Special Report

Special Focus Issue: Sample Extraction Techniques 

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An update on solid phase-supported liquid

extraction

Solid phase-supported liquid extraction (SLE) is a technique almost 40 years old being Luigi Silvestro*,1
rediscovered in the last few years due to its simplicity, optimal for automation and & Simona Rizea Savu1
giving very clean extracts with minimal matrix effects when analyzed by techniques
1
3S-Pharmacological Consultation
& Research GmbH, Koenigsberger
like HPLC–MS/MS, GC–MS/MS, CE–MS/MS. In the next paragraphs the evolution of
Strasse 1 27243 Harpstedt, Germany
SLE, according to literature, will be presented first, followed by some considerations *Author for correspondence:
on the SLE material now available and a typical protocol of work. To conclude, Tel.: +49 424 495 083
considerations based on the author’s practical experiences with SLE will be done, as dreispharmasl@aol.com
well as few remarks on potential future areas of SLE development.

It is first of all important to clarify the mean-
ing of SLE in the context of this article: this
acronym in fact is often used to indicate,
in a wide sense, a solid phase liquid extrac-
tion. Procedures of this kind are very old and
widespread, used not only in chemistry but
also to prepare perfumes, to obtain herbal
extracts and in the daily life to brew a coffee
or prepare a tee. It is clear that this is not the
target of this article where, more specifically,
applications of solid phase-supported
liquid extraction methods will be dis-
cussed in view of analytical applications; no
special acronym was introduced for this tech-
nique and SLE is normally accepted risking
to create some confusion. In these methods,
as previously indicated, liquid extractions are
carried-out with the help of a solid media
employed as support for the liquid sample to
be extracted with an appropriate solvent.
It is important to briefly discuss the dif-
ference between SLE and SPE which also
employs a solid phase media. In the case
of SLE the solid phase has only the aim to
adsorb the liquid sample, ideally without
interactions with the analyte to be extracted,
creating a thin layer of sample that can be
extracted with a nonmiscible solvent without
leaking the sample itself. The solid phases of
SPE on the contrary are developed to selec-
tively bind the analyte/s without retain-
ing the sample matrix that can be easily
washed away before eluting the analyte with
an appropriate solvent. It is so far clear that
the difference of solid phase, sample and
analyte/s interaction make the two-sample
preparation approach completely different.
It is finally interesting to see the main
similitudes and differences of SLE with the
more classical LLE:
• Similitudes:
–– In both cases a liquid sample (free
or absorbed on particles) is extracted
with a nonmiscible solvent;
–– The extraction efficiency is determined
in both cases by the analyte solubility
in the extraction solvent, in case of ion-
izable analyte/s pH conditions being
important to modify the partition
between phases getting a quantitative
extraction. In principle the solid phase
of SLE is not relevant in the extraction
selectivity even if experimental results
show sometime particular interactions
with matrix components;
–– Clean samples, generally with less
matrix (compared with protein pre-
cipitation or SPE), are normally
part of
obtained;

