Strategies for the Assessment of Matrix Effect in

Quantitative Bioanalytical Methods Based on HPLC-

MS/MS
In recent years, high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS)
detection has been demonstrated to be a powerful technique for the quantitative determination of drugs and
metabolites in biological fluids. However, the common and early perception that utilization of HPLC-MS/MS
practically guarantees selectivity is being challenged by a number of reported examples of lack of selectivity
due to ion suppression or enhancement caused by the sample matrix and interferences from metabolites. In
light of these serious method liabilities, questions about how to develop and validate reliable HPLC-MS/MS
methods, especially for supporting long-term human pharmacokinetic studies, are being raised. The central
issue is what experiments, in addition to the validation data usually provided for the conventional bioanalytical
methods, need to be conducted to confirm HPLC-MS/MS assay selectivity and reliability. The current
regulatory requirements include the need for the assessment and elimination of the matrix effect in the
bioanalytical methods, but the experimental procedures necessary to assess the matrix effect are not detailed.
Practical, experimental approaches for studying, identifying, and eliminating the effect of matrix on the results
of quantitative analyses by HPLC-MS/MS are described in this paper. Using as an example a set of validation
experiments performed for one of our investigational new drug candidates, the concepts of the quantitative
assessment of the “absolute” versus “relative” matrix effect are introduced. In addition, experiments for the
determination of, the “true” recovery of analytes using HPLC-MS/ MS are described eliminating the
uncertainty about the effect of matrix on the determination of this commonly measured method parameter.
Determination of the matrix effect allows the assessment of the reliability and selectivity of an existing HPLC-
MS/MS method. If the results of these studies are not satisfactory, the parameters determined may provide a
guide to what changes in the method need to be made to improve assay selectivity. In addition, a direct
comparison of the extent of the matrix effect using two different interfaces (a heated nebulizer, HN, and ion
spray, ISP) under otherwise the same sample preparation and chromatographic conditions was made. It was
demonstrated that, for the investigational drug under study, the matrix effect was clearly observed when ISP
interface was utilized but it was absent when the HN interface was employed.
High-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection is now considered
the method of choice for the quantitative determination of drugs and metabolites in biological fluids. The methodology
achieved its preferred status because it has been perceived that MS/MS detection was highly selective and thus effectively
eliminated interference by endogenous impurities. Even without any cleanup or extraction of samples and with very little or
no chromatographic separation, endogenous impurities from biofluids were not detected, and the only MS/MS signal
observed in control biofluids originated from the desired analyte. Therefore, a common perception was that utilization of
HPLC-MS/MS practically guaranteed method selectivity and both sample extraction and chromatography could be
simplified or even eliminated. Chromatographic run times of 0-3 min using short (e2 cm) HPLC columns were commonly
utilized, allowing high-throughput (20-40 samples/ h) determination of analytes in complex biological matrixes. Contrary to
this common belief, the reliability of quantitative assays using HPLC-MS/MS and the integrity of resulting pharmacokinetic
(PK) data may not be absolute. Results may be adversely affected by lack of selectivity due to ion suppressions caused by
the sample matrix, interferences from metabolites, and “cross-talk” effects. Remarkable examples of the importance of
eliminating matrix effect and ion suppression during the development of quantitative methods based on HPLC-MS/MS for
two compounds studied in our laboratories were reported by us earlier.1,2 Coeluting, undetected matrix components may
reduce or enhance the ion intensity of the analytes and affect the reproducibility and accuracy of the assay. The degree of ion
suppression for an analyte and an internal standard may be different in different lots of the same biofluid (for example, urine
or plasma), originating from different subjects and over a prolonged period of time required, for example, for completion of
multiple dose clinical study, adversely affecting the reliability of determination and the integrity of PK data. Some additional
examples are available in the literature illustrating the need for careful assessment of HPLC-MS/MS assay selectivity
including evaluation of selectivity in postdose biological fluids in the presence of metabolites and the need for an efficient
extraction of analytes from biological materials and chromatographic separation.2 The matrix effect phenomenon was
originally described by Kebarle and Tang,8 who showed that electrospray responses of organic bases decreased with an
increase in concentrations of other organic bases. However, in the context of quantitative bioanalysis of drugs and
metabolites, present in the same type of matrix (i.e., human urine or plasma) but originating from different sources
(subjects), the matrix effect issue was not sufficiently studied and addressed. In addition, the recently issued U.S. Food and
Drug Administration’s (FDA) Guidance for Industry on Bioanalytical Method Validation9 and a Conference Report from the
workshop held on the same subject in Arlington, VA, in January 2000, 10 clearly indicate the need for the assessment of
matrix effect during development and validation of HPLC-MS/MS methods “to ensure that precision, selectivity, and
sensitivity will not be compromised”.9,10 However, in both of these documents, the experiments necessary to demonstrate the
presence or absence of matrix effect in a given bioanalytical method are not described or suggested. Qualitatively,
experiments confirming the presence of matrix effect in biological matrixes in comparison with the MS/MS response in neat
solvents or HPLC mobile phases were proposed,11-14 but they do not provide a guidance of how to evaluate and determine
whether an existing analytical method or a method being developed is selective or suffers from the lack of selectivity due to
the effect of matrix. Therefore, a need exists to develop an experimental protocol to demonstrate during assay development
and validation the absence or presence of matrix effect in a newly developed bioanalytical method and use this information
as guidance for making changes and corrections, if any, to the original method that would allow the establishment of a truly
selective method free of matrix effect interferences. Experimental strategies that allow this type of method evaluation are
described in this paper. These strategies will be illustrated using as an example the experimental data obtained during
development of bioanalytical methods for a selected drug candidate studied recently in our laboratories. The mechanism and
the origin of the matrix effect is not fully understood,8,12 but it may originate from the competition between an analyte and
the coeluting, undetected matrix components reacting with primary ions formed in the HPLC-MS/MS interface. Depending
on the environment in which the ionization and ion evaporation processes take place, this competition may effectively
decrease (ion suppression) or increase (ion enhancement) the efficiency of formation of the desired analyte ions present at
the same concentrations in the interface. To determine an analyte by HPLC-MS/MS, the uncharged molecules of this analyte
need to be transformed to ions that are later analyzed by MS/MS according to their mass-to-charge (m/z) ratios. The HPLC-
MS/MS interface can be considered as a “chemical reactor” in which primary ions react with analyte molecules in a very
complex series of charge-transfer and ion-transfer reactions. The rate and efficiency of these reactions are highly dependent
on the relative ionization energies, proton affinities, or both of the molecules present in the “reactor” at any given time. It is
intuitively clear that the efficiency of formation of the desired ions must be very much matrix-dependent due to the
competition between the molecule of interest and a number of other undetected but coeluting molecules present in the system
that are capable of reacting with primary ions. This effect may reduce or increase the intensity of analyte ions and affect the
reproducibility and accuracy of the assay. Unfortunately, most of the HPLC-MS/MS methods published in the literature do
not address the matrix effect issue although eliminating this effect is critical in establishing reliable methods. Ignoring this
effect may adversely affect the reliability of determination of analyte concentrations and the integrity of PK data generated.
