Fast Extraction of VMA, 5-HIAA, and HVA from Synthetic Urine

Introduction

Analytical measurements that target the metabolites of serotonin probe defects in the biochemical systems related to the progressive and degenerative conditions of the gastroin- testinal system as well as cognitive disorders are required in clinical laboratories. The selected metabolites homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA) and vanil- lylmandelic acid (VMA) are endogenous metabolites found in urine. This application note describes a method developed to extract the targeted metabolites from urine using a mixed- mode anion exchange retention mechanism, prior to analysis via LC-MS/MS. In the absence of a true blank with real clinical specimens, the method described in this report was developed using synthetic urine EVOLUTE EXPRESS 96-well SPE plates are optimised for quicker and easier SPE processing. Using a load-wash-elute procedure, traditional conditioning and equilibration steps are eliminated, reducing solvent costs and increasing sample throughput.

Figure 1. Structures of 5-HIAA, HVA and VMA

Analytes

5-hydroxyindoleacetic acid (5-HIAA); homovanillic acid (HVA); vanillylmandelic acid (VMA)

Sample Preparation Procedure

Format:
EVOLUTE EXPRESS AX 96-well plate, part number 603-0030-PX01. Sample Pre-Treatment:
Dilute synthetic urine 1:1 (v/v) with HPLC grade water. Sample Loading:
Load pre-treated sample (1 mL) into wells. Apply positive pressure (PRESSURE+ 96 Positive Pressure Manifold PPM-96) to maintain a flow rate of 1 mL/min (10-12 drops/min). Note: no conditioning or equilibration steps are required. Wash:
Wash with HPLC grade water/methanol (75:25, v/v, 1 mL). Analyte Elution:
Elute analytes of interest with methanol/formic acid (98:2, v/v, 1 mL). Post Extraction:
Evaporate sample to dryness (SPE Dry Dual) and reconstitute in mobile phase (1 mL). Sample Throughput:
Sample plates can be loaded onto a Positive Pressure+ 96 manifold and processed in 1 hour or less

HPLC Conditions

Instrument: Agilent 1200 Liquid Handling System (Agilent Technologies, Berksire, UK) Column: Restek Organic Acids 150 mm x 4.6 mm (5 μm) (catalog # 9165565) Mobile Phase: A: 1% Formic acid in Water B: Acetonitrile Injection Volume: 20 μL Temperature: 35 °C

>> Download the full Application Note as PDF

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