10.4155/bio.15.144 © 2015 Future Science Ltd Bioanalysis (2015) 7(17), 2177–2186 ISSN 1757-6180 2177
Special Report  Silvestro & Savu
Key terms
Solid phase-supported liquid extraction: Liquid
extraction method in which a solid media is used to
support the liquid samples.
High-throughput sample processing: Sample
preparation method permitting the fast preparation of a
large series of analytical samples.
Diatomaceous earth: Most commonly used solid media
for solid phase-supported liquid extraction. It is formed by
fossil diatom.
Matrix effect: Influence of sample components, beside
the analytes of interest, on sample detection, in particular
analyte ionization in the case of MS.
–– As expected, analytes not soluble in organic
solvents are not separated by both type of
extractions.
• Differences:
–– Separation of the extraction solvent from the
samples and a careful removal are typically
needed in case of classic LLE. This tedious and
slow step is not needed in SLE;
–– Some samples tend to form emulsions or
gelify when mixed with certain extraction sol-
vents making difficult phases separation. The
problem is minimal in case of SLE;
–– SLE operations can be easily performed in
automated liquid handling units, ideal for
high-throughput sample processing,
while LLE, in case a total recovery is needed,
are very difficult to automatize;
–– Consequently, classical LLE is more time
consuming, requires manual handling and
preparation error can appear more often.
A literature review on SLE
At the beginning of the 19th century (1836–1837)
diatomaceous earth, a natural product, was discov-
ered in Germany. This kind of material, formed by fos-
silized diatoms, gained rapidly a lot of attention for its
unique porous properties ideal for filtration as well as
liquid adsorption [1] ; it is clear that this is not the date
of SLE discovery but it is an important step aiming to
an adequate solid support on which to adsorb liquid
samples. In the second half of the 20th century the
development of analytical chemistry and chromato-
graphic separation techniques had a very important
role. Sample clean-up and extraction methods gained a
lot of attention because complex samples like the bio-
logical ones cannot be, in general, directly analyzed
especially in case of hyphenated methods with a first
2178 Bioanalysis (2015) 7(17)
separative step: LLE as well as protein precipitation
were first employed.
Making a search on the web for supported liquid
extraction, and/or Extrelut (the first commercially
available SLE product developed by Merck, Dar-
mastadt, Germany) shows in 1976 [2] an interesting
article presenting the application of Extrelut columns
for the extraction of drugs from biological samples
(plasma and urines) in comparison with the more clas-
sical LLE; this kind of extraction was also subject for
an article in 1978 [3] describing a method to analyze
antiepileptic drugs by GC. It is noteworthy that SLE
can be used also with other analytical methods beside
chromatography as it is presented in a study of 1980 [4]
describing a new radioimmunoassay method for testos-
terone employing this extraction to pretreat the sam-
ples. Due to the very good extractions achievable by
SLE, applications in whole blood and gastric juice were
also reported [5] . In the same period, a paper dealing
with cyclosporine analytic [6] showed that SLE, apart
from facilitating extraction procedures, permitted to
get cleaner samples compared with other preparation
methods. Applications on metabolites identification
were also published showing that several analytes with
different polarity can be extracted in a single step [7] .
Already in 1986, interesting SLE methods were devel-
oped for veterinary applications in complex matrixes
like tissues [8] ; in the same field SLE techniques to
detect growth promoters (e.g., clenbuterol), at low
concentrations were validated [9] .
A comparison between SLE and a column switch
method was published in 1991 putting in evidence the
need for fast, robust, reliable analytical methods and
the positive aspects of supported liquid extraction [10] .
Whole blood samples extraction, always technically
quite difficult, was again object of a study in 1992
focused on benzodiazepines detection [11] . SLE was
also successfully employed to determine molecules of
toxicological interest in man like cotinine and nico-
tine (urine samples) in 1998 [12] or buprenorphine
and its main metabolite from postmortem samples in
1997  [13] ; in the same year this extraction approach
was also introduced for cotinine and nicotine in hair
samples after digestion [14] . An application of SLE
to determine pesticide in a very difficult matrix like
fruit/vegetables appeared in 1999 [15] . A quite interest-
ing paper of 2001, despite devoted to purification of
compound libraries, shows a nice comparison between
SLE and LLE presenting beautiful data on selectivity
and solvent selection [16] .
In the following years applications of SLE further
expanded even if the wider diffusion of LC–MS/MS,
by means of its high specificity, permitted to use less
selective extraction procedures as, for example, protein
future science group

