I think that in order to get a truly selective and quantitative measurement of cysteine in a complex mixture such as animal feed, some sort of separation method will have to be used to isolate the signal for cysteine from signals due to other substances.

A very simple colorimetric method for measuring the concentration of total thiol groups, including cysteine (but not cystine or cysteic acid), is to use Ellman's reagent. For this procedure, however, you will have to produce a clear extract of the feed. Also, it seem likely to me that cysteine in animal feed would be oxidized, so would not be present in the reduced form and would therefore not react with Ellman's reagent. In that case, you would need a method that can measure the oxidized forms of cysteine.

Here is a fluorescence-based assay kit for cysteine thiol that is not affected by other thiol-containing amino acids. It is intended for use with serum or other biological fluid.

http://www.abcam.com/cysteine-assay-kit-fluorometric-ab211099.html

To measure both cysteine and cystine (and other thiols), you could first reduce the cystine to cysteine using TCEP, then react the thiols with a fluorogenic maleimide such as coumarin phenyl maleimide (https://www.thermofisher.com/order/catalog/product/D346), which does not require removal of the TCEP.

I think that in order to get a truly selective and quantitative measurement of cysteine in a complex mixture such as animal feed, some sort of separation method will have to be used to isolate the signal for cysteine from signals due to other substances.

A very simple colorimetric method for measuring the concentration of total thiol groups, including cysteine (but not cystine or cysteic acid), is to use Ellman's reagent. For this procedure, however, you will have to produce a clear extract of the feed. Also, it seem likely to me that cysteine in animal feed would be oxidized, so would not be present in the reduced form and would therefore not react with Ellman's reagent. In that case, you would need a method that can measure the oxidized forms of cysteine.

Here is a fluorescence-based assay kit for cysteine thiol that is not affected by other thiol-containing amino acids. It is intended for use with serum or other biological fluid.

http://www.abcam.com/cysteine-assay-kit-fluorometric-ab211099.html

To measure both cysteine and cystine (and other thiols), you could first reduce the cystine to cysteine using TCEP, then react the thiols with a fluorogenic maleimide such as coumarin phenyl maleimide (https://www.thermofisher.com/order/catalog/product/D346), which does not require removal of the TCEP.

Thank you for your valuable inputs. Will take into consideration of all your suggestion.

By separation method, what would you suggest? Will a simple centrifugation or filtration works? Or some sort of chemical separation need to be done?

To begin, you will have to use some sort of digestion and extraction procedure to separate the amino acids from the feed. Then the liquid part will have to be separated from the solid part by centrifugation or filtration. Finally, you will probably need to use chromatography to separate the small molecules from each other.

Amended on 7 May 2018

Please use reliable and precise HPLC-photometric method. I have recently found that HPLC-photometric method gives c.a. 2-fold larger determination values as compared to direct-photometric method (please see Fig. 10 of the file; JCB Fucoidan transport).

Pyridylethylation (PE) and PTC (phenylthiocarbamyl) derivatization of cysteine has been reported (please see file; PTH Roma RP-HPLC). PTC-Cys is a little difficult to measure than PTH-PeC, and please use a short column (3 cm column-length). PTC-Cysteine and PTC-Cystine may be separated in the RP-HPLC, and both Cysteine and Cystine may be measurable simultaneously.

I have recently borrowed Shimadzu's HPLC system from ORIX Leasing Co.. (HPLC-pump + Injector + HPLC-photometer + Data processor (Chromatopac)), please do not use the secondhand pump.

New pump gives sufficiently reliable performance, therefore high-performance liquid chromatography (HPLC) is achieved.

ORIX has Thai branch.

Thai ORIX Leasing Co., Ltd. (TOLC)

http://www.orix.co.th/about-us/

Leasing cost has been 120,000 Yen/month or 1,200 USD/month in my case.

Some technical advices.

To avoid possible thiol oxidation, the procedure could be done inside "the anoxic hood" if available (?).

Inside such the hood

you can homogenize "animal feed"

add the Ellman reagent

spin down the "solids"

measure Abs in supernatant

My experience - Ellman reagent reacts also with dissolved sulfide...

Thank you all for your input.

Dear Dr. Shapiro,

By digestion, you mean something like performic acid digestion under vacuum condition right? Unfortunately, in my understanding of the protocol, the lab lacks the necessary glasswares and micro-pump for that purpose.

Assuming that I'm studying spray-coating of ready-made animal feeds with cysteine solution. Do you think simple feed blending using blender and re-suspension of small amount of sample in deionized water will work with the colorimetric method I asked earlier? Since I'm detecting free cysteine additionally coated instead of cysteine already present in the feed?

I think this is the critical question to whether if I can do this in the lab here or not.

Dear Dr. Hayakawa,

Thank you for your input. Indeed that HPLC will yield better and more reliable method than any direct UV measurement, but we may be under budget constraint. Your suggestion of lending a HPLC unit is a highly possible workaround, we will give it a thought. Appreciate your idea.

Dear Dr. Galushko,

I'm interested in your suggestion of using Ellman's reagent for direct UV detection. Some reading on the internet suggested that Ellman only works for reduced cysteine, together with your mention of anoxic hood, I suppose it will not work with oxidized cystine, right?

So one more question in this reply. I know that cysteine solution is easily oxidized. Can anyone, by experience or literature, tell me exactly how "fast" is this oxidation of cysteine solution? I mean after I dissolved the chemical in water. This is crucial to me as it will determine how should I worked with this chemical in the lab for my study.

Once again, thank you to everyone.

If you are only interested in the amount of cysteine on the surface of the feed, as opposed to the total cysteine content of the feed, then perhaps it would be sufficient to extract the surface cysteine by soaking the feed pieces in water, and measuring the cysteine content of the resulting supernatant, after filtering out the solids.

Agree with Adam B Shapiro.

Kind regards

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