Introduction
25-hydroxyvitamin D (25-OH VD), including 25-hydroxyvitamin D2 (25-OH VD2) and 25-hydroxyvitamin D3 (25-OH VD3), is one of the main metabolic forms of vitamin D. It is considered as testing marker of vitamin D for its long-life and biological stability. The determination of vitamin D in human body has attracted more attentions from researchers of pharmaceutical and clinical field. LC-MS/MS is regarded as the “Gold Standard” of evaluating the nutritional status of vitamin D due to the high accuracy and reproducibility. In this application, an effective sample preparation procedure was developed for the extracting of 25-OH VD in serum using Cleanert PEP Micro Plate by LC-MS/MS.
Figure 1 Structure of 25-OH VD2 Figure 2 Structure of 25-OH VD3
Experimental
Materials and Reagents
Cleanert PEP Micro Plate (5mg/well), Cleanert PPT protein precipitation plate, SPE-M96 positive pressure device, Cleanert 96-well collection plate, Unisol C18 (2.1 × 50mm, 3 mm) were purchased from Bonna-Agela Technologies; 25-OH VD2, 25-OH VD3, d6-25-OH VD3 were purchased from Sigma-Aldrich; Calibration samples were purchased from RECIPE;
Sample Preparation
Transfer 25 mL zinc sulfate solution (0.2 M) and 200 mL methanol/acetonitrile (1/1) containing internal standard into the Cleanert PPT micro plate, add 100 mL serum, vortex and wait for 5 min. Put Cleanert PPT and collection plate on the Cleanert M96 orderly, adjust pressure to 4 psi, collect the extract and wait for further SPE preparation.
Cleanert PEP Micro Plate (5 mg/ well) used for further purification. The steps are as follow:
Condition: 200 mL methanol, 200 mL water of 60% methanol;
Loading: Add 200 mL solvent from Cleanert PPT;
Washing: Add 200 mL 5% methanol in water, 200mL 60% methanol in water.
Elution: Add 100 mL methanol/ isopropanol (95/5). Collect eluent and add 40 mL water in it. Shake it and analyze by LC-MS/MS.
Instrumentation
Detector: LCMS/MS, QTRAP 5500. SCIEX
Column: Unisol C18, 2.1 × 50 mm, 3 mm
Temperature: 40 °C
Injection: 20 mL
Mobile phase: A: 0.02% formic acid-water; B: 0.02% formic acid-methanol
Ion source: ESI+
Scan mode: MRM
Table 1 Gradient
Step | Total Time(min) | Flow Rate(µL/min) | A | B |
0 | 0 | 800 | 30 | 70 |
1 | 0.2 | 800 | 30 | 70 |
2 | 1.5 | 800 | 0 | 100 |
3 | 2.3 | 800 | 0 | 100 |
4 | 2.31 | 800 | 30 | 70 |
5 | 3 | stop |
Table 2 MS Parameters
Compounds | RT/min | Q1 | Q3 | DP | CE |
25-OH VD2 | 1.62 | 413.3 | 337.3 | 80 | 15 |
413.3 | 355.4 | 80 | 14 | ||
25-OH VD3 | 1.59 | 401.3 | 257.3 | 80 | 20 |
401.3 | 365.2 | 80 | 16 | ||
d6-25-OH VD3 | 1.59 | 407.3 | 371.4 | 80 | 19 |
Results and Discussion
Linear Range and Sensitivity
Replace blood sample with 6% BSA solution to make a standard curve which contains 6 points. The 25-OH VD2 concentrations were 1, 2, 5, 10, 20 and 40 ng/mL and the 25-OH VD3 concentrations were 5, 10, 25, 50, 100 and 200 ng/mL. Table 3 shows the curve equation of the extracts.
Table 3 Linear Range and Sensitivity
Compounds | 25-OH VD2 | 25-OH VD3 |
Regression Equation | y=0.00852x+0.000384 | y=0.0111x+0.00697 |
R2 | 0.9962 | 0.9990 |
LOD(ng/mL) | 1 | 0.67 |
Recoveries of spiking sample
Human serum was used to inspect the feasibility of this method. Serum samples of 10 patients were mixed due to the existence of determinated in human serum. The measured concentrations of 25- OH VD2 and 25-OH VD3 in the mixed serum were 1.26 ng/mL and 36.6 ng/mL respectively and these data were used to deduct background in computing the recovery. Table 4 shows the experiment results.
Table 4 Recoveries of spiked serum samples
25-OH VD2 | 25-OH VD3 | |||||
Spiked | Measured | Recoveries | Spiked | Measured | Recoveries | |
Spiked sample 1 in low concentration | 2 | 2.8 | 77.00% | 10 | 47.4 | 108.30% |
Spiked sample 2 in low concentration | 2 | 2.9 | 82.00% | 10 | 46 | 94.30% |
Spiked sample 1 in high concentration | 10 | 12.2 | 109.40% | 50 | 85.5 | 97.90% |
Spiked sample 2 in high concentration | 10 | 12.5 | 112.40% | 50 | 88.8 | 104.50% |
Notes: The value of low concentration 25-OH VD2 sample was equivalent to that of the background of mixed serum, resulting in large error and low recovery rate.
Test Result of RECIPE
Calibration and QC samples, bought from RECIPE, were used to test the accuracy of this method, the analytical results are as follows:
Table 5 Accuracy of RECIPE test
25-OH VD2 | 25-OH VD3 | ||||||
Label concentration | Measured concentration | Accuracy | Label concentration | Measured concentration | Accuracy | ||
level-1 | 2.5 | 2.46 | 98.4 | 0.97 | 0.97 | 100 | |
level-2 | 8.2 | 8.57 | 105 | 9.35 | 9.3 | 99.4 | |
level-3 | 24.8 | 25.8 | 104 | 27.9 | 28.4 | 102 | |
level-4 | 68.5 | 63.6 | 92.8 | 77.3 | 76.3 | 98.8 | |
QC-level 1 | 16.3 | 15.1 | 92.6 | 20.5 | 23 | 112 | |
QC-level 2 | 36.6 | 37.8 | 103.3 | 44.3 | 48 | 108.3 |
Figure 3 Chromatography of 25 ng/mL 25-OH VD3 in serum
Figure 4 Chromatography of 5 ng/mL 25-OH VD2 in serum
Conclusion
This experiment developed a quick method to purify the 25-OH VD2 and 25-OH VD3 from serum. First use the Cleanert PPT to precipitate protein of serum, then transfer the solvent into Cleanert PEP MicroPlate and add only 100μL solvent to eluent. The method should be saving time by no need for further concentration. So this method can be used in clinical study for detection of 25-OH VD in serum.
Ordering Information
Products | Specification | Cat.No |
Cleanert PEP MicroPlate | 96-well plates | PE00501-MW |
Cleanert PPT | 2.2 mL Square well | 96CD2025-Q |
Unisol C18 | 2.1 × 50mm, 3 μm | VA930502-0 |
Cleanert® M96 Positive Pressure Device | Adapt to 96-well plate | SPE-M96 |
96-well Collection Plate | 1.0mL, 8×12, round well and round bottom | 96SP1036-Y |
96-well Mat | 8×12, silica, round well, piecible | 96GP2036-M |