TY - JOUR
AU1 - Mulder, Erik, J
AU2 - Oosterloo-Duinkerken,, Alida
AU3 - Anderson, George, M
AU4 - , De Vries, Elisabeth GE
AU5 - Minderaa, Ruud, B
AU6 - Kema, Ido, P
AB - Abstract Background: Quantification of 5-hydroxyindole-3-acetic acid (5-HIAA) in urine is useful in diagnosing and monitoring of patients with carcinoid tumors and in the study of serotonin (5-hydroxytryptamine) metabolism in various disorders. We describe an automated method that incorporates on-line solid-phase extraction (SPE) and HPLC to measure urinary 5-HIAA. Methods: Automated prepurification of urine was accomplished with HySphere-resin GP SPE cartridges containing strong hydrophobic polystyrene resin. The analyte (5-HIAA) and internal standard [5-hydroxyindole-3-carboxylic acid (5-HICA)] were eluted from the SPE cartridge, separated by reversed-phase HPLC, and detected fluorometrically with a total cycle time of 20 min. Urinary excretion of 5-HIAA was measured in a group of patients with known and suspected carcinoid tumors (n = 63) and in 20 patients with autism. Results: The internal standard (5-HICA) and 5-HIAA were recovered in high yields (87.2%–114%). Within- and between-series CVs for the measurement of 5-HIAA in urine ranged from 1.2% to 3.9% and 3.2% to 7.6%, respectively. For urine samples from patients with known or suspected carcinoid tumors, results obtained by the automated method were highly correlated (r = 0.988) with those from an established manual extraction method. For samples from autistic patients, urinary excretion of 5-HIAA was similar to that reported for healthy individuals. Conclusion: This SPE-HPLC method demonstrated lower imprecision and time per analysis than the manual solvent extraction method. The neurotransmitter/neurohormone serotonin [5-hydroxytryptamine (5-HT)]1 is synthesized from the essential amino acid tryptophan in the enterochromaffin cells of the gut and in serotonergic neurons in the central nervous system (1)(2). Peripheral serotonin is metabolized mainly in the lung and the liver through the action of monoamine oxidase-A. 5-Hydroxyindole-3-acetic acid (5-HIAA) is the predominant end-product of serotonin metabolism and is subsequently excreted in urine (3). Serotonin is clearly involved in carcinoid syndrome and is hypothesized to play a role in schizophrenia, depression, migraine, and autism (4)(5)(6). The most pronounced deviations in serotonin metabolism are found in patients with carcinoid tumors, which can secrete large amounts of serotonin (7). Quantification of urinary 5-HIAA is important in the diagnosis and follow-up of such patients. Diagnosis can be established by measuring urinary 5-HIAA or platelet serotonin. When serotonin production of the tumor exceeds platelet storage capacity, however, changes in production will not be reflected in platelet serotonin concentrations, whereas changes in the amount of 5-HIAA excreted in urine will still occur (8). Increased platelet serotonin has also been observed in autistic patients, with typical reported group mean increases of 25%–50% (5). For the study of peripheral serotonin metabolism in autism, measurement of 5-HIAA in urine is useful to evaluate a possible role of altered production or catabolism in observed changes in platelet serotonin (5). Since the early 1950s, many analytical methods have been described to measure 5-HIAA in urine (9). The first methods used colorimetric methodology, paper and thin-layer chromatography, or gas chromatography. Later, various HPLC analyses were reported that used colorimetric, fluorometric, or electrochemical detection (9). Recently, liquid chromatographic–tandem mass spectrometric methods have been reported for urine and whole blood (10)(11). We developed an automated method with on-line solid-phase extraction (SPE) and HPLC with fluorometric detection to measure urinary 5-HIAA and applied this method to the determination of urinary 5-HIAA excretion rates in individuals with known or suspected carcinoid tumors and in individuals