Quantitative Bioanalysis Research Articles

Fully-Automated Approach for Online Dried Blood Spot Extraction and Bioanalysis by Two-Dimensional-Liquid Chromatography Coupled with High-Resolution Quadrupole Time-of-Flight Mass Spectrometry

An integrated automated approach has been developed for the direct determination of drug concentrations using a SCAP DBS system for online extraction and analysis of dried blood spots (DBS) from DBS paper cards to a multidimensional liquid chromatography system coupled to a high-resolution QTOF mass spectrometry (LC-HRMS). An accurate, precise, selective, and sensitive two-dimensional liquid chromatography-high-resolution mass spectrometry (2D LC-HRMS) assay was developed and validated using small volumes of rat blood (approximately 1.25 μL) from a 2 mm DBS punch. The methodology was validated according to internationally accepted regulated bioanalysis acceptance criteria in order to establish the validity of the combination of online DBS assay and use of HRMS for quantitative bioanalysis. The fully automated procedure exhibited acceptable linearity (r(2) > 0.997) over the concentration range of 5 to 1000 ng/mL. Intra- and interday precision and accuracy runs indicated relative errors less than 20% at the LLOQ level and less than 15% at all other levels. The direct extraction and analysis of DBS samples resulted in a 5-fold improvement in assay sensitivity compared to conventional off-line extraction of punched DBS samples. In addition, the impact of blood hematocrit (Hct) on accurate quantification of the studied drugs also was evaluated, comparing Hct values of 30% and 60% against standards prepared at 45%. Hematocrit experiments show that Hct can influence the accuracy of drugs quantified by DBS and needs to be thoroughly evaluated prior to committing to validating a DBS assay. The online DBS system coupled to the LC-HRMS was then successfully applied to a pharmacokinetic study performed on male Sprague-Dawley rats after administration of a single dose of 5 and 10 mg/kg for midazolam and desipramine, respectively.

Bimetallic gold–silver nanoplate array as a highly active SERS substrate for detection of streptavidin/biotin assemblies

The silver-modified gold nanoplate arrays as bimetallic surface-enhanced Raman scattering (SERS) substrates were optimized for the surface-enhanced Raman detection of streptavidin/biotin monolayer assemblies. The bimetallic gold–silver nanoplate arrays were fabricated by coating silver nanoparticles uniformly on the gold nanoplate arrays. Depending on silver nanoparticle coating, the localized surface plasmon resonance (LSPR) peak of the bimetallic gold–silver nanoplate arrays blue-shifted and broadened significantly. The common probe molecule, Niel Blue A sulfate (NBA) was used for testing the SERS activity of the bimetallic gold–silver nanoplate arrays. The SERS intensity increased with the silver nanoparticle coating, due to a large number of hot spots and nanoparticle interfaces. The platforms were tested against a monolayer of streptavidin functionalized over the bimetallic gold–silver nanoplate arrays showing that good quality spectra could be acquired with a short acquisition time. The supramolecular interaction between streptavidin (strep) and biotin showed subsequent modification of Raman spectra that implied a change of the secondary structure of the host biomolecule. And the detection concentration for biotin by this method was as low as 1.0nM. The enhanced SERS performance of such bimetallic gold–silver nanoplate arrays could spur further interest in the integration of highly sensitive biosensors for rapid, nondestructive, and quantitative bioanalysis, particularly in microfluidics.

Assessment of The Within- and Between-Lot Variability of Whatman™ Fta ® DMPK and 903 ® DBS Papers and Their Suitability for The Quantitative Bioanalysis of Small Molecules

To ensure that PK data generated from DBS samples are of the highest quality, it is important that the paper substrate is uniform and does not unduly contribute to variability. This study investigated any within and between lot variations for four cellulose paper types: Whatman™ FTA(®) DMPK-A, -B and -C, and 903(®) (GE Healthcare, Buckinghamshire, UK). The substrates were tested to demonstrate manufacturing reproducibility (thickness, weight, chemical coating concentration) and its effect on the size of the DBS produced, and the quantitative data derived from the bioanalysis of human DBS samples containing six compounds of varying physicochemical properties. Within and between lot variations in paper thickness, mass and chemical coating concentration were within acceptable manufacturing limits. No variation in the spot size or bioanalytical data was observed. Bioanalytical results obtained for DBS samples containing a number of analytes spanning a range of chemical space are not affected by the lot used or by the location within a lot.

