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HPLC Workflow

Sample Preparation Technical Library

Application Notes

  • PDF: Multisample filtration of small volumes using the Samplicity® System
  • PDF: Low-extractable Millex Samplicity® filters are ideal for preparing samples for sensitive HPLC detectors
  • PDF: Filtration of viscous / particle-laden samples using the Samplicity® Filtration System
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Featured Application: Avoid, Identify and Minimize LC-MS Contaminants

Sample preparation for HPLC is particularly important when you use a sensitive downstream detection method, such as mass spectrometry (MS). Liquid chromatography-mass spectrometry (LC-MS) is widely used to analyze complex samples, such as plasma, urine, food samples, and wastewater. Of the key parameters determining LC-MS success (sensitivity, resolution, reproducibility, efficiency, selectivity, speed, column lifetime), sensitivity, reproducibility, and column lifetime are negatively affected by the presence of contaminants.

Parameters negatively affected by contaminantsWhat it isHow to minimize contaminants
Sensitivity Lowest level of analyte detectable above background; sensitivity is reduced by ion suppression Wash columns to mitigate column bleeding
Effective sample preparation
Use certified mobile phase
Reproducibility Can mean column-to-column
reproducibility or run-to-run reproducibility
Prepare sample with devices that do not introduce extractable impurities
Run sufficient controls to verify run-to-run stability within a batch
Remove sample components that interfere with separation, ionization, and fragmentation
Use high-quality HPLC columns
Column Lifetime Number of injections on a column without change in selectivity and efficiency Eliminate sample contaminants that adsorb strongly or ionize easily. Avoid polymers that, when  fragmented, result in multiple peaks of varying m/z.
Fully elute/clean column after each sample
Use guard column and proper sample preparation (such as centrifugation, filtration, and extraction) to remove particles and extend column lifetime.

Sample Components That Can Interfere with LC-MS Results

Metabolites, detergents, salts, buffer components, and counterions are among the components of complex samples that can interfere with LC-MS performance. Some biological matrices, such as plasma, contain high amounts of phospholipids. If not removed prior to chromatography, separating phospholipids from analytes of interest can require long chromatography run times and high concentrations of organic solvents. Furthermore, phospholipids can build up on analytical column, and unexpectedly elute in future runs. Drug formulation agents, such as polysorbitans and polyethylene glycol, can also interfere and cause ionization suppression.

Besides sample-derived contaminants, additional sources of contamination are sampling devices, solvent impurities, containers, sample preparation devices, volatile organics introduced as a result of handling personal care products, and even columns themselves. Plasticizers from labware can interfere with LC-MS, resulting in the need to lengthen the chromatography run in order to resolve these peaks from analyte peaks.

How to Quickly Identify Sources of LC-MS Contamination

Analysis software supporting many LC-MS instruments may help you identify signals in your mass spectrum that are likely to represent contamination. We have also compiled a list of common mass spectrometry contaminants, their monoisotopic masses, and the sources of these contaminants.

Browse this list in the appendices of our technical bulletin, "LC-MS Contaminants."

Merck:/Freestyle/WRKFLW/lc-contaminants.jpg Featured Technical Bulletin:
LC-MS Contaminants

You thought your LC-MS analysis would be straightforward, but there are peaks you didn't expect.

In this technical bulletin, you will learn some tips on identifying LC-MS contaminants and avoiding contamination. Our 110 years of separation expertise, combined with our precision manufactured products, give you the greatest chance of obtaining reproducible, clean data.

Download Technical Bulletin

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