Vitamins B1 and B6 in Whole Blood, with 96 Well Filter Plates – HPLC Reagent Kit

Order No.: 52752/F, for 960 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

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, and of vitamin B6, pyridoxal 5‘-phosphate, in human whole blood 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

 

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 plates/seals included
• No excess quantities of mobile phase and calibrators

52052-F-BK Basic Kit
For 960 sample preparations

52744 Internal Standard Mix, 50 ml (1x)
52705 Extraction Reagent, 75 ml (2x)
52706 Prep Solution, 25 ml (3x)
52707 Finisher 1, 5 x 0.25 ml (lyoph.) (2x)
52708 Finisher 2, 50 ml (1x)
52709 Dilution Buffer, 100 ml (1x)
52057 96 Well Filter Plates, 5 pcs. (2x)
52058 Collection Plates, 10 pcs. (2x)
52059 Pierceable Adhesive Seals, 5 pcs. (2x)

Reagent Kit
• Mobile phase and calibrator included
 

52752/F Reagent Kit
For 960 tests

52744 Internal Standard Mix, 50 ml (1x)
52705 Extraction Reagent, 75 ml (2x)
52706 Prep Solution, 25 ml (3x)
52707 Finisher 1, 5 x 0.25 ml (lyoph.) (2x)
52708 Finisher 2, 50 ml (1x)
52709 Dilution Buffer, 100 ml (1x)
52057 96 Well Filter Plates, 5 pcs. (2x)
52058 Collection Plates, 10 pcs. (2x)
52059 Pierceable Adhesive Seals, 5 pcs. (2x)
52100 HPLC Column, 1 pc. (1x)
52001 Mobile Phase A, 1000 ml (10x)
52022 Mobile Phase B, 1000 ml (10x)
52003 Whole Blood Calibration Standard, 5 x 1.0 ml (lyoph.) (5x)
More Information
Method of Analysis HPLC
Number of Tests 960
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 4.2 – 750 µg/l (whole blood)
PLP 1.1 – 500 µg/l (whole blood)

Specimen Whole Blood
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.

  • Pipette 100 µl whole blood (calibrator, control, samples) into a 96 Well Collection Plate.
  • Add 50 µl Internal Standard Mix + 150 µl Extraction Reagent, mix 4 min.
  • Separate precipitate by centrifugation (3 min).
  • Transfer 75 µl of the supernatant into a 96 Well Filter Plate.
  • Add 75 µl Prep Solution + 50 µl finisher solution, mix 30 s.
  • Put 96 Well Filter Plate onto a new 96 Well Collection Plate.
  • Separate precipitate by centrifugation (2 min), discard 96 Well Filter Plate.
  • Incubate the solution obtained at +60 °C for 45 min.
  • Add 100 µl Dilution Buffer, mix 3 min.
  • Seal the plate with an adhesive seal and transfer to the autosampler.
  • Inject 25 µl eluate into the HPLC system.
Run Time 9 min
Injection Volume 25 µl
Flow Rate 1.5–2.3 ml/min
Column Temperature +20 to +25 °C
Wavelengths

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

Additional Info Every 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 and Vitamin B6 in Whole Blood – HPLC Reagent Kit (52752/F)
For 960 tests

Order No. Reagent Kit components (Quantities in kit)
52001 Mobile Phase A, 1000 ml (10x)
52022 Mobile Phase B, 1000 ml (10x)
52003 Whole Blood Calibration Standard, 5 x 1.0 ml (lyoph.) (5x)
52744 Internal Standard Mix, 50 ml (1x)
52705 Extraction Reagent, 75 ml (2x)
52706 Prep Solution, 25 ml (3x)
52707 Finisher 1, 5 x 0.25 ml (lyoph.) (2x)
52708 Finisher 2, 50 ml (1x)
52709 Dilution Buffer, 100 ml (1x)
52057 96 Well Filter Plates, 5 pcs. (2x)
52058 Collection Plates, 10 pcs. (2x)
52059 Pierceable Adhesive Seals, 5 pcs. (2x)
52100 HPLC Column, 1 pc. (1x)

 

Order No. Required components (not included in the Reagent Kit)
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.)

