Impurity determination for hepcidin by liquid chromatography-high resolution and ion mobility mass spectrometry for the value assignment of candidate primary calibrators

Bros, Pauline; Josephs, R D; Stoppacher, Norbert; Cazals, Guillaume; Lehmann, Sylvain; Hirtz, Christophe; Wielgosz, Robert; Delatour, Vincent

doi:10.1007/s00216-017-0202-4

Cited by 18 publications

(17 citation statements)

References 12 publications

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“…Ion mobility spectrometry (IMS) studies of two Hep-25 standards from two synthesis pathways showed different mobility profiles. Although the different mobilograms obtained were not further assigned, the authors suggested that multiple distinct conformers were present in these peptide standards [39]. …”

Section: Discussionmentioning

confidence: 99%

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

Abbas

Vranić

Hoffmann

et al. 2018

IJMS

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Hepcidin-25 was identified as the main iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II)-binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandem mass spectrometry (MS/MS), high-resolution mass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D) model of hepcidin-25 with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or reference material comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.

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Section: Discussionmentioning

confidence: 99%

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

Abbas

Vranić

Hoffmann

et al. 2018

IJMS

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“…Commercially obtained synthetic hepcidin was used as candidate pRM (cpRM). Amino-acid analysis and comprehensive impurity analysis of the cpRM, performed by liquid chromatography high-resolution tandem MS, were performed for value assignment [13].…”

Section: Overview Study Designmentioning

confidence: 99%

Provisional standardization of hepcidin assays: creating a traceability chain with a primary reference material, candidate reference method and a commutable secondary reference material

Diepeveen

Laarakkers

Martos

et al. 2018

Clinical Chemistry and Laboratory Medicine (CCLM)

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Background Hepcidin concentrations measured by various methods differ considerably, complicating interpretation. Here, a previously identified plasma-based candidate secondary reference material (csRM) was modified into a serum-based two-leveled sRM. We validated its functionality to increase the equivalence between methods for international standardization. Methods We applied technical procedures developed by the International Consortium for Harmonization of Clinical Laboratory Results. The sRM, consisting of lyophilized serum with cryolyoprotectant, appeared commutable among nine different measurement procedures using 16 native human serum samples in a first round robin (RR1). Harmonization potential of the sRM was simulated in RR1 and evaluated in practice in RR2 among 11 measurement procedures using three native human plasma samples. Comprehensive purity analysis of a candidate primary RM (cpRM) was performed by state of the art procedures. The sRM was value assigned with an isotope dilution mass spectrometry-based candidate reference method calibrated using the certified pRM. Results The inter-assay CV without harmonization was 42.1% and 52.8% in RR1 and RR2, respectively. In RR1, simulation of harmonization with sRM resulted in an inter-assay CV of 11.0%, whereas in RR2 calibration with the material resulted in an inter-assay CV of 19.1%. Both the sRM and pRM passed international homogeneity criteria and showed long-term stability. We assigned values to the low (0.95±0.11 nmol/L) and middle concentration (3.75±0.17 nmol/L) calibrators of the sRM. Conclusions Standardization of hepcidin is possible with our sRM, which value is assigned by a pRM. We propose the implementation of this material as an international calibrator for hepcidin.

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“…Four or five mass fraction levels were selected to establish external calibration curves according to the impurity levels present in the AVP study material. The assigned mass fraction for an impurity detected by LC/HRMS is calculated by the equation given by Bros et al 19 …”

Section: Resultsmentioning

confidence: 99%

Impurity identification and quantification for arginine vasopressin by liquid chromatography/high‐resolution mass spectrometry

et al. 2020

Rapid Comm Mass Spectrometry

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Rationale:For pharmaceutical quality control, impurities may have unexpected pharmacological or toxicological effects on quality, safety, and efficacy of drugs.Arginine vasopressin (AVP) is an important cyclic peptide drug that is mainly used for the treatment of diabetes insipidus and esophageal varices bleeding. With the advancement made in analytical techniques, liquid chromatography/high-resolution mass spectrometry (LC/HRMS) has emerged as a critical technique for the identification and quantification of structurally related peptide impurities in AVP.Methods: An LC/HRMS/MS-based method using a quadrupole ion trap-Orbitrap mass spectrometer operated in the positive ion electrospray ionization mode was developed for the determination and quantification of structurally related peptide impurities in AVP. Results:Under optimized experimental conditions, three deamidation products, ([Glu 4 ]AVP, [Asp 5 ]AVP, and AVP acid), two amino acid deletion impurities (des-Pro 7 -AVP and des-Gly 9 -AVP), one amino acid insertion impurity (endo-Gly 10a -AVP), one end chain reaction product (N-acetyl-AVP), and one AVP isomer were detected. Subsequent quantification using an external standard method estimated the total mass fraction of all structurally related peptide impurities in the AVP study material to be 30.3 mg/g with an expanded uncertainty of 3.0 mg/g (k = 2). Conclusions:This study complements the AVP impurity profile and improves the separation and discovery of other potential impurities in vasopressin analogues.

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Impurity determination for hepcidin by liquid chromatography-high resolution and ion mobility mass spectrometry for the value assignment of candidate primary calibrators

Cited by 18 publications

References 12 publications

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

Provisional standardization of hepcidin assays: creating a traceability chain with a primary reference material, candidate reference method and a commutable secondary reference material

Impurity identification and quantification for arginine vasopressin by liquid chromatography/high‐resolution mass spectrometry

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