Shun-Hsin Liang (Presenter)
Restek Corporation
Bio: Shun-Hsin Liang, Ph.D. Senior LC Applications Chemist Restek Inc.
Authorship: Shun-Hsin Liang, Frances Carroll, Sharon Lupo, Ty Kahler, Carrie Sprout, Paul Connolly
Restek Corporation
| Short Abstract Vanillylmandelic acid (VMA), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) are final metabolites of epinephrine, dopamine, and serotonin, respectively. Measurement of these metabolites in urine is used for the diagnosis of carcinoid and neuroendocrine tumors including neuroblastoma, pheochromocytoma, and paraganglioma. The development of a simple dilute-and-shoot and yet accurate, specific, and fast LC-MS/MS analysis would greatly reduce the time and cost for this type of clinical test. Using a Raptor™ Biphenyl column, it was demonstrated that VMA, HVA, and 5-HIAA can be simultaneously analyzed in human urine without extensive sample clean up and resulted in accurate and reproducible quantitation. |
Long Abstract
Vanillylmandelic acid (VMA), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) are final metabolites of epinephrine, dopamine, and serotonin, respectively. Measurement of these metabolites in urine is used for the diagnosis of carcinoid and neuroendocrine tumors including neuroblastoma, pheochromocytoma, and paraganglioma. Several commercial products are available for simultaneous analysis of these three compounds in urine. These commercial kits all require extensive sample preparation and chromatographic analysis times up to 16 minutes. The development of a simple and fast methodology would greatly reduce the time and cost for this type of clinical test. In this study, a dilute-and-shoot LC-MS/MS method was developed for accurate and specific quantitation of VMA, HVA, and 5-HIAA simultaneously in urine using a Raptor™ Biphenyl column.
A 10-fold dilution was performed by mixing 40µl of urine sample with 360µl of water and 10µl of internal standard solution (33.3µg/mL of VMA-d3, 5-HIAA-d5, and HVA-d5 in methanol) in a Thomson 0.45µm PVDF filter vial and injected for analysis after filtration. The analysis was performed on a Shimadzu UFLCXR System coupled with a Sciex API 4000™ mass spectrometer.
The linearity range was from 0.2-100 µg/mL for all three analytes. Eight standard solutions (0.2, 0.4, 1.0, 5.0, 10, 25, 50, and 100 µg/mL) were prepared in the synthetic human urine (Surine Negative Urine Control, Cerilliant) and diluted per the sample preparation procedure described above. It was determined that a 1/x weighted quadratic regression is the best fit for the standard curve for all three analytes. The deviations were all ≤10% (the lowest concentration was ≤15%) and the R-square was 0.999-1.000. Estimated from the signal-to-noise value of the 0.2 µg/mL standard injection, the LODs are 0.007, 0.03, and 0.02 µg/mL for VMA, 5-HIAA, and HVA, respectively.
Two levels of urine control samples (Bio-Rad Lyphochek Quantitative Urine Control) were used as QC samples for accuracy and precision analysis. The control level 1 and 2 were indicated as normal and abnormal levels by Bio-Rad and a concentration range for VMA, 5-HIAA, and HVA was determined by Bio-Rad reversed-phase HPLC method. Three sets of analyses were performed and the method accuracy was demonstrated to be acceptable as the quantitative concentrations fell within the nominal concentration of both low and high urine control samples. The %RSD ranged from 0.2-2.8% and 0.2-2.9% for intra-day and inter-day, respectively, indicating an acceptable method precision.
It was demonstrated that the Raptor™ Biphenyl column is excellent for rapid and accurate analysis of VMA, 5-HIAA, and HVA in human urine. With the established dilute-and-shoot method, these catecholamine and serotonin metabolites can be measured with 5 minutes of analysis time. The analytical method is applicable to the clinical analysis of VMA, 5-HIAA, and HVA using a combination of fast analysis times and low-cost sample preparation procedures.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Restek Corporation |
| Board Member | no | |
| Stock | yes | Restek Corporation |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |