Normally glucosinolates that haven't been desulfated, are eluted earlier in the chromatogram. Could you please give the concentration of the sulfatase solution and the incubation time used?

(Not a glucosinolate specialist but...) How is the saturation of your column? Is it possible that considering the concentration of your analites, they fail to elute at their normal retention time?   

Not a specialist of glucosinolate, but find in attached files some publications about extraction of glucosinolate in different substrats

Our glucosinolate specialist has retired but I can contact him if you are still stuck. Best, Nick

Don Cipollini at Wright State University has been measuring glucosinolates from garlic mustard for a number of years. He may be a good resource for discussion of various extraction protocols and their potential pitfalls.

Try Atle Bones  in Norway (Trondheim University). They have good analysis systems for glucosinolates.

Thank you for all your really helpful comments. For clarification, we use a 0.2% (m:v) concentration of the sulfatase and incubate 15h. Does this change any of your suggestions? Thank you very much and 

Best wishes

Although not being a specialist, I believe that your analytes haven't been desulfated, probably due to insufficient sulfatase activity (U/ mL). I hope the link below or an expert could help you

http://wrap.warwick.ac.uk/3913/1/WRAP_THESIS_Issa_2010.pdf

If the sinigrin standard is still intact (i.e. glucosinolate form) then the desulfonation step hasn't worked properly. Try using fresh sulfatase and repeat the procedure. If the problem persists the  following paper contains an alternative method which might be of use;

Valverde, J., Reilly, K., Villacreces, S., Gaffney, M., Grant, J.  & Brunton, N. (2014). Variation in bioactive content in broccoli (Brassica oleracea var. italica) grown under conventional and organic production systems. Journal of the Science of Food and Agriculture.

Sulfatase (Type H-1 from Helix pomatia; Sigma, St Louis, MO, USA) was purified by dissolving the sulfatase powder (70 mg) in deionised water (3 mL) and adding ethanol (3 mL). This solution was centrifuged (18 000 g, 10 min, room temperature) and to the supernatant ethanol (9 mL) was added after which the solution was centrifuged (18 000 g, 10 min, room temperature) again. The pellet was dissolved in deionised water (2 mL) and this sulfatase solution was subsequently passed through a 0.5 mL DEAE Sephadex A-25 and a 0.5 mL SP Sephadex C-25 column. This solution was collected in a vial and kept at −80 °C until use.

An aliquot (1 mL) of glucosinolate extract was applied to a DEAE Sephadex A-25 column (0.5 mL) and the unbound material was removed by washing with deionised water (2 × 1 mL) and sodium acetate buffer (2 × 0.5 mL, 20 mmol L−1, pH 5.0). After washing, purified sulfatase solution prepared as above was added and the columns were incubated overnight at room temperature. After overnight incubation, the desulfoglucosinolates (dGLS) were eluted from the columns with deionised water (3 × 1 mL). The collected eluate was dried under constant N2 flow and re-dissolved in deionised water (200 µL).

You could also try to analyse the intact glucosinolates rather than isothiocyanate form. However, as they are eluting quite quickly you may need to change to a HILIC column and experiment with the gradient. 

I tried to attach a file wilt glucosinolate extarctio technique to to you but I  don't know if it works

Article Release of Allyl Isothiocyanate from Mustard Seed Meal Powder

Article Can mustard seed meal increase attacks by Aleochara spp. on ...

Article Structural features of an l-arabinan derived from mustard seed meal

Andre, for what you describe it seems that what you have in your chromatograms is intact glucosinolates and not the desulfated form.

But, if you say that the sulfatase is working properly, there are other issues that I think could explain your results. Did you hydrate the DEAE with water instead an acidic buffer? Because that can affect both glucosinolate binding to the DEAE and the desulfation reaction. Did you check the pH of the incubation buffer for the desulfation step? If you are using the proper buffer for the incubation, maybe the activity of your enzyme is affected, try to increase enzyme concentration . If this does not solve the problem then check the elution step from the DEAE. We usually elute desulfoglucosinolates from the DEAE Sephadex with MilliQ water, to avoid eluting intact glucosinolates and other compounds that have not been desulfated. If you are eluting the DEAE with a buffer instead of water, maybe you are eluting glucosinolates that have not been desulfated at all. If you are eluting whit water, I think that if the sulfatase wasn't working properly, you should not have been able to elute the glucosinolates from the DEAE Sephadex and you should not have any peaks in your chromatogram. So, if the sulfatase is working properly maybe the problem is in the HLPC column, did you check the column performance with metabolites other than glucosinolates? Did you change the mobile phase composition or its pH?

I hope you can solve the problem, best wishes!

This does sound like HPLC problem. Can you try a new column? or an old desulfated sample on the same column? Is the column temparture controled? Also make sure your A and B solvents were not replaced by mistake.

Yoram

I have a related question. I am trying to analyze myrosinase from Thlaspi arvense using a microplate assay testing for glucose. I'm getting glucose in my sample controls, which suggests to me that glucosinolates are getting through my columns. I've been eluting the columns with Tris-EDTA buffer pH 5.5 (same buffer used for the myrosinase extraction step). I used 0.5M acetic acid buffer pH 5 to swell the sephadex.  I'm wondering if I should be using DI water both for the swelling and eluting. Any suggestions?

Robin, what is the column used for? Purification of intact glucosinolates?

I'm trying to isolate myrosinase, so the column is to trap the glucosinolates. I then quantify amount of myrosinase by adding a known amount of substrate (sinigrin). Differences in glucose concentration are indicative of different levels of myrosinase. I need to remove the endogenous glucosinolates so that they don't interfere with the quantification of myrosinase.