Holly D. Cox

Interlaboratory Agreement of Insulin-like Growth Factor 1 Concentrations Measured by Mass Spectrometry

BACKGROUND Insulin-like growth factor 1 (IGF-1)(7) is a key mediator of growth hormone (GH) action and a well-characterized biomarker of GH abuse. Current immunoassays for IGF-1 suffer from poor concordance between platforms, which makes comparison of results between laboratories difficult. Although previous work has demonstrated good interlaboratory imprecision of LC-MS/MS methods when plasma is supplemented with purified proteins, the interlaboratory imprecision of an endogenous protein in the nanogram-per-milliliter concentration range has not been reported. METHODS We deployed an LC-MS/MS method to quantify serum IGF-1 in 5 laboratories using 5 different instruments and analyzed 130 healthy human samples and 22 samples from patients with acromegaly. We determined measurement imprecision (CV) for differences due to instrumentation, calibration curve construction, method of calibration, and reference material. RESULTS Instrument-dependent variation, exclusive of digestion, across 5 different instrument platforms was determined to be 5.6%. Interlaboratory variation was strongly dependent on calibration. Calibration materials from a single laboratory resulted in less variation than materials made in individual laboratories (CV 5.2% vs 12.8%, respectively). The mean imprecision for 152 samples between the 5 laboratories was 16.0% when a calibration curve was made in each laboratory and 11.1% when a single-point calibration approach was used. CONCLUSIONS The interlaboratory imprecision of serum IGF-1 concentrations is acceptable for use of the assay in antidoping laboratories and in standardizing results across clinical laboratories. The primary source of variability is not derived from the sample preparation but from the method of calibration.

Sensitive quantification of IGF-1 and its synthetic analogs in dried blood spots

BACKGROUND Dried blood spot sample collection could improve detection of the misuse of IGF-1, its analogs and growth hormone. An LC-MS/MS method was developed to measure two IGF-1 peptides and one analog peptide after trypsin digestion. In addition to standard method validation parameters, the effect of hematocrit on cysteine alkylation, trypsin digestion and the selection of internal standard were evaluated. RESULTS Quantification of IGF-1 peptides was possible with an LLOQ of 25 ng/ml and imprecision of less than 15%. CONCLUSION While the effects of hematocrit must be evaluated empirically for each method, dried blood spots are a suitable matrix for the measurement of IGF-1 and its analogs by MS.

Detection of human insulin‐like growth factor‐1 in deer antler velvet supplements

RATIONALE Reported incidents of the use of nutritional supplements containing deer antler velvet by athletes has increased significantly in recent years. The supplements have been reported to contain insulin-like growth factor-1 (IGF-1), which is a banned substance included on the World Anti-Doping Agency (WADA) prohibited list. The presence of deer and human IGF-1 was tested in six commercially available supplements. METHODS IGF-1 was extracted from the six deer antler velvet supplements using chloroform and acetonitrile precipitation methods. Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) methods were developed to measure intact IGF-1 protein and IGF-1 trypsin peptides using a triple quadrupole mass spectrometer. Five deer-specific and five human-specific multiple-reaction monitoring (MRM) transitions for intact IGF-1were measured as well as six deer-specific and seven human-specific MRM transitions for an IGF-1 trypsin peptide. RESULTS The peak area from each MRM transition was used to calculate the product ion ratios relative to the most abundant transition. Product ion ratios measured in the supplements were matched to ratios measured in purified protein standards. A match to human IGF-1 was identified for all the MRM transitions measured in four of the supplements tested. CONCLUSIONS The presence of a pharmaceutical protein, human IGF-1, was confirmed in four commercially available products sold as all natural, nutritional supplements. These methods can be used to screen additional products to further prevent the illegal sale of adulterated supplements.

Investigation of the metabolites of the HIF stabilizer FG-4592 (roxadustat) in five different in vitro models and in a human doping control sample using high resolution mass spectrometry

HIGHLIGHTSMetabolites from the HIF stabilizer FG‐4592 were investigated.Five in vitro methods and a human doping control sample were used as models.Twelve different metabolites were detected using UHPLC‐QTOF‐MS.Three of the in vitro models had one metabolite in common. ABSTRACT FG‐4592 is a hypoxia‐inducible factor (HIF) stabilizer, which can increase the number of red blood cells in the body. It has not been approved by regulatory authorities, but is available for purchase on the Internet. Due to its ability to improve the oxygen transportation mechanism in the body, FG‐4592 is of interest for doping control laboratories, but prior to this study, little information about its metabolism was available. In this study, the metabolism of FG‐4592 was investigated in a human doping control sample and in five in vitro models: human hepatocytes and liver microsomes, equine liver microsomes and S9 fraction and the fungus Cunninghamella elegans. By using liquid chromatography coupled to a Q‐TOF mass spectrometer operated in MSE and MSMS modes, twelve different metabolites were observed for FG‐4592. One monohydroxylated metabolite was detected in both the human and equine liver microsome incubations. For the fungus Cunninghamella elegans eleven different metabolites were observed of which the identical monohydroxylated metabolite had the highest response. This rich metabolic profile and the higher levels of metabolites produced by Cunninghamella elegans demonstrates its usefulness as a metabolite producing medium. In the doping control urine sample, one metabolite, which was the result of a direct glucuronidation, was observed. No metabolites were detected in neither the human hepatocyte nor in the equine liver S9 fraction incubates.

Mass Spectrometry Method to Measure Membrane Proteins in Dried Blood Spots for the Detection of Blood Doping Practices in Sport

The dried blood spot (DBS) matrix has significant utility for applications in the field where venous blood collection and timely shipment of labile blood samples is difficult. Unfortunately, protein measurement in DBS is hindered by high abundance proteins and matrix interference that increases with hematocrit. We developed a DBS method to enrich for membrane proteins and remove soluble proteins and matrix interference. Following a wash in a series of buffers, the membrane proteins are digested with trypsin and quantitated by parallel reaction monitoring mass spectrometry methods. The DBS method was applied to the quantification of four cell-specific cluster of differentiation (CD) proteins used to count cells by flow cytometry, band 3 (CD233), CD71, CD45, and CD41. We demonstrate that the DBS method counts low abundance cell types such as immature reticulocytes as well as high abundance cell types such as red blood cells, white blood cells, and platelets. When tested in 82 individuals, counts obtained by the DBS method demonstrated good agreement with flow cytometry and automated hematology analyzers. Importantly, the method allows longitudinal monitoring of CD protein concentration and calculation of interindividual variation which is difficult by other methods. Interindividual variation of band 3 and CD45 was low, 6 and 8%, respectively, while variation of CD41 and CD71 was higher, 18 and 78%, respectively. Longitudinal measurement of CD71 concentration in DBS over an 8-week period demonstrated intraindividual variation 17.1-38.7%. Thus, the method may allow stable longitudinal measurement of blood parameters currently monitored to detect blood doping practices.

Evaluation of serum markers for improved detection of autologous blood transfusions

Autologous blood transfusion is used by athletes to enhance performance by increasing oxygen transport to muscles. It is the only form of blood doping that is currently not detectable using a direct method. Autologous blood transfusions are detected by an indirect method which monitors the