• Isotopically labelled internal standard

• Multilevel calibrators

• Online sample preparation

• Upgrade available for the dermination of 3-epi-25-OH-Vitamin D3/D2

Vitamin D has been widely recognised as a biomarker in relation to osteoporosis and osteopenia. It has also been shown to play a prominent role in immune response, cardiovascular disease and certain types of cancer. Vitamin D analysis, therefore, carries significance in several respects.

25-OH-vitamin D3 constitutes the principal diagnostic target and is the main metabolite of vitamin D in humans; it is the precursor of the physiologically most active compound 1,25-(OH)2-Vitamin D3. Vitamin D deficiency is treated with vitamin D3 or vitamin D2 supplementation. Vitamin D2 is easier to produce synthetically and, where prescribed, its monitoring is also necessary.

This reagent kit allows for the specific analysis of 25-OH-vitamin D3 and 25-OH-vitamin D2 in serum and plasma by tandem mass spectrometry. Manual sample preparation is minimised and limited to simple and effective protein precipitation. A subsequent online trap column concentrates the analytes and separates interfering substances. A six-port-valve connects the trap column to an HPLC column which provides further purification. The use of atmospheric pressure chemical ionisation (APCI) and an isotopically labelled internal standard assures precision and robustness and minimises ion suppression effects. The 3PLUS1® multilevel calibrators offer additional precision.

The upgrade to MassChrom® 25-OH-vitamin D3/D2 analysis allows for the separation of the main metabolites of vitamins D3 and D2, 25-Hydroxycholecalciferol and 25-Hydroxyergocalciferol, from their epimeric forms 3-epi-25-OH-vitamin D3 and 3-epi-25-OH-vitamin D2, hence allowing for separate quantification by LC MS/MS.

Standard method

Upgrade for determination of
diastereomeric forms

Specifications:

Linearity:	up to 250 µg/l
Limit of quantification:	25-OH-Vitamin D3:	2.0-3.0 µg/l	3-epi-25-OH-Vitamin D3:	2.0 µg/l
	25-OH-Vitamin D2:	1.0-2.2 µg/l	3-epi-25-OH-Vitamin D2:	1.0 µg/l
Intra-assay:	CV < 5 %
Inter-assay:	CV < 7 %
Recovery:	25-OH-Vitamin D3:	93-104 %	3-epi-25-OH-Vitamin D3:	102-106 %
	25-OH-Vitamin D2:	91-101 %	3-epi-25-OH-Vitamin D2:	104-105 %
Run time:	25-OH-Vitamin D3/D2:	5 min	3-epi-25-OH-Vitamin D3/D2:	8.5-10 min

HPLC parameters

Injection volume:	10 to 50 µl

Pump A delivers 100 % Mobile Phase A for loading the trap column.

Flow:

1.00 ml/min

Pump B delivers 100 % Mobile Phase B for separating analytes on the analytical column.

Flow:

0.60 ml/min

The trap column and the analytical column are linked to the mass spectrometer through a 6-port valve.

LC-MS/MS parameters

Ionisation:	APCI positive
MS/MS-mode:

 

Place 100 µl sample into a reaction vial.

Add 25 µl Precipitation Reagent.

Add 200 µl Internal Standard and vortex 20 s.

Incubate 10 min at +4 °C.

Centrifuge 5 min at 15000 g.

Transfer 200 µl supernatant into an autosampler vial.

Order no.	Product
62000	MassChrom® Reagent kit for the LC-MS/MS analysis of 25-OH-Vitamin D3 and D2 in serum/plasma For 200 analyses
62000/1000	MassChrom® Integrated kit for the LC-MS/MS analysis of 25-OH-Vitamin D3 and D2 in serum/plasma, For 1000 analyses, complete with: Analytical column Trap-column 3PLUS1® Calibrators MassCheck® Controls
62000/1000/F	MassChrom® Integrated kit for the LC-MS/MS analysis of 25-OH-Vitamin D3 and D2 in serum/plasma, For the automated sample preparation with 96 well filter plates. For 1000 analyses, complete with: Analytical column Trap-column 3PLUS1® Calibrators MassCheck® Controls
	Components available separately:
62001	Mobile Phase A, 1000 ml
62002	Mobile Phase B, 1000 ml
62009	Rinsing Solution, 1000 ml
62003	Precipitation Reagent, 5 ml
62004	Internal Standard, 40 ml
3006	Reaction vials, 100 pcs./td>
	Multilevel Calibrators and Startup Accessories:
62028	3PLUS1® Multilevel Serum Calibrator Set 25-OH-Vitamin D3/D2, 4 x 1 ml
62039	6PLUS1® Multilevel Serum Calibrator Set 25-OH-Vitamin D3/D2, 7 x 1 ml
62110	Trap column, 1 pc.
62100	Analytical column, 1 pc.
62015	Tuning Mix 25-OH-Vitamin D3/D2, 2 ml
15010	PEEK prefilter housing, 1 pc.
15011	PEEK-encased Prefilter, 2 µm, 5 pcs.
	Chromsystems MassCheck® Controls (lyoph.):
0221	MassCheck® 25-OH-Vitamin D3/D2 Serum Control, Bi-Level (I+II), 2 x 5 x 1 ml
0222	MassCheck® 25-OH-Vitamin D3/D2 Serum Control, Level I, 5 x 1 ml
0223	MassCheck® 25-OH-Vitamin D3/D2 Serum Control, Level II, 5 x 1 ml
0256	MassCheck® 25-OH-Vitamin D3/D2 Serum Control, Level III, 5 x 1 ml
	Accessories for sample preparation with 96 well filter plates:
62057	96 Well filter plates, 3 pcs.
62058	Collection plates, 3 pcs.
62059	Pierceable adhesive seals, 3 pcs.
	Upgrade accessories for the analysis of diastereomeric forms of 25-OH-Vitamin D3/D2:
62044	Internal Standard Mix (for 200 analyses), 40 ml
62029	3PLUS1® Multilevel Serum Calibrator Set 3-epi-25-OH-D3/D2 and 25-OH-D3/D2 (lyoph.), 4 x 1 ml
0310	MassCheck® 3-epi-25-OH-D3/D2 and 25-OH- D3/D2 Serum Control, Bi-Level (I + II) (lyoph.), 2 x 5 x 1 ml
0311	MassCheck® 3-epi-25-OH-D3/D2 and 25-OH- D3/D2 Serum Control, Level I (lyoph.), 5 x 1 ml
0312	MassCheck® 3-epi-25-OH-D3/D2 and 25-OH-D3/D2 Serum Control, Level II (lyoph.), 5 x 1 ml
62120	Analytical column for determination of epimers (equilibrated with test chromatogram), 1 pc.
62110/Epi	Trap column for determination of epimers (equilibrated with test chromatogram), 1 pc.
62016	Tuning Mix, contains 25-OH-Vitamin D3/D2 and ISTD 1, 1 ml