LC-MS Bioanalysis Research Articles

Inclusion of dilution quality control samples in quantitative LC-MS bioanalysis.

In LC-MS bioanalysis, sample dilution plays various roles, including bringing analyte concentrations within the validated/qualified dynamic range or alleviating matrix effect for accurate determination of the target analyte(s) in the intendedstudy samples. Adherence to health authority requirements, incorporating good dilution practices, and timely demonstration of dilution integrity whenever samples are diluted in an analytical run are essential to ensure the reliability of bioanalytical results.

LC-MS bioanalysis of protein biomarkers and protein therapeutics in formalin-fixed paraffin-embeddedtissue specimens.

Formalin-fixed paraffin-embedded (FFPE) is a form of preservation and preparation for biopsy specimens. FFPE tissue specimens are readily available as part of oncology studies because they are often collected for disease diagnosis or confirmation. FFPE tissue specimens could be extremely useful for retrospective studies on protein biomarkers because the samples preserved in FFPE blocks could be stable for decades. However, LC-MS bioanalysis of FFPE tissues poses significant challenges. In this Perspective, we review the benefits and recent developments in LC-MS approach for targeted protein biomarker and protein therapeutic analysis using FFPE tissues and their clinical and translational applications. We believe that LC-MS bioanalysis of protein biomarkers in FFPE tissue specimens represents a great potential for its clinical applications.

QbD-Driven Development and Validation of a Bioanalytical LC–MS Method for Quantification of Paliperidone in Human Plasma

This paper discusses how a Quality by Design (QbD) strategy was used to develop and test an HPLC-MS bioanalytical method for detecting plasma Paliperidone concentration. A C18 column and an isocratic mobile phase of organic solvents and water were improved for chromatographic separation. To optimise method performance, Box-Behnken design was used to study column temperature, mobile phase composition, and flow rate. The research used a logical QbD methodology to build an optimised HPLC-MS technology that meets US-FDA standards. Validation studies evaluated selectivity, sensitivity, carryover effects, matrix factor determination, linearity, accuracy and precision testing, recovery, dilution veracity, ruggedness, stability, and reinjection reproducibility, with supporting documentation. The validation studies showed the method's suitability and met acceptance requirements. The QbD framework was successfully used to build and validate an HPLC-MS bioanalytical technique for Paliperidone measurement in human plasma. This method improves chromatographic performance and Paliperidone quantification in clinical and pharmacokinetic studies.

Optimisation of Solid-Phase Extraction and LC-MS/MS Analysis of Six Breast Cancer Drugs in Patient Plasma Samples.

In the development of bioanalytical LC-MS methods for the determination of drugs in plasma samples in a clinical setting, adequate sample preparation is of utmost importance. The main goals are to achieve the selective extraction of the analytes of interest and attain thorough matrix removal while retaining acceptable ecological properties, cost-effectiveness, and high throughput. Solid-phase extraction (SPE) offers a versatile range of options, from the selection of an appropriate sorbent to the optimisation of the washing and elution conditions. In this work, the first SPE method for the simultaneous extraction of six anticancer drugs used in novel therapeutic combinations for advanced breast cancer treatment-palbociclib, ribociclib, abemaciclib, anastrozole, letrozole, and fulvestrant-was developed. The following sorbent chemistries were tested: octylsilyl (C8), octadecylsilyl (C18), hydrophilic-lipophilic balance (HLB), mixed-mode cation-exchange (MCX and X-C), and mixed-mode weak cation-exchange (WCX), with different corresponding elution solvents. The samples were analysed using LC-MS/MS, with a phenyl column (150 × 4.6 mm, 2.5 μm). The best extraction recoveries (≥92.3%) of all analytes were obtained with the C8 phase, using methanol as the elution solvent. The optimised method was validated in the clinically relevant ranges, showing adequate precision (inter-day RSD ≤ 14.3%) and accuracy (inter-day bias -12.7-13.5%). Finally, its applicability was successfully proven by the analysis of samples from breast cancer patients.

How does the order of sample analysis influence the matrix effect during LC-MS bioanalysis?

