Quantification of insulin-like growth factor-I by liquid chromatography-mass spectrometry.

We describe three methods of sample preparation for the liquid chromatography coupled with mass spectrometric measurement of insulin-like growth factor-I concentration in blood. One method involves trypsin digestion, the second involves intact protein quantification and the third method is a combination of the first two. Step-by-step directions are provided for sample collection and handling including transport, storage conditions as well as detailed instructions for preparation for analysis, which can be modified for larger or smaller sample volumes as needed. A fully 15 N-labelled internal standard is used and the merits of a single-point calibrator are discussed. Example instrumental conditions are presented for both intact and digest methods.

A Targeted Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification of Peptides from the Carboxyl-terminal Region of Type III Procollagen, Biomarkers of Collagen Turnover.

BACKGROUND: The development of analytical approaches to help reduce the risk of growth hormone (GH) doping is important to fair competition and the health of athletes. However, the reliable detection of GH use remains challenging. The identification of novel biomarkers of GH administration could lead to a better understanding of the physiological response to GH, more sensitive detection of the illicit use of GH in sport, and better management of patients treated for GH disorders. METHODS: We developed a targeted liquid chromatography-tandem mass spectrometry method to simultaneously quantify the carboxyl-terminal propeptide of type III procollagen (P-III-CP) and type III collagen degradation products in human serum. Following proteolysis, we instituted a simple acid precipitation step to reduce digested sample complexity before peptide immunoenrichment, which improved the recovery of one target peptide from serum. We evaluated the concentration of each biomarker at different age ranges and after GH administration in healthy participants. RESULTS: The assay was linear over an estimated concentration range of 0.3 to1.0 nM and 0.1 to 0.4 nM for each surrogate peptide of P-III-CP and collagen fragments, respectively. Intra-day and inter-day coefficients of variation were ≤15%. Biomarker concentrations appeared to vary with age and to reflect age-specific collagen turnover. Moreover, their concentrations changed after GH administration. CONCLUSIONS: Our method quantifies the proteins belonging to the family of P-III-CP and type III collagen degradation products in human serum, which could be used to detect GH administration in athletes and better understand diseases involving GH therapy or altered type III collagen turnover.

Procollagen type III amino-terminal propeptide and insulin-like growth factor I as biomarkers of growth hormone administration.

The acceptance in 2012 by the World Anti-Doping Agency (WADA) of the biomarker test for human growth hormone (hGH) based on procollagen type III amino-terminal propeptide (P-III-NP) and insulin-like growth factor I (IGF-I) was perhaps the first time that such a method has been used for forensic purposes. Developing a biomarker test to anti-doping standards, where the strict liability principle applies, is discussed. An alternative WADA-accepted approach is based on the measurement of different hGH isoforms, a method that suffers from the very short half-life of hGH limiting the detection period. Modification or withdrawal of the immunoassays, on which the biomarker measurements largely depend, has necessitated revalidation of the assays, remeasurement of samples and adjustment of the decision limits above which an athlete will be assumed to have administered hGH. When a liquid chromatography coupled mass spectrometry (LC-MS) method became a reality for the measurement of IGF-I, more consistency of results was assured. Measurement of P-III-NP is still dependent on immunoassays although work is underway to develop an LC-MS method. The promised long-term detection time for the biomarker assay does not appear to have been realised in practice, and this is perhaps partly the result of decision limits being set too high. Nevertheless, more robust assays are needed before a further adjustment of the decision limit is warranted. In the meantime, WADA is considering using P-III-NP and IGF-I as components of a biomarker passport system recording data from an individual athlete, rather than the population. Using this approach, smaller perturbations in the growth hormone (GH) score would mandate an investigation and possible action for hGH administration.

Bioanalysis Rising Star Award 2020: announcing our finalists.