Systematic Investigation of Orthogonal SPE Sample Preparation for The LC–MS/MS Bioanalysis of A Monoclonal Antibody After Pellet Digestion

Increasing assay sensitivity is critical for promoting the application of LC-MS/MS quantitative bioanalysis of therapeutic proteins. A sample processing method that can selectively remove the abundant background peptides in the serum tryptic digests and retain the target peptides can greatly improve the assay sensitivity. Mixed-mode strong-cation exchange SPE was systematically investigated as an orthogonal sample separation technique to reversed-phase UHPLC for the analysis of a test monoclonal antibody, BMS-986012, in monkey serum after pellet digestion. Strong cation exchange SPE efficiently removed most of the background peptides and reduced the matrix effect and background level in the monitored mass transition channels. As a result, improved sensitivity was observed for the surrogate peptides VVSV and SLIY. This orthogonal approach provides a simple and easy-to-develop sample preparation method that can selectively remove most background peptides and extract the target peptides, therefore, improving the LC-MS/MS assay sensitivity.

What are The Challenges of Stability Investigations and Sample Stabilization in Regulated Bioanalysis?

What are The Challenges of Stability Investigations and Sample Stabilization in Regulated Bioanalysis?

Hemato-Critical Issues in Quantitative Analysis of Dried Blood Spots: Challenges and Solutions

Dried blood spot (DBS) sampling for quantitative determination of drugs in blood has entered the bioanalytical arena at a fast pace during the last decade, primarily owing to progress in analytical instrumentation. Despite the many advantages associated with this new sampling strategy, several issues remain, of which the hematocrit issue is undoubtedly the most widely discussed challenge, since strongly deviating hematocrit values may significantly impact DBS-based quantitation. In this review, an overview is given of the different aspects of the 'hematocrit problem' in quantitative DBS analysis. The different strategies that try to cope with this problem are discussed, along with their potential and limitations. Implementation of some of these strategies in practice may help to overcome this important hurdle in DBS assays, further allowing DBS to become an established part of routine quantitative bioanalysis.

Determination of Rosiglitazone and 5-Hydroxy Rosiglitazone in Rat Plasma Using LC–HRMS by Direct and Indirect Quantitative Analysis: A New Approach for Metabolite Quantification

With recent advances in mass spectrometry instrumentation, HRMS is of increasing interest for quantitative bioanalysis due to its high sensitivity, rapid acquisition of full scan data, and advanced software for metabolite identification. In particular, there is strong interest in use of HRMS for simultaneous quantification of parent drug and metabolites without authentic metabolite standard materials. Rosiglitazone and 5-hydroxy rosiglitazone in rat plasma were analyzed using LC-Q-TOF by both direct and indirect quantitative analysis. Direct quantitative analysis used an authentic metabolite standard (5-hydroxy rosiglitazone). Indirect quantitative analysis firstly used the parent drug (rosiglitazone) calibration curve to provide a semiquantitative measure of metabolite concentration. A correction factor was then applied to the original data to re-calculate the 5-hydroxy rosiglitazone metabolite concentration. The ratio of the calibration curve slope of rosiglitazone to that of 5-hydroxy rosiglitazone was determined to be 2.09 ± 0.28 using different batches of mobile phases and columns. The correction factor 2.09 was then used to correct for the 5-hydroxy rosiglitazone concentrations found from the semiquantitative results using the rosiglitazone calibration standard curve. The concentrations of 5-hydroxy rosiglitazone found by direct and indirect quantitative analysis were highly comparable (within ±8%). Indirect quantitative analysis provides an alternative approach for metabolite quantification for discovery PK studies.

High-Resolution MS in Regulated Bioanalysis: Where are We Now and Where Do We Go from Here?

