High Throughput UHPLC, combined Vitamin B1/B6 Analysis - UHPLC
UHPLC separation in less than 3 min
Each parameter safeguarded by a specific internal standard
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.
- High throughput due to separation in less than 3 minutes
- Reliable and reproducible results with a specific internal standard for each parameter
- Easy sample preparation with Pre-mixed Tubes
The new UHPLC kit facilitates the combined analysis of vitamin B1 in whole blood and vitamin B6 in whole blood or plasma. Due to faster analysis times in less than 3 minutes, this method fulfils the requirements of clinical laboratories with higher sample throughput. The easy and efficient sample preparation consists of a single precipitation step and a final derivatisation in a reaction vial. The internal standard for each of the two parameters and special quality controls ensure precise and reliable quantification.
Chromsystems also offers two more kits that feature determination using HPLC: a standard version as well as one with pre-mixed tubes.
An automated method using HPLC is also available for laboratories with higher sample throughput.
Mobile Phase A, 2 x 1000 ml
Mobile Phase B, 1000 ml
Whole Blood Calibration Standard (lyoph.), 5 x 5 x 1 ml
Internal Standard, 10 x 10 ml
Precipitation Reagent, 10 x 30 ml
Pre-mixed Neutralisation Tubes, 10 x 100 pcs.
Derivatisation Reagent 1 (lyoph.), 10 x 2 x 0.3 ml
Derivatisation Reagent 2 , 10 x 15 ml
Reaction vials, amber coloured (light protection), 10 x 100 pcs.
UHPLC column, 1 pc.
|Number of Tests||1000|
|Limit of quantification||PLP 5 μg/l (whole blood) TPP 5 μg/l (whole blood) PLP 1.4 μg/l (plasma)|
|Linearity||Vit. B6 (PLP) 500 μg/l Vit. B1 (TPP) 750 μg/l|
|Intraassay||CV ≤ 3.8 %|
|Interassay||CV ≤ 6 %|
|Analysis Time||approx. 3 min|
|Sample Stability||The prepared samples are stable at room temperature for 1 day, at +2 to +8 °C for 7 days. For longer storage (up to 2 weeks) keep samples below -18 °C|
|Pre-analytic Treatment||The patient samples are stable at +2 to +8 °C for 1 week, light-protected in a closed vial. For longer storage (up to two weeks) keep samples below -18 °C.|
|Injection Volume||2.5–10 μl|
|Flow rate||0.7 ml/min|
|Column temperature||ambient (~ 25°C)|
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.|
|Method of Analysis||UHPLC|
|Parameter||Vitamin B1 (TPP, thiamine pyrophosphate), Vitamin B6 (PLP, pyridoxal 5’-phosphate)|
|Additional Info||Any major UHPLC system with binary pump and programmable fluorescence detector is suitable.|
|Pressure||minimum 450 bar|