Urine testing is commonly used to determine illegal drug use and the presence of prescription drugs, as well as to identify possible contamination from environmental toxins. Current urine testing methods, which focus on both free analytes and the glucuronide conjugates formed by the liver as the organ processes these chemicals, are mostly based on LC/MS/MS.
These traditional methods consist of lengthy sample handling steps, and include hydrolysis, centrifugation, sample cleanup, and concentration before analysis. Automating sample preparation steps would increase reproducibility, boost processing throughput, and reduce cost per sample. Results reporting would be more efficient, and in emergency situations, might even be life-saving.
Researchers developed and tested an automated “Prep-and-Shoot” workflow that allowed fast (15 minute) hydrolysis, and injection and analysis of more than 200 samples in only one day. The researchers’ work appears in the GERSTEL application note, “Prep-and-Shoot’: The Completely Automated Workflow for the Hydrolysis and Analysis of Urine Samples by LC/MS/MS.”
The researchers used 45 neat reference standards, including eight glucuronide conjugates, and analogue solutions of drug classes, including glucuronide conjugates of morphine, oxymorphone, codeine, tapentadol, buprenorphine, oxazepam, lorezapam and THC-COOH. They then set up a workflow that used a GERSTEL MultiPurpose Sampler (MPS) with a well plate incubating station that was configured with an LC/MS/MS system. 96-well plates were loaded into the MPS, which mixed sample buffers, enzymes and the internal standard.
Hydrolysis of more than 95 percent was observed for most compounds—only codeine-6-glucuronide required longer incubation than 15 minutes to achieve complete deconjugation. In addition, the analytical column showed no adverse pressure increase (due to possible fouling) even after 960 injections of urine samples (ten 96-well plates). Small injection volumes and the use of diverter valves also helped maximize the analytical column’s lifetime and kept the MS inlet clean. The method resulted in high reproducibility, with percentage relative standard deviation (RSD) values no higher than 10.4 percent.
The combination of automated hydrolysis, injection and analysis provided by the GERSTEL MPS could then process more than 200 samples, with hydrolysis efficiencies of at least 80 percent (more than 95 for most compounds). These results demonstrated that a lower cost, less labor-intensive method for urine analysis is available to a wide range of analytical laboratories.
You can read the full application note byclicking here.
After reading the details, feel free to reach out to us for a deeper conversation about improving the efficiency and productivity of sample preparation and analysis in your own studies.