Developing Robust UHPLC Methods for Clinical, Forensic and Bioanalytical Samples
Using HybridSPE, Titan C18 UHPLC Columns and Other Products for the LC-MS Workflow

By: Craig Aurand, Senior Application Chemist and Jennifer Claus, Product Manager, Reporter US Vol 32.1

HybridSPE-PLus plates remove endogenous phospholipids for consistently high analyte response compared to protein precipitation plates. Combined with Titan UHPLC columns, they provide a powerful analytical pair for robust UHPLC-MS of biological samples.


As the limits of method speed, sensitivity and specificity are pushed using modern UHPLC systems and mass spectrometric detection, compromise of data quality and throughput from sample matrix contamination cannot be tolerated. Researchers and analysts looking for small molecule analytes in biological sample matrices face challenges from matrix interferences that can impact the robustness, accuracy and sample throughput of their analytical method.

Goal of the Study

The purpose of this study was to evaluate the improvement in method robustness when utilizing the HybridSPER-PLus phospholipid depletion plate in a realistic bioanalytical application with a Titan UHPLC sub-2.0 μm particle column. The goal was to document control response variability and demonstrate the impact of the sample preparation technique on method performance when using the electrospray ionization (ESI+) source of the LC-MS system.
Monitoring the control response is also useful for determining maintenance intervals for cleaning of the ESI source.

Rationale for Using MS Response Over Column
Pressure to Assess Performance

Though monitoring column pressure is one common means of assessing the impact of sample cleanup technique on column performance, extracted sample matrix can also have a dramatic impact on performance of the electrospray ionization (ESI) source. Sample sprayed within the desolvation chamber causes deposition of sample matrix onto ionization source surfaces, potentially decreasing the efficiency of the source. This can be further compounded by ionizable species that compete with target analytes during the ionization process, thus forming a charge competition within the source. Problems associated with this type of matrix interference are further amplified as a result of concentration variability of the competing ionizable species within the sample population.


Fluoxetine and tamoxifen (Figure 1) were chosen as controls to monitor ESI source changes throughout the spiked plasma sample series. Two 96-well sample prep methods were explored, protein precipitation plates and HybridSPE-PLus phospholipid depletion plates. Titan C18 UHPLC columns were used for efficient and rapid separation. Detection was via TOF-MS in ESI(+) mode. System performance was established initially, and then at intervals of 300, 600 and 1,000 injected samples. Plasma samples were first precipitated using 1% formic acid:acetonitrile at a ratio of 3:1 solvent to plasma. Samples were then vortex agitated and centrifuged for 4 minutes at 1,000 rpm. A 300 μL aliquot of the sample supernatant was applied to a 96-well plate, either protein precipitation (Seahorse 400 μL, 1.0 μm GF) or HybridSPE-PLus. (Note that protein precipitation can also be performed in the HybridSPE plates for a truly one-step sample prep method. However, users often prefer to carry out the protein precipitation as a separate step.) Vacuum was applied and the resulting eluent was collected in a 96-well collection plate and injected directly into the LC-MS system. Chromatographic conditions are listed in Figure 2.

Figure 1. Structure of Control Analytes

Figure 2. Gradient Analysis of Fluoxetine and Tamoxifen on Titan™ C18 1.9 μm

Comparing Effectiveness of Sample Prep Methods

System pressure was consistent across the study, even with the protein precipitation plates, with less than 7 bar increase across the 1,000 sample series. This demonstrates the ability of the Titan UHPLC columns to stand up to bioanalytical samples, irrespective of the sample prep method. However, baseline and analyte response was affected by the sample prep method. Column and system performance data are presented in Figures 3 through 5.

Improvement in Analyte Response

Using the HybridSPE-PLus plates, 300 ng/mL control response samples had a variability of less than 10% over the 1,000 samples (Figure 3). This consistency is due to the ability of the HybridSPE-PLus plate to deplete the phospholipid matrix from the samples that would otherwise interfere with analyte response.

In contrast, with the protein precipitation plates the response of the control samples dropped to less than 70 ng/mL by the 300 sample series (Figure 4). Tamoxifen control values continued to drop over the injection series, with a final value of less than 40 ng/mL at the 1,000 sample series. This dramatic drop in tamoxifen control response after only a few hundred samples would signal a need to halt the analysis, clean the source and recalibrate the instrument, dramatically impacting sample throughput. Although the control response for fluoxetine did not decrease, it was more erratic throughout the 1,000 sample series when compared to samples processed using the protein precipitation technique alone versus samples processed using the HybridSPE-PLus phospholipid depletion plate. This further exemplifies the need for sufficient sample preparation to avoid sample matrix interference which is both varied and compound dependent.

Figure 3. Control Response Over 1,000 Injections for Samples Processed with HybridSPE-PLus Phospholipid Depletion Plates

Figure 4. Control Response Over 1,000 Injections for Samples Processed with Protein Precipitation Alone

The results can be visualized in Figure 5 which compares the monitoring of the phospholipid matrix after the HybridSPE-Plus and protein precipitation techniques across the 1,000 sample series. Samples processed using the HybridSPE-PLus plates presented a clean and consistent background over the 1,000 samples studied. However, the protein precipitation plates provided an initially well-defined elution region for several of the phospholipid species. As the number of injected samples increased, the total amount of phospholipid background continued to rise. Instead of a defined phospholipid peak, a broader total background increase was observed. As previously mentioned, a dramatic decrease in tamoxifen response was coincident with this phospholipid background increase over the 1,000 sample series for samples processed using only the protein precipitation technique.

Figure 5. Phospholipid Matrix for Samples Processed with Protein Precipitation Plates vs. HybridSPE-PLus Plates


This study demonstrates that monitoring column pressure is not always a sufficient assessment of analytical assay robustness. Rather, specifically for LC-MS applications, routine monitoring of control sample response provides an effective means for establishing the long-term accuracy of a method. Likewise, taking steps to ensure sufficient sample cleanup, not just particulate removal, can greatly aid in accurate sample assessment and decreased instrument downtime to improve sample throughput and assay robustness.


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