The Substance Abuse and Mental Health Services Administration (SAMHSA) reported that about 21.8 million Americans said they were using illegal drugs in 2009, compared to the 20 million that were estimated to have used illegal drugs in 2006.1 Western Europe now accounts for 60% of the world's heroin consumption.2
As drug use increases, so does the need for drug abuse screening. Patient samples such as urine, blood, plasma, and saliva are all used to test for drugs of abuse, but these matrices can be difficult to work with if not prepared correctly. For this reason, the toxicology industry is demanding quicker and more robust means to prepare and analyze samples, making sample preparation one of the most important steps in the confirmational drug testing procedure.
Typically, a positive sample via immunoassay screening is routed for further analysis by GC/MS (Gas Chromatography-Mass Spectrometry) to confirm the positive result. GC/MS has long been, and still is, the gold standard for confirmational analysis. However, more recently, a growing number of laboratories have been looking to adopt Liquid Chromatography—Mass Spectrometry and Liquid Chromatography—Tandem Mass Spectrometry (LC/MS/MS) technology for confirmational testing. LC/MS/MS has the advantage of increased sensitivity, and for the most part it eliminates the need for derivitization, which is commonly required for analysis by GC/MS.
Both analytical platforms are viable options, but both also require good sample preparation practices to produce reliable results. GC/MS, in order to detect at the biological drug concentrations, requires a sample preparation method that both concentrates the target analytes and reduces background noise from matrix interferences. LC/MS/MS, although a very sensitive platform, is prone to matrix effect. Matrix effect is defined as suppression or enhancement of the signal due to endogenous matrix components. Suppression or enhancement of target analytes due to the matrix will be variable among patients and thus can lead to imprecision in the data. In order to reduce matrix effect, proper sample preparation is necessary.
The most imperative goals for sample preparation when working with drugs of abuse are to clean up, concentrate, and exchange the original sample matrix with a solvent that is amenable to GC/MS or LC/MS/MS analysis. Although there are many sample preparation options, the most often used approaches are protein precipitation (PPT), liquid/liquid extraction (LLE), and solid phase extraction (SPE).
Protein precipitation is specific to protein rich matrices such as plasma/serum or whole blood. A reagent, typically an organic solvent, is added to the matrix to induce denaturing of the protein and resulting precipitation. The protein precipitate can then either be filtered or centrifuged to remove it from the sample. This is a simplistic and rapid sample preparation approach, but this technique does not concentrate the sample; instead it dilutes the sample. PPT also does not remove matrix interferences other than proteins and does achieve the solvent switching goal, resulting in a sample that may need to be blown down and reconstituted. At best, PPT will extend analytical column lifetime, but will not do much to increase sensitivity or address matrix effect.
Liquid/liquid extraction (LLE) is an approach that is applicable to all aforementioned matrices. An immiscible organic solvent is added to the sample. The solvent forms a separate layer from the sample, and target analytes migrate from the original sample matrix to the organic layer. The organic layer is then transferred to another sample container for further manipulation. LLE does remove protein, provides some cleanup, and also achieves the goal of solvent switching into a more amenable solvent. The drawbacks are that it does not concentrate the sample, requires use of hazardous organic solvents, and requires labor-intensive transfer steps. Additionally, emulsions can form, making the desired organic layer difficult to identify, which results in incomplete diffusion of target analyte and difficult solvent transfers. LLE is a step above PPT in effectiveness, but it does require more user intervention.
The last of these three approaches is solid phase extraction (SPE). It is essentially a chromatographic media packed into a disposable syringe barrel or 96-well plate. Different chemistries are bonded to either a silica- or polymeric-based particle. These chemistries are chosen to selectively target the drugs of abuse and remove the matrix interferences that cause problems with GC/MS or LC/MS/MS analysis. The sample is loaded onto the SPE media, where the target drugs are retained by the specifically chosen chemistry. The sample matrix is allowed to flow through the sorbent to waste. Now retained, the target drugs are subjected to a series of washing steps using solvents intended to remove many of the matrix interferences including salts, proteins, phospholipids, and other endogenous compounds. After washing, the target compounds are reclaimed or eluted back off the chemistry and are collected in a clean vial/tube or collection plate. Of the three techniques discussed, SPE is the only technique that will clean up, concentrate, and produce a final sample that is in an amendable solvent for further analysis. Thus, SPE is often the approach preferred by the drug-testing laboratorians because it addresses the needs for both GC/MS and LC/MS/MS to produce accurate and precise data.
This is not to say the benefits of SPE do not come at a cost. Traditional SPE methods are regarded as lengthy and labor-intensive, and they require more time for method development. This is most likely due to the lack of specialization of SPE products designed for the modern drugs-of-abuse testing laboratory. With the demand for increased testing and fewer resources to meet the need, this has driven manufacturers to provide better solutions.
In 2011, Phenomenex introduced a family of products that are specifically designed for drugs-of-abuse testing. The design objectives of these products were to provide a means to realize the benefits of SPE, and at the same time address the drawbacks of conventional SPE technologies. The family was developed with prepacked procedures for many drug classes to minimize method development time. The procedures are consolidated and require a minimal number of steps to process samples.
As drug use continues to rise, drugs-of-abuse testing will surely increase over the years. In response, it is expected that more and more technologies designed specifically for drugs-of-abuse testing will be introduced in an effort to create a more streamlined and sensitive testing method.
Michael Rummel is Sample Preparation Product Manager for California-based Phenomenex. During his tenure, he has worked throughout the world developing, troubleshooting, and optimizing extraction protocols across industries.
References
- Hanamel S. Illegal drug use is higher than in nearly a decade, report finds. Associated Press. September 16, 2010.
- International Narcotics Control Board Annual Report. March 2, 2011.
