High Throughput Reliable Quantitation of 25-hydroxyvitamin D in Serum by Offline
Sample Preparation and a LC-MS/MS Instrument
Sha Joshua Ye1; Changtong Hao1; June Zang2, Javier Ramirez2, Asha A. Oroskar2
1IONICS Mass Spectrometry, Bolton, Ontario, Canada. 2Orochem, Naperville, IL, U.S.A.

RESULTSINTRODUCTION
Vitamin D is a group of fat-soluble hormones, which have the two
major forms: D2 (ergocalciferol) and D3 (cholecalciferol). The
metabolites of vitamin D have a critical physiological function tomaintain calcium and phosphate homeostasis. Vitamin D deficiency
can be best diagnosed using 25(OH) vitamin D versus the othervitamin D metabolites because 25(OH) vitamin D levels in serum
reflect the body’s storage levels of vitamin D and correlate with theclinical symptoms of vitamin D deficiencies.[1,2] A simple and fast
offline sample preparation coupled to a sensitive LC-MS/MS tandem
mass spectrometer has been developed to simultaneously measure
25(OH) vitamin D3 and 25(OH) vitamin D2 over a commercial level Ito IV analytical concentration range in human serum.

The extraction recovery rate on samples using PURITY Phospholipid
Depletion Kit 96-well plate are about 60 and 65% for 25(OH) VD3and 25(OH) VD2, respectively. Overall the extraction efficiency isabout 50% for serum samples. Summary of the extraction
performance is shown in Table 4:
Table 4: Sample Extraction Performance.

%

25-(OH)-VD3

25-(OH)-VD2

Recovery rate

57.9

64.6

Matrix effect

87.5

74.9

Process efficiency

METHOD

Quantitation Results

Sample Extraction Results

50.6

The calibration curves generated for 25-hydroxyvitamin D2(413.2/355.2) and 25-hydroxyvitamin D3 (401.3/257.2) show
injections which covers a concentration range of nearly 2 orders ofmagnitude from 1.1 to 73.4 ng/mL for 25-hydroxyvitamin D3(413.2/355.2) and from 3.9 to 63.6 for 25-hydroxyvitamin D2(401.3/257.2) (Figure 4a-b, respectively). The linear regression hasa weighting factor, 1/x. Good linearity (R2>0.994) was found for bothanalytes. Level I and II Recipe quality controls results with 3
injections were found to be excellent as shown in summary Table 5.

a) 25OH-VD348.4Extracted Ion Chromatograms (EICs)

Chemicals and Solvents
25(OH) vitamin D was purchased from Sigma (Milwaukee, WI) andvitamin D free human serum was purchased from Golden Western
Biologicals (Temecula, CA). Serum level I to IV and Recipe 25(OH)vitamin D quality controls were purchased from IRIS (Olathe, KS). Allof the chemicals were stored in the freezer. No IS was used.

EIC Chromatogram in serum blank and spiked one with 0.25 and 0.5
ng/mL 25 -Hydroxyvitamin D3 and D2 Is shown in Figure 1a-b & 2a-b.

a) blank

Sample Extraction
Sample preparation was carried out with the Orochem (Naperville,IL) PURITY Phospholipid Depletion Kit 96-well plate. The eluting step
was performed with an Orochem EzpressTM positive pressure
manifold. Refer to Table 1 for the steps used.

b) 25OH-VD2b) 0.25ng/mL

Table 1: Steps & Procedure
Step
Load 1
Load 2ElutionProcedure
300 µL of Vitamin D commercial precipitation reagent
100 µL of serum sample, wait for 5 minutes,
apply a few pressure pulse until all solution passes throughFig. 1. Chromatograms of 25 -Hydroxyvitamin D3 for blank and 0.25ng/mL.

a) blank
Fig. 4. Calibration curves of 25 -Hydroxyvitamin D3 and D2 .

Mass Spectrometry Conditions
The LC-MS/MS analysis was performed using IONICS 3Q 220 triple
quadrupole mass spectrometer. Table 2 outlines the MS instrumental
source parameter settings. The optimized MRM transition parametersfor 25(OH) vitamin D are shown in Table 3.
Table 2: MS Conditions
ESI Voltage (V)HSID Temp (oC)
Nebulizer Gas Setting
Drying Gas Setting
Source Temp (oC)

Table 5: Level I and II QCs quantification results (n=3 ).

b) 0.5ng/mL

Table 3: Optimized MRM Parameters
5050
175450120350

Compound Name Precursor Fragment CCL
(m/z)
(m/z)
2CE

401.3
401.3
413.3
413.3

2313161325(OH) VD325(OH) VD2

257.2
383.2
355.2
395.2

-51-60-55-60

Fig. 2. Chromatograms of 25 -Hydroxyvitamin D2 for blank and 0.5ng/mL.

LC Conditions
Shimadzu UFLCxr system was used with a Imtakt Cadenza C18 –HT(2.1X 50mm) 3 µm particle size column. The LC was run with agradient flow with a run time of 5min and the following conditions:

Extracted Ion Chromatograms for QC samples
Representative chromatograms of 25 -Hydroxyvitamin D3 and D2 fora Recipe level I serum control (20.5 and 16.3 ng/mL, respectively) inthis study, are shown in Figure 3a-b.

a) 20.5ng/mL

A (H2O, 0.1% Formic Acid, 5mM NH4OAc)
B (MeOH,0.1% Formic Acid, 5mM NH4OAc)
0.6 mL/min
10 µL
32 0C

Mobile Phase:
Flow Rate:
Injection Volume:
Column Temperature:b) 16.3ng/mL
Time(min)B%

0.1
100.570

2.81003.11003.21025 -Hydroxyvitamin D325 -Hydroxyvitamin D2Conc.
(ng/mL)

Avg.
accuracy(%)

Conc.
Avg.
CV (%)(ng/mL) accuracy(%)

20.5

92.6

8.3

16.3

95.7

4.0

44.3

101.3

1.1

36.6

101.0

1.9

CV (%)

CONCLUSION
A 5-min, sensitive, and reliable LC-MS/MS method wasdeveloped for quantitative determination of 25(OH) vitamin D in
human serum. The LLOQ achieved by IONICS 3Q 220 triple
quadrupole mass spectrometer for 25-OH-D3 and 25-OH-D2 inhuman serum are 0.25 and 0.5 ng/mL, respectively. The load,filter two-step simple method showed no signs of interferences.
The results show a good linearity and selectivity over level I to IV
Recipe calibrators. The offline sample preparation for this LCMS/MS method is simple and well suited for routine clinicalanalysis of 25(OH) vitamin D.

REFERENCES[1] Reinhold Vieth, Am J Clin Nutr 1999;69:842–56.
[2] Robert P. Heaney, Clin J Am Soc Nephrol 3: 1535–1541, 2008.

510Fig. 3. Chromatograms of 25 -Hydroxyvitamin D quality control level I.

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