with autism. Materials and Methods reagents and stock solutions HPLC-grade acetonitrile and methanol were obtained from Rathburn; 5-HIAA and disodium EDTA were from Sigma; tetrabutylammonium bromide was from Romil; dipotassium EDTA was from Brunschwig; 5-hydroxyindole-3-carboxylic acid (5-HICA) was from Janssen Chimica; and acetic acid, sodium metabisulfite (Na2S2O5), ascorbic acid, phosphoric acid, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate were from Merck. All reagents were of at least analytical reagent grade. Reagent-grade water, obtained from a Barnstead system, was used throughout. Stock solutions containing 0.34 mmol/L 5-HICA or 2.1 mmol/L 5-HIAA were prepared in water containing 20 mL/L glacial acetic acid. The 5-HICA stock solution was stored protected from light at −20 °C for a maximum of 1 year. The 5-HIAA stock solution was prepared on the day of analysis. Saturated stock solutions containing ascorbic acid (300 g/L), dipotassium EDTA (750 g/L), and Na2S2O5 (750 g/L) were prepared in water and stored at room temperature for 3 weeks. samples and participants Patient urine samples were collected into brown polypropylene bottles (Sarstedt) containing ∼250 mg each of Na2S2Os and disodium EDTA. Urine samples were acidified to pH 4 with acetic acid and stored at −20 °C. Before analysis, aliquots of thawed urine samples (50 μL) were mixed with 300 μL of antioxidant solution (100 μL of ascorbic acid stock solution, 100 μL of dipotassium EDTA stock solution, and 100 μL of Na2S2O5 stock solution) and 100 μL of internal standard stock solution (0.34 mmol/L 5-HICA). The mixture was then diluted with 1.2 mL of water, and 20 μL of each sample was injected into the SPE-HPLC system as described below. This injection volume was equivalent to 0.61 μL of urine. For method-comparison studies, we used spot urine samples from 63 patients with suspected or known carcinoid tumors and 24-h urine specimens from 20 individuals with autism (12). analysis and quantification Instrumentation. The Prospekt-2 (Spark Holland) on-line SPE unit consisted of 3 modules: an SPE controller unit (automated cartridge exchanger), a solvent delivery unit (high-pressure dispenser), and an autosampler (Triathlon). A SparkLink software package (Ver. 2.10) was required for control of the Prospekt-2 modules. The main task of the automated cartridge exchangers was exchange of disposable SPE cartridges. The high-pressure dispenser had a single-syringe configuration and delivered solvents for conditioning, sample application, and clean up of SPE cartridges. HySphere-resin GP cartridges (10 × 2 mm; Spark Holland) were used for cleaning up samples and concentrating analytes. Samples were injected with a Triathlon autosampler. The HPLC pump used was a Gynkotek Series P580A binary high-pressure gradient pump (Softron); detection was with a Waters 474 spectrofluorometer (excitation wavelength, 280 nm; emission wavelength, 360 nm). The temperature of the column was regulated with a Mistral column oven (Spark Holland). For analytical HPLC, a 100 × 4.6 (i.d.) mm Brownlee Spheri-5 RP-18 column filled with 5-μm spherical particles (Inacom) was used. The analytical column was preceded by a 15 × 3.2 (i.d.) mm Aquapore RP18 ODS guard column filled with 7-μm spherical particles (Inacom). Detector output was integrated by ChromPerfect (Ver. 3.51) integration software (Justice Innovations). Eluents. The isocratic system consisted of eluent B (11 mmol/L dipotassium hydrogen phosphate and 3 mmol/L tetrabutylammonium bromide in water adjusted to pH 6.0 with phosphoric acid, added to 80 mL of acetonitrile to a total eluent volume of 1 L). Eluent A (22 mmol/L potassium dihydrogen phosphate in water adjusted to pH 2.0 with phosphoric acid, added to 10 mL of acetonitrile to a total eluent volume of 1 L) was delivered to the high-pressure dispenser. Before use, eluent B was filtered through a 0.45 μm membrane filter (Schleicher & Schuell) and was degassed before entering the HPLC pump. The flow