Mass spectrometry coupled with liquid chromatography is a valuable tool for drug development and personalised drug therapy. The matrix effect is caused by enhancing or suppressing the analyte signal intensity by the interfering compounds of biological fluids. The matrix effect may influence the reliability of the quantitative results. Thus, its evaluation is a critical part of bioanalytical method validation. Identified factors affecting the matrix effect are the physicochemical properties of the analyte, type of biological material, analytical conditions, the ion source construction and calculation method. The order of analysis of test samples (pure solutions and post-extraction spiked samples) is another factor possibly affecting quantifying the matrix effect variability between sources. Our primary goal was to find which experimental design – interleaved or set of blocks – is more sensitive to detect matrix effect variability. Additionally, to better understand the reason of variability, we evaluated the influence of chromatographic elution and the type of plasma (normal, lipemic or hemolyzed), co-elution, and carry-over of phospholipids. We used chemometric methods: Principal Component Analysis and Partial Least-Squares Discriminant Analysis. Although a comparable (but statistically different) matrix effect (%RSDMF) is observed using the interleaved and block schemes, for some compounds, the order of the samples strongly influences the results. The interleaved scheme was generally more sensitive in detecting the matrix effect than the block scheme. Thus, reporting the order of samples is needed to ensure the repeatability of experiments. Chemometrics suggests that lipemic samples analyzed in isocratic conditions are most prone to the matrix effect. Different compositions of matrix lots of the same type – especially lipemic – may influence method reliability. Thus, evaluating more than one source of lipemic and hemolyzed plasma is recommended.

Abstract 3581: Differentiation of niraparib and olaparib brain penetration in healthy rhesus macaque monkeys

Abstract Purpose: There remains an unmet need to provide effective treatment therapy for patients with primary and metastatic brain tumors; lack of drug penetration across the blood brain barrier (BBB) is a key factor. Synthetic lethality is an attractive mechanism in treating brain tumors post radiotherapy. Here we investigate the brain penetration of niraparib and olaparib in healthy monkeys to understand the potential of each to cross the intact BBB. Experimental Procedures: Four healthy male Rhesus macaque monkeys were dosed daily via oral gavage for five days with either niraparib (n=2; 6 mg/kg) or olaparib (n=2; 10 mg/kg). Pre-dose blood was collected daily and terminal blood, cerebrospinal fluid (CSF), and brain tissue was collected at necropsy. Coronal brain sections were analyzed by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) to quantitatively assess the tissue distribution of the dosed compounds. Blood, CSF, and bulk homogenate of brain tissue were analyzed by LC-MS bioanalysis. Summary of Data: Greater brain penetration was observed for niraparib when compared to olaparib in healthy Rhesus macaque monkeys following five days oral administration. The unbound brain-to-plasma partition coefficient (Kp,uu,brain) was 15x higher for niraparib compared to olaparib. Quantitative MALDI IMS of coronal brain sections from monkeys administered niraparib showed consistent concentrations distributed throughout the brain parenchyma. Olaparib was not detected by MALDI IMS in any of the coronal brain sections collected from the monkeys administered olaparib. Similar plasma and CSF concentrations were observed between the monkey’s administered niraparib vs olaparib, highlighting the unique ability of niraparib to cross the intact BBB of monkeys and distribute throughout the brain. Conclusions: Niraparib showed markedly higher brain penetration than olaparib in healthy Rhesus macaque monkeys demonstrating enhanced ability to cross intact BBB. Summary of Quantification Results Displayed as Total Conc. with Adjusted Free Conc. in Parentheses Compound Animal LC-MS Pre-Dose Plasma Days 1-5 (ng/mL) LC-MS Pre-Dose Plasma Days 1-5 (ng/mL) LC-MS Terminal CSF (ng/mL) LC-MS Bulk Brain Homogenate (ng/g) MALDI IMS Brain Section Average (ng/g) Kp,uu Brain/Plasma Mean Kp, uu Brain/Plasma Niraparib Monkey 1 BLQ, 28 (10), 49 (18), 59 (22), 72 (26) 84 (31) 9 378 (14) 283 (10) 0.45 0.30 Niraparib Monkey 2 BLQ, 58 (21), 87 (32), 74 (27), 66 (24) 436 (160) 25 797 (29) 684 (25) 0.16 Olaparib Monkey 3 BLQ, 27 (8), 26 (8), 33 (10), 33 (10) 322 (99) 13 12 (2) BLQ 0.02 0.02 Olaparib Monkey 4 BLQ, 51 (16), 17 (5), 53 (16), 45 (14) 254 (78) 6 11 (2) BLQ 0.03 Citation Format: Mark Reid Groseclose, Jeremy Barry, Yongle Pang, Jennifer Deutsch, Shannon Berry, Elaina McCormick, David K. Lim, Hoang Tran, Sean Maguire, Hasan Alsaid, Amine Aziez, Elaine Paul, Keyur Gada. Differentiation of niraparib and olaparib brain penetration in healthy rhesus macaque monkeys. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3581.