Our earlier report and observations of others indicated that the extent of matrix effect may be dependent on the HPLC-MS
interface employed in a given method (atmospheric pressure chemical ionization, APCI, vs electrospray ionization, ESI).
The ionization mechanism is different when these different interfaces are used, which may affect the efficiency of formation
of the desired ions in the presence of the same coeluting compounds. To address this issue, a detailed comparison of the
matrix effect under otherwise the same sample extraction and HPLC conditions was made using a heated nebulizer (HN)
versus ion spray (ISP) interface that are utilized in the Sciex HPLC-MS/MS systems commonly used for quantitative
bioanalysis. EXPERIMENTAL SECTION Materials. Compound 1 and the internal standard (IS, 2, Figure 1) were
synthesized at Merck Research Laboratories (Rahway, NJ). All solvents and reagents were of HPLC or analytical grade and
were purchased from Fisher Scientific (Fair Lawn, NJ). The different lots of drug-free human heparinized plasma originated
from Biological Specialties Corp. (Lansdale, PA). Nitrogen (99.999%) was purchased from West Point Supply (West Point,
PA). Instrumentation. A Perkin-Elmer (PE) Sciex (Thornhill, ON, Canada) API 3000 tandem mass spectrometer equipped
with a heated nebulizer or an ion spray interface, a PE 200 autoinjector, and a PE 200 quaternary pump were used for all
HPLC-MS/MS analyses. The data were processed using MacQuan software (PE Sciex) on a MacIntosh Quadra 900
microcomputer. Standard Solutions. A stock solution of 100 íg/mL for standards 1 and 2 were prepared in the mobile
phase. A 10 íg/ mL stock solution containing 1 was then prepared by serial dilution. This solution was then diluted further
with the mobile phase to give a series of working standards of 0.005-5.0 íg/mL. The 100 íg/mL stock solution of the internal
standard 2 was serially diluted with the mobile phase to yield a working standard of 0.3 íg/mL. Chromatographic
Conditions. Chromatographic separation of 1 and 2 was performed on a Keystone Scientifics Hypersil BDS C-18 (50 _ 4.6
mm 3 ím, Keystone Scientific, Bellefonte, PA) analytical column with a mobile phase consisting of 80% acetonitrile and
20% water containing 0.1% formic acid, pumped at a flow rate of 1 mL/min. The total run time was 6 min. Both analytes
were baseline separated. The retention times of 1 and 2 were about 2.4 and 1.2 min corresponding to capacity factors (k¢) of
3.8 and 1.4, respectively. When the HN interface was utilized, the total eluent from the column (1 mL/min) was directed to
the interface, whereas in the case of the ISP interface, the flow was split 95:5; the flow directed to the ISP interface was
equivalent to 50 íL/min. HPLC-MS/MS Conditions. A PE Sciex triple quadrupole mass spectrometer (Sciex API 3000) was
interfaced via a Sciex HN or ISP probe with the HPLC system. The HN probe was maintained at 500 °C, and gas-phase
chemical ionization was effected by a corona discharge needle (+4 íA) using positive ion APCI. The nebulizing gas (N2)
pressure was set for the HN and ISP interfaces at 80 and 40 psi, respectively. The auxiliary flow was 2.0 (HN) and 0.0 L/min
(ISP), the curtain gas flow (N2) was 0.9 L/min, and the sampling orifice potential was set at +50 V, for both HN and ISP
interfaces. The dwell time was 400 ms, and mass analyzers Q1 and Q3 were operated at unit mass resolution. The mass
spectrometer was programmed to admit the protonated molecules [M + H]+ at m/z 394 for 1 and m/z 358 for 2 via the first
quadrupole filter (Q1). Collision-induced fragmentation at Q2 (collision gas N2, 275 _ 1013 atoms cm-2) yielded the product
ions at Q3 of m/z 326 and 290 for 1 and 2, respectively. Peak area ratios (1/2) obtained from selective reaction monitoring of
the analytes (m/z 394 f 326)/(m/z 358 f 290) were utilized for the construction of calibration lines, using weighted (1/x2)
linear least squares regression of the plasma concentrations and measured peak area ratios. Data collection, peak integration,
and calculations were performed using MacQuan PE-Sciex software. Sample Preparation. Three sets of five standard lines
were prepared to evaluate the assay accuracy, precision, recovery, and absence or presence of matrix effect. The first set of
five standard lines (set 1) was prepared to evaluate the MS/MS response for neat standards of two analytes (1 and 2) injected
in the mobile phase. The second set (set 2) was prepared in plasma extracts originating from five different sources and
spiked after extraction. The third set (set 3) was prepared in plasma from the same five different sources as in set 2, but the
plasma samples were spiked before extraction. By comparing the absolute areas of peaks 1, 2, peak areas ratios, and slopes
of the standard lines between these three different sets of standard lines, the absence or presence of matrix effect on the
quantification of 1 and 2 was assessed. In addition, precision and accuracy of the method and recovery of analytes were also
determined. Set 1. Five standard lines were constructed using neat solutions of 1 and 2 in the mobile phase. The samples
were prepared by placing 100 íL of the appropriate standards of 1, 100 íL of the 0.3 íg/mL stock solution of 2, and 100 íL of
the mobile phase (total volume 300 íL) into 15-mL centrifuge tubes. After mixing, the solutions were transferred into
autosampler vials and 50 íL was injected directly into the HPLC-MS/MS system. Set 2. Five standard lines were constructed
in five different lots of plasma by placing 1 mL of plasma in 15-mL centrifuge tubes followed by the addition of 200 íL of
the mobile phase (to simulate the addition of standard solutions of 1 and 2 that were each added in set 3 in 100 íL of the
mobile phase). After vortexing, the plasma was basified with pH 9.8 carbonate buffer (1 mL) and extracted with 7 mL of
methyl tert-butyl ether. The tubes were capped with Teflon-lined caps, rotate-mixed for 15 min, centrifuged at 3000 rpm
