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Analysis of 25-OH Vitamin D2/D3 in Serum by LC-MS/MS with full-automated sample preparation First Edition: October , 2016 Conclusion • Fully Automated sample preparation procedure resulted suitable for the quantitation o…
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Analysis of 25-OH Vitamin D2/D3 in Serum by LC-MS/MS with
full-automated sample preparation
Fig. 3 Calibration Curves (L1-L4) and MRM Chromatograms (L1)
The calibration curves showed good linearity (R^2>0.999)
over a clinical relevant range of 4.10 - 68.5 μg/L for
25-OH Vitamin D2 and 4.68 - 77.3μg/L for 25-OH
Vitamin D3 (Fig 3). The reproducibility (N=7) at three
concentrations, including LLOQ, of each compounds was
excellent (CV<6.5%). Different day reproducibility (N=7)
for 3 days at three concentrations as well (CV<7.2%)
(Table 2).
Linearity, Precision
Result and discussion
Column Temp. : 40 ºC
Injection Volume : 30 μL
[LC] NexeraX2 System
Ionization : APCI Positive
Source conditions :
Nebulizer Gas : 1.5 L/min
Interface temperature : 375 °C
Desolvation Line : 225 °C
Heat Block temperature : 250 °C
Drying Gas : Off
Scan Type : MRM
[MS] LCMS-8050
Table 1 Analytical Condition
0 10 20 30 40 50 60 Conc. Ratio
0.00
0.25
0.50
0.75
1.00
Area Ratio
2.2 2.3 2.4 2.5 2.6 2.7
0
2500
5000
7500
10000
1:395.40>269.20(+)
1:395.40>209.20(+)
25-OH Vitamin D2
r
2
=0.999
0 10 20 30 40 50 60 70 Conc. Ratio
0.00
0.25
0.50
0.75
1.00
Area Ratio
2.3 2.4 2.5 2.6 2.7
0
2500
5000
7500
10000
2:383.40>211.20(+)
2:383.40>257.40(+)
r
2
=0.999
25-OH Vitamin D3
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Analysis of 25-OH Vitamin D2/D3 in Serum by LC-MS/MS with
full-automated sample preparation
Fig. 4 Data correlation between Automatic sample preparation (CLAM-2000) and
Manual method (n=30 human serum samples + 2 reference material, Recipe)
Comparison of 25-OH Vitamin D3 concentration between manual sample preparation (following Recipe
specications) and automated sample preparation shows good agreement as highlighted by Passing and Bablok plot
and scores (Fig. 4).
Data correlation with Manual method
Table 2 Intra-assay and Inter-assay Precision (* n=7 ; ** 3 days)
Intra-assay*
Inter-assay**
CV%
4.4%
5.2%
LOW
(5.7μg/L)
2.3%
4.7%
Medium
(17.1μg/L)
1.1%
3.7%
High
(40.0μg/L)
25-OH Vitamin D2
Intra-assay*
Inter-assay**
CV%
2.9%
5.1%
LOW
(6.3μg/L)
5.1%
5.3%
Medium
(18.9μg/L)
2.4%
5.2%
High
(43.0μg/L)
25-OH Vitamin D3
Intercept A (95%CI)
Slope (95%CI)
Corr. Coeff. (P<0,0001)
0,41
1,006
0,98
CLAM-2000
Manual Sample Preparation
Analysis of 25-OH Vitamin D2/D3 in Serum by LC-MS/MS with
full-automated sample preparation
First Edition: October, 2016
Conclusion
• Fully Automated sample preparation procedure resulted suitable for the quantitation of 25-OH Vitamin D by
elimination of all manual preparation steps.
• The automation of the method increases the analytical performance, reduces the risk for human operators and,
due to the reduced reagent consumption, reduces also the cost of the analysis.