Abstract 3582: Differentiation of niraparib and olaparib brain penetration in a mouse brain metastatic tumor model

Abstract Purpose: There remains an unmet need to provide effective treatment therapy for patients with primary and metastatic brain tumors; lack of drug penetration across the blood brain barrier (BBB) is a key factor. Synthetic lethality remains an attractive mechanism in treating brain tumors post radiotherapy. Here, we evaluated brain penetration and distribution of niraparib and olaparib in a mouse brain tumor model. Experimental: Female mice (CrTac:NCr-Foxn1nu; 6 w/o) received 2.5E5 luciferase transfected human breast cancer line (MDA 231-BRM2-831) via intracardiac injection. Mice were imaged twice/week using bioluminescence imaging (BLI) to monitor tumor growth. On day 35, mice with brain metastases (BM) were treated via oral gavage once daily for 5 days with either niraparib (35 mg/kg, n=4 BM, n=3 control), olaparib (50 mg/kg, n=3 BM, n=3 control), or vehicle (n=3 control). Terminal blood samples and brains were collected 2 hr post final dose. Serial tissue sections were collected for MALDI-IMS, H&E and IHC staining from 5 distinct horizontal planes in the brain. Tissue collected between each imaging plane was homogenized for LC-MS bioanalysis. Summary of Data: In vivo BLI imaging was used to identify mice with BM and tumor presence was confirmed ex vivo using IHC. Quantitative MALDI IMS of coronal brain sections collected from mice administered niraparib showed consistent concentrations distributed throughout the brain parenchyma with locally higher concentrations detected from tumor regions. Table 1 summarizes the LC-MS bioanalysis concentrations in plasma and bulk brain homogenates and brain section concentrations detected by MALDI IMS. The estimated mean unbound brain-to-plasma partition coefficient (Kp,uu,brain) was 3.0x and 5.6x higher for niraparib compared to olaparib in control and BM mice, respectively. Conclusions: Herein, we demonstrated that niraparib has a higher brain penetration and distribution compared to olaparib in both control mice and mice with BM. Summary of Quantification Results Displayed as Total Conc. with Adjusted Free Conc. in Parentheses Compound Group Terminal Plasma Mean (ng/mL) LC-MS Bulk Brain Homogenate Mean (ng/g) MALDI IMS Brain Section Mean (ng/g) Kp,uu Brain Niraparib Control 3847 (769) 658 (111) 532 (89) 0.18 Niraparib BM 2535 (507) 543 (91) 473 (80) 0.14 Niraparib Vehicle BLQ BLQ Olaparib Control 226 (68) 6 (2) BLQ 0.06 Olaparib BM 119 (36) 5 (2) BLQ 0.03 Olaparib Vehicle BLQ BLQ Citation Format: Mark Reid Groseclose, Jeremy Barry, Tinamarie Skedzielewski, Gerald McDermott, Chakravarthi Balabhadrapatruni, Bill Benson, Yongle Pang, David K. Lim, Hoang Tran, Mike Ringenberg, Keyur Gada, Amine Aziez, Elaine Paul, Hasan Alsaid. Differentiation of niraparib and olaparib brain penetration in a mouse brain metastatic tumor model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3582.

Single-day protein LC-MS bioanalysis: can next-generation trypsins cut it?

Aims: The drive toward more sensitive LC-MS assays has resulted in long, complex methods. We assessed next-generation trypsins to identify a suitable candidate to integrate into protein LC-MS method development strategies, to simplify methods and increase throughput. Materials & methods: The performance of commercially available next-generation trypsins was assessed based on the digestion of protein standards in buffer and complex matrix by LC-high-resolution MS. Results: The performance of all next-generation trypsins assessed exceeded that of an overnight tryptic digest in a fraction of the time. Performing reduction and alkylation prior to digestion with heat-stable trypsins may be beneficial and should be investigated. Conclusion: Promega Rapid-Digestion Trypsin is the best-performing next-generation trypsin, surpassing an overnight tryptic digestion.

