Today’s drug testing facilities often rely on urinary analysis because these samples are readily available and collection is relatively non-invasive as compared to other matrices such as blood. Although sample collection is easy, downstream challenges arise because many drugs undergo glucuronidation prior to excretion, meaning a hydrolysis step must be performed prior to HPLC or GC analysis.
Hydrolysis can be performed in two ways: acid hydrolysis or enzymatic hydrolysis. While acid hydrolysis is an efficient process, the resulting samples can corrode metal components of the analytical instruments and can also degrade target analytes such as 6-monoacetylmorphine (6-MAM). For this reason, enzymatic hydrolysis using ß-glucuronidase has become popular among drug-testing laboratories. However, it has its own associated challenges. For one, the enzyme must be removed from the sample prior to analysis in order to halt its reaction with target drug compounds. Perhaps the most common issue that labs encounter when using ß-glucuronidase is an increase in HPLC/UHPLC column backpressure due to incomplete removal of the enzyme prior to injecting the sample onto the column. This article proposes a rapid and simple method to completely remove ß-glucuronidase from urine samples in an effort to improve downstream HPLC/UHPLC methods as well as to reduce the time required to process samples in a high-throughput environment: protein precipitation (PPT).
The demand for drug testing, particularly pain management drug testing, has grown substantially in the past few years. As the demand increases, so does the need for less expensive, quicker, and more reliable analytical methods. To increase throughput, many labs choose to process 96 samples at once and have adopted a “dilute-and-shoot” approach to urine analysis. However, a hydrolysis step must still be performed, meaning that ß-glucuronidase enzyme will still be present. The sample is often centrifuged in an effort to remove ß-glucuronidase; however, this is difficult to perform when working with 96-well plate formats because the maximum spin rate is not always fast enough to fully pellet the enzyme. Suspended enzyme is then injected onto the HPLC/UHPLC column, where it can precipitate on the head of the column, resulting in a rapid decrease in column lifetime and an increase in backpressure.
When this method was investigated in one study, column failure due to increased backpressure occurred after only 15 to 20 injections (Figure 1). This is considered unacceptable in a high-throughput laboratory because it increases cost per sample due to instrument downtime during column changes, a decrease in the number of samples processed per column, and an increase in the amount of system maintenance required. Therefore, laboratories must find a way to effectively remove the ß-glucuronidase from their samples prior to injection without introducing significant cost or time requirements.
|Figure 1. Premature column death after 15 injections
Protein precipitation has always been a rapid and cost-effective cleanup technique for bioanalytical samples, but it is rarely used to clean up urine samples because urine has a relatively lower protein concentration compared to serum or plasma. However, the introduction of ß-glucuronidase to facilitate enzymatic hydrolysis makes the hydrolyzed urine sample a perfect candidate for cleanup via PPT. In the study, 100 µL of hydrolyzed urine was added to 300 µL of acetonitrile that was preloaded into a protein precipitation 96-well plate. Upon sample introduction, the plate was then vortexed for two minutes to facilitate mixing between the urine sample and the acetonitrile. Once mixed, vacuum was applied, and clean eluent was collected in a collection plate, leaving the precipitated ß-glucuronidase within the wells of the protein precipitation plate. The clean sample was dried down and reconstituted prior to injection onto an HPLC column.
The samples that were subjected to protein precipitation showed stable pressure readings in excess of 500 injections. When this method was transferred to a high-throughput laboratory, more than 1,000 injections were achieved without an increase in backpressure. The extended column lifetime reduced the amount of instrument downtime that was previously required to change failed columns and also contributed to significant cost savings by reducing the cost per sample.
|Figure 2. Stable pressure after clean-up using protein precipitation plates
By adapting a sample preparation technique that is traditionally used for bioanalytical samples such as plasma and whole blood to urine samples that have undergone an enzymatic hydrolysis, laboratories can improve their HPLC analysis by extending column lifetime and reducing their cost per sample. In addition to extended column lifetime, the technique can be easily automated for high-throughput processing using protein precipitation 96-well plates. This allows laboratories to effectively process urine samples via enzymatic hydrolysis without compromising their downstream analysis.