rate of eluent B was 1.0 mL/min, and chromatography was performed at 20 °C. On-line SPE. On-line SPE was performed according to the schedule specified in Table 11 and as depicted schematically in Fig. 11 . The system was designed to proceed automatically through a series of programmable routines during which the SPE cartridge was loaded, purged for clean up, and eluted to the analytical column. During the loading routine, the SPE cartridge was conditioned sequentially with methanol, eluent A, 5 g/L dipotassium EDTA in water, and eluent A, all provided by the high-pressure dispenser at flow rates of 2000 μL/min. The mixing coil between the high-pressure dispenser and the autosampler facilitated mixing of the eluents before they reached the cartridge. After the mixed eluents reached the cartridge, the autosampler injected the sample, thereby loading the cartridge with the sample, using eluent A. The high-pressure dispenser then continued to wash the loaded cartridge with eluent B, thus cleaning the cartridge; the analytes were then eluted from the cartridge to the analytical column with eluent B in 1.5 min. After elution from the cartridge, chromatographic separation on the analytical column occurred. During this separation, the next sample was loaded on the cartridge and washed. When the pump was ready, this sample was eluted to the analytical column. If necessary, cartridges could be changed automatically. 5-HIAA was quantified by calculation of the peak-area ratios in samples relative to those of the internal standard (5-HICA). Sample peak-area ratios were compared with the peak-area ratios obtained for the calibration solutions at 6 different concentrations, which were prepared by addition of known amounts of 0.21 mmol/L 5-HIAA calibrator solution (prepared fresh daily by diluting the stock solution 10-fold with water). Urine creatinine was measured by a picric acid method on a Merck Mega Analyzer. analytical characteristics In on-line SPE, detection limits depend on the extent of sample preconcentration on the SPE cartridge. For urine, we determined detection limits by injecting samples with decreasing concentrations of 5-HIAA. The detection limit was defined as the injected amount that produced a signal-to-noise ratio of 3. We estimated the percentage of carryover between sequential analyses performed on a single SPE cartridge by alternating injections of blanks and urine samples with high concentrations of 5-HIAA. quality control and validation of the automated urinary 5-hiaa method Recoveries were estimated by the addition of 5-HIAA in 3 different concentrations (160, 350, and 520 μmol/L) to 1 urine specimen. Recoveries were measured in 6 replicates of these samples and calculated relative to the recovery of the internal standard; mean recoveries and ranges are reported. Within- and between-series precision was determined by use of 3 samples with 5-HIAA concentrations in the low, medium, and high ranges. Within-series precision was assessed in 6 replicates analyzed in a single series; between-series precision was assessed on 6 different days over a 1-month period. Experiments were performed using 1 SPE cartridge per series. The identities of sample 5-HIAA peaks were verified by addition of calibrator (standard addition) and by observation of retention time changes of the sample peak under different chromatographic conditions. The automated urinary 5-HIAA method was also validated by comparison of its results with those obtained by our routinely used manual ether extraction method (13). statistics We compared methods by Deming regression analysis using the slope and intercept; we also calculated the correlation coefficient and the standard error of prediction (Sy|x). The Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) EP-6P protocol (14) was applied to test the linearity of the method. Results chromatography The chromatographic separation of 2 calibrators by automated on-line SPE and HPLC with fluorometric detection is shown in Fig. 