LC-UV tells if I have diabetes!

Diabetes is a disease that affects over  800 million people world wide. If someone is suspected of having diabetes, their doctor will do a dipstick test on their urine for the presence of ketones.  

Ketones in the urine can be caused by several things, such as a low carbohydrate diet (keto diet), but the most likely cause is diabetes. The ketones arise due to the body using fat, rather than glucose, for energy.  If too many ketones are present in the blood, called ketoacidosis, there could be a build-up of acid, which is a serious medical issue, and requires immediate medical attention.

In the past I have used the Lucidity HPLC to test various types of samples for ketones, and I wondered if this method could be used to test for ketones in urine.

The procedure was simple, I used synthetic urine for my sample that was either free of ketones, or spiked with 1 mg/mL of acetoacetate, the same ketone that the dipstick test measures. I then made a standard using the same concentration, 1 mg/mL, of the acetoacetate and ran them on the Lucidity LC-UV using the method below.

Before I could run the samples, I had to derivatize the samples using a 2,4-dinitrophenylhydrazine (DNPH) solution.  The solution was made by adding 0.2 g of DNPH into a 1:1 solution of acetonitrile:methanol, and mixing until all the DNPH was dissolved. This solution is only useable for about 48 hours, so the solution was made fresh just before the samples were to be run on the LC-UV.  Once ready to run the batch on the LC-UV, 600 μL of sample and 600 μL of the DNPH solution were added to a sample vial, shaken vigorously for 30 second and put onto the autosampler tray.

I ran a blank, standard, and two urine samples and collected the chromatograms.

The overlaid chromatograms of the two urine samples and the standard.

There are two major peaks, the derivatized acetoacetate peak and the excess DNPH solution.  Looking at the chromatograms, the urine 1 sample had no acetoacetate peak and the largest DNPH peak.

Acetoacetate standard chromatogram

Urine sample 1 shows only the DNPH peak, having no sign of ketones in the sample

Urine sample 2 has a significant acetoacetate peak, showing signs of ketoacidosis

Overall, the peak shape of the ketone wasn’t the best.  This could be due to the column selection. Normally, I would use either a 250 mm column or a smaller particle size to get better peak shapes. Also, I did no optimization of the mobile phase. Most likely, a gradient would have been a much better choice in running these samples, but the point of this experiment was to see if it could be done, and the LC-UV was able to correctly determine which urine sample was spiked with acetoacetate.

If you would like to learn more or to see the LC-UV in a virtual demo, click the links below to find out how!