Minimizing Matrix Effects While Preserving Throughput in LC–MS/MS Bioanalysis

Ye, Zhengqi; Tsao, Hong; Gao, Hong; Brummel, Christopher L.

doi:10.4155/bio.11.141

Cited by 27 publications

(20 citation statements)

References 17 publications

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“…In addition, the screening assays were performed at relatively high test compound concentrations (e.g., 8 mM) to increase the assay sensitivity and to ensure the capture of any potential reactive compounds, even though the same peptide-compound conjugates were detected when the assays were conducted at lower compound concentrations (e.g., 80 µM), which is comparable to the compound concentrations in GSH-trapping assays (e.g., 50 µM). Methods using LC-HRMS and LC-HRMS/MS found great utility in studies of biomarkers, 14,15 biotransformation, 16,17 and bioanalysis to minimize or eliminate interferences 18,19 in drug discovery and development. High-resolution mass analyzers, such as TOF and Orbitrap-based mass spectrometers, have made data acquisition by LC-HRMS analysis routine.…”

Section: Discussionmentioning

confidence: 99%

Screening and Characterization of Reactive Compounds with In Vitro Peptide-Trapping and Liquid Chromatography/High-Resolution Accurate Mass Spectrometry

et al. 2014

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The present study describes a novel methodology for the detection of reactive compounds using in vitro peptidetrapping and liquid chromatography-high-resolution accurate mass spectrometry (LC-HRMS). Compounds that contain electrophilic groups can covalently bind to nucleophilic moieties in proteins and form adducts. Such adducts are thought to be associated with drug-mediated toxicity and therefore represent potential liabilities in drug discovery programs. In addition, reactive compounds identified in biological screening can be associated with data that can be misinterpreted if the reactive nature of the compound is not appreciated. In this work, to facilitate the triage of hits from high-throughput screening (HTS), a novel assay was developed to monitor the formation of covalent peptide adducts by compounds suspected to be chemically reactive. The assay consists of in vitro incubations of test compounds (under conditions of physiological pH) with synthetically prepared peptides presenting a variety of nucleophilic moieties such as cysteine, lysine, histidine, arginine, serine, and tyrosine. Reaction mixtures were analyzed using full-scan LC-HRMS, the data were interrogated using postacquisition data mining, and modified amino acids were identified by subsequent LC-HRMS/mass spectrometry. The study demonstrated that in vitro nucleophilic peptide trapping followed by LC-HRMS analysis is a useful approach for screening of intrinsically reactive compounds identified from HTS exercises, which are then removed from follow-up processes, thus obviating the generation of data from biochemical activity assays.

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Section: Discussionmentioning

confidence: 99%

Screening and Characterization of Reactive Compounds with In Vitro Peptide-Trapping and Liquid Chromatography/High-Resolution Accurate Mass Spectrometry

et al. 2014

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“…PLs are a major source of ion suppression and have been a hot topic in bioanalytical literature over the past few years [8][9][10]; however, to confirm that PLs were the cause of the ion suppression in this case, a QC high sample (120 ng/ml) was extracted via Evolute ABN using only a 30% MeCN wash. The resulted extract was then analyzed using the previously published LC-MS/MS methodology [4] with the following additional MRM transitions added to monitor for common cholinecontaining PLs and lyso-PLs found in human plasma: 16:0 lyso-phosphatidylcholine (PC; m/z 496→184), 18:0 lyso-PC (m/z 524→184), d34:2 PC (m/z 758→184), d36:3 PC (m/z 784 →184), d36:2 PC (m/z 786→184) and d38:6 PC (m/z 806→184).…”

Section: Recovery From Spementioning

confidence: 96%

“…In recent years monitoring PLs as potential sources of ion suppression has become a standard process in bioanalytical laboratories [8][9][10]. Whilst examples of monitoring other endogenous lipids do exist in the literature [8], neutral lipids, such as di-and tri-acylglycerols are more challenging due to the need to monitor a specific ion transition for a specific molecular species.…”

Section: Conclusion and Practical Tipsmentioning

confidence: 99%

Modification Without Impact: A Case Study in Clinical Assay Failure Due to Lipemia

2012

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After obtaining his PhD in Bioorganic Chemistry from the University of Southampton in 1999, Graeme T Clark has spent the past 13 years working in the field of analytical chemistry. He has held posts of increasing responsibility in academia, biotech, large pharma and contract research where he has both implemented novel technologies and managed functions as diverse as lipidomics, small-molecule bioanalysis (Discovery to Phase IIb clinical trials), biotransformation and LC–MS/MS-based large-molecule analysis. He has published over 18 peer-reviewed articles on subjects covering lipid profiling and biomarkers, microsampling, dried blood spot bioanalysis and novel analytical solutions to peptide bioanalysis. Graeme currently leads up the Bioanalysis and Metabolite Identification group at Cyprotex focussing on in vitro and in vivo analysis. A previously validated LC–MS/MS method for the analysis of H.g.-12 (a steroid glycoside found in Hoodia gordonii) in patients failed when clinical plasma samples from the target population were collected. The failure was identified as a result of much higher lipid concentrations (lipemia) than previously observed, and through the course of the method redevelopment it was further clarified as excessive neutral lipids. The sample preparation element was focused on as not only did the assay fail acceptance criteria, but attempted analysis of the clinical samples resulted in routine system overpressures and physical failure of the LC–MS/MS. The original method was based on liquid–liquid extraction with tertiary-butyl methyl ether and here we present the systematic assessment of SPE, supported liquid extraction as well as pretreatment and multistage sample preparation approaches to solve the bioanalytical challenge.

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“…In general, matrix effects are usually caused by endogenous interferences [25]. Phospholipids are known to be the major cause for ion suppression or enhancement in LC-MS/MS bioanalysis [26]. Thus, monitoring phospholipids has been found to be helpful to avoid matrix effects.…”

Section: Matrix Effect Assessment and Eliminationmentioning

confidence: 99%

LC-MS/MS analysis of pramipexole in mouse plasma and tissues: Elimination of lipid matrix effects using weak cation exchange mode based solid-phase extraction

Guo

Yang

et al. 2015

Journal of Chromatography B

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Minimizing Matrix Effects While Preserving Throughput in LC–MS/MS Bioanalysis

Cited by 27 publications

References 17 publications

Screening and Characterization of Reactive Compounds with In Vitro Peptide-Trapping and Liquid Chromatography/High-Resolution Accurate Mass Spectrometry

Screening and Characterization of Reactive Compounds with In Vitro Peptide-Trapping and Liquid Chromatography/High-Resolution Accurate Mass Spectrometry

Modification Without Impact: A Case Study in Clinical Assay Failure Due to Lipemia

LC-MS/MS analysis of pramipexole in mouse plasma and tissues: Elimination of lipid matrix effects using weak cation exchange mode based solid-phase extraction

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