Anal Bioanal Chem (2012) 403:21552172
DOI 10.1007/s00216-012-6035-2
REVIEW
Aspects of matrix effects in applications of liquid
chromatographymass spectrometry to forensic and clinical
toxicologya review
Frank T. Peters & Daniela Remane
Received: 9 December 2011 / Revised: 22 February 2012 / Accepted: 6 April 2012 / Published online: 2 May 2012
# Springer-Verlag 2012
Abstract In the last decade, liquid chromatography coupled
to (tandem) mass spectrometry (LCMS(MS)) has become
a versatile technique with many routine applications in
clinical and forensic toxicology. However, it is well-known
that ionization in LCMS(MS) is prone to so-called matrix
effects, i.e., alteration in response due to the presence of co-
eluting compounds that may increase (ion enhancement) or
reduce (ion suppression) ionization of the analyte. Since the
first reports on such matrix effects, numerous papers have
been published on this matter and the subject has been
reviewed several times. However, none of the existing
reviews has specifically addressed aspects of matrix effects
of particular interest and relevance to clinical and forensic
toxicology, for example matrix effects in methods for multi-
analyte or systematic toxicological analysis or matrix effects
in (alternative) matrices almost exclusively analyzed in clini-
cal and forensic toxicology, for example meconium, hair, oral
fluid, or decomposed samples in postmortem toxicology. This
review article will therefore focus on these issues, critically
discussing experiments and results of matrix effects in LC
MS(MS) applications in clinical and forensic toxicology.
Moreover, it provides guidance on performance of studies
on matrix effects in LCMS(MS) procedures in systematic
toxicological analysis and postmortem toxicology.
Published in the special issue Young Investigators in Analytical and
Bioanalytical Science with guest editors S. Daunert, J. Bettmer, T.
Hasegawa, Q. Wang and Y. Wei.
Electronic supplementary material The online version of this article
(doi:10.1007/s00216-012-6035-2) contains supplementary material,
which is available to authorized users.
F. T. Peters (*) : D. Remane
Institute of Forensic Medicine, Jena University
HospitalFriedrich Schiller University Jena,
07740 Jena, Germany
e-mail: frank.peters@med.uni-jena.de
Frank T. Peters
is Head of Toxicology at the Insti-
tute of Forensic Medicine at the
University Hospital Jena. He has
received the Young Scientist Best
Published Paper Award and the
Achievement Award of The Inter-
national Association of Forensic
Toxicologists (TIAFT), the Young
Scientist Award of the Society of
Toxicological and Forensic Chemis-
try (GTFCh), and the Young Inves-
tigator Award of the International
Association of Therapeutic Drug
Monitoring and Clinical Toxicology
(IATDMCT). Current projects of his workgroup include: studies on the
metabolism of drugs and poisons by microorganisms colonizing corpses;
metabolism, toxicological analysis, and prevalence of new designer drugs;
and development and validation of mass spectrometry-based methods for
application in forensic and clinical toxicology.
Keywords Liquid chromatography . Mass spectrometry .
Matrix effect . Ion suppression . Ion enhancement
Introduction
Qualitative and quantitative toxicological analysis is the
basis of competent toxicological judgment and consultation
in clinical and forensic toxicology. The reliability of analyt-
ical data is of utmost importance in these fields, because
unreliable results may lead to over or underestimation of
effects, to false interpretations, or to unwarranted conclu-
sions. In the worst case, this might result in incorrect treat-
ment of a patient or unjustified legal consequences for a
defendant.
Because of the very high separating power of gas chro-
matography (GC) and the high selectivity of electron
2156
ionization mass spectrometry (MS), GCMS used to be the
unambiguous gold standard technique for reliable com-
pound identification in clinical and forensic toxicology
[14]. However, one of the limitations of GCMS always
has been that hydrophilic, thermolabile, and non-volatile
analytes are amenable to analysis by this technique only
after extensive sample preparation, or not at all. The cou-
pling of liquid chromatography (LC) to MS or tandem MS
(MSMS), which became available as an experimental tech-
nique in the early 1990s has helped to close this analytical
gap. Since then, LCMS(MS) has become increasingly
important in clinical and forensic toxicology and is now a
robust and established routine analytical technique [58].
However, LCMS(MS)-based analysis is also associat-
ed with pitfalls [9], the most important probably being its
susceptibility to so-called matrix effects (MEs), which Tay-
lor [10] therefore called the Achilles heel of this tech-
nique. Shah et al. [11] defined MEs as the direct or indirect
alteration or interference in response due to the presence of
unintended analytes (for analysis) or other interfering sub-
stances in the sample. This alteration in response is attrib-
utable to interference with the ionization process and may
either lead to increased intensity (ion enhancement) or to
reduced intensity (ion suppression) [10, 1215]. It is impor-
tant to note that the definition above explicitly includes
effects caused by any co-eluting compound and is not lim-
ited to those caused by the sample matrix itself as the term
matrix effect may suggest.
Owing to the complexity of the ionization process in LC
MS(MS) analysis, it can be affected by several conditions for
example sample matrix, sample preparation, chromatographic
separation, and ionization technique [10, 1215]. The molec-
ular mechanisms of ion suppression/enhancement effects are
not yet fully understood but interactions can take place in the
solution or the gas phase. Trufelli et al. [15] recently catego-
rized the most relevant mechanisms as follows:
1. competition of analyte and co-eluting compound for the
available charges and access to the droplet surface;
2. reduced efficiency of droplet formation because of in-
creased viscosity and surface tension of the droplet;
3. formation of solid analyte inclusion particles with non-
volatile additives; and
4. ion pair formation of analyte and co-eluting (matrix)
compounds or mobile phase additives.
More detailed discussions on mechanisms of MEs can be
found elsewhere [10, 1218]. In any case, electrospray
ionization (ESI) seems to be more prone to this phenomenon
than atmospheric-pressure chemical ionization (APCI) [12,
17, 1926].
Since the first report on ion suppression observed after
co-infusion of organic bases into an ESI mass spectrometer
F.T. Peters, D. Remane
in 1990 [27], numerous reports on MEs have been published
and reviewed several times in respect of the mechanisms
and factors affecting MEs, and strategies to avoid them [10,
1215, 28]. However, MEs in the LCMS(MS) methods
for systematic toxicological analysis (STA) or multi-analyte
assays often used in forensic and clinical toxicology have
not been systematically addressed in these reviews. More-
over, aspects relating to the sample matrices almost exclu-
sively used in these fields, for example hair, oral fluid,
sweat, postmortem samples, or meconium, have not
been covered in detail. Therefore, this review article
will provide an overview on those aspects of MEs that
are of particular relevance to clinical and forensic tox-
icology. It focuses on LCMS(MS)-based multi-analyte
and STA procedures which were specifically developed
for application in clinical and/or forensic toxicology,
and on target analytes relevant to these fields, and
includes papers which were published in the last ten
years.
General aspects of matrix effects
Testing for matrix effects
Two basic approaches have been developed to study MEs.
The first, which will henceforth be called the post-column
infusion approach, was initially proposed by Bonfiglio et al.
[29]. It is based on constant infusion of an analyte solution
into the eluent from the analytical column, via a post-
column tee connection, by use of a syringe pump. In the
absence of MEs, the continuous post column infusion leads
to a constant signal in the detector. However, elution of
compounds that can suppress or enhance ionization will
lead to decreased or increased detector response, respective-
ly. Thus, monitoring the detector response after injection of
blank matrix extracts or other compounds possibly causing
ion suppression or enhancement can be used to check for the
presence of such effects and where in the chromatogram
they have to be expected. This is particularly helpful in the
method development phase and may also provide informa-
tion about which compounds are responsible for the ob-
served MEs.
The second strategy, which will henceforth be referred to
as the post-extraction addition approach, was published by
Buhrman et al. as early as 1996 [30] and later modified and
expanded by Matuszewskis group [22, 31]. In principle, it
involves determination of analyte peak areas in three differ-
ent sets of samples, one consisting of neat standards (set 1),
one consisting of blank matrix extracts from different sour-
ces and spiked after extraction (set 2), and one consisting of
these blank matrix sources spiked before extraction (set 3).
From the resulting data, MEs can be calculated by use of the
Matrix effects in clinical and forensic toxicology
equation:
 