2A2 . Total cycle time was ∼20 min (Table 11 ). Chromatograms of urine samples obtained from a healthy adult and from a patient with a carcinoid tumor are shown in panels B and C, respectively, of Fig. 22 (note the different sensitivity scales). Complete separation of the analytes from interferents was achieved within 16 min. Comparison of the 2 chromatograms shows markedly increased 5-HIAA in the sample from the patient. analytical characteristics A signal-to-noise ratio of 3 was achieved at a 5-HIAA concentration of 0.8 μmol/L. Analysis of a split urine specimen with increasing amounts of added 5-HIAA calibrator demonstrated that the assay was linear to 5-HIAA concentrations of at least 2000 μmol/L. The percentage carryover between sequential analyses performed on a single SPE cartridge was <1%. quality control and validation of the automated urinary 5-hiaa method The results of the recovery and precision experiments are presented in Table 22 . The recovery data represent recoveries for 6 replicates of each of 3 samples analyzed in 1 analysis time series with 1 SPE cartridge. Recoveries ranged from 87.2% to 114.0%. Within-series imprecision (as CVs) based on 6 measurements of 3 different urine samples were 3.9%, 1.6%, and 1.2% in samples with mean (SD) 5-HIAA concentrations of 2.3 (0.1), 25.0 (0.4), and 147.9 (1.7) μmol/L, respectively. The between-series imprecision (CVs) for analyses performed across 6 series were 7.6%, 3.7%, and 3.2% in samples with 5-HIAA concentrations of 2.5 (0.2), 26.3 (1.0), and 154.8 (3.2) μmol/L, respectively. As seen in Fig. 33 , 5-HIAA values determined in 63 clinical samples by the automated SPE method were highly correlated with results obtained by the manual ether extraction method (13). Deming regression gave a slope of 0.9996 (range, 0.9811–1.0181) and a y-intercept of −3.50 μmol/L (95% confidence interval, −9.92 to 2.92 μmol/L). The Spearman correlation coefficient was 0.988 (P <0.0001), and the SE of prediction (Sy|x) was 3.21 μmol/L. The lack of linear fit test according to the CLSI EP-6P protocol indicated no significant deviance from linearity (F = 0.22; P = 0.80). In the autism group, mean (SD) 24-h urinary 5-HIAA excretion [0.0165 (0.0086) mmol/24 h or 1.74 (0.79) mmol/mol creatinine] was similar to that reported previously for control groups and autistic patients (9)(15)(16)(17). Discussion A variety of methods for urine 5-HIAA analysis have been described, including several HPLC methods (9). The automated SPE-HPLC method we developed reduced the per sample analysis time considerably, with a concomitant increase in sample throughput. The CVs for this method were ∼50% lower than those for the manual method. We found that use of the on-line SPE system allows regeneration of SPE cartridges up to 40 times when small amounts of urine or plasma are extracted (18). This can be done with negligible loss of accuracy. During the 2-year period that this method has been in operation, interferences from drugs and other substances have been extremely rare (and discernable). The group mean urine concentrations and excretion rates reported here for 5-HIAA were within the reference intervals established at the University Hospital Groningen and were in excellent agreement with previous reports (9)(15)(16)(17). In summary, our automated SPE-HPLC method for the determination of urinary 5-HIAA appears to offer advantages in time and precision compared with manual chromatographic methods. Table 1. Summary of the sequence of events used for on-line SPE, as described in Materials and Methods. Step . Action . Details . . Comment . 01 New cartridge Left clamp Put cartridge in clamp 02 Solvation Methanol (2000 μL at 2000 μL/min) SSM 1A Activate cartridge with methanol 03 Equilibration Eluent A (2000 μL at 2000 μL/min) SSM 1B Condition cartridge with eluent A 04 Equilibration 5 g/L dipotassium EDTA (2000 μL at 2000 μL/min) SSM 1D Condition cartridge with dipotassium EDTA 05 Equilibration Eluent A (2000 μL at 2000 μL/min) SSM 1B Condition cartridge with eluent A 06 Start autosampler Start loopfill Fill loop autosampler with sample 07 Sample application Eluent A (2000 μL at 2000 μL/min) SSM 1B Inject sample (load sample on cartridge using eluent A) 08 Wash cartridge Eluent B (560 μL at 1000 μL/min) SSM 1C Wash cartridge with eluent B (removal of interferents) 09 Inputs Input 1 high Pump ready? 