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

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1 Clinical Biochemistry 38 (2005) Review Matrix effects: The Achilles heel of quantitative high-performance liquid chromatography electrospray tandem mass spectrometry Paul J. Taylor* Department of Clinical Pharmacology and Australian Bioanalytical Services Pty Ltd, 3rd Floor-R wing, Building One, Princess Alexandra Hospital, Ipswich Road, Brisbane, QLD 4102, Australia Received 16 June 2004; received in revised form 17 November 2004; accepted 17 November 2004 Available online 25 January 2005 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. D 2004 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Electrospray; Tandem mass spectrometry; Matrix effects; Quantitation Contents Introduction What are matrix effects? How can matrix effects be assessed? Strategies to overcome matrix effects Method validation Conclusions Acknowledgments References Introduction * Fax: address: PTaylor@soms.uq.edu.au. 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 /$ - see front matter D 2004 The Canadian Society of Clinical Chemists. All rights reserved. doi: /j.clinbiochem

2 P.J. Taylor / Clinical Biochemistry 38 (2005) 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 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 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 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. 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 et al. [23] have shown through a series of experiments that matrix effects are the result of competition between nonvolatile matrix components and analyte ions for access to the droplet surface for transfer to the gas phase. Although they conclude that the exact mechanism of the alteration of analyte release into the gas phase by these nonvolatile components is unclear. They postulate b...a likely list of effects relating to the attractive force holding the drop together and keeping smaller droplets from forming should account for a large proportion of the ionization suppression observed with electrospray ionizationq. Depending on the environment in which the ionization and ion evaporation processes take place, this competition may effectively decrease (commonly known as ion suppression) or increase (ion enhancement) the efficiency of formation of the desired analyte ions present at the same concentrations in the interface. Thus the efficiency of analyte ions to form is very much dependent on the matrix entering the electrospray ion source. Matrix effects are also compound dependent. Bonfiglio et al. [24] reported that the chemical nature of a compound has a significant effect on the degree of matrix effects. In a study of four compounds of different polarities under the same mass spectrometric conditions, the most polar was found to have the largest ion suppression and the least polar was affected less by ion suppression. These findings of differential matrix effects have important ramifications particularly when selecting a suitable internal standard for quantification purposes. For example, if a drug and a glucuronide metabolite were quantified by internal standardization against a close analogue of the parent drug and matrix effects were slightly different between samples, then the change in ionization of the more polar glucuronide metabolite would probably not be compensated by the internal standard. Thus if there are multiple analytes to be quantified, with varying degrees of polarity, there may be requirements for multiple internal standards [25]. The importance of matrix effects on the reliability of HPLC ESI MS/MS has been shown in terms of accuracy and precision [26], and when ion suppression occurs, the sensitivity and lower limit of quantification of a method

3 330 P.J. Taylor / Clinical Biochemistry 38 (2005) may be adversely affected [27]. Thus to develop a reliable HPLC ESI MS/MS method, experiments should be performed to understand these matrix effects. 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 28]. The difference in response between the postextraction sample and the pure solution divided by the pure solution response determines the degree of matrix effect occurring to the analyte in question under chromatographic conditions. An example of calculations from postextraction addition experiments is shown in Table 1. These data are adapted from a study by Buhrman et al. [27] who showed using this technique the various amounts of matrix effects that may occur using different sample preparation methods of a platelet-activating factor receptor antagonist from human plasma. The example shown is for the liquid liquid extraction of this compound using hexane. The matrix effect was determined as 26%. This value represents a loss of 26% of the analyte signal (ion suppression) due to alterations in ionization efficiency. A calculated value of 0% would represent no matrix effects, the ideal scenario. Additional data from this experiment can be obtained from the comparison of the ion intensity obtained from extracts with analyte added preextraction with pure solutions to give an overall value termed bprocess efficiencyq (Table 1). Process efficiency represents the combination of matrix effects and recovery of the analyte from the matrix by the sample extraction process. The absolute recovery in the above example is 63% (4050/ %), but the process efficiency is only 47%. Low process efficiency can be deleterious to the accuracy and in particular the lower limit of quantification of a method [26]. Fierens et al. [29] reported preliminary data on the measurement of urinary C-peptide by HPLC ESI MS/ Table 1 Calculation of matrix effects, recovery, and process efficiency using the postextraction addition technique Experiment Ion