B
ME  1  100%
A in clinical and forensic toxicology in which samples arise
where ME is the matrix effect (ion suppression/ enhance-
ment), B is the peak area for the sample from set 2 (spiked
blank matrix extract), and A is the peak area for the sample
from set 1 (neat standard).
The equation is a modification of the original equation
reported by Matuszweski et al. [22] which yields ME results
expressed as a percentage. With the above equation, ME
results are obtained as percent difference from 100 %. Ac-
cordingly, negative results indicate ion suppression, whereas
positive results indicate ion enhancement.
In addition to ME, the extraction recovery and so-called
process efficiency (combination of ME and extraction re-
covery) can be calculated by comparing peak areas for
sample sets 2 and 3 or 1 and 3, respectively [22, 32, 33].
The full experimental design proposed in Ref. [22] involves
analysis of 105 samples (three sets with seven concentration
levels and five different sources of blank matrix per level).
On the one hand, the resulting data provide valuable infor-
mation about the performance of the studied assay, includ-
ing a quantitative assessment of MEs. On the other hand,
analysis of such large numbers of samples is time-
consuming and expensive. For this reason, a number of
simplified experimental designs have also been included in
Ref. [22]. In any case, the post-extraction addition approach
enables quantitative assessment of the extent and variability
of MEs between samples. It is, therefore, generally preferred
during validation, especially of quantitative methods.
Matrix effects and bioanalytical method validation
Evaluation of MEs is regarded as an essential part in the
validation of any LCMS-based method [1012, 22,
3436]. Recommendations on acceptance criteria for MEs
only exist for the post-extraction addition approach. A con-
sensus document to the Third American Association of
Pharmaceutical Scientists (AAPS)/Food and Drug Admin-
istration (FDA) Bioanalytical Workshop [34] includes a
recommendation to limit the variability of MEs between
samples rather than the average extent of MEs. A relative
standard deviation (RSD) of MEs <15 % between six dif-
ferent blank matrix lots was considered acceptable in this
document. It seems reasonable to also use this latter recom-
mendation as guidance for quantitative methods in clinical
and forensic toxicology.
The above consensus document further states that evalu-
ation of six different blank matrix lots can be waived if a
stable-isotope-labeled (SIL) analogue of the analyte is used
2157
as internal standard (IS). Although this may be acceptable
for bioavailability, bioequivalence, and pharmacokinetic
studies performed under controlled conditions on a well-
defined population of study subjects, it seems unacceptable
from a wide variety of subjects with regard to age, dietary
habits, and pathological conditions, or even from decom-
posed bodies.
In the latter fields it also seems important to define an
acceptance limit for the average extent of MEs, because
major ion suppression leads to reduced sensitivity of the
method and may therefore lead to false negative results.
Consequently, the German speaking Gesellschaft fr Toxiko-
logie und Forensische Chemie (GTFCh, Society of Toxico-
logical and Forensic Chemistry) recommended an acceptance
interval of 25 % for average MEs for five different blank
matrix samples [36]. In the same document, variability of MEs
is regarded as acceptable when RSD is <15 % (RSD <20 %
near the lower limit of quantification) for analytes without a
stable-isotope-labeled internal standard (SIL-IS) or <25 % for
analytes for which a SIL analogue is used as IS. The GTFCh
document further requires evaluation of MEs at two concen-
trations, low and high with regard to the calibration range, to
account for the fact, that MEs may be concentration-
dependent [22, 37].
Specific aspects of matrix effects with particular
relevance to clinical and forensic toxicology
Clinical and forensic toxicology covers a wide range of
toxicological problems including in-utero drug exposure,
drug abuse and driving under influence of drugs (DUID),
acute poisoning of the living, and postmortem toxicology.
Depending on the analytical problem, biological matrices
different from classical blood or urine are frequently used,
for example meconium for detection of in utero drug expo-
sure, oral fluid (in DUID and workplace drug testing), hair
(chronic or past drug exposure), or various postmortem
biofluids and tissues for postmortem toxicology.
Clinical and forensic toxicologists are routinely faced
with the problem that the compounds involved in a partic-
ular case are unknown. Analytical methods for STA ideally
covering hundreds of relevant drugs, poisons, and/or their
metabolites are therefore important in these fields. Apart
from that, multi-analyte procedures covering all relevant
analytes from one or several drug classes are often used to
reduce the number of methods to be established in the
laboratory. Information on analytes, MEs, sample prepara-
tion, and the type of interface for selected multi-analyte
procedures in urine [3848], blood plasma or serum
[4961], whole blood, including postmortem blood [44,
6274], oral fluid [57, 7586], hair [53, 8793], and a
2158 F.T. Peters, D. Remane

variety of other matrices [9497] are listed in Tables 1, 2, 3,
4, 5 and 6, respectively. Data on STA procedures [73, 92,
98104] are summarized in Table 7 and will be dis-
cussed further below. Additional information on the
chromatography and mass spectrometers used in these
studies is available in the Electronic Supplementary
Material (Table S1).
Matrix effects in multi-analyte procedures
To some extent, ME experiments were performed in all the
references listed in Tables 1, 2, 3, 4, 5 and 6. As expected for
methods with a primarily quantitative character and a limit-
ed number of analytes, the post-extraction addition approach
was used in all of the listed references with the exception of
[47, 48, 5053, 58, 60, 83, 95]. Some of the methods used
online extraction/desorption making post-extraction spiking
impossible [50, 51, 60, 95]. In the other references, the
reasons for preferring post-column infusion to the better
suited post-extraction addition approach are not clear. At
least in some, however, no relevant matrix effects were
observed with post-column infusion, so the authors proba-
bly did not see any need to perform additional post-
extraction addition experiments [48, 58, 83].
The need to consider at least five to six different blank
matrix sources in the ME experiments has been taken into
account by most authors. However, in quite a number of
studies fewer matrix sources [44, 53, 59], or replicates of
only a single blank matrix source [74, 75] have been used.
Another problem is that the number of matrix sources and/or
replicates have not been specified in many publications, so it
remains unclear what exactly the reported results represent
[40, 45, 51, 52, 57, 58, 60, 76, 78, 81, 8388, 97].
It is remarkable that in more than two thirds of the
methods listed in Tables 1, 2, 3, 4, 5 and 6 substantial
MEs of 20 % or more were reported for at least one
analyte. In more than half of the methods listed in these
tables, MEs for at least one analyte would even be outside
the acceptable range of 25 % proposed by the GTFCh [36].
Maximum MEs ranged from almost complete suppression
of ionization (cocaethylene in oral fluid) [84] to enormous
ion enhancement of over 1,000 % (bupropione in plasma)
[61]. The extreme ion suppression in Ref. [84] can be easily
attributed to the fact that sample preparation consisted of
simple dilution, which has been associated with extensive
MEs [19, 24, 105]. The large ion enhancement reported in
Ref. [61], however, emphasizes that strong MEs may still
occur after extraction. In fact, large MEs have even
been observed after more sophisticated, or even two-
step, sample-preparation procedures, for example incu-
bation of hair with methanol and subsequent mixed-
mode solid-phase extraction (SPE) [88] or pre-extraction
of meconium with methanol followed by mixed-mode SPE
[96]. This shows, that especially for such very complex
matrices MEs cannot be fully eliminated by sample
preparation.

