Anal. Chem.
2003, 75, 3019-3030
Strategies for the Assessment of Matrix Effect in
Quantitative Bioanalytical Methods Based on
HPLC-MS/MS
B. K. Matuszewski,* M. L. Constanzer, and C. M. Chavez-Eng
Merck Research Laboratories, West Point, Pennsylvania 19486
In recent years, high-performance liquid chromatography
(HPLC) with tandem mass spectrometric (MS/MS) detec-
tion has been demonstrated to be a powerful technique
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for the quantitative determination of drugs and metabo-
lites 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
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reported examples of lack of selectivity due to ion sup-
pression 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, identify-
ing, 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 assess-
ment of the “absolute” versus “relative” matrix effect are
introduced. In addition, experiments for the determina-
tion 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 selectiv-
ity of an existing HPLC-MS/MS method. If the results of
these studies are not satisfactory, the parameters deter-
mined 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 dem-
onstrated that, for the investigational drug under study,
the matrix effect was clearly observed when ISP interface
10.1021/ac020361s CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/04/2003
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 de-
tected, and the only MS/MS signal observed in control biofluids
originated from the desired analyte. Therefore, a common percep-
tion was that utilization of HPLC-MS/MS practically guaranteed
method selectivity and both sample extraction and chromatogra-
phy 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
* Corresponding author. Fax: (215) 652-8548. E-mail: bogdan_matuszewski@
merck.com.
(1) Fu, I.; Woolf, E. J.; Matuszewski, B. K. J. Pharm. Biomed. Anal. 1998, 18,
347-357.
(2) Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M. Anal. Chem.
1998, 70 (5), 882-889.
Analytical Chemistry, Vol. 75, No. 13, July 1, 2003 3019
illustrating the need for careful assessment of HPLC-MS/MS
assay selectivity3 including evaluation of selectivity in postdose
biological fluids in the presence of metabolites4-6 and the need
for an efficient extraction of analytes from biological materials2,7
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.
(3) Clarke, S. D.; Hill, H. M.; Noctor, T. A. G.; Thomas, D. Pharm. Sci. 1996,
2, 203-207.
(4) Constanzer, M. L.; Chavez, C. M.; Matuszewski, B. K.; Carlin, J.; Graham,
D. J. Chromatogr., B 1997, 693, 117-129.
(5) Matuszewski, B. K.; Chavez, C. M.; Constanzer, M. L. J. Chromatogr., B
1998, 716, 195-208.
(6) Jemal, M.; Xia, E.-Q. Rapid Commun. Mass Spectrom. 1999, 13, 97-106.
(7) Buhrman, D.; Price, P.; Rudewicz, P. J. Am. Soc. Mass Spectrom. 1996, 7,
1099-1105.
(8) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A-986A.
(9) Department of Health and Human Services, Food and Drug Administration,
Guidance for Industry on Bioanalytical Method Validation. Fed. Regist. 2001,
66 (100), 28526 (Docket No. 98D-1195).
(10) Shah, V. P.; Midha, K. K.; Findlay, J. W. A.; Hill, H. M.; Hulse, J. D.;
McGilveray, I. J.; McKay, G.; Miller, K. J.; Patnaik, R. N.; Powell, M. L.;
Tonelli, A.; Viswanathan, C. T.; Yacobi, A. Pharm. Res. 2000, 17 (12), 1551-
1557.
(11) Bonfiglio, R.; King, R. C.; Olah, T. V.; Merkle, K. Rapid Commun. Mass
Spectrom. 1999, 13, 1175-1185.
(12) King, R.; Bonfiglio, R.; Fernandez-Metzler, C.; Miller-Stein, C.; Olah, T. J.
Am. Soc. Mass Spectrom. 2000, 11 (11), 942-950.
(13) Choi, B. K.; Hercules, D. M.; Gusev, A. I. J. Chromatogr., A 2001, 907,
337-342.
(14) Choi, B. K.; Hercules, D. M.; Gusev, A. I. Fresenius’ J. Anal. Chem. 2001,
369, 370-377.
3020 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003
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 inter-
face. Depending on the environment in which the ionization and
ion evaporation processes take place, this competition may
effectively decrease (ion suppression) or increase (ion enhance-
ment) 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 un-
detected 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 deter-
mination of analyte concentrations and the integrity of PK data
generated.
Our earlier report2 and observations of others12 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.

