The level of 25-hydroxyvitamin D2 (25-OH VD2) and D3 (25-OH VD3) in human body is an important factor for clinical application. Recently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been accepted as a standard method for determination of 25-OH VD2/VD3 in plasma and serum. It is no doubt a clean-up procedure is necessary to remove the interferences from biosamples prior to LC-MS/MS.
Figure 1 chemical structure of 25-OH VD2 Figure 2 chemical structure of 25-OH VD3
25-hydroxyvitamin D2/D3 and internal standard substance were purchased from Altascientific. Methanol, formic acid and isooctane were HPLC grade. Sodium hydroxide was analytically pure. Serum was purified by Cleanert® SLE plate (200mg/well) which was from Bonna-Agela Technologies. The SPE M96 positive pressure device was used to collect elute solution and Cleanert® V96 nitrogen evaporator was used to concentrate the elute solution.
The LC-MS/MS system was consisted of ExionLC™ AC HPLC system with a binary pump and an auto-injector coupled with a triple quadrupole tandem mass spectrometer API 5500 (SCIEX). Data acquisition and processing were performed using Analyst software (version 1.6.2) from SCIEX. Chromatographic separation was achieved on a Durashell C18(L) column (particle size 5 μm, pore size 150 Å, 3.0 mm×50 mm, Bonna-Agela Technologies) at a column temperature of 30 °C. The experiment adopted isocratic elution which contained 0.1% formic acid in methanol /0.1% formic acid in water (90/10, v/v), the flow rate was 500 μL/min. The injection volume was 5 μL. Quantification of 25-OH VD3 and 25-OH VD2 were conducted with electrospray ionization MS/MS in the multiple-reaction monitoring mode. The MS/MS transitions and optimal MS parameters were listed in Table 1.
Table 1 MS parameter of 25-OH VD2 and 25-OH VD3
A simplified clean-up procedure utilizing supported liquid extraction (SLE) plates was developed. 10μL of internal standard (hexadeuterated 25-hydroxyvitamin D3 and Trideuterated 25-hydroxyvitamin D2) was added to 100 μL serum and vortexed for 30s. The serum was diluted with 100μL of 50mM NaOH. The mixture was loaded onto Cleanert® SLE plate and wait for 5 min for fully adsorption of the aqueous phase on the solid phase of the plate. The target compounds were eluted with 4 x 500 μL of isooctane and collected into a deep well plate. Cleanert® M96 positive pressure device was used to remove the residue of organic phase from Cleanert® SLE plate. The fractions in collection plate were evaporated to dryness and reconstituted with 200 μL of methanol / water (80/20，v/v) containing 0.1% of formic acid. The treated samples were detected by LC-MS/MS coupled with a Durashell C18-L HPLC column.
Results and Discussion
Linear Range and Sensitivity
A series concentration of standard solution was prepared with serum which contained no target compounds. The concentration of 25-OH VD2 was 2、4、10、30、60、120、200、400、800、1000 ng/mL, and concentration of 25-OH VD3 was 1、2、5、15、30、60、100、200、400、500 ng/mL. The samples were prepared and analyzed following by the procedure which described above.
Table 2 Linear Range and Sensitivity
Accuracy and Precision
In order to evaluate the method, different concentration grade of spiked sample was prepared and purified by the method. Parallel sample of each concentration was 5 samples.
Table 3 Accuracy and Precision
Chromatogram of spiked sample were shown in figure 3~4. The abundant phospholipid in serum was essential reason of matrix effect in LC-MS/MS. Characteristic MRM transitions of phospholipid, 496/184 and 524/184, was used to evaluate the purify capability of PPT, LLE and Cleanert® SLE. Chromatogram of phospholipid was listed in figure 5~6.
Figure 3 Chromatogram of 25-OH VD3 in spiked sample (spiked concentration 15 ng/mL)
Figure 4 Chromatogram of 25-OH VD2 in spiked sample (spiked concentration 30 ng/mL)
Figure 5 Comparison chromatogram of phospholipid - PPT-LLE-SLE(496/184)
Figure 6 Comparison chromatogram of phospholipid - PPT-LLE-SLE (524/184)
The application presents a simple and efficiency method for determination of 25-OH VD2 and 25-OH VD3. Cleanert SLE plate was suitable for removing the matrix from serum which might interfere in analysis of 25-OH VD2 and 25-OH VD3 by LC-MS/MS. The usage of 96-well plate is sufficient for high throughput sample clean – up procedure prior to LC-MS/MS. The researcher could substitute Cleanert® SLE for liquid –liquid extraction in clinical research and DMPK which is time–consuming and inefficiency.
96-well Collection plate
2.2 mL Square well
Cleanert® M96 Positive Pressure Device
Adapt to 96-well plate
96-well Vacuum Manifold
Adapt to 96-well plate
3.0 × 50mm, 5 μm, 150 Å