(3056g) for 5 min, and placed in a dry ice-acetone mixture. The organic layer was separated, placed in clean 15-mL
centrifuge tubes, and evaporated to dryness under a stream of nitrogen in a 50 °C water bath, and the residue was
reconstituted in 100 íL of the mobile phase, 100 íL of the 0.3 íg/mL stock solution of 2, and 100 íL of the appropriate
standards of 1 (total volume 300 íL). The extracts from the control samples (blanks) were reconstituted in 300 íL of the
mobile phase. A total of 50 íL of the extracts was injected into the HPLC-MS/MS system. In set 2, the analytes were spiked
after extraction into different plasma extracts, whereas in set 3 (below), the analytes were spiked into different plasmas
before extraction. Set 3. Five standard lines were constructed in five different lots of plasma (same plasmas as in set 2) by
placing 1 mL of plasma in 15-mL centrifuge tubes to which 100 íL of the appropriate standards of 1 and 100 íL of the 0.3
íg/mL stock solution of 2, both in the mobile phase, were added before extraction. The control (blank) tubes had 1 mL of
plasma to which 200 íL of the mobile phase was added. After vortexing, the plasma was basified and analytes were extracted
in the same manner as in set 2. The residues were reconstituted in 300 íL of the mobile phase, and 50 íL was injected into the
HPLC-MS/MS system. Samples from sets 1-3 were analyzed using ISP interface first, and as soon as all samples were
analyzed, the same samples were injected into the same HPLC-MS/MS system equipped with the HN interface. The order of
injection was as follows: samples from set 1 were injected first (five standard lines, each line from a low to a high
concentration), followed by sets 2 and 3 injected in the same order (low to high concentrations) for plasma lot 1, followed by
lot 2, etc. Precision, Accuracy, and Recovery. The precision of the method was determined by the replicate analyses (n ) 5,
set 3) of human plasma containing 1 at all concentrations utilized for the construction of calibration curves. The linearity of
each standard curve was confirmed by plotting the peak area ratio of 1 to 2 versus drug concentration. The sample
concentrations were calculated from the equation y ) mx + b, as determined by weighted (1/x2) linear regression of the
standard line. The accuracy of the method was expressed by [(mean observed concentration)/(spiked concentration] _ 100.
The recovery was determined by comparing the mean peak areas of 1 and 2 obtained in set 3 to those in set 2. Assessment of
Matrix Effect. The assessment of matrix effect and assay reliability is critical when homologues rather than stable isotope-
labeled analytes are utilized as internal standards. By comparing the peak areas of the analyte standards, standards spiked
before and after extraction into different lots of plasma, and the peak area ratios of analytes to an IS, the recovery and ion
suppression or enhancement associated with a given lot of plasma were assessed. Assessment of Assay Selectivity. The
assay selectivity was assessed by analyzing extracts from five lots of plasma from different sources. Endogenous peaks at the
retention time of the analytes of interest were not observed in any of the plasma lots evaluated. In addition, the “cross-talk”
between MS/MS channels used for monitoring 1 and 2 for both analytes was assessed by the following: (1) separately
injecting 1 at the highest concentration on the standard line (200 ng/mL) and monitoring the response in the IS channel and
(2) by injecting a plasma sample spiked only with the IS (2) and monitoring the response in the drug channel at the
sensitivity (y-axis) required for monitoring 1 at the lowest limit of quantification. No “cross-talk” was observed. RESULTS
Evaluation of the Matrix Effect and Assay Validation Using HN Interface. The matrix effect and the possibility of
ionization suppression or enhancement for 1 and 2 was evaluated by comparing the results of analysis of three sets of
samples (set 1, set 2, set 3) prepared as described in the Experimental Section. These three sets corresponded to three types
of system evaluation. In the first set (set 1), standards of the analytes present in the neat reconstitution solvent (HPLC mobile
phase used in the assay) were analyzed directly at seven concentrations and analyses were repeated five times at each
concentration (35 samples). The results of analyses of set 1 provided a good insight into the overall HPLCMS/ MS system
reproducibility in measuring the absolute peak areas on consecutive injections, the performance of the detector, and the
chromatographic system as a whole. In the second set (set 2), plasma samples from five different plasma lots were first
extracted and spiked after extraction with the analytes 1 and 2 in the same solvent (mobile phase) as in set 1. Any additional
variability of the peak areas for the analytes than those observed in set 1, as demonstrated by an increase in the coefficients
of variation (CV) at each concentration, would be indicative of an effect of sample matrix since analytes at the same
concentrations were spiked into plasma extracts. In set 3, analytes were spiked before extraction into plasma samples
originating from five different sources as in set 2. The variability in CV values here would reflect a combined effect of a
sample matrix and potential differences in recovery of analytes from different plasma lots. In all three cases (sets 1-3), five
standard lines were constructed (total of 3 _ 35 ) 105 samples). In a typical, conventional method validation, only set 3
samples (35 samples) with analytes spiked before extraction into a single lot of a biological fluid are usually analyzed. The
results of the analyses for sets 1-3 are summarized in Tables 1 and 2. The results obtained in this manner allow determination
of the matrix effect (ME), recovery (RE) of the extraction procedure, and overall “process efficiency” (PE) by comparing the
absolute peak areas for 1 obtained in sets 1-3 (Table 2). If one depicts the peak areas obtained in neat solution standards in