Table 1 Analytes, testing approach, results of matrix effect experiments, sample preparation, and ionization technique for quantitative multi-
analyte applications in urine using liquid chromatographymass spectrometry

Matrix Analytes Test Results Sample preparation Ionization Ref.

technique

Urine 7 Amphetamines PEA, 1 conc. k09 13.9 % to 10.7 %, LLE ESI [38]
RSD 5.6 % to 10.3 %
Urine Opiates PCI, 1 conc. PCI 35 %, PEA 37 %, Dilution ESI [39]
PEA, 1 conc. k08 RSD 12.3 %
Urine Multiple drugs of abuse and metabolites PEA 66 % to +292 %, RSD Automated SPE ESI [40]
not given (HLB)
Urine 12 Illicit drugs of abuse PEA, 1 conc. k010 43 % to 19 %, RSD (Enzymatic hydrolysis), ESI [41]
3.4 % to 29.3 % SPE (HLB)
Urine Opiates and cocaine PEA, 2 conc. k08 29 % to 5 %, RSD SPE (HCX) ESI [42]
2.5 % to 11 %
Urine Multiple hallucinogens, PEA, 1 conc. k06 46.1 % to 34.0 %, SPE (HCX) ESI [43]
chlorpheniramine, RSD 5.3 % to 12.1 %
ketamine, ritalinic acid, and metabolites
Urine 5 Toxic alkaloids PEA, 3 conc. k03 18.4 % to 9.3 %, SPE (HCX) ESI [44]
RSD not given
Urine 10 Hallucinogenic designer drugs PEA, 3 conc. k not given <10 % (Enzymatic hydrolysis) APCI [45]
SPE (HCX)
Urine 19 Drugs of abuse PEA, 1 conc. k05 49.1 % to 24.8 %, SPE (C18) ESI [46]
RSD not given
Urine 21 Benzodiazepines PCI, 1 conc. k05 No significant effects, SPE (HLB) ESI [47]
20 % for alprazolam
Urine 6 Dialkylphosphate pesticides PCI, 1 conc. k05 No effects detected LLE ESI [48]

Abbreviations: PEA, post-extraction addition; PCI, post-column infusion; conc., concentration; k, number of different matrix sources used; n,
number of replicates; x, not defined, if different source or replicate; RSD, relative standard deviation; LLE, liquidliquid extraction; SPE, solid-
phase extraction; ESI, electrospray ionization; APCI, atmospheric-pressure chemical ionization
Matrix effects in clinical and forensic toxicology 2159

Table 2 Analytes, testing approach, results of matrix effect experiments, sample preparation, and ionization technique for quantitative multi-
analyte applications in serum and plasma using liquid chromatographymass spectrometry

Matrix Analytes Matrix effect test Results Sample preparation Ionization Ref.
technique

Serum Designer amphetamines, tryptamines, PEA, 1 conc. k05 35 % to +18 %, SPE (HCX) ESI [49]
and piperazines RSD 2.4 % to 19.4 %
Serum 13 Antidepressants and metabolites PCI, 1 conc. k010 No effect detected Online extraction ESI [50]
for analytes (turbular flow)
Serum 18 Basic drugs and PCI, PEA, x04 ca 45 % to no effect, Online ESI [51]
metabolites RSD not given extraction (PFP)
Plasma 9 Benzodiazepines PCI, 1 conc. x05 No effects detected LLE ESI [52]
Plasma 26 Benzodiazepines and metabolites, PCI, 1 conc. k03 From 1.7 min to 6.0 min Basic LLE with APCI [53]
zolpidem, and zopiclone, ME, 90 % to 20 %, 1-chlorobutane
but analytes elute
later than 6 min
Plasma 9 Toxic alkaloids PEA, 2 conc. k05 APCI 30 % to +11 %, RSD SPE (HCX) ESI [54]
2.7 % to 18.7 %
ESI 28 % to no effect, RSD
1.9 % to 9.3 %
Plasma 9 Herbal phenylalkylamines PEA, 2 conc. k05 27 % to +3 %, RSD SPE (HCX) ESI [55]
2.0 % to 13.3 %
Plasma 10 Antiarrhythmics PEA, 3 conc. x06 10.2 % to 1.9 %, RSD Protein precipitation ESI [56]
not given
Plasma 13 Drugs and drugs of abuse Comparison calibration <20 % Dilution with MeOH ESI [57]
curves in solvent to and filtration
matrix
Plasma 6 AT II receptor antagonist+1 PCI, 2 conc. Effects seen at 1 min and Protein precipitation ESI [58]
metabolite 10 min retention time,
analytes elute between
2.4 and 7.3 min
Plasma 43 Benzodiazepines and metabolites, PEA, 2 conc. k03 <10 %, except 7- SPE (polymer) ESI [59]
zolpidem, and zopiclone aminoflunitrazepam, 80 %,
RSD not given
Plasma 14 Antidepressants and metabolites PCI, 2 conc. x03 17.7 % to 0.5 %, RSD not Online SPE (HCX) ESI [60]
sodium citrate, x03 given
sodium fluoride
PEA, 1 conc. x03
Plasma 136 Analytes (antidepressants, PEA, 2 conc. x06 54 % to +1082 %, CV 2 % LLE butyl acetateethyl APCI [61]
neuroleptics,benzodiazepines, to 121 % acetate
betablockers, oral diabetics);
analytes measured in the context of
brain death analysis

Abbreviations: PEA, post-extraction addition; PCI, post-column infusion; conc., concentration; k, number of different sources used; n, number of
replicates; x, not defined, if different source or replicate; RSD, relative standard deviation; LLE, liquidliquid extraction; SPE, solid-phase
extraction; ESI, electrospray ionization; APCI, atmospheric-pressure chemical ionization

Apart from sample preparation, modification and optimi-
zation of chromatographic separation is believed to be help-
ful in avoiding MEs [12, 14, 15]. Because LCMS(MS)
requires a mobile phase with volatile buffers, modification is
limited in comparison with conventional HPLC. Chromato-
graphic optimization would therefore largely depend on the
stationary phase used. However, when looking at Tables 1,
2, 3, 4, 5 and 6 one can see that, with some exceptions,
conventional reversed-phase C18 packing was used. The
stationary phases used in the other references were small-
particle C18 phases with greater separation power [43, 46,
59, 65, 73, 80, 84, 85], or other types of stationary phase, for
example PFP [51, 86], SCX [55], phenylhexyl [58], C8 [97],
cyano [64], phenyl [93], or zwitterionic hydrophilic interac-
tion liquid chromatography (ZIC-HILIC) phases [66, 95].
However, these were not specifically used to avoid MEs, but
rather to achieve adequate separation of the multiple analy-
tes covered by the respective assays.
A very similar observation can be made when looking at
the ionization techniques. Although it is well-known that
ESI is much more susceptible to MEs than APCI, the vast
majority of authors nevertheless used ESI in their methods,
most probably because of higher sensitivity for the studied
analytes. Of the comparatively few studies using APCI [19,
45, 54, 61, 71, 84, 88], the latter was systematically com-
pared with ESI in four publications. Dams et al. [19] found
less pronounced MEs for urine, plasma, and oral fluid when
using APCI rather than ESI, after different work-up proce-
dures. In contrast with this study and the general opinion in
the literature, Wang et al. [84] found ESI to be somewhat
2160 F.T. Peters, D. Remane

