HybridSPE-Phospholipid Technology

Effective Removal of Phospholipids and Proteins for Accurate and Reproducible LC-MS Analysis

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NEW Try our HybridSPE® DPX 96-well tips for completely automated phospholipid removal in seconds
Impact of Phospholipids on Accuracy and Reproducibility of LC-MS Data
Limitations of Traditional Sample Prep Methodologies for Biological Samples
The Unique Chemistry and Basic Methodology of HybridSPE®-phospholipids Technology
Technical Resources - Articles & Applications

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HybridSPE®-PLus Plates - the Next Generation in HybridSPE®-Phospholipid Technology back to top

HybridSPE-PLus platesOur new HybridSPE®-PLus plates offer the same time-proven chemistry of our traditional HybridSPE®-Phospholipid plates coupled with plate manufacturing enhancements that stem from years of production experience. These manufacturing enhancements have allowed us to increase well-to-well flow consistency and reduce sample hold-up volumes, further improving analyte recoveries and assay reproducibility. We will continue to offer and support our traditional square-well HybridSPE®-Phospholipid plates for those customers who have developed methods on them, but we invite those customers developing new methods to take advantage of the newest technology: HybridSPE®-PLus.

Impact of Phospholipids on Accuracy and Reproducibility of LC-MS Data back to top

Phospholipids: A Concern for LC-MS Analysis of Small Molecules in Biological Matrices
Cell MembranePhospholipids are present as a major component of all cell membranes. They are therefore present in all biological sample matrices including serum, plasma and whole blood and can be a problem in LC-MS analysis of small molecules because they often co-elute and ionize along with the analytes of interest. This co-elution results in ion suppression (an erroneous decrease) of the mass spec signal that can cause variability and impact LC-MS result accuracy (Figure 1).

Figure 1. Phospholipid Effect on Ionization of Clonidine
Phospholipid Effect on Ionization of Clonidine

Importance of Accurate Results and Fast Answers in Bioanalysis
At Supelco, we understand that the results of your analyses can have a significant impact on the lives of many others. This puts pressure on you to ensure the data you produce is as accurate as possible. Not only do you need quick answers, you need answers you can trust. The complexity of the types of samples with which you work does not make your job any easier. Proteins and phospholipids inherently present, to varying degrees in your samples, can add variability to your analytical results when using sensitive techniques such as LC-MS or LC-MS/MS. At Supelco, we have over four decades of chromatography expertise to help you navigate the complexity of your sample prep options.

Limitations of Traditional Sample Prep Methodologies for Biological Samples back to top

Limitations of Traditional Biological Sample Cleanup Methods
Most clinical and biological researchers use traditional methods such as protein precipitation and liquid-liquid extraction to cleanup their samples prior to analysis. While these techniques allow for inexpensive and quick removal of proteins, they completely fail to address the problem of phospholipid-induced ion suppression.

Even if phospholipids do not co-elute with the analyte of interest, they can accumulate on your analytical column and elute from the column sporadically in downstream analyses. This can cause unpredictable ion suppression and poor reproducibility, thereby putting the accuracy of your results at risk (Figure 2).

Figure 2. Gradient RP LC-MS of Blank Plasma Samples Prepared by Standard Protein PPT vs. HybridSPE®-Phospholipid

Background Interference Observed after Multiple Injections Using Std. Protein PPT
Standard Protein PPT

Background Interference Observed after Multiple Injections Using HybridSPE®-Phospolipid Technology
HybridSPE-Phospolipid Technology

Back Pressure Generated Within C18 1.8 µm Column (5 cm x 2.1 mm) after Multiple InjectionsBack Pressure Generated

Phospholipid Removal Techniques
To overcome the problem of phospholipid-induced ion suppression, some analysts try traditional SPE. Traditional SPE often requires time-consuming and complex method development, but still only removes nominal amounts of phospholipids. Remaining phospholipids can impact the accuracy of your results, accumulate on your analytical column to impact future analyses, add to column replacement costs and increase instrument downtime.

A variety of products designed specifically for the removal of both proteins and phospholipids are now commercially available, including HybridSPE® plates and cartridges. Most of these products are simple, fast and easy-to-use, offering fairly standardized methods with minimal method development. Most, however, use a hydrophobic retention mechanism to separate phospholipids from analytes of interest in the sample. This poses a problem if the analytes of interest are also hydrophobic because they will be retained and removed along with the hydrophobic phospholipids. This results in decreased analyte recovery and inaccurate results. HybridSPE®Phospholipid Technology is different in that it completely removes both proteins and phospholipids from the sample without retaining other hydrophobic compounds.

The Unique Chemistry and Basic Methodology of HybridSPE®-Phospolipid Technology back to top

How Is HybridSPE®-Phospholipid Technology Different Than Other Phospholipid Removal Products?
The first of its kind, HybridSPE®-Phospholipid technology was introduced in 2008. It fuses the simple, standardized methodology of traditional protein precipitation with the specificity of solid phase extraction (SPE) for the simultaneous removal of proteins and phospholipids from biological samples prior to LC-MS analysis. Unlike other phospholipid removal products that use a hydrophobic retention mechanism to remove phospholipids from biological samples, HybridSPE®-Phospholipid technology uses a unique retention mechanism (Figure 3). This allows it to separate phospholipids from even very hydrophobic analytes, such as vitamins which are often retained along with phospholipids on competitive products.

