Received: 28 January 2021 Revised: 25 May 2021 Accepted: 27 May 2021

DOI: 10.1002/wfs2.1436

OVERVIEW

An overview of sample preparation in forensic toxicology

Sabra Jones | Courtney McGowan | Sarah Boyle | Yiling Ke |

Chi Hin Marco Chan | Hajin Hwang

Biomedical Forensic Sciences, Boston

University School of Medicine, Boston,
Abstract
Massachusetts, USA Sample preparation is an important step in forensic toxicological analysis.
With the technological advancements and the availability of mass spectrome-
Correspondence
Sabra Jones, Biomedical Forensic ters with increasing sensitivities, the need to remove potential interferences,
Sciences, Boston University School of such as matrix components or non-relevant analytes to the analysis, is essen-
Medicine, Boston, MA.
tial to optimizing an analytical method. Forensic toxicological analysis
Email: [email protected]
involves diverse analytes with a variety of chemical properties that need to be
Edited by Simon Elliott, Editor considered when selecting an appropriate sample preparation technique. The
push for broad standards and best practice documents for forensic toxicology
demonstrates the need for laboratories to ensure they meet the required capa-
bilities to not only detect specific classes of drugs commonly encountered in
forensic case work, but also ensure method validity. The objective of this arti-
cle is to provide the forensic toxicology community a high-level overview of
biological matrices and their relevant components that should be considered
during sample pre-treatment as well as traditional and non-traditional sample
preparation techniques and present general applications.

This article is categorized under:

Toxicology > Drug Analysis

KEYWORDS
analytical chemistry, biological matrices, forensic toxicology, sample preparation

1 | INTRODUCTION

Forensic toxicology involves the analysis of biological matrices such as blood and urine, for the detection and
quantitation of drugs as well as other compounds including poisons (“Princ. Forensic Toxicol.”; Levine &
Kerrigan, 2020). There are numerous techniques that range from simple to rigorous to prepare samples for analysis.
These include dilutions (e.g., dilute and shoot), phospholipid depletion, liquid–liquid, supported liquid, and solid phase
extractions as well as others. Even within each type of extraction, there is variability in the complexity and number of
steps involved as well as amenability to automation. These techniques can be scaled to accommodate the matrix type
and sample volume. Further, as the complexity of the matrix increases from simple fluids (e.g., urine) to solids
(e.g., tissue or bone), the process can be adjusted accordingly to disrupt the matrix so that the target analytes are
released and can be recovered.
Specific compounds may exhibit enhanced extraction recovery with one technique over another. Certain techniques
can be utilized to target diverse groups of compounds while others will be more compound or class specific. Choosing
the ideal sample preparation technique for the situation helps to ensure successful analysis (Table 1). Typical biological

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TABLE 1 Critical applications in sample preparation in forensic toxicology

Sample preparation
method Critical applications
Liquid–Liquid • Traditional sample preparation technique employed for the analysis of acidic, neutral and alkaline drugs
from biological fluids that is based on the analyte's chemical properties (Juhascik & Jenkins, 2009;
Wells, 2018).
Solid phase • Extensive sample preparation technique to remove matrix and other interferences.
• Used for targeted drug analysis in general unknown screening (Wells, 2018).
• Efficient alternative sample preparation method to LLE in targeted drug analysis of postmortem samples
such as blood, urine, bile and/or tissues (Juhascik & Jenkins, 2009; Wells, 2018).
Supported liquid • Efficient and effective sample preparation technique with limited steps.
• Extraction of THC and metabolites from whole blood and oral fluid for LC–MS/MS analysis (Sauve
et al., 2012).
• Extraction of a drugs of abuse panel from oral fluid using supported liquid extraction (Sauve et al., 2012).
• Extraction of opiates and benzodiazepines from whole blood (Sauve et al., 2012).
Phospholipid depletion • Minimal sample preparation technique that utilizes a solid sorbent bed for filtration with or without a
preceding protein precipitation step.
• Profiling phospholipids in biological fluids using HybridSPE-PL for enrichment in pathological studies (Lu
& Ye, 2021).
• HybridSPE is used for sample preparation on plasma specimens to reduce the interference of phospholipid
matrix in HILIC LC–MS analysis (Aurand, 2021).
Dilution • Using dilute and shoot method to extract commonly prescribed antihypertensive medicine from urine
(Lawson et al., 2016).
• Detection of polar drugs in horse plasma and urine with dilute and shoot method as sample preparation
(Kwok et al., 2016).
• Dilute and shoot sample preparation method coupled with LC–MS analysis is mostly used in detection and
quantitation for stimulants and narcotics for urine in doping control (Deventer et al., 2014).

