Matrix Effect and Ion Suppression

A caveat for aU direct sample injection assays is an understanding of the analyte chemical stability in the biological fluid during the analysis period. Nonetheless, an increasingly growing body of literature is suggestive that direct injection of post-dose biological fluids for quantification purposes has become a routine and efficient procedure. 

Ion suppression or so-called matrix effect is a common problem in atmospheric pressure ionization (API) mass spectrometry [29-40]. There are differences in opinion as to the amount of ion suppression that is acceptable for an analytical method. 

Validation of quantitative LC/MS methods used in the determination of small-molecule drugs and/or their metabohtes in biological fluids is of paramount 

Moreover, in introduction of generic or new formulations and/or to establish bioequivalence (BE) between two products, certain guidehnes are followed to compare the systematic exposure of the test article to that of a 

Column FluoroSep RP Phenyl, 2.1 x 50 mm, 5 pm (ES Industries) Mobile Phase A = 5 mM aq. ammonium fonnate B = Methanol Flow Rate 400 pL/mln. 

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Matrix Effects. The matrix effects (ion suppression or ion enhancement) can compromise the selectivity and sensitivity of LC/MS/MS methods for the determination of drug concentrations in biological matrices. One common matrix effect is ion suppression due to co-eluting components that can affect the ionization efficiency of the analyte of interest. The matrix effects are major causes for errors in precision, accuracy, linearity, and reproducibility of the quantitation methods based on LC/MS/MS [104,108,114-118]. It is critical to overcome such effects in quantitative bioanalysis by LC/MS/MS. 

King, R., Ion formation from complex solutions Understanding matrix effects and ionization suppression, Presented at the Ninth Annual Symposium on Chemical and Pharmaceutical Structure Analysis, Princeton, NJ, October 17-19, 2006. 

Stolker et al. " described an analytical method based on TFC-LC-MS/MS for the direct analysis of 11 veterinary drugs (belonging to seven different classes) in milk. The method was applied to a series of raw milk samples, and the analysis was carried out for albendazole, difloxacin, tetracycline, oxytetracycline, phenylbutazone, salinomycin-Na, spiramycin, and sulfamethazine in milk samples with various fat contents. Even without internal standards, results proved to be linear and quantitative in the concentration range of 50-500 (xg/1, as well as repeatable (RSD<14% sulfamethazine and difloxacin <20%). The limits of detection were between 0.1 and 5.2 xg/l, far below the maximum residue limits for milk set by the EU. While matrix effects, namely, ion suppression or enhancement, were observed for all the analytes, the method proved to be useful for screening purposes because of its detection limits, linearity, and repeatability. A set of blank and fortified raw milk samples was analyzed and no false-positive or falsenegative results were obtained. 

In the manuscript that we are describing in depth in this chapter [73], repeatability, reproducibility, and accuracy were checked, and the calibration curves were established, allowing the calculation of the detection and quantification limits. RSD values for repeatability were lower than 7.01% accuracy values oscillated between 97.2% and 102.0% and limits of detection were low, ranging from 1.64 to 730.54 ppb (negative polarity) and from 0.51 to 310.23 ppb (positive polarity). Neither matrix effect nor ion suppression were detected. The authors proved that the method developed for UHPLC-UV-ESI-TOF MS was a very valuable tool, because it was able to determine a wide number of metabolites in a single run and it was reliable from an analytical point of view. For this reason, it was also applied for the quantitative analysis of the 26 samples under study. 

Ismaiel OA, Halquist MS, Elmamly MY et al (2008) Monitoring phospholipids for assessment of ion enhancement and ion suppression in ESI and APCI LC/MS/MS for chorpheniramine in human plasma and the importance of multiple source matrix effect evaluation. J Chromatogr B 875 333-343... 

A mandatory requirement when validating an LC-MS(-MS) based method according to the FDA guidelines is to evaluate and attempt to minimize the incidence of matrix effects [58], Matrix effects refer to the direct or indirect alteration or interference in response due to the presence of interfering substances in the sample [52], It can either reduce the analyte response (ion suppression) or enhance it (ion enhancement). Both can considerably compromise the accuracy of quantification and ion suppression may in the worst case even lead to decrease sensitivity and to false negative results. 

Because of the widespread use of LC/MS/MS for drug discovery bioanalysis, there is currently less of a necessity for finely tuned solid-phase extractions than there once was in this area. Instead, generic solid-phase extraction conditions that can accommodate many different analyte structures using the same extraction conditions have become more interesting. To make a solid-phase sample preparation useful for LC/MS/MS it must remove as much of the sample salts as possible in order to reduce the effects of ion suppression [47,51—52] and it must remove as many nonvolatile matrix components as possible so that the instrument ion source is not quickly fouled. Because the LC/MS/MS instrument is inherently so selective, added assay selectivity, per se, is no longer an objective of solid-phase extraction. 

