Ion suppression in liquid chromatography–mass spectrometry

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Ion suppression in LC-MS and LC-MS/MS refers to reduced detector response, or signal:noise as a manifested effect of competition for ionisation efficiency in the ionisation source, between the analyte(s) of interest and other endogenous or exogenous (e.g. plasticisers extracted from plastic tubes, [1] mobile phase additives) species which have not been removed from the sample matrix during sample preparation. Ion suppression is not strictly a problem unless interfering compounds elute at the same time as the analyte of interest. In cases where ion suppressing species do co-elute with an analyte, the effects on the important analytical parameters including precision, accuracy and limit of detection (analytical sensitivity) can be extensive, severely limiting the validity of an assay's results. [2]

Contents

History

At its inception as a tool of analytical chemistry, LC-MS/MS spread rapidly and indeed continues to do so in (amongst others) bioanalytical fields, owing to its selectivity for analytes of interest. Indeed, in many cases this selectivity can lead to a misconception that it is always possible to simplify or (on occasion) almost completely remove the necessity for extensive sample preparation. Consequently, LC-MS/MS has become the analytical tool of choice for bioanalysis owing to its impressive sensitivity and selectivity over other, more conventional chromatographic approaches. However, during and after uptake by bioanalytical laboratories worldwide, it became apparent that there were inherent problems with detection of relatively small analyte concentrations in the complex sample matrices associated with biological fluids (e.g. blood and urine). [3]

Proposed mechanisms of ion suppression

Put simply, ion suppression describes the adverse effect on detector response due to reduced ionisation efficiency for analyte(s) of interest, resulting from the presence of species in the sample matrix which compete for ionisation, or inhibit efficient ionisation in other ways. Use of MS/MS as a means of detection may give the impression that there are no interfering species present, since no chromatographic impurities are detected. However, species which are not isobaric may still have an adverse effect on the sensitivity, accuracy and precision of the assay owing to suppression of the ionisation of the analyte of interest. [4]

Although the precise chemical and physical factors involved in ion suppression are not fully understood, it has been proposed that basicity, high concentration, mass and more intuitively, co-elution with the analyte of interest are factors which should not be ignored. [5]

The most common atmospheric pressure ionisation techniques used in LC-MS/MS are electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). APCI is less prone to pronounced ion suppression than ESI, [6] an inherent property of the respective ionisation mechanisms.

In APCI, the sole source of ion suppression can be attributed to the change of colligative properties in the solute during evaporization (King et al, J. Am. Soc. Mass Spectrom 2000, 11, 942-950).

ESI has a more complex ionisation mechanism, relying heavily on droplet charge excess and as such there are many more factors to consider when exploring the cause of ion suppression. It has been widely observed that for many analytes, at high concentrations, ESI exhibits a loss of detector response linearity, perhaps due to reduced charge excess caused by analyte saturation at the droplet surface, inhibiting subsequent ejection of gas phase ions from further inside the droplet. Thus competition for space and/or charge may be considered as a source of ion suppression in ESI. Both physical and chemical properties of analytes (e.g. basicity and surface activity) determine their inherent ionisation efficiency. Biological sample matrices naturally tend to contain many endogenous species with high basicity and surface activity, hence the total concentration of these species in the sample will quickly reach levels at which ion suppression should be expected.

Another explanation of ion suppression in ESI considers the physical properties of the droplet itself rather than the species present. High concentrations of interfering components give rise to an increased surface tension and viscosity, giving a reduction in desolvation (solvent evaporation), which is known to have a marked effect of ionisation efficiency.

The third proposed theory for ion suppression in ESI relates to the presence of non-volatile species which can either cause co-precipitation of analyte in the droplet (thus preventing ionisation) or prevent the contraction of droplet size to the critical radius required for the ion evaporation and/or charge residue mechanisms to form gas phase ions efficiently.

It is worthwhile to consider that the degree of ion suppression may be dependent on the concentration of the analyte being monitored. A higher analyte/matrix ratio can give a reduced effect of ion suppression. [7]

Assessment of ion suppression during method validation

Since it is accepted that ion suppression has the potential to affect the other analytical parameters of any assay, a prudent approach to any LC-MS method development should include an evaluation of ion-suppression. There are two accepted protocols by which this may be achieved, described as follows.