To learn more about HybridSPE®-Phospholipid technology and view a video of the product in action, Visit HybridSPE®-PL

Figure 3. Unique Retention Mechanism of HybridSPE®-Phospholipid Technology Allows for Separation of Even Very Hydrophobic Analytes from Phospholipid Contaminants in Biological Sample Matrices
Unique Retention Mechanism of HybridSPE®-Phospholipid Technology

A Newer, Better “Go-to” Sample Prep Method for Phospholipid Removal
Labs working with biological samples often choose to perform protein precipitation prior to LC-MS analysis, using it as their “go-to” sample prep method. They view phospholipid removal as more costly, more time-consuming and unnecessary if the specific analytes of interest do not co-elute with the phospholipids in their sample. This is not the case.

Labs can actually reduce overall costs and increase overall throughput while generating more accurate data by using HybridSPE®-Phospholipid technology to remove both proteins and phospholipids from all biological samples prior to small molecule LC-MS analysis.

Simple and Fast Phospholipid Removal
The 96-well plate protocol involves just a few simple steps (Figures 4 and 5), and plates can be used right out of the package with no pre-conditioning step required. The sample (containing internal standards if desired) and a precipitation solvent are first added to the well plate, followed by a mixing step and vacuum application to collect the sample. Collected samples are then ready to be analyzed.

Figure 4. Depiction of Basic HybridSPE®-Phospholipid Sample Prep Workflow

Depiction of Basic HybridSPE-Phospholipid

Figure 5. HybridSPE® 96-well Plate Protocol

  1. Add Sample
    Pipette 100 µL plasma or serum to the HybridSPE® plate followed by 300 µL precipitation solvent. Add internal standards as necessary.
  1. Mix
    by vortexing/shaking HybridSPE® plate or by aspirating/dispensing with 0.5–1 mL pipette tip.
  1. Apply vacuum
    The packed-bed filter/frit assembly acts as a depth filter for the concurrent physical removal of precipitated proteins and chemical removal of phospholipids. Small molecules (e.g., pharma compounds and metabolites) pass through unretained.
  1. Collect Sample
    Resulting filtrate/eluate is free of proteins and phospholipids and ready for immediate LC-MS/MS analysis.

Technical Resources back to top
Category / Title Type Size (kb) No. Pages I.D.
Instructions for HybridSPE®-PLus 96-Well Plates Data Sheet 105 2 T713202
Instructions & Troubleshooting for HybridSPE®-Phospholipid Ultra Cartridge Data Sheet 350 2 T710124
Investigating Matrix Interference in Analysis of Antiarrhythmic Cardiac Drugs in Plasma (Reporter 31.1) Article - - -
Isolation and LC-MS Characterization of Illicit Bath Salts in Urine (Reporter 30.3) Article - - -
Meeting the LC-MS Needs of a High-Throughput Clinical Lab
(60 min; Bioanalysis Zone recorded webinar; registration required)
Webinar - - -
HybridSPE®-Precipitation Technology Product Info. 483 2 T408102
Enrichment of Phospholipids in Biological Samples Using HybridSPE® (Reporter 28.3) Article - - T210003
Minimizing Phospholipid Matrix Effects in HILIC LC-MS using HybridSPE®-Precipitation Small Volume Plates (Reporter 28.2) Article - - T210002
Increased Bioanalytical Throughput and Recovery Utilizing HybridSPE®-PPT Small Volume Plates (Reporter 28.1) Article - - T210001
Troubleshooting Analyte Recovery when Using HybridSPE®-Precipitation Technology (Reporter 27.2) Article - - T209002
Increase Bioanalytical Assay Speed via Phospholipid Removal using HybridSPE™-Precipitation Technology (Reporter 26.5) Article - - T208005
High Recovery Method of HybridSPE®-Phospholipid for Cleanup of Biological Samples Prior to LC-MS Analysis Presentation 465 22 T411056H
High Throughput Bioanalysis Utilizing Fused-Core™ Particle and HybridSPE®-PPT Technology (HPLC 2010) Presentation 494 19 T410086H
Alternative Method of Hybrid SPE Sample Preparation for High Recovery LC-MS Analysis of Pharmaceutical Compounds in Plasma Presentation 712 17 T409228H
Comparison of Sample Preparation Techniques for Reduction of Matrix Interference in Biological Analysis by LC-MS-MS Presentation 216 22 T408163H
Depletion of Phospholipid Matrix Interference when Dealing with Small Volume Plasma Samples Presentation 587 20 T410128H
High Throughput Removal of Both Phospholipids and Proteins in Bioanalytical Sample Preparation Presentation 257 20 T409038H
Impact of Ion-Suppression Due to the Presence of Phospholipids on the Enantiomeric LC-MS Analysis of Clenbuterol Presentation 185 17 T409108H
Increased Bioanalytical Throughput Utilizing Fused-Core™ Particles with Selective Phospholipid Depletion Presentation 532 21 T409189H
Recovery of Pharmaceutical Drugs From Small Volume Biological Sample Using HybridSPE®-PPT 96-well Plate Presentation 646 22 T410048H
Selective Depletion of Phospholipids Interference Utilizing HybridSPE™-Precipitation Technology Presentation 658 49 T408181H

SelectScience Award back to top
HybridSPE®-PPT Technology – Winner of the SelectScience.net Scientists’ SelectScience Award
Choice Award for Best New Separations Product!

The award was presented at Pittcon 2009.