10 Outputs Auxiliaries 1 and 2 Start pump and start data 11 Elution 1.5 min Elution of sample from cartridge to analytical column 12 Move cartridge Left to tray Cartridge back to tray Step . Action . Details . . Comment . 01 New cartridge Left clamp Put cartridge in clamp 02 Solvation Methanol (2000 μL at 2000 μL/min) SSM 1A Activate cartridge with methanol 03 Equilibration Eluent A (2000 μL at 2000 μL/min) SSM 1B Condition cartridge with eluent A 04 Equilibration 5 g/L dipotassium EDTA (2000 μL at 2000 μL/min) SSM 1D Condition cartridge with dipotassium EDTA 05 Equilibration Eluent A (2000 μL at 2000 μL/min) SSM 1B Condition cartridge with eluent A 06 Start autosampler Start loopfill Fill loop autosampler with sample 07 Sample application Eluent A (2000 μL at 2000 μL/min) SSM 1B Inject sample (load sample on cartridge using eluent A) 08 Wash cartridge Eluent B (560 μL at 1000 μL/min) SSM 1C Wash cartridge with eluent B (removal of interferents) 09 Inputs Input 1 high Pump ready? 10 Outputs Auxiliaries 1 and 2 Start pump and start data 11 Elution 1.5 min Elution of sample from cartridge to analytical column 12 Move cartridge Left to tray Cartridge back to tray Table 1. Summary of the sequence of events used for on-line SPE, as described in Materials and Methods. Step . Action . Details . . Comment . 01 New cartridge Left clamp Put cartridge in clamp 02 Solvation Methanol (2000 μL at 2000 μL/min) SSM 1A Activate cartridge with methanol 03 Equilibration Eluent A (2000 μL at 2000 μL/min) SSM 1B Condition cartridge with eluent A 04 Equilibration 5 g/L dipotassium EDTA (2000 μL at 2000 μL/min) SSM 1D Condition cartridge with dipotassium EDTA 05 Equilibration Eluent A (2000 μL at 2000 μL/min) SSM 1B Condition cartridge with eluent A 06 Start autosampler Start loopfill Fill loop autosampler with sample 07 Sample application Eluent A (2000 μL at 2000 μL/min) SSM 1B Inject sample (load sample on cartridge using eluent A) 08 Wash cartridge Eluent B (560 μL at 1000 μL/min) SSM 1C Wash cartridge with eluent B (removal of interferents) 09 Inputs Input 1 high Pump ready? 10 Outputs Auxiliaries 1 and 2 Start pump and start data 11 Elution 1.5 min Elution of sample from cartridge to analytical column 12 Move cartridge Left to tray Cartridge back to tray Step . Action . Details . . Comment . 01 New cartridge Left clamp Put cartridge in clamp 02 Solvation Methanol (2000 μL at 2000 μL/min) SSM 1A Activate cartridge with methanol 03 Equilibration Eluent A (2000 μL at 2000 μL/min) SSM 1B Condition cartridge with eluent A 04 Equilibration 5 g/L dipotassium EDTA (2000 μL at 2000 μL/min) SSM 1D Condition cartridge with dipotassium EDTA 05 Equilibration Eluent A (2000 μL at 2000 μL/min) SSM 1B Condition cartridge with eluent A 06 Start autosampler Start loopfill Fill loop autosampler with sample 07 Sample application Eluent A (2000 μL at 2000 μL/min) SSM 1B Inject sample (load sample on cartridge using eluent A) 08 Wash cartridge Eluent B (560 μL at 1000 μL/min) SSM 1C Wash cartridge with eluent B (removal of interferents) 09 Inputs Input 1 high Pump ready? 