precipitation. SLE remained, however, an interesting
extraction procedure when clean extracts were critical
for the separation methods, as in the case of chiral deter-
minations  [17,18] . The application of SLE gained also
interest due to the possibility to obtain a high-through-
put samples processing facilitated by the introduction
of 96-well plates ideal for extraction processing [19] , as
presented in a 2009 paper on hydrocortisone [20] and
another of 2010 on fluoxetine [21] .
In the last 10 years the so called ‘matrix effect,’
typically due to peaks from unknown substances pres-
ent in biological samples coeluting with the analytes to
measure, in LC–MS/MS [22] obliged to reconsider the
extraction methods in order to minimize the matrix
effect; in this context SLE has proved to be a quite effi-
cient approach to minimize such problem as discussed
in a 2010 article [23] and exemplified in a 2012 study
using ten molecules as model and comparing different
extraction approaches [24] . Recently, the application of
SLE showed a growing interest in all domains when
specificity and high-throughput sample processing are
important as presented in a 2010 article [25] on Erlo-
tinib and in a 2013 work on elvitegravir and ritona-
vir [26] ; in the first paper a comparison of SLE with LLE
was also carried-out showing a better recovery with the
first method. Several clinical chemistry applications of
LC–MS have been also presented like aldosterone in
plasma  [27,28] and estrogens [29] showing optimal sen-
sitivity and reproducibility. Interesting research papers
have also presented high-throughput applications in
toxicology and forensic chemistry [30,31] . Besides the
presented, new applications are constantly appear-
ing and being published demonstrating the growing
relevance of this extraction approach.
Type of columns commercially available
This section does not intend to be a guide to pur-
chase SLE material while it will present the relevant
and important features of some products commercially
available to may have them in mind when developing a
new SLE; a more complete list can be found in a recent
(2012) paper [32] .
As already mentioned, Extrelut (Merck Millipore)
was the first commercial SLE product and for many
years remained the only one. In the following years
Agilent has launched a similar product called Hydro-
matrix, Thermo a product named HyperSep SLE and
Biotage other products marketed as Isolute SLE+ and
HMN; some other companies are also present with
SLE material on the market but it is not the scope
of the paper to make a full list of them. It is first of
all important to observe that, differently form SPE,
where a large number of solid phases are available, the
SLE materials are quite similar, being diatomaceous
future science group
An update on solid phase-supported liquid extraction Special Report
earth the common constituent in all cases. However,
ISOLUTE columns (Biotage), differently from other
products, are packed with two different kinds of dia-
tomaceous earth with coarser or smaller particles and
a narrower distribution of particle size. The HMN
material is closer to the packing of other products on
the market while the newer type, called SLE+, is based
on smaller particles. As a consequence of the larger
surface of the smaller particles for the same weight,
when compared with HMN, higher volume of buff-
ered plasma can be loaded on SLE+ then on HMN.
It is clear that in case of SLE+, due to higher flow
resistance of this packed material, the column loading
and elution need almost invariably positive or negative
pressure while in HMN it is not mandatory. Another
feature that can be observed in some SLE columns, like
the ones of Agilent, is the availability of diatomaceous
earth washed, before the final drying, with different
buffer. This treatment has been introduced in order
to help keeping the added sample at a pH optimal for
the solvent extraction; it is however clear that the sam-
ples can be premixed with a buffer at the desired pH
obtaining the same final results. The columns for SLE
are available in a wide range of formats going from 96
well plates to large (up to 60 ml) columns, the later
are in some cases of glass to minimize contaminants
coming from plasticware. The type of columns, first
to support the packing and the level of contaminants
present in the diatomaceous earth can be quite differ-
ent between producers and in case of difficult extrac-
tion can be worthy to test different materials. Finally
shall be considered also that some producers are offer-
ing bulk material in case really large extractions has to
be performed.
A standard protocol of SLE
The steps normally followed in a SLE, based on
instructions of columns by the producers mentioned
in the previous paragraph, are presented in Figure 1
and are the following (note: remember that no column
preconditioning is requested):
• Sample preparation, spiking with IS: assuming that
an IS is used, as normally performed for any extrac-
tion, it is a good practice to add it in the biologi-
cal samples in separate tubes leaving an adequate
time of mixing (i.e., 10 min) for equilibration in
the matrix. An addition of IS in an aqueous phase
or diluted biological matrix is preferable to avoid
risks of sample precipitation by solvent;
• Sample preparation, dilution or sample buffering: in
samples rich in proteins, like plasma, a dilution 1/1
with water or a buffer is recommended to make eas-
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Special Report  Silvestro & Savu
Supported liquid extraction process
Step 1: Step 2:
Plasma + buffer + IS Add mix from step 1
Sample absorbed
onto column
(no flow through)
Figure 1. Main steps of a solid phase-supported liquid extraction.
ier the loading on the column. Higher dilutions are
also OK but it must be considered that the volume
of liquid to adsorb is limited and corresponds to the
total volume of plasma + IS + diluting solution (or
buffer) and so far excessive dilutions will decrease
sensitivity. In case acidic or basic analyte/s have to
be extracted, buffers are added in this step to maxi-
mize the solvent extraction as performed in a clas-
sic LLE. In case of samples with low proteins, like
urine, the dilution is not needed but the addition of
IS and buffering, if necessary, has to be performed;
• Column loading: at this point, the samples are
ready for column loading. The maximum load-
ing volume recommended by the column producer
has to be respected in order to avoid a leaking of
the unextracted samples by the column; the use
of smaller volumes on the contrary is possible. In
this step, clear differences exist between columns
depending from the kind of solid support. Diato-
maceous earth with large particles permits a sam-
ples loading just by gravity while materials with
small particles need the use of negative or positive
pressure in order to get the samples permeate the
column packing. Interestingly, the packing are
retaining quite well the samples and generally also
with a prolonged application of positive or negative
pressure there is no leaking of the aqueous sample;
instructions from the producer are again relevant
to select the right conditions;
• Equilibration step: it is important to consider that
an equilibration step of at least 5 min is generally
suggested after full penetration of the sample in the
column;
• Samples extraction: at this point the columns can
be finally extracted with an appropriate nonmis-
2180 Bioanalysis (2015) 7(17)
Step 4:
Add organic
solvent
Step 3:
Rest 5 min
cible solvent. Solvent volumes two- to four-times
the volume of the samples (plus dilution buffer
and IS) are used, smaller and larger volumes can
be employed. Also in this step, positive or negative
pressures are often needed to facilitate the process;
• Evaporation: the solvent extracts can be finally
dried with a stream of gas or vacuum and then
redissolved for the chromatographic analyses.
In all these procedures, if a LLE method already
exist, it can be generally transferred directly to the
SLE method keeping in mind that volume optimiza-
tion of buffer and solvents may be needed, most often
reducing the extraction solvent volume; also in this
case indications by the column producers are a good
starting point.
As a last remark the use of high density organic sol-
vent like chloroform, that is sometime strongly recom-
mended [32] , permits an easier filtration but, especially
if positive or negative pressure are employed to facili-
tate filtration, is not mandatory and any other solvent
is allowed.
Our direct SLE experience
Despite the relevant number of papers published on
SLE and beside materials from different SLE produc-
ers, no comprehensive review is available in literature;
it has been considered so far interesting to introduce a
summary on methods developed by the authors on a
wide range of pharmaceuticals in biological samples.
After a few experiences on clenbuterol, back in 1991,
and on betahystine in 1996, several methods have
been developed in our HPLC–MS unit for PK stud-
ies beginning with 2008 when it has been decided to
implement liquid handling robotic systems, in our case
STARlet models by Hamilton, and the automatiza-
tion of classic LLE proved to be quite difficult. The
main problems in these applications were the difficulty
future science group
 An update on solid phase-supported liquid extraction Special Report