set 1 as A, the corresponding peak areas for standards spiked after extraction into plasma extracts as B (set 2), and peak areas
for standards spiked before extraction as C (set 3), the ME, RE, and PE values can be calculated as follows: The terms
“process efficiency”, “extraction efficiency”, and “ion suppression” were originally introduced by Buhrman et. al. 7 In their
study, ion suppression was defined as (100 - B/A _ 100), and the potential for ion enhancement was not considered. To
account for both ion suppression and ion enhancement and to avoid negative values in the case of ion enhancement, the ratio
(B/A _ 100) is defined here generally as a matrix effect. The ME calculated in this manner may be referred to as an
“absolute” matrix effect since the signal response of the standard present in the plasma extract is compared to the response of
a standard made directly in a neat mobile phase. Although the presence of this absolute matrix effect may be of some
concern (vide infra), the more important parameter in the evaluation and validation of a bioanalytical method in biofluids is
the demonstration of the absence of a “relative” matrix effect, the word relative referring to the comparison of ME values (eq
1) between different lots (sources) of biofluids. This aspect of the matrix effect assessment that is highly relevant for the
development of selective HPLCMS/ MS methods and the detailed comparison of the recovery versus process efficiency will
be discussed in more detail in the Discussion section. In Table 2, the ME, RE, and PE values for 1 and 2 are presented. The
mean values of slopes of standard lines in five different plasma lots for standards spiked before and after plasma extraction
are presented in Table 3, together with the analogous mean slope value of five standard lines for standards injected directly
in the mobile phase. A comparison of the absolute peak areas of 1 and 2 spiked post extraction (set 2) into different plasma
lots for both HN and ISP (see below) is illustrated in Figure 2. Evaluation of the Matrix Effect and Assay Validation
Using ISP Interface. To compare the performance of the ISP interface with the HN interface under otherwise the same
extraction and chromatographic conditions, similar data, as presented in Tables 1 and 2 were obtained during analyses of 1
and 2 using the ISP interface. The same extracts as those utilized during evaluation of the HN interface were injected. The
results of the analyses are presented in Tables 4 and 5 and in Figure 2. The comparison between the slopes of the standard
lines obtained in five different lots of plasma (set 3) using the ISP versus HN interface is illustrated in Figure 3. In addition,
to demonstrate the difference in validation results between the commonly performed assay validations in a single (n ) 5)
source of plasma versus validation performed in five different plasma lots, the results of the precision and accuracy
determinations using the ISP interface in a single plasma lot are also presented in Table 4 (set 3a). These results may be
directly compared with similar data presented in Table 4 (set 3) where, under otherwise the same conditions, validation was
attempted in five different plasma lots. The precision of the assay using the ISP interface measured in a single plasma lot
versus in five different lots is illustrated in Figure 4. DISCUSSION The evaluation of the matrix effect on the results of
quantitative determination of drugs and metabolites in biological fluids is an important and often overlooked element of
assay validation and is explicitly required not only by the current validation standards but, more importantly, is critical to
generate reliable PK data. It is a common practice that assay validation experiments and daily or run-to-run standard curves
are prepared in a single lot of a biofluid and the response of analytes in this particular fluid (lot) is then used to calculate an
analyte response in biofluid samples originating from a large number of different subjects, from multidays and multiweeks
multiple dose, food effect, drug interaction studies, etc. Seemingly the same biofluid (for example, plasma) from these
studies may contain different endogenous compounds that were not present in the plasma lot used during assay validation or
in plasma used for constructing a daily standard line. If unseen, undetected, endogenous compounds present in these
different plasma samples coelute with analytes of interest, they may affect the efficiency of ionization of analytes leading to
the decrease or an increase in the MS response. Therefore, elimination of the matrix effect is critical to generate reliable
bioanalytical and PK data. It is clearly impossible to generate standard lines for an analyte in exactly the same postdose
plasma samples containing the same endogenous compounds and metabolites but without the presence of an analyte of
interest. However, some initial effort during assay validation may increase considerably the probability of the method to be
much more reliable by eliminating a matrix effect when at least control biofluids (plasma, for example) from different
sources or subjects are evaluated. In the experiments presented in this paper, assay validations were performed in five
different lots of plasma instead in a single lot. In the course of utilization of a method for longterm bioanalytical support,
hundreds or even thousands of different subjects may participate in these studies and the molecular content of their plasmas,
urines, or both (for example) may be widely different. By eliminating matrix effects in at least plasmas or urines originating
from five different sources, the likelihood of providing more accurate bioanalytical and PK data may dramatically increase.
Problems with matrix effect may not be limited to bioanalytical methods based on HPLC-MS. They may be of concern also
in all other more conventional bioanalytical methods based, for example, on HPLC/FLU, HPLC/UV, or HPLC/EC detection.
