Hydroxypyridinium Crosslinks HPLC Assay


The Hydroxypyridinium Crosslinks HPLC Assay kit is intended for the quantitative determination of the hydroxy-pyridinium crosslinks in urine. The Hydroxypyridinium Crosslinks HPLC Assay kit is for research use only and not to be used in diagnostic procedures.

Hydroxypyridinium Crosslinks HPLC Assay

The Hydroxypyridinium Crosslinks HPLC Assay is For Research Use Only

Size: 100 tests
Incubation Time: 10 minutes
Sample Type: Lithium Heparinate Plasma
Sample Size: 200 µL

Product Developed and Manufactured in Germany
Product Support in the USA

Assay Background

Pyridinium and deoxypyridinium are built as intramolecular crosslinkers during the maturation of collagen. During bone resorption collagen is degraded and the pyridinium-crosslinks are excreted. They are not re-utilized by the body and excreted in the urine. The crosslinks are not released during the de novo synthesis of collagen. They are specific for resorptive processes. Neither PYD nor DPD are taken up by nutrition. Therefore the determination of the crosslinks is independent from the diet. PYD is found in cartilage, ligaments and bone, whereas DPD is rather specific for bone.

The crosslink excretion shows a strong circadian rhythm with a maximum in the early morning hours. Reliable data can be obtained with a 24h or a creatinine corrected morning urine. An elevated crosslink excretion is shown in osteoporotic women. Crosslink levels decrease during treatment with estrogen (Seibel et al. 1994). Therefore they are suited for therapy monitoring (Ganero et al. 1994). The measurement of pyridinium crosslinks is an independent predictor for future bone fractures (Ganero et al. 1995). Increased crosslink concentrations are also described in patients with Paget’s disease, primary hyperparathyroidism and bone metastasis.

Hydroxypyridinium Crosslinks HPLC Assay kit application for the pyridinium crosslinks makes it possible to determine pyridinium and deoxypyridinium in an easy, fast and precise method. The Hydroxypyridinium Crosslinks HPLC Assay kit includes all reagents in a ready to use form for preparation and separation of the samples with exception of the columns (IC3201rp) and the controls (IC3201ko). Both can be supplied separately by Eagle Biosciences. Beside the complete test kits it is possible to order all components separately. Please request our single component price list.

Related Products

Hydroxypyridinium Crosslinks HPLC Control Set
Hydroxypyridinium Crosslinks HPLC Column
Malondialdehyde HPLC Assay Kit

Additional Information

Assay Principle

For the determination of the pyridinium crosslinks, the samples are hydrolyzed and extracted on solid phase extraction cartridges. The probe is centrifuged and 100 µl of the supernatant are injected into the HPLC system. The isocratic separation is via HPLC at 30°C using a “reversed phase” column. One run lasts 15 minutes. The chromatograms are recorded by a fluorescence detector. The quantification is performed with the delivered plasma calibrator; the concentration is calculated via integration of the peak areas or heights.

Assay Procedure

  1. Pipette into glass tubes: 1 ml sample, CAL or CTRL + 1 ml HYDRO (caution: Hydrochloric acid).
  2. Mix well. Leave the tubes for 4 hours at 120°C.
  3. Soak 1 ml WASH1 through the SPE cartridge by vacuum.
  4. Mix 0.5 ml WASH1 + 0.5 ml hydrolyzed cold urine + 3 ml REAC.
  5. Shake very well, pipette in the SPE cartridge and let it soak through by mild vacuum.
  6. Rinse the cartridge with 6 ml WASH1 under mild vacuum.
  7. Dry the cartridge by vacuum for 10 min.
  8. Rinse the cartridge with 1 ml WASH2 under mild vacuum.
  9. Dry the cartridge by vacuum for 10 min.
  10. Elute the sample with 0.5 ml ELUSO under vacuum.
  11. Centrifuge and inject 100 µl of the supernatant for chromatography into the HPLC-system.


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