Figure 1. Chemical structures of compound 1 and an internal
standard 2.
Standard Solutions. A stock solution of 100 µg/mL for
standards 1 and 2 were prepared in the mobile phase. A 10 µg/
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 µg/mL.
The 100 µg/mL stock solution of the internal standard 2 was
serially diluted with the mobile phase to yield a working standard
of 0.3 µg/mL.
Chromatographic Conditions. Chromatographic separation
of 1 and 2 was performed on a Keystone Scientifics Hypersil BDS
C-18 (50 × 4.6 mm 3 µm, Keystone Scientific, Bellefonte, PA)
analytical column with a mobile phase consisting of 80% acetoni-
trile 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 µL/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 µA) 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 µL of the appropriate standards of 1, 100
µL of the 0.3 µg/mL stock solution of 2, and 100 µL of the mobile
phase (total volume 300 µL) into 15-mL centrifuge tubes. After
mixing, the solutions were transferred into autosampler vials and
50 µL 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 µL of the mobile phase (to simulate
the addition of standard solutions of 1 and 2 that were each added
in set 3 in 100 µL 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 µL of
the mobile phase, 100 µL of the 0.3 µg/mL stock solution of 2,
and 100 µL of the appropriate standards of 1 (total volume 300
µL). The extracts from the control samples (blanks) were
reconstituted in 300 µL of the mobile phase. A total of 50 µL 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 µL of the
appropriate standards of 1 and 100 µL of the 0.3 µg/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 µL 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 µL of the mobile
phase, and 50 µL 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
Analytical Chemistry, Vol. 75, No. 13, July 1, 2003 3021
same order (low to high concentrations) for plasma lot 1, followed Table 1. Precisiona (CV, %) of Determination of Peak
by lot 2, etc. Areas of 1, Internal Standard (2), and the Peak Area
Precision, Accuracy, and Recovery. The precision of the Ratios (1/2) in Sets 1,b 2,c and 3d Using Heated
method was determined by the replicate analyses (n ) 5, set 3) Neublizer Interface
of human plasma containing 1 at all concentrations utilized for precision (CV, %)
the construction of calibration curves. The linearity of each peak area 1 peak area 2 peak area ratio 1/2
nominal
standard curve was confirmed by plotting the peak area ratio of concn set set set set set set set set set accuracye
1 to 2 versus drug concentration. The sample concentrations were (ng/mL) 1 2 3 1 2 3 1 2 3 (%)
calculated from the equation y ) mx + b, as determined by 0.5 4.0 7.9 10.2 4.0 5.2 9.3 0.9 4.7 2.9 99
weighted (1/x2) linear regression of the standard line. The 1.0 5.0 6.0 8.5 3.2 4.4 7.7 2.5 3.3 2.6 101
5.0 4.7 5.0 5.2 5.3 2.3 4.7 1.6 4.1 2.9 103
accuracy of the method was expressed by [(mean observed 10.0 4.2 6.0 5.6 5.2 4.1 6.8 1.6 4.4 1.9 101
concentration)/(spiked concentration] × 100. The recovery was 50.0 3.1 5.3 4.3 3.0 4.0 4.7 0.7 3.9 1.3 100
100.0 2.0 6.9 3.9 3.1 4.7 3.6 2.7 3.4 2.6 99
determined by comparing the mean peak areas of 1 and 2 200.0 3.2 5.2 6.8 4.1 4.1 5.1 2.0 2.7 1.9 97
obtained in set 3 to those in set 2.
column A B C D E F G H I J
Assessment of Matrix Effect. The assessment of matrix effect
a n ) 5. b 1 and 2 standards in mobile phase. c 1 and 2 spiked after
and assay reliability is critical when homologues rather than stable
extraction into extracts from five different plasma lots. d 1 and 2 spiked
isotope-labeled analytes are utilized as internal standards. By before extraction into extracts from five different plasma lots. e Ex-
comparing the peak areas of the analyte standards, standards pressed as the[(mean observed concentration)/(nominal concentra-
tion)] × 100.
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. sources as in set 2. The variability in CV values here would reflect
Assessment of Assay Selectivity. The assay selectivity was a combined effect of a sample matrix and potential differences in