One sample multi-point calibration curve as a novel approach for quantitative LC-MS analysis: The quantitation of six aflatoxins in milk and oat-based milk as an example

Preparing of calibration curves are critical steps for accurate quantitative LC-MS bioanalysis. Traditional multi-sample external calibration curve (MSCC) is labor-intensive and prone to error. In this study, a novel strategy of one sample multi-point calibration curve (OSCC) using multiple isotopologue reaction monitoring (MIRM) was proposed and validated using LC-MS for the quantitation of six aflatoxins in milk and oat-based milk samples. The developed MIRM-OSCC methodology is comprehensively validated and the results indicated that the established method exhibits good performance in selectivity, sensitivity, accuracy and precision. Furthermore, the OSCC could realize sample dilution by monitoring the MIRM channel with less intensity for samples beyond the upper limit of quantification, without the need of sample dilution, which improves the assay throughput. Considering the advantages of excluding the MSCC preparation and sample dilution in OSCC, this strategy can be widely applied in various fields such as drugs, food safety and environmental analysis.

The Origin-Adjusted Approach for Reliable Quantification of Endogenous Analytes in Mass Spectrometric Bioanalysis.

The reliably accurate and precise quantification of biomarkers is a priceless objective in the drug development and diagnostic arenas. To employ a technique that brings such reliability and furthermore involves a simpler, faster, and inexpensive regime would only underline the potential importance of the concept and technique. To the existing established approaches for biomarker quantification in bioanalytical LC-MS, surrogate matrix (SUR-M) and surrogate analyte (SUR-A), in this Letter we present an approach that fulfills the aforementioned advantages. The concept builds on the historic method of standard addition (SA), in which one source of biological matrix is spiked with analyte to form a calibration curve. With the SA curve back-calculated, the heart of this procedure is the subsequent adjustment of the intercept to zero, the origin, and using only the slope of the curve for interpolation giving calculated sample concentrations. In SA, the concentration axis intercept indicates the endogenous analyte concentration, and our zeroing of this is equivalent to removing the endogenous level. This key shift of the calculated line to the origin unveils our novel origin-adjusted (OA) approach. It enables use akin to a regular xenobiotic method, with no need to ultimately account for the endogenous analyte level in the control matrix used for calibrants. We present a comparison of OA against the control approach of SUR-M in a representative application for kynurenine and tryptophan in human plasma by LC-MS. A numerical performance analysis performed is demonstrative of equivalence between the two approaches for both analytes.

Bioanalysis of Oligonucleotide by LC–MS: Effects of Ion Pairing Regents and Recent Advances in Ion-Pairing-Free Analytical Strategies

Oligonucleotides (OGNs) are relatively new modalities that offer unique opportunities to expand the therapeutic targets. Reliable and high-throughput bioanalytical methods are pivotal for preclinical and clinical investigations of therapeutic OGNs. Liquid chromatography-mass spectrometry (LC-MS) is now evolving into being the method of choice for the bioanalysis of OGNs. Ion paring reversed-phase liquid chromatography (IP-RPLC) has been widely used in sample preparation and LC-MS analysis of OGNs; however, there are technical issues associated with these methods. IP-free methods, such as hydrophilic interaction liquid chromatography (HILIC) and anion-exchange techniques, have emerged as promising approaches for the bioanalysis of OGNs. In this review, the state-of-the-art IP-RPLC-MS bioanalytical methods of OGNs and their metabolites published in the past 10 years (2012-2022) are critically reviewed. Recent advances in IP-reagent-free LC-MS bioanalysis methods are discussed. Finally, we describe future opportunities for developing new methods that can be used for the comprehensive bioanalysis of OGNs.

Pharmacokinetic-Pharmacometabolomic Approach in Early-Phase Clinical Trials: A Way Forward for Targeted Therapy in Type 2 Diabetes.

