Challenges and Recommendations in Developing LC–MS/MS Bioanalytical Assays of Labile Glucuronides and Parent Compounds in the Presence of Glucuronide Metabolites

Yuan, Long; Xu, Xiaohui; Ji, Qin

doi:10.4155/bio-2020-0055

Cited by 12 publications

(8 citation statements)

References 31 publications

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“…The intensity of the precursor ion of a putative metabolite is vital for identification. Some types of metabolites such as glucuronide conjugate are liable to undergo ISF 34,35 . The metabolites of natural compounds that contain fragile sites may also suffer from ISF, resulting in low intensity of the precursor ion of metabolites.…”

Section: Resultsmentioning

confidence: 99%

“…Interestingly, dehydroandrographolide also underwent severe ISF, and its precursor ion intensity was dramatically decreased (Table 1), which may be more likely to cause ISF. 34,35 The metabolites of natural compounds that contain fragile sites may also suffer from ISF, resulting in low intensity of the precursor ion of metabolites. Consequently, it may cause a false negative in identification.…”

Section: Interference Of Isf In Compound Identificationmentioning

confidence: 99%

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Widespread occurrence of in‐source fragmentation in the analysis of natural compounds by liquid chromatography–electrospray ionization mass spectrometry

Chen

Pan

Zhai

et al. 2023

Rapid Comm Mass Spectrometry

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Rationale:The in-source fragmentation (ISF) of analyte or co-eluting substances produces unintentional fragment ions, which hampers identification and quantification by liquid chromatography-mass spectrometry (LC/MS). Natural compounds derived from plants also contain fragile moieties that may undergo ISF.However, the characteristics of ISF of natural compounds in LC/MS are still unclear. Methods:The ISF behavior of 214 natural compounds was assayed in LC with Q/orbitrap MS in electrospray ionization (ESI) mode and the extent of ISF was evaluated.Results: Up to 82% of tested compounds underwent ISF and half of the tested natural compounds that contain more than one fragile moiety underwent successive and severe ISF to generate serial structurally related ISF products. The major ISFaltering moieties for natural compounds were hydroxyl, lactone, glycosyl and ether, resulting in neutral loss of H 2 O or CO, deglycosylation or cleavage of ether bond, respectively. Some compounds such as terpenoids underwent severe ISF and less than 1% parent form can be observed. For natural compounds, ISF products with similar structures are more likely to cause interference in analysis because the ISF products may share identical mass-to-charge ratio and similar MS 2 fragmentation patterns with precursor ions of the homologs in plants. Furthermore, severe ISF may cause a false negative in the identification of the parent form.Conclusions: In summary, ISF was a highly frequent phenomenon for analysis of natural compounds by LC/ESI-MS, and extensive and successive ISF of natural products may cause misannotation and misidentification with homologs in plants.The study should raise awareness of ISF interference during the analysis of natural compounds. | INTRODUCTIONLiquid chromatography-mass spectrometry (LC/MS) is a powerful and versatile analytical tool for quantitative and qualitative applications.The inherent advantage of the LC/MS method is the high selectivity due to the chromatographic separation in the LC system and ion separation in the MS monitor. In combination with the high sensitivity, resolution and available structural information, LC/MS is extensively used to identify and quantify multiple components in a complex matrix.The mass-to-charge ratio (m/z value) of precursor ion, chromatographic retention time (RT), and precursor ion-based MS/MS

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Section: Resultsmentioning

confidence: 99%

Section: Interference Of Isf In Compound Identificationmentioning

confidence: 99%

Widespread occurrence of in‐source fragmentation in the analysis of natural compounds by liquid chromatography–electrospray ionization mass spectrometry

Chen

Pan

Zhai

et al. 2023

Rapid Comm Mass Spectrometry

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“…Moreover, the precursor ion for CA4G at m/z 510 could be easily broken into fragment ion in MS2 mode with low CE of 19 V. The product ion for CA4G was successfully obtained and monitored at m/z 317, whereas m/z 286 and m/z 290 were generated for CA4 and IS respectively. Glucuronide conjugates are easier to undergo in‐source fragmentation during MS ionization to form the same ions as those of the intact compounds, which could cause interference to the analysis of the intact compounds (Yuan et al, 2020). In the current study, MS parameters and chromatographic separation of CA4G were optimized using standard material.…”

Section: Resultsmentioning

confidence: 99%

A high‐throughput and simultaneous determination of combretastatin A‐4 phosphate and its metabolites in human plasma using HPLC–MS/MS: Application to a clinical pharmacokinetic study

