Strategies for the Detection and Elimination of Matrix Effects in Quantitative LC–MS Analysis

Jan 14, 2014

Currently available methods for the detection of matrix effects in liquid chromatography–mass spectrometry (LC–MS) are tedious and complex; therefore, a simpler method is required. Although there are no methods to completely eliminate matrix effects, the most well-recognized technique available to correct for matrix effects is that of internal standardization using stable isotope–labeled versions of the analytes. As this method can prove expensive, an alternative method of correction is likely to be useful. In this study, a simple method based on recovery is assessed for the detection of matrix effects. Two alternative methods for the rectification of matrix effects in LC–MS are also assessed: standard addition and the coeluting internal standard method.

High performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) has become the predominant analytical method for the quantitative determination of analytes in biological matrices because of its high specificity, sensitivity, and throughput (1–3). However, matrix effects have become a major concern in quantitative liquid chromatography–mass spectrometry (LC–MS) because they detrimentally affect the accuracy, reproducibility, and sensitivity (3). Matrix effects occur when compounds that are coeluted with the analyte interfere with the ionization process in the MS detector, thereby causing ionization suppression or enhancement (2–7). Compounds with high mass, polarity, and basicity are possible candidates to cause matrix effects (4–8). However, the mechanisms involved in matrix effects have not been fully explored. One of the proposed theories to explain matrix effects is that the coelution of interfering compounds, especially basic compounds, may deprotonate and neutralize the analyte ions and, thus, reduce the formation of protonated analyte ions (2,4). Another theory postulates that less-volatile compounds may affect the efficiency of droplet formation and reduce the ability of charged droplets to convert into gas-phase ions (2–4,8). In addition, matrix effects may also be caused by high viscosity interfering compounds that could possibly increase the surface tension of the charged droplets and reduce the efficiency of droplet evaporation (2,4,6).

Several methods have been proposed for the detection and assessment of matrix effects, including postextraction spike and postcolumn infusion methods. The postextraction spike method evaluates matrix effects by comparing the signal response of an analyte in neat mobile phase with the signal response of an equivalent amount of the analyte in the blank matrix sample spiked post-extraction. The difference in response determines the extent of matrix effect (2,3,9). The major drawback of this method is that for endogenous analytes such as metabolites (for example, creatinine) blank matrix (urine or plasma) is not available. The postcolumn infusion method assesses matrix effects qualitatively. A constant flow of analyte is infused into the HPLC eluent, followed by injection of the blank sample extract. A variation in signal response of the infused analyte caused by coeluted interfering compounds indicates ionization suppression or enhancement (3,10). By identifying the ionization suppression or enhancement regions of the chromatogram, analytical methods can be developed to eliminate matrix effects by preventing the elution of the analyte peak in regions where matrix effects occur. However, the process of postcolumn infusion is time consuming and requires additional hardware, and it is not appropriate for multianalyte samples. Considering these drawbacks of existing methods, we propose a simple, fast, and reliable method to detect matrix effects that can be applied to any analyte including endogenous compounds such as creatinine and to any matrix without requiring any additional hardware.

To obtain accurate and reliable LC–MS data, several methods have been suggested to reduce or eliminate matrix effects. Matrix effects can be reduced simply by injecting small amounts of samples or by diluting samples (11,12). However, this approach can only be feasible when the sensitivity of the assay is very high (12). Methods to reduce or eliminate matrix effects include optimizing sample preparation to remove interfering compounds from the samples (1,9,10,13), changing chromatographic parameters to avoid coelution of analytes and interfering compounds (4,14–17), and changing MS conditions to reduce the occurrence of matrix effects in the ion source. However, these methods are not without their limitations. Most of the sample cleanup methods fail to remove impurities that are similar to the analyte and, hence, likely to be coeluted with the analyte (11,18). Modifying chromatographic conditions can be time-consuming, and some of the additives used in the mobile phase to improve separation have been found to suppress the electrospray signal of the analytes (3,4,9,15). Furthermore, even when the sample is devoid of coeluted substances, trace impurities present in the mobile phase can significantly suppress the analyte peak (19).

It is clear from the above that matrix effects in LC–MS cannot be completely eliminated. Therefore, the only option available is the rectification of data to eliminate the matrix effects. Calibration techniques such as the external-matched standards method, the echo-peak technique, and the most commonly used approach, the internal standard method, have been developed to correct the data (15,18–21). However, these calibration techniques also have their drawbacks. For example, the matrix-matching technique requires many blank matrices and appropriate blank matrices are not always available for the preparation of external standards (9,11,18,23). It is also impossible to match the matrix of the calibration standards with each of the samples exactly, as each sample has coeluting, interfering compounds that are thereby exposed to a different extent of ionization suppression (18). Echo-peak does not compensate for matrix effects completely because both standard and analyte peaks are not eluted at the exact same retention time (11). The stable isotope–labeled internal standards (SIL-IS) approach is the best available option but it is expensive and standards are not always commercially available for the analyte of interest (4,9,23).

The standard addition method for correcting matrix effects is widely used in spectrophotometric analysis, especially in atomic spectroscopy (24–27). However, this method is less well documented with other analytical techniques and currently there is no record of its practical use in compensating matrix effects in LC–MS. Standard addition does not require a blank matrix and is therefore appropriate for compensating matrix effects for any analyte including endogenous metabolites in biological fluids (20,28,29). In this study, we investigated the possibility of using the method of standard addition in routine LC–MS analysis to compensate for matrix effects and to thereby obtain improved data. We also investigated the use of a coeluting structural analogue of the analyte as the internal standard as an alternative to the expensive and often unavailable stable isotope–labeled internal standard for correcting matrix effects. Although coeluting structural analogue compounds are used to extend the linear range of calibration curves (30), there have been no reports of their use in compensating matrix effects in routine LC–MS analysis. In addition, we report a simple and effective method for the detection as well as the correction of matrix effects in routine LC–MS. All studies were carried out using a creatinine assay applied to human urine samples.