While triple quadrupole MS remains the workhorse of bionanalytical laboratories, LC coupled with high-resolution MS (LC-HRMS) is making headway in drug discovery. LC-HRMS is well suited for quantitative bioanalysis with the inherent advantage of post-acquisition data mining, which is not possible with triple quadrupole systems operated in SRM mode. LC-HRMS can, thus, accomplish the core task of a bioanalytical laboratory--accurate determination of a targeted analyte--with the added bonus of being able to monitor other compounds of interest either at the time of sample analysis, or as an afterthought, after sample analysis, with no additional effort in sample preparation, chromatographic optimization or sample reinjection. Despite these advantages, LC-HRMS has not been broadly adopted in regulated bioanalytical laboratories. The slow progress in embracing the technology may be due, in part, to difficulties in replacing an entire fleet of triple quadrupole MS. Additional reasons are that data mining is of less benefit in development, especially late-stage, than in discovery and that the technical and regulatory challenges associated with the change of platform are perceived to be significant. In addition, the current platform of LC-HRMS introduced by instrument companies has not been tailored to the core responsibility of the bioanalytical community. In marketing current LC-HRMS systems, there is a tendency to combine the needs of the bioanalytical community with those of the drug metabolism community, despite their inherent differences. As a result, the current HRMS systems available lack some basic features desired for bioanalysis, but include features that are not important for bioanalysis making the systems unnecessarily complex and expensive. A simple, cost effective, ideal HRMS system for a bioanalytical laboratory would provide HRMS with high resolving power (the higher the better), no MS/MS capability, and with software suitable for quantitative analysis and appropriate for use in regulated laboratories. Under this scenario, one can foresee a future where part of the regulated bioanalytical work will be accomplished using LC-HRMS, reserving triple quadrupole-based LC-MS/MS for assays that require exquisite sensitivity.

Applications of High-Resolution MS in Bioanalysis

High-resolution MS (HRMS) in conjunction with LC (LC-HRMS) has become available to many laboratories in the pharmaceutical industry. Due to its enhanced, though sometime perceived, specificity using the high-resolution power and its capability of simultaneous quantitation and structural elucidation using the post-acquisition data mining feature, utilization of LC-HRMS for bioanalysis could lead to potential rapid and reliable method development as well as sample analysis, thus generating both cost and resource savings. Here, we would like to share our perspectives about several current and future applications of LC-HRMS in bioanalysis. We will also discuss the factors influencing the quality of method establishment and potential pitfalls that need to be considered for the utilization of LC-HRMS in the field of regulated bioanalysis. We believe when utilized appropriately, LC-HRMS will play a significant role in the future landscape of quantitative bioanalysis.

A Novel Approach to Capillary Plasma Microsampling for Quantitative Bioanalysis

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. 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. A simple and robust novel approach for the collection of small plasma volumes from rodent TK studies has been demonstrated.

Performance Assessment of Microflow LC Combined with High-Resolution MS in Bioanalysis

There continues to be consistent pressure for bioanalytical scientists to achieve lower limits of quantitation. The reasons range from smaller sample volumes available for analysis, to more potent analytes and the growth of biologics in drug development. This has led scientists to investigate alternative LC techniques, including microflow and nanoflow. These techniques have been shown to increase sensitivity of electrospray methods and reduce ionization matrix effects. Because high-resolution MS has significant benefits for the analysis of biologics, this type of mass spectrometer is becoming increasingly important in bioanalysis. For microflow analysis, a new ion source and significant extra sample preparation or chromatographic separation are not required. However, increased sensitivity and reduced matrix effects were consistently demonstrated when compared with UHPLC flow rates. The extent of matrix effects observed were compound dependent. This paper presents the utility of combining high-resolution/accurate mass with microflow LC from a quantitative standpoint. This includes evaluating the typical quantitative parameters of sensitivity, linearity/dynamic range, precision and accuracy. It also includes the evaluation of changes in signal suppression using microflow LC and microspray ionization. The benefits and disadvantages of using the combination of these two technologies for quantitative bioanalysis are also discussed.

Evaluation of mobile phase composition for enhancing sensitivity of targeted quantification of oligonucleotides using ultra-high performance liquid chromatography and mass spectrometry: Application to phosphorothioate deoxyribonucleic acid

