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Proteomic absolute quantitation strategies mainly rely on the use of synthetic stable isotope-labeled peptides or proteins as internal standards, which are highly costly and time-consuming to synthesize. To circumvent this limitation, we recently developed a coulometric mass spectrometry (CMS) approach for absolute quantitation of proteins without the use of standards, based on the electrochemical oxidation of oxidizable surrogate peptides, followed by mass spectrometry measurement of the peptide oxidation yield. Previously, CMS was only applied for single-protein quantitation. In this study, first, we demonstrated absolute quantitation of multiple proteins in a mixture (e.g., β-lactoglobulin B, α-lactalbumin, and carbonic anhydrase) by CMS in one run, without using any standards. The CMS quantitation result was validated with a traditional isotope dilution method. Second, CMS can be used for absolute quantitation of a low-level target protein in a mixture; for instance, 500 ppm of PLBL2, a problematic host cell protein (HCP), in the presence of a highly abundant monoclonal antibody (mAb) was successfully quantified by CMS with no use of standards. Third, taking one step further, this study demonstrated the unprecedented quantitative analysis of deamidated peptide products arising from the mAb heavy chain deamidation reaction. In particular, absolute quantitation of the deamidation succinimide intermediate which had not been performed before due to the lack of standard was conducted by CMS, for the first time. Overall, our data suggest that CMS has potential utilities for quantitative proteomics and biotherapeutic drug discovery.

To solve long-term lack of traceability of commercial calibrator kits and standardize clinical routine assays, we developed a human serum matrix-based unconjugated estriol (uE3) reference material (RM) with five concentration gradients. The RMs of uE3 were certified by the National Institute of Metrology (NIM) with the codes of GBW (E) 091048, GBW (E) 091049, GBW (E) 091050, GBW (E) 091051, and GBW (E) 091052. The RMs were determined by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference method which was developed in our group and recommended by the Joint Committee on Traceability on Laboratory Medicine (JCTLM). GBW09224 is intended for use as a primary reference material to enable the SI-traceable measurement of uE3. This study describes the development process of these certified RMs. The candidate material was prepared by collecting from the remaining serum samples after routine clinical testing. Satisfactory homogeneity and stability were shown in these RMs. They are also commutable between the reference method and the three routine clinical immunoassay systems. To improve the accuracy of value assignment, a collaborative study in nine reference laboratories was conducted which was performed according to ISO/WD 15725-1 and all of the reference laboratories have been confirmed by China National Accreditation Service for Conformity Assessment (CNAS). The raw results were statistically analyzed and processed, coupled with uncertainty evaluation, to obtain the certified value: GBW (E) 091048 is 22.1 ± 1.3 nmol/L, GBW (E) 091049 is 33.6 ± 1.6 nmol/L, GBW (E) 091050 is 10.4 ± 0.8 nmol/L, GBW (E) 091051 is 15.5 ± 1.0 nmol/L, GBW (E) 091052 is 47.0 ± 2.0 nmol/L. The preparation process of human serum matrix-based reference material and the lack of these type of secondary (commutable) reference material of unconjugated estriol lead to the interruption of its traceability chain, which is a problem to be solved in its standardization as mentioned in the metrological traceability in ISO 17511, 2020., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Adulteration/unintentional contamination of milk with melamine could have negative health and economic implications especially in the developing countries due to insufficient laboratory support and surveillance. This paper presents an Isotope Dilution Liquid Chromatography-Mass Spectrometry (ID LC-MS) method developed for detection of melamine in powdered milk. The rapid sample preparation involved dissolution of 1g of milk powder in 2.5% formic acid, precipitation of protein with acetonitrile, spiking of samples with melamine (triamine- 15 N 3 ) at 200 µg L -1 and detection of intrinsic 14 N-melamine molecular ratio to the spike. The isotope dilution calibration procedure was free from matrix effects, unlike other methods where the detector sensitivity can fluctuate up to several orders of magnitude. Limit of detection of the method was 13 µg kg -1 , and the recovery of melamine at 50, 100, and 250 µg kg -1 was 78.7-126.3%. The method was used to determine melamine levels in 22 milk powder products (local and imported) available in Sri Lanka. Melamine was detected in all the samples (range = 0.33-0.96 mg kg -1 ). Full cream milk powders (both local and imported) contained melamine in the range of 0.39-0.84 mg kg -1 , and various health and pregnancy formulas contained <0.5 mg kg -1 of melamine. Two imported infant formula samples contained the highest levels of melamine (0.96 and 0.94 mg kg -1 ). Although these melamine levels are below the regulatory limit in Sri Lanka (1 mg kg -1 ), a monitoring programme would ensure consumer safety.