Achieving excellent drugs-of-abuse analysis starts with the proper sample preparation technique

Jan. 18, 2015

Drugs-of-abuse analysis involves the collection of many different types of sample matrices of varying complexity, including urine, whole blood, hair, saliva, and more. Each of these matrices has its own inherent challenges, making it important to choose the proper sample preparation technique to ensure accurate downstream analysis. There are many sample preparation techniques to choose from, so it is important to first decide what your goals are.

When working with extremely complex matrices such as whole blood, your goal will most likely be to clean up your sample as much as possible to remove the wide variety of contaminants present. In these cases, solid phase extraction (SPE) is perhaps the best sample preparation solution because it is extremely effective and results in ultraclean samples. However, when working with less complex matrices such as urine, an in-depth sample preparation technique such as SPE may not be necessary, and simpler techniques such as supported liquid extraction (SLE) can be employed. 

Solid phase extraction (SPE)

SPE is one of the most selective sample preparation techniques. Using sorbents with varying chemistries, a particular analyte or compound class can be targeted, cleaned up, and concentrated. SPE follows five steps: conditioning, equilibrating, loading, washing, and eluting (Figure 1). Target analytes bind to the SPE sorbent while contaminants are washed away using a solvent that does not disrupt the binding of the target analytes to the sorbent. Target analytes are then released from the sorbent by applying a solvent that disrupts the interaction between the analytes and the sorbent. This method is extremely effective, but it will require a decent amount of method development time, especially when working with a large panel of target analytes.

Figure 1. SPE protocol.

When analyzing pain management drugs from whole blood, an SPE method may be optimal because of the complexity of the sample matrix. In one case, target analytes included 39 basic and neutral compounds from various classes, including benzodiazepines, opiates, synthetic opiates, amphetamines, analgesics, and illicit drugs. Because the compounds were both neutral and basic in nature, an SPE sorbent that has hydrophobic and strong cation-exchange properties was chosen (Figure 2). Recoveries of >88 percent for all 39 compounds at a concentration of 20 ng/mL were achieved by following the SPE method which is displayed in Table 1.

Figure 2. Structure of a sorbent.
Table 1. Optimized SPE method for 39 pain management drugs from whole blood.

Supported liquid extraction (SLE)

SLE is gaining popularity among labs that are looking for a high-throughput sample cleanup with minimal method development. The technique is not as clean and selective as SPE, but it does remove phospholipids, proteins, and salts from samples, making it an excellent cleanup method for sample matrices that are less complex such as plasma and urine. The procedure follows the same principles as a liquid-liquid extraction (LLE); a water-immiscible organic solvent is added to the aqueous-based sample and target analytes partition into the water-immiscible organic solvent, which is then collected for further analysis. The technique simplifies the extraction process but utilizes a solid support that distributes the aqueous sample, creating a larger surface area upon which the aqueous and water-immiscible organic solvent interact and thus resulting in a more efficient extraction (Table 2). The technique also eliminates emulsions that can form when performing LLE, which can make LLE difficult to automate and can also lead to analyte loss.

Table 2. Simplified liquid extraction (SLE) procedure.

Traditional SLE products rely on a natural resource, diatomaceous earth, as the solid support; however, recent advances have introduced a new synthetic SLE material. The synthetic material alleviates the challenges associated with natural resources such as availability and consistency because the sorbent is lab-manufactured and undergoes strict QC guidelines. In one case, a new synthetic sorbent was used to extract 18 pain management drugs from a urine sample. Following the method in Table 3, 96 samples processed in less than 15 minutes, and recoveries of >82 percent for all 18 compounds were achieved. (Table 4). 

With such a large variety of sample matrices to work with, drug testing labs must carefully examine various sample preparation techniques in order to determine the most appropriate technique for their samples. While reducing the amount of time spent preparing samples is beneficial in terms of throughput and cost per sample, it is important that a reduction in the amount of sample preparation does not negatively affect downstream analysis. More complex matrices such as whole blood will require more selective techniques such as SPE, while less complex matrices such as urine can be processed using rapid, less selective techniques such as SLE. Understanding the inherent challenges associated with the sample matrix at hand is the key to determining the most effective sample preparation approach.

Table 3. Optimized SLE protocol for 18 pain management drugs from urine using 96-well plates.
Table 4. Recoveries of pain management drugs from urine using SLE.