Introduction
Serotonin is an indoleamine molecule that is derived from the amino acid tryptophan. Its biological function is complex, and it impacts multiple aspects in our lives, such as our mood, our memory, and even undesirable effects such as vomiting. The determination of the serotonin concentration in a serum sample is used as a biomarker for the diagnosis of carcinoid syndrome and other clinical disorders.
Our goal for this application note is to use an automated sample preparation method for the quantification of serotonin in serum using a single operation in LDTD-MS/MS.
LDTD-MS/MS offers specificity combined with an ultra-fast analysis for an unrivaled quantification method. To develop this application, we focused on performing a quick and simple sample preparation. Serotonin is analyzed and results are obtained in less than 8 seconds per sample.
Sample Preparation Method
Automated Sample Extraction
Serum samples were transferred into barcoded tubes, readable by the Azeo Liquid Handler.
Each barcoded vial was scanned by the Azeo Liquid Handler and an automatic batch file was created. The Azeo extraction system (Figure 3) is used to extract the samples using the following conditions:
- 100 µL of serum sample were transferred from the vials to a deep-well plate placed in the Lumo Vortexer.
- 100 µL of Internal standard (Serotonin-d4, 100 ng/mL in water) were added to each sample.
- Mix
- 200 µL of extraction buffer and 300 µL acetonitrile were added into a deep-well plate.
- Mix
- Centrifuge 2 minutes/5000 rpm
- Spot 4 µL Butylated Hydroxytoluene (BHT, 100 µg/mL) followed by 4 µL upper layer phase onto a LazWell™ 96 plate.
- Dry 4 minutes at 40°C in the Aura LazWell Dryer.
LDTD®-MS/MS Parameters
LDTD
Model: Luxon S-960, Phytronix
Carrier gas: 3 L/min (air)
Laser pattern: 3-second ramp to 45% power
MS/MS
MS model: Q-Trap System® 5500, Sciex
Scan Time: 50 msec
Total run time: 8 seconds per sample
Ionization: APCI
Analysis Method: Negative MRM mode
Table 1 – MRM transitions for LDTD-MS/MS
| Transition | CE (V) | |
|---|---|---|
| Serotonin | 174 → 144 | -25 |
| Serotonin-d4 | 178 → 146 | -25 |
Results and Discussion
Validation Test
Calibration curves ranging from 10 to 500 ng/mL were prepared in a BSA solution (30 mg/mL) and three human serum samples were used as QC (endogenic concentration values were evaluated with a reference method and used as a nominal value). Replicate extractions were deposited onto a LazWell™ plate and dried before analysis. The peak area against the internal standard (IS) ratio was used to normalize the signal.
Linearity
The calibration curves were plotted using the peak area ratio and the nominal concentration of standards. For the linearity test, the following acceptance criteria was used:
- Linear regression (r) must be ≥ 995
Table 2 shows the inter-day correlation coefficients for serotonin. Values greater than 0.999 are obtained. Figure 4 shows a typical calibration curve result for serotonin.
Table 2 – Inter-day calibration curve correlation coefficients
| Serotonin | |
|---|---|
| Curve 1 | 0.99991 |
| Curve 2 | 0.99988 |
| Curve 3 | 0.99969 |
| Curve 4 | 0.99917 |
| Curve 5 | 0.99973 |
Precision and Accuracy
For the accuracy and precision evaluation, the following acceptance criteria were used:
- Each concentration must not exceed 15% CV.
- Each concentration must be within 100 ± 15% of the nominal concentration.
For the inter-run precision and accuracy experiment, each standard was analyzed in triplicate, on five different days. Table 3 shows the inter-run precision and accuracy results for serotonin. The obtained %CV was below 15% and the accuracy was within 15% of the nominal value.
Table 3 – Inter-Run Precision and Accuracy of Serotonin
| Serotonin | QC1 | QC2 | QC3 |
|---|---|---|---|
| Conc (ng/mL) | 71.9 | 120.4 | 148.1 |
| N | 15 | 15 | 15 |
| Mean (ng/mL) | 81.3 | 127.2 | 162.3 |
| SD | 3.50 | 2.98 | 7.42 |
| %CV | 4.3 | 2.3 | 4.6 |
| %Nom | 113.0 | 105.6 | 109.6 |
Wet Stability of Sample Extracts
Following the extraction, sample extracts are kept at 4°C in closed containers. After 1 day, sample extracts are spotted on a LazWell™ plate, dried and analyzed. Precision and accuracy of QC samples are reported in Table 4. All the results are within the acceptable criteria range for 1 day at 4°C.
Dry Stability of Samples Spotted in LazWell™
Extracted samples are spotted onto a LazWell™ plate, dried and kept at room temperature for 1 hour before analysis. Serotonin must be stabilized with BHT to avoid drug degradation on the LazWell™ plate. The precision and accuracy results of QC samples are reported in Table 4. All the results are within the acceptable criteria range for 1 hour at room temperature. An important signal lost is observed after 1 hour. Analysis within 30 minutes after sample evaporation is strongly recommended.
Table 4 – Wet and Dry Stability of Serotonin
| Parameters | Dry stability (1 hour / RT) | Wet stability (1 day / 4°C) | ||||
|---|---|---|---|---|---|---|
| QC | QC1 | QC2 | QC3 | QC1 | QC2 | QC3 |
| Conc. (ng/mL) | 71.9 | 120.4 | 148.1 | 71.9 | 120.4 | 148.1 |
| N | 3 | 3 | 3 | 3 | 3 | 3 |
| Mean (ng/mL) | 82.3 | 135.4 | 168.4 | 71.7 | 121.5 | 149.6 |
| %CV | 0.9 | 3.1 | 1.4 | 2.1 | 1.4 | 1.0 |
| %Nom | 114.4 | 112.5 | 113.7 | 99.8 | 100.9 | 101.0 |
Cross validation study
Real patients’ serum samples (N=12) have been tested with this method to correlate with results obtained by traditional LC-MS/MS. The percentage of difference between the values are evaluated. A difference below 15% is obtained. Results are reported in Table 5.
Table 5 – Comparison between serotonin concentration values
| Serotonin | LC (ng/mL) |
LDTD (ng/mL) |
%Diff (%) |
|---|---|---|---|
| M1 | 148.1 | 157.0 | -5.8% |
| M2 | 71.9 | 82.2 | -13.3% |
| M3 | 120.4 | 125.6 | -4.2% |
| M4 | 197.6 | 205.3 | -3.8% |
| M5 | 119.6 | 138.1 | -14.3% |
| M6 | 169.3 | 180.4 | -6.4% |
| M7 | 49.2 | 55.6 | -12.3% |
| M8 | 80.4 | 85.2 | -5.8% |
| M9 | 64.3 | 71.9 | -11.1% |
| M10 | 93.8 | 99.3 | -5.7% |
| M11 | 80.2 | 85.5 | -6.4% |
| M12 | 107.1 | 117.1 | -8.9% |
Conclusion
The Luxon Ion Source® combined with Sciex Q-Trap 5500 mass spectrometer system allows ultra-fast (8 seconds per sample) analysis of serotonin in serum.