Overcoming challenges associated with the bioanalysis of cysteine-conjugated metabolites in the presence of antibody-drug conjugates.

Aim: Investigations have shown that for the antibody-drug conjugate (ADC) belantamab mafodotin, concentrations of the cysteine-conjugated metabolite, Cys-mcMMAF, were overestimated in the presence of the ADC during sample processing when utilizing a historical SPE method. Results: A new assay was developed utilizing an acidic protein precipitation to remove the ADC early in the extraction process, thus eliminating the risk of overestimating Cys-mcMMAF in the presence of belantamab mafodotin. In vitro experiments demonstrated a linear relationship between the concentration of belantamab mafodotin and the release of Cys-mcMMAF. Extensive stability assessments were performed to cover storage of study samples. Conclusion: This work emphasized the critical importance of understanding the performance of a bioanalytical method for free toxic payload in the presence of the ADC.

Microflow UPLC and high-resolution MS as a sensitive and robust platform for quantitation of intact peptide hormones.

Aim: Recent advances in microflow ultra performance liquid chromatography (UPLC) systems offer higher sensitivity with robustness to meet the routine bioanalytical demands. Modern high-resolution mass spectrometers (HRMS) enable the development of highly selective methods with broad dynamic range. Results: The quantitative performances of tandem quadrupole MS and HRMS were comprehensively compared using seven intact peptide hormones up to 9.4 kDa. Results show comparable performance between two platforms in sensitivity, accuracy and linearity. For some peptides, HRMS provided lower background interference. The benefit of increased sensitivity using microflow UPLC was also demonstrated. Conclusion: HRMS is a versatile platform capable of both basic characterization and reliable quantitation in complex matrices. Microflow UPLC provides lower LLOQs than conventional flow systems, even with less sample volume injected.

The importance of evaluating the chemical structures and strategies to avoid pitfalls in quantitative bioanalysis.

Quantitative bioanalytical data are crucial in pharmaceutical research and development, allowing project teams to make informed scientific decisions on the progression of candidate molecules to medicines. Many challenges are often encountered during the bioanalysis of drugs in biological matrices which require resolution in a timely manner. In this publication, guidance is provided to bioanalytical scientists on how to identify potential problems before they become an obstacle for the drug development and to share our experiences dealing some of most common problems encountered in the bioanalytical laboratory. Relevant topics in bioanalysis such as stabilization approaches for glucuronides (Acyl and N-); prodrugs (phosphate and esters), amides, amines, N-oxides; bioanalysis of light sensitive molecules, halogenated drugs and lactones are discussed in this publication.

Drug monitoring by volumetric absorptive microsampling: method development considerations to mitigate hematocrit effects.

AIM: GSKA is a compound that was in development in clinical trials. A bioanalysis method to quantify GSKA using volumetric absorptive microsampling (VAMS) was developed and hematocrit (HCT) related assay bias was investigated. METHODOLOGY: After accurate sampling of 10 µl blood, VAMS tips were air dried approximately 18 h and desorbed by an aqueous solution containing internal standard. The recovered blood underwent liquid-liquid extraction in ethyl acetate to minimize matrix suppression. Assay accuracy, precision, linearity, carryover, selectivity, recovery, matrix effects, HCT effects and long-term quality control stability were evaluated. CONCLUSION: HCT-related assay bias was minimized in 30-60% blood HCT range, and all validation parameters met acceptance criteria. The method is suitable for quantitative analysis of GSKA in human blood.

Overcoming bioanalytical challenges associated with the separation and quantitation of GSK1278863, a HIF-prolyl hydroxylase inhibitor, and its 14 stereoisomeric metabolites.

GSK1278863 is an investigative drug under investigation for treatment of anemia associated with chronic kidney disease. Its metabolism is primarily metabolized by P450 enzymes where 19 unique metabolic species have been identified. These include multiple products of mono-, di-, and tri-oxygenation. Initially, two separate and complex ultra high performance liquid chromatography (UHPLC) reverse phase methodologies were developed, validated and applied to measure parent and various predominant and circulating metabolites in numerous clinical studies. However, 5 of the 6 oxidative metabolites may exist in different stereoisomeric forms, resulting in 14 separate species; therefore a chiral methodology was required to determine which stereoisomeric forms circulated in human. A variety of conventional approaches were explored, where in the end a supercritical fluid chromatography (SFC) method was required to separate this complex mixture of 14 stereoisomeric metabolites; data from these experiments provided important information on which species circulate in human. The details of these methodologies will be discussed herein.

