Optimizing Sample Preparation Methods for Comprehensive Drug Research Panels from Urine

Gluca-what? Hydrolysis? Beta-something? While the terminology for urine drug testing can be confusing, the methods should not be. Improve upon traditional urine testing by learning the science behind the steps and simplifying the sample preparation.

When drugs are ingested, they are tagged with a glucuronic acid during the metabolism process, which helps change the polarity of the drug compound and aids in absorption into the kidneys. Once the drugs exit the body through urine, they are still in their glucuronide form. Before chromatographic analysis can occur, the glucuronide bond must be cleaved through hydrolysis to return the drugs to their native state for proper chromatographic detection and analysis. Enzymatic hydrolysis (using β-glucuronidase) is preferred over acid hydrolysis because the bond is cleaved according to stereochemistry, meaning it is more specific than acid hydrolysis and yields less downstream complications. The sample now contains drug compounds and residual β-glucuronidase enzyme, which if the enzyme is not removed can precipitate out during LC analysis. If this occurs, the column’s selectivity and lifetime is negatively affected and can result in buildup in the mass spectrometer (MS).

While “dilute-and-shoot” is a common method used to prepare hydrolyzed urine samples for LC-MS analysis, it can cause issues with sensitivity because it dilutes the sample 10 to 30-fold before injection into the column. The β-Gone™ β-Glucuronidase Removal product is used to remove the large enzyme in a single step and in less than 1 minute, making it a great alternative to dilute-and-shoot.

We compared signal responses and recoveries of various classes of drugs of abuse using β-Gone Removal products with our standard protocol, which utilizes a 0.1% formic acid in methanol dilution, and another protocol without a dilution prior to filtration. Both procedures were also compared to the dilute-and-shoot protocol to further understand how dilution affects sensitivity.

The β-Gone Protocol
B-gone9

Using β-Gone β-Glucuronidase Removal product aids in the improvement of sensitivity in comparison to the dilute-and-shoot method. Focusing on a notoriously low responding compound, buprenorphine, the chromatogram shows the improvements in sensitivities compared to dilute-and-shoot. Table 1 below shows the average recovery values (n=8) using β-Gone for a large panel of drug compounds. All % CVs are below 6 % and most recoveries are near 100 %. β-Gone offers the possibility of an increased throughput for labs that need to save time on running multiple samples, automation potentials and a simple protocol that is complete in under 1 minute.

b-gone6

Figure 1: Buprenorphine (view our LC conditions here)
b-gone5

While the method is easy and the improvements in recovery are obvious, there is a way to further optimize these methods. Most toxicology labs are aware that there are two types of enzymatic hydrolysis they can perform: recombinant enzymes and non-recombinant enzymes. Next, we compared signal responses and recoveries of various classes of drugs of abuse using both types of enzymatic β-Gone β-Glucuronidase Removal products (one that is specific to recombinant enzymes and the other which is specific to non-recombinant enzymes). Both products were compared using a standard protocol of 0.1 % formic acid in methanol dilution and a protocol with no methanol prior to filtration and then compared to the dilute-and-shoot protocol to further understand how dilution affects sensitivity.

Figure 2
b-gone4
B-gone3
Figure 3
b-gone8

The recovery charts reveal that for each type of technique, the methanol dilution is not necessary when using IMCSzyme® (recombinant enzyme). Recovery was lower compared to using methanol, however, the chromatogram showed better results in sensitivity for all compounds in this suite, excluding THC-COOH and buprenorphine. Both analytes show a better response with the standard protocol involving a 40% dilution with 0.1% formic acid in methanol. With no methanol dilution, both compounds still provide sensitivity comparable to dilute-and-shoot.

Therefore, most comprehensive drug research panel suites hydrolyzed by IMCSzyme can be used with a recombinant β-Gone β-Glucuronidase Removal product effectively with or without the dilution that is recommended in the general protocol. By contrast, when working with the Campbell Enzyme (non-recombinant enzyme), simply loading the hydrolysate solution onto the β-Gone plate (no dilution) yields an increase in sensitivity for all compounds relative to the ones prepared by the standard dilution protocol, except for THC-COOH, which shows no recovery without the methanol dilution. When working with the Campbell Enzyme in a suite that contains THC-COOH, a 40 % dilution with 0.1 % formic acid in methanol is necessary to achieve acceptable results.

More questions? Check out our β-Gone β-Glucuronidase Removal FAQs page

 

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s