10 Outputs Auxiliaries 1 and 2 Start pump and start data 11 Elution 1.5 min Elution of sample from cartridge to analytical column 12 Move cartridge Left to tray Cartridge back to tray Figure 1. Open in new tabDownload slide Schematic representation of the on-line SPE system coupled to HPLC with fluorescence detection. (A), solvation and equilibration step, showing the system during conditioning of the cartridge. (B), filling of the autosampler loop, showing the sample as it is processed by the autosampler. (C), flow of sample to cartridge, showing the system as the sample is on its way to the cartridge, where it will be washed. (D), elution of sample from the cartridge to the column, showing the system during the elution of the cartridge. After this, the system can be programmed to change the cartridge before the next sample is introduced. For additional details, see Table 11 and the Materials and Methods. HPD, high-pressure dispenser; LC, liquid chromatography; P, pump; Det, detector; ACE, automated cartridge exchanger. Figure 1. Open in new tabDownload slide Schematic representation of the on-line SPE system coupled to HPLC with fluorescence detection. (A), solvation and equilibration step, showing the system during conditioning of the cartridge. (B), filling of the autosampler loop, showing the sample as it is processed by the autosampler. (C), flow of sample to cartridge, showing the system as the sample is on its way to the cartridge, where it will be washed. (D), elution of sample from the cartridge to the column, showing the system during the elution of the cartridge. After this, the system can be programmed to change the cartridge before the next sample is introduced. For additional details, see Table 11 and the Materials and Methods. HPD, high-pressure dispenser; LC, liquid chromatography; P, pump; Det, detector; ACE, automated cartridge exchanger. Figure 2. Open in new tabDownload slide Chromatograms of urine from a healthy adult and a patient with carcinoid tumor, as obtained by on-line SPE coupled to HPLC with fluorescence detection. Excitation wavelength, 280 nm; emission wavelength, 360 nm. (A), separation of 5-HIAA and the internal standard 5-HICA (injected amounts, 0.34 nmol for both). (B), 5-HIAA in urine from a healthy adult (note the 10-fold increase in sensitivity). (C), 5-HIAA in urine obtained from a patient with carcinoid tumor. Chromatograms in B and C were obtained with 20-μL injections of diluted urine (equivalent to 0.61 μL of urine). Calculated 5-HIAA concentrations in the urine samples were 8.75 and 506 μmol/L for B and C, respectively. Figure 2. Open in new tabDownload slide Chromatograms of urine from a healthy adult and a patient with carcinoid tumor, as obtained by on-line SPE coupled to HPLC with fluorescence detection. Excitation wavelength, 280 nm; emission wavelength, 360 nm. (A), separation of 5-HIAA and the internal standard 5-HICA (injected amounts, 0.34 nmol for both). (B), 5-HIAA in urine from a healthy adult (note the 10-fold increase in sensitivity). (C), 5-HIAA in urine obtained from a patient with carcinoid tumor. Chromatograms in B and C were obtained with 20-μL injections of diluted urine (equivalent to 0.61 μL of urine). Calculated 5-HIAA concentrations in the urine samples were 8.75 and 506 μmol/L for B and C, respectively. Table 2. Recovery and precision of the on-line SPE method for urinary 5-HIAA excretion. Recovery1 . . . . Added,2 μmol/L . . % (range) . . 160 94.0 (87.2–98.9) 350 99.9 (95.0–109.1) 540 102.1 (95.8–114.0) Recovery1 . . . . Added,2 μmol/L . . % (range) . . 160 94.0 (87.2–98.9) 350 99.9 (95.0–109.1) 540 102.1 (95.8–114.0) Precision3 . . . . Within-series . . Between-series . . Mean (SD) 5-HIAA, μmol/L . CV, % . Mean (SD) 5-HIAA, μmol/L . CV, % . 2.3 (0.1) 3.9 2.5 (0.2) 7.6 25.0 (0.4) 1.6 26.3 (1.0) 3.8 147.9 (1.7) 1.2 154.8 (3.2) 3.2 Precision3 . . . . Within-series . . Between-series . . Mean (SD) 5-HIAA, μmol/L . CV, % . Mean (SD) 5-HIAA, μmol/L . CV, % . 2.3 (0.1) 3.9 2.5 (0.2) 7.6 25.0 (0.4) 1.6 26.3 (1.0) 3.8 147.9 (1.7) 1.2 154.8 (3.2) 3.2 1 Recovery data represent data from 6 measurements of each supplemented sample made in a single series on 1 SPE cartridge. 2 The mean (SD) endogenous concentration of 5-HIAA in the urine sample used in the recovery experiments was 29.2 (1.2) μmol/L. 3 Within-series precision data are derived from 6 measurements of 3 samples (low, medium, and high concentrations) made in a single series. Between-series data are from measurements of the same 3 samples made in 6 different series with 1 SPE cartridge per series. Table 2. Recovery and precision of the on-line SPE method for urinary 5-HIAA excretion. Recovery1 . . . . Added,2 μmol/L . . % (range) . . 