Pharmacometabolomics in early phase clinical trials demonstrate the metabolic profiles of a subject responding to a drug treatment in a controlled environment, whereas pharmacokinetics measure the drug plasma concentration in human circulation. Application of the personalized peak plasma concentration from pharmacokinetics in pharmacometabolomic studies provides insights into drugs’ pharmacological effects through dysregulation of metabolic pathways or pharmacodynamic biomarkers. This proof-of-concept study integrates personalized pharmacokinetic and pharmacometabolomic approaches to determine the predictive pharmacodynamic response of human metabolic pathways for type 2 diabetes. In this study, we use metformin as a model drug. Metformin is a first-line glucose-lowering agent; however, the variation of metabolites that potentially affect the efficacy and safety profile remains inconclusive. Seventeen healthy subjects were given a single dose of 1000 mg of metformin under fasting conditions. Fifteen sampling time-points were collected and analyzed using the validated bioanalytical LCMS method for metformin quantification in plasma. The individualized peak-concentration plasma samples determined from the pharmacokinetic parameters calculated using Matlab Simbiology were further analyzed with pre-dose plasma samples using an untargeted metabolomic approach. Pharmacometabolomic data processing and statistical analysis were performed using MetaboAnalyst with a functional meta-analysis peaks-to-pathway approach to identify dysregulated human metabolic pathways. The validated metformin calibration ranged from 80.4 to 2010 ng/mL for accuracy, precision, stability and others. The median and IQR for Cmax was 1248 (849–1391) ng/mL; AUC0-infinity was 9510 (7314–10,411) ng·h/mL, and Tmax was 2.5 (2.5–3.0) h. The individualized Cmax pharmacokinetics guided the untargeted pharmacometabolomics of metformin, suggesting a series of provisional predictive human metabolic pathways, which include arginine and proline metabolism, branched-chain amino acid (BCAA) metabolism, glutathione metabolism and others that are associated with metformin’s pharmacological effects of increasing insulin sensitivity and lipid metabolism. Integration of pharmacokinetic and pharmacometabolomic approaches in early-phase clinical trials may pave a pathway for developing targeted therapy. This could further reduce variability in a controlled trial environment and aid in identifying surrogates for drug response pathways, increasing the prediction of responders for dose selection in phase II clinical trials.

Novel hydrophilic-phase extraction, HILIC and high-resolution MS quantification of an RNA oligonucleotide in plasma.

Aim: In the theme of quantitative LC-MS bioanalysis of oligonucleotides free of ion-pairing, a 22-mer RNA oligonucleotide took center stage. The focus was on a unique polar-based retention scheme to produce a high-recovery extraction presenting a high-performance alternative extraction means, also there was the opportunity to involve hydrophilic-interaction liquid chromatography and contemporary high-resolution MS as the end point. Results: Original LC-MS methodology was developed for the oligonucleotide and the performance was robust for both nominal and accurate mass detection, the latter affording 10× improvement in sensitivity and 4000-fold linear dynamic range, 500pM to 2000nM. Conclusion: A novel means of solid-phase extraction is exhibited within a robust pair-free methodology, reaching pM sensitivity with the demonstrably beneficial accurate mass platform.

LC-MS bioanalysis of targeted nasal galantamine bound chitosan nanoparticles in rats' brain homogenate and plasma.

Validated LC-MS method for the direct quantitative analysis of galantamine (acetylcholinesterase inhibitor) was developed in rat cerebrospinal fluid and brain homogenate besides rat plasma, utilizing structurally close nalbuphine as an internal standard. After a simple protein precipitation step, samples are separated on 2-μm C18 column kept at 40 °C, using isocratic flow of 80% methanol in pH 9.5 ammonium formate buffer, and retention times were about 1.8 and 2.9 min for galantamine and nalbuphine, respectively. Mass detection with electrospray ionization (ESI) and positive polarity was able to detect 0.2 ng mL-1 galantamine using single ion monitoring mode (SIM) at m/z 288 for galantamine and m/z 358 for nalbuphine. The method showed linearity within the range of 0.5 - 300 ng mL-1. The proposed method was validated according to FDA guidelines. Trueness and precision showed acceptable values at all quality control levels, and recoveries were within 85.6 - 114.3% in all matrices at all runs and with relative standard deviations within 0.2 - 12.4%. The method was used to study in vivo brain uptake and pharmacokinetics of galantamine from brain homogenate and plasma samples following the administration of nasal galantamine-bound chitosan nanoparticles compared to oral and nasal galantamine solutions, in scopolamine-induced Alzheimer's disease rat model.