 

Order No. Lab equipment
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:
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, and of vitamin B6, pyridoxal 5‘-phosphate, in human whole blood 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

 

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 plates/seals included
• No excess quantities of mobile phase and calibrators

52052-F-BK Basic Kit
For 960 sample preparations

52744 Internal Standard Mix, 50 ml (1x)
52705 Extraction Reagent, 75 ml (2x)
52706 Prep Solution, 25 ml (3x)
52707 Finisher 1, 5 x 0.25 ml (lyoph.) (2x)
52708 Finisher 2, 50 ml (1x)
52709 Dilution Buffer, 100 ml (1x)
52057 96 Well Filter Plates, 5 pcs. (2x)
52058 Collection Plates, 10 pcs. (2x)
52059 Pierceable Adhesive Seals, 5 pcs. (2x)

Reagent Kit
• Mobile phase and calibrator included
 

52752/F Reagent Kit
For 960 tests

52744 Internal Standard Mix, 50 ml (1x)
52705 Extraction Reagent, 75 ml (2x)
52706 Prep Solution, 25 ml (3x)
52707 Finisher 1, 5 x 0.25 ml (lyoph.) (2x)
52708 Finisher 2, 50 ml (1x)
52709 Dilution Buffer, 100 ml (1x)
52057 96 Well Filter Plates, 5 pcs. (2x)
52058 Collection Plates, 10 pcs. (2x)
52059 Pierceable Adhesive Seals, 5 pcs. (2x)
52100 HPLC Column, 1 pc. (1x)
52001 Mobile Phase A, 1000 ml (10x)
52022 Mobile Phase B, 1000 ml (10x)
52003 Whole Blood Calibration Standard, 5 x 1.0 ml (lyoph.) (5x)
More Information
Method of Analysis HPLC
Number of Tests 960
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 4.2 – 750 µg/l (whole blood)
PLP 1.1 – 500 µg/l (whole blood)

Specimen Whole Blood
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.

  • Pipette 100 µl whole blood (calibrator, control, samples) into a 96 Well Collection Plate.
  • Add 50 µl Internal Standard Mix + 150 µl Extraction Reagent, mix 4 min.
  • Separate precipitate by centrifugation (3 min).
  • Transfer 75 µl of the supernatant into a 96 Well Filter Plate.
  • Add 75 µl Prep Solution + 50 µl finisher solution, mix 30 s.
  • Put 96 Well Filter Plate onto a new 96 Well Collection Plate.
  • Separate precipitate by centrifugation (2 min), discard 96 Well Filter Plate.
  • Incubate the solution obtained at +60 °C for 45 min.
  • Add 100 µl Dilution Buffer, mix 3 min.
  • Seal the plate with an adhesive seal and transfer to the autosampler.
  • Inject 25 µl eluate into the HPLC system.
Run Time 9 min
Injection Volume 25 µl
Flow Rate 1.5–2.3 ml/min
Column Temperature +20 to +25 °C
Wavelengths

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

Additional Info Every 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 and Vitamin B6 in Whole Blood – HPLC Reagent Kit (52752/F)
For 960 tests

Order No. Reagent Kit components (Quantities in kit)
52001 Mobile Phase A, 1000 ml (10x)
52022 Mobile Phase B, 1000 ml (10x)
52003 Whole Blood Calibration Standard, 5 x 1.0 ml (lyoph.) (5x)
52744 Internal Standard Mix, 50 ml (1x)
52705 Extraction Reagent, 75 ml (2x)
52706 Prep Solution, 25 ml (3x)
52707 Finisher 1, 5 x 0.25 ml (lyoph.) (2x)
52708 Finisher 2, 50 ml (1x)
52709 Dilution Buffer, 100 ml (1x)
52057 96 Well Filter Plates, 5 pcs. (2x)
52058 Collection Plates, 10 pcs. (2x)
52059 Pierceable Adhesive Seals, 5 pcs. (2x)
52100 HPLC Column, 1 pc. (1x)

 

Order No. Required components (not included in the Reagent Kit)
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.)

 

Order No. Lab equipment
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:
As a customer please login or register to gain full access.