Integrated hemolysis monitoring for bottom-up protein bioanalysis.

Background: Hemolysis can result in analyte suppression or enhancement and it can affect the extraction efficiency and analyte stability. Triskelion developed an LC-MS method to monitor hemolysis. The concept can be integrated into existing and new quantitative protein LC-MS methods and can be validated according to the most appropriate tier. Results/methodology: In this proof of concept study, the tryptic target LLVVYPWTQR was used to quantify hemoglobin. The peptide target has only few variations considering the most common (laboratory) animals and is thus nearly generic. It was shown that LC-MS is a suitable technique for the quantification of hemoglobin in hemolyzed samples and that the signals are not affected by lipemia. Conclusion: LC-MS exhibited the best performance to monitor hemolysis when the results were compared with UV-VIS and visual inspection, especially when samples were lipemic.

Current State of Oligonucleotide Characterization Using Liquid Chromatography-Mass Spectrometry: Insight into Critical Issues.

As interests increase in oligonucleotide therapeutics, there has been a greater need for analytical techniques to properly analyze and quantitate these biomolecules. This article looks into some of the existing chromatographic approaches for oligonucleotide analysis, including anion exchange, hydrophilic interaction liquid chromatography, and ion pair chromatography. Some of the key advantages and challenges of these chromatographic techniques are discussed. Colloid formation in mobile phases of alkylamines and fluorinated alcohols, a recently discovered analytical challenge, is discussed. Mass spectrometry is the method of choice to directly obtain structural information about oligonucleotide therapeutics. Mass spectrometry sensitivity challenges are reviewed, including comparison to other oligonucleotide techniques, salt adduction, and the multiple charge state envelope. Ionization of oligonucleotides through the charge residue model, ion evaporation model, and chain ejection model are analyzed. Therapeutic oligonucleotides have to undergo approval from major regulatory agencies, and the impurities and degradation products must be well-characterized to be approved. Current accepted thresholds for oligonucleotide impurities are reported. Aspects of the impurities and degradation products from these types of molecules are discussed as well as optimal analytical strategies to determine oligonucleotide related substances. Finally, ideas are proposed on how the field of oligonucleotide therapeutics may improve to aid in future analysis.

The Role of Fluorinated Alcohols as Mobile Phase Modifiers for LC-MS Analysis of Oligonucleotides.

Hexafluoroisopropanol (HFIP) has been widely used as an acidic modifier for mobile phases for liquid chromatography-mass spectrometry (LC-MS) analysis of oligonucleotides ever since the first report of its use for this purpose. This is not surprising, considering the exceptional performance of HFIP compared with carboxylic acids, which cause significant MS signal suppression in electrospray ionization. However, we have found that other fluorinated alcohols can also be utilized for mobile phase preparation and the choice of optimal fluorinated alcohol is determined by the ion-pairing (IP) agent. Although HFIP is a very good choice to be used alongside less hydrophobic IP agents, other fluorinated alcohols such as 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFMIP) can significantly outperform HFIP when used with more hydrophobic IP agents. We also found that more acidic fluorinated alcohols assist with the transfer of oligonucleotides with secondary structure (e.g., folded strands and hairpins) into the gas phase. Graphical Abstract ᅟ.

Quantitative bottom up analysis of infliximab in serum using protein A purification and integrated µLC-electrospray chip IonKey MS/MS technology.

BACKGROUND: TNO Triskelion has applied its general workflow for the development of quantitative LC-MS methods for proteins in biological matrices to the quantification of infliximab in rat serum using bottom up µLC-MS/MS. Results/methodology: The general workflow consists of sample purification, analyte processing and LC-MS analysis. In the development of a quantitative µLC-MS/MS method for infliximab in rat serum the analyte processing part and the LC-MS part were optimized, in order to meet the different sample requirements of µLC-MS as compared with UPLC-MS. Using the optimized µLC-MS/MS method the LOQ was 75 ng/ml. CONCLUSION: The present study showed that it is possible to gain sensitivity when going to smaller scale LC-MS (UPLC-MS to µLC-MS). Due to the combination of a modified sample preparation approach and the application of µLC-MS a lower LOQ could be achieved for infliximab compared with a previously developed UPLC-MS method.