Monitoring of detector response under constant infusion

The more comprehensive approach to assessment of ion suppression is to constantly infuse an appropriate concentration into the mobile phase flow, downstream from the analytical column, using a syringe pump and a 'tee union'. A typical sample should then be injected through the HPLC inlet as per the usual analytical parameters.

Monitoring of detector response during this experiment should yield a constant signal appropriate to the concentration of infused species. Once the sample has been injected, a drop in signal intensity (or a negative response) should be observed any time a species is ionised in the ion source. This should allow the retention time of any such species under the analytical parameters of the assay to be determined. Any species causing a negative response may be considered to be contributing to ion suppression, but only if such species co-elute with the analyte of interest.

It is also important to consider that species contributing to ion suppression may be retained by the column to a much greater extent than the analyte of interest. To this end, the detector response should be monitored for several times the usual chromatographic run time to ensure that ion suppression will not affect subsequent injections.

Preparation of spiked plasma samples

Another approach to evaluation of ion suppression is to make a comparison between:

Approaches to negating ion suppression

There are several strategies for removal and/or negation of ion suppression. These approaches may require in-depth understanding of the ionisation mechanisms involved in different ionisation sources or may be completely independent of the physical factors involved.

Chromatographic separation

If the chromatographic separation can be modified to prevent coelution of suppressing species then other approaches need not be considered. The effect of chromatographic modification may be evaluated using the detector response monitoring under constant infusion approach described previously.

Sample preparation

An effective sample preparation protocol, usually involving either liquid-liquid extraction (LLE) or solid phase extraction (SPE) and frequently derivatisation can remove ion suppressing species from the sample matrix prior to analysis. These common approaches may also remove other interferences, such as isobaric species.

Protein precipitation is another method that can be employed for small molecule analysis. Removal of all protein species from the sample matrix may be effective in some cases, although for many analytes, ion suppressing species are not of protein origin and so this technique is often used in conjunction with extraction and derivatisation.

Sample concentration and mobile phase flow rate

Dilution of sample or reducing the volume of sample injected may give a reduction of ion suppression by reducing the quantity of interfering species present, although the quantity of analyte of interest will also be reduced, making this an undesirable approach for trace analysis.

Similar is the effect of reducing the mobile phase flow rate to the nanolitre-per-minute range since, in addition to resulting in improved desolvation, the smaller droplets formed are more tolerant to the presence of non-volatile species in the sample matrix.

Calibration techniques to compensate for ion suppression

It is not always possible to eliminate ion suppression by sample preparation and/or chromatographic resolution. In such cases it may be possible to compensate for the effects of ion suppression on accuracy and precision (although not for analytical sensitivity) by adopting complex calibration strategies.

Matrix matched calibration standards

Using matrix matched calibration standards can compensate for ion suppression. Using this technique, calibration standards are prepared in identical sample matrix to that used for analysis (e.g. plasma) by spiking a normal sample with known concentrations of analyte. This is not always possible for biological samples, since the analyte of interest is often endogenously present in a clinically significant, albeit normal, quantity. For matrix matched calibration standards to be effective in compensating for ion suppression, the sample matrix must be free of the analyte of interest. Additionally, it is important that there is little variation in test sample composition since both the test sample and the prepared calibration sample must be affected in the same way by ion suppression. Again, in complex biological samples from different individuals, or even the same individual at a different time, there may be large fluctuations in the concentrations of ion suppressing species.

Standard addition

The standard addition approach involves spiking the same sample extract with several known concentrations of analyte. This technique is more robust and effective than using matrix matched standards but is labor-intensive since each sample must be prepared several times to achieve a reliable calibration.

Internal standard

In this approach, the sample is spiked with a species (internal standard) which is used to normalise the response of analyte, compensating for variables at any stage of the sample preparation and analysis, including ion suppression.

It is important that the internal standard displays very similar (ideally identical) properties, with respect to detector response (i.e. ionisation), as the analyte of interest. To simplify the selection of internal standard, most laboratories use an analogous stable isotope in an isotope dilution type analysis. The stable isotope is almost guaranteed to be chemically and physically as close as possible to the analyte of interest, hence producing an almost identical detector response in addition to behaving identically during sample preparation and chromatographic resolution. To this end, the ion suppression experienced by both the analyte and the internal standard should be identical. It is important to note that an excessively high concentration of stable isotope internal standard may cause ion suppression itself, since it will co-elute with the analyte of interest. Hence, the internal standard should be added at an appropriate concentration.

