| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1 Biochemical Genetics Laboratory, Department of Laboratory Medicine & Pathology, Mayo Clinic and Foundation, Rochester, MN 55905
aaddress correspondence to this author at: Biochemical Genetics Laboratory, Hilton 360, Department of Laboratory Medicine & Pathology, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905; fax 507-266-2888, e-mail rinaldo{at}mayo.edu
5-Hydroxyindole-3-acetic acid (5-HIAA), the primary metabolite of the neurotransmitter serotonin, is one of the biochemical markers measured in urine by different methods [colorimetric, fluorometric, HPLC, gas chromatography, and immunoassay (1)(2)] in the identification and surveillance of intestinal carcinoid syndrome (3). Usually, 1–3% of dietary tryptophan is metabolized to serotonin. However, as much as 60% of tryptophan is converted to serotonin in patients with the intestinal carcinoid syndrome. Modestly increased urinary 5-HIAA has been seen in Whipple disease, celiac disease, tropical sprue, and some noncarcinoid pancreatic islet tumors. Diet is also a factor affecting urinary excretion of 5-HIAA (4)(5). Our laboratory is actively involved in the conversion to tandem mass spectrometry (MS/MS) of assays currently based on other chromatographic technologies to improve quality and to reduce turnaround time, supply costs, and personnel needs in a high-volume testing environment (6)(7)(8)(9)(10). We directed our efforts toward developing a liquid chromatography (LC)-MS/MS method to replace an interference-prone HPLC procedure currently used for the routine determination of urinary 5-HIAA (2).
We purchased 5-HIAA from Sigma. HIAA-d6 was custom-synthesized by Cambridge Isotope Laboratories (Andover, MA). Working solutions of 5-HIAA (0.5–150 mg/L) were prepared in acetic acid (10 mL/L) from an aqueous stock solution (1 g/L). A working solution of HIAA-d6 (10 mg/L) was prepared in acetic acid (10 mL/L) from a methanol stock solution (1 g/L). Bond-Elut® C18 solid-phase extraction (SPE) cartridges were obtained from Varian Inc. All other chemicals and solvents were of the highest purity available from commercial sources and were used without further purification.
We mixed 0.5 mL of urine with 0.5 mL of internal standard solution (HIAA-d6; 5 µg) in a 10 x 75 mm glass culture tube and placed the tube in a tray on the processor. Reversed-phase C18 SPE was performed on a Gilson ASPECTM after the SPE cartridges were preconditioned with 1 mL of methanol, followed by 1 mL of reverse osmosis water. The 1-mL urine–internal standard mixture was added to the cartridge, which was then washed with 0.5 mL of reverse osmosis water; 5-HIAA and HIAA-d6 were then eluted in 1 mL of LC mobile phase [200 mL/L acetonitrile–800 mL/L aqueous formic acid (0.5 g/L)]. Cycle time for the sample preparation was 7 min/specimen. The elution tubes were removed from the processor and mixed, and aliquots were transferred to autosampler vials for analysis. Calibrators were prepared in 10 mL/L acetic acid by the addition of stock solution (1 g/L) corresponding to a blank and 5-HIAA concentrations of 0.5, 5.0, 12.5, 25, 50, 100, and 150 mg/L and were extracted as described.
We used an API 2000 tandem mass spectrometer (Applied Biosystems/MDS SCIEX) operated with a pneumatically assisted electrospray ionization source in the negative-ion mode. A selected reaction monitoring (SRM) experiment was optimized to monitor the m/z 190
146 and m/z 196152 transitions for 5-HIAA and HIAA-d6, respectively. LC injection volume was 10 µL with the LC flow rate set at 1 mL/min. Separation of 5-HIAA and HIAA-d6 from the specimen matrix was achieved by use of a Discovery® RP Amide C16 HPLC column (50 x 4.6 mm; 5-µm bead size; Supelco) with the LC column effluent flow split to deliver 200 µL/min to the tandem mass spectrometer. 5-HIAA and HIAA-d6 coeluted with a retention time of 1.5 min; the run time was 2 min/sample.
Careful choice of the LC-MS/MS mobile phase was necessary, as acidification is required to separate 5-HIAA from the specimen matrix but also causes some signal suppression. At low concentrations, a urine specimen in which the calculated 5-HIAA concentration was 0.6 mg/L exhibited a signal-to-noise ratio for the extracted 5-HIAA SRM signal of 8.3:1 (injected amount equal to 6 ng).
The interassay (n = 8) variability of calibration data obtained over concentrations from 0.5 to 150.0 mg/L was examined. The mean slope, intercept, and coefficient of linear regression (R2) were 1.78 (95% confidence interval, 1.63–1.93), 0.05 mg/L (-0.09 to 0.2 mg/L), and 0.9997, respectively. The lower limit of detection, defined as a minimum signal-to-noise ratio of at least 3:1 for the extracted 5-HIAA SRM signal, was 0.03 mg/L, with linear response extending to at least 250 mg/L.
