Robotic Extraction & Analysis of Insulin Analogs [Agilent]Julian Lee
Insulin, like other drugs and chemical agents, may be implicated or suspected as a cause of death, though toxicological analysis of insulin remains a challenge due to complexities associated with traditional immunoassay screening (cross-reactivity between endogenous and pharmaceutical analogs). Recently, multiple liquid chromatography tandem mass spectrometry (LC-MS/MS) based assays have been developed to alleviate these concerns. However, complex multistage sample preparation techniques (e.g., protein precipitation coupled to solid phase extraction or antibody immunopurification), as well as difficulties with mass analysis (i.e., poor fragmentation, low specificity transitions, analog/isotope coelution, and a reliance on low flow microbore or nanobore chromatography), remain a challenge. Consequently, the determination of insulin in postmortem cases is not routinely performed.
The analytical workflow described in this webinar enables unambiguous differentiation of human insulin as well as five pharmaceutical analogs, including insulin Glargine (Lantus®), Glulisine (Apidra®), Lispro (Humalog®), Aspart (NovoLog®), Human (Humulin®), and Detemir (Levemir®), using robotic immuno-microchromatography coupled with insulin β-chain analysis by LC-MS/MS. Briefly, insulin extraction was performed on the Agilent AssayMap Bravo with Protein-G cartridges. Before extraction, human vitreous humor was diluted with PBS buffer and fortified with porcine insulin as an internal standard. Cartridges were primed and conditioned with PBS buffer prior to loading two mouse anti-Insulin monoclonal antibodies to generate anti-insulin immunoaffinity microchromatography cartridges. Diluted vitreous humor was then loaded onto the immunoaffinity cartridges, washed sequentially, and eluted at low pH. Following a brief incubation, insulin beta chains were analyzed in positive MRM mode on an Agilent 6495 triple quadrupole mass spectrometer coupled with a 1290 series UHPLC. Chromatographic separation was performed using an Agilent RRHD 300Å SB-C18 1.8 µm, 2.1 x 50 mm analytical column with a stepwise separation at 0.4 mL/min over 9 minutes.
Analytical method validation was performed in accordance with the SWGTOX guidelines for Standard Practices for Method Validation in Forensic Toxicology. Parameters evaluated included linear range, limit of quantitation, limit of detection, accuracy & precision, interference, carryover, and stability (4°C and – 20°C up to 30 days). Aside from remaining challenges with stability, all analogs performed within criteria for acceptable performance. In addition to the validation results, samples from several cases involving a suspected death by insulin have been analyzed. A summary of the case history and results, as well as an interpretation of findings, will be disclosed.
Detailed Learning Objectives:
1. Describe the chemistry of insulin and the challenges surrounding traditional and modern analytical approaches.
2. Demonstrate basic proficiency in large molecule mass spectrometry and the advantages of automated sample preparation.
3. Implement a validated workflow for the quantitation of insulin from post-mortem samples.
Price: Funding for this course has been provided by Agilent Technologies
Course Type: On-Demand Presentation
Section: Forensic toxicology
Length: Approximately 60 minutes
Audience: Forensic Practitioners
Course Accreditation: Certificate of Attendance