However, contrary to the conventional methods, HPLC-MS/MS assays are commonly developed in a relatively short time
period (days or weeks), and due to apparent selectivity of the MS/MS detection, extraction procedures are very simplified (or
not utilized at all), and chromatographic conditions with very little retention and separation of analytes from endogenous
compounds are employed. Therefore, the matrix effect issue became more apparent and of more concern in the HPLC-
MS/MS methods in comparison with the seemingly less selective conventional methods. The overall precision and accuracy
of any bioanalytical method, as determined usually using experimental data obtained in set 3, are dependent on many factors
including the overall performance of the chromatographic system, reproducibility of the detector response, reproducibility of
sample preparation procedures, consistency of recovery of analytes from different sources of a biofluid, and, finally, absence
of a matrix effect on the quantification. When typical validation is performed in a single lot of a biofluid and satisfactory
precision and accuracy data are obtained in set 3, the performance of all individual system components and the overall
performance of the system are confirmed. However, the issues of potential recovery differences in different biofluid lots
originating from different subjects and the potential of matrix effect on analyte quantification are not addressed. To study
these effects and quantitatively assess their individual importance to the overall method precision and accuracy, analyses of
sets 1 and 2, in addition to set 3, should be considered. From data obtained in set 1, the overall chromatographic system and
detector performance can be assessed. From data in set 2, both absolute and relative matrix effect can be ascertained, and
finally, from set 3, the overall effect of matrix and recoveries on method performance may be assessed when a biofluid
(plasma) from different sources, instead from a single source, is used in sets 2 and 3 for accuracy and precision
determination. The experiments described in this paper and their results provide detailed guidance of how the evaluation of
the matrix effect may be performed and the validity of a bioanalytical method confirmed. Other, simplified approaches,
described and discussed in the second part of the Discussion section, may also be considered. However, to introduce the
overall concept of matrix effect evaluation, a detailed discussion of the results of the analysis of samples, prepared and
analyzed in sets 1-3 (above), using two different HPLC-MS/MS interfaces, is made first. Matrix Effect. The matrix effect
during validation of analytical methods in biological fluids may be best examined by comparing the MS/MS response (peak
areas or peak heights) of an analyte at any given concentration spiked postextraction into a biological fluid extract (B, eq 1),
to the MS/MS response (A, eq 1) of the same analyte present in the “neat” mobile phase. Also, the B values at any given
concentration may be compared between different plasma lots. In the former case, the value (B/A _ 100), obtained according
to eq 1, may be considered as an absolute matrix effect, indicating a comparison is made here between the MS/MS response
of an analyte present in the mobile phase (or other solvent) containing a plasma extract to the response from the same analyte
present in the “neat” mobile phase or a solvent but not “contaminated” with compounds extracted from a biofluid. In the case
of a direct comparison of B values obtained in different sources of a biofluid, a relative matrix effect between different lots
may be ascertained. Large differences between B values indicate that the MS/MS response originating from the same amount
of an analyte is different in different lots of a biofluid, and unless an internal standard exhibit the same relative matrix effect
profile, the method should not be considered as valid. The mean absolute matrix effect, calculated according to eq 1, was 135
and 127%, for 1 and 2, respectively, when the HN interface was utilized (Table 2, columns G and H), and 104 and 92%, for
1 and 2, respectively, when the ISP interface was employed (Table 5, columns G and H). A value of 100% indicates that the
response in the mobile phase and in the plasma extracts were the same and no absolute matrix effect was observed. A value
of >100% indicates an ionization enhancement and a value of <100% indicates an ionization suppression. It is interesting to
note that, when the HN interface was employed, an ionization enhancement for both 1 and 2 (135 and 127%, respectively)
was observed, whereas in the case of the ISP interface, a very small ionization enhancement for 1 (104%) and an ionization
suppression for 2 (92%) were observed under the same chromatographic and extraction conditions. This confirms that the
mechanism of ionization for 1 and 2 may be different between HN (APCI) and ISP (ESI) interfaces, and the efficiency of
formation of analyte ions in the presence of the same coeluting undetected compounds extracted from a biofluid may be
different. The assessment of the presence of a relative matrix effect can be made based on direct comparison of the MS/MS
responses (peak areas) of an analyte spiked into extracts originating from different lots (sources) of a biofluid (B, eq 1). The
variability in these responses, expressed as CVs (%), may be considered as a measure of the relative matrix effect for a given
analyte. The precision of the determination of B at different concentrations varied from 5.0 to 7.9% and 2.3 to 5.2% (Table 1,
columns B and E) for 1 and 2, respectively, when a HN interface was utilized. This variability seemed to be comparable or
only slightly higher to the precision of determination of standards injected directly in the mobile phase (2.0-5.0% and 3.0-
5.3% for 1 and 2, respectively, Table 1, columns A and D). These data confirm that the relative matrix effect for 1 and 2 was
practically absent when the HN interface was utilized (Figure 2). On the other hand, the analogous B values for the ISP
interface were higher and varied from 11.6 to 23.8% for 1 and 4.6 to 11.3% for 2 (Table 4, columns B and F), as compared
to the similar values for the directly injected standards (5.8-12.3% and 6.1-13.5% for 1 and 2, respectively, Table 4, columns
A and E). A careful examination of the absolute peak areas for 1 in set 2 clearly indicated that the peak area of 1 in one
plasma lot (lot 4) at all concentrations studied was consistently much higher (Figure 2) than in all other plasma lots, leading
to the high CV values for 1. On the other hand, the peak areas for 2 were much more consistent between different plasma
lots (Figure 2). These data clearly indicated that the relative matrix effect may be different for different analytes (a drug and
an internal standard), affecting greatly the precision of the determination of the ratio 1/2 (Table 4, column J). In addition, the
relative matrix effect may be observed for the same set of samples when one interface is utilized (ISP) and be absent when
the same samples are analyzed using a different interface (HN). The absence of any significant relative matrix effect for 1
and 2 when HN interface was employed is also illustrated in Figure 2. The presence of an absolute or even a relative matrix
effect for a given analyte (for example, a drug) does not necessarily indicate that the bioanalytical method may not be valid.
Assuming the relative matrix effect exhibits the same pattern for the drug and the internal standard in all lots studied, the
drug-to-internal standard ratio (1/2), a measure of the drug concentration, should not be affected. As illustrated in Table 1
(column H), the CV of the ratio of 1/2 for samples spiked postextraction into extracts from five different lots of plasma was
small and varied from 2.7 to 4.7% at different concentrations. This variability was only slightly higher than the CV of the
similar ratio for standards injected directly (n ) 5) in the mobile phase (0.7-2.7%, Table 1, column G), confirming that the
absolute and relative matrix effects for 1 and 2 have practically no effect on quantification of 1 spiked into five different lots
of plasma when the HN interface was used. On the other hand, in the case of the ISP interface, the CV of the ratio of 1/2 for
standards injected directly (n ) 5) in the mobile phase was much lower (2.1-5.4%, Table 4, column I) than the analogous CVs
for samples spiked postextraction into extracts from five different lots of plasma (10.1-19.7%, Table 4, column J), indicating
that the method employing the ISP interface may suffer from a significant overall matrix effect and may not be suitable for
quantification of analytes from different plasma sources. When actual validation of a bioanalytical method is performed, the
samples are spiked before extraction into biological fluids, the MS/MS responses C (eq 3) for the drug and the internal
standard are measured, and the precision and accuracy of the method are determined. When this is done in a single source of
a biofluid (n ) 5), extraction recovery is the same and the potential variability in matrix effect and in recovery of analytes, as
it may be the case when plasma from different lots are utilized, does not effect the precision and accuracy of the method.
