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Access Agilent eNewsletter, August 2014

Mixed-mode solid phase extraction for forensics applications

By Mike Chang
Agilent Sample Prep Applications Chemist

Mixed-mode solid phase extraction is an effective method for forensic and toxicology applications, such as the analysis of drugs and alcohol in bodily fluids, among others. Whether you are testing blood, urine, oral fluids, and other biological materials, illicit or prescription drug samples, foods, or other substances, Agilent provides versatile solutions that can help you achieve your analytical goals with accuracy, timeliness, and cost-effectiveness.

When you extract compounds from complex matrixes, you want to make sure that your sample prep is effective, consistent, has a proven history, and saves money. The Agilent Bond Elut Certify family has been helping analysts meet these needs for over 20 years. As well as being versatile and flexible, you can easily transfer robust Certify methods from Gas Chromatography (GC)/Mass Spectrometry (MS) and Liquid Chromatography (LC)/MS, because the elution solvent works with both. This means you use the same elution solvents and methods with different instruments.

Agilent Bond Elut Certify detects illicit drugs in urine samples

In this application, we examine the use of Agilent Bond Elut Certify for the extraction of phencyclidine (PCP) from urine, with consideration given to method development.


Figure 1. LC/MS/MS chromatograms of phencyclidine eluates from all stages of a solid phase extraction process.

To verify method optimization, every stage – from sample loading to elution – needs to be sampled and analyzed by chromatography. Figure 1 shows chromatograms of phencyclidine by LC/MS/MS for all eluates collected during solid phase extraction of PCP with Bond Elut Certify.

It is clear that PCP (RT = 1.35 min) came through the sorbent in the sample loading step. The chromatograms from washes 1 to 3 also confirm that PCP was still present. The elution step should contain PCP and, as the chromatograms show from eluates 1 to 3, it came off the sorbent. The wash 3 vial was visually clear and the chromatogram from wash 3 did not have the target compound peak. Therefore, wash 3 can be skipped. In addition, eluate 3 did not have a significant amount of PCP, which indicates that eluate 2 and 3 could be combined as a single elution step.

Different elution solvents and injection volumes also can be tested for further elution step optimization. For further details, see Agilent publication 5991-4695EN.

Keys to sample prep method development

There is no straightforward shortcut to a new sample prep method. Ultimately, a good balance between the removal of interference and retention of the target compounds, followed by elution optimization for cleaner extracts, is essential for successful method development in solid phase extraction.

The conventional solid phase extraction workflow consists of conditioning, equilibration, sample loading, washing, elution, evaporation, and reconstitution. Some of these steps may be eliminated to reduce process time. In the procedure, the solid phase extraction sorbent retains the compounds of interest until the elution step, while other interferences are removed in the wash step. The first three steps (condition, equilibrate, sample load) typically do not require much variation to optimize the performance of the extraction.

Occasionally, the conditioning and equilibration steps can be eliminated for some polymeric solid phase extraction sorbents. The most effective optimization can be done in the washing and elution steps. During sample loading, compounds of interest bind to the sorbent material along with interferences. The purpose of the washing step is to remove these interferences. The elution step then recovers the target compound by interrupting the interaction between it and the solid phase extraction sorbent.

Agilent Bond Elut Certify for simple, rapid, and reproducible extraction

The Agilent Bond Elut Certify family uses mixed-mode sorbents with non-polar, polar, and ion-exchange properties to ensure rapid, reproducible, simple, and clean extraction of many drug classes. The Certify range exhibits a variety of sorbent-analyte interactions when testing for drugs of abuse in biological fluids, and so it can be used either for general clean-up across a range of drug classes, or for specific extractions for instrumental confirmation of drugs and metabolites.

The bonded phase of Bond Elut Certify contains a medium-length hydrocarbon chain that allows some exposure of the polar silica surface. This optimizes polar and non-polar interactions of the drugs and matrix interferences with the sorbent. The second bonded phase, a strong ion-exchanger, is optimized for capacity. Too many ion-exchange sites results in high background and difficult elution, whereas too few produce low recoveries. Because the three modes of retention – polar, non-polar, and ion-exchange – are precisely matched, the Bond Elut Certify range is ideal for general drug analysis or the extraction of specific basic, acidic, or neutral drugs.

Bond Elut Certify uses a non-polar C8 sorbent and a strong cation-exchanger to extract basic (cationic) drugs from urine and blood, but it is also very effective for extraction of many compounds from a diverse range of aqueous matrixes.

Bond Elut Certify II was developed specifically for the rapid and effective extraction of acidic drugs and metabolites from urine and other biological matrixes. Bond Elut Certify II has non-polar C8 and strong anion exchange sorbents. It is optimized for acidic drugs such as 11-nor-Δ-9-tetrahydrocannibinol-carboxylic acid, salicylic acid, ibuprofen, acetaminophen, and other compounds that possess both non-polar and anionic characteristics.

Check out the new Agilent Bond Elut Certify Method Development Guide and video, as well as other sample prep solutions for forensics, to see what Bond Elut Certify can do for you.

For Forensic Use.

Figure 1.

LC/MS/MS chromatograms of phencyclidine eluates from all stages of a solid phase extraction process.