Choice of ionization source

APCI generally suffers less ion suppression than ESI, as discussed previously. Where possible, if ion suppression is unavoidable it may be advisable to switch from ESI to APCI. If this is not possible, it may be useful to switch the ESI ionisation mode from positive to negative. Since fewer compounds are ionisable in negative ionisation mode, it is entirely possible that the ion suppressing species may be removed from the analysis. However, it should also be considered that the analyte of interest may not be ionised effectively in negative mode either, rendering this approach useless.

Related Research Articles

In chemical analysis, Chromatography is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. The different constituents of the mixture have different affinities for the stationary phase. The different molecules stay longer or shorter on the stationary phase, depending on their interactions with its surface sites. So, they travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.

Inductively coupled plasma mass spectrometry

Inductively coupled plasma mass spectrometry (ICP-MS) is a type of mass spectrometry that uses an inductively coupled plasma to ionize the sample. It atomizes the sample and creates atomic and small polyatomic ions, which are then detected. It is known and used for its ability to detect metals and several non-metals in liquid samples at very low concentrations. It can detect different isotopes of the same element, which makes it a versatile tool in isotopic labeling.

High-performance liquid chromatography Technique used in analytical chemistry

High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column.

Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures.

Ion source Device that creates charged atoms and molecules (ions)

An ion source is a device that creates atomic and molecular ions. Ion sources are used to form ions for mass spectrometers, optical emission spectrometers, particle accelerators, ion implanters and ion engines.

Electrospray ionization Technique used in mass spectroscopy

Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized. ESI is different from other ionization processes since it may produce multiple-charged ions, effectively extending the mass range of the analyser to accommodate the kDa-MDa orders of magnitude observed in proteins and their associated polypeptide fragments.

Calibration curve Method for determining the concentration of a substance in an unknown sample


In analytical chemistry, a calibration curve, also known as a standard curve, is a general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration. A calibration curve is one approach to the problem of instrument calibration; other standard approaches may mix the standard into the unknown, giving an internal standard.

Gas chromatography Type of chromatography

Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.

Gas chromatography–mass spectrometry Analytical method

Gas chromatography–mass spectrometry (GC-MS) is an analytical method that combines the features of gas-chromatography and mass spectrometry to identify different substances within a test sample. Applications of GC-MS include drug detection, fire investigation, environmental analysis, explosives investigation, and identification of unknown samples, including that of material samples obtained from planet Mars during probe missions as early as the 1970s. GC-MS can also be used in airport security to detect substances in luggage or on human beings. Additionally, it can identify trace elements in materials that were previously thought to have disintegrated beyond identification. Like liquid chromatography–mass spectrometry, it allows analysis and detection even of tiny amounts of a substance.

Peptide mass fingerprinting

Peptide mass fingerprinting (PMF) is an analytical technique for protein identification in which the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with a mass spectrometer such as MALDI-TOF or ESI-TOF. The method was developed in 1993 by several groups independently. The peptide masses are compared to either a database containing known protein sequences or even the genome. This is achieved by using computer programs that translate the known genome of the organism into proteins, then theoretically cut the proteins into peptides, and calculate the absolute masses of the peptides from each protein. They then compare the masses of the peptides of the unknown protein to the theoretical peptide masses of each protein encoded in the genome. The results are statistically analyzed to find the best match.

Liquid chromatography–mass spectrometry Analytical chemistry technique

Liquid chromatography–mass spectrometry (LC–MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry (MS). Coupled chromatography - MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides structural identity of the individual components with high molecular specificity and detection sensitivity. This tandem technique can be used to analyze biochemical, organic, and inorganic compounds commonly found in complex samples of environmental and biological origin. Therefore, LC-MS may be applied in a wide range of sectors including biotechnology, environment monitoring, food processing, and pharmaceutical, agrochemical, and cosmetic industries.