When a similar validation is performed in five different lots of a biofluid, the variability in recoveries of both the drug and
the internal standard may contribute, in addition to the matrix effect, to the overall method precision and accuracy. This
variable recovery contributions may be assessed by comparing the overall CVs of the 1/2 ratios for samples spiked before
extraction (for example, for the HN CV values listed in column I, Table 1) with the analogous CVs for samples spiked after
extraction (in this case, CV values listed in Table 1, column H). If the range of these two sets of values is similar, the
variability in the recovery on the overall method precision may be considered negligible. If the overall precision of the
method for samples spiked after extraction is better than a similar set of CVs for sample spiked before extraction, the poorer
precision may be attributed to the variability in recoveries between different plasma lots in addition to the matrix effect.
However, if the overall precision of the method for samples spiked before extraction is actually better than for samples
spiked after extraction, the difference in recovery between different plasma lots may have had some compensating effect,
and the large variability in matrix effect between plasmas may have been minimized by the differences in recoveries. The
inspection of data in Table 1 (columns H and I) indicates that the range of CV values was 2.7- 4.7% and 1.3-2.9% for
samples spiked after and before extraction, respectively, when the HN interface was utilized, indicating that any variability
in recoveries between different plasma lots for both 1 and 2 had a minimal impact on the overall method precision. This
absence of a significant contribution of recoveries to the overall precision of the methods was confirmed by inspecting
similar data for the ISP interface (Table 4, columns J and K). Although imprecision of the ISP methodology was high due to
a significant matrix effect, the range of CV values for samples spiked after and before extraction were comparable (10.1-
19.7% and 11.1-27.8%, respectively), confirming the relatively small, if any, effect of recoveries on the overall precision of
the method. Recovery vs Process Efficiency. It is a common practice to determine the recovery of a compound extracted
from a biofluid by comparing the response (for example, peak areas) of a compound spiked into a biofluid, extracted,
reconstituted in a solvent (for example, HPLC mobile phase), and injected with the corresponding peak areas of the same
compound injected directly in the same solvent (mobile phase). Using the terminology presented in eqs 1-3, this practice
would be equivalent to the recovery being determined as a value of (C/A _ 100) depicted in eq 3. However, the recovery
calculated from eq 3 may not be correct since it does not take into account the matrix effect that may greatly influence this
ratio. Therefore, the recovery (RE) should be determined as a ratio of (C/B _ 100) (eq 2), a “true” recovery value that is not
affected by the matrix. The value (C/A _ 100) (eq 3) may be instead considered as the overall process efficiency, and it can
be calculated by multiplying the value of the absolute matrix effect by the recovery (eq 3). It is quite common in the
literature to see recovery values reported as being >100%, a clear indication that matrix effect was not taken into account in
the calculations, and that the ratio C/A _ 100 (eq 3) rather than C/B _ 100 (eq 2) was utilized. In the case of 1 and 2, due to
the observed ionization enhancement for both 1 and 2 (135 and 127%, respectively, Table 2, columns G and H), the (C/A _
100) values were >100% (130 and 118% for 1 and 2, respectively, Table 2, columns K and L). The “true” recovery values,
free from matrix effect contributions, and calculated using the ratio (C/B _ 100) (eq 2), were 97 and 93% for 1 and 2,
respectively (Table 2, columns I and J) when the HN interface was utilized. The recovery values calculated according to eq 2
should be independent of the HPLC-MS/MS interface employed. However, they calculated lower when the ISP interface was
utilized (77 and 83% for 1 and 2, respectively, Table 5, columns I and J). The origin for this apparent discrepancy is, at
present, unknown. However, since absolute peak areas were used for calculations, any instrument or interface instability
during analyses of samples in set 2 (that were analyzed first) and set 3 (analyzed at the end of the run) may have led to the
decrease of the absolute peak areas of C in comparison with B lowering the RE values. Such a relative decrease in response
may be especially noticeable when the ISP interface is utilized. In this case, 95:5 splitting of the flow from the column to the
interface was employed, and any partial clogging of the splitter may have decreased the absolute response for both analytes
at the end of the run when samples from set 3 were analyzed. The matrix effect may also affect the reliability of
determination of recovery values in conventional bioanalytical methods. Based on the data presented here, the determination
of recoveries in all of these methods should be performed using eq 2 instead of eq 3. Method Validation and Matrix
Effect. Evaluation of the Assay Precision and Accuracy in Single versus Multiple Sources of a Biofluid. The importance and
the necessity of evaluating the matrix effect during method development and validation is best illustrated by the comparison
of the results of a typical validation experiments performed in a single lot of plasma versus the same validation experiment
performed in five different plasma lots. The precision and accuracy values obtained in a single plasma lot using the ISP
interface were highly satisfactory and ranged from 4.2 to 8.5%, and 96 to 108%, respectively, at all concentrations utilized
for constructing the standard line ((Table 4, columns L and N). In this case, even the precision values for the determination
of the absolute peak areas for both 1 and 2 were excellent and varied from 3.3 to 8.5% for 1 and 2.1-6.0% for 2 (data not
shown in the tables). Based on these results, the ISP method would have been considered as valid and adequate for
supporting PK studies. However, when the same validation was attempted in five different plasma lots, the precision values
were unacceptably high (11.1- 27.8%, Table 4, column K) under otherwise identical extraction and chromatographic
conditions as in the validation experiment utilizing a single plasma lot. The reason for this significant method imprecision
(Figure 4) was the presence of high relative matrix effect when the ISP interface was employed, as indicated by the high CV
values for peak areas of 1 and 2 in five different plasma lots (11.6-23.8% and 4.6-11.3%, respectively, Table 4, columns B
and F). In a single plasma lot, the analogous CV values were in the range of 3.3-8.5% for 1 and 2.1-6.0% for 2. Despite this
high relative matrix effect in five different lots of plasma for the drug, the method would have been validated if the relative
matrix effect for the IS had the same pattern as for the drug. In such a case, the 1/2 ratio would not have been affected.