Table 3 Analytes, testing approach, results of matrix effect experiments, sample preparation, and ionization technique for quantitative multi-
analyte applications in whole blood or postmortem blood using liquid chromatographymass spectrometry

Matrix Analytes Matrix effect test Results Sample preparation Ionization Ref.
technique

Postmortem 7 low-dosage anti PEA, 1 conc. k05 36 % to 5 %, Basic LLE with ESI [64]
blood psychotic drugs RSD 7.4 % methyl t-butyl
to 40.4 % ether
Whole Blood 23 Benzodiazepines PEA, 1 conc. k06 15.8 % to +20 %, Basic LLE with ESI [65]
and metabolites, RSD not given ethyl acetate
zopiclone, and
zaleplone
Whole blood and Metformine, PEA, 1 conc. k020, antemortem 2 % to no LLE with ACN, ESI [66]
postmortem phenformine, blood and postmortem effect, RSD<6 % SPE (WCX)
blood and buformine blood Postmortem +2 % to
+5 %, RSD<9 %
Whole blood 19 Drugs of abuse and PEA, 1 conc. k06 Enhancement for some Automated SPE ESI [68]
metabolites analytes +18 % to
+64 %, RSD <15 %,
35 % for zopiclone
Blood 5 Toxic alkaloids PEA, 3 conc. k03 16.7 % to 8.2 % blood; SPE (HCX) ESI [44]
RSD not given
Postmortem 14 Cardiovascular drugs PEA, 3 conc. k06 Height evaluated, Automated SPE ESI [69]
blood 6 % to +14 %, (HCX)
RSD 3.1 % to 12 %
Postmortem 25 Opioids Comparing relative RSD mainly at the same Basic LLE with ESI [70]
blood standard deviation level between and within ethyl acetate
within and between samples, except for
authentic samples, norfentanyl 31 %,
1 conc. naloxone 14 %,
k015 heroin 14 %,
Whole blood 8 Benzodiazepines PEA, k06 +15 % at higher conc., LLE APCI [71]
no effect on
quantitative results
Blood 19 Antipsychotics PEA, 1 conc. k05 Depending on extraction Basic LLE with ESI [72]
antemortem, and sample used, 30 % chlorobutane
k05 non-decomposed to +57 %, or SPE
postmortem, k05
decomposed postmortem
Blood 5 Model drugs PEA, 1 conc. pool of blood Blood bank blood, 28.3 % SPE (Clean Screen ESI [73]
(diltiazem, quetiapine, bank blood, k05 non to +8.8 %, RSD<4.2 % ZSDAU020)
venlafaxincitalopram, decomposed postmortem, Postmortem non-decomposed,
tramadol) k05 decomposed 21.9 % to +13.8 %,
postmortem RSD<7.3 %
Postmortem decomposed,
23.3 % to +10.8 %,
RSD<10.6 %
Blood 8 Quaternary PEA, 1 conc. n05 35 % to +44.7 %, SPE (weak ESI [74]
ammonium drugs RSD not given cation exchange)
and 3 quaternary
herbicides

Abbreviations: PEA, post-extraction addition; PCI, post-column infusion; conc., concentration; k, number of different sources used; n, number of
replicates; x, not defined, if different source or replicate; RSD, relative standard deviation; LLE, liquidliquid extraction; SPE, solid-phase
extraction; ESI, electrospray ionization; APCI, atmospheric-pressure chemical ionization

less susceptible to MEs than APCI, but the observed effects
were large with both ionization techniques. Beyer et al. [54]
reported comparable matrix effects in ESI and APCI.
Remane et al. [61] observed ion suppression for only one
of 14 tested standards in APCI mode whereas in ESI mode
significant ion suppression was observed for 12 analytes and
ion enhancement for another.
In summary, these findings show that MEs cannot be
fully eliminated by sample preparation, at least not when
analyzing very complex matrices such as hair or meconium.
In multi-analyte procedures, it is also difficult to optimize
chromatography to avoid suppression zones in the chro-
matogram, because separation of multiple analytes is the
primary objective in such methods. Finally, using APCI
instead of ESI may not be possible with some instruments,
because of lower sensitivity in APCI mode. Moreover, use
of APCI may not always eliminate MEs. Other issues that
must be considered in multi-analyte procedures are mutual
Matrix effects in clinical and forensic toxicology 2161

Table 4 Analytes, testing approach, results of matrix effect experiments, sample preparation, and ionization technique for quantitative multi-
analyte applications to oral fluid using liquid chromatographymass spectrometry

Matrix Analytes Matrix effect test Results Sample preparation Ionization Ref.
technique

Oral fluid 32 Drugs PEA, n010 oral 76 % to +123 % LLE with ammonium ESI [75]
fluid, n05 mobile (mobile phase), carbonate buffer and
phase, n05 neat 50 % to +221 % ethyl acetateheptane
calibrator (neat calibrator),
RSD 2 % to 63 %
Oral fluid 13 Drugs of abuse Comparison <20 % Dilution with MeOH ESI [57]
and metabolites calibration curves and filtration
in solvent to matrix
Oral fluid Amphetamines, cocaine PEA, 1 conc. x04 13 % to 12 %, RSD SPE (MCX) ESI [76]
and metabolite, opiates PCI, oral fluid before not given
and after extraction
Oral fluid 23 Illicit and medicinal PEA, 1 conc. k010, 2 % to 56 %, except SPE ESI [77]
drugs, and metabolites k010 preserved THC +100 %,
RSD<23 %, RSD
37.2 % for
methamphetamine
Oral fluid 17 Benzodiazepines, zaleplon, PEA, 1 conc. x03 <10 %, 93 % for zopiclone LLE with phosphate ESI [78]
zopiclone, zolpidem buffer and
dichloromethane
diethyl ether
Oral fluid Opiates, amphetamines, PEA, 1 conc. k09 73.8 % to 12 %, Automated SPE (HLB) ESI [79]
cocaine and matrix, n03 large for amphetamines,
metabolite, THC mobile phase RSD 12.8 % to 21 %
Oral fluid 29 Drugs of abuse PEA, 1 conc. k06 Pure oral fluid 35 %, Automated SPE (HCX) ESI [80]
(5 different, 1 pool), Mixed oral fluid 39 %,
for pure oral fluid RSDs not given
and oral fluid mixed
with Stat-Sure buffer
PSI, k05
Oral fluid Antidepressants and PEA, x06 ME<15 %, +45 % Automated SPE (MCX) ESI [81]
metabolites for norfluoxetine,
+30 % for paroxetine
PCI, 2 conc. x06 No effects with PCI
Oral fluid Buprenorphine, PEA, 3 conc. k010 56.4 % to 315.5 %, Protein precipitation ESI [82]
methadone, cocaine, less than 29.4 %. with cold ACN
opiates, nicotine,
and metabolites
Oral fluid Benzodiazepines PCI No significant effects Basic LLE with ESI [83]
1-chlorobutane
Oral fluid Opiates, amphetamines, PEA, 1 conc. x04 ESI 98.0 to +28.7 %, Dilution ESI, APCI, APPI [84]
cocaine and metabolites, RSD<10 %
benzodiazepines APCI 99.8 to +46.8 %,
RSD<9 %,
APPI 99.9 to +74.4 %,
RSD<5 %
Oral fluid Synthetic cannabinoids PEA, 1 conc. x03 32 % to 11 %, SPE (Trace-N solid phase) ESI [85]
RSD not given
Oral Fluid 9 Benzodiazepines PCI, 1 conc. x05 No effects detected LLE ESI [52]
Oral fluid 21 Drugs of abuse PEA, 1 conc. x05 57.2 % to 7.9 %, Automated SPE (HCX) ESI [86]
RSD not given