Atmospheric-pressure chemical ionization

Atmospheric pressure chemical ionization (APCI) is an ionization method used in mass spectrometry which utilizes gas-phase ion-molecule reactions at atmospheric pressure (105 Pa), commonly coupled with high-performance liquid chromatography (HPLC). APCI is a soft ionization method similar to chemical ionization where primary ions are produced on a solvent spray. The main usage of APCI is for polar and relatively less polar thermally stable compounds with molecular weight less than 1500 Da. The application of APCI with HPLC has gained a large popularity in trace analysis detection such as steroids, pesticides and also in pharmacology for drug metabolites.

Surface-enhanced laser desorption/ionization (SELDI) is a soft ionization method in mass spectrometry (MS) used for the analysis of protein mixtures. It is a variation of matrix-assisted laser desorption/ionization (MALDI). In MALDI, the sample is mixed with a matrix material and applied to a metal plate before irradiation by a laser, whereas in SELDI, proteins of interest in a sample become bound to a surface before MS analysis. The sample surface is a key component in the purification, desorption, and ionization of the sample. SELDI is typically used with time-of-flight (TOF) mass spectrometers and is used to detect proteins in tissue samples, blood, urine, or other clinical samples, however, SELDI technology can potentially be used in any application by simply modifying the sample surface.

Desorption electrospray ionization

Desorption electrospray ionization (DESI) is an ambient ionization technique that can be coupled to mass spectrometry for chemical analysis of samples at atmospheric conditions. Coupled Ionization sources-MS systems are popular in chemical analysis because the individual capabilities of various sources combined with different MS systems allow for chemical determinations of samples. DESI employs a fast-moving charged solvent stream, at an angle relative to the sample surface, to extract analytes from the surfaces and propel the secondary ions toward the mass analyzer. This tandem technique can be used to analyze forensics analyses, pharmaceuticals, plant tissues, fruits, intact biological tissues, enzyme-substrate complexes, metabolites and polymers. Therefore, DESI-MS may be applied in a wide variety of sectors including food and drug administration, pharmaceuticals, environmental monitoring, and biotechnology.

Sample preparation for mass spectrometry is used for the optimization of a sample for analysis in a mass spectrometer (MS). Each ionization method has certain factors that must be considered for that method to be successful, such as volume, concentration, sample phase, and composition of the analyte solution. Quite possibly the most important consideration in sample preparation is knowing what phase the sample must be in for analysis to be successful. In some cases the analyte itself must be purified before entering the ion source. In other situations, the matrix, or everything in the solution surrounding the analyte, is the most important factor to consider and adjust. Often, sample preparation itself for mass spectrometry can be avoided by coupling mass spectrometry to a chromatography method, or some other form of separation before entering the mass spectrometer. In some cases, the analyte itself must be adjusted so that analysis is possible, such as in protein mass spectrometry, where usually the protein of interest is cleaved into peptides before analysis, either by in-gel digestion or by proteolysis in solution.

Two-dimensional chromatography

Two-dimensional chromatography is a type of chromatographic technique in which the injected sample is separated by passing through two different separation stages. Two different chromatographic columns are connected in sequence, and the effluent from the first system is transferred onto the second column. Typically the second column has a different separation mechanism, so that bands that are poorly resolved from the first column may be completely separated in the second column. Alternately, the two columns might run at different temperatures. During the second stage of separation the rate at which the separation occurs must be faster than the first stage, since there is still only a single detector. The plane surface is amenable to sequential development in two directions using two different solvents.

Direct electron ionization liquid chromatography–mass spectrometry interface

A direct electron ionization liquid chromatography–mass spectrometry interface is a technique for coupling liquid chromatography and mass spectrometry (LC-MS) based on the direct introduction of the liquid effluent into an electron ionization (EI) source. Library searchable mass spectra are generated. Gas-phase EI has many applications for the detection of HPLC amenable compounds showing minimal adverse matrix effects. The direct-EI LC-MS interface provides access to well-characterized electron ionization data for a variety of LC applications and readily interpretable spectra from electronic libraries for environmental, food safety, pharmaceutical, biomedical, and other applications.