LC-MS-based quantification of intact proteins: perspective for clinical and bioanalytical applications.

Bioanalytical LC-MS for protein quantification is traditionally based on enzymatic digestion of the target protein followed by absolute quantification of a specific signature peptide relative to a stable-isotope labeled analog. The enzymatic digestion, nonetheless, limits rapid method development, sample throughput and turnaround time, and, moreover, makes that essential information regarding the biological function of the intact protein is lost. The recent advancements in high-resolution MS instrumentation and improved sample preparation techniques dedicated to protein clean-up raise the question to what extent LC-MS can be applied for quantitative bioanalysis of intact proteins. This review provides an overview of current and potential applications of LC-MS for intact protein quantification as well as the main limitations and challenges for broad application.

Conference report: moving forward together: "we are making progress".

The 6th European Bioanalysis Forum Open Meeting 20-22 November 2013, Hesperia Tower Hotel, Barcelona, Spain At the 6th European Bioanalysis Forum Open Meeting, held from 20-22 November 2013 in Hesperia Tower Hotel, Barcelona, Spain, bioanalytical experts from pharmaceutical industry, academia, contract laboratories and regulatory bodies discussed current topics of interest in bioanalysis. 450 delegates from more than 170 institutes and companies participated in 75 open and stimulating presentations regarding the new US FDA Guidance for industry, technology updates, including liquid chromatography-mass spectrometry of proteins and antibody-drug conjugates, dried blood spots, sampling and extraction and regulatory aspects of, for example, flow cytometry, parallelism, and interferences in ligand-binding assays. This article aims to provide an overview of the highlights discussed at the meeting.

Identification of drug metabolites in human plasma or serum integrating metabolite prediction, LC-HRMS and untargeted data processing.

BACKGROUND: Comprehensive identification of human drug metabolites in first-in-man studies is crucial to avoid delays in later stages of drug development. We developed an efficient workflow for systematic identification of human metabolites in plasma or serum that combines metabolite prediction, high-resolution accurate mass LC-MS and MS vendor independent data processing. Retrospective evaluation of predictions for 14 (14)C-ADME studies published in the period 2007-January 2012 indicates that on average 90% of the major metabolites in human plasma can be identified by searching for accurate masses of predicted metabolites. Furthermore, the workflow can identify unexpected metabolites in the same processing run, by differential analysis of samples of drug-dosed subjects and (placebo-dosed, pre-dose or otherwise blank) control samples. To demonstrate the utility of the workflow we applied it to identify tamoxifen metabolites in serum of a breast cancer patient treated with tamoxifen. RESULTS & CONCLUSION: Previously published metabolites were confirmed in this study and additional metabolites were identified, two of which are discussed to illustrate the advantages of the workflow.

Bioanalytical LC-MS/MS of protein-based biopharmaceuticals.

Biotechnology increasingly delivers highly promising protein-based biopharmaceutical candidates to the drug development funnel. For successful biopharmaceutical drug development, reliable bioanalytical methods enabling quantification of drugs in biological fluids (plasma, urine, tissue, etc.) are required to generate toxicokinetic (TK), pharmacokinetic (PK), and bioavailability data. A clear observable trend is that liquid chromatography coupled to (tandem) mass spectrometry (LC-MS(/MS)) is more and more replacing ligand binding assays (LBA) for the bioanalytical determination of protein-based biopharmaceuticals in biological matrices, mainly due to improved selectivity and linear dynamic ranges. Practically all MS-based quantification methods for protein-based biopharmaceuticals traditionally rely on (targeted) proteomic techniques and include "seven critical factors": (1) internal standardization, (2) protein purification, (3) enzymatic digestion, (4) selection of signature peptide(s), (5) peptide purification, (6) liquid chromatographic separation and (7) mass spectrometric detection. For this purpose, the variety of applied strategies for all "seven critical factors" in current literature on MS-based protein quantification have been critically reviewed and evaluated. Special attention is paid to the quantification of therapeutic monoclonal antibodies (mAbs) in serum and plasma since this is a very promising and rapidly expanding group of biopharmaceuticals. Additionally, the review aims to predict the impact of strategies moving away from traditional protein cleavage isotope dilution mass spectrometry (PC-IDMS) toward approaches that are more dedicated to bioanalysis.

