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Hydroxyproline contamination or interference with 1290 infinity HPLC-DAD

Question asked by simon on Nov 30, 2017
Latest reply on Jan 17, 2018 by jhagel

We have a contamination or interference for the hydrolysate sample of  hydroxyproline analysis. We think it is the product that we use that is the problem, because we can see a peak in the retention time of the hydroxyproline in a method blank. We have an 30-40 fold overestimation of our hydrolysate sample. We can see a shoulder peak or a split peak in those samples. Do you have any suggestion what is the best company to use to make sure that I don't have any contamination that come from the product mention below :


1.  Hydrochloric acid 34-37%

2.  Phenol 99%

3.  Ammonium hydroxide 28-30% ammonia solution

4.  Formic acid 88%

5.  Phosphoric acid 85%

6.  Sodium phosphate dibasic heptahydrate

7.  Sodium tetraborate decahydrate

8.  Dowex 50wx8 200-400

9.  Fiber glass filter 1.2 um

10.Regenerated Cellulose Membrane filter 0.2 um

11.Filter hplc vial PVDF 0.2 um

12.Hplc vial and insert

13.Syringes 10 mL

14.Syringes Filter PVDF 0.2 um


If you have any other suggestion why I have a peak of hydroxyproline  in a method blank, can you share it with me.


My method that I use is :

  1. The acid hydrolysis with 6 N HCl + 1% phenol for 24 hours at 110°C. 
  2. SPE with the Dowex resin.
  3. Analyze with the 1290 infinity HPLC-DAD with an OPA and FMOC derivatization.

          Column : InfinityLab Poroshell HPH-C18 4.6 * 150 mm 2.7-micron

           Mobile phase A : 10 mM Na2HPO4 7H20 and 10 mM Na2B4O7 10H20

           Mobile phase B : ACN:MeOH:H20 (45:45:10).