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Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a key comparison, CCQM-K115.b, was coordinated by the Bureau International des Poids et Mesures (BIPM) and the Chinese National Institute of Metrology (NIM). Seven Metrology Institutes or Designated Institutes and the BIPM participated. Participants were required to assign the mass fraction of oxytocin (OXT) present as the main component in the comparison sample for CCQM-K115.b. The comparison samples were prepared from synthetic OXT purchased from a commercial supplier and used as provided without further treatment or purification. OXT was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of chemically synthesized peptides of known sequence, with one cross-link and up to 5 kDa. It was anticipated to provide an analytical measurement challenge representative for the value-assignment of compounds of broadly similar structural characteristics. The majority of participants used amino acid analysis (PICAA) or quantitative nuclear magnetic resonance (PICqNMR) spectroscopy with a correction for structurally-related peptide impurities approach as the amount of material that has been provided to each participant (25 mg) is insufficient to perform a full mass balance based characterization of the material by a participating laboratory. The coordinators, both the BIPM and the NIM, were the laboratories to use the mass balance approach as they had more material available. It was decided to propose KCRVs for both the OXT mass fraction and the mass fraction of the peptide related impurities as indispensable contributor regardless of the use of PICAA, PICqNMR or mass balance to determine the OXT purity. This allowed participants to demonstrate the efficacy of their implementation of the approaches used to determine the OXT mass fraction. In particular, it allows participants to demonstrate the efficacy of their implementation of peptide related impurity identification and quantification. More detailed studies on the identification/quantification of peptide related impurities and the hydrolysis efficiency revealed that the integrity of the impurity profile of the related peptide impurities obtained by the participant is crucial for the impact on accuracy of the OXT mass fraction assignment. The assessment of the mass fraction of peptide impurities is based on the assumption that only the Largest Consistent Subset (LCS) of results is taken for the calculation of the KCRVPepImp by use of the weighted mean. The KCRVPepImp of 31.6 mg/g is associated with a small corresponding expanded uncertainty of ±1.4 mg/g (k =2) providing a more realistic basis of evaluation for the capabilities of the participants to identify/quantify peptide related impurities. Inspection of the degree of equivalence plots for the mass fraction of peptide impurities and additional information obtained from the peptide related impurity profile indicates that in many cases the major related peptide impurities have been identified and quantified. The approach selected to obtain a KCRVOXT for the mass fraction of OXT is based on random-effects meta-analysis (DerSimonian-Laird (DSL) variance-weighted mean). The DSLmean takes into account the uncertainties of the results while introducing sufficient excess variance to allow for their observed dispersion resulting in a larger expanded uncertainty U(KCRVOXT). The KCRVOXT for CCQM-K115.b is 787.2 mg/g with a corresponding expanded uncertainty of the KCRVOXT of ±12.9 mg/g. All OXT mass fraction results except the result of NMIM are in agreement with the KCRVOXT. It should be pointed out that the mass balance approaches show smaller uncertainties than PICAA or PICqNMR approaches. Mass balance approaches seem to produce slightly higher OXT mass fractions while PICAA approaches deliver slightly lower OXT mass fractions. Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

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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β-casein, a protein in milk and dairy products, has two main variant forms termed as A1 and A2. A1 β-casein may have adverse effects on humans. The fact that there is only one amino acid variation at the 67th position between A1 and A2 β-casein makes it difficult to distinguish between them. In this study, a novel method using characteristic thermolytic peptides is developed for the determination of A1 and A2 β-casein in milk. Firstly, caseins extracted from milk samples are thermolytic digested at 60 °C without any denaturing reagents required for unfolding proteins, which simplifies the sample pretreatment procedure. The characteristic thermolytic peptides (i.e., fragments 66–76 and 59–76 for A1 and A2 β-casein, respectively) selected to specifically distinguish A1 and A2 β-casein only have eleven or eighteen amino acid moieties. Compared with tryptic characteristic peptides with a length of 49 amino acid moieties, these shorter thermolytic characteristic peptides are more suitable for LC-MS analysis. This novel method, with the advantages of high specificity, high sensitivity, and high efficiency, was successfully applied for the analysis of six milk samples collected from a local supermarket. After further investigation, it is found that this method would contribute to the development of A2 dairy products for a company and the quality inspection of A2 dairy products for a government.