LC–MS based assays are a promising approach for the bioanalysis of oligonucleotide therapeutics due to their selectivity and structure identification capabilities. However, the lack of sensitivity and complicated sample preparation procedures remain a barrier for application of LC–MS based assays to preclinical and clinical studies. Numerous studies have shown that the mobile phase composition, especially organic solvent type, has a significant impact on the MS sensitivity of oligonucleotides. In this study, we systematically investigated the type of organic solvents and concentration of organic modifiers for their effect on electrospray desorption efficiency, chromatographic separation and LC–MS signal intensity and provide mechanisms for these effects. 25mM HFIP, 15mM DIEA and the use of ethanol as an organic solvent were observed to achieve a two order of magnitude increase in LC–MS signal intensity when compared to the most commonly used LC–MS mobile phase composition. Phenol–chloroform LLE in combination with ethanol precipitation was demonstrated to be effective for quantitative bioanalysis of therapeutic oligonucleotides. Various conditions for ethanol precipitation were evaluated and >75% absolute recovery was achieved using an optimized extraction procedure. No increase in column pressure or deterioration of separation was observed for >500 injections of biological samples. The method run time was 5min and the LOQ was 2.5ng/ml. The accuracy (% error) and precision (%RSD) are <5.09% and <10.56%, respectively, over a dynamic range of 2.5–1000ng/ml. The assay was applied to a proof of concept animal study and similar PK parameters to previous studies were obtained.

An Alternative Approach for Quantitative Bioanalysis using Diluted Blood to Profile Oral Exposure of Small Molecule Anticancer Drugs in Mice

A quantitative bioanalytical method for pharmacokinetic studies using diluted whole blood from serially bled mice was developed. Oral exposure profiles in mice for five model anticancer compounds dacarbazine, gefitinib, gemcitabine, imatinib, and topotecan were determined following discrete and cassette (five-in-one) dosing. Six micro blood samples per animal were collected and added to a fixed amount of water. This dilution served several purposes: the red blood cells were lysed; an anticoagulant was unnecessary and the fluid volume of diluted sample was sufficient for bioanalytical assays. AUC values obtained from blood concentrations were within twofold for discrete and cassette dosing except for imatinib (2.1-fold difference) and in agreement with those obtained from plasma concentrations after discrete dosing. All compounds were stable in plasma and diluted blood samples for at least 2 weeks at approximately -80°C. Matrix and intermatrix effects were evaluated to ensure robustness and integrity of the bioanalytical assays. This method provides significant process improvement by enhancing efficiency for sample collection and processing and reducing resources (e.g., reduced compound, cost, and animal requirement) compared with conventional methods. Our study demonstrates the applicability of using diluted micro blood samples for small molecule quantitative bioanalysis to support mouse studies in drug discovery.

Simple And Efficient Digestion of A Monoclonal Antibody in Serum Using Pellet Digestion: Comparison With Traditional Digestion Methods in Lc–MS/MS Bioanalysis

Fast, efficient and reproducible digestion is critical for LC-MS/MS quantitative bioanalysis of therapeutic proteins. Traditional digestion methods require a pretreatment, such as sequential denaturation, reduction and alkylation, which are very time-consuming. Pellet digestion, which does not require the serial pretreatments of denaturation, reduction and alkylation, was evaluated using a test monoclonal antibody (mAb) with 16 disulfide bonds, and compared with traditional digestion methods. For the test mAb, pellet digestion provided much better digestion efficiency compared with direct digestion, and provided similar or better digestion efficiency compared with digestion-with-pretreatment. In particular, for two peptides with very low digestion efficiency under direct digestion, pellet digestion improved the digestion yield by approximately 30-fold, which was similar to or better than what digestion-with-pretreatment offered. This method was then successfully applied to an LC-MS/MS assay of the test mAb in monkey serum. Pellet digestion will be a very useful technique for high-throughput and reliable LC-MS/MS bioanalysis of mAbs and other large proteins, including ones with multiple disulfide bonds.

Exploring Rolled-up Au–Ag Bimetallic Microtubes for Surface-Enhanced Raman Scattering Sensor

A technique to design and fabricate Au–Ag bimetal microtubes for the investigation of curvature-dependent localized surface plasmon modes is demonstrated. Highly surface-enhanced Raman scattering (SERS) is observed that illustrates the distribution of localized surface plasmon modes leading to an enhanced electromagnetic field. A finite-difference time-domain method is also employed to simulate the electromagnetic field properties on the metal surface. The enhanced SERS performance of such noble bimetal microtubes could spur further interest in the integration of highly sensitive biosensors for rapid, nondestructive, and quantitative bioanalysis, particularly in microfluidics.