Atmospheric pressure photoionization Soft ionization method

Atmospheric pressure photoionization (APPI) is a soft ionization method used in mass spectrometry (MS) usually coupled to liquid chromatography (LC), Ionization of molecules is achieved using a vacuum ultraviolet (VUV) light source operating at atmospheric pressure, either by direct absorption followed by electron ejection or through ionization of a dopant molecule that leads to chemical ionization of target molecules. The sample is usually a solvent spray that is vaporized by nebulization and heat. The benefit of APPI is that it ionizes molecules across a broad range of polarity and is particularly useful for ionization of low polarity molecules for which other popular ionization methods such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are less suitable. It is also less prone to ion suppression and matrix effects compared to ESI and APCI and typically has a wide linear dynamic range. The application of APPI with LC/MS is commonly used for analysis of petroleum compounds, pesticides, steroids, and drug metabolites lacking polar functional groups and is being extensively deployed for ambient ionization particularly for explosives detection in security applications.

Probe electrospray ionization (PESI) is an electrospray-based ambient ionization technique which is coupled with mass spectrometry for sample analysis. Unlike traditional mass spectrometry ion sources which must be maintained in a vacuum, ambient ionization techniques permit sample ionization under ambient conditions, allowing for the high-throughput analysis of samples in their native state, often with minimal or no sample pre-treatment. The PESI ion source simply consists of a needle to which a high voltage is applied following sample pick-up, initiating electrospray directly from the solid needle.

Laser diode thermal desorption

Laser diode thermal desorption (LDTD) is an ionization technique that is coupled to mass spectrometry to analyze samples with atmospheric pressure chemical ionization (APCI). It uses a laser to thermally desorb analytes that are deposited on a stainless steel sheet sample holder, called LazWell. The coupling of LDTD and APCI is considered to be a soft-ionization technique. With LDTD-APCI, it is possible to analyze samples in forensics, pharmaceuticals, environment, food and clinical studies. LDTD is suitable for small molecules between 0 and 1200 Da and some peptides such as cyclosporine.

References

  1. ↑ Mei, H; Nardo C; Xu X; Wang S; Ng K; Korfmacher WA (November 2002). "Investigation of matrix effects in bioanalytical high-performance liquid chromatography/tandem mass spectrometric assays: application to drug discovery". Rapid Communications in Mass Spectrometry. 17 (1): 97–103. Bibcode:2003RCMS...17...97M. doi:10.1002/rcm.876. PMID   12478560.
  2. ↑ Furey, Ambrose; Merisa Moriarty; Vaishali Bane; Brian Kinsella; Mary Lehan (October 2013). "Ion suppression; A critical review on causes, evaluation, prevention and applications". Talanta. 115: 104–122. doi:10.1016/j.talanta.2013.03.048. PMID   24054567.
  3. ↑ Jessome, Lori Lee; Volmer D (May 2006). "Ion Suppression: A Major Concern in Mass Spectrometry". LCGC North AMerica. 24 (5).
  4. ↑ Buhrman, Deborah L; Price PI; Rudewicz PJ (November 1996). "Quantitation of SR 27417 in Human Plasma Using Electrospray Liquid Chromatography-Tandem Mass Spectrometry: A Study of Ion Suppression". J Am Soc Mass Spectrom. 7 (11): 1099–1105. doi: 10.1016/s1044-0305(96)00072-4 . PMID   24203071.
  5. ↑ Annesley, Thomas M (July 2003). "Ion Suppression in Mass Spectrometry". Clinical Chemistry. 49 (7): 1041–1044. doi: 10.1373/49.7.1041 . PMID   12816898.
  6. ↑ Bruins CH, Jeronimus-Stratingh CM, Ensing K, van Dongen WD, de Jong GJ (November 1999). "On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples. I. Determination of clenbuterol in urine". J Chromatogr A. 863 (1): 115–122. doi:10.1016/S0021-9673(99)00959-0. PMID   10591469.
  7. ↑ Van Hout, MW; Hofland CM; Niederländer HA; de Jong GJ (2000). "On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples. II. Determination of clenbuterol in urine using multiple-stage mass spectrometry in an ion-trap mass spectrometer". Rapid Communications in Mass Spectrometry. 14 (22): 2103–2111. doi:10.1002/1097-0231(20001130)14:22<2103::AID-RCM138>3.0.CO;2-V. PMID   11114016.
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