Amino Acid Analysis in Supplements
Amino acids are the building blocks of life. Every living thing uses amino acids to build the proteins needed to do various functions within the organism. Where do amino acids come from and how many are needed to function? For humans, there are a total of 20 amino acids that are necessary for your body to function properly, of these 20 only 11 are made within the body and the rest must come from your diet. The nine amino acids that are not made by the human body are called essential amino acids.
I have decided to focus on two amino acids, they are Glutamine and Leucine. Looking around samples at the store, I came across a sample that contained many amino acids including the two that I was focused on. The sample is a mixture marketed to promote healthy skin and nails. The analysis will be carried out on the LC-UV. The supplement will be dissolved in HPLC grade water.
A standard was made in a concentration of 300 mg/L, made from 1000 mg/L stock solution. Then, 1 gram of the powdered sample was added to 50 mL of water.
Example of the 1 gram sample
Next, each standard and sample needed to be derivatized because amino acids don’t show up using UV light, so something that does needs to be attached to the amino acid. I have opted to use o-Phthaldialdehyde or OPA reagent. This reagent works well on primary amino acids.
For each, 14 uL of each was pipetted to a 1.5 mL LC vial, then 255 uL of OPA reagent is added to the vial, followed by 731 uL of HPLC grade water to dilute the samples.
The mobile phases for the HPLC are a 0.05 M sodium dihydrogen phosphate solution adjusted to pH 7.8 using 10 M sodium hydroxide and a 45:45:10 Acetonitrile:methanol:water solutions. The method for the LC-UV is outlined below.
| Lucidity LC-UV Method | |
|---|---|
| Flow Rate | 2.0 mL/min |
| Column | Agilent Eclipse AAA 150 x 4.6 mm, 5μm |
| Loop Size | 20 μL |
| Oven Temperature | 40 ℃ |
| Wavelength | 338 nm ±1 |
| Flow | Isocratic – 20 minutes |
| Mobile Phase A | 6.2 g Sodium Dihydrogen Phosphate in 1 L water, pH 7.8 |
| Mobile Phase B | Acetonitrile:Methanol:Water (45:45:10) |
| Gradient Program | Time (min) % B
0.0 2 2.0 2 12.0 57 17.0 90 25.0 90 26.0 2 35.0 2 |
Each of the standards was run individually at first to obtain an elution order for a mixed standard that will be used for the samples. The mixed standard consists of Leucine, Glutamate and Lysine. The sample did not contain lysine so it was dropped from future testing in this study.
Retention time check for Leucine
The large peak to toward the end of the chromatogram is due to the mobile phase changing from buffer to organic phases and back again. To clear things up and make them easier to see I will zoom in on the peaks of interest.
Zoomed in chromatogram of Leucine
Chromatogram of Glutamine
Once, all of the standards elution times were confirmed a mixed standard was run. This was done to ensure that the total amount of OPA reagent was enough to derivatize all of the amino acids in the standard and in the sample.
Chromatogram of the three standards used, Glutamine, Lysine and Leucine
After a blank, the sample was run. The same procedure that was applied to the standards was applied to the sample, although the sample was filtered through a 0.45 μm filter before being derivatized.
Chromatogram of the mixed Amino Acid sample
The Lucidity UV-LC was easily able to separate out all of the amino acids in the mixture and pick out the Glutamine and Leucine. This was just a qualitative analysis to ensure that the sample could be derivatized and show the different amino acids within. Stayed tuned for our more in depth study on amino acids where we quantify the amount of amino acids in different samples and compare against the label claim.