Abbreviations: PEA, post-extraction addition; PCI, post-column infusion; conc., concentration; k, number of different sources used; n, number of
replicates; x, not defined, if different source or replicate; RSD, relative standard deviation; LLE, liquidliquid extraction; SPE, solid-phase
extraction; ESI, electrospray ionization; APCI, atmospheric-pressure chemical ionization

ion-suppression of unlabeled analytes and the respective
SIL-IS, and of co-eluting analytes, which will be discussed
further below.
The variability of MEs among samples from different
matrixes is regarded the most important aspect of MEs;
in contrast with the recognized need to study matrix
effects, however, this has not yet been acknowledged
by all authors. In many references [40, 44, 46, 47, 51,
56, 59, 60, 65, 71, 74, 7678, 80, 81, 8588, 93], no
information about the variability of MEs is reported,
although the described validation experiments included
different blank matrix sources or replicates of the same
2162 F.T. Peters, D. Remane

Table 5 Analytes, testing approach, results of matrix effect experiments, sample preparation, and ionization technique for quantitative multi-
analyte applications in hair using liquid chromatographymass spectrometry

Matrix Analytes Matrix effect test Results Sample preparation Ionization Ref.
technique

Hair 28 Benzodiazepines PEA, 3 conc. x03 55.4 % to +5.3 %, Decontamination, overnight ESI [87]
mainly for early eluting incubation in phosphate
7-aminonitrazepam buffer, extraction with
and 7-aminoclonazepam, dichloromethanediethyl
RSD not given ether
Hair 4 Opiates, cocaine, PEA, 1 conc. x06 31.8 % to +167.1 %, Incubation, extraction with APCI [88]
and metabolites RSD not given MeOH, SPE (Clean Screen)
Hair 21 Benzodiazepines PEA, 2 conc. k05 89 % to +43 %, Decontamination, incubation ESI [89]
and 3 z-drugs RSD 3.6 % to 44.3 % with MeOH, extraction with
MeOH and AF
Hair 17 Drugs of abuse PEA, 1 conc. k06 36.5 % to +35.6 %, SPE (Clean Screen ZSDAU020) ESI [90]
RSD 3.4 % to 66.1 %
Hair 26 Benzodiazepines and PCI, 1 conc. k03 From 1.7 min to 6.0 min ME, Decontamination, incubation ESI [53]
metabolites, zolpidem, 90 % to 20 %, but with MeOH, basic LLE with
and zopiclone, compounds elute later 1-chlorobutane
than 6 min
Hair 27 Benzodiazepines and PEA, 2 conc. k05 42 % to 110 % (mean), Decontamination, incubation ESI [91]
metabolites, zolpidem RSD<50 % with MeOH, filtration
Hair 24 illegal drugs, their PEA, 2 conc. k06 57.6 % to +6.8 %, Decontamination, incubation ESI [92]
metabolites and RSD<20 % with MeOHACN AF in
benzodiazepines duplicate
Hair 22 Illicit and medicinal drugs PEA, 1 conc. k05 21 % to +23 %, Decontamination, extraction ESI [93]
RSD not given with ACN, FA

Abbreviations: PEA, post-extraction addition; PCI, post-column infusion; conc., concentration; k, number of different sources used; n, number of
replicates; x, not defined, if different source or replicate; RSD, relative standard deviation; LLE, liquidliquid extraction; SPE, solid-phase
extraction; ACN, acetonitrile; AF, ammonium formate; ESI, electrospray ionization; APCI, atmospheric pressure chemical ionization

blank sample. Of the references in Tables 1, 2, 3, 4, 5
and 6 which include data on the variability of MEs,
quite a number [41, 64, 70, 75, 82, 89, 91] report RSD
values outside the acceptance limits proposed in the
above-mentioned validation guidelines [34, 36]. Quanti-
fication of the respective analytes was not compromised
when their SIL analogues were used as IS [41, 75, 82,
89, 91]. However, accuracy and/or precision data out-
side the generally applied acceptance limits were
reported for some analytes for which a SIL analogue
was either not available or not used in the respective
methods [64, 89]. In any case, SIL analogues are
regarded as ideal IS in LCMS-based methods and
should be used whenever possible [106].

Table 6 Analytes, testing approach, results of matrix effect experiments, sample preparation, and ionization technique for quantitative multi-
analyte applications to different matrices using liquid chromatographymass spectrometry

Matrix Analytes Matrix effect test Results Sample preparation Ionization Ref.
technique

Sweat Buprenorphine, methadone,
cocaine, opiates, nicotine,
and metabolites
Dried blood spots Cyclodextrins, cannabinoids,
benzodiazepines,
buprenorphine, opiates
and metabolites
Meconium 20 Drugs of abuse
Meconium Fatty acid ethyl esters
PEA, 3 conc. k09 63.3 % to+42.6 %, Patch extraction with ESI [94]
RSD<15 %, except for aqueous sodium acetate
EDDP and ecgonine buffer and subsequent
methyl ester SPE (Strata-XC)
PCI, 1 conc. No effect stated, whereas Online desorption ESI [95]
intensity shift over whole
run is seen in figure
PEA, 3 conc. n05, 40.4 % to +305.7 %, Extraction with MeOH, ESI [96]
variability 1 conc. variations between different SPE (Clean Screen
k07 pools depend on analyte ZSDAU020)
PEA, QC samples, No significant ion suppression Extraction with acetone ESI [97]
number not given (less than 10 % analytical hexane, SPE
signal suppression because (aminopropylsilica)
to matrix effect)

Abbreviations: PEA, post-extraction addition; PCI, post-column infusion; conc., concentration; k, number of different sources used; n, number of
replicates; x, not defined, if different source or replicate; RSD, relative standard deviation; LLE, liquidliquid extraction; SPE, solid-phase
extraction; MeOH, methanol; ESI, electrospray ionization; APCI, atmospheric-pressure chemical ionization
Matrix effects in clinical and forensic toxicology 2163

Table 7 Analytes, testing approach, results of matrix effect experiments, sample preparation, and ionization technique for systematic toxicological
analysis methods using liquid chromatographymass spectrometry

Matrix Model compounds Matrix effect test Results Sample preparation Ionization Ref.
technique

Blood 5 Model drugs PEA, 1 conc., blank Blood bank blood, SPE (Clean Screen ESI [73]
blood bank blood 28.3 % to +8.8 %, ZSDAU020)
RSD<4.2 %
k05 non-decomposed Postmortem non-
postmortem, k05 decomposed,
decomposed postmortem 21.9 % to +13.8 %,
RSD<7.3 %
Postmortem decomposed
23.3 % to +10.8 %,
RSD<10.6 %
Serum, urine, 20 Model compounds PCI, 1 conc. k06 No effect detected for SPE (HLB) ESI [98]
whole blood these compounds
Hair Model compounds, PCI, k and concentration Suppression between Decontamination, ESI [99]
number not given not given 0.7 and 1.7 min, 17 % digestion, SPE
to 57 %
Hair 24 Model compounds PEA, 2 conc. k06 57.7 % to 8.8 %, Decontamination, two ESI [92]
RSD 2.4 % to 21.7 % incubations with
MeOHACN
2 mmol L1 AF
Vitreous humor 7 Model compounds PCI, 1 conc. pooled blank 18 % to +12 %, RSD SPE (HCX) ESI [100]
vitreous humor, number of IS 18 % to 33 %
not given, variability of
IS, k05, n010
Urine 22 Model compounds PEA, 1 conc. k06 34 % to +60 %, Protein precipitation ESI [101]
RSD 6 % to 85 %
Urine 16 Model compounds PEA, 1 conc. k06 43 % to +37 %, Protein precipitation ESI [102]
RSD 5 % to 66 %
Urine 29 Model compounds PEA, 1 conc. k08 40 % to +110 %, LLE ESI [103]
RSD 4 % to 40 %
Urine 47 Model compounds PCI, k06, modified 40.4 % to no effect, Online extraction APCI [104]
post-extraction addition RSD 2.0 % to 15.7 %

