Vitamin B1 in Whole Blood and Vitamin B6 in Whole Blood / Plasma – HPLC Reagent Kit

Order No.: 52052/Premix, for 100 tests
Parameters:
Vitamin B1 (TPP, thiamine pyrophosphate), Vitamin B6 (PLP, pyridoxal 5’-phosphate)

Each parameter safeguarded by a specific internal standard
No post-column derivatisation required
One sample preparation and one run for both parameters
Simplified sample prep with Pre-mixed Neutralisation Tubes

Assay validated according to IVDR (=> Declaration of Conformity)

Vitamin B1 (TPP, thiamine pyrophosphate)

Vitamin B6 (PLP, pyridoxal 5’-phosphate)

Clinical relevance
This assay allows the quantitative determination of the physiologically active forms of vitamin B1, thiamine pyrophosphate, in human whole blood samples and of vitamin B6, pyridoxal 5‘-phosphate, in human whole blood or plasma samples.

It is intended to be used for for screening and/or monitoring of vitamin B1 and/or B6 levels where indicated
• in patients with suspected vitamin B1 and/or B6 deficiency,
• in patients with suspected vitamin B1 and/or B6 excess,
• in patients under vitamin B1 and/or B6 supplementation therapy.

 

Product advantages
• Specific internal standard for each parameter
• No post-column derivatisation required
• One sample preparation and one run for both parameters
• Simplified sample prep with Pre-mixed Neutralisation Tubes

 

Assay characteristics
Thiamine pyrophosphate (TPP) and pyridoxal 5’-phosphate (PLP) can be determined in one run, with one sample preparation and without complex post-column derivatisation. Sample preparation is restricted to a single precipitation step and subsequent derivatisation and is possible with Reaction Vials (assays 52052 and 52052/Premix) or 96 Well Filter Plates (assay 52752/F). Pre-mixed Neutralisation Tubes simplify sample preparation when performing the 52052/Premix assay. Chromatographic separation is carried out with two Mobile Phases by HPLC. The derivatised analytes are determined by fluorescence detection with wavelength switching. The use of two internal standards assures high precision and safety in quantifying the analytes.

 

Two types of kits available

Basic Kit
• Only sample preparation reagents and vials included
• No excess quantities of mobile phase and calibrators

52052-PREMIX-BK Basic Kit
For 200 sample preparations

52044 Internal Standard, 10 ml (2x)
52005 Precipitation Reagent, 30 ml (2x)
52906 Pre-mixed Neutralisation Tubes, 100 pcs. (2x)
52007 Derivatisation Reagent 1, 2 x 0.3 ml (lyoph.) (2x)
52008 Derivatisation Reagent 2, 15 ml (2x)
33005 Reaction Vials, 1.5 ml, amber colour (light protection), 100 pcs. (2x)

Reagent Kit
• Mobile phase and calibrator included
 

52052/PREMIX Reagent Kit
For 100 tests

52044 Internal Standard, 10 ml (1x)
52005 Precipitation Reagent, 30 ml (1x)
52906 Pre-mixed Neutralisation Tubes, 100 pcs. (1x)
52007 Derivatisation Reagent 1, 2 x 0.3 ml (lyoph.) (1x)
52008 Derivatisation Reagent 2, 15 ml (1x)
33005 Reaction Vials, 1.5 ml, amber colour (light protection), 100 pcs. (2x)
52001 Mobile Phase A, 1000 ml (1x)
52022 Mobile Phase B, 1000 ml (1x)
52003 Whole Blood Calibration Standard, 5 x 1.0 ml (lyoph.) (1x)
More Information
Method of Analysis HPLC
Number of Tests 100
Please note The information listed here, including the sample preparation, is not sufficient for using the product. Please read the information provided in the instruction manual, which includes detailed information on limitations associated with the use of the product in line with its intended purpose. Detailed performance evaluation data for this assay can be found in the appendix of the instruction manual.
Lower and Upper Limit of Quantitation

TPP 3.5 – 750 µg/l (whole blood)
PLP 4.0 – 500 µg/l (whole blood)
PLP 0.5 – 500 µg/l (plasma)

Specimen Whole Blood / Plasma
Sample Preparation

The information on the sample preparation presented here is not sufficient for use in the laboratory. For a detailed step by step description, please refer to the instruction manual.

