Three different immunological methods at four different locations were used in our laboratory for determination of vitamin D. Comparison of results showed some discrepancies in methods.

Objective

Results and discussion References

Standards and controls were made in-house in 5% albumin solved in Krebs-Ringer buffer. A five point calibration curve was used and it covered 10 to 360 nmol\L for both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. 25-hydroxyvitamin D2-d6 and 25-hydroxyvitamin D3-d6 was used as internal standards.

Department of Medical Biochemistry, Vestre Viken Hospital Trust, Drammen, Norway

Per Olov Nordstrøm, Sylwia Wolosowska, Vivi-Ann Tennfjord and Trine Lauritzen

Development of an automated high throughput HPLC-ESI-MS method for determination of 25-OH vitamin D2 and D3 in serum

The figures above shows product ion scan for 25-OH vitamin D2 and D3. The known vitamin D quantitation methods without derivatization, use different ions and collision energies for quantification [4,5,6]. The transitions found for each compound are presented in Table 3. The quantitation was performed using Shimadzu LCMS instrument which allows making a total ion chromatogram (TIC) for each compound. The aim of using TIC was to increase the sensitivity and improve the repeatability for the method. However, our study showed only a minor impact on these parameters.

The purpose of this project was to develop a LCMS procedure that could replace these methods. It should be fully automated and be able to handle up to 600 samples per day on one LCMS instrument.

Table 1. LC configuration and parameters

LC system Shimadzu Nexera HPLC with Shimadzu trippel quadrupole MS 8050

Column Raptor biphenyl (30 x 2.1 mm )

Column temp 40 °C

Flow rate 0.8 mL/min

Mobile phase 0.1 % Formic acid in water : Acetonitile (45:55); isocratic

Injection volume 15 µL

Run time 1 min

Sample preparation was performed on a pipetting robot. A precipitation reagent containing internal standard and magnesium sulphate was used for protein crash from serum. Hexane was added for vitamin D liquid–liquid extraction. The hexane layer was then transferred to a new sample plate, evaporated to dryness and the samples were reconstituted in a solvent compatible to the LCMS method. Similar method of sample purification has been used by others [2,3,4]. The details of sample preparation is given in Figure 1. The separation was performed on a Restek’s biphenyl column. Mass spectrometry was conducted using positive electrospray ionization. MS conditions are presented in Table 2. Run time:

One minute!

Table 4. Representative accuracy and precision for analytes

25-hydroxyvitamin D3 25-hydroxyvitamin D2

CV (%) Accuracy (%) CV (%) Accuracy (%)

10 nmol/L 6.4 129 13.6 116

50 nmol/L 9.1 100 7.0 106

260 nmol/L 3.5 97 3.1 100

Conclusions

We have developed a high throughput LCMS method for analyzing 25-OH vitamin D2 and D3 in serum that is well suited for large samples amount. The procedure includes an automated liquid-liquid extraction performed on a pipetting robot. The method was accurate and reproducible and was successfully applied to routine analysis.

Table 3. MS parameters (* qualifier)

Compound Precursor

ion Fragment

ion Q1 Pre Bias Q3 Pre Bias CE

25-OH vit D2 395,20 259.40 -14 -29 -14

119.00* -27 -12 -21

25-OH vit D3 383,00 229.30 -30 -11 -20

185.20* -30 -12 -21

297.50 -30 -29 -17

211.20 -30 -10 -23

128.10 -30 -24 -55

25-OH vit D2-d6

401,20 119.20* -28 -22 -23

269.20 -19 -30 -23

25-OH vit D3-d6

389,40 303.30 -11 -30 -18

128.10* -14 -28 -55

Measuring vitamin D has been increasingly popular over the last decade. This is probably caused by the large number of studies suggesting links between vitamin D status and body functions other than the well established bone related. There have been found links to the immune system, cardiovascular conditions and certain types of cancer etc [1].

25-hydroxyvitamin D2 and D3 calibrations curves were constructed based on biologically relevant concentrations of 10, 25, 60, 150 and 390 nmol/L (standard curve fit type: quadratic, weighting method 1/c˄2). This calibration model worked well with a coefficient of determination (r2) 0.994 and 0.993 for 25-OH vitamin D2 and D3 respectively. The limit of quantitation for both analytes were established at 10 nmol/L.