to avoid emulsion or gel formation and then how to extraction solvent previously used. The LLOQ were
adequately aspirate samples detecting the phases inter- at least the same of liquid–liquid methods but most
face. The pipetting of solvents with very low boiling often better and the extraction solvent volumes were
point, like hexane, ethyl ether and dichloromethane, 30–50% less by SLE. As it can be seen only in four
was another challenging task. methods, the extractions were performed in 1-ml col-
Table 1 summarizes the methods we developed, umn while 96-well plates, ideal for automation and
using ISOLUTE SLE+ (Biotage) plates and columns, high-throughput sample processing, were adequate
the relevant information regarding the methods as for the remaining molecules. The reported volumes
well as the LLOQ achieved. In 30% of molecules of plasma were always below the generally recom-
we had a previous LLE method that could be easily mended  [32] volume ratio of 1 ml plasma or aqueous
transformed in SLE; in such cases we always kept the sample per G of sorbent but it must be considered that
Table 1. Application of solid phase-supported liquid extraction to PK plasma samples.
Analyte LLOQ (pg/ml) Isolute SLE+ sorbent (mg) Plasma volume (μl) Extraction solvent
Acenocoumarol 5000 200 150 Diethyl ether
Alverine 5 200 150 TBME
Amlodipine 50 200 125 Ethyl acetate\hexane 1\1
Anastrozole 200 200 150 Dichloromethane\diethyl ether 3\7
Aripiprazole 250 200 150 Diethyl ether
Budesonide 5 400 300 Dichloromethane
Buprenorphine 6 400 300 Ethyl acetate\hexane 1\1
Chlormadinone acetate 66.666 200 150 Diethyl ether
Cyproterone acetate 300 400 250 Diethyl ether
Desloratadine 25 200 150 TBME
Desogestrel 100 1000 500 Hexane
Dienogest 500 400 250 Diethyl ether
Donepezil chiral 50 400 150 TBME
Drospirenone 500 200 200 Diethyl ether
Ebastine 35 200 150 Dichloromethane\diethyl ether 1\1
Esomeprazole 5000 200 200 Diethyl ether
Ethinylestradiol 2 1000 700 Diethyl ether
Etonogestrel 30 1000 500 Hexane
Exemestane 111 200 200 Diethyl ether
Ezetimibe 50 200 130 TBME
Fluticasone 2 400 300 Dichloromethane
Hydrochlorothiazide 1 200 100 Ethyl acetate\diethyl ether 2\1
Isradipine 33.333 400 250 TBME
Lorazepam 500 200 100 Dichloromethane\toluene 3\7
Paliperidone 200 400 100 TBME
Phenprocoumon chiral 2500 400 250 Diethyl ether
Progesterone 50 1000 500 Hexane
Rivastigmine 50 400 300 TBME
Salmeterol 1.5 400 300 Diethyl ether
Solifenacin 200 200 150 TBME
Zolmitriptan\N-demethyl 100 200 200 Ethyl acetate\dichloromethane 4\1
zolmit
SLE: Solid phase-supported liquid extraction; TBME: Tert-butyl-methyl-ether.

