Cory Muraco , Stacy Shollenberger , Hillel Brandes
MilliporeSigma, Bellefonte, PA USA
Insulin is an approximately 6000 Da peptide hormone used to regulate the glucose concentration in blood. There are several different forms of insulin that are both natural and recombinant; the recombinant forms have been engineered to improve pharmacokinetic properties.1 A quick and reliable chromatographic technique was developed to identify and detect a mixture of insulin variants in an unknown sample. This is of utmost importance to pharmaceutical quality control (QC) labs as some formulations of insulin-containing drugs needs to include only a single, correct form of insulin for the drug to elicit its biotherapeutic effects or not to elicit an allergic response from the patient.2
A mixture of six insulin variants (insulin-bovine, insulin-human, insulin-porcine, insulin-lispro, insulin-Asp, and insulin-glargine, 100 µg/mL, 0.1% aqueous trifluoroacetic acid) was analyzed chromatographically on a BIOshell™ A160 Peptide C18 HPLC column using a Thermo Ultimate™ BioRS 3000 UHPLC (UV detection) and an Agilent® 1290/6530 Q-TOF system (mass spectral detection). All analyses were performed in triplicate and the elution order of the insulin variants was confirmed by mass spectral analysis.
Figure 1. Separation of Insulin Variants by RPC
Figure 2. Resolution of Insulin Variants: Effect of Flow Rate
Figure 3.Peak Width, Half Height Versus Flow Rate for the Six Insulin Variants
Figure 4. Analysis of Insulin Variants by LC/MS using DFA
Figure 5. Analysis of Insulin Variants by LC/MS Using 8 mM Ammonium Formate, pH 2.6
*Determined by deconvolution of raw, mass spectral profile data.
Figure 6. Comparison of Signal Intensities Between Two LC/MS Methods
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Thermo Ultimate is a trademark of Thermo Fisher Scientific Inc.