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Direct qualitative methods that allow the rapid screening and identification of insulin products during early stages of the drug development process and those already in the market can be of great utility for manufacturers and regulatory agencies and the recent scientific literature describes several methods. Herein, a qualitative proteomic method is presented for the identification of recombinant human insulin and all marketed biosynthetic analogues -insulin lispro, aspart, glulisine, glargine, detemir and degludec- via tryptic digestion and identification of proteotypic peptides for each insulin. Individual insulins were first denatured under reducing conditions and the cysteine residues blocked by iodoacetamide. The proteins were then digested with trypsin and the peptide products separated by reversed phase liquid chromatography on an Ascentis® Express ES-C18 column and detected by positive polarity ESI-MS/MS. The digestion peptides were characterized using a multiplexed MRM approach that monitors the fragmentation of the doubly charged unlabeled precursor ion of each peptide into a collection of signature y and b ions. The MRM transitions for the individual peptides were optimized to allow maximal ionization on a standard triple quadrupole mass spectrometer. All products of the digestion procedure for all insulins were detected with adequate signal intensity except for the C-terminal B30Thr whenever it was present and cleaved and the tryptic B1-3 tripeptide of insulin glulisine. The unique proteotypic peptides identified for each of the insulin analogues coupled with their signature y and b ions permitted the unambiguous verification of all sequence variations and chemical modifications. The elution of the A polypeptide chain for all insulins and the tryptic peptides of the B chain, with the exception of a very few, occurred around the same time point. This underscores the close similarity in the physicochemical properties between the digestion peptides and is consistent with the subtle variations in amino acid sequence among the various insulins. Therefore, the identification and distinction of the different types of insulin based solely on the chromatographic retention time of their respective proteolytic products can be deceptive without proper mass spectrometric analysis and may result in false positives.
This article was published in the following journal.
Name: Journal of pharmaceutical and biomedical analysis
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A syndrome with excessively high INSULIN levels in the BLOOD. It may cause HYPOGLYCEMIA. Etiology of hyperinsulinism varies, including hypersecretion of a beta cell tumor (INSULINOMA); autoantibodies against insulin (INSULIN ANTIBODIES); defective insulin receptor (INSULIN RESISTANCE); or overuse of exogenous insulin or HYPOGLYCEMIC AGENTS.
Diminished effectiveness of INSULIN in lowering blood sugar levels: requiring the use of 200 units or more of insulin per day to prevent HYPERGLYCEMIA or KETOSIS. It can be caused by the presence of INSULIN ANTIBODIES or the abnormalities in insulin receptors (RECEPTOR, INSULIN) on target cell surfaces. It is often associated with OBESITY; DIABETIC KETOACIDOSIS; INFECTION; and certain rare conditions. (from Stedman, 25th ed)
Regular insulin preparations that contain the HUMAN insulin peptide sequence.
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Insulin formulation containing substance which delays or retards time period of the absorption of insulin.
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