected as generic extraction approach, resulting in recoveries from plasma >70%. Extraction from tissue homogenates showed often more variation and lower recoveries. A proof of concept of a novel tailored hybridization extraction is demonstrated for two 16-mer reference OGNs. Conclusion: Two methods for extraction of OGNs were investigated and applied for quantitative analysis. The AEX-SPE is considered a more generic approach preferred when multiple compounds are evaluated. Hybridization extraction has great potential but critical reagents per analyte are needed.

Comparison of toxicokinetic parameters of a drug and two metabolites following traditional and capillary microsampling in rat.

Aim: Following the request of a regulatory authority, a rat study was conducted to compare pharmacokinetic parameters from traditional large volume sampling and capillary microsampling. Materials & methods: Rats were dosed with a proprietary compound in three dose groups and blood samples were collected via capillary microsampling (32 µl), immediately followed by traditional large volume sampling (300 µl) up to 24 h postdose. Resulting plasma samples were analyzed for parent drug and two metabolites. AUCs were compared between sampling techniques. Results: There was no statistical difference between AUCs from traditional and microsampling across different doses and analytes. Conclusion: Toxicokinetic parameters generated from plasma collected as a capillary microsample or traditional large volume sample are highly comparable.

Strategies and analytical workflows to extend the dynamic range in quantitative LC-MS/MS analysis.

Aim: To evaluate alternative analytical strategies to extend the dynamic range in quantitative LC-MS/MS. Methods & results: Two approaches based on prior or no prior knowledge of expected exposure levels were evaluated. These approaches make use of two analytical strategies, which include the use of more than one injection volume or dilution of sample extract with solvents or solvent mixtures. A total of 16 compounds with varying logP values were classified into polar and nonpolar groups and used in this evaluation. From the two analytical strategies, three workflows were derived. Conclusion: All three workflows were successfully evaluated and resulted in good accuracy (80-120%) for all the compound groups.

Montmorillonite-surfactant hybrid particles for modulating intestinal P-glycoprotein-mediated transport.

In the small intestine, P-glycoprotein (P-gp) may limit the permeability of its substrates, which lead to reduced oral absorption. To circumvent the effect of P-gp, a nanocomposite material termed montmorillonite-surfactant hybrid particles was developed. The particles consisted of montmorillonite, the P-gp-inhibiting, nonionic surfactant, polysorbate 20, and the P-gp substrate, digoxin. The present study aimed to investigate if montmorillonite-surfactant hybrid particles could modulate the absorption of digoxin in vivo. Montmorillonite-surfactant hybrid particles were prepared by lyophilising an aqueous suspension of the constituents. Scanning electron microscopy, thermogravimetric analysis, and powder X-ray diffraction revealed an altered surface morphology, decreased water content, and intercalation of polysorbate 20 between montmorillonite layers. The particles were administered orally to Sprague Dawley rats, and digoxin was quantified by liquid chromatography-tandem mass spectrometry. Control digoxin-containing montmorillonite decreased the exposure of digoxin. In contrast, montmorillonite-surfactant hybrid particles increased AUC and Cmax by 31 and 91%, respectively, compared to digoxin in solution. It was hypothesised that montmorillonite-surfactant hybrid particles increased digoxin exposure by forming mucosa-localised elevated concentrations of polysorbate 20 and digoxin, which enhanced the inhibitory effect of polysorbate 20 on P-gp.

Feedback from the European Bioanalysis Forum liquid microsampling consortium: capillary liquid microsampling and assessment of homogeneity of the resultant samples.

Following the completion of a detailed experimental protocol into the potential inhomogeneity of capillary liquid microsamples, which was performed at seven European Bioanalysis Forum member companies, the summary and conclusion on the data are reported here. It has been demonstrated that it is possible to generate homogeneous samples using these microsampling techniques; that the resultant microsamples can be accurate and precise and that capillary liquid microsampling data can be consistent with conventional larger volume plasma samples. However, the data contain some variability which is contributed to by the different range of experiences that each investigating site had with these techniques. Therefore, knowledge of the compounds, well-designed experiments and experience with these techniques are essential for the delivery of high quality data.