Crosslinks in Urine - HPLC

Crosslinks in Urine - HPLC

Order no.: 48000/RUO
100 Tests
  • Deoxypyridinoline (DPD)
  • Pyridinoline (PYD)

Hydrolysis resistant internal standard
Easy sample preparation
Accurate and reliable calibration

Description

The high sensitivity of the Chromsystems method permits both the total contents of pyridinoline (PYD) and deoxypyridinoline (DPD), as well as the concentration of the free pyridinolines (PYD, DPD) in urine to be determined. The use of a hydrolysis-stable internal standard (IS) allows possible losses during sample preparation to be compensated, thus maximising performance of the determination. Interfering fluorophores are removed by solid-phase extraction and then PYD, DPD and IS are eluted together. The chromatographic separation is isocratic with subsequent fluorescence detection.

Chromatogram

Technical Data

Method of Analysis

HPLC

Number of Tests

100

Parameter

Deoxypyridinoline (DPD), Pyridinoline (PYD)

Analysis Time

approx. 15 min

Column temperature

ambient (~25 °C)

Flow rate

1.2 ml/min

Specimen

Urine

Pre-analytic Treatment

 

 

Sample Preparation

  • Add 50 μl Internal Standard to 250 μl urine and hydrolyse with 250 μl 12 N HCl in screw capped glass tubes overnight (12–16 h) at 100°C.
  • Add 2.5 ml Extraction Buffer, mix briefly.
  • To condition the SPE-columns, apply 2.0 ml Wash Buffer and drain by centrifugation.
  • Pipette entire research sample volume to conditioned columns, drain by centrifugation at approx. 170 x g, discard the effluents.
  • Pipette to each column 2.5 ml Wash Buffer, drain sharply by centrifugation at approx. 700 x g, discard the effluents, repeat this step twice.
  • Change the collection tubes.
  • Pipette 1.0 ml Elution Buffer to each column and drain completely by centrifugation at approx. 700 x g, collect each eluate and mix well.
  • Inject 50 μl of each eluate into the HPLC system.

Sample Stability

The research samples are stable up to 7 days at +4°C. For longer periods of storage, deepfreeze them (below -18°C).

Injection Volume

50 µl

Additional Info

The analysis of the free and/or total pyridinium crosslinks requires a simple, isocratic system with an HPLC pump, an injector and a fluorescence detector.

Wavelength

EX 290 nm
EM 395 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

Mobile Phase


Order no.: 48001/RUO

Mobile Phase, 10 x 1000 ml


Order no.: 48002/RUO

Internal Standard


Order no.: 48004/RUO

Extraction Buffer


Order no.: 48005/RUO

Wash Buffer


Order no.: 48006/RUO

Elution Buffer


Order no.: 48007/RUO

Sample Clean Up Columns


Order no.: 48008/RUO

Calibrators

Calibrators available separately

Controls

Controls available separately

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