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5 Integration of steroids analysis in serum using LC-MS/MS with full-automated sample preparation We found that the sample preparation time was reduced from 60 minutes to 6 minutes by the automated system. Thus sample pr…

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Integration of steroids analysis in serum using LC-MS/MS
with full-automated sample preparation
Fig. 3 Calibration Curves (L1-L7) and MRM Chromatograms (L1) of 10 Steroids
Result and discussion
We evaluated this system using calibrator and control
serum spiked with 10 steroids contained in the kit and
carried out concurrent analysis over a range of
concentrations for each steroid: cortisol (1.51-320
ng/mL), aldosterone (0.03-1.14 ng/mL), 11-deoxycortisol
(0.08-18 ng/mL), corticosterone (0.29-62 ng/mL), 17-OHP
(0.12-26 ng/mL), androstenedione (0.08-18 ng/mL),
DHEA (0.31-65 ng/mL), DHEAS (12.9-2750 ng/mL),
progesterone (0.12-26.5 ng/mL) and testosterone
(0.03-7.2 ng/mL). The calibration curves that were
generated had linear regression values of r2 >0.997 for
each curve. The reproducibility (N=3) at seven
concentrations, including LLOQ of each compounds was
excellent (CV<10%).
Aldosterone Cortisol DHEAS Coricosterone 11-Deoxycortisol
Androstenedione Testosterone 17-OHP DHEA Progesterone
0.00 0.25 0.50 0.75 Conc. Ratio
0.00
0.25
0.50
0.75
Area Ratio
0 100 200 Conc. Ratio
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
Area Ratio
0 1000 2000Conc. Ratio
0
10
20
30
40
50
60
Area Ratio
0.0 25.0 Conc. Ratio
0.0
2.5
5.0
7.5
10.0
12.5
Area Ratio
0.0 5.0 10.0 Conc. Ratio
0
1
2
3
4
5
6
7
Area Ratio
0.0 5.0 10.0 Conc. Ratio
0
5
10
15
20
25
30
35
40
Area Ratio
0.0 2.5 5.0 Conc. Ratio
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
Area Ratio
0 10 Conc. Ratio
0
5
10
15
20
Area Ratio
0.0 25.0 Conc. Ratio
0
1
2
3
4
5
Area Ratio
0 10 20Conc. Ratio
0
10
20
30
40
Area Ratio
Aldosterone
(0.03 ng/mL)
Cortisol
(1.51 ng/mL)
DHEAS
(12.9 ng/mL)
Coricosterone
(0.29 ng/mL)
11-Deoxycortisol
(0.08 ng/mL)
Androstenedione
(0.08 ng/mL)
Testosterone
(0.03 ng/mL)
17-OHP
(0.12 ng/mL)
DHEA
(0.31 ng/mL)
Progesterone
(0.12 ng/mL)
Aldosterone (neg)
(0.03 ng/mL)
DHEAS (neg)
(12.9 ng/mL)
5.0 6.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
(x10,000)
361.20>343.00(+)
361.20>315.20(+)
5.0 6.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
(x100,000)
363.40>97.00(+)
363.40>121.10(+)
4.0 4.5 5.0 5.5
0.00
0.25
0.50
0.75
1.00
1.25
(x100,000)
271.20>197.10(+)
271.20>213.20(+)
6.0 7.0
0.00
0.25
0.50
0.75
1.00
(x100,000)
347.20>97.15(+)
347.20>121.15(+)
6.0 7.0
2.5
5.0
7.5
(x10,000)
347.20>97.05(+)
347.20>109.10(+)
8.0 9.0
0.0
0.5
1.0
1.5
2.0
(x100,000)
287.10>109.15(+)
287.10>97.10(+)
4.0 4.5 5.0 5.5
0.0
0.5
1.0
1.5
2.0
(x1,000,000)
367.10>97.10(-)
5.0 6.0
0.0
0.5
1.0
1.5
2.0
2.5
(x1,000)
359.20>331.35(-)
359.20>189.25(-)
9.0 10.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
(x100,000)
315.20>109.10(+)
315.20>97.05(+)
7.06.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
(x10,000)
271.20>253.15(+)
271.20>213.20(+)
6.5 7.0 7.5 8.0
2.5
5.0
7.5
(x10,000)
331.10>109.00(+)
331.10>97.00(+)
7.0 8.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
(x10,000)
289.10>109.05(+)
289.10>97.15(+)
r
2
=0.997
r
2
=0.999
r
2
=0.999
r
2
=0.998
r
2
=0.997
r
2
=0.999
r
2
=0.999
r
2
=0.998 r
2
=0.999
r
2
=0.999
CV=6.3%
(N=3)
1.6%
3.9%
4.6%
5.5%
4.6%
3.7%
6.2% 6.5%
3.9%
6.6%
2.1%
5
Integration of steroids analysis in serum using LC-MS/MS
with full-automated sample preparation
We found that the sample preparation time was reduced from 60 minutes to 6 minutes by the automated system. Thus
sample preparation and LC-MS/MS analysis can be performed in parallel to accelerate throughput.
Fig. 4 Comparison with a time required for sample preparation
Fig. 5 Analytical Flow with Parallel Processing
LC/MS/MS analysis
12min
LC/MS/MS analysis
12min
12min 12min
Sample
injection
Sample
Preparation
6min
Sample
Preparation
6min
60min
6min
• Traditional sample preparation (protein precipitation)
• Automated sample preparation process by CLAM-2000
Add ACN with IS
→
Shake for 15 min
→
Centrifuge for 30 min
→
Transfer the supernatants
→
Dry for >30 min
→
Reconstitute
Add ACN with IS
→
Shake for 3 min
→
Filtrate for 2 min
Sample
injection
Sample
injection
Sample
Preparation
6min
Integration of steroids analysis in serum using LC-MS/MS
with full-automated sample preparation
First Edition: October, 2016
Conclusion
We completed steroid analysis using the automated sample preparation system coupled to LC-MS/MS. The results
shows the capability of the system for large sample set analyses with improved accuracy and precision by eliminating
human error associated with manual sample handling.