Online extraction of 5-hydroxyindole acetic acid from urine for analysis by liquid chromatography-tandem mass spectrometry

Background: Urinary 5-hydroxyindole acetic acid (5-HIAA) is a useful marker for the turnover of tryptophan metabolites in the diagnosis and monitoring of carcinoid tumours and the carcinoid syndrome. We have developed a simple and cost-effective assay for urinary 5-HIAA using liquid chromatography-tandem mass spectrometry (LC-MS/MS) incorporating an online sample clean-up process to replace a liquid chromatography electrochemical (LC-EC) technique. Methods: Acidified urine was serially diluted in ammonium acetate buffer followed by ammonium acetate buffer enriched with 5-hydroxyindole-3-acetic-2,2-D2 acid internal standard. A 2.1 x 10 mm C18 column was used for primary online clean-up and eluted with 100% methanolic mobile phase onto a second dC18 Atlantis 2.1 x 20 mm column. Analytes were detected by mass spectrometry using transitions 192.1 > 146.3 and 194.1 > 148.0 for 5-HIAA and deuterated analyte, respectively. Results: Run time was 3 min with 5-HIAA eluting at 1.37 min. The inter and intra-assay imprecision and accuracies of the three levels of inhouse quality control (QC) (30, 300 and 600 mu mol/L) were acceptable with coefficient of variations (CVs) and deviation from target values < 12% (n = 15). The average recovery of 5-HIAA spiked into urine was 93.7% with no ion suppression observed. The limit of detection was 2.8 and lower limit of quantification 4.0 mu mol/L. Passing-Bablok regression of LC-EC with LC-MS/MS results showed good agreement between the methods, the relationship described as LC-MS/ MS = 1.01(LC-EC)-1.22. No systematic or proportional biases were observed over the working range of the method. The assay was linear to at least 2000 mu mol/L. Conclusions: We have developed a robust method offering a more than six-fold improvement in linearity compared to the existing LC-EC method.