Aela Agela Technologies

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   concentration   /ng/mL	Measured   concentration   /ng/mL	Recoveries   /%	Spiked   concentration   /ng/ mL	Measured   concentration   /ng/ mL	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   /ng/mL	Measured concentration   /ng/ mL	Accuracy   /%		Label concentration   /ng/mL	Measured concentration   /ng/ mL	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