Methods for quantifying insulin-like growth factor-1 and insulin-like growth factor-2
Inventors
Dunbar, Carmen • Bajaj, Amol O. • Kushnir, Mark M.
Assignees
University of Utah • University of Utah Research Foundation Inc
Publication Number
US-12105102-B2
Publication Date
2024-10-01
Expiration Date
2043-03-30
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Abstract
Disclosed herein are methods of determining a concentration of IGF-1 and/or IGF-2 with improved sensitivity using high performance liquid chromatography (HPLC) and mass spectrometry (MS). The methods can achieve improved sensitivity by using a supercharging reagent as part of a mobile phase of the HPLC, which can increase the amount of specific charge states of IGF-1 and/or IGF-2 ions detected by MS. An example method includes subjecting a sample to HPLC, wherein the HPLC comprises a mobile phase including a supercharging reagent and an organic acid; ionizing the sample to produce one or more ions detectable by mass spectrometry; determining amounts of IGF-1 ions in an 8+ charge state by mass spectrometry; and relating the amount of the determined IGF-1 ions in the 8+ charge state to the concentration of IGF-1 in the sample.
Core Innovation
The invention discloses methods for the quantification of insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-2 (IGF-2) in a sample with improved sensitivity using high performance liquid chromatography (HPLC) and mass spectrometry (MS). The core of the innovation involves the use of a supercharging reagent, such as dimethyl sulfoxide (DMSO), in the HPLC mobile phase. Inclusion of this supercharging agent leads to enhanced formation of IGF-1 and/or IGF-2 ions in specific higher charge states (notably the 8+ charge state), which are more readily detected and quantified by MS.
The problem addressed by the invention is the limited sensitivity in existing methods for quantifying IGF-1 and IGF-2, which is essential for improved diagnosis and treatment of various diseases. Higher sensitivity allows detection of lower concentrations of IGF proteins, which is critical in biological and clinical settings.
The disclosed method includes subjecting the sample to HPLC with a mobile phase containing both a supercharging reagent and an organic acid, followed by ionization to generate detectable ions for MS analysis. The abundance of IGF-1 and/or IGF-2 ions in the 8+ charge state—determined through MS—is then directly related to the concentration of the IGF in the sample. This approach achieves a lower limit of detection (LOD) and enhances quantitative accuracy compared to methods lacking a supercharging reagent in the mobile phase.
Claims Coverage
There are two independent claims, each directed to a method for determining the concentration of IGF-1 using HPLC and mass spectrometry with enhanced sensitivity involving DMSO in the mobile phase.
Use of DMSO in HPLC mobile phase for IGF-1 quantification
A method of determining a concentration of IGF-1 in a sample comprising: - Subjecting the sample to high performance liquid chromatography (HPLC) with a mobile phase comprising DMSO at about 1% by volume. - Ionizing the sample to produce ions detectable by mass spectrometry, with the result that the amount of IGF-1 ions in an 8+ charge state is greater than the amount of IGF-1 ions in a 6+ or 7+ charge state. - Determining the amount of IGF-1 ions in the 8+ charge state by mass spectrometry. - Relating the amount of IGF-1 ions in the 8+ charge state to the concentration of IGF-1 in the sample. - The method achieves a limit of detection (LOD) of less than or equal to 4 ng/ml.
Method with DMSO and organic acid in the mobile phase
A method of determining a concentration of IGF-1 in a sample comprising: - Subjecting the sample to HPLC utilizing a mobile phase comprising DMSO at about 1% by volume and an organic acid. - Ionizing the sample to produce ions detectable by mass spectrometry, resulting in an amount of IGF-1 ions in an 8+ charge state that is greater than that in a 6+ or 7+ charge state. - Determining the amount of IGF-1 ions in the 8+ charge state by mass spectrometry. - Relating the amount determined to the concentration of IGF-1 in the sample.
The independent claims broadly protect the use of HPLC with a DMSO-containing (and optionally organic acid-containing) mobile phase to increase the abundance of IGF-1 ions in the 8+ charge state for sensitive quantification by mass spectrometry.
Stated Advantages
The methods provide improved sensitivity in quantifying IGF-1 and IGF-2 by increasing the abundance of ions in the 8+ charge state, enabling detection and quantification at lower concentrations.
The use of DMSO as a supercharging reagent in the HPLC mobile phase results in lower limits of detection and quantification for IGF-1 and IGF-2 compared to current LC-MS methods.
The method increases absolute signal intensity for IGF ions in higher charge states, allowing for more accurate and reliable measurement.
The method enables strong correlation and comparability between quantification using the new 8+ charge state and existing charge state measurements.
Documented Applications
The method can be used for determining IGF-1 and IGF-2 concentrations in biological samples, such as plasma or serum.
Measurement of circulating IGF-1 and IGF-2 levels is a useful tool in the management of various growth hormone-related disorders, clinical evaluation, epidemiological research, and clinical trials.
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