Human insulin‐like growth factor 1 (IGF‐I) is the primary mediator of the effects of the growth hormone (GH). Therefore, it has been used as a biomarker to detect the abuse of GH in sports. The measurement of IGF‐I relies on mass‐based and immunological approaches to analysis. Among the mass‐based analysis methods, liquid chromatography–mass spectrometry (LC–MS) has a number of functional advantages. LC–MS measurements based on the quantification of IGF‐I, according to trypsin digestion, are used in the most common method of analyzing doping. However, this method is time‐consuming and subject to experimental variability. In this study, we optimized a rapid method for detecting IGF‐I without the trypsin digestion step. This method of analysis uses an ultra‐centrifugal filter and an LC‐HRMS through narrow‐range mass scan method. To verify the validity of this method, eight categories of validation testing were applied with the following results: linearity, R2 > 0.99; limit of detection, 15 ng/ml; limit of quantification, 20 ng/ml; accuracy, >99%; recovery rate, >95%; carryover, <0.03; and inter‐ and intra‐day precision values, %CV < 2% and %CV < 6%, respectively. Furthermore, we discussed the correlation of the quantified concentration from two other methods, immunoradiometric assay (IRMA) and parallel reaction monitoring method, using 209 serum samples. In conclusion, although both mass spectrometry‐based methods worked equally well in terms of analytical performance and correlation with IRMA results, narrow‐range mass scan method had several advantages, such as time and cost savings and reliable reproducibility, over the existing methods.

Erythropoietin (EPO) is a glycoprotein hormone that stimulates red blood cell production. It is produced naturally in the body and is used to treat patients with anemia. Recombinant EPO (rEPO) is used illicitly in sports to improve performance by increasing the blood’s capacity to carry oxygen. The World Anti-Doping Agency has therefore prohibited the use of rEPO. In this study, we developed a bottom-up mass spectrometric method for profiling the site-specific N-glycosylation of rEPO. We revealed that intact glycopeptides have a site-specific tetra-sialic glycan structure. Using this structure as an exogenous marker, we developed a method for use in doping studies. The profiling of rEPO N-glycopeptides revealed the presence of tri- and tetra-sialylated N-glycopeptides. By selecting a peptide with a tetra-sialic acid structure as the target, its limit of detection (LOD) was estimated to be < 500 pg/mL. Furthermore, we confirmed the detection of the target rEPO glycopeptide using three other rEPO products. We additionally validated the linearity, carryover, selectivity, matrix effect, LOD, and intraday precision of this method. To the best of our knowledge, this is the first report of a doping analysis using liquid chromatography/mass spectrometry-based detection of the rEPO glycopeptide with a tetra-sialic acid structure in human urine samples.