Camphanic acid chloride: a powerful derivatization reagent for stereoisomeric separation and its DMPK applications.

BACKGROUND: Camphanic acid chloride has proven to be an efficient chiral derivatization reagent for determination of stereoisomers. RESULTS: The utility of chemical derivatization of various stereoisomers containing hydroxy functional groups with camphanic acid chloride in the presence or absence of a base is highlighted. This procedure is shown to be relatively simple, fast and a cost-effective method of separating racemic drugs and stereoisomeric metabolites in biological matrices. Camphanic derivatives contain two additional chirogenic centers, which are found to enhance stereoisomeric separation on both traditional and chiral stationary phases. CONCLUSION: Four methodologies described herein for separation of multiple stereoisomers in biological samples confirm camphanic acid chloride to be a powerful chiral reagent for stereoisomeric resolution for drug metabolism and PK applications.

A novel approach to capillary plasma microsampling for quantitative bioanalysis.

BACKGROUND: A novel device and procedure for the collection and isolation of microvolumes of plasma have been developed and two pilot rodent PK studies have been completed. RESULTS: This method involves collection of blood into a plastic-wrapped, EDTA-coated capillary tube, containing a small amount of a thixotropic gel and a porous plug. Following blood collection, the capillary is placed into a secondary labeled container suitable for centrifugation and plasma is generated. During centrifugation, the thixotropic gel isolates the plasma from the red blood cells and creates a physical barrier between the two matrices. The plasma is then dispensed from the capillary tube into a separate container for storage or processing. CONCLUSION: A simple and robust novel approach for the collection of small plasma volumes from rodent TK studies has been demonstrated.

Evaluation of glucuronide metabolite stability in dried blood spots.

BACKGROUND: Stabilization of phase II metabolites is an important consideration during bioanalytical method development, method validation and sample analysis. Generic approaches to stabilize these metabolites during storage in liquid-based matrices include pH adjustment of samples prior to storage and/or temperature control; although a variety of other compound-specific stabilization techniques exist. Dried blood spot (DBS) technology is becoming a popular alternative to liquid matrix sampling in many preclinical and clinical applications. However, concerns remain regarding the stability of metabolites stored under ambient conditions using DBS. RESULTS: Experimental data have shown that, under ambient storage conditions, the stability of the glucuronides investigated herein stored as DBS is equivalent to that of liquid samples stored at -80°C. CONCLUSION: The decision to employ DBS technology for a given study needs to be considered on a case-by-case basis with an understanding of compound-specific metabolism characteristics and clinical study design.

Rapid analysis of dried blood spot samples with sub-2-µm LC-MS/MS.

The use of dried blood and dried plasma spots for storage and transportation of samples derived from clinical trials holds the promise to reduce cost, simplify storage and shipping as well as reducing animal usage. From the bioanalysts' point of view, these dried-paper samples add an extra layer of complexity to the analysis introducing extra matrix effects from the paper itself and sometimes from antiviral treatments applied to the card. In this article we demonstrate the use of the sub-2-µm particle LC-MS/MS for the bioanalysis of samples derived from a dried blood spot. The higher resolution provided by these small-particle separations allowed for greater resolution of the analyte from the endogenous components in blood samples and from the card-treatment chemicals. The method-development process was enhanced by the use of MS, which could simultaneously acquire full scan and multiple reaction monitoring data, allowing resolution from metabolites and endogenous matrix components. The use of this approach produced sensitivity levels in the 50-100 pg/ml range and analysis times in the 1-2 min range, which was five-times more sensitive and three-times faster than HPLC. This throughput and sensitivity makes this approach ideal for the analysis of preclinical and clinical studies derived from dried blood spots.

Application of DBS for quantitative assessment of the peptide Exendin-4; comparison of plasma and DBS method by UHPLC-MS/MS.

BACKGROUND: An investigation was performed in order to establish if dried blood spots (DBS) could be applied to the quantitation of biopharmaceuticals in biological matrices and perform equivalently in terms of accuracy, precision and stability to traditional plasma methods. RESULTS: A method was successfully validated for the peptide Exendin-4 (39 amino acids in length) utilizing DBS technology. The validated DBS method resulted in a more sensitive and simplistic method than an existing monkey plasma method and required tenfold less sample volume. The final DBS method resulted in a 10-2000-ng/ml linear calibration range using approximately 5 µl of dried blood, compared with the plasma method in which 150 µl of plasma coupled with SPE sample preparation resulted in a 20-2000-ng/ml linear calibration range. Although not needed for DBS, SPE was required for the plasma method to reduce endogenous matrix interferences and achieve desired LLOQ. Matrix stability was also enhanced by the implementation of the DBS platform when compared with either plasma or whole blood. CONCLUSION: DBS technology can be utilized for the quantitation of biopharmaceuticals and offer advantages over traditional plasma-based methods.