intensity (cps) Parameter Calculation and result (%) Preextraction addition 4050 Matrix effect ( )/ = 26 Postextraction 6390 Process 4050/ = 47 addition efficiency Pure solution 8580 Data modified from Buhrman et al. [27]. MS. Using aqueous standards, it was determined that by direct injection of urine a limit of detection of 0.2 ng could be achieved. Comparing these standards with urine, it was found that the signal was suppressed by 70 85%. Thus without removing these matrix effects, via sample preparation, the desired lower limits of detection could not be attained. The postextraction addition technique can be considered a static technique that only provides information about matrix effects at the point of elution of the analyte of interest. A more dynamic technique for determining matrix effects is postcolumn infusion [23,24,30 32]. An infusion pump is used to deliver a constant flow of analyte into the HPLC eluent at a point after the chromatographic column and before the mass spectrometer ionization source (Fig. 1). A sample extract (without added analyte) is injected under the desired chromatographic conditions and the response from the infused analyte recorded. The postinfusion technique enables the influence of the matrix on analyte response to be investigated over the entire chromatographic run. One point taken into consideration is that the concentration of the infused analyte should be in the analytical range being investigated as too much added analyte may overwhelm the electrospray s ability to generate ions and thus give misleading results. Results of postcolumn infusion experiments enable the scientist to evaluate the influence of different sample extraction techniques on matrix effects, the appropriate analytical column, where matrix effects occur and are absent during a chromatographic run, the mechanistic aspect of matrix effects, and the influence of mobile additives on response. As an example, Fig. 2 shows a comparison of an injection of (A) mobile phase, (B) a whole blood sample prepared by protein precipitation, and (C) a whole blood sample by solid phase extraction [33]. The analyte sirolimus, an immunosuppressant drug (50 Ag/L), was infused (10 AL/min) postcolumn under the chromatographic conditions described in Taylor and Johnson [33]. Sirolimus was monitored by selected reactant monitoring using the following mass transition: m/z 931.6Y The retention time of sirolimus under these chromatographic conditions would be approximately 6 min. For mobile phase injection (Fig. 2A), no change in signal was observed throughout the chromatogram. For the sample treated with acetonitrile to precipitate proteins (Fig. 2B), the signal is suppressed for the majority of the chromatogram. While the sample prepared by solid phase extraction (Fig. 2C) showed minimal suppression in the solvent front but constant and maximum signal at the retention time of the analyte. 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

4 P.J. Taylor / Clinical Biochemistry 38 (2005) Fig. 1. Schematic of the postcolumn infusion system. 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 are modification to the sample extraction methodology and improved chromatographic separation [34]. These two parameters are linked together in developing a quantitative HPLC ESI MS/MS method. There are several sample preparation options that have been used for many years with HPLC and studies have evaluated the degree of matrix effects for these different techniques [24,27,29,30,32]. It is considered that protein precipitation using an organic solvent or dilute and shoot are the bdirtiestq sample preparation techniques and thus produce the most matrix effects compared to solid phase or liquid liquid extraction [24,30]. More recently, on-line two-dimensional chromatography, using a column-switching device, has been shown to provide an alternate sample preparation that gives minimal matrix effects [35]. A clear advantage of this technique is the ability to automate the analytical process but column breakthrough will limit how much crude sample can be loaded. The selection of which extraction technique to use for an application is not just limited to providing the cleanest sample but linked also to the chromatographic separation when optimizing a method. The majority of matrix effects occur in the solvent front of a chromatographic run [30]. Thus intuitively if the analytes can be retained chromatographically to some degree then matrix effects can be minimized. Data obtained from postcolumn infusion experiments can provide data on where matrix effects are occurring. Thus using these data to move the analytes away from these affected areas provides for a more stable method. These changes may limit the aim of high-throughput analysis. An alternative approach is to apply a rapid gradient or bballistic gradientq to separate the analytes from the solvent front while maintaining high throughput [6]. It has been shown, for specific compounds, that atmospheric pressure chemical ionization is less prone to matrix effects than electrospray [36,37]. The presence of excess reagent ions to produce charged species means the ionization process of atmospheric pressure chemical ionization is less susceptible to matrix effects. Therefore, the problem of matrix effects can be removed by changing the ionization source, although some differences in matrix effect for atmospheric pressure chemical ionization have been found between manufacturer s ion sources [37]. Another limitation being that the analytes must be thermally stable and readily ionized by this technique. 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 validation. The experiments described in the following are from a recent paper by Matuszewski et al. [21] and the author s personal experience.