  • In a light protected vial mix 200 μl whole blood or plasma with 100 μl Internal Standard and 300 μl Precipitation Reagent, mix for at least 30 s (vortex).
  • Centrifuge for 5 min at 14000 x g.
  • Add 100 μl of Derivatisation Mix and 250 μl of supernatant to a labeled Premixed Neutralisation Tube, mix briefly.
  • Incubate for 25 min at 60 °C (water bath).
  • Cool sample for 10 min at +2 to +8°C, then centrifuge for 2 min at 14000 x g.
  • Transfer supernatant into a light protected autosampler vial, inject 25 μl into the HPLC system.
Run Time 7 min (plasma), 9 min (whole blood)
Injection Volume 25 μl
Flow Rate 1.5–2.3 ml/min
Column Temperature +20 to +25 °C
Gradient binary
Wavelengths

Start EX 320 nm, EM 415 nm,
after approx. 3.8 min switch to EX 367 nm, EM 435 nm

Additional Info Any common HPLC system with binary pump and programmable fluorescence detector is suitable.
Parameters Vitamin B1 (TPP, thiamine pyrophosphate), Vitamin B6 (PLP, pyridoxal 5’-phosphate)

Vitamin B1 in Whole Blood and Vitamin B6 in Whole Blood / Plasma – HPLC Reagent Kit (52052/PREMIX)
For 100 tests

Order No. Reagent Kit components (Quantities in kit)
52001 Mobile Phase A, 1000 ml (1x)
52022 Mobile Phase B, 1000 ml (1x)
52003 Whole Blood Calibration Standard, 5 x 1.0 ml (lyoph.) (1x)
52044 Internal Standard, 10 ml (1x)
52005 Precipitation Reagent, 30 ml (1x)
52906 Pre-mixed Neutralisation Tubes, 100 pcs. (1x)
52007 Derivatisation Reagent 1, 2 x 0.3 ml (lyoph.) (1x)
52008 Derivatisation Reagent 2, 15 ml (1x)
33005 Reaction Vials, 1.5 ml, amber colour, 100 pcs. (2x)

 

Order No. Required components (not included in the Reagent Kit)
52100 HPLC Column, equilibrated, with test chromatogram, 1 pc.
   
  Required for the analysis of vitamin B1 and vitamin B6 in whole blood:
0165 Vitamin B1/B6 Whole Blood Control, Level I, 5 x 2.0 ml (lyoph.)
0167 Vitamin B1/B6 Whole Blood Control, Level II, 5 x 2.0 ml (lyoph.)
   
  Required for the analysis of vitamin B6 in plasma:
36005 Plasma Calibration Standard, 5 x 1.0 ml (lyoph.)
0038 Vitamin B6 Plasma Control, Level I, 5 x 2.0 ml (lyoph.)
0039 Vitamin B6 Plasma Control, Level II, 5 x 2.0 ml (lyoph.)

 

Order No. Lab equipment
33005 Reaction Vials, 1.5 ml, amber colour (light protection), 100 pcs.
15010 PEEK Prefilter Housing, 1 pc.
15011 PEEK-encased Prefilter, 2 µm, 5 pcs.
18001 Precolumn Cartridge Holder 4/10, 1 pc.
18010 Precolumn Cartridge 4/10, 1 pc.
The following components are included in the kit:
The following products are not included in the kit but are required for the application of the method:
As a customer please login or register to gain full access.

Vitamin B1 (TPP, thiamine pyrophosphate)

Vitamin B6 (PLP, pyridoxal 5’-phosphate)

Clinical relevance
This assay allows the quantitative determination of the physiologically active forms of vitamin B1, thiamine pyrophosphate, in human whole blood samples and of vitamin B6, pyridoxal 5‘-phosphate, in human whole blood or plasma samples.

It is intended to be used for for screening and/or monitoring of vitamin B1 and/or B6 levels where indicated
• in patients with suspected vitamin B1 and/or B6 deficiency,
• in patients with suspected vitamin B1 and/or B6 excess,
• in patients under vitamin B1 and/or B6 supplementation therapy.

 

Product advantages
• Specific internal standard for each parameter
• No post-column derivatisation required
• One sample preparation and one run for both parameters
• Simplified sample prep with Pre-mixed Neutralisation Tubes

 

Assay characteristics
Thiamine pyrophosphate (TPP) and pyridoxal 5’-phosphate (PLP) can be determined in one run, with one sample preparation and without complex post-column derivatisation. Sample preparation is restricted to a single precipitation step and subsequent derivatisation and is possible with Reaction Vials (assays 52052 and 52052/Premix) or 96 Well Filter Plates (assay 52752/F). Pre-mixed Neutralisation Tubes simplify sample preparation when performing the 52052/Premix assay. Chromatographic separation is carried out with two Mobile Phases by HPLC. The derivatised analytes are determined by fluorescence detection with wavelength switching. The use of two internal standards assures high precision and safety in quantifying the analytes.

