LC column and sample preparation considerations for LC-MS/MS drug analysis in clinical research

Introduction

Accurate identification and quantification of drug analytes in biological samples is a crucial component of clinical research. Prescribed drugs are analyzed to optimize dosing for drug therapy, ensure efficacy and minimize adverse effects. Drugs of abuse testing is used to monitor compliance with therapy, address addiction concerns and determine toxicity or overdose. Traditionally, many labs have used immunoassay (IA) for drug analysis. Still, IA methods often lack specificity, and many identify a drug class, not specific analytes. They are also prone to false positives that arise from cross-reactivity with a non-targeted compound.

Mass Spectrometry (MS) paired with the separation power of chromatography offers enhanced solutions for drug analysis with improved specificity compared to IA methods. Gas chromatography-mass spectrometry (GC-MS) offers better specificity than IA methods but often requires derivatization of non-volatile analytes. Over the last decade, use of liquid chromatography-mass spectrometry (LC-MS) techniques have continued to revolutionize drug testing in clinical research laboratories. LC-MS techniques offer superior specificity, selectivity and sensitivity compared to IA and GC-MS. Labs are implementing high resolution MS (HR-MS) or tandem MS (LC-MS/MS) platforms to conduct drug analysis for therapeutic drug monitoring (TDM) and drugs of abuse testing. Although there have been advances in MS instrumentation, effective chromatography and sample preparation are still critical for sensitive, specific, reproducible and robust clinical research LC-MS methods.

HPLC column considerations

Advancements in HPLC column technology have contributed to improvements in drug testing. Superficially porous “core-shell” columns offer increased efficiency compared to fully porous columns, yielding faster but highly efficient separations for large drug panels. While the “go to” LC column in most laboratories is a reversed-phase C18 column, this might not be the best choice for drug analysis. Reversed-phase LC stationary phases that incorporate aromatic ligands can offer enhanced selectivity for aromatic compounds through pi-pi interactions between analytes and the LC column (Figure 1). Phenomenex™ Core-shell Kinetex™ Biphenyl and Phenyl-Hexyl LC columns provide fast, efficient separations with complementary selectivity for drug analysis compared to a C18 column.

Figure 1. Representation of Phenomenex Kinetex C18, Phenyl-hexyl and Biphenyl column stationary phases.

Sample preparation considerations

Protein Precipitation with Phospholipid Removal

Biological matrices must be cleaned up prior to introduction to an LC-MS system. Simple techniques like protein precipitation can be effective, but this is considered a minimal cleanup technique. Phospholipids and other endogenous compounds that can cause matrix effects and interfere with accurate LC-MS/MS quantitation remain in the sample after protein precipitation. Protein precipitation with phospholipid removal (PLR), using products like Phenomenex Phree™, removes not only precipitated proteins but also phospholipids from blood samples (Figure 2). This simple extraction workflow requires very little method development, and can be used for whole blood, serum and plasma. A crash solvent like methanol or acetonitrile with 1% formic acid is added to the PLR well plate or cartridges. Next serum, plasma or lysed whole blood samples are added and mixed with the crash solvent to induce protein precipitation. Last, vacuum or positive pressure is applied to the well plate or cartridges. Precipitated proteins are retained by a filter and phospholipids are retained by the SPE sorbent. The extracted samples are collected in a collection plate or vials for preparation for LC-MS/MS analysis. Figure 3 shows 39 drug analytes extracted by PLR using Phree and separated on a Kinetex Biphenyl LC column, detected using a SCIEX™ QTRAP™ 6500+.

Phree Well Plate and Tube

Figure 2. Chromatogram comparing phospholipid content of a sample extracted by protein precipitation and phospholipid removal (PLR) using Phree

Top Graph: Phospholipid from serum extracted by protein precipitation

Bottom Graph: Phospholipid from serum extracted by Phree

Figure 3. Separation of 39 drug analytes extracted using Phree PLR on a Kinetex Biphenyl column.

Solid phase extraction

For applications where cleaner extracted samples are required, solid phase extract (SPE) is recommended. SPE extraction products are available in cartridges or 96-well plates. Polystyrene-divinylbenzene (PSDVB) polymeric sorbents, like Phenomenex Strata™-X, retain by hydrophobic (non-polar or reversed-phase) interactions. Strong or weak cation or anion exchange moieties can be incorporated into the polymer. This results in an SPE sorbent that retains and elutes analytes based on both non-polar and ionic mechanisms, so they are called mixed-mode SPE sorbents.

In SPE, samples are pretreated to optimize solubility and retention on the chosen SPE sorbent, then loaded onto the cartridge or well plate. Next, an aqueous wash is done to remove water-soluble endogenous matrix components that could interfere with analyte detection. This is followed by an organic solvent wash to remove more hydrophobic interfering matrix compounds. Target analytes are eluted from the sorbent and collected. The extracted sample is then evaporated and reconstituted for LC-MS/MS analysis. Often a lower reconstitution volume is used compared to the original sample volume to concentrate the sample for increased sensitivity. Because each step can be optimized for maximum retention of target analytes and removal of endogenous interferences, SPE provides the cleanest extracted samples for LC-MS/MS analysis. However, it also requires more method development and more time and labor in a clinical research workflow. One way to speed up SPE method development is the use of a method development plate. Four SPE chemistries are screened at once under multiple sets of conditions. This single plate experiment can identify the best SPE sorbent for a group of targeted analytes. Phenomenex has a technical note describing the use of an SPE method development plate for drug analysis.

Microelution SPE

The trend towards smaller specimen volumes opens the door to the use of microelution SPE. Microelution SPE has a 2 mg sorbent bed mass packed in a unique geometry. The amount of sorbent used is much lower than traditional SPE products that usually have 10 or 30 mg of sorbent per well or cartridge. The unique geometry has a higher bed height and allows higher sample load volumes in addition to lower wash and elution volumes when compared to traditional SPE formats. With microelution SPE, load volumes are between 10 and 375 µL of pretreated sample. Typical wash volumes are 50-100 µL, and elution volumes are typically 25 -50 µL. The lower volumes used means less organic solvent is required which makes microelution SPE methods more sustainable. It also means that extracted samples can often be diluted for analysis, compared to evaporation and reconstitution of samples required with traditional SPE protocols.

Conclusions

LC-MS techniques continue to replace IA and GC-MS in clinical research drug analysis. Optimized chromatography and sample preparation methods are critical for robust, reproducible results. Core-shell columns with phenyl-hexyl or biphenyl stationary phases offer complementary, improved selectivity for drug analysis. Extraction that combines protein precipitation with phospholipid removal (PLR) provides cleaner samples than protein precipitation alone. SPE provides superior sample cleanliness, and the use of a method development plate can streamline method development for clinical research workflows. Microelution SPE using smaller solvent volumes is more sustainable and eliminates evaporation and reconstitution of the extracted sample.

For more information on LC columns and solutions for sample preparation including phospholipid removal and traditional and microelution SPE method development and implementation, visit the Phenomenex Drug Analysis and Research Page.

References

Strata, Phree, and Kinetex are Trademarks of Phenomenex, Inc. QTRAP is a trademark of SCIEX, Inc.

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