Utility of dried blood spot sampling and storage for increased stability of photosensitive compounds.

BACKGROUND: Compound stability remains a major point of concern within pharmaceutical development. In attempts to minimize degradation, scientists may utilize acidification of samples prior to storage, dark chambers, decreased freezer temperatures and a variety of other stabilization techniques. All of these steps require additional procedures, increased costs and increased validation steps. Dried blood spots (DBS) are becoming a popular alternative to plasma sampling in many small- and even large-molecule applications. An investigation was performed in order to establish if DBS would provide storage advantages over liquid-based matrices for two light-sensitive compounds, nifedipine and omeprazole, to prevent or minimize photodegradation. RESULTS: Experimental data has shown, through forced and natural photodegradation experiments, that the compounds nifedipine and omeprazole exhibit increased photostability when spotted and stored on various DBS paper, when compared with water, plasma or whole blood. For omeprazole, between 40 and 90% loss was observed in liquid matrices, while photodegradation was negligible when utilizing DBS. Some loss of nifedipine is noted during exposure conditions on DBS; however, photodegradation in liquid matrices is far more severe. CONCLUSION: Within the experimental compound set, DBS technology offers a significant reduction in the photodegradation process when compared with the liquid matrices water, plasma or blood.

Development and validation of a sensitive and selective UHPLC-MS/MS method for simultaneous determination of both free and total eicosapentaeonic acid and docosahexenoic acid in human plasma.

A sensitive, selective, and quantitative method for the simultaneous determination of free and total eicosapentaeonic acid (EPA) and docosahexenoic acid (DHA) has been developed and validated in human plasma using fatty acid free human serum albumin as a surrogate matrix. Clean-up for free EPA and DHA employs a liquid-liquid extraction with hexane to remove plasma interferences and provide for cleaner chromatography. The method for total EPA and DHA requires a digestion of the triglycerides followed by liquid-liquid extraction with hexane. Ultra high performance liquid chromatography (UHPLC) technology on a BEH C18 stationary phase column with 1.7 µm particle size was used for chromatographic separation, coupled to tandem mass spectrometry (UHPLC-MS/MS). The method for free EPA and DHA was validated over the concentration range of 0.05-25 µg/mL, while total EPA and DHA concentration range was 0.5-250 µg/mL. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies. To our knowledge, this work represents the first UHPLC-MS/MS based method that combines both free and total EPA and DHA with a relatively small sample volume (25 µL aliquot) and a run time of 1.5 min, facilitating automation and high throughput analysis.

Direct analysis of dried blood spots utilizing desorption electrospray ionization (DESI) mass spectrometry.

A novel approach to the quantitative determination of xenobiotics in whole blood samples without sample preparation or chromatography is described. This method is based on direct analysis of microlitre volumes of blood which are spotted onto specialized paper cards and dried, with the resulting dried blood spots (DBS) analyzed directly via desorption electrospray ionization (DESI) mass spectrometry (MS). Using sitamaquine, terfenadine, and prazosin as model compounds with verapamil as a common internal standard, this methodology demonstrated detection of each compound down to 10 ng mL(-1) from DBS where standard calibration curves show linearity from 10-10,000 ng mL(-1) with r(2) > 0.99. Three (3) different untreated types of filter papers (Whatman 903 and 31ETF as well as Ahlstrom 237) and two (2) treated types of filter paper (Whatman FTA and FTA Elute) were examined and the effect of each surface on the recovery of each analyte was evaluated. The results show that the untreated papers provide the best substrates for DBS analysis by DESI. A more in depth study of the quantitation of sitamaquine on 31ETF paper stock provided bias and error measurements of less than 20%. The promising results shown in this study may have important implications in the areas of therapeutic drug monitoring (TDM), clinical and forensic toxicology, and pharmacology.

Potent and selective small-molecule human urotensin-II antagonists with improved pharmacokinetic profiles.

Lead compound 1 was successfully redesigned to provide compounds with improved pharmacokinetic profiles for this series of human urotensin-II antagonists. Replacement of the 2-pyrrolidinylmethyl-3-phenyl-piperidine core of 1 with a substituted N-methyl-2-(1-pyrrolidinyl)ethanamine core as in compound 7 resulted in compounds with improved oral bioavailability in rats. The relationship between stereochemistry and selectivity for hUT over the kappa-opioid receptor was also explored.

