Simultaneous Analysis of Intact Human Insulin and Five Analogs in Human Plasma Using μElution SPE and a CORTECS UPLC Column

IN T RO DU C T IO N Insulin is perhaps one of the best known and earliest peptide therapeutics. Multiple long and fast-acting analogs have also been developed, and a patient may often be prescribed one of each for diabetes control. Quantification of biologics, such as insulins, has historically been performed using ligand binding assays (LBAs) such as ELISAS. LC/MS/MS, however, has certain advantages over LBAs, such as shorter development times, higher accuracy and precision, the ability to multiplex, no cross-reactivity, and the ability to readily distinguish between closely related insulins. Intact insulins are particularly difficult to analyze by LC/MS/MS, as MS sensitivity is low due to poor transfer into the gas phase, and poor fragmentation patterns exist due to the presence of multiple stabilizing disulfide bonds. In addition, insulin and its analogs suffer from nonspecific binding and poor solubility, making LC and sample preparation method development difficult. A few LC/MS/MS methods do exist; however, most of those methods involve time-consuming and laborious immunoaffinity purification and/or nano-flow LC. Distinguishing between human insulin and insulin lispro (Humalog) is a very specific challenge for quantifying insulins, as they differ by a simple reversal in the position of two amino acids. Only a single low-molecular weight fragment differentiates the two, making selective sample preparation and chromatography critical.