Abbreviations: PEA, post-extraction addition; PCI, post-column infusion; conc., concentration; k, number of different sources used; n, number of
replicates; x, not defined, if different source or replicate; RSD, relative standard deviation; LLE, liquidliquid extraction; SPE, solid-phase
extraction; ACN, acetonitrile; AF, ammonium formate; AA, ammonium acetate; FA, formic acid, MeOH, methanol; QQQ, triple quadrupole;
QQQ/LIT, hybrid triple quadrupole/linear ion trap; TOF, time-of-flight; ESI, electrospray ionization; APCI, atmospheric-pressure chemical
ionization; MRM, multiple-reaction monitoring; SIM, selected-ion monitoring

Matrix effects from stable-isotope-labeled internal standards
(SIL-IS)
SIL analogues of the analytes are generally regarded as ideal
internal standards for MS-based methods [76, 106]. Because
of their almost identical physicochemical properties, they
can ideally compensate for variability resulting from sample
preparation and instrumental analysis including MEs. Al-
though the latter is true for the vast majority of applications,
examples have been published in which the SIL-IS did not
fully compensate for MEs, because a slight difference in
retention time led to different ion suppression for analyte
and SIL-IS [107, 108].
Slight differences in retention do not usually lead to
different MEs. Because of co-elution of analyteSIL-IS
pairs, potential affects on the ionization of each other should
rather be considered. In fact, mutual ion suppression/en-
hancement has been studied and reported by several groups
[20, 61, 109]. Sojo et al. [109] studied four such pairs and
for all of these a continuous decrease of SIL-IS peak area
with increasing analyte concentration was observed in ESI
mode. Despite this phenomenon, these authors still obtained
linear calibration lines when plotting peak-area ratios (ana-
lyte vs. SIL-IS) against analyte concentration and hence
expected quantitative performance of the respective meth-
ods not to be compromised. However, they cautioned
against using too high concentrations of SIL-IS because
reciprocal suppression of the analyte signal by the SIL-IS
might have a detrimental effect on the limit of detection
(LOD) of the method. Similar findings were reported by
Liang et al. [20], who studied nine analyteSIL-IS pairs in
APCI and ESI modes. In ESI mode, mutual ion suppression
of the analytes and the respective SIL-IS was observed,
whereas seven out of nine analyteSIL-IS pairs enhanced
each others ionization in APCI mode. Again, linear cali-
bration curves could be established despite these effects, if
the SIL-IS concentration was carefully chosen; potentially
negative effects on the assay sensitivity were also discussed.
2164
Remane et al. [61] studied 14 analyteSIL-IS pairs in both
ESI and APCI modes. With APCI, statistically significant
ion suppression was observed for one of the 14 tested stand-
ards only, whereas in ESI mode statistically significant
suppression effects were observed for 12 and significant
enhancement was observed for another. These authors there-
fore chose APCI for further development of their multi-
analyte assays [110, 111].
Seen together, these findings show that negative effects
of potential mutual ion suppression/enhancement of analy-
tes and their respective SIL-IS on sensitivity and quantita-
tive performance can be avoided or at least controlled by
careful selection of ionization mode and SIL-IS concentra-
tions. However, such effects can become a problem for
multi-analyte procedures in which the SIL-IS is not only
used as IS for the respective unlabeled analogue, but also for
a number of other analytes of the same or similar drug class.
For such assays, it is common practice for calibrators to
contain a mixture of the unlabeled analogue of the SIL-IS
and the additional analytes for which it is used as IS. For
routine samples, however, the unlabeled analogue will gen-
erally not be present when one of the other analytes is
detected. This means that ion suppression/enhancement of
the SIL-IS would be present in the calibrators but not in such
real samples, so substantial quantification bias may result.
For such multi-analyte procedures it is, therefore, essential
to either demonstrate that mutual ion suppression/enhance-
ment does not occur or to work with two separate sets of
calibrators, one containing only the unlabeled analogue of
the SIL-IS and one containing only the additional analytes
for which the latter is used as IS. The SIL-IS is added to
both sets of calibrators.
Matrix effects from co-eluting analytes
Ideally, multi-analyte procedures should enable separation
of all included analytes in a very short time; in practice,
however, it is generally necessary to make a compromise
between separation and run-time, meaning that overlap or
co-elution of some peaks has to be accepted. Given the high
selectivity of (tandem) MS, such overlap or co-elution is
generally not a problem from the perspective of identifica-
tion. However, co-elution could be a problem if it causes ion
suppression/enhancement of the co-eluting compounds. The
reasons are similar to those discussed for SIL-IS in the previ-
ous section. Any change of ionization caused by co-eluting
compounds present in a calibrator but not in a routine sample
could be detrimental to accurate quantification.
A systematic study on mutual ion suppression/enhance-
ment by co-eluting analytes was published by Remane et al.
[25], who tested for such effects among 47 co-eluting ana-
lyte pairs within six different drug classes (antidepressants,
F.T. Peters, D. Remane
neuroleptics, benzodiazepines, beta-blockers, oral antidia-
betics, and drugs measured in the context of brain-death
analysis) and 22 additional co-eluting drugs among these
groups. Analyte peaks were considered overlapping when
the overlapping peak area was estimated to exceed 25 %.
Each compound was analyzed in the absence and presence
of the co-eluting partner and absolute responses were com-
pared. All experiments were performed in the ESI and APCI
modes. In the within-group tests, five and six analytes were
affected by ion enhancement or ion suppression >25 %,
respectively, in APCI mode. The maximum effects were
large, ranging from 71 % to 190 %. In ESI mode, 16
analytes were affected by ion suppression effects >25 %
(maximum 73 %). In the between-group experiments, sig-
nificant ion suppression was observed for 13 out of 34
analytes in APCI mode and for 24 analytes in ESI mode.
In the latter mode, significant ion enhancement was ob-
served for one other analyte.
Another example of mutual ion suppression by co-eluting
analytes was described by Egge-Jacobson et al. [112]. Other
groups tested for such effects but did not observe any [68,
113115]. Nevertheless, it seems important that in multi-
analyte assays with co-eluting analytes the co-eluting analyte
pairs should be checked for mutual ion suppression or en-
hancement. If such effects are found, either separation must be
optimized in respect of these compounds or different sets of
calibrators must be used, each containing only one of the co-
eluting analytes. If the co-eluting analytes are coincidentally
present in the same routine sample, interpretation of quantita-
tive results for these analytes may be considerably biased.
Matrix effect studies in systematic toxicological analysis
(STA) procedures
STA for identification of toxicologically relevant com-
pounds in biological matrices is an important part of daily
routine work. Analytical methods for STA should, ideally,
cover hundreds of relevant drugs, poisons, and/or their
metabolites [4, 116, 117]. Screening procedures covering
at least hundreds of analytes relevant to clinical and forensic
toxicology have been published and reviewed several times
[4, 6, 8, 118120]. It is obvious that evaluation of MEs for
each of these compounds would be associated with an
unrealistically high workload and hence it is not surprising
that the ME studies in these methods were performed with a
subset of analytes more or less representative of the com-
plete set of analytes covered by the respective methods.
Weinmanns group described, and later updated, a screen-
ing method for serum and urine samples using a hybrid
triple-quadrupolelinear ion-trap instrument equipped with
an ESI source [121123]. In an earlier study by the same
group, MEs after liquidliquid extraction and silica-based
Matrix effects in clinical and forensic toxicology
and polymer-based solid-phase extraction (SPE) had been
systematically studied by use of the post-column infusion
approach [105]. Codeine and glafenine were chosen as
model compounds for positive and negative ionization, re-