assessed by analyzing extracts from five lots of plasma from recovery of analytes from different plasma lots. In all three cases
different sources. Endogenous peaks at the retention time of the (sets 1-3), five standard lines were constructed (total of 3 × 35
analytes of interest were not observed in any of the plasma lots ) 105 samples). In a typical, conventional method validation, only
evaluated. In addition, the “cross-talk” between MS/MS channels set 3 samples (35 samples) with analytes spiked before extraction
used for monitoring 1 and 2 for both analytes was assessed by into a single lot of a biological fluid are usually analyzed. The
the following: (1) separately injecting 1 at the highest concentra- results of the analyses for sets 1-3 are summarized in Tables 1
tion on the standard line (200 ng/mL) and monitoring the and 2.
response in the IS channel and (2) by injecting a plasma sample The results obtained in this manner allow determination of the
spiked only with the IS (2) and monitoring the response in the matrix effect (ME), recovery (RE) of the extraction procedure,
drug channel at the sensitivity (y-axis) required for monitoring 1 and overall “process efficiency” (PE) by comparing the absolute
at the lowest limit of quantification. No “cross-talk” was observed. 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,
RESULTS the corresponding peak areas for standards spiked after extraction
Evaluation of the Matrix Effect and Assay Validation Using into plasma extracts as B (set 2), and peak areas for standards
HN Interface. The matrix effect and the possibility of ionization spiked before extraction as C (set 3), the ME, RE, and PE values
suppression or enhancement for 1 and 2 was evaluated by can be calculated as follows:
comparing the results of analysis of three sets of samples (set 1,
set 2, set 3) prepared as described in the Experimental Section. ME (%) ) B/A × 100 (1)
These three sets corresponded to three types of system evaluation.
In the first set (set 1), standards of the analytes present in the RE (%) ) C/B × 100 (2)
neat reconstitution solvent (HPLC mobile phase used in the assay)
PE (%) ) C/A × 100 ) (ME × RE)/100 (3)
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 HPLC- The terms “process efficiency”, “extraction efficiency”, and “ion
MS/MS system reproducibility in measuring the absolute peak suppression” were originally introduced by Buhrman et. al.7 In
areas on consecutive injections, the performance of the detector, their study, ion suppression was defined as (100 - B/A × 100),
and the chromatographic system as a whole. In the second set and the potential for ion enhancement was not considered. To
(set 2), plasma samples from five different plasma lots were first account for both ion suppression and ion enhancement and to
extracted and spiked after extraction with the analytes 1 and 2 avoid negative values in the case of ion enhancement, the ratio
in the same solvent (mobile phase) as in set 1. Any additional (B/A × 100) is defined here generally as a matrix effect. The
variability of the peak areas for the analytes than those observed ME calculated in this manner may be referred to as an “absolute”
in set 1, as demonstrated by an increase in the coefficients of matrix effect since the signal response of the standard present in
variation (CV) at each concentration, would be indicative of an the plasma extract is compared to the response of a standard made
effect of sample matrix since analytes at the same concentrations directly in a neat mobile phase. Although the presence of this
were spiked into plasma extracts. In set 3, analytes were spiked absolute matrix effect may be of some concern (vide infra), the
before extraction into plasma samples originating from five different more important parameter in the evaluation and validation of a
3022 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003
Table 2. Matrix Effect (ME), Recovery (RE), and Process Efficiency (PE) Data for 1 and 2 in Five Different Lots of
Human Plasma Using HN Interface

