Development and Validation of a LC-MS/MS Method for Plasma Analysis of the Serotonin Metabolite: 5-Hydroxyindoleacetic acid (5-HIAA)

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1 W Ward, B Begley, R Friley, M Barringer,, J Nofsinger, M Allen, D Hudson, D Witt Tandem Labs, Laboratory Corporation of America Holdings P Rosner, A Wilson, S Wason,, J Townsend, L Law, QM Yang Lexicon Pharmaceuticals, Inc. Development and Validation of a LC-MS/MS Method for Plasma Analysis of the Serotonin Metabolite: 5-Hydroxyindoleacetic acid (5-HIAA) Purpose Serotonin (5-HT) plays a critical role in regulating several major physiological processes of the gastrointestinal (GI) tract, including aspects of secretion, motility, inflammation, and sensation. Enterochromaffin (EC) cells release 5-HT when the intestinal wall is stimulated by intraluminal pressure or chemicals. Through multiple classes of receptors, 5-HT is believed to initiate directly or facilitate peristaltic and secretory reflexes. There is increasing evidence that 5-HT dysregulation is involved in the etiology of irritable bowel syndrome (IBS). Plasma 5-HT levels were found to be higher in diarrheapredominant IBS subjects. 1 Increased EC cell population was noted in the colon of IBS subjects. 2 Furthermore, serotonergic agents, such as 5-HT3 antagonists have been shown to be effective in the treatment of IBS-D. 3,4,5 5-Hydroxyindoleacetic acid (5-HIAA) (Figure 1) is a metabolite of serotonin which is under investigation for use as a biomarker in the diagnosis and treatment of certain GI conditions, including carcinoid tumors and IBS. The detection of 5-HIAA is viewed as a surrogate marker for inhibition of serotonin production. Performance requirements for biomarker assays intended for diagnostic and therapeutic purposes must be carefully evaluated to optimize for clinical accuracy. Fundamental factors considered in development of a method for plasma 5-HIAA included precision and interference testing. Due to the potential of some therapeutics to lower plasma 5-HIAA, a calibration range with an LLOQ lower than nominal endogenous levels was needed. The validation data for the 5-HIAA assay presented herein supports the method s reliability, selectivity, and robustness. W Ward et al. (2014) 1

2 Methods Samples were added to a 96-well plate containing stable-label internal standard in 90/10 ACN/MeOH. Samples were mixed, centrifuged, and supernatants were transferred to a filtration plate. Filtrates were evaporated at 50 C, reconstituted and analyzed using a 2.8 min gradient elution (Table 1) onto a XSELECT HSS T3 Column an AB Sciex API 5000 operated in electrospray mode for positive ion detection (Table 2). See Figures 4-7 for example chromatograms. Area ratios for the standards were used to create a linear calibration curve using 1/x 2 weighted least-squares regression analysis. Table 1. HPLC/MS Conditions Table 2. Mass Transitions W Ward et al. (2014) 2

3 Results The method was validated over a linear range of ng/ml in human plasma (Figure 3). Calibration standards and LLOQ/Low QCs were prepared in charcoal stripped human K 2 EDTA plasma. Additional plasma QCs were analyzed at 4 higher concentrations, including endogenous QCs. Analytical performance and method validation tests were conducted to evaluate assay specificity, precision, accuracy, reproducibility, linearity, concentration range, dilution, extraction recovery, carryover, matrix effects (including hemolysis, hyperlipidemia, and icteric effects), interference, in-process stability (bench-top, freeze/thaw), extract stability, whole blood stability, freezer storage stability, and solution stability. Results from these validation tests confirm the analytical method is acceptable for use in a clinical study. Twenty consecutive AP runs met intra/interassay acceptance criteria (Table 4, Table 5). For QCs in unstripped plasma, intra-assay precision ranged from 0.7 to 4.7%, while inter-assay precision ranged from 3.7 to 5.5% (Figure 2). The intra-assay accuracy ranged from -7.3 to 6.9% and the inter-assay accuracy ranged from -2.6 to 1.3%. Bench-top and freeze-thaw matrix stability were demonstrated for 71 hours and 4 freeze/thaw cycles, respectively. Reinjection reproducibility was established for 103 hours (4 C). See Table 3 for a summary of the validation results. Figure 2. QC Performance for 20 Accuracy and Precision Runs Figure 3. Representative Calibration Curve W Ward et al. (2014) 3

4 Results continued Table 3. 5-HIAA Validation Results W Ward et al. (2014) 4

5 Results continued Table 4. Inter-run Statistics of Calibration Standards for 20 Accuracy and Precision Runs Table 5. Inter-run Statistics Quality Control Samples for 20 Accuracy and Precision Runs W Ward et al. (2014) 5

6 Results continued Figure 4. Representative Low Standard (0.400 ng/ml) Figure 6. Representative Matrix Blank (Charcoal Stripped Plasma) Figure 5. Representative High Standard (100 ng/ml) Figure 7. Representative Endogenous QC W Ward et al. (2014) 6

7 Conclusions The method was successfully validated using criteria and test requirements, meeting or exceeding the principles outlined in the U.S. Code of Federal Regulations Titles 21 Part 58, and 42 Part 493 (GLP and CLIA). The acceptance criteria for this method validation were more stringent than for standard bioanalytical methods (15% and 10% vs. 20% and 15%). Some proprietary therapeutics have shown as little as 30% difference between pre- and post-treatment, so stringent criteria are necessary to optimize clinical accuracy, as method precision will help ensure accuracy of inhibition effects of various therapeutics. References 1. Houghton, et al. Increased platelet depleted plasma 5-hydroxytryptamine concentration following meal ingestion in symptomatic female subjects with diarrhea predominant irritable bowel syndrome. Gut 2003; 52: Spiller, et al. Increased rectal mucosal enterochromaffin cells, T lymphocytes, and increased gut permeability following acute Campylobacter enteritis and in postdysenteric irritable bowel syndrome. Gut 2000; 47: Kerckhoffs, et al. Trypsinogen IV, serotonin transporter transcript levels and serotonin content are increased in small intestine of irritable bowel syndrome subjects. Neurogastroenterol Motil 2008; 20: Lembo, et al. Alosetron controls bowel urgency and provides global symptom improvement in women with diarrhea-predominant irritable bowel syndrome. Am J Gastroenterol 2001;96: Rahimi, et al. Efficacy and tolerability of alosetron for the treatment of irritable bowel syndrome in women and men: a meta-analysis of eight randomized, placebo-controlled, 12-week trials. Clin Ther 2008; 30: W Ward et al. (2014) 7