160 94.0 (87.2–98.9) 350 99.9 (95.0–109.1) 540 102.1 (95.8–114.0) Recovery1 . . . . Added,2 μmol/L . . % (range) . . 160 94.0 (87.2–98.9) 350 99.9 (95.0–109.1) 540 102.1 (95.8–114.0) Precision3 . . . . Within-series . . Between-series . . Mean (SD) 5-HIAA, μmol/L . CV, % . Mean (SD) 5-HIAA, μmol/L . CV, % . 2.3 (0.1) 3.9 2.5 (0.2) 7.6 25.0 (0.4) 1.6 26.3 (1.0) 3.8 147.9 (1.7) 1.2 154.8 (3.2) 3.2 Precision3 . . . . Within-series . . Between-series . . Mean (SD) 5-HIAA, μmol/L . CV, % . Mean (SD) 5-HIAA, μmol/L . CV, % . 2.3 (0.1) 3.9 2.5 (0.2) 7.6 25.0 (0.4) 1.6 26.3 (1.0) 3.8 147.9 (1.7) 1.2 154.8 (3.2) 3.2 1 Recovery data represent data from 6 measurements of each supplemented sample made in a single series on 1 SPE cartridge. 2 The mean (SD) endogenous concentration of 5-HIAA in the urine sample used in the recovery experiments was 29.2 (1.2) μmol/L. 3 Within-series precision data are derived from 6 measurements of 3 samples (low, medium, and high concentrations) made in a single series. Between-series data are from measurements of the same 3 samples made in 6 different series with 1 SPE cartridge per series. Figure 3. Open in new tabDownload slide Comparison of 5-HIAA results for 63 urine samples as derived from the automated SPE-HPLC method (y axis) and the ether extraction method (x axis). The main panel shows values from 0 to 100 μmol/L. Equation for the line: y = 0.996x − 3.50 μmol/L (r = 0.988; P <0.0001). The inset shows values from 100 to 1500 μmol/L. Figure 3. Open in new tabDownload slide Comparison of 5-HIAA results for 63 urine samples as derived from the automated SPE-HPLC method (y axis) and the ether extraction method (x axis). The main panel shows values from 0 to 100 μmol/L. Equation for the line: y = 0.996x − 3.50 μmol/L (r = 0.988; P <0.0001). The inset shows values from 100 to 1500 μmol/L. 1 Nonstandard abbreviations: 5-HT, 5-hydroxytryptamine (serotonin); 5-HIAA, 5-hydroxyindole-3-acetic acid; SPE, solid-phase extraction; 5-HICA, 5-hydroxyindole-3-carboxylic acid; and CLSI, Clinical Laboratory Standards Institute. 1 Tyce GM. Biochemistry of serotonin. Vanhoutte PM eds. 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Liquid-chromatographic determination of urinary 5-hydroxy-3-indoleacetic acid, with fluorescence detection. Clin Chem 1982 ; 28 : 207 -208. 14 Passey RB, Bee DE, Caffo A, Erikson JM. Evaluation of the linearity of quantitative analytical methods; proposed guideline. NCCLS Document 6[18] 1986 NCCLS (Clinical Laboratory Standards Institute) Villanova, PA. . 15 Anderson GM, Minderaa RB, Cho SC, Volkmar FR, Cohen DJ. The issue of hyperserotonemia and platelet serotonin exposure: a preliminary study. J Autism Dev Disord 1989 ; 19 : 349 -351. 16 Minderaa RB, Anderson GM, Volkmar FR, Akkerhuis GW, Cohen DJ. Urinary 5-hydroxyindoleacetic acid and whole blood serotonin and tryptophan in autistic and normal subjects. Biol Psychiatry 1987 ; 22 : 933 -940. 17 Launay JM, Ferrari P, Haimart M, Bursztejn C, Tabuteau F, Braconnier A, et al. Serotonin metabolism and other biochemical parameters in infantile autism. A controlled study of 22 autistic children. Neuropsychobiology 1988 ; 20 : 1 -11. 18 Kema IP, Meijer WG, Meiborg G, Ooms B, Willemse PH, De Vries EG. Profiling of tryptophan-related plasma indoles in patients with carcinoid tumors by automated, on-line, solid-phase extraction and HPLC with fluorescence detection. Clin Chem 2001 ; 47 : 1811 -1820. © 2005 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Automated On-Line Solid-Phase Extraction Coupled with HPLC for Measurement of 5-Hydroxyindole-3-acetic Acid in Urine
JF - Clinical Chemistry
DO - 10.1373/clinchem.2005.050062
DA - 2005-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/automated-on-line-solid-phase-extraction-coupled-with-hplc-for-K7FtS0zTJd
SP - 1698
VL - 51
IS - 9
DP - DeepDyve
ER -