Development of potent and selective small-molecule human Urotensin-II antagonists.

This work describes the development of potent and selective human Urotensin-II receptor antagonists starting from lead compound 1, (3,4-dichlorophenyl)methyl{2-oxo-2-[3-phenyl-2-(1-pyrrolidinylmethyl)-1-piperidinyl]ethyl}amine. Several problems relating to oral bioavailability, cytochrome P450 inhibition, and off-target activity at the kappa opioid receptor and cardiac sodium channel were addressed during lead development. hUT binding affinity relative to compound 1 was improved by more than 40-fold in some analogs, and a structural modification was identified which significantly attenuated both off-target activities.

Dual negative precursor ion scan approach for rapid detection of glutathione conjugates using liquid chromatography/tandem mass spectrometry.

Screening for conjugates formed by the tripeptide glutathione with new chemical entities is an essential step during the drug discovery process, as the formation of these conjugates serves as an indicator for the presence of reactive electrophilic intermediates. To increase the selectivity and throughput of this analysis, various mass spectral scan types have evolved over time. Historically, samples were analyzed under positive ionization conditions for the neutral loss of m/z 129 (loss of the pyroglutamic acid moiety from glutathione); however, more recently, negative precursor ion scanning for the loss of m/z 272 (deprotonated gamma-glutamyl-dehydroalanyl-glycine from glutathione) has emerged as a more selective tool. Further increasing the selectivity, we report on an extension of this methodology by incorporating a simultaneous dual negative precursor ion scan for two commonly observed ion fragments from glutathione conjugates, m/z 272 and 254 (the dehydrated form of m/z 272). This negative dual precursor ion scan methodology was first validated using substrates known to undergo metabolic bioactivation (diclofenac, carbamazepine, and 3-methyl indole) and has then been applied to the routine analysis of proprietary compounds undergoing active lead optimization. In comparison to alternative scan methodologies, the increased selectivity offered by this simultaneous dual precursor method results in a reduction in the generation of false positive results as well as reduced data interpretation time.

No difference in the skeletal muscle angiogenic response to aerobic exercise training between young and aged men.

Ischaemia-induced skeletal muscle angiogenesis is impaired in aged compared with young mice. In humans, vascular endothelial growth factor (VEGF) mRNA and protein following an acute exercise bout are lower in aged compared with young untrained men. We hypothesized that exercise-induced skeletal muscle angiogenesis would be attenuated in aged compared with young men. In eight aged (mean age: 64 years) and six young (mean age: 25 years) sedentary men, muscle biopsies were obtained from the vastus lateralis prior to (Pre), after 1 week and after 8 weeks of an aerobic exercise training program for the measurement of capillarization and VEGF mRNA. Dialysate VEGF protein collected from the muscle interstitial space was measured at rest and during submaximal exercise at Pre, 1 week and 8 weeks. Exercise training increased capillary contacts (CC) and capillary-to-fibre perimeter exchange index (CFPE) of type I and IIA fibres similarly in young and aged. The CC of type IIA and IIB fibres was lower in aged compared with young independent of training status. Exercise-induced interstitial VEGF protein was lower in aged compared with young independent of training status. In untrained, greater exercise-induced interstitial VEGF protein during exercise was associated with greater type I, IIA and IIB CC. Exercise training increased VEGF mRNA similarly in young and aged. These results demonstrate that the angiogenic response to aerobic exercise training is not altered during the ageing process in humans. In addition, muscular activity-associated increases in interstitial VEGF protein may play an important role in the maintenance of skeletal muscle capillarization across the life span.

Use of a variably sensitive selected reaction monitoring method to extend the linear dynamic range for quantitative high-performance liquid chromatography/tandem mass spectrometry.

A method has been devised with the capacity to extend the linear dynamic range of a triple quadrupole mass spectrometer operated in the selected reaction monitoring (SRM) mode of analysis. This extended range experiment can be realized by simultaneously acquiring variably sensitive data, via collision energy adjustment, for the same precursor-to-product ion transition within a single SRM method. While this method can be applied universally to many different study types without any detrimental effect to the analysis or throughput, it was applied herein to acquire and quantify, within a single analysis, the concentrations of GSK-A in a multiple-dose rodent study, that previously required a dilution scheme. Using this methodology, the linear dynamic range of GSK-A was increased over traditional methods by nearly two orders of magnitude, from 2.00-10,000 ng/mL to 0.500-100,000 ng/mL.