Optimization, validation, and comparison of a rapid method for the quantification of insulin‐like growth factor 1 in serum using liquid chromatography–high‐resolution mass spectrometry

Abstract

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.

Synthetic Peptides in Doping Control: A Powerful Tool for an Analytical Challenge

“…97 These same guidelines were used by Seo et al for the validation of a method for the quantification of insulin growth factor I (IGF-I), a biomarker for GH misuse, in human serum samples using LC-HRMS following a rapid sample preparation method. 98 In this regard, they validated the linearity (correlation r 0.9994), selectivity, intra-and interday precision (CV < 2% and CV < 6% respectively), recovery (>95%), accuracy (>99%), LOQ (20 ng/mL), and LOD (15 ng/mL), and compared the method with those usually applied for detecting IGF-I, finding that their method was faster and cheaper and required a low sample volume. 98 The use of Eurachem validation guidelines was reported to determine the LOD, selectivity, and specificity of desmopressin in 10 human plasma samples and in 10 urine samples.…”

“…98 In this regard, they validated the linearity (correlation r 0.9994), selectivity, intra-and interday precision (CV < 2% and CV < 6% respectively), recovery (>95%), accuracy (>99%), LOQ (20 ng/mL), and LOD (15 ng/mL), and compared the method with those usually applied for detecting IGF-I, finding that their method was faster and cheaper and required a low sample volume. 98 The use of Eurachem validation guidelines was reported to determine the LOD, selectivity, and specificity of desmopressin in 10 human plasma samples and in 10 urine samples. 99 For human plasma samples, the LOD was established at 50 pg/mL, and additionally it was found that the method exhibited good selectivity, since an adequate separation was achieved between desmopressin, the ISs, and the carrier peptide.…”

“…Seo et al reported the use of 209 serum samples collected from athletes to establish and compare IGF-1 concentration levels. Although the method was successfully validated and compared with current methodologies, the authors report that a limitation in these assays involved the lack of samples with high concentrations of IGF-1 …”

“… 98 In this regard, they validated the linearity (correlation r 0.9994), selectivity, intra- and interday precision (CV < 2% and CV < 6% respectively), recovery (>95%), accuracy (>99%), LOQ (20 ng/mL), and LOD (15 ng/mL), and compared the method with those usually applied for detecting IGF-I, finding that their method was faster and cheaper and required a low sample volume. 98 …”

Development and single laboratory validation of a targeted liquid chromatography-triple quadrupole mass spectrometry-based method for the determination of insulin like growth factor-1 in different types of milk samples

“…Moreover, the concordances between the manufacturers' reference intervals and those obtained in the study were generally poor ( Chanson et al, 2016 ). An alternative to immunoassays is liquid chromatography-tandem mass spectrometry (LC-MS/MS), already used for IGF-1 quantification mainly in serum ( Ketha and Singh, 2015 , Cox et al, 2014 , Such-Sanmartín et al, 2015 , Lopes et al, 2014 , Bronsema et al, 2018 , Thevis et al, 2011 , Cox et al, 2014 , Mongongu et al, 2021 , Seo et al, 2021 , Coppieters et al, 2020 ). In particular, Bronsema et al (2018) developed and validated an LC-MS/MS targeted method for the detection of IGF-1 in serum in the 10–1000 ng/mL concentration range by selecting two signature peptides; an urea-based sample preparation procedure was proposed and no analyte enrichment step was required.…”