Abstract
This technical note describes a simple dilution sample preparation method and LC-MS/MS analysis for vanillylmandelic acid in urine samples, achieving a 0.025 μg/mL limit of quantitation (LOQ). Using the QTRAP 6500+ system and 20 μL injection volume, the method demonstrated good quantitative performance in urine-matrix spiked calibration and quality control (QC) standards. Specifically, the 0.025 μg/mL LOQ standard showed a mean accuracy of 111% and precision of 8.7%CV (n=3). Urine-matrix QC standards (n=5) were evaluated at 0.3, 30 and 60 μg/mL and showed mean accuracy between 93.4% and 100.4% with mean precision <3%CV. Good linearity was observed in the urine-spiked calibration standards with an r² value of 0.990 for the quantifier transition across the 0.025- 70 μg/mL calibration range. Finally, the Phenomenex Synergi Polar-RP column showed good peak shape and analyte retention using the 5 min linear gradient.
Key features of the QTRAP 6500+ system for the quantitation of vanillylmandelic acid in urine samples
- Sub-ng/mL sensitivity in urine-matrix calibration standards: Using the QTRAP 6500+ system, the in-sample LOQ was 0.025 μg/mL in the urine-spiked calibrators with mean accuracy of 111% and precision of 8.7%CV (n=3)
- Good quantitative performance in spiked urine QC standards: The QC standards were evaluated at 0.3, 30 and 60 μg/mL (n=5) and showed mean accuracies ranging from 93.4% to 100.4% and mean precision ranging from 1.2%CV to 2.4%CV
- Good analyte peak shape and retention: The combination of Phenomenex Synergi Polar-RP column and gradient conditions showed good peak shape, retention and void volume separation with a retention factor (k’) 1.48 within the 5 min runtime.
Introduction
Vanillylmandelic acid (VMA) is the terminal metabolite of the catecholamine hormones, epinephrine and norepinephrine, and is primarily excreted in the urine. 1 Elevated urinary VMA levels are associated with catecholamine-secreting tumors, including neuroblastoma, pheochromocytoma, and paraganglioma in children.2 Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used for the quantitation of urinary VMA levels due to its high specificity, sensitivity, capacity for multiplexed small-molecule biomarkers, and suitability for high-throughput workflows. In this technical note, a simple dilution-based sample preparation procedure and an LC-MS/MS method using the QTRAP 6500+ system were developed for the rapid quantitation of VMA in human urine.
Methods
Reagent and standard preparation: The analyte and internal standard (IS) were purchased from LGC Standards. Intermediate stock solutions were prepared in methanol and stored at -20°C. The IS working solution was prepared at 20 ng/mL in methanol.
Urine-spiked calibration standards and QC sample preparation: The blanks, calibration standards (n=3), and QC samples (n=5) were prepared in acidified control urine using the Sigmatrix Urine Diluent (Millipore Sigma) as the matrix. To prepare the acidified urine, 0.125 mL of acetic acid was added to 50 mL of the Sigmatrix Urine Diluent. Urine-spiked calibration standard and QC samples were prepared using the scheme shown in Table 1.
Sample preparation: The double blanks, blanks, calibration standards, and QC samples were prepared by 1000-fold dilution in two stages. First, 980 µL of HPLC-grade water was aliquoted into 2 mL microcentrifuge tubes. For the calibration standards and QC samples, 20 μL of the corresponding urine-spiked calibration standard or QC sample was added, and the tubes were vortexed for 10 s. For the double blank and blank samples, 20 μL of the acidified control urine was added, and the tubes were vortexed for 10 s. Second, the blank, calibration standard and quality control samples were further diluted by aliquoting 50 μL of the initial dilution sample into a clean tube containing 900 μL of HPLC-grade water and vortexed for 10 s. Then, 50 µL of the IS solution was added, and the tubes were vortexed again. The double blank samples were diluted by aliquoting 50 μL of the initial dilution into a clean tube containing 950 μL of HPLC grade water and vortexed. Finally, all samples were transferred to autosampler vials for LC-MS/MS analysis.
