Structurally related peptide impurity identification and accurate quantification for synthetic oxytocin by liquid chromatography-high-resolution mass spectrometry.

Oxytocin (OXT) is an important peptide that is mainly used as a therapeutic drug to induce labor or strengthen uterine contractions, or to control bleeding after childbirth. OXT has also been reported as a biomarker linked to emotion, and as a potential biomarker for cancer diagnosis. The accurate purity characterization of OXT calibrators is critical for quality control of pharmaceuticals and the development of reference measurement systems for this analyte in laboratory medicine. OXT possesses the particular analytical measurement challenge of a disulfide bond. Accurate value assignment of the purity of oxytocin calibrators can be carried out by applying the mass balance approach or alternative approaches such as amino acid analysis, quantitative nuclear magnetic resonance spectrometry, and nitrogen determination. In order to avoid biases, all these approaches require a correction for structurally related peptide impurities. Structurally related peptide impurities present in a synthetic OXT material have been identified and quantified by a newly developed and in-house-validated liquid chromatography-high-resolution mass spectrometry (LC-hrMS) method. This method was adopted for the measurement of the study material used for an international comparison evaluating the competencies of laboratories to perform peptide characterization. Eighteen structurally related impurities were identified, confirmed, and accurately quantified in the OXT study material by using LC-hrMS. The study material contained a total mass fraction of 31.1 mg/g structurally related OXT impurities with an associated expanded uncertainty of 1.7 mg/g.

The BIPM Mycotoxin Metrology Capacity Building and Knowledge Transfer Program: Accurate Characterization of a Pure Aflatoxin B1 Material to Avoid Calibration Errors.

Background: The contamination of food and feed by mycotoxins, Aflatoxin B1 (AfB1) being one of the most prominent examples, is of imminent concern to many countries. Regulatory limits for mycotoxins have been implemented, and these need to be supported by a sound measurement infrastructure for mycotoxin analysis in order to enforce and verify products, protect populations, and avoid technical barriers to trade in food stuffs. Objective: A Capability Building and Knowledge Transfer program on Metrology for Safe Food and Feed in Developing Economies was started at the International Bureau of Weights and Measures to allow National Metrology Institutes or Designated Institutes to work together to strengthen their national mycotoxin metrology infrastructure. Methods: Knowledge transfer to scientists is provided to enable the characterization of selected pure mycotoxin materials and the production of corresponding certified reference material solutions. Results: This higher-order measurement capability can in turn support mycotoxin testing laboratories within their countries through the provision, for example, of standard solutions of critical analytes that are traceable to the International System of Units (SI). Conclusions and Highlights: The purity characterization and value assignment for a high-purity AfB1 material (979.6 ± 2.3 mg/g, k = 2) intended to be used for the gravimetric production of SI traceable calibration solutions for AfB1 is described using an approach combining quantitative NMR and LC-diode array detection-tandem MS for the correction of the mycotoxin-related impurity content.

Identification and accurate quantification of structurally related peptide impurities in synthetic human C-peptide by liquid chromatography-high resolution mass spectrometry.

Peptides are an increasingly important group of biomarkers and pharmaceuticals. The accurate purity characterization of peptide calibrators is critical for the development of reference measurement systems for laboratory medicine and quality control of pharmaceuticals. The peptides used for these purposes are increasingly produced through peptide synthesis. Various approaches (for example mass balance, amino acid analysis, qNMR, and nitrogen determination) can be applied to accurately value assign the purity of peptide calibrators. However, all purity assessment approaches require a correction for structurally related peptide impurities in order to avoid biases. Liquid chromatography coupled to high resolution mass spectrometry (LC-hrMS) has become the key technique for the identification and accurate quantification of structurally related peptide impurities in intact peptide calibrator materials. In this study, LC-hrMS-based methods were developed and validated in-house for the identification and quantification of structurally related peptide impurities in a synthetic human C-peptide (hCP) material, which served as a study material for an international comparison looking at the competencies of laboratories to perform peptide purity mass fraction assignments. More than 65 impurities were identified, confirmed, and accurately quantified by using LC-hrMS. The total mass fraction of all structurally related peptide impurities in the hCP study material was estimated to be 83.3 mg/g with an associated expanded uncertainty of 3.0 mg/g (k = 2). The calibration hierarchy concept used for the quantification of individual impurities is described in detail. Graphical abstract ᅟ.