Over the past 10 years, Bioanalysis and Bioanalysis Zone have been proud to host the Bioanalysis Rising Star Award (formerly the New Investigator Award), to recognize and showcase the most promising early-career scientists in our community. The time has now come for you to select your winner for the Bioanalysis Rising Star Award 2020. We are delighted to present our judges' selection of finalists (in alphabetical order): Ashley Ross, University of Cincinnati (OH, USA) Chris Williams, QPS (Groningen, The Netherlands) Danielle Moncrieffe, King's College London (UK) Omar Barnaby, Amgen (CA, USA) Sooraj Baijnath, University of KwaZulu-Natal (South Africa) Sumit Kar, Celerion (NE, USA).

Inter-Laboratory Agreement of Insulin-like Growth Factor 1 Concentrations Measured Intact by Mass Spectrometry.

BACKGROUND: Insulin-like growth factor-I (IGF-1) is measured mainly by immunoassay for the diagnosis and treatment of growth hormone (GH) disorders, and to detect misuse of GH in sport. Immunoassays often have insufficient inter-laboratory agreement, especially between commercial kits. Over the expected range of IGF-1 in blood (∼50-500 ng/mL), in an inter-laboratory study we previously established a measurement imprecision of 11% (%CV) for the digested protein analyzed by LC-MS. Measuring intact IGF-1 by LC-MS should be simpler. However, no inter-laboratory agreement has been published. METHODS: Intact and trypsin-digested IGF-1 in 32 serum samples from healthy volunteers and human growth hormone administration studies were analyzed by LC-MS using different instruments in five laboratories, as well as by immunoassay in a single laboratory. Another 100 samples were analyzed for IGF-1, both intact and after trypsin-digestion, in each laboratory by LC-MS. The statistical relationship between measurements and the imprecision of each assay group was assessed. RESULTS: An intra-laboratory variability of 2-4% CV was obtained. Inter-laboratory variability was greater at 14.5% CV. Orthogonal regression of intact versus trypsin-digestion methods (n = 646) gave a slope of 1.01 and intercept of 2.05 ng/mL. CONCLUSIONS: LC-MS measurements of IGF-1 by intact and trypsin-digestion methods are not statistically different and each is similar to immunoassay. The two LC-MS approaches may be used interchangeably or together to eliminate concerns regarding an immunoassay IGF-1 measurement. Because intact and digested IGF-1 measurements generally agreed within 20% of each other, we propose this as a criterion of assay acceptability.

Peptide selection for the quantification of P-III-NP in human serum by mass spectrometry.

RATIONALE: Procollagen III amino-terminal propeptide (P-III-NP) is currently monitored in human doping control as a biomarker for growth hormone administration and also in clinical diagnostics using immunoassays. Drawbacks to this approach have been highlighted and research is ongoing to develop a mass spectrometric method to complement these methods. However, a lack of traceable reference material, the presence of post-translational modifications (PTMs), and small blood concentration complicate the development of targeted analytical methods for P-III-NP quantification. METHODS: Tryptic digest products of P-III-NP were assessed by liquid chromatography/mass spectrometry (LC/MS). In silico digestion was used to predict P-III-NP peptides for MS analysis; however, these excluded PTMs. With a priori knowledge of PTMs, we associated experimental P-III-NP peptides with those derived by in silico digestion. Synthesized P-III-NP peptides, hT1 (human) and T5 (human/bovine), were used to develop sensitive micro- and nano-flow LC/MS methods to analyse P-III-NP originating from human serum semi-quantitatively. RESULTS: P-III-NP peptides, T1 and T5, were identified using high-resolution accurate MS (HRAMS). PTMs modified the mass of observed peptides. N-terminal pyroglutamation (pE) in T1 and several hydroxylated prolines (hP) in T5 (G-X-hP motif) were observed. With PTM, hT1 and T5 were observed in a digest of immuno-captured P-III-NP by LC/MS. Using a semi-quantitative approach, hP-III-NP at basal concentrations of 2 ng/mL (50 pmol) could be estimated from a 200-µL sample volume. CONCLUSIONS: Consideration of PTMs is needed to identify P-III-NP peptides produced by digestion with trypsin. The information presented here now gives the most appropriate peptide sequences for synthesizing suitable reference materials required for quantification of human P-III-NP in blood and evidences methodology that is sufficiently sensitive to develop a quantitative method.