Experimental

Sample preparation

50,0 75,0 100,0 125,0 150,0 175,0 200,0 225,0 250,0 275,0 300,0 325,0 350,0 375,0 m/z

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

Inten.(x100,000)

395

378

9177115

17611391

126

12877 11591

142

55

153 189

177217

115

91

129

16555 79

178

141

191

212

105

55

6791 128

141

165

208178

195223

11955

95

69

107

169

145

155197

212

251224

269

281

395119

37783

269

137

251

161

197 229211 335

107

171

57 69325

310283

50,0 75,0 100,0 125,0 150,0 175,0 200,0 225,0 250,0 275,0 300,0 325,0 350,0 375,0 m/z0,00

0,25

0,50

0,75

1,00

1,25

1,50

1,75

2,00

2,25

2,50

2,75

3,00

3,25

3,50

3,75

4,00

Inten.(x1,000,000)

126 17777 1551035742 14114112877 16515210355 65

11577

14115265 16655

102

91

128

141

55

65 153105 165

91

128

55141

105

67

153

167

79143

55 93 157119

183169

21169

237225

365

383

185229

211

171253121

135

29781 159 323

95

57271

283

383

365

Figure 3. Chromatogram of 25-OH vitamin D2 and D3

25-hydroxyvitamin D3

25-hydroxyvitamin D2

The sample preparation was performed using a robot with a maximum capacity of 384 samples per one setup. Preparation time for one sample set is approximately three hours. Maximum capacity required in our laboratory is about 600 samples per day. Further work is planned to increase the sample preparation capacity.

25-hydroxyvitamin D3

25-hydroxyvitamin D2

The Restek’s biphenyl column was used for the separation. 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 were almost fully separated and eluted at 0.55 and 0.65 minute in LC respectively (Figure 3). The total time per sample on the instrument was 1.5 minute. Equally separation were not achievable with the commonly used C18 columns.

The precision was determined by analyzing six replicates at each level. Results are presented in table 4. An external control from Chromsystems (MassCheck level II, c = 100 nmol/L) is analyzed on a daily basis. The reproducibility is 7.4% and 6.0% and the accuracy 110% and 103%, for 25-OH vitamin D2 and D3 respectively (results of the last seven months). We also participate in the DEQAS quality control program for vitamin D. The accuracy on the latest round was between 82% to 108% (average 95%, n=5) for 25-OH vitamin D3. The method has been in use in our laboratory for ten month with good results.

Acknowledgement

1. Teresa Kulie et al, Vitamin D: An Evidence-Based Review, JABFM, 2009, vol. 22 no 6 2. Naesgaard Patrycja A et al, Serum 25(OH)D Is a 2-Year Predictor All-Cause Mortality, Cardiac

Death and Sudden Cardiac Death in Chest Pain Patints from Northen Argentina, PLOS ONE 2012 Vol 7 Issue 9

3. Netzel Brian C et al, Increasing Liquid Chromatography-Tandem mass Spectrometry Throughput by mass Tagging: A Sample-Multiplexed High- Throughput Assay for 25-Hydroxyvitamin D2 and D3, Clinical Chemistry 57:3 (2011) p. 431-440

4. Maunsell Zoë et al, Routin Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Measurement of the 25-Hydroxy Metabolites of Vitamin D2 and D3, Clinical Chemistry 521:9 (2005) p. 1683-1690

5. Shah Iltaf et al, Misleading measures Vitamin D analysis: A novel LC-MS/MS assay to account for epimers and isobars, Nutrition Journal 2011, 10:46

6. Calton Lisa J. et al, The Analysis for 25-Hydroxyvitamin D in serum using UPLC/MS/MS, Waters application note # 720002748 (2008)

Thanks for support and good cooperation to Shimadzu Europa GMBH, Restek and Teknolab AS.

Table 2. MS/MS conditions Ion mode Positive Interface temperature 300 °C DL temperature 125 °C Nebulizer gas flow 3 L/min Heating gas flow 8 L/min Heat block temperature 250 °C Drying gas flow 8 L/min

Figure 1. Sample preparation

Figure 2. Product ion scan of 25-OH vitamin D2 and D3

Sample: 50 µL serum

Add 75µL of precipitation reagent with IS

Add 100µL of MgSO4

Add 600 µL of hexan

Shaking Centrifuge

Transfer 450 µL of hexane phase

Evaporation to dryness

Reconstitution in 200 µL of solvent

Analyze by LCMSMS

Mixing