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Special Report  Silvestro & Savu

1000 1000
960 960
920 920
880 880
840 840
800 800
760 760
720 720
0.32
680 680
640 640
Intensity (cps)

600 600
560 560
0.21
520 520
480 480
440 440
0.91
400 400
360 360
0.43
320 320
280 0.47 0.90 280
240 1.41 240
200 200
0.55 0.69 0.80 1.21
160 160
120 120
80 80
40 40
0 0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 0.2 0.4 0.6 0.8 1.0 1.2 1.4
Time (min) Time (min)
Blank LLE Blank SLE
0.93
2000
2000
1900
1900
1800
1800
1700 1700
1600 1600
1500 1500
0.29
1400 1400
1300 1300
Intensity (cps)

1200 1200
1100 1100
1000 1000
900 900
800 0.89 800
700 700
600 0.47 600
500 0.03 500 0.22
400 400
1.33
300 0.80 300 0.44

200 200
100 100
0 0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 0.2 0.4 0.6 0.8 1.0 1.2 1.4
Time (min) Time (min)
LLOQ LLE LLOQ SLE
Figure 2. MS/MS chromatograms of rivastigmine following LLE or solid phase-supported liquid extraction of
blank plasma or spiked at LLOQ level (see facing page).
SLE: Solid phase-supported liquid extraction.

2182 Bioanalysis (2015) 7(17) future science group

 An update on solid phase-supported liquid extraction Special Report

300 300
290 290
280 280
270 270
260 260
250 250
240 240
230 230
220 220
210 210
200 200
190 190
Intensity (cps)

180 180
170 170
160 160
150 150
140 140
130 130 3.37
120 120
1.32 1.93
110 110
100 100 0.48 2.18
90 90 0.42 0.91 1.58
80 3.86 80 0.17 3.56
70 70 1.75 3.19
3.61
0.75
60 60 3.72
2.67
50 50 3.03
40 2.23
40
30 3.78
30
1.80
20 0.51 0.99 1.361.70 1.87 2.42 20
0.32 0.75 1.08 2.04 2.812.933.153.28
3.55
10 10
0 0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Time (min) Time (min)
Blank LLE EE Blank SLE EE
330 300
320 290
310 280
300 270
290 260
280
250
270 1.94
240
260
250 230
240 220
230 3.96 210
220 200
210 190
Intensity (cps)