spectively. MEs were only reported for the first two minutes
of the chromatogram. However, it is not clear if these results
are still representative for the current version of the method
[122] which uses simple protein precipitation or an alterna-
tive LLE method different from that described by Mueller et
al. [105].
Sauvage et al. [98] also used a similar instrument for their
STA procedure covering more than 1,000 drugs, toxic com-
pounds, and some metabolites. They used the post-column
infusion approach to check for potential MEs from polymer-
based SPE extracts of six different serum, urine, and whole
blood samples. The experiments were performed by simul-
taneous post-column infusion of 20 model compounds cov-
ering a wide range of retention times (5.5 to 18.5 min) and
polarity. No ion suppression was observed for any of these
20 compounds, but, as stated by the authors themselves, this
does of course not guarantee absence of MEs for all other
analytes. Another issue that might be raised at this point is
that the compounds being infused as a mixture may have
affected each others ionization. However, it seems unlikely
that relevant MEs arising because of such interactions would
have been overlooked.
In an LCtime of flight (TOF)-MS based method for
screening of basic drugs in the hair of drug addicts, Pelander
et al. [99] studied MEs by post-column infusion of selected
substances, but neither the number of substance nor the
basis for their selection was specified. Strong MEs were
reported for the early eluting analytes metformine and mor-
phine. In a similar method for screening of drugs in vitreous
humor, the same groups performed post-column infusion
experiments using seven model compounds of different
polarity and retention behavior and an extract of pooled
vitreous humor [100]. The observed MEs were moderate,
ranging from 18 % to +12 %, but no information was given
about the variability of matrix effects in vitreous humor
from different sources.
In an STA procedure using LCquadrupole-TOF-MS,
Broecker et al. [92] performed a full validation for 24
selected amphetamines, opiates/opioids, and benzodiaze-
pines, and for cocaine and its major metabolites. The vali-
dation procedure included evaluation of MEs using the post-
extraction addition approach and six different blank hair
samples. Interestingly, extensive suppression (58 % to
28 %) was observed only for the benzodiazepines alprazo-
lam, bromazepam, lorazepam, nordazepam, and oxazepam,
for which fairly large inter-sample variability (>15 % RSD)
was also observed. The authors of this study attributed this
phenom enon to the less basic properties of the
2165
benzodiazepines making them more prone to ion suppression,
which is clearly supported by the fact that no such effects were
observed for the more basic benzodiazepine metabolite 7-
aminoflunitrazepam, despite its otherwise very similar
structure.
Wissenbach et al. [101, 102] published an STA procedure
for urine covering more than 900 drugs and toxic com-
pounds and 2,300 metabolites. In both reports the authors
used Matuszewskis [22] simplified version of the post-
extraction addition approach. Six different blank urine sam-
ples were used in both studies. Altogether, 36 model com-
pounds covering the entire chromatographic run time were
used in these experiments. Most were parent drugs or phase
I metabolites of common antidepressants or drugs of abuse,
but five phase II metabolites were also included, so the
polarity of the compounds also covered a wide range. Major
MEs were observed for 13 of the 36 model compounds.
Most of these eluted early, confirming earlier observations
that MEs are most likely at short retention times [17, 19, 24,
29, 105]. However, some of the affected compounds eluted
later in the chromatogram emphasizing earlier observations,
that MEs may occur at any time during a chromatographic
run [19, 24].
In an LCTOF-MS-based screening procedure for urine
covering more than 300 common drugs and metabolites,
Lee et al. [103] also looked for MEs using the post-
extraction addition approach. Blank sample from eight dif-
ferent volunteers and 29 model compounds from different
drug classes and covering the entire retention time range
were used in their experiments. Major ion suppression was
observed only for the first-eluting analyte ecgonine methyl
ester, but it is remarkable that substantial and, in part,
extensive ion enhancement (up to +110 %) was observed
for several drugs eluting in the last quarter of the chromato-
gram. The variability of MEs between samples was gener-
ally low, but the compounds with major ion suppression or
enhancement also had higher variability of MEsup to
40 % RSD.
Extensive ME experiments for an LCMSn-based screen-
ing procedure were reported by Mueller et al. [104]. In their
fully automated procedure using online extraction with
turbulent-flow chromatography for toxicological screening
of urine, these authors used post-column infusion and a
modified post-extraction addition approach. In both experi-
ments, six different blank urine samples were used, includ-
ing five representative of pathological conditions to cover
the entire range of expected routine samples. Forty-seven
compounds not only covering the entire retention time and
mass range, but also representing multiple drug classes were
infused in groups of eight in the post-column infusion
experiments. The post-extraction addition experiments had
to be modified in comparison to the procedure originally
2166
proposed by Matuszewski [22], because spiking after ex-
traction is not possible with online extraction. Instead, a set
of spiked matrix samples and a second set of neat standards
were injected into the online extraction system. Thus, ex-
traction losses would be similar in both sets and, hence, the
presence and absence of matrix would be the only difference
between the two sets. With post-column infusion, MEs were
only observed in urine samples containing ketone bodies in
high concentrations and primarily at the beginning of the
chromatogram. Also with post-extraction addition, MEs
were only observed for a few analytes (five out of 47) and
were most pronounced for morphine (40 %). Moreover,
the effects observed were fairly consistent from sample to
sample and, if at all, were only marginally outside the
recommended acceptance limit of 15 % RSD [34, 36],
which seems acceptable for a purely qualitative method.
An LCMSMS method for screening of more than 710
drugs and metabolites in postmortem blood samples was
reported by Rosano et al. [73], who used the post-
extraction addition method for evaluation of MEs. More
details and results will be discussed below.
Other authors describing STA methods either have not
addressed the issue of MEs [124127] or have only dis-
cussed potential MEs without reporting any systematic
experiments to check for them [122, 128131]. Although
this may be acceptable for early publications, when aware-
ness of MEs was still limited, it seems very problematic for
more recent procedures, because potential problems associ-
ated with MEs should now be common knowledge among
analysts in clinical and forensic toxicology. Clearly, ion
suppression can lead to a higher limit of detection and hence
also compromise the performance of qualitative methods. In
the authors opinion, it should therefore be mandatory for
future publications on LCMS-based STA procedures that
experiments on MEs have been performed.
As long as the methods are purely qualitative in charac-
ter, the post-column infusion approach seems the most ap-
propriate, because it provides information over the entire
chromatogram, whereas the information obtained by post-
extraction addition is inherently limited to the retention
times of the test compounds. In any case it seems important
that ME experiments are performed with a sufficiently large
number of model compounds, at least 2030, that represent
the different classes of compounds. When using the post-
extraction addition approach, it is also essential the com-
pounds cover the entire retention time range. With regard to
acceptance criteria for MEs in such purely qualitative meth-
ods, the requirements for variability between samples could
be less strict than the 15 % RSD (20 % RSD near the lower
limit of quantification) proposed for quantitative methods [34,
36], although it might be important to stipulate a limit for the
average extent of ME to minimize the risk of analyte signals
being suppressed below the limit of detection (LOD).
F.T. Peters, D. Remane
Matrix effects in postmortem toxicology
In forensic postmortem toxicology, the complexity of post-