mean peak areaa

nominal 1 2 MEb (%) REc (%) PEd (%)

concn (ng/mL) set 1 set 2 set 3 set 1 set 2 set 3 1 2 1 2 1 2
0.5 1.94 2.56 2.57 49.16 61.77 58.81 132 126 101 95 133 120
1.0 3.78 5.03 4.88 48.23 59.44 55.44 133 123 97 93 129 115
5.0 19.16 25.87 25.93 48.96 61.25 58.01 135 125 100 95 135 118
10.0 39.66 52.62 49.70 50.36 62.22 57.01 133 124 94 92 125 113
50.0 194.29 252.38 251.35 48.34 60.53 57.92 130 125 100 96 130 120
100.0 390.24 553.14 498.21 48.07 66.07 58.13 142 137 90 88 128 121
200.0 756.25 1046.41 978.84 48.99 64.23 58.19 138 131 94 91 129 119
mean 135 127 97 93 130 118
column A B C D E F G H I J K L
a In arbitrary units, ×104, n ) 5. b Matrix effect expressed as the ratio of the mean peak area of an analyte spiked postextraction (set 2) to the
mean peak area of the same analyte standards (set 1) multiplied by 100. A value of >100% indicates ionization enhancement, and a value of <100%
indicates ionization suppression. c Recovery calculated as the ratio of the mean peak area of an analyte spiked before extraction (set 3) to the mean
peak area of an analyte spiked postextraction (set 2) multiplied by 100. d Process efficiency expressed as the ratio of the mean peak area of an
analyte spiked before extraction (set 3) to the mean peak area of the same analyte standards (set 1) multiplied by 100.

Table 3. Slopesa of the Standard Lines for Neat Standards (Set 1), in Five Different Lots of Plasma Spiked after
(Set 2) and before Extraction (Set 3), and in a Single Lot of Plasma Spiked before Extraction (Set 3a) Using HN and
ISP Interface

plasma extracts plasma spiked single lot of plasma

neat standards spiked after extraction before extraction spiked before extraction
set 1 set 2 set 3 set 3a
HN interface
slopeb 0.0792 0.0840 0.0866
SD 0.00100 0.00297 0.000897
CV (%) 1.3 3.5 1.0
ISP interface
slopeb 0.223 0.253 0.231 0.253c
SD 0.00195 0.03781 0.03039 0.00608
CV (%) 0.9 14.9 13.2 2.4
column A B C D
a Calculated from the equation y ) mx + b; each standard line was constructed using 1 at seven different concentrations. b Mean values of five
slopes each obtained in a different plasma lot. c Mean values of five slopes obtained in a single plasma lot.

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 HPLC-
MS/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
postextraction (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
Analytical Chemistry, Vol. 75, No. 13, July 1, 2003 3023
Figure 2. Normalized peak areas of 1 and 2 spiked postextraction (set 2) into extracts originating from five different lots of human plasma and
analyzed using the HN and ISP interfaces. The peak areas for 1 at all seven concentrations obtained in a each plasma lot were normalized to
the concentration of 10 ng/mL, whereas for 2 peak areas were normalized to 1 ng/mL.

Table 4. Precisiona (CV, %) of Determination of Peak Areas of 1, Internal Standard (2), and the Peak Area Ratios
(1/2) in Set 1,b Set 2,c Set 3,d and Set 3ae Using ISP Interface