fluids analyzed for forensic toxicological analysis are blood and urine, however, other biological matrices may be ana-
lyzed depending on a number of factors including: availability of specimen, interpretation, target analytes, sample
acquisition (antemortem vs. postmortem), sample suitability, and stability. Further, in postmortem forensic toxicologi-
cal analysis the state of decomposition may limit available specimens for testing. In some cases, the only viable matrices
may be tissue samples and in extreme cases bone (Mella et al., 2018).
Numerous drugs have been identified in forensic toxicological case work (e.g., drug facilitated sexual assaults, drug
overdoses, postmortem, and driving while under the influence of drugs; Darke & Hall, 2003; Drummer, 2006; ElSohly &
Salamone, 1999; Lipari et al., 2013; Poklis et al., 1987; Wylie et al., 2005). Aside from adequate staff, there are ways that
a laboratory can increase sample throughput and those include utilizing more efficient sample preparation techniques
and instrumentation. In forensic toxicology casework, testing for multiple drugs at once can expedite analysis and pre-
vent or aid in addressing case backlogs. Automation has also been implemented in laboratories with large caseloads to
increase sample throughput and decrease the amount of time analysts spend on sample preparation and thereby freeing
them up to perform other laboratory or administrative duties. Further, automation can aid in reducing human errors
by limiting human interaction. Although automation may be of benefit in many situations, it is challenging to imple-
ment when working with more complex matrices such as tissues or bone. High sample request volumes, along with the
need to identify and quantitate a wide range of analytes and their metabolites at low concentrations, make these factors
some of the most important and challenging stages in method development. As technology advances, manufacturers
continue to provide analytical instruments with greater sensitivity and faster analysis. Although these instruments are
now capable of rapidly delivering optimum results at low concentrations, the variability and quantity of target analytes
has resulted in a bottleneck during the sample preparation stage in analysis. This step is typically followed by general
screening of unknown analytes or targeted determination of a predetermined analyte(s).
Issues such as reaching required limits of detection/quantitation and ensuring the quality of these results is greatly
dependent upon the sample preparation technique chosen. During this stage of the analytical process, potential inter-
ferences from the matrix may be removed and the analyte of interest is concentrated resulting in enhanced selectivity
and reproducibility. Determining low amounts of analyte in biological matrices can be arduous especially with the
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continual influx of matrix effects encountered during instrumental analysis, and therefore increases the need for
cleaner extracts.
This article presents an overview of common biological matrices and sample preparation techniques utilized in rou-
tine toxicological analysis.

2 | CHARACTERISTICS OF BIOLOGICAL MATRICES

2.1 | Blood and blood components (whole blood, plasma, and serum)

