Quantitation of insulin by high resolution/high accuracy mass spectrometry using the 6+ multiply charged insulin ion

A method for determining the amount of insulin in a human plasma or serum sample by high resolution/high accuracy mass spectrometry, the method comprising:

• (a) subjecting the sample to solid phase extraction, to form an extracted insulin sample;
• (b) purifying the extracted insulin sample by high performance liquid chromatography to form a purified insulin sample;
• (c) subjecting the purified insulin sample to an ionization source under acidic conditions suitable to generate a multiply charged insulin ion, wherein the multiply charged insulin ion is detectable by mass spectrometry;
• (d) determining the amount of the one or more multiply charged insulin ion by high resolution/high accuracy mass spectrometry conducted at a FWHM of 10,000 or higher, and a mass accuracy of 50 ppm or less;
• wherein the multiply charged insulin ion is a 6+ charged insulin ion with a mass to charge ratio (m/z) of about 968.8±1.5;
• and
• (e) using the amount of the 6+charged insulin ion to determine the amount of insulin in the sample.