200 180
190 1.95
170
180 1.08
160
170 150
160
140
150 2.68 3.82
140 130
130 0.44 1.65 120
120 110
110 100
1.30 1.59
100 90
90 3.30 3.37 3.97
80 0.87
80 1.27 2.34 2.44 2.22
1.52
70 0.47 2.80 3.22 3.55
70 0.25 2.68 2.93 3.60
3.04 60
60 0.92 2.86
50 3.00 3.67
3.60
50 0.21
40
40 3.48
30 30
20 20
10 10
0 0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Time (min) Time (min)
LLOQ LLE EE LLOQ SLE EE

Figure 3. MS/MS chromatograms of ethinyl estradiol following LLE or solid phase-supported liquid extraction of
blank plasma or spiked at LLOQ level.
SLE: Solid phase-supported liquid extraction.

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Special Report  Silvestro & Savu
IS and/or buffer were always added reaching the above
optimal ratio. Only with six molecules dichlorometh-
ane was employed, alone or in a solvent mixture, due
to solubility characteristics of the analytes confirming
that efficient extractions can be performed also with
low-density solvents.
In several cases SLE presented a cleaner back-
ground and/or a better signal-to-noise ratio due to
matrix effect reduction improving the LLOQ deter-
mination. Figures 2 & 3 show the results obtained, by
HPLC–MS/MS, on rivastigmine (Figure 2) and ethi-
nyl estradiol (Figure 3) determined in plasma blank or
spiked at LLOQ levels following LLE or SLE. As it
can be clearly seen, the signal-to-noise ratio in the SLE
samples is clearly better at LLOQ level most probably
due to a reduction of matrix effect as well to a cleaner
background.
All together the results obtained pushed us to
abandon completely LLE in favor of SLE performed
with a liquid handling system or, during method
development, manually.
Conclusion
At the end of this presentation it can be concluded
that SLE has all advantages of LLE, in particular
clean extract and the possibility to pre-concentrate
samples, with the advantage to may be easier adapted
to high-throughput sample processing. The simple
transfer from LLE to SLE, without complex devel-
opment experiments to find adequate solvents and
conditions, is another relevant point in favour of
SLE especially in comparison to a change from LLE
to SPE. Solvent consumption is generelly reduced in
comparison to LLE and the overall costs per samples
are comparable to LLE, when considering also the
operator time, or SPE.
Executive summary
• Solid phase-supported liquid extraction is a technique almost 40 years old going through a phase of rediscovery.
• An optimal and simple way to bring the well known advantages of LLE in automation friendly procedures.
• LLE procedures can be transferred to SLE without complicate experimental work.
• SLE means reduced use of solvents in comparison to LLE.
• Samples extracted by SLE have, in general, less matrix effects than those prepared by other common extraction
procedures.
References
1 Hull WO, Keel H, Kenney J, Gamson BW. Staff-industry
collaborative report diatomaceous earth. Ind. Eng. Chem.
45(2), 256–269 (1953).
2 Breiter J, Helger R, Lang H. Evaluation of column
extraction: a new procedure for the analysis of drugs in body
fluids. Forensic Sci. 7(2), 131–140 (1976).
3 Schweizer K, Wick H, Brechbühler T. An improved method
for preparation of samples for the simultaneous assay of some
2184 Bioanalysis (2015) 7(17)
All together SLE represents a step forward to high-
throughput sample processing without compromise
analytical performances.
Future perspective
Possible future improvements to SLE might be in the
following areas:
• Development of new solid phases for SLE, probably
replacing diatomaceous earth with synthetic mate-
rials, with optimized absorption and, hopefully,
more reproducible results.
• Improvement of available packing materials,
optimized for automatic processing.
• Chemical modification of the solid-phase to
increase the removal of phospholipids and other
matrix components responsible of ionization
suppression.
• On-line SLE, at the moment may looks a dream
but it cannot be excluded.
We hope that these goals can be one day achieved;
in the meantime, it can be expected a continuous
development of new analytical methods based on solid
phase-supported liquid extraction.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involve-
ment with any organization or entity with a financial inter-
est in or financial conflict with the subject matter or mate-
rials discussed in the manuscript. This includes employment,
consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this
manuscript.
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