mortem matrices, for example organ tissues, or the state of
decomposition of the samples to be analyzed poses a par-
ticular challenge to any analytical toxicologist. It is therefore
surprising that, despite the increasing use and importance of
LCMS methods in postmortem toxicology, few authors
have specifically addressed the issue of MEs in postmortem
samples.
Saar et al. [72] systematically compared MEs of 19 anti-
psychotics drugs in human antemortem and postmortem
blood after nine different LLE procedures (combination of
three buffers and three extraction solvents) and one SPE
method. The postmortem blood samples were further clas-
sified as non-decomposed or severely decomposed, depend-
ing on the state of decomposition. The final LLE method
combining Trizma buffer and 1-chlorobutane as extrac-
tion solvent and the SPE method were further compared
with regard to extraction efficiencies and MEs. The
experimental set-up was a classical post-extraction addi-
tion approach and five individual blank samples for
each antemortem, non-decomposed, and decomposed
postmortem blood were used for spiking. Median MEs
observed for antemortem and non-decomposed postmor-
tem blood were essentially the same after LLE, and
ranged from 30 to +59 % and from 29 to +45 %,
respectively. Although median MEs for the decomposed
postmortem blood were similar (23 to +25 %), the
variability between the individual decomposed samples
was much higher than for antemortem and non-
decomposed blood. After SPE, median MEs for ante-
mortem and postmortem blood were, again, more or less
comparable whereas the variability of the effects for
decomposed postmortem samples was much higher than
for the other two sample types.
Gergov et al. [70] checked for combined MEs and ex-
traction efficiency by analyzing duplicates of 15 different
autopsy blood samples spiked with a mixture of 25 opioid
drugs at medium concentrations. Thereafter, they compared
the variability of the determined concentrations within and
between samples. MEs were considered to be present when
one-way analysis of variance (ANOVA) indicated that the
RSD between samples was significantly larger than within
samples. Although this approach does cover the most im-
portant property, namely the variability of MEs from sample
to sample, it does not enable differentiation of effects caused
by extraction from those caused by ion suppression or
enhancement. Moreover, it does not provide any information
about the absolute extent of ion suppression or enhancement.
Rosano et al. [73] compared MEs for blood bank samples
with those for blank blood from five non-decomposed and
five decomposed cases. Five drugs from different drug
Matrix effects in clinical and forensic toxicology
classes were used as model drugs. Average ion suppression
tended to be higher for postmortem samples than for blood
bank samples. Moreover, the variability of MEs between
samples tended to be higher for postmortem samples, espe-
cially decomposed samples.
Sorensen et al. [66] compared MEs for metformine,
phenformine, and buformine in antemortem and postmortem
whole blood samples after protein precipitation followed by
weak cation-exchange SPE. These authors also used the
post-extraction addition approach and 20 blank samples of
each antemortem and postmortem blood. Only very minor,
if any, MEs were observed for both sample types, the
explanation of which is most certainly that the samples were
thoroughly cleaned up during two-step sample preparation
involving both protein precipitation and SPE.
In their method for determination of benzodiazepines
and the so-called z-drugs zopiclone and zaleplone in
whole blood, Simonsen et al. [65] also studied MEs
using the post-extraction addition approach and a total
of six different authentic antemortem and postmortem
whole-blood samples. The average MEs were generally
much lower than 20 %. However, the meaning of these
findings for postmortem toxicology in general remains un-
clear, because neither the number of postmortem samples nor
their decomposition status was specified. Moreover, no infor-
mation on the variability of MEs between samples was
reported.
Kristoffersen et al. [69] used post-extraction addition and
six different lots of autopsy blood to study ME during
quantification of 14 cardiovascular drugs. Again the average
MEs were rather low, ranging from 6 % to +14 %. The
between-sample variability was within the proposed limit
of <15 % RSD. The respective RSD values, after taking into
account the IS diazepam-d5, were only marginally and cer-
tainly not significantly lower, which is not surprising con-
sidering that the IS was not the SIL analogue of any of the
analytes.
Taylor et al. [67] published a method for determination of
morphine and its glucuronides in whole blood using LCMS
MS. The validation study included experiments on MEs in
which the post-extraction addition approach and blank blood
from five different postmortem cases were used. No major
MEs above 15 % were observed in this study, although sample
preparation involving SPE on a C18 cartridge was not very
selective and retention times were short. In contrast, Herrin et
al. [128] reported 97 % reduction of the morphine signal in
screening for 400 drugs in whole blood.
Finally, Roman et al. [64] reported particularly strong
MEs for one of five tested postmortem blood samples after
LLE of seven neuroleptic drugs with tert-butyl methyl ether
and LCMSMS analysis. Seen together, the results of these
systematic evaluations of MEs for postmortem blood show
that methods intended for postmortem toxicology should
2167
consider a much higher risk of MEs. In the authors opinion,
validation of LCMS(MS) methods intended for use in
postmortem toxicology should, therefore, always include a
thorough investigation of potential MEs using authentic
postmortem material. Ideally, non-decomposed and decom-
posed samples should be treated as different matrices and
hence be evaluated separately.
Entomotoxicology, i.e., toxicological analysis of the dif-
ferent developmental stages of insects feeding on cadavers,
is a specific area of postmortem toxicology that can be
applied when no or only severely decomposed tissue is
available for analysis. Gosselin et al. [132] recently de-
scribed a method for determination of methadone and its
main metabolite EDDP in third instar larvae, pupae, and
adults of Lucilla seratica, a carrion-feeding fly relevant to
forensic entomology. The study included evaluation of MEs
for all three developmental stages using both the post-
column infusion and post-extraction addition approaches.
Although no significant ion suppression was observed in
the post-column infusion experiments, minor to moderate
ion suppression was observed in the post-extraction addition
experiments. Inter-sample variability was remarkably low
which is most probably attributable to the controlled labo-
ratory conditions under which the larvae, pupae, and adult
flies were kept.
Conclusions and perspectives
Although the message that study of matrix effects is an
essential part of the development and validation of LC
MS(MS)-based methods has been received by most clini-
cal and forensic toxicologists, the number of blank matrix
sources in the respective experiments is often below the
recommended minimum number of five or six. Moreover,
there seems to be no general awareness that the variability of
MEs between samples is at least as important as the average
extent of these effects.
Concerning issues of particular relevance to clinical and
forensic toxicology, this overview shows that avoiding ma-
trix effects can be difficult for multi-analyte and STA pro-
cedures, because even sophisticated sample-preparation
techniques do not always eliminate such effects. In addition,
chromatographic separation is generally predetermined by
the requirement to separate multiple analytes, leaving less
freedom for optimization of the separation to minimize
matrix effects. SIL-IS generally compensate efficiently for
the detrimental effects of MEs on quantification, but do not
compensate for their detrimental effects on method sensitiv-
ity. Problems with such standards may occur if they are
simultaneously used for the respective unlabeled analyte
and for other analytes. Co-elution of analytes must be taken
into account for any multi-analyte procedure. Finally,
2168
development and validation of STA methods should include
matrix-effect experiments with selected analytes from differ-
ent drug classes covering the entire retention time range.
Acknowledgement The authors thank Dirk K. Wissenbach for his
assistance.
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