An integrated outsourcing practice of nonclinical LC-MS bioanalysis and toxicokinetics at Novartis small molecule drug development.

With decommissioning of internal regulated bioanalytical (BA) and toxicokinetic (TK) capabilities, Novartis has relied on external service providers (ESPs) for all nonclinical LC-MS BA and majority of the associated TK work since 2017. This paper outlines an integrated outsourcing practice of the Novartis nonclinical LC-MS BA/TK group, which covers the roles and responsibilities of Novartis nonclinical LC-MS BA/TK expert scientific monitors, selection of ESPs for Novartis nonclinical LC-MS BA/TK studies, qualification of BA/TK ESPs, study conduct and completion, ESP oversight and evaluation, issue mitigation, and future perspectives.

Tandem mass spectrometry of small-molecule signal transduction inhibitors: Accurate-m/z data to adapt structure proposals of product ions

Protein kinases inhibitors or, more generally, signal transduction inhibitors (STIs) can be used to treat diseases in which deregulation of the protein kinase activity plays a role, such as in cancer. A wide variety of drugs has been developed and/or is under investigation to act as protein kinase inhibitors, especially in tyrosine kinase inhibition. The bioanalysis of STIs has received considerable attention in the past 20 years. Liquid chromatography–tandem mass spectrometry (LC–MS–MS) in selected-reaction monitoring (SRM) mode is the method-of-choice in such studies. In several of these studies from us and others, structures are proposed for the product ions applied in SRM. A critical review of these proposed structures is presented using accurate-m/z data, which we have now generated with a linear-ion-trap–Orbitrap hybrid mass spectrometer. This led to adaptation and new structural proposals of 18 product ions for 13 STIs. Our investigation endorses the power of accurate-m/z analysis in structure elucidation of product ions in bioanalytical LC–MS–MS studies and for which the SRM mode in tandem-quadrupole instruments is apparently less suitable.

Improving data integrity in regulated bioanalysis: proposal for a generic data transfer process for LC-MS from the European Bioanalysis Forum.

In this paper, the European Bioanalysis Forum reports back from the discussions with software developers, involved in regulated bioanalysis software solutions, on agreeing to data transfer specification in the bioanalytical labs' LC-MS workflows as part of today's Data Integrity (DI) challenges. The proposed specifications aim at identifying what consists of a minimum dataset, that is, which are the pre-identified fields to be included in DI proof bidirectional data transfer between LC-MS and information management systems. The proposal is an attempt from the European Bioanalysis Forum to facilitate new software solutions becoming available to increase compliance related to DI in today's LC-MS workflows. The proposal may also serve as a template and inspiration for new data transfer solutions in other workflows.

Wenkui Li, Wenying Jian, and Yunlin Fu (Eds.): Sample preparation in LC-MS bioanalysis.

Wenkui Li, Wenying Jian, and Yunlin Fu (Eds.): Sample preparation in LC-MS bioanalysis.

LC–MS bioanalysis of intact proteins and peptides

Bioanalysis assays that reliably quantify biotherapeutics and biomarkers in biological samples play pivotal roles in drug discovery and development. Liquid chromatography coupled with mass spectrometry (LC-MS), owing to its superior specificity, faster method development and multiplex capability, has evolved as one of the most important platforms for bioanalysis of biotherapeutics, particularly new scaffolds such as half-life extension platforms for proteins and peptides, as well as antibody drug conjugates. Intact LC-MS analysis is orthogonal to bottom-up surrogate peptide approach by providing whole molecule quantitation and high-level sequence and structure information. Here we review the latest development in LC-MS bioanalysis of intact proteins and peptides by summarizing recent publications and discussing the important topics such as the comparison between top-down intact analysis and bottom-up surrogate peptide approach, as well as simultaneous quantitation and catabolite identification. Key bioanalytical issues around intact protein bioanalysis such as sensitivity, data processing strategies, specificity, sample preparation and LC condition are elaborated. For peptides, topics including quantitation of intact peptide vs. digested surrogate peptide, metabolites, sensitivity, LC condition, assay performance, internal standard and sample preparation are discussed.

Recommendations and best practices for calibration curves in quantitative LC-MS bioanalysis.

Recommendations and best practices for calibration curves in quantitative LC-MS bioanalysis.