The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies while providing lower storage and shipping costs, because cold-chain logistics can be eliminated. Thus, precise, sensitive, and multiplexed methods for measuring protein concentrations in DBSs can be used for de novo biomarker discovery and for biomarker quantification or verification experiments. To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBSs. The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, whereas the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%. The majority (88%) of the assays displayed parallelism, whereas the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles. For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months. Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals. Graphical Abstract Highlights Repeatable quantification of 200 proteins in dried blood spots. Determined lower limit of quantification, repeatability, parallelism and stability. Protein stability in DBS stored at ambient temperatures for up to 2 months. Concentration ranges for 200 proteins in 20 healthy individuals.

Rationale: Hepcidin is a peptide hormone that plays a central role in regulating iron metabolism. It is a potential biomarker for the diagnosis, monitoring and treatment of iron metabolism disorders. Serum hepcidin level can differ by 3 orders of magnitude depending on the patient's condition. Existing liquid chromatography/mass spectrometry (LC/MS) assays lack clinical sensitivity or require costly sample preparation steps. A simple, sensitive, robust and cost‐effective assay for serum hepcidin quantitation in routine clinical laboratories is needed. Methods: A high‐performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method was developed to quantify hepcidin in human serum using chemically synthesized hepcidin as a standard and stable‐isotope‐labeled hepcidin as the internal standard. The method was validated according to CLSI‐C62A guidelines. Calibrators were prepared with hepcidin‐free serum. Clinical samples were separately processed and compared using solid‐phase extraction (SPE) and acetonitrile (ACN) protein precipitation. Results: The calibration curve was validated over the range of 0.1–100 nmol/L with R ² >0.99. Both the SPE and the ACN precipitation methods had excellent and comparable reproducibility. The intra‐day and inter‐day coefficients of variation (CVs) were <3% and <6%. There was 89% and 88% hepcidin recovery by SPE and ACN preparation. Measurement of secondary reference material using non‐traceable calibrators yielded up to 30% positive bias, comparable with values obtained by an external comparator. Hepcidin was stable in serum at ambient temperature and at 4°C. The relative errors (REs) were ≤1.2% and ≤4.4%, respectively. The freeze–thaw (−70°C) stability after 3 cycles showed a relative error (RE) of ≤1.8%. The impact on hepcidin recovery due to hemolysis (4+), lipemia (4+) and Icterus (4+) was <3%. Conclusions: We have developed and validated a simple, sensitive, robust and cost‐effective HPLC/MS/MS method for the quantitation of serum hepcidin. The method uses ACN protein precipitation for sample preparation and reversed‐phase normal‐flow HPLC. Sample preparation is inexpensive; it can be automated with a liquid handling system to allow high‐throughput application.

An analytical benchmark for high-sensitivity cardiac troponin (hs-cTn) assays is to achieve a coefficient of variation (CV) of ≤ 10.0 % at the 99th percentile upper reference limit (URL) used for the diagnosis of myocardial infarction. Few prospective multicenter studies have evaluated assay imprecision and none have determined precision at the female URL which is lower than the male URL for all cardiac troponin assays. Human serum and plasma matrix samples were constructed to yield hs-cTn concentrations near the female URLs for the Abbott, Beckman, Roche, and Siemens hs-cTn assays. These materials were sent (on dry ice) to 35 Canadian hospital laboratories (n = 64 instruments evaluated) participating in a larger clinical trial, with instructions for storage, handling, and monthly testing over one year. The mean concentration, standard deviation, and CV for each instrument type and an overall pooled CV for each manufacturer were calculated. The CVs for all individual instruments and overall were ≤ 10.0 % for two manufacturers (Abbott CV_pooled = 6.3 % and Beckman CV_pooled = 7.0 %). One of four Siemens Atellica instruments yielded a CV > 10.0 % (CV_pooled = 7.7 %), whereas 15 of 41 Roche instruments yielded CVs > 10.0 % at the female URL of 9 ng/L used worldwide (6 cobas e411, 1 cobas e601, 4 cobas e602, and 4 cobas e801) CV_pooled = 11.7 %). Four Roche instruments also yielded CVs > 10.0 % near the female URL of 14 ng/L used in the United States (CV_pooled = 8.5 %). The number of instruments achieving a CV ≤ 10.0 % at the female 99th-percentile URL varies by manufacturer and by instrument. Monitoring assay precision at the female URL is necessary for some assays to ensure optimal use of this threshold in clinical practice.