[Bitty]

I am using a Megazyme Lactic Acid Kit to determine L-Lactic acid in animal feed.  Basically I'm using L-lactate dehydrogenase to convert Lactate to pyruvate and NADH. The reaction is driven to the right by trapping pyruvate with glutamate-pryuvate tranaminase.

The Lactic acid concentration is determined by absorbency of NADH at 340 nm. My problem is that the absorbance seems to keep increasing indefinitely. It says in the SOP that the reaction should take approx 10 min, after which the absorbance should level off.

Anyone have any idea why the absorbance is increasing indefinitely? I've tried using readings at 10 min, 20 min, 30 min and extrapolating to t=0 and got bogus values.

Thanks!

oh yeah, the method SOPs are available here:

http://secure.megazyme.com/downloads/en/data/K-LATE.pdf

[Arkcon]

A quick look at your method shows a lot of steps, and since I don't know your technical level, I can't guess where the problems might be.  You'll have to satisfy for yourself that you're following all procedures.

Do you see visual changes in your sample over time?  Turbidity or cloudyness can show up in any sample, and of course, scatter all light, including your chosen wavelength.

Are you satisfied that your optical instrument is warmed up, and in good working condition?  Do you have a test sample, usually a neutral density filter (a piece of colored glass or plastic) and does it give stable results?  Can you find a standard that will be specific for 340 nm, to be sure your instrument is up to snuff?

Do you have a lactic acid standard?  That is pure L-lactic acid, to react with this kit to see if your system behaves properly with an executed kit, with no possibility of other feed components interfering.

What about the possibility that the feed contains so much, or so little, L-lactic acid that the reaction is saturating, or failing to react.  You may have to try a number of sample sizes, to learn how this kit works with some feeds.

[Bitty]

Thanks for the help Arkcon. I do in fact have a 0.15 g/L L-lactic acid standard which I have used to calibrate my equipment. I ran into the same problem using the standard as with the extracted feed samples.  I suspect there may be a problem with the Spec.

I graphed the absorbances vs.  reaction time and the plot was linear enough to extrapolate the reaction time required to obtain the correct concentration. It ended up being 52 min. Although I'm sceptical of waiting 52 min per sample until I can figure out why this is happening. Also I have over 150 samples to analyze so that could be a bit time consuming!


I do expect the feed samples to contain very little lactic acid, near the lower detection limit. However I am encountering the same problem using standards and blanks.

I'll take a look around the lab for a neutral density filter, hopfully that will diagnose the issue. Thanks again.

 

[Arkcon]

I'll take a look around the lab for a neutral density filter, hopfully that will diagnose the issue. Thanks again.

Yes, the N.D. filter will help remove ambiguous chemistry from the mix, you can see if your instrument needs tweaking, or repair, or simply a longer warm-up time.

[Barry McCleary]

Hello Bitty

I hope that you resolved the problem with our L-lactic acid kit. I suspect the problem is related to sample preparation and possibly interfering chemicals in the sample. If you want specific help, please contact me at   xxx

Barry

Edit: asked on forums, answerd on forums.