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Figure 2: Good linear response was achieved for vitamin
D metabolites using the Raptor™ ARC-18 column.
y = 0.00984x + -0.00225 (r = 0.9992)
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
Analyte Conc./ IS Conc.
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
AnalyteArea / ISArea
A: 25-Hydroxyvitamin D2
B: 25-Hydroxyvitamin D3
y = 0.0201 x + 0.00229 (r=0.9989)
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
AnalyteConc./ IS Conc.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
AnalyteArea / ISArea
Table I: Excellent results for method accuracy and precision provide confidence in data quality.
Low Fortification (5 ng/mL) Mid Fortification (25 ng/mL) High Fortification (100 ng/mL)
Analyte Conc.
(ng/mL)
Accuracy
(%Recovery)
Precision
(%RSD)
Conc.
(ng/mL)
Accuracy
(%Recovery)
Precision
(%RSD)
Conc.
(ng/mL)
Accuracy
(%Recovery)
Precision
(%RSD)
25-Hydroxyvitamin D2 5.4 107.3 10.7 25.3 101.1 3.9 101.5 101.5 1.6
25-Hydroxyvitamin D3 4.5 92.6 8.5 25.6 102.4 0.3 107.1 107.1 1.4
Table values are averages of replicate samples.
Figure 3: Good separation of 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 from matrix components ensures more
accurate results.
0.2
1
2
3
0.0 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0.2 0.0 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8
1
2
Time (min)
LC_CF0601
Conc. Precursor Product Qualifier
Peaks tR(min) (ng/mL) Ion Ion Ion
Blank Plasma
1. 25-Hydroxyvitamin D3-d6 1.93 25 407.3 389.5 –
2. 25-Hydroxyvitamin D3 1.95 unknown 401.3 383.5 365.4
25 ng/mL Plasma
1. 25-Hydroxyvitamin D3-d6 1.92 25 407.3 389.5 –
2. 25-Hydroxyvitamin D3 1.95 25 401.3 383.5 365.4
3. 25-Hydroxyvitamin D2 2.05 25 413.3 395.5 355.4
LC_CF0602
Column:Raptor™ ARC-18 (cat.# 9314A12); Dimensions: 100 mm x 2.1 mm ID;
Particle Size: 2.7 μm; Pore Size: 90 Å; Temp.: 40 °C; Inj. Vol.: 5 μL;Mobile Phase: A.
0.1% Formic acid in water, B. 0.1% Formic acid in methanol; Gradient (%B): 0.00 min
(85%), 3.00 min (96%), 3.01 min (85%), 5.00 min (85%); Flow: 0.5 mL/min;
Detector: MS/MS; Ion Mode: ESI+; Instrument: HPLC.
Blank Plasma 25 ng/mL Plasma
Good Accuracy and Precision Ensure Reliable Results
In order to evaluate method accuracy and precision in matrix, rep-
licate charcoal-stripped rat plasma samples were fortified at 5, 25,
and 100 ng/mL with 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin
D3. Quantitation was performed using calibration standards ranging
from 1 to 150 ng/mL that were prepared in 4% human albumin in PBS
solution. Eight calibration concentrations were used for 25‐hydroxyvi-
tamin D2 and seven were used for 25‐hydroxyvitamin D3. Both
the fortified samples and standards were extracted using a simple
liquid-liquid extraction method with 25‐hydroxyvitamin D3-d6 as the
internal standard. Visit for the full sample
preparation procedure.
Linearity was evaluated and good response curves were obtained
for both metabolites (Figure 2). Using 1/x weighting, the correla-
tion coefficients (r) were 0.9992 (25‐hydroxyvitamin D2) and 0.9989
(25‐hydroxyvitamin D3), and the deviations were ≤10% for both com-
pounds. Blanks and fortified samples were also analyzed to evaluate
accuracy and precision. Since the extracted blank plasma samples
contained 25‐hydroxyvitamin D3 (Figure 3), blank values were sub-
tracted from fortified samples to improve quantitative accuracy.
As Table I shows, excellent results for accuracy and precision were
obtained for both compounds at all three fortification levels, with an
overall range of 92.6-107.3% recovery for accuracy and 0.3-10.7 % RSD
for precision.
Summary
Designed specifically for use on LC-MS/MS systems, the Raptor™
ARC-18 column is the cornerstone of this high-throughput LC-MS/
MS method for analysis of vitamin D metabolites in plasma. This new
column from Restek delivers the fast analysis times needed to improve
sample throughput and lab productivity along with the accurate, pre-
cise performance needed to ensure data quality.
Time (min.) Time (min.)