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.

Bioanalytical LC-MS of therapeutic oligonucleotides.

Therapeutic oligonucleotides (OGNTs) are important biopharmaceutical drugs for the future, due to their ability to selectively reduce or knockout the expression of target genes. For the development of OGNTs, reliable and relatively high-throughput bioanalytical methods are required to perform the quantitative bioanalysis of OGNTs and their metabolites in biological fluids (e.g., plasma, urine and tissue). Although immunoaffinity methods, especially ELISA, are currently widely applied for this purpose, the potential of LC-MS in OGNT analysis is under investigation. Owing to its inherent ability to monitor the individual target OGNTs as well as their metabolites, LC-MS is now evolving into the method-of-choice for the bioanalysis of OGNTs. In this paper, the state-of-the-art of bioanalytical LC-MS of OGNTs and their metabolites in biological fluids is critically reviewed and its advantages and limitations highlighted. Finally, the future perspective of bioanalytical LC-MS, that is, lower detection levels and potential generic LC-MS methodology, is discussed.

Simultaneous determination of endogenous deoxynucleotides and phosphorylated nucleoside reverse transcriptase inhibitors in peripheral blood mononuclear cells using ion-pair liquid chromatography coupled to mass spectrometry.

Nucleoside reverse transcriptase inhibitors (NRTIs) are activated intracellularly to their triphosphate (TP) form, which compete with endogenous deoxynucleotide-triphosphates (dNTP) as substrate for HIV reverse transcriptase. The activity of NRTIs is thus described by the NRTI-TP-to-dNTP ratio in relevant cell types. Therefore, we developed an ion-pair (IP) LC-MS method for the simultaneous analysis of the mono-, di-, and TP forms of NRTIs and endogenous deoxynucleosides in peripheral blood mononuclear cells (PBMC). The IP-LC method was applied on an IT mass spectrometer using the MS-mode as well as on a triple quadrupole mass spectrometer using the MS/MS mode. The MS/MS approach on the triple quadrupole mass spectrometer demonstrated the best clinical applicability due to its higher sensitivity. The LOD (minimum amount on column) were 25 fmol for the TP forms of zidovudine, lamivudine, and stavudine, as well as for their endogenous dNTP counterparts. The linearity (R(2) ) of the calibration curves were>0.99. The obtained LOD readily allow for clinical applications using just one million PBMC obtained from HIV-infected patients under therapy.

Generic solid phase extraction-liquid chromatography-tandem mass spectrometry method for fast determination of drugs in biological fluids.

A generic method was developed for the fast determination of a wide range of drugs in serum or plasma. The methodology comprises generic solid-phase extraction, on-line coupled to gradient HPLC with tandem mass spectrometric detection (SPE-LC-MS/MS). The individual components of the SPE-LC-MS/MS system were optimized in an integrated approach to maximize the application range and minimize the method development time. The optimized generic SPE-LC-MS/MS protocol was evaluated for 11 drugs with different physicochemical properties. Good quantification for 10 out of 11 of the pharmaceuticals in serum or plasma could be readily achieved. The quantitative assays gave recoveries better than 95%, lower quantification limits of 0.2-2.0 ng/ml, acceptable precision and accuracy and good linearity over 2-4 orders of magnitude. Carry-over was determined to be in the range of 0.02-0.10%, without optimization.