5 332 P.J. Taylor / Clinical Biochemistry 38 (2005) Fig. 2. Comparison of (A) mobile phase, (B) whole blood sample prepared by protein precipitation, and (C) a whole sample prepared by solid phase extraction [33] by the postcolumn infusion method. The areas influenced by matrix effects are shown in B and C. The solid lines indicate the regions of altered ionization due to matrix effects. Most if not all method validations are performed using standard and quality control samples prepared from a pooled source of matrix (whole blood, urine, etc.). In such a case, the matrix is homogenous and thus can be assumed that prepared samples will have the same matrix effects. Using these homogenous samples for validation does not take into account the inter- and intrapatient matrix variability that is present in the clinical setting. It is highly likely that an extract of a plasma sample collected from a burn s patient will be of a different composition to a recent liver transplant recipient and certainly to a healthy subject and thus may have variable matrix effects. This interpatient variability is a typical problem faced but seldom addressed by the clinical scientist establishing an HPLC ESI MS/MS method. Matuszewski et al. [21] have elegantly shown the influence of different subjects on assay imprecision for a quantitative HPLC ESI MS/MS method by comparing standard samples prepared from one pool of plasma with that from multiple sources. Standard curves were prepared in pooled plasma and also different individuals, subjected to the same liquid liquid extraction and chromatographic conditions with multiple extractions performed for each concentration (n = 5). For the standards prepared from pooled plasma, across an analytical range of Ag/L,

6 P.J. Taylor / Clinical Biochemistry 38 (2005) Table 2 Validation data of an HPLC ESI MS/MS method, for the measurement of cyclosporin, using the pre- and postextraction technique Parameter F standard Cyclosporin concentration (Ag/L) Internal standard deviation (300 Ag/L) Matrix effect (%) 7.5 F F F F 7.5 Absolute recovery (%) 75.7 F F F F 6.7 Process efficiency (%) 69.8 F F F F 9.3 Intersubject variability a (%) b Cyclosporin parameters are calculated on five measurements and internal standard parameters on 15 measurements. Methodology reported in Taylor et al. [40]. a Expressed as coefficient of variation (n = 5). b Not applicable. the variability at each concentration in terms of coefficient of variation was %. This variability would be considered acceptable for method validation based on current opinion [39]. For the standard samples prepared from different lots of plasma, across this same concentration range, the variability was between 11.6% and 23.8%. A significant increase in variability due to intersubject differences in matrix effects was reported; with this decrease in performance, the method would now fail validation. It can be speculated that many HPLC ESI MS/MS quantification methods may fall into this same scenario if validation is solely based on one pooled matrix. It is clear that at some stage during method validation the variability of a method must be assessed in samples taken from a variety of subjects in the patient population of interest. Further, it has been shown that endogenous sources of matrix effects may come from the types of specimen containers and preservatives within these containers [37]. Thus matrix effect studies during validation must be carried out in the collection tube used for the method to be established. If different types of anti-coagulants are used, all of these should be evaluated. While it is not practical to prepare all calibration standard and quality control samples from individual sources, some assessment of patient variability must be undertaken. The following approach has been used in the author s laboratory. This protocol assesses the performance of a method using blood from subjects who are the subject of the clinical investigation being studied (i.e., transplant recipients, HIV patients, uremic patients, etc). Three quality control concentrations, over the analytical range, in replicates of five are prepared using different individuals for each aliquot (i.e., each individual is used only once). The analyte of interest and internal standard are added pre- and postextraction and prepared in pure solution according to the protocol of Buhrman et al. [27], previously described above. Using this protocol of 45 injections, matrix effects, absolute recovery, process efficiency, and most importantly intersubject variability can be calculated. As an example, data from an HPLC ESI MS/MS method for the measurement of cyclosporin in whole blood that used solid phase sample preparation are shown in Table 2 [40]. Three concentrations of cyclosporin at 30, 400, and 1500 Ag/L were assessed using blood obtained from transplant recipients not receiving this drug. Matrix effects were minimal with less than 7% ion suppression. The intersubject variability, expressed as coefficient of variation, was b3% for the three concentrations. These data compared favorably with the variability obtained from pooled blood of b5% coefficient of variation (data not shown). The results of this experiment suggest that matrix effects will have minimal influence on the results of this method. If the intersubject variability was greater than 15%, changes to the extraction procedure and chromatography would have to be undertaken. It is hoped that this type of procedure will be common practice in the validation of HPLC ESI MS/MS methods. Conclusions HPLC ESI MS/MS is a powerful tool for the scientist to utilize for quantitative clinical investigations. This technique is not a bturn keyq 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 assessment systems into their validation protocols. If these procedures are not performed, there can be doubt in the validity of results produced. Acknowledgments The author thanks Mr. Paul Salm MBA, Australian Bioanalytical Services Pty Ltd, for providing the matrix effect data on the cyclosporin method. References [1] Covey TR, Lee ED, Henion JD. High-speed liquid chromatography/ tandem mass spectrometry for the determination of drugs in biological samples. Anal Chem 1986;58: [2] Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. Electrospray ionization for mass spectrometry of large biomolecules. Science 1989;246:64 71.

