13 Instrument imprecision, determined by repetitive injection of the same sample (n=8) 100 25 Hydroxy Vitamin D2, Daughters of 413.2 % 82.9 112.9 80.9 59.1 0 100 383.1 25 Hydroxy Vitamin D3 Daughters of 401.2 % 365.2 108.9 0 100 104.9 %
81. 90.90 78.9
107.0 131.0 117.0 159.0 145.0 159.9 0 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 m/z Figure 1. Daughter Scans Conclusion
We have developed a robust, cost effective automated on line sample preparation method for the sensitive simultaneous measurement of 25 Hydroxy vitamin D2 and D3, on 50 µl of serum. The required limit of detection of 1ng/mL is easily achieved. It has the potential to analyse 300 samples in a twenty four hour period. Sample
preparation time takes approximately 60 mins and post analytical processing can be completed in 30 mins. Analysis using both a primary quantifier ion and a secondary qualifier ion (Figures 2 and 3), when compared to an established HPLC method showed excellent agreement.
20.0 40.0 60.0 80.0 100.0 120.0
0.0 0.0 20.0 40.0 60.0 HPLC UV 80.0 100.0 120.0
10 15 20 25 30 35
0 5
0 5 10 15 20 HPLC Consensus Method Mean 25 Internal Standard, Daughter of 407.5 245.0 257.3 271.1 94.8 134.9 151.0 188.9 217.1 229.0 271.1 255.1 273.0 395.3
showed a CV of 5.1% for 25(OH)D2 at a concentration of 2.3ng/mL and a CV of 2.5% for 25(OH)D3 at a concentration of 4.7ng/mL.
337.3 325.2 355.2 377.2 396.2
Mean recovery of the sample extraction at 5ng/mL and 50 ng/mL of 25(OH)D2 was 91.2% and that of 25(OH)D3 was 90.4% when compared to equivalent methanolic non-extracted standards at equivalent concentrations. Between batch
(n=12) CVs for 25(OH)D2 were 5.5% and 4.2% at concentrations of 1.4 and 13.5 ng/mL respectively. Between batch (n=12) CVs for 25(OH)D3 were 4.5% and 5.7% at concentrations of 4.9 and 19.7ng/mL respectively.
This technology and application moves the measurement of 25 OH D forward in the clinical arena to meet the stringent demands of both physicians investigating and treating patients and researchers requiring consistent measurement of large numbers of samples.
y = 1.0465x + 0.2291 R2 = 0.9505 n=16
30
35
Figure 4. Comparison with HPLC DEQAS Consensus Method Mean
413.5 > 83.0 y = 1.0378x + 4.0468
R2 = 0.944
413.5 > 395.3 y = 1.0217x + 2.4162 R2 = 0.9738
100 120 140 160 180 200
413.5>83.0 413.5>395.3
20 40 60 80
0 0 20 40 60
80 100 120 140 160 180 200 HPLC UV
25 Hydroxy Vitamin D3 Patient Comparison (ng/mL)
y = 0.9459x + 1.9363 R2 = 0.9695 n=200
20.0 40.0 60.0 80.0 100.0 120.0 140.0 160.0 180.0 200.0
0.0 0.0 50.0 100.0 HPLC UV 150.0 200.0
Figure 5. Comparison of LCMSMS method Vs established HPLC UV method
401.5>383.3 y = 0.9329x + 4.4012
R2 = 0.9657
401.5>107.0 y = 0.9197x + 3.7258
R2 = 0.9659 References
[1]Holick MF. Vitamin D: a D-Lightful health perspective. Nutr Rev 2008 October;66(10 Suppl 2):S182-S194.
401.5>383.3 401.5>107.0
[2]Carter GD, Carter R, Jones J, Berry J. How accurate are assays for 25-hydroxyvitamin D? Data from the international vitamin D external quality assessment scheme 2. Clin Chem 2004 November;50(11):2195-7.
[3]Glendenning P, Taranto M, Noble JM, Musk AA, Hammond C, Goldswain PR et al. Current assays overestimate 25-hydroxyvitamin
D3 and underestimate 25-hydroxyvitamin D2 compared with HPLC: need for assay-specific decision limits and metabolite-specific assays. Ann Clin Biochem 2006 January;43 (Pt 1):23-30.
Figures 2. (top) and 3 (bottom) Ion ratios of primary and secondary transitions
indicated excellent selectivity for both 25(OH)D2 and 25(OH)D3, as indicated in Figures 2 and 3.
The accuracy of the assay was assessed using sixteen DEQAS samples and comparing them to the consensus method means Figure 4. Two hundred patient samples were also compared to an established HPLC method with UV detection Figure 5.
[4]Carter GD, Jones JC. Use of a common standard improves the performanc of liquid chromatography-tandem mass spectrometry methods for serum 25-hydroxy vitamin-D. Ann Clin Biochem 2009 January;46 (Pt1): 79-81.
[5]Fraser WD. Standardization of vitamin D assays: art or science? Ann Clin Biochem 2009 January;46(Pt 1):3-4.
LCMSMS LCMSMS
LC MSMS
LC MSMS
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