Normal phase-liquid chromatography-tandem mass spectrometry with atmospheric pressure photoionization for the purity assessment of 17ß-estradiol.

A normal phase-liquid chromatography-hybrid tandem mass spectrometry (NP-LC-MS/MS) method utilizing atmospheric pressure photoionization (APPI) without dopant has been developed and implemented for the simultaneous determination of several estrogenic steroid hormones. The combination of both NP-LC and APPI-MS/MS tolerates the use of solvents that have the advantages of being self-doping for APPI and, at the same time, inhibit the in situ formation of estrogen dimers as frequently observed for conventional reversed phase (RP)-LC methods. The NP-LC-APPI-MS/MS method has been validated in-house, and its performance characteristics (linearity, repeatability, limits of detection, etc.) were assessed for use in the quantification of estrogens. Moreover, the method was used to characterize and determine the inherent related structure impurities in batches of ß-estradiol, required for the establishment of reference measurement systems for clinical chemistry and laboratory medicine, which served as candidate reference material for an organic purity assessment interlaboratory study (CCQM-K55.a) organized by the International Bureau of Weights and Measures (BIPM) Chemistry Department and carried out within the framework of the Organic Analysis Working Group (OAWG) of the Consultative Committee for Amount of Substance-Metrology in Chemistry (CCQM).

Mass balance method for the SI value assignment of the purity of organic compounds.

A mass balance method is described for determining the mass fraction of the main component of a high purity organic material. The resulting assigned value is established to be traceable to the SI and can be determined with a small associated measurement uncertainty. Pure organic materials with values and uncertainties determined in this way are necessary as primary calibrators of reference measurement systems in order to underpin the metrological traceability of routine measurement results. The method has been applied to materials in which the main components were respectively theophylline, digoxin, 17ß-estradiol, and aldrin. Its performance has been validated in international comparisons coordinated by the BIPM and is in principle applicable to a wide structural range of stable, nonvolatile organic compounds. It has been successfully applied to mass fraction assignments when the main component is present in the range of (950-1000) mg/g and can achieve associated standard uncertainties ranging from 0.5 mg/g (for high purity materials or those containing well-characterized, stable minor components) to 2 mg/g (materials with a significant number or variety of impurities). It is in principle equally applicable to materials with a smaller mass fraction content of the main component.

Simultaneous determination of various cardiac glycosides by liquid chromatography-hybrid mass spectrometry for the purity assessment of the therapeutic monitored drug digoxin.

A high performance liquid chromatography-hybrid tandem mass spectrometry (LC-MS(n)) method utilising electrospray ionisation has been developed and implemented for the simultaneous determination of several cardiac glycosides (CGs) as well as their corresponding aglycones formed by and extracted from herbaceous plants of the genus Digitalis. The method has been validated in-house and its performance characteristics (linearity, repeatability, limits of detection, etc.) were assessed for use in the quantification of CGs and their corresponding aglycones. LODs from 38 to 936pgg(-1) in solution, corresponding to mass fraction impurity levels from 0.0009 (or 0.00008%) to 0.019mgg(-1) (or 0.0019%) detectable in the pure materials have been realized. Moreover, the method was used to characterize and to determine the inherent CG impurities in batches of the therapeutic monitored drug digoxin which served as candidate reference material for an organic purity assessment inter-laboratory study (CCQM-P20.f) organised by the BIPM Chemistry Section and carried out within the framework of the Organic Analysis Working Group (OAWG) of the Consultative Committee for Amount of Substance - Metrology in Chemistry (CCQM). Digoxin was selected from materials required for the establishment of reference measurement systems for clinical chemistry and laboratory medicine.