Serotonin in Serum as a Biomarker of Clinical Disorders

Authors: Pierre Picard, Jean Lacoursière and Serge Auger
Themes: High-Throughput, Serum, Serotonin, LDTD-MS/MS
From: Phytronix Technologies, Québec, Canada
Download PDF

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:

 

 

Azeo
Figure 3 – Automated extraction system

 

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:

 

 

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.

 

serotonin-curve
Figure 4 – Serotonin calibration curve

 

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:

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.