7 334 P.J. Taylor / Clinical Biochemistry 38 (2005) [3] Bruins AP. Liquid chromatography mass spectrometry with ionspray and electrospray interfaces in pharmaceutical and biomedical research. J Chromatogr 1991;554: [4] Kebarle P. A brief overview of the present status of the mechanisms involved in electrospray mass spectrometry. J Mass Spectrom 2000;35: [5] Brewer E, Henion J. Atmospheric pressure ionization LC/MS/MS techniques for drug disposition studies. J Pharm Sci 1998;87: [6] Hopfgartner G, Bourgogne E. Quantitative high-throughput analysis of drugs in biological matrices by mass spectrometry. Mass Spectrom Rev 2003;22: [7] Bayliss MK, Little D, Mallett DN, Plumb RS. Parallel ultra-high flow rate liquid chromatography with mass spectrometric detection using a multiplex electrospray source for direct, sensitive determination of pharmaceuticals in plasma at extremely high throughput. Rapid Commun Mass Spectrom 2000;14: [8] Grant RP, Cameron C, Mackenzie-McMurter S. Generic serial and parallel on-line direct injection using turbulent flow liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 2002;16: [9] Dooley KC. Tandem mass spectrometry in the clinical chemistry laboratory. Clin Biochem 2003;36: [10] Chace DH. Mass spectrometry in the clinical laboratory. Chem Rev 2001;101: [11] Fredline VF, Taylor PJ, Dodds HM, Johnson AG. A reference method for the analysis of aldosterone in blood by high performance liquid chromatography atmospheric pressure chemical ionisation tandem mass spectrometry. Anal Biochem 1997;252: [12] Johnson DW. Dimethylaminoethyl esters for trace, rapid analysis of fatty acids by electrospray tandem mass spectrometry. Rapid Commun Mass Spectrom 1999;13: [13] Casetta B, Tagliacozzi D, Shushan B, Federici G. Development of a method for rapid quantitation of amino acids by liquid chromatography tandem mass spectrometry. Clin Chem Lab Med 2000;38: [14] Kushnir MK, Urry FM, Frank EL, Roberts WL, Shushan B. Analysis of catecholamines in urine by positive ion electrospray tandem mass spectrometry. Clin Chem 2002;48: [15] Tai SS-C, Sniegoski LT, Welch MJ. Candidate reference method for total thyroxine in human serum: use of isotope-dilution liquid chromatography mass spectrometry with electrospray ionization. Clin Chem 2002;48: [16] Taylor PJ. Therapeutic drug monitoring of immunosuppressant drugs by high-performance liquid chromatography mass spectrometry. Ther Drug Monit 2004;26: [17] Volosov A, Alexander C, Ting L, Soldin SJ. Simple rapid method for quantification of antiretrovirals by liquid chromatography tandem mass-spectrometry. Clin Biochem 2002;35: [18] Muller C, Vogt S, Goerke R, Kordon A, Weinmann W. Identification of selected pyschopharmaceuticals and their metabolites in hair by LC/ ESI CID/MS and LC/MS/MS. Forensic Sci Int 2000;113: [19] Marquet P. Progress of liquid chromatography mass spectrometry in clinical and forensic toxicology. Ther Drug Monit 2002;24: [20] Annesley TM. Ion suppression in mass spectrometry. Clin Chem 2003;49: [21] Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC MS/MS. Anal Chem 2003;75: [22] Tang L, Kebarle P. Dependence of ion intensity in electrospray mass spectrometry on the concentration of the analytes in the electrosprayed solution. Anal Chem 1993;65: [23] King R, Bonfiglio R, Fernandez-Metzler C, Miller-Stein C, Olah T. Mechanistic investigation of ionization suppression in electrospray ionization. J Am Soc Mass Spectrom 2000;11: [24] Bonfiglio R, King RC, Olah TV, Merkle K. The effects of sample preparation methods on variability of the electrospray ionization response for model drug compounds. Rapid Commun Mass Spectrom 1999;13: [25] Lagerwerf FM, van Dongen WD, Steenvoorden RJJM, Honing M, Jonkman JHG. Exploring the boundaries of bioanalytical quantitative LC MS MS. TrAC, Trends Anal Chem 2000;19: [26] Matuszewski BK, Constanzer ML, Chavez-Eng CM. Matrix effect in quantitative LC/MS/MS analyses of biological fluids: a method for determination of finasteride in human plasma at picogram per millilitre concentrations. Anal Chem 1998;70: [27] Buhrman D, Price P, Rudewicz P. Quantitation of SR in human plasma using electrospray liquid chromatography tandem mass spectrometry: a study of ion suppression. J Am Soc Mass Spectrom 1996;7: [28] Fu I, Woolf EJ, Matuszewski BK. Effect of the sample matrix on the determination of indinavir in human urine by HPLC with turbo ion spray tandem mass spectrometric detection. J Pharm Biomed Anal 1998;18: [29] Fierens C, Thienport LM, Stfckl D, Leenheer AP. Matrix effect in the quantitative analysis of urinary C-peptide by liquid chromatography tandem mass spectrometry. Rapid Commun Mass Spectrom 2000; 14: [30] Mqller C, Sch7fer P, Stfrtzel M, Vogt S, Weinmann W. Ion suppression effects in liquid chromatography electrospray ionisation transport-region collision induced dissociation mass spectrometry with different serum extraction methods for systematic toxicological analysis with mass spectral libraries. J Chromatogr, A 2002;773: [31] Mallet CR, Lu Z, Mazzeo JR. A study of ion suppression effects in electrospray ionization from mobile phase additives and solid-phase extracts. Rapid Commun Mass Spectrom 2004;18: [32] Dams R, Huestis MA, Lambert WE, Murphy CM. Matrix effect in bio-analysis of illicit drugs with LC MS/MS: influence of ionization type, sample preparation, and biofluid. J Am Soc Mass Spectrom 2003;14: [33] Taylor PJ, Johnson AG. Quantitative analysis of sirolimus (Rapamycin) in blood by high performance liquid chromatography electrospray tandem mass spectrometry. J Chromatogr, B 1998; 718: [34] Avery M. Quantitative characterization of differential ion suppression on liquid chromatography/atmospheric pressure ionisation mass spectrometric bioanalytical methods. Rapid Commun Mass Spectrom 2003;17: [35] Pascoe R, Foley JP, Gusev AI. Reduction in matrix-related signal suppression effects in electrospray ionization mass spectrometry using on-line two-dimensional liquid chromatography. Anal Chem 2001;73: [36] Schuhmacher J, Zimmer D, Tesche F, Pickard V. Matrix effects during analysis of plasma samples by electrospray and atmospheric pressure chemical ionization mass spectrometry: practical approaches to their elimination. Rapid Commun Mass Spectrom 2003;17: [37] Mei H, Hsieh Y, Nardo C, et al. Investigation of matrix effects in bioanalytical high-performance liquid chromatography/tandem mass spectrometric assays: application to drug discovery. Rapid Commun Mass Spectrom 2003;17: [38] Anon. Guidance for industry. Bioanalytical method validation. www. fda.gov/cder/guidance/guidance.htm. Accessed May 28, [39] Shah VP, Midha KK, Findlay JW, et al. Bioanalytical method validation A revisit with a decade of progress. Pharm Res 2000; 17: [40] Taylor PJ, Lynch SV, Pillans PI. A high-throughput HPLC mass spectrometry method suitable for the TDM of cyclosporin [abstract]. Ther Drug Monit 2003;25:506.