Quantification of Free Estrone in Serum Using Luxon Ion Source

Authors: Serge Auger, Pierre Picard and Jean Lacoursière
Themes: High-Throughput, Estrone, Serum, LDTD®-MS/MS
From: Phytronix Technologies, Québec, Canada
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Introduction

The rapid quantification of free estrone in serum can be useful to monitor estrogen levels and help explain abnormal menstrual cycles, symptoms of menopause, abnormal or heavy bleeding, infertility or any other hormonal alterations.

Our goal for this application note is to develop a diagnostic tool to rapidly do the quantification of free estrone in serum, which will allow the rapid diagnosis of abnormal disorders.

LDTD®-MS/MS offers specificity combined with an ultra-fast analysis for an unrivaled quantification method. To develop this application, we focused on performing an efficient sample preparation, which will increase the speed of the analysis process and confirm diagnoses more quickly.

Sample Preparation Method

A stock solution of Estrone was prepared in methanol at 1 mg/mL. Then, a bovine serum albumin solution (20 mg/mL in PBS) was spiked to generate a calibration curve and QCs.

Automated Sample Extraction

An automated system (Figure 3) is used to extract the samples using the following conditions:

Figure 3 – Automated extraction system – Azeo Liquid Handler

 

LDTD®-MS/MS Parameters

LDTD

Model: Luxon S-960, Phytronix

Carrier gas: 5.0 L/min (air)

Laser pattern:

MS/MS

MS model: QTrap® System 5500, Sciex

IonSpray Voltage: -5500

Temperature: GS1 and GS2 set to zero.

CUR: 20

Scan Time: 50 msec

Analysis Method: Negative MRM mode

 

Transition CE (V)
Estrone 269.0 → 145.0 -50
Estrone-d4 273.0 → 147.0 -50

 

Results and Discussion

Validation Test

Calibration curves ranging from 10 to 250 pg/mL were prepared in Bovine serum albumin (20 mg/mL dilute in PBS buffer). A set of QCs were prepared in same matrix. 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 Estrone. Values greater than 0.995 are obtained. Figure 4 shows a typical calibration curve result for Estrone.

 

Figure 4 – Estrone calibration curve

 

Table 2 – Inter-day calibration curve correlation coefficients
R
Curve 1 0.99763
Curve 2 0.99848
Curve 3 0.99748
Curve 4 0.99811

Precision and Accuracy

For the accuracy and precision evaluation, the following acceptance criteria were used:

For the intra and inter-run precision and accuracy experiment, each replicate of QC was analyzed on four different runs. Table 3 and 4 show the intra and inter-run precision and accuracy results for Estrone. The obtained %CV was below 15% and the accuracy was within 15% of the nominal value.

Table 3 – Intra-Run Precision and Accuracy of Estrone
Estrone LLOQ QC-L QC-M QC-H ULOQ
Conc (ng/mL) 10 30 125 187.5 250
N 6 6 6 6 6
Mean (ng/mL) 10.6 31.0 115.3 196.2 261
%CV 12.9 5.5 3.7 3.1 4.1
%Nom 105.7 103.2 92.2 104.6 104.4

 

Table 4 – Inter-Run Precision and Accuracy of Estrone
Estrone QC-L QC-M QC-H
Conc (ng/mL) 30 125 187.5
N 15 15 15
Mean (ng/mL) 31.3 115.4 197.2
%CV 5.9 4.1 3.1
%Nom 104.2 92.3 105.2

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. The precision and accuracy results of QCs are reported in Figure 5 and Figure 6. All the results are within the acceptable criteria range for 1 day at 4°C.

Dry Stability of Samples Spotted on LazWell™

Extracted samples are spotted onto a LazWell plate, dried and kept at room temperature for 2 hours before analysis. The precision and accuracy results of QCs are reported in Figure 5 and Figure 6. All the results are within the acceptable criteria range for 2 hours at room temperature.

Figure 5 – Precision of sample after stability tests

 

Figure 6 – Accuracy of sample after stability tests

Cross Validation Study

Serum samples from real patients were tested with this method to correlate with results obtained by traditional LC-MS/MS. The percentage difference between the values ​​is evaluated. A difference of less than 20% is obtained. The results are reported in Table 5.

Table 5 – Cross validation results
LC

(pg/mL)

Luxon

(pg/mL)

%Diff
M1 64.1 64.5 0.6
M2 55.1 61.2 10.4
M3 18.2 19.5 7.1
M4 18.9 20.1 6.3
M5 12.7 13.3 4.3
M6 12.9 15.3 16.9

Conclusion

The Luxon Ion Source combined with the Sciex QTrap® 5500 mass spectrometer system enables the rapid quantification of free estrone in serum. This method of analysis can thus be applied at the clinical level in order to diagnose different estrone disorders.