However, this was not the case for 1 and 2, and the high CV values of the ratio of 1/2 (11.1-27.8%, Table 4, column K) had
its origin in a significant variability in the MS/MS response for the same concentrations of 1 in different plasma lots
accompanied by relatively the same responses for the IS (Figure 2). Contrary to the results obtained using the ISP interface,
the precision and accuracy of the method using the HN interface in five different lots of plasma was excellent, as illustrated
by the precision and accuracy values ranging from 1.3 to 2.9% and 97 to 103%, respectively (Table 1, columns I and J). The
CVs of the peak area ratios of 1/2 were smaller (Table 1, column I) than the CVs of the peak areas of 1 and 2 (Table 1,
columns C and F, respectively), confirming the desired compensating effect of the presence of internal standard on the
precision and reliability of quantification of 1. When such highly satisfactory data are obtained in five different lots of a
biofluid, there is no need to assess the overall system and assay performance in a single lot of a biofluid, and such
experiment was not repeated here. Evaluation of Slopes of Standard Lines. The lot-to-lot variability in the determination of
1/2 ratios, a measure of drug concentration, is best evaluated by comparing the slopes of standard lines constructed in
different plasma lots. The high variability (CV) of these slopes is indicative of the overall effect of the sample matrix on the
drug/IS ratio rather than on the individual responses of 1 and 2, when slopes obtained in set 2 (spiked plasma extracts) are
compared. Any variability in the analogous slopes obtained in set 3 in five different lots of plasma may reflect the combined
differences in the effect of matrix and a variable recovery in different plasma lots (Figure 3). In the case of the HN interface,
the variability in standard line slopes (3.5% for set 2 and 1.0% for set 3, Table 3, columns B and C) were very small and
comparable to the variability in similar five slopes constructed directly from standards (set 1, CV ) 1.3%, Table 3 column A).
The very small variability of slopes obtained in set 1 was a direct indicator of an excellent overall HPLC-MS/MS system
reproducibility and performance. Together, these results indicated that the matrix effect, if any, had no effect on the
determination of 1/2 ratios in different plasma lots. A small improvement in the CV values obtained in set 3 versus set 2 (1.0
vs 3.5%) may be indicative of a possible and favorable compensating effect of the small difference in recoveries for 1 and 2
in different plasma lots on the 1/2 ratios. Contrary to the HN interface, the analysis of similar slope data for sets 1-3 obtained
using the ISP interface (Table 3) clearly indicated that due to a significant matrix effect the variability in slopes between
different plasma lots was quite significant (for example, _44% increase in slope was observed between lots 3 and 4 in set 2;
data not shown). The CV values for sets 2 and 3 were high and comparable (14.9 and 13.2%, Table 3, columns B and C),
indicating that the overall high variability of the method was due to the matrix effect rather than any potential differences in
recoveries between different plasma lots for both 1 and 2. The variability of slopes obtained in set 1 (0.9%, Table 3, column
A) was negligible and was similar to analogous values obtained for set 1 when the HN interface was employed (1.3%, Table
3, column A), confirming that the overall ISP system reproducibility and performance was maintained. To additionally
confirm that inadequate assay precision and accuracy of the ISP method was due to the effect of matrix and not, for example,
due to the inadequate overall system performance, the experiments similar to those performed in set 3 but using a single
instead of five different lots of plasma were repeated and five slopes of standard lines in the same single plasma lot were
determined. The small CV value (2.4%) obtained for slopes in set 3a (Table 3, column D) clearly indicated that the overall
system performance was highly adequate and the variability in slope values (13.2%) observed when plasma from five
different sources was utilized (Table 3, column C) was due primarily to the effect of matrix. Simplified Approaches for
Assessing the Matrix Effect. The approach described in this paper allows a detailed quantitative assessment of the matrix
effect during method validation and a full examination of the validity of the bioanalytical method. However, some
simplified, alternative approaches for the assessment of matrix effect listed below may be considered. (1) Since the
knowledge of the absolute matrix effect is not necessary to establish the method validity, analyses of samples in set 1 may
not be required. In addition, the data obtained in set 3 are reflective of the combination of two effects: the effect of sample
matrix and recoveries of analytes. To determine the relative matrix effect only, samples in set 2 prepared in a biofluid
originating from five different sources need only to be analyzed. Careful examination of the peak areas (heights) of the drug
and an IS spiked into different biofluid extracts, the degree of variability (CV) of the absolute responses, and drug/IS ratios
are all indicative of the presence or absence of matrix effect. Although the knowledge of an absolute matrix effect is, in
principle, not necessary to establish method validity, it is important to have an idea about the extent of detector sensitivity on
the matrix in which analytes are injected since the possibility of a more pronounced relative matrix effect may increase with
the increase in the absolute matrix effect. If a large absolute matrix effect is observed, the likelihood for greater variability in
the detector response from a biofluid originating from large number of different subjects (instead of just five plasma lots
studied in a typical validation experiment) participating in long-term clinical studies may increase. The elimination of a
relative matrix effect in such cases may be especially important and may require much more attention than in cases where
the absolute matrix effect is relatively small or negligible. (2) Instead of analyses of a full set 2 samples (7 _ 5 ) 35 samples),
repeat analyses of standards spiked into a biofluid extract from five different sources but only at two or three concentrations
(2 _ 5 or 3 _ 5 ) 10 or 15 samples) may be performed and data analyzed as in case 1 above. (3) Both the absolute and relative
matrix effects can be determined by analyzing samples in sets 1 and 2 at two or three concentrations (2 _ 2 _ 5 ) 20 or 2 _ 3
_ 5 ) 30 samples) instead at all seven concentrations. Again, the presence of the absolute and even a relative matrix effect for
the individual analytes (drug and an IS) does not preclude the method to be valid. If the pattern of variability for the drug and
the IS is the same, the ratio of the drug/IS, a measure of drug concentration, may not be affected, and the CVs of the ratio
would be much smaller than the CVs of the individual responses for the drug and the IS. Such decrease in the CV values
would confirm the compensating effect of the presence of internal standard on the precision and reliability of quantification
of the drug. (4) To assess absolute and relative matrix effect and recoveries of analytes in an abbreviated fashion, samples in
sets 1-3 but only at two or three concentrations instead at all concentrations (seven) on the standard line need to be analyzed.