Mass spectrometry: Samples were analyzed using the QTRAP 6500+ system with electrospray ionization operating in negative polarity mode. Data was acquired using multiple reaction monitoring (MRM) with the optimized source gas parameters shown in Table 3 and the compound-specific parameters in Table 4. Two MRMs per compound were monitored.
Data processing: Data acquisition and processing were performed using the SCIEX OS software (version 3.4.5). The raw peak area count was normalized to the IS response.
Sensitivity, accuracy and precision in urine-spiked calibration standards
The chromatographic conditions were extensively optimized to achieve good peak shape, retention, and separation of the analyte from the void volume. Further, using the Phenomenex Synergi Polar-RP column, the analyte retention time was ~1.1 min, with a retention factor (k’) of 1.48, during the 5 min runtime. The enhanced sensitivity of the QTRAP 6500+ system enabled a 1000-fold dilution, reducing matrix effects and improving data quality while maintaining low detection limits.
Triplicate samples of each urine-spiked calibration standard were used to evaluate the method sensitivity, accuracy, precision and linearity. The in-sample LOQ was 0.025 μg/mL, and linearity ranged from 0.025 to 70 μg/mL in the urine-spiked calibrators (see Figure 1 for the LOQ XIC and calibration curve). The LOQ selection was based on the following criteria for both the quantifier and qualifier transitions: signal-to-noise (S/N) ratio ≥ 10 for both MRMs, accuracy of (±30%), precision %CV (<15%) and ion ratio tolerance of ± 30%. The LOQ mean accuracy was 111%, and the mean precision was 8.7%CV. The matrix-spiked calibration standards yielded a linear dynamic range (LDR) with 3 orders of magnitude and an r²-value of 0.99 with the 1/x² weighting factor. The internal standard normalized mean accuracy of the full calibration standard set ranged from 77.3% to 119%. The quantitative data for the quantifier transition of the calibration standards are shown in Table 5.
Carry-over was evaluated by analyzing a double blank immediately after the highest calibration standard (70 μg/mL). Across the batch, no vanillylmandelic acid peak was detected in the double blank (Figure 2), demonstrating negligible carryover in the LC-MS/MS system.
Quantitative performance in urine-spiked QC standards
The QC standards (n=5) were prepared at 0.3 μg/mL (low). 30 μg/mL (mid), and 60 μg/mL (high) to further evaluate the method performance. The QC standards were quantified against the matrix-spiked calibration standards. The IS normalized mean accuracy ranged from 93.4% to 100.4% and the mean precision ranged from 1.2% to 2.4% CV. The urine-spiked QC standard recovery from the quantifier MRM transition were shown in Table 6. Overall, these results illustrate the sensitivity and capability of the QTRAP 6500+ system for the quantitative analysis of vanillylmandelic acid in urine with good accuracy and precision.
Conclusions
This technical note demonstrated:
- An LC-MS/MS method, with a dilution-based sample preparation procedure, for the quantitation of vanillylmandelic acid in urine using the QTRAP 6500+ system
- Excellent peak shape and retention from the void volume using the Phenomenex Synergi Polar-RP column with a short 5 min linear gradient
- An in-sample LOQ of 0.025 μg/mL in the urine spike calibration standards (n=3) with a mean accuracy of 111% and mean precision of 8.7% CV
- The urine-spiked calibration curve showed an r² value of 0.990 across the 0.025-70 μg/mL range
- Good quantitative performance in the urine matrix QC standards (n=5); mean accuracy was 93.4-100.4%, precision <3% CV
References
- Eisenhofer, G.; Kopin, I.J.; Goldstein, D.S. Catecholamine metabolism: a contemporary view with implications for physiology and medicine. Pharmacol. Rev. 2004, 56(3), 331-349. DOI: 10.1124/pr.56.3.1
- Eisenhofer, G.; Peitzsch, M.; Bechmann, N.; Huebner, A. Biochemical diagnosis of catecholamine-producing tumors of childhood: neuroblastoma, pheochromocytoma and paraganglioma. Front. Endocrinol. 2022, 13, 901760. DOI: 10.3389/fendo.2022.901760