52952-UHPLC-F kit vitamin B1 vitamin B6 whole blood

Vitamin B1/B6 in Whole Blood, fully automated analysis - UHPLC

Order no.: 52952/UHPLC/F
960 Tests
  • Vitamin B1
  • Vitamin B6

Significantly reduced hands-on time for sample preparation
UHPLC separation in less than 3 min
Accurate and fast results for both vitamins
Each parameter safeguarded by a specific internal standard

Description

Clinical relevance

Vitamin B1 (thiamine) plays an important role in deriving energy from carbohydrates and fats. The active form is thiamine pyrophosphate (TPP), a coenzyme for some enzymatic reactions in carbohydrate metabolism and for pyruvate decarboxylase. TPP is also significantly involved in the oxidative degradation of glucose. The determination of the TPP concentration is more significant than measurements of total thiamine, as by this method only the physiologically active form is taken into account.

Vitamin B6 comprises pyridoxine, pyridoxamine and pyridoxal, as well as their phosphate derivatives. It is ingested with food and transferred via several enzymatic conversions into its active form, pyridoxal-5’-phosphate (PLP). PLP is involved in nearly one hundred different enzymatic reactions, among other things, as co-factor in amino acid metabolism, and in the formation of haemoglobin or neurotransmitters in the brain.

 

Product advantages

  • Maximum throughput: Automated sample preparation and fast UHPLC in less than 3 minutes
  • Reliable and reproducible results
  • Each parameter safeguarded by a specific internal standard

 

This UHPLC kit facilitates the combined analysis of vitamin B1 in whole blood and vitamin B6 in whole blood or plasma. Sample preparation is automated in 96 well filter plates for maximum output and minimal workload. UHPLC requires less than 3 minutes per sample. The internal standard for each of the two parameters and special quality controls ensure precise and reliable quantification.

Chromsystems also offers two kits that feature manual determination using HPLC: a standard version as well as a kit for simplified sample preparation with pre-mixed tubes.

Alternatively, an automated method using HPLC is also available.

 

Kit content for 960 analyses:

Mobile Phase A (for UHPLC), 2 x 1000 ml
Mobile Phase B (for UHPLC), 1000 ml
UHPLC column, 1 pc.
Vitamins B1/B6 Whole Blood Calibration Standard (lyoph.), 5 x 5 x 1 ml
Internal Standard Mix for automated sample preparation, 50 ml
Extraction Reagent for automated sample preparation, 2 x 75 ml
Prep Solution for automated sample preparation, 3 x 25 ml
Finisher 1 for automated sample preparation (lyoph.), 2 x 5 x 0.25 ml
Finisher 2 for automated sample preparation, 50 ml
Dilution Buffer for automated sample preparation, 100 ml
96 well filter plates, 2 x 5 pcs.
96 well collection plates, 2 x 10 pcs.
Pierceable adhesive seals for 96 well plates, 2 x 5 pcs.

Chromatogram

Technical Data

Method of Analysis

UHPLC

Number of Tests

960

Parameter

Vitamin B1, Vitamin B6

Analysis Time

3 min

Limit of quantification

PLP 2.5 μg/l
TPP 4.0 μg/l

Linearity

PLP 500 μg/l
TPP 750 μg/l

Intraassay

CV < 5 %

Interassay

CV < 6 %

Column temperature

ambient (~ 25°C)

Flow rate

0.7 ml/min

Specimen

Whole Blood

Pre-analytic Treatment

Light protected in a closed vial and stored at +2 to +8 °C patient samples are stable up to 1 week. For longer storage (up to 2 weeks) patient samples should be frozen below -18 °C.

Sample Preparation

  • Add 100 μl whole blood/calibrator/control and 50 μl Internal Standard Mix in a 96 Well Collection Plate.
  • Add 150 μl Extraction Reagent.
  • Shake for 4 min.
  • Remove precipitate by centrifugation (3 min at 2000 x g).
  • Transfer 75 μl of supernatant in a 96 Well Filter Plate.
  • Add 75 μl Prep Solution and 50 μl of Finisher Solution.
  • Mix for 30 s.
  • Remove precipitate by centrifugation (2.5 min at 1500 x g).
  • Incubate supernatant for 45 min at 60 °C
  • Add 100 μl Dilution Buffer, mix briefly (30 s).
  • Seal plate and transfer to autosampler.
  • Inject 2.5–5 μl of the eluate into the UHPLC system.

Sample Stability

Up to 1 week at +2 to +8 °C and up to 2 weeks at below -18 °C. Protect from light.

Injection Volume

2.5–5 μl

Wavelength

Start EX 320 nm, EM 415 nm,

after approx. 1.6 min switch to EX 367 nm, EM 435 nm

Please note

The freely available information on this website, in particular on the sample preparation, are not sufficient to work with our products. Please read instructions and warning notices on products and/or instruction manuals.

Kit Components

Components available separately

Calibrators

Calibrators available separately

Controls

Controls available separately

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