precision (CV, %)
accuracyf
nominal peak area 1 peak area 2 peak area ratio 1/2 (%)
concn (ng/mL) set 1 set 2 set 3 set 3a set 1 set 2 set 3 set 3a set 1 set 2 set 3 set 3a set 3 set 3a
0.5 8.5 14.2 32.8 5.6 7.6 4.6 16.3 6.0 3.6 13.9 27.8 5.3 99 96
1.0 10.7 15.0 26.5 8.5 6.1 5.2 17.3 5.0 5.4 15.1 24.3 4.8 100 108
5.0 12.3 16.0 19.7 6.1 10.9 6.1 12.6 4.0 2.1 16.1 14.0 8.5 105 101
10.0 5.8 13.9 22.3 7.0 7.3 6.6 14.6 3.9 2.3 13.5 15.1 5.0 103 100
50.0 10.5 11.6 19.6 5.1 10.6 7.3 10.8 2.1 2.4 10.1 14.0 4.2 104 101
100.0 11.7 20.4 24.0 3.3 13.5 11.3 11.0 3.8 2.4 19.7 14.4 4.5 97 96
200.0 10.7 23.8 24.9 4.0 8.5 9.5 14.5 2.7 3.9 16.9 11.1 4.4 91 98
column A B C D E F G H I J K L M N
a n ) 5. b 1 and 2 standards in mobile phase. c 1 and 2 spiked after extraction into extracts from five different plasma lots. d 1 and 2 spiked
before extraction into five different plasma lots. e 1 and 2 spiked before extraction into a single plasma lot (n ) 5). f Expressed as the [(mean observed
concentration)/(nominal concentration)] × 100.

important and often overlooked element of assay validation and
is explicitly required not only by the current validation standards9,10
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 inter-
action 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
3024 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003
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 me-
tabolites 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
Table 5. Matrix Effect (ME), Recovery (RE), and Process Efficiency (PE) Data for 1 and 2 in Five Different Lots of
Human Plasma Using ISP Interface

mean peak areaa

nominal 1 2 MEb (%) REc (%) PEd (%)

concn (ng/mL) set 1 set 2 set 3 set 1 set 2 set 3 1 2 1 2 1 2
0.5 4.25 4.19 3.60 38.08 33.13 29.55 99 87 86 89 85 78
1.0 8.54 8.38 6.64 35.52 32.84 28.08 98 92 79 85 78 79
5.0 41.95 42.07 35.11 35.20 33.13 28.75 100 94 83 87 84 82
10.0 79.54 84.67 65.82 33.82 32.56 27.62 106 96 78 85 83 82
50.0 389.00 392.71 299.31 33.55 33.37 24.85 101 99 76 74 77 74
100.0 779.76 884.84 587.34 35.78 32.93 26.15 113 92 66 79 75 73
200.0 1445.70 1555.74 1069.30 37.54 31.55 25.17 108 84 69 80 74 67
mean 104 92 77 83 79 76
column A B C D E F G H I J K L
a In arbitrary units, ×104, n ) 5. b-d See Table 2.

Figure 3. Comparison of slopes of the standard lines in five different lots of plasma for samples spiked before extraction (set 3) using the ISP
vs HN interface.

in a single lot. In the course of utilization of a method for long-
term 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 meth-
ods.
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 quantifica-
tion. 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
Analytical Chemistry, Vol. 75, No. 13, July 1, 2003 3025
Figure 4. Precision of the assay determined in a single plasma lot and in five different plasma lots (set 3) using the ISP interface.
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 perfor-
mance 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,
3026 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003
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 enhance-
ment 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, respec-
tively, 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 extrac-
tion, 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 indepen-
dent 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 instru-
Analytical Chemistry, Vol. 75, No. 13, July 1, 2003 3027
ment 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,
3028 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003
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 concentra-
tion, 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 perfor-
mance, 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 assess-
ment 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 impracti-
cal.
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
Analytical Chemistry, Vol. 75, No. 13, July 1, 2003 3029
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 HPLC-
MS interface, and internal standards. The decision about perform-
ing simultaneous assays for a number of analytes using HPLC-
MS/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
(15) Chavez-Eng, C. M.; Constanzer, M. L.; Matuszewski, B. K. J. Chromatogr.,
B 2002, 767, 117-129.
3030 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003
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 bioanaly-
sis.15 They include the problems with isotopic purity of com-
pounds, “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 HPLC-
MS/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 condi-
tions, 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.
ACKNOWLEDGMENT
The authors thank Mr. M. Schwartz for his help in graphical
presentations of some data. Thanks are also due to Dr. E. J. Woolf
for the critical review of the manuscript.
Received for review May 31, 2002. Accepted April 10,
2003.
AC020361S