Recovery experiments were conducted with unused portions of urine specimens submitted for 5-HIAA determination. We added 2.2 mg/L (n = 4), 4.4 mg/L (n = 4), or 8.8 mg/L (n = 4) 5-HIAA to the specimens and analyzed them in single determinations. Recoveries ranged from 100% to 118% over the range of concentrations. Intraassay precision was determined by replicate analyses in a single run at three concentrations (n = 20). Interassay precision was determined by analysis of four concentrations over 5 weeks (n = 22). The precision data are shown in Table 1
.
|
For method comparison, unused portions of 232 specimens routinely analyzed for 5-HIAA by HPLC (based on dilution, HPLC separation, and electrochemical detection) were reanalyzed with the new LC-MS/MS method. Major limitations of the HPLC method include the long analytical time (13 min/sample), lack of positive peak identification, and incomplete resolution from interfering compounds. Of the original 263 specimens analyzed for method comparison, HPLC determinations for 21 specimens were precluded by chromatographic interference, and 10 specimens were beyond the linearity limit of the HPLC and were repeated as a dilution. The method-comparison statistics were generated from the remaining 232 specimens. The regression equation for the LC-MS/MS method (y) and the HPLC method (x) was: y = (1.003 ± 0.009)x - (0.12 ± 0.08); R2 = 0.98. Comparison of the LC-MS/MS vs HPLC methods by a Bland–Altman plot (11) showed a mean difference for HIAA values of -0.1 mg/L (Fig. 1
).
|
Bench-top LC-MS/MS systems are finding wider use and application in modern clinical laboratories. Improvements in analytical methods that enable the unambiguous identification of low-molecular-weight analytes with automated sample preparation can yield significant gains in efficiency and effective utilization of laboratory resources. Accordingly, we have developed and validated a LC-MS/MS method for the determination of 5-HIAA in urine, using a stable-isotope-labeled internal standard. Our method uses automated SPE, isocratic LC elution, and quantification against a stable-isotope-labeled internal standard. The SPE can be set up to process samples unattended and off shift. Elution with LC mobile phase eliminates the need to evaporate and reconstitute the SPE eluate and provides an eluate containing 5-HIAA, whereas more nonpolar components are retained on the C18 SPE cartridge. During a large-scale analyses, the presence of these nonpolar components in the SPE eluate could lead to their irreversible retention on the LC column, or elution as "ghosts", causing signal suppression in subsequent patient analyses. Urine collected at acidic pH is necessary to suppress the in situ ionization of the carboxyl moiety and facilitates retention of 5-HIAA on the reversed-phase SPE stationary matrix. Preparation of the internal standard (HIAA-d6) in 10 mL/L acetic acid ensures that every specimen has an adequately acidic pH, eliminating the need to verify and adjust the pH of urine specimens before HPLC analysis.
The rate of repeat analyses for the HPLC method was 15%. Implementation of the LC-MS/MS method with an extended calibration range (0–150 mg/L) reduced the number of repeat analyses from 4% to <1%. The remaining 11% of HPLC repeat analyses were performed as mostly unsuccessful attempts to resolve chromatographic interferences caused by unidentified compounds. Under these circumstances, the specimen was deemed unsatisfactory, and a repeat urine collection was requested. As described for the method comparison, with the LC-MS/MS method, we were able to provide interference-free analytical results for 100% of specimens that had to be excluded because of chromatographic interference in the HPLC analysis.
In summary, we have developed a method for the routine determination of urinary 5-HIAA that overcomes the major limitations of an existing HPLC procedure. In particular, sample preparation is fully automated and analytical time is considerably shorter (2 vs 13 min/sample) with virtually no need for repeat analyses because of chromatographic interference or dilutions.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  H. Perry and B. Keevil Online extraction of 5-hydroxyindole acetic acid from urine for analysis by liquid chromatography-tandem mass spectrometry Ann Clin Biochem, March 1, 2008; 45(2): 149 - 152. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. J. Mulder, A. Oosterloo-Duinkerken, G. M. Anderson, E. G.E. De Vries, R. B. Minderaa, and I. P. Kema Automated On-Line Solid-Phase Extraction Coupled with HPLC for Measurement of 5-Hydroxyindole-3-acetic Acid in Urine Clin. Chem., September 1, 2005; 51(9): 1698 - 1703. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Magera, A. L. Thompson, D. Matern, and P. Rinaldo Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Vanillylmandelic Acid in Urine Clin. Chem., May 1, 2003; 49(5): 825 - 826. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