Wang

et al. 2021

Biomedical Chromatography

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To investigate the clinical pharmacokinetics of CA4P, a high‐throughput high‐performance liquid chromatography–tandem mass spectrometry assay with an identical positive electrospray ionization (ESI) mode was developed for the simultaneous determination of CA4P, its active metabolite CA4, and CA4 glucuronide in human plasma. CA4P and CA4 were easier to protonate in positive ESI mode, whereas CA4G was reported to produce deprotonated ion in negative ESI mode. Because the baseline separation of CA4P and CA4G could not be achieved, using MS positive/negative ion switching is not feasible. In this study, an abundant ammonium adduct ion of CA4G in ESI+ was observed as an ideal precursor ion. The final precursor/product transition pairs chosen for CA4P, CA4, and CA4G were at m/z 397/350, 317/286, and 510/317, respectively. To the best of our knowledge, it is the first report on the simultaneous quantification of CA4P, CA4, and CA4G in biological samples. The proposed method was validated, which showed a wide linear dynamic range, high selectivity and sensitivity, good repeatability, and a short run time. Compared with the literatures, the lower limits of quantification were five‐ and two‐fold more sensitive for CA4G and CA4, respectively. Therefore, this method was successfully applied to the pharmacokinetic study of CA4P in phase I clinical trial.

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“…All can cause false elevation of drug concentrations in validated bioanalytical LC−MS assays without due care for these issues and without predetermined knowledge of the extent or structure of glucuronide conjugates. 5 IMMS offers one potential solution to these issues. Briefly, analytes may be separated through their mobilities based on charge distribution, size and shape 6,7 which provides a drift time, the time taken to traverse the ion mobility cell.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Phase II conjugated metabolites can be particularly problematic to analyze by LC–MS due to their labile dissociation to parent drug within the MS ion source, poor chromatographic retention on separation methods optimized for parent drug, and identical, and hence uninformative, product ion spectra. All can cause false elevation of drug concentrations in validated bioanalytical LC–MS assays without due care for these issues and without predetermined knowledge of the extent or structure of glucuronide conjugates …”

Section: Introductionmentioning

confidence: 99%

Investigation into Small Molecule Isomeric Glucuronide Metabolite Differentiation Using In Silico and Experimental Collision Cross-Section Values

Connolly

Munoz-Muriedas

Lapthorn

et al. 2021

J. Am. Soc. Mass Spectrom.

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Identifying isomeric metabolites remains a challenging and time-consuming process with both sensitivity and unambiguous structural assignment typically only achieved through the combined use of LC–MS and NMR. Ion mobility mass spectrometry (IMMS) has the potential to produce timely and accurate data using a single technique to identify drug metabolites, including isomers, without the requirement for in-depth interpretation (cf. MS/MS data) using an automated computational pipeline by comparison of experimental collision cross-section (CCS) values with predicted CCS values. An ion mobility enabled Q-Tof mass spectrometer was used to determine the CCS values of 28 (14 isomeric pairs of) small molecule glucuronide metabolites, which were then compared to two different in silico models; a quantum mechanics (QM) and a machine learning (ML) approach to test these approaches. The difference between CCS values within isomer pairs was also assessed to evaluate if the difference was large enough for unambiguous structural identification through in silico prediction. A good correlation was found between both the QM- and ML-based models and experimentally determined CCS values. The predicted CCS values were found to be similar between ML and QM in silico methods, with the QM model more accurately describing the difference in CCS values between isomer pairs. Of the 14 isomeric pairs, only one (naringenin glucuronides) gave a sufficient difference in CCS values for the QM model to distinguish between the isomers with some level of confidence, with the ML model unable to confidently distinguish the studied isomer pairs. An evaluation of analyte structures was also undertaken to explore any trends or anomalies within the data set.

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Challenges and Recommendations in Developing LC–MS/MS Bioanalytical Assays of Labile Glucuronides and Parent Compounds in the Presence of Glucuronide Metabolites

Cited by 12 publications

References 31 publications

Widespread occurrence of in‐source fragmentation in the analysis of natural compounds by liquid chromatography–electrospray ionization mass spectrometry

Widespread occurrence of in‐source fragmentation in the analysis of natural compounds by liquid chromatography–electrospray ionization mass spectrometry

A high‐throughput and simultaneous determination of combretastatin A‐4 phosphate and its metabolites in human plasma using HPLC–MS/MS: Application to a clinical pharmacokinetic study

Investigation into Small Molecule Isomeric Glucuronide Metabolite Differentiation Using In Silico and Experimental Collision Cross-Section Values

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