Tissue Bioanalysis of Biotherapeutics And Drug Targets to Support Pk/Pd

Contemporary drug discovery leverages quantitative modeling and simulation with increasing emphasis, both to gain deeper knowledge of drug targets and mechanisms as well as improve predictions between preclinical models and clinical applications, such as first-in-human dose projections. Proliferation of novel biotherapeutic modalities increases the need for applied PK/PD modeling as a quantitative tool to advance new therapies. Of particular relevance is the understanding of exposure, target binding and associated pharmacology at the target site of interest. Bioanalytical methods are key to informing PK/PD models and require assessment of both PK and PD end points. Where targets are sequestered in tissues (noncirculating), the ability to quantitatively measure drug or biomarker in tissue compartments becomes particularly important. This perspective provides an overview of contemporary applications of quantitative bioanalysis in tissue compartments as applied to PK and PD assessments associated with novel biotherapeutics. Case studies and key references are provided.

Strategies for Improving the Quantitative Bioanalytical Performance of LC-MS in Pharmacokinetic Studies

Quantitative bioanalysis is urgently required for the evaluation of pharmacokinetic properties of a drug and to demonstrate the body exposure to the parent drug and/or metabolite for interpretation of the efficacy and toxicity. New trends in drug discovery and development will be always posing challenges on LC-MS-based quantitative bioanalysis. The focus of this minireview is to highlight the commonly used strategies for improving the quantitative bioanalytical performance including overcoming matrix effects and improving MS detectability. "LC-electrolyte effects" and "pulse gradient chromatography" proposed by our group are new approaches that have also showed potential efficiencies on improving overall bioassay performance, including lowering lower limit of quantification (LLOQ), enlarging upper limit of quantification (ULOQ), decreasing matrix effects, and overcoming elutropic effects, etc.. They should also work well in metabolic profiling studies and other important analytical fields, such as food pesticide residue analysis, environmental analysis, clinical and forensic toxicology, doping control, and so on.

Lc–Ms Systems For Quantitative Bioanalysis

LC-MS has become the method-of-choice in small-molecule drug bioanalysis (molecular mass <800 Da) and is also increasingly being applied as an alternative to ligand-binding assays for the bioanalytical determination of biopharmaceuticals. Triple quadrupole MS is the established bioanalytical technique due to its unpreceded selectivity and sensitivity, but high-resolution accurate-mass MS is recently gaining ground due to its ability to provide simultaneous quantitative and qualitative analysis of drugs and their metabolites. This article discusses current trends in the field of bioanalytical LC-MS (until September 2012), and provides an overview of currently available commercial triple quadrupole MS and high-resolution LC-MS instruments as applied for the bioanalysis of small-molecule and biopharmaceutical drugs.

In Silico Prediction For The Investigation of Comedication Interferences in Quantitative Lc–Ms Detection in The Srm Mode

LC-SRM/MS method validation in quantitative bioanalysis requires screening for potential interferences caused by the coelution of comedications or their metabolites. Current approaches are time-consuming, difficult to transfer to other experimental systems and not comprehensive. We propose an in silico strategy based on predicted LC retention time and MS precursor interferences to rank compounds that could potentially interfere with the analyte of interest, followed by a more focused experimental verification. The suggested screening strategy was applied to investigate 129 potential comedications in everolimus patient samples analyzed with a validated LC-SRM/MS assay. A mixture of analytes with the same nominal mass was also investigated to illustrate the interference issues in SRM method development. A strategy was developed that allows the rapid screening of comedications, which is scalable to any analyte and transferable to any other LC-MS system.

Recent Advances in Metabolite Identification And Quantitative Bioanalysis By Lc–Q-Tof Ms

The need for rapid, sensitive and effective identification and quantitation of drugs and metabolites to accelerate drug discovery and development has given MS its central position in drug metabolism and pharmacokinetic research. This review attempts to orient the readers with respect to hybrid Q-TOF MS, which enables accurate mass measurement and generates information-rich datasets. The key properties of the Q-TOF MS system, including mass accuracy, resolution, scan speed and dynamic range, are herein discussed. Developments on tandem separation techniques (e.g., UHPLC(®) and ion mobility spectrometry), data acquisition and data-mining methods (e.g., mass defect, product/neutral loss, isotope pattern filters and background subtraction) that facilitate qualitative and quantitative analysis are then examined. The performance and versatility of LC-Q-TOF MS are thoroughly illustrated by its applications in metabolite identification and quantitative bioanalysis. Future perspectives are also discussed.