 

Two types of kits available

Basic Kit
• Only sample preparation reagents and vials included
• No excess quantities of mobile phase and calibrators

52052-PREMIX-BK Basic Kit
For 200 sample preparations

52044 Internal Standard, 10 ml (2x)
52005 Precipitation Reagent, 30 ml (2x)
52906 Pre-mixed Neutralisation Tubes, 100 pcs. (2x)
52007 Derivatisation Reagent 1, 2 x 0.3 ml (lyoph.) (2x)
52008 Derivatisation Reagent 2, 15 ml (2x)
33005 Reaction Vials, 1.5 ml, amber colour (light protection), 100 pcs. (2x)

Reagent Kit
• Mobile phase and calibrator included
 

52052/PREMIX Reagent Kit
For 100 tests

52044 Internal Standard, 10 ml (1x)
52005 Precipitation Reagent, 30 ml (1x)
52906 Pre-mixed Neutralisation Tubes, 100 pcs. (1x)
52007 Derivatisation Reagent 1, 2 x 0.3 ml (lyoph.) (1x)
52008 Derivatisation Reagent 2, 15 ml (1x)
33005 Reaction Vials, 1.5 ml, amber colour (light protection), 100 pcs. (2x)
52001 Mobile Phase A, 1000 ml (1x)
52022 Mobile Phase B, 1000 ml (1x)
52003 Whole Blood Calibration Standard, 5 x 1.0 ml (lyoph.) (1x)
More Information
Method of Analysis HPLC
Number of Tests 100
Please note The information listed here, including the sample preparation, is not sufficient for using the product. Please read the information provided in the instruction manual, which includes detailed information on limitations associated with the use of the product in line with its intended purpose. Detailed performance evaluation data for this assay can be found in the appendix of the instruction manual.
Lower and Upper Limit of Quantitation

TPP 3.5 – 750 µg/l (whole blood)
PLP 4.0 – 500 µg/l (whole blood)
PLP 0.5 – 500 µg/l (plasma)

Specimen Whole Blood / Plasma
Sample Preparation

The information on the sample preparation presented here is not sufficient for use in the laboratory. For a detailed step by step description, please refer to the instruction manual.

  • In a light protected vial mix 200 μl whole blood or plasma with 100 μl Internal Standard and 300 μl Precipitation Reagent, mix for at least 30 s (vortex).
  • Centrifuge for 5 min at 14000 x g.
  • Add 100 μl of Derivatisation Mix and 250 μl of supernatant to a labeled Premixed Neutralisation Tube, mix briefly.
  • Incubate for 25 min at 60 °C (water bath).
  • Cool sample for 10 min at +2 to +8°C, then centrifuge for 2 min at 14000 x g.
  • Transfer supernatant into a light protected autosampler vial, inject 25 μl into the HPLC system.
Run Time 7 min (plasma), 9 min (whole blood)
Injection Volume 25 μl
Flow Rate 1.5–2.3 ml/min
Column Temperature +20 to +25 °C
Gradient binary
Wavelengths

Start EX 320 nm, EM 415 nm,
after approx. 3.8 min switch to EX 367 nm, EM 435 nm

Additional Info Any common HPLC system with binary pump and programmable fluorescence detector is suitable.
Parameters Vitamin B1 (TPP, thiamine pyrophosphate), Vitamin B6 (PLP, pyridoxal 5’-phosphate)

Vitamin B1 in Whole Blood and Vitamin B6 in Whole Blood / Plasma – HPLC Reagent Kit (52052/PREMIX)
For 100 tests

Order No. Reagent Kit components (Quantities in kit)
52001 Mobile Phase A, 1000 ml (1x)
52022 Mobile Phase B, 1000 ml (1x)
52003 Whole Blood Calibration Standard, 5 x 1.0 ml (lyoph.) (1x)
52044 Internal Standard, 10 ml (1x)
52005 Precipitation Reagent, 30 ml (1x)
52906 Pre-mixed Neutralisation Tubes, 100 pcs. (1x)
52007 Derivatisation Reagent 1, 2 x 0.3 ml (lyoph.) (1x)
52008 Derivatisation Reagent 2, 15 ml (1x)
33005 Reaction Vials, 1.5 ml, amber colour, 100 pcs. (2x)

 

Order No. Required components (not included in the Reagent Kit)
52100 HPLC Column, equilibrated, with test chromatogram, 1 pc.
   
  Required for the analysis of vitamin B1 and vitamin B6 in whole blood:
0165 Vitamin B1/B6 Whole Blood Control, Level I, 5 x 2.0 ml (lyoph.)
0167 Vitamin B1/B6 Whole Blood Control, Level II, 5 x 2.0 ml (lyoph.)
   
  Required for the analysis of vitamin B6 in plasma:
36005 Plasma Calibration Standard, 5 x 1.0 ml (lyoph.)
0038 Vitamin B6 Plasma Control, Level I, 5 x 2.0 ml (lyoph.)
0039 Vitamin B6 Plasma Control, Level II, 5 x 2.0 ml (lyoph.)

 

Order No. Lab equipment
33005 Reaction Vials, 1.5 ml, amber colour (light protection), 100 pcs.
15010 PEEK Prefilter Housing, 1 pc.
15011 PEEK-encased Prefilter, 2 µm, 5 pcs.
18001 Precolumn Cartridge Holder 4/10, 1 pc.
18010 Precolumn Cartridge 4/10, 1 pc.
The following components are included in the kit:
The following products are not included in the kit but are required for the application of the method:
As a customer please login or register to gain full access.