Home    /   News

Common problems with LC-MS


While LC-MS offers numerous advantages for trace analysis in complex matrices, several precautions have to be taken to overcome the following potential issues while using this technique.


The selectivity, sensitivity, reproducibility, and resolution of analysis may be obstructed by contaminants, such as phthalates, metal ions, polyethylene glycol (PEG) or dosing vehicle components for in vivo PK assessments, water, slip agents, endogenous matrix components, and particles entering the system from various sources such as reagents and solvents, water used for preparing buffers, chemicals leaching from glassware, microcentrifuge tubes, inlet filters, solvent lines, instrument parts, such as pump seals, the sample itself, gases used for desolvation of the eluent in the source and the collision cell.
These contaminants can inhibit the analysis by directly overlapping, suppressing, or enhancing the ionization of analyte(s) in the source, forming adducts with the analytes or altering ionization potentials, masking the analyte peaks, changing signal intensities, or appearing as ghost peaks in the chromatograms, making the baseline noisy, fouling the system, causing failure to meet analytical specifications, and contaminating the LC column, requiring frequent maintenance and replacement of parts.

To Minimize Contamination

High-purity solvents, water, and reagents should be used to prepare mobile phases; freshly prepared mobile phases essential be used to minimize the unintended microbial contamination of the aqueous mobile phase and polymerization of acetonitrile (ACN).
The usage of detergents/soaps should be avoided to clean glassware as they can be hard to remove and cause interference during analysis. High purity gases and plumbing are required (e.g., commonly used nitrogen gas of purity > 99%, cleaned burnished brass and Swagelok fittings). Nitrogen generators must be well-maintained, and the gas cylinder must be replaced when the pressure is reduced below the acceptable level. Analytes must be extracted from the matrix and chromatographic parameters enhanced to improve the resolution of analyte peaks from interfering peaks. Considerations should also be made if particular contaminants interfere with the assay. For example, if an analyte of interest overlaps a known phthalate (plasticizer), plastics may be avoided, or if interference is seen from a drug’s dosing vehicle, dilution or other techniques may be necessary to ensure there is no signal interference from components present in selected samples.

Matrix Effects

While evaluating biological samples, other sample constituents can suppress or enhance the ionization of the analyte in the source. Analytes of interest should be isolated to minimize the matrix effect. Therefore, sample preparation is an essential prerequisite of LC-MS analysis. While this decreases matrix effects, it cannot be easy to extract only the analyte(s) from the matrix. The chromatographic parameters can be optimized to prevent the co-elution of interfering compounds. Preparation of standard solutions in an analyte-free matrix (matrix matching) also helps normalize the analysis for matrix effects. Known concentrations of isotopically-labeled internal standards (IS), which experience similar ionization suppression or enhancement, additionally compensate for matrix effects.


Analyte peaks may appear at the analyte retention time in blank injections run after high concentration samples due to sample analytical carryover. Carryover typically presents in the autosampler or LC column. Carryover may be addressed by using needle, solvent line, and column cleaning procedures, such as repetitive blank injections, column conditioning, and increased needle washes with multiple solvents to confirm the sensitivity and accuracy of the analysis are sustained.

Sample Loss

Adsorption of analytes influences the precision and accuracy of the assay. Analytes, such as DNA and proteins, may be diminished due to non-specific binding to lab consumables, such as the inner surface of centrifuge tubes. Analyte loss can be minimized using containers with low surface bonding, for example, silanized glass. Usage of the blocking agents also minimizes the analyte’s interaction with the containers’ inner surfaces.

Mobile Phase Buffer Selection

As the column eluent has to be detached before mass spectrometry analysis, only volatile buffers such as ammonium acetate or ammonium formate, which will not precipitate in the source, can be used to prepare mobile phases. Volatile acids include formic and acetic acid and volatile bases include ammonium hydroxide.


Perform routine maintenance of the mass spectrometer as per a pre-determined schedule based upon vendor specifications and local standard operating procedures to ensure the instrument’s accuracy, reproducibility, and trouble-free operation and minimize unplanned downtime.

Back to List