A unique property of LC/API/MS is the extent to which the analyte signal is affected by the sample matrix or the existence of co-eluting analytes. This property can have a profound influence on sensitivity and assay reproducibility. Because of matrix-ion suppression, it is not possible to estimate extraction recovery by comparison of the signal from a neat sample to an extracted sample. This is because the reduction in signal represents the combined effects of recovery and ion suppression. As first shown by Buhrman et al., quantitative assessment of extraction efficiency is made by spiking the neat sample into an extracted blank and comparison of the result to a similar sample spiked before extraction [120]. Conversely, the extent of ion suppression is obtained by the comparison of the signals for a neat unextracted sample to the same neat solution spiked into an extracted matrix blank. 

The suitability of MS detectors for quantitative analyses is debated. For example, ESI is a competitive process and, occasionally, matrix (background) material interferes with the ionization of the analyte [40]. These effects of ion suppression are especially aggravated when several species coelute, such as in the case of biological extracts or with direct infusion (without previous separation). Generally, hydrophilic species are more sensitive to ion suppression than hydrophobic ones, which tend to concentrate on the droplet surface during ESI [41]. In MALDI, the sample dispersion is often inhomogeneous or the matrix crystals unevenly distributed on the surface. A truthful representation of the sample composition is obtained exclusively upon thorough laser desorption of the entire spot. In addition, with some instruments the transmission of ions in the mass... 

An important issue regarding selectivity concerns the assessment and quantitation of the matrix effects. Because UHPLC yields peaks with bases as narrow as 1 s, an overall enhancement of the chromatographic resolution is obtained. The potential co-elution and ion suppression are thus reduced, which enhances the sensitivity and reliability of the MS. However, while UHPLC improves the separation throughput and resolution, practical issues with using MS may arise because acquiring sufficient data points (>15 points per peak) is essential to ensure reliable quantitation. As previously mentioned (Section 4.2.2), instruments with high acquisition rates and low dwell times are, therefore, preferentially selected for quantitative determination. 

Regular LLE procedures are mostly implemented prior to UHPLC and present several benefits in terms of sample cleanup and enrichment. This technique is well adapted to LC-MS analysis because proteins and salts are extensively excluded, which minimizes any matrix effect and/or ion suppression and allows for very low detection limits with good recovery and precision (45). However, LLE usually includes long handling steps, which are not in line with UHPLC throughput. 

Desorption/ionisation techniques such as LSIMS are quite practical, as they give abundant molecular ion signals and fragmentation for structural information. In the conditions of Jackson et al. [96], all the molecular ion and/or protonated molecule ion species were observed in the LSIMS spectrum when only 1 pmol of each additive was placed on the probe tip. However, as mentioned above, in LSIMS/MS experiments the choice of the matrix (e.g. NBA, m-nitrobenzylalcohol) is very important. Matrix effects can lead to suppression of the generation of molecular ions for some additives. LSIMS is not ideal for the quantitative detection of polymer additives, as matrix effects are very important [96]. 

Matrix effect is a phrase normally used to describe the effect of some portion of a sample matrix that causes erroneous assay results if care is not taken to avoid the problem or correct for it by some mechanism. The most common matrix effects are those that result in ion suppression and subsequent false negative results. Ion enhancement may lead to false positive results.126 127 Several reports about matrix effects include suggestions on what can cause them and how to avoid them.126-147 While various ways to detect matrix effects have been reported, Matuszewski et al.140 described a clear way to measure the matrix effect (ME) for an analyte, recovery (RE) from the extraction procedure, and overall process efficiency (PE) of a procedure. Their method is to prepare three sets of samples and assay them using the planned HPLC/MS/MS method. The first set is the neat solution standards diluted into the mobile phase before injection to obtain the A results. The second set is the analyte spiked into the blank plasma extract (after extraction) to obtain the B results. The third set is the analyte spiked into the blank plasma before the extraction step (C results) these samples are extracted and assayed along with the two other sets. The three data sets allow for the following calculations ... 

For PK assays, it is generally believed that most matrix effects are due to the sample matrix (typically plasma). While this is correct in many cases, this assumption has some exceptions (vide infra). One of the most useful tools for avoiding matrix effects is studying the sample matrix and proposed assay by using the post-column infusion technique described by Bonfiglio et al.14S This technique allows visualization of the portion of the chromatographic step affected by ion suppression.161721 Xu et al.101 recommended inclusion of this step in the method development process for drug discovery PK assays. 

Total or partial ion suppression is a well-known LC-MS effect, which is induced by coeluting matrix components that can have a dramatic effect on the intensity of the analyte signal. As can be observed in Fig. 1, analyte suppression occurs as a consequence of the different matrix interferences present in waste-water samples, making the identification and/or quantification process difficult or unfeasible. Even when working under selection ion monitoring (SIM) conditions, these matrix effects can cause ion suppression in the detection of some analytes that are present at low levels of concentration, as seen in this figure. Several papers have reported this effect [30-32] and different alternatives to overcome these problems, such as the inclusion of a size-exclusion step [33] or sequential SPE [28], have been applied for the determination of pesticides in... 

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