The total number of samples would be 2 _ 3 _ 5 ) 30 or 3 _ 3 _ 5 ) 45. Assuming the absence of the matrix effect is
demonstrated, the validation experiments (set 3, 35 samples) need to be later performed. (5) Slopes of five standard lines
obtained in set 3 may be compared (vide infra). If these slopes in five different sources of a biofluid are practically the same
(CV < 4-5%), the absence of any significant matrix effect on quantification may be considered as being confirmed.
Recommendations for Bioanalytical Method Validation. The current common practice in method validations involves the
analyses of samples in set 3 only and in a single source of a biofluid. As clearly demonstrated in this paper, the validation
data obtained in this manner (Table 4, column L and Figure 4) may be totally misleading since they do not take into account
the possibility of a severe matrix effect on quantification. When the same method was attempted to be validated in the same
biofluid (plasma) but originating from five different sources, unacceptable precision values were obtained (Table 4, column
K and Figure 4) and the method could not be considered as valid. The best way to perform method validation and to assess
the combined effect of sample matrix and variable recoveries on assay results for both the drug and an IS is to determine
precision and accuracy of the method (set 3) in biofluid samples originating from at least five different sources instead from a
single source. The slopes of the standard lines constructed in these different biofluid lots may then be compared. These
slopes should be practically the same, which would indicate that sample matrix and any potential differences in recoveries do
not affect the precision and accuracy of the method. The method may be considered valid and no further evaluation of the
matrix effect may be necessary when validation is performed in five different lots of a biofluid, and the precision and
accuracy values for set 3 and slopes of the standard lines are as good as those illustrated in Table 1 (column I) and Table 3
(column C) (HN interface). Similar data obtained in a single lot of a biofluid are not sufficient for the method to be
considered valid, and the absence of matrix effect needs to be demonstrated. A question may be asked why validation
experiments are recommended in five instead of in any other number of different biofluid lots. The only reason for this
recommendation is to take into account the practical aspects of method validation procedures that usually involve repeat
analyses of five samples at each concentration. Without increasing overall number of samples analyzed, the matrix effect in
five different lots of a biofluid may then be assessed simultaneously with the determination of the precision and accuracy of
the method. It would be highly desirable to assess matrix effect in as many different biofluid sources as possible, but this is
difficult experimentally and is highly impractical. General Comments and Other Possible Implications of Matrix Effect
on the Results of Quantitative Bioanalysis. 1. Matrix Effect and Drug Interaction Studies. A different form of matrix effect
may be encountered during drug interaction studies. In these studies, a three-way crossover experiment is usually performed
in which subjects are treated with drug A in the first part (1), with drug B, that may potentially interact with A, in the second
part (2) of the study and, finally, in the third part (3), with drugs A + B combined. The bioanalytical and PK data obtained in
these drug interaction studies are usually obtained based on the analyses of biofluid samples (for example, plasma) generated
in parts 1 and 3 from a number of subjects using a bioanalytical method developed for drug A in a control plasma. However,
matrix effect may also originate from the presence of B and its metabolites that are present only in samples from part 3 of the
study but not in samples from part 1. Therefore, compound A may be considered as being present in two different matrixes
in plasma samples from part 1 and part 3 of the study. To confirm the selectivity of the method for A in the presence of B
and its metabolites, one can consider pooling postdose plasma samples from part 2 of the study and constructing a standard
line for A in this pooled plasma containing B and its metabolites. When the slopes of the standard line constructed in a
control plasma and in pooled plasma from part 2 are practically the same, the matrix effect from B and its metabolites on the
quantification of A may be considered as negligible. This procedure is routinely utilized in our laboratories to assess matrix
effect in drug interaction studies when samples from part 2 of the study are available. 2. Matrix Effect and Assay of Multiple
Analytes. The matrix effect issues in quantitative HPLC-MS/MS may be especially complex when bioanalysis of multiple
analytes in the same analytical run are considered. The absence of matrix effect for all individual analytes may need to be
demonstrated. The development of methods free from matrix effect for multiple analytes may require some considerable
effort in designing proper chromatographic conditions, sample extraction procedure, proper choice of HPLCMS interface,
and internal standards. The decision about performing simultaneous assays for a number of analytes using HPLCMS/ MS
should not be made easily since the data generated may not be as reliable as it is commonly perceived. 3. Elimination of
Matrix Effect. Matrix effect may be eliminated or minimized by the following: (1) changing and improving sample
extraction procedure and by eliminating undetected matrix interferences, (2) performing the assay under more efficient
chromatographic conditions to separate analytes of interest from undetected endogenous compounds that may affect the
efficiency of ionization of analytes, and (3) evaluating and changing the HPLC-MS interface and the mechanism of
ionization of analytes. Whenever any change in the above parameters is made, the matrix effect should be reevaluated and its
absence should be confirmed before analysis of “real” samples is undertaken. 4. Matrix Effect and the Use of Stable Isotope-
Labeled Internal Standards. Use of stable isotope-labeled analogues as internal standard is highly recommended since matrix
effect should not affect the relative efficiency of ionization of the drug and its stable isotope-labeled IS. There are a number
of analytical issues connected with the use of labeled internal standards in bioanalysis. 15 They include the problems with
isotopic purity of compounds, “cross-contamination” or “cross-talk” between MS/MS channels used for monitoring the drug
and IS, isotopic integrity of the label in biological fluid and during sample processing, etc. These issues need to be carefully
addressed and require separate studies. CONCLUSIONS Careful assessment of matrix effect should constitute an integral
and important part of validation of any quantitative HPLCMS/ MS method utilized for supporting PK studies. The precision
and accuracy of the method should be assessed using biofluids from different sources (subjects), and a relative matrix effect
should be evaluated by analyzing biofluid extracts from different sources (lots) spiked with analytes after extraction. The
extent of matrix effect seems to be highly dependent on the mechanism of ionization in the HPLC-MS interface. Under
otherwise identical sample extraction and chromatographic conditions, the relative matrix effect for compounds studied in
this paper was not observed when the APCI (HN) interface was utilized but was very significant when the ESI (ISP)
interface was employed. The validity and integrity of quantitative data obtained using HPLC-MS/MS should be carefully
verified by demonstrating the absence of matrix effect, interference from metabolites in postdose samples, and absence of
“cross-talk” effect. The strategies described in this paper may provide guidance for establishing selective, quantitative
bioanalytical methods based on HPLC-MS/ MS.