48000 HPLC kit crosslinks urine

Crosslinks in Urine - HPLC

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

Hydrolysis resistant internal standard
Easy sample preparation
Accurate and reliable calibration

Description

Clinical relevance

Pyridinoline (PYD) and deoxypyridinoline (DPD) are produced to provide structural stability in the form of crosslinks when collagen fibrils are created. In pathological bone degeneration, the concentrations of PYD and DPD increase in the urine. This is the case with osteoporosis or various bone cancers, for example. Thus PYD and DPD are direct biomarkers for diagnosing post-menopausal osteoporosis and monitoring therapy after intake of drugs. A significant drop in crosslinks in the urine can be observed after just a few months of successful treatment. Furthermore, crosslink concentrations in the urine are subject to ongoing monitoring in patients at greater risk of fracture, so that suitable measures can be taken right away if the levels are already high.

 

Product advantages

  • Hydrolysis-resistant internal standard
  • Easy sample preparation
  • Accurate and reliable calibration

 

This kit allows simple and reliable simultaneous quantification of pyridinoline (PYD) and deoxypyridinoline (DPD) in urine using a simple isocratic HPLC system. Chromatographic separation is performed at an RP column with subsequent fluorescence detection. The use of an acidic hydrolysis-resistant internal standard maximises the precision and reliability of the determination. Interfering fluorophores are removed using solid phase extraction, and then PYD, DPD, and internal standard are eluted in one run.

Chromatogram

Technical Data

Method of Analysis

HPLC

Number of Tests

100

Parameter

Deoxypyridinoline (DPD), Pyridinoline (PYD)

Analysis Time

< 15 min

Limit of quantification

Pyridinoline: 15 pmol/ml
Deoxypyridinoline: 15 pmol/ml 

Linearity

Pyridinoline: up to 3200 pmol/ml
Deoxypyridinoline: up to 1200 pmol/ml

Recovery

Pyridinoline: 95.5 % (CV: 1.4 %); Deoxypyridinoline: 93.6 % (CV: 2.0 %)

Intraassay

Pyridinoline: CV = 3.8 %
Deoxypyridinoline: CV = 5.9 %

Interassay

Pyridinoline: CV = 5.1 %
Deoxypyridinoline: CV = 8.9 %

Specimen

Spontaneous urine or collected for a period

Pre-analytic Treatment

Patient samples can be transported at room temperature. For further storage (up to 7 days), storage at +4 °C is recommended. For longer periods, samples should be stored below -18 °C. Protect against incident light!

Sample Preparation

  • Add 50 µl Internal Standard to 250 µl urine and hydrolyse with 250 µl 12 N HCl in screw capped glass tubes over night (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 sample volume to conditioned column, drain by centrifugation at approx. 170 x g, discard the effluent.

  • 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 prepared samples are stable for 2 days at room temperature and for 4 days at +2 to +8 °C

Additional Info

Any isocratic HPLC system with fluorescence detector is suitable.

Injection Volume

50 µl

Flow rate

1.2 ml/min

Gradient

isocratic

Column temperature

ambient (~ 25 °C)

Wavelength

EX 290 nm
EM 395 nm

Please note

The product information provided shows typical values as an example only. Please review the information provided in instruction manual and product information leaflets.

Kit Components

Components available separately

Mobile Phase


Order no.: 48001

Internal Standard


Order no.: 48004

Extraction Buffer


Order no.: 48005

Wash Buffer


Order no.: 48006

Elution Buffer


Order no.: 48007

Sample Clean Up Columns


Order no.: 48008

Calibrators

Calibrators available separately

Controls

Controls available separately