The LC Analysis of Trending Weight Management Drugs: Semaglutide, Retatrutide, Tirzepatide

The latest class of weight management drugs is trending everywhere. By now I’m sure you’ve heard the stories of incredible weight loss experienced by celebrities or maybe even someone you know by using these drugs. But what are these drugs and how can we analyze them? Semaglutide, Retatrutide, and Tirzepatide belong to a class of drugs called Glucagon-like peptide-1 (GLP-1) receptor agonists. These drugs reduce blood sugar by activating the glucagon-like peptide-1 receptor (GLP1R) that controls blood sugar levels by insulin secretion. This can help to mimic insulin production which reduces hyperglycemia in patients with type 2 diabetes. Additionally, these drugs have found a popular alternative use as they also help to simulate fullness which helps to decrease food intake, and as such is great for weight management. 

These compounds are peptides composed of amino acids linked by peptide bonds ranging from 4113.6- 4813.5 Da. LC-UV methodology was investigated first since this detector is robust and cost-effective. A method was developed with excellent resolution of the three target analytes with a cycle time of only 4.5 minutes using a Raptor Biphenyl column. 

Next, LC-MS was investigated for the detection of these compounds. The single quadruple mass spectrometer that was used in these experiments has a mass range from 30 – 1250 m/z. Typically when analyzing peptides, polypeptides, or large molecules by ESI (+) MS, the analyte has multiple charges on its structure that are facilitated by the mobile phase additives and ESI source to have a m/z ratio that falls within the detectors range. 

During method development, a scan was performed ranging from 500 – 1250 m/z to determine that dominant charge state for each of the peptides using 0.5% formic acid in both water and acetonitrile as the mobile phases. It was determined that the +4 was the dominant charge state for each of the analytes monitored: 

Retatrutide 4731.3 Da= 1184 m/z
Semaglutide 4113.6 Da= 1029 m/z
Tirzepatide 4813.5 Da= 1204 m/z

The charge state was determined by the following equation:

[4731.3 Dɑ+4] 4

= 1183.8 m/z for Retatrutide

[4113.6+4] 4

= 1029.4 m/z for Semaglutide

[4813.5+4] 4

= 1204.4 m/z for Tirzepatide

The same method used for the LC-UV analysis was able to be applied to MS to achieve better sensitivity. 

Next, Ozempic, a solution of Semaglutide, was obtained and prepared for analysis. A 4 mg/3 mL solution of Semaglutide was extracted from the syringe. 37.5 µL of the solution was aliquoted into a vial followed by 962.5 µL of 50/50 water/ACN for a final concentration of 50 ppb. The following chromatograms were obtained using the previously outlined analytical method above and Semaglutide was detected by either UV or MS. 

With the popularity of GLP-1 drugs skyrocketing, it is likely that the analysis of these drugs will be more prolific than ever. Is your lab interested in implementing this test or other peptide analysis? Let us know in the comments!