Can any one give a verified method for estimation of urinary citrate or Oxalate or tartarate by HPLC?

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Bruce Neagle

Try this method.

0.5835-jecm.omu.31.04.007-190427.pdf

Parmita Kar

i have read this method but it does not explain appropriately what proportion they have used trioctylamine chloroform and phosphoric acid. Can u suggest anything??

Nope! I have never used it. You will have to experiment. Try 50:50 then 25:75 and finally 75:25. Two of the chromatograms will be really bad while the other will be so so and give you a direction to go.

Actually i had tried a 30:70 ratio of trioctylamine chloroform !! But not phosphoric acid!! Many other protocols suggest acidification if urine with phosphoric acid .. do u think acidification works in this case as the mobile phase is also acidified. Also should I make my standards in urine or mobile phase?? Any suggestions

As always in HPLC make your standards and your diluted samples in mobile phase. Pure 85% ortho-phosphoric acid (with Potassium hydrogen phosphate) can be used to acidify your samples (at pH 3) and is also almost UV invisible. Some urine samples can be alkaline due to medical conditions.

Narong Chamkasem

https://www.researchgate.net/publication/21168223_Ion-chromatographic_determination_of_L-tartrate_in_urine_samples

Thanks Bruce Neagle

Thanks Narong Chamkasem ! I am new to HPLC and require some tips on standardisation. The article u sent shows a lot of peaks. it is difficult to identify one peak of interest . Also it has a different mobile phase that most of the papers mention as KH2PO4 and TBA pH2. Please suggest a step waise approach how to go for it

Parmita,

A pH buffer in the mobile phase has several conditions to ameliorate. Its primary one is to ensure only one ionic form (or peak) is present. Your urinary acids can be present as an anion or acid (tartrate anion or tartaric acid for example). By ensuring your mobile phase is acidic you will only have one peak (the protonated acid). Many HPLC chemists (back in the dark ages) used slightly acidic conditions so the simple ODS bond was not broken which would shorten the lifespan of a 'very' expensive column. However, now we have columns that can tolerate alkaline conditions up to pH 12.

The next question is buffer strength. Typically the buffer is 50-100 mM or enough (10x) to ionize your your molecule of interest. However, your pH buffer may be consumed by some other molecule in the sample and thus become ineffective in the chromatographic separation. Thus usually samples are pretreated or diluted with the mobile phase just prior to injection.

KH2PO4 alone is slightly acidic since it has 3 infection points and TBA is slightly alkaline thus you will have to use 85% Phosphoric Acid as your counter ion to get to pH 2.

First, I sent you the same paper three times because the software did not response that it was sent, so I keep sending it. Secondly, the paper shows that the ion chromatoghy column method can handle many acidic compounds, including your analytes. All you do is looking at the method and test your three compund to see ifnit will work from you for the start.

Thanks Narong Chamkasem

I could get quite a few hints for my experiments!! Thanks for the detailed suggestions !! I shall incorporate your suggestions and post my results soon

First, you work with std to see if the chromatography is good for you the way you like i.e. retention time and peak shape and see if the sensitvity fit your need. That is only half of the story. Next is work with the sample to see if the dilute and shoot will work ir need the sample cleanup. Depending on the detectir you are using.

After quite a few sample preparation methods and standardisations I finally got my specific peak at desired time point and linearity in standard dilutions.

But still facing one issue. My sample showed a peak more area than standard peaks area.

As first correction, tried to dilute my samples and checked spiking my standards with 20 times diluted samples. Diluting the samples also diluted the concentration by the same amount. But still multiplying by the dilution factor will yeild the same result. The range of sample I am getting is beyond the physiological range. Thus I feel dilution is not the correct option

As second correction I tried treating my standards the same way as I treated my samples To check wether the derivitisation process did make a difference.. But dat did not work

I use potassium dihydrogen phosphate and TBA at ph2 as mobile phase.

can anyone please help give suggestions what should I do

I don't know what is your detector (UV or MS). I would inject the blank sample that has no target analyte to see if you see any peak at the same retention time as your analyte to prove that you do not see a false positive. Because if you see the peak, it may add to the area of spiked sample. If you use MS and see nothing in the blank, you may have matrix effect. Prove by adding the same amount of the analyte in the final sample just before injection. Do the same but using solvent. If you have no matrix effect, the area of the two injections should be the same. If the spiked blank has more area than the std in solvent, you have matrix enhancement. If you have less, you have matrix suppression. As I said before, you need to play with std in solvent to get the chromatography done. Then you will have to deal with the sample preparation to minimize matrix issue. You may have either the bad blank containing the analytes or matrix effect. Since I have a limited information, I cannot help you much i.e. sample preparation, type of sample, instrument condition blah blah blah......