Elsevier

Clinical Biochemistry

Volume 38, Issue 4, April 2005, Pages 328-334
Clinical Biochemistry

Review
Matrix effects: the Achilles heel of quantitative high-performance liquid chromatography–electrospray–tandem mass spectrometry

https://doi.org/10.1016/j.clinbiochem.2004.11.007Get rights and content

Abstract

High-performance liquid chromatography coupled by an electrospray ion source to a tandem mass spectrometer (HPLC–ESI–MS/MS) is the current analytical method of choice for quantitation of analytes in biological matrices. With HPLC–ESI–MS/MS having the characteristics of high selectivity, sensitivity, and throughput, this technology is being increasingly used in the clinical laboratory. An important issue to be addressed in method development, validation, and routine use of HPLC–ESI–MS/MS is matrix effects. Matrix effects are the alteration of ionization efficiency by the presence of coeluting substances. These effects are unseen in the chromatogram but have deleterious impact on methods accuracy and sensitivity. The two common ways to assess matrix effects are either by the postextraction addition method or the postcolumn infusion method. To remove or minimize matrix effects, modification to the sample extraction methodology and improved chromatographic separation must be performed. These two parameters are linked together and form the basis of developing a successful and robust quantitative HPLC–ESI–MS/MS method. Due to the heterogenous nature of the population being studied, the variability of a method must be assessed in samples taken from a variety of subjects. In this paper, the major aspects of matrix effects are discussed with an approach to address matrix effects during method validation proposed.

Introduction

The development of atmospheric pressure ionization techniques (electrospray, atmospheric pressure chemical, and atmospheric pressure photo-ionization) has enabled the coupling of high-performance liquid chromatography with mass spectrometry (HPLC–MS). The use of hydrophobic separation combined with selective mass spectrometric detection makes this a versatile analytical tool. Electrospray is currently the most widely used ionization source. The electrospray interface produces singly or multiply charge ions directly from an aqueous–organic solvent system by creating a fine spray of highly charged droplets in the presence of a strong electric field, with the assistance of heat or pneumatics. The successful formation of ions using electrospray ionization requires two steps: the transfer of the compound of interest into the gas phase and the addition of a charge to the analyte if it is not already in a charged state [1], [2], [3], [4].

HPLC–MS systems using an electrospray ion source coupled with tandem mass analyzers (HPLC–ESI–MS/MS) have been applied to a wide variety of studies in pharmaceutical analysis and life sciences. With HPLC–ESI–MS/MS now considered the benchmark for measurement of drugs and their metabolites in biological matrices [5], the high selectivity of tandem mass spectrometry, with successive mass filtrations, leads to little or no observed interference even though there may be relatively high concentrations of coextracted and coeluted matrix components present. These characteristics have led to a growing trend of high-throughput analysis that incorporates little or no sample preparation and minimal chromatographic retention [6], [7], [8].

With HPLC–ESI–MS/MS having these characteristics of high selectivity, sensitivity, and throughput, it is not surprising that this technology is being increasingly used in the clinical laboratory. A recent review by Dooley [9] reported an exponential growth from 1991 to 2001 in clinical chemistry papers that mention tandem mass spectrometry. It is now accepted that HPLC–ESI–MS/MS is the method of choice for screening for inherited metabolic disorders [10], but it can be expected that many more applications will follow. While many biochemical markers such as steroids, fatty acids, amino acids, catecholamines, and thyroxine have been measured by this analytical technique [11], [12], [13], [14], [15]. Specific examples of clinical laboratories applying HPLC–ESI–MS/MS to drug quantification are for the therapeutic drug monitoring of immunosuppressant drugs [16] and protease inhibitors [17], and toxicological investigations [18], [19].

While HPLC–ESI–MS/MS offers much promise for clinical laboratories, one issue that must be addressed in method development, validation, and routine use is matrix effects. Matrix effects are the alteration of ionization efficiency by the presence of coeluting substances. A recent paper by Annesley [20] highlighted the importance of understanding matrix effects in clinical mass spectrometry applications, and although critical to the success of an HPLC–ESI–MS/MS analytical method, few published methods adequately address this problem [21]. The aim of this report is to provide an overview of matrix effects and from a clinical laboratory perspective show how this issue should be addressed for quantitative HPLC–ESI–MS/MS methodologies.

Section snippets

What are matrix effects?

Matrix effects occur when molecules coeluting with the compound/s of interest alter the ionization efficiency of the electrospray interface. This phenomenon was first described by Tang and Kebarle [22] who showed that electrospray responses of organic bases decreased as the concentrations of other organic bases were increased. The exact mechanism of matrix effects is unknown, but it probably originates from the competition between an analyte and the coeluting, undetected matrix components.

King

How can matrix effects be assessed?

The two main techniques used to determine the degree of matrix effects on an HPLC–ESI–MS/MS method are postextraction addition and postcolumn infusion. The postextraction addition technique requires sample extracts with the analyte of interest added postextraction compared with pure solutions prepared in mobile phase containing equivalent amounts of the analyte of interest [20], [21], [26], [27], [28]. The difference in response between the postextraction sample and the pure solution divided by

Strategies to overcome matrix effects

As discussed previously, the presence of coeluting compounds causes matrix effects. Thus, to obtain a robust HPLC–ESI–MS/MS method, there is a need to remove or minimize their presence. The source of these interfering matrix components must also be considered. The interference may come from the current sample being injected, a previously injected sample (as a late eluting interference), or build-up and overload of the analytical column [25]. Two approaches to remove or minimize matrix effects

Method validation

The experiments described above can be considered as semiquantitative and confirm the presence or absence of matrix effects and aid in minimizing their influence on results. But these data do not provide evidence that a validated analytical method is acceptable in terms of these effects. While the US Food and Drug Administration states that there is a need to investigate matrix effects for HPLC–MS methodologies [38], there is no clear guidance on how this should be performed during method

Conclusions

HPLC–ESI–MS/MS is a powerful tool for the scientist to utilize for quantitative clinical investigations. This technique is not a “turn key” solution to analytical problems, as matrix effects can be its' Achilles heel. But by careful assessment of matrix effects and judicial use of the appropriate sample preparation coupled with adequate chromatography, HPLC–ESI–MS/MS can provide a robust analytical platform. Clinical scientists in developing methods must acknowledge matrix effects and build

Acknowledgments

The author thanks Mr. Paul Salm MBA, Australian Bioanalytical Services Pty Ltd, for providing the matrix effect data on the cyclosporin method.

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