MSACL 2016 US Abstract

Development and Validation of an LC-MS/MS Sulfonylurea Assay for Hypoglycemia Cases in the Emergency Department

He Yang (Presenter)
UCSF General Hospital

Bio: I am currently a clinical chemistry fellow at UCSF. I had five-year medical school in China, and received my PhD from Northwestern University. In my current position, my training focuses on clinical application of mass spectrometry, toxicology and case analysis in collaboration with the CA Poison Control Center.

Authorship: He S. Yang, Alan HB Wu, Kara L. Lynch
Department of Laboratory Medicine, University of California San Francisco, San Francisco General Hospital, San Francisco, CA 94110.

Short Abstract

Detection of the presence of sulfonylurea-type oral antidiabetics allows for the differential diagnosis of hypoglycemia of unknown origin and rules out other pathophysiology conditions. We developed and validated a qualitative LC-MS/MS assay to detect sulfonylureas in serum. Compounds were identified by retention time, two MRM transitions and an ion ratio. Linearity, LOD, precision, matrix effect, recovery, carry-over and stability of the final method were evaluated. Method comparison studies with an outside reference laboratory using 51 authentic patient samples revealed a correlation coefficient of 0.99. The assay had been utilized in 19 hypoglycemia cases in the ED and assisted differential diagnosis.

Long Abstract

Background: Sulfonylurea derivatives are a class of antidiabetic drugs that are commonly used in the treatment of type-II diabetes mellitus. Sulfonylurea medications inhibit ATP-sensitive potassium channels in pancreatic beta cell membrane and increase endogenous insulin release. Unlike other classes of oral antidiabetic drugs such as the biguanides and thiazolidinediones, sulfonylurea drugs are more likely to causes hypoglycemia with overdose or when ingested by nondiabetic patients. In most cases, patients with sulfonylurea poisoning present with undifferentiated hypoglycemia, with symptoms including confusion, difficulty speaking, hemiparesis, anxiety, sweating, palpation, seizures and coma. However, the signs and symptoms can also be caused by other diseases that increase insulin secretion or decrease insulin availability, such as insulinoma or insulin antibodies. In addition, sepsis, hepatic failure, and Addison’s disease could also lower blood glucose to dangerous levels. Therefore, detection of the presence of sulfonylurea allows for the correct identification of the underlying cause of hypoglycemia and rule out the other causes.

Methods: 250 μL of serum samples, calibrators and controls spiked with 10 ul internal standard (glipizide-d11, 100 μg/mL) were extracted by protein precipitation using 750 μl of acetonitrile. The mixture was agitated for 15 s, and then centrifuged at 10,000 rpm for 10 min. The supernatant was separated from the protein pellet and evaporated under nitrogen in a 37°C water bath. The drugs were reconstituted in 100 μL of the LC-MS/MS buffer (80% mobile phase A and 20% mobile phase B). Separation of eight sulfonylurea compounds was performed using an Agilent high-performance liquid chromatography (HPLC) with a Phenomenex Luna C8(2) 100Å, 50×2.0 mm LC column. The column was used at 50 °C with a gradient elution at a flow rate of 0.4 mL/min. Mobile phase A consisted of 20 mM ammonium acetate in 95:5 water/methanol, and mobile phase B consisted of 100% methanol. The gradient started at 30% B, was linearly increased to 50% B in 1 min, then increased from 50 - 85% B in 4.5 min, held at 95% B for 2 min, and re-equilibrated at initial condition for additional 2.5 min. An ABSciex 3200 Qtrap was utilized with an electrospray ionization source (ESI) operated in the positive mode. Validation of the final method included determining the lower limit of detection (LOD), linearity, precision, matrix effect, recovery, carryover and processed sample stability for each analyte. 51 serum samples from the patients who had been prescribed for sulfonylurea were tested by the newly developed LC-MS/MS assay, and sulfonylurea concentrations in the positive samples were quantified from the standard curve. These samples were then sent to an outside reference laboratory for testing. Deming regression analysis was employed to compare the two different methods.

Results: Calibration curves of each analyte exhibited consistent linearity and reproducibility in the range of 100 – 10,000 and 5 – 1,000 ng/mL for the first- and second-generation sulfonylureas, respectively. Regression coefficients (r) are higher than 0.990 for all analytes. The LOD of each analyte was 1 ng/mL with the signal-to-noise ratio higher than 10:1. The cutoffs for clinical reporting were selected to be 100 and 5 ng/ml for the first and second generation drugs, respectively, which were consistent with other reference lab cutoffs. Extraction recovery of this assay ranged from 81 – 94%, with an average of 91%. The mean ion suppression at 100 and 500 ng/mL were 91 and 98%, respectively. Intra-assay precision studies yielded CVs ranging from 7 – 11%, 8 – 13%, and 4 – 6% using the three levels of quality control samples at 200/20, 2000/200 and 8000/800 ng/mL, respectively. Inter-assay precision studies yielded CVs ranging from 12 – 15%, 10 – 14%, and 10 – 13% at the three concentration levels. In addition, no carryover was observed after injection of samples that contained high concentrations of compounds. The sulfonylureas were found to be stable in the samples that were tested in three consecutive days after extraction.

51 samples from the patients who had been prescribed for sulfonylureas were tested by the LC-MS/MS method. 25 samples were positive for glipizide, one was positive for glimepiride, and three were positive for glyburide. The concentrations of glipizide ranged between 11.6 and 1260 ng/mL. All the peaks were confirmed by the method of the reference laboratory, and it detected an additional glipizide peak (6 ng/mL) and a glyburide peak (7 ng/mL). The correlation across the concentration range yielded a Deming regression slope of 1.24 (95% Cl: 1.17 to 1.32), and intercept of -37.9 (95% Cl: -71.7 to -4.1), and r = 0.99. Across the range of glipizide concentrations, the newly developed LC-MS/MS method showed a mean percent bias of 11.9%.

Between April 2014 and September 2015, the presented analytical method had been used to screen for sulfonylureas in 19 hypoglycemia cases in the emergency department, in which four cases were positive for glipizide and one case was positive for glimepiride. The turnaround time was less than 12 hours.

Conclusion: We have developed and validated a sensitive and rapid LC-MS/MS assay for the detection of sulfonylurea in serum. The assay was fully validated and correlated well with the method of an outside reference laboratory. It was successfully utilized in hypoglycemia cases in the ED and provided real-time information to assist differential diagnosis.

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