Blood or what is often referred to in forensic toxicology as “whole blood” is most often encountered as a liquid where
blood cells (red and white), platelets, cellular debris/fragments, and other molecules are suspended in plasma and
accounts for approximately 8% of human body weight (ASH, 2009; Basu & Kulkarni, 2014; Burkhart et al., 2012;
Dean, 2005). The average adult has a blood volume of approximately five liters or 1.3 gallons. The ratio of components
are approximately 45% whole blood, approximately 54% plasma, and greater than 1% white cells/platelets. Due to the
large percentage of hemoglobin, blood appears red, and this intensity of the color can vary due to the interaction of
heme groups with other components and oxygenation of blood. Deoxygenated blood is characterized as having a dark
red color. The color of blood can be indicative of toxic substances (Hirsch et al., 1977; Wirthwein & Pless, 1996). For
example, due to the formation of carboxyhemoglobin following exposure to carbon monoxide a bright red color may be
observed. Further, cyanide poisoning may result in a distinct red color as the blood will remain oxygenated (Panigrahi
et al., 2019). The detection and quantitation of drugs and metabolites in blood is dependent on a number of factors and
can include the therapeutic half-life of the compounds and metabolites, route of administration, previous exposure to
the compound (i.e., acute versus chronic use), pharmacogenomics, and more.
During sample collection and depending on the collection tube utilized, whole blood or blood components may be
obtained and can include plasma or serum (Basu & Kulkarni, 2014; Mathew & Varacallo, 2020). The site of collection
(i.e., arterial or venous femoral or cardiac blood) will be important for interpretation (Dinis-Oliveira et al., 2016).
Plasma is the pale-yellow liquid component of blood in which blood cells are suspended unless removed. Serum is the
liquid component of blood after it is allowed to clot and the sample is centrifuged then the liquid portion, serum, is
transferred to a new tube. Other isolated components of blood may potentially be encountered, however whole blood
and plasma/serum are the most common in forensic toxicological analysis. Further, there are several sample preserva-
tion and anticoagulants that may be included in the collection tube including potassium oxalate, sodium fluoride, or
ethylenediaminetetraacetic acid or EDTA which may need to be considered when preparing a sample for analysis or in
interpretation of the results (Mathew & Varacallo, 2020).

2.2 | Urine

Urine is the liquid waste that is produced by the kidneys and stored in the bladder (Fogazzi et al., 2008). Normal urine
is a clear, transparent fluid which is yellow in color. In healthy adults, approximately 40–60 ounces of urine is excreted
within 24 h. Urine is composed of water, urea, uric acid, and salts at a ratio of 960:40 (water:solid particles) (Rohrig &
Kunia, 2019; Verhamme et al., 2008). In unhealthy individuals it may contain sugars, albumin, bile pigments, or abnor-
mal quantities of its normal components.
Urine is collected into a specimen container either from living individuals through a witnessed (e.g., workplace drug
testing) or unwitnessed collection (Caplan & Goldberger, 2001). In healthy adults, the bladder can hold up to 16 ounces
of urine from 2 to 5 h or longer (Moeller et al., 2008). In postmortem cases, urine may be obtained by a puncture to the
bladder and utilizing a syringe for collection and then transfer to a collection container (Fogazzi et al., 2008).

2.3 | Oral fluid

Oral fluid (saliva) is produced by three main glands in the mouth: the parotid, submandibular, and sublingual
(Drummer, 2006; Humphrey & Williamson, 2001). It is a cleaner matrix when compared with blood with a low protein
content of approximately 0.3% (Humphrey & Williamson, 2001). The collection of oral fluid is less invasive compared
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with that of blood or urine. Unlike urine, it typically cannot be substituted by common liquids or chemicals. Oral fluid
analysis has become a growing area of interest for testing drugs of abuse. Screening and confirmation testing has been
conducted using oral fluid in several countries (Van der Linden et al., 2012). In the United States, oral fluid drug testing
was added to the Mandatory Guidelines for Federal Workplace Drug Testing by the Substance Abuse and Mental
Health Services Administration and is growing in prevalence for routine drug testing (SAMHSA, 2019).
Collection of oral fluid can be conducted utilizing several different approaches. A person can simply expectorate or
spit into a collection tube, but this may result in a viscous fluid with food contaminants. Commercial collection devices
that use a diluent such as an extraction buffer can help create an easier matrix to work with (Quintela et al., 2006). Cer-
tain commercial devices may have better recovery for specific drugs or classes of drugs than others. Oral fluid produc-
tion can be stimulated by agents such as gum or citric acid which can also affect the pH or concentration of the drug in
the mouth. Additionally, certain drugs can increase or decrease oral fluid production such as codeine, amphetamines,
and antidepressants. Ultimately, interindividual variability in saliva production can result in sample volume variations
(Bosker & Huestis, 2009; Crouch, 2005; Drummer, 2005, 2006).
The concentration of drugs in oral fluid has been reported to be similar to that of blood and plasma but varies with
different factors (Crouch, 2005; Drummer, 2006; Drummer et al., 2007). The detection of drugs in oral fluid can be
affected by route of administration, pKa, molecular size, degree of protein binding, and lipophilicity of the drug
(Desrosiers & Huestis, 2019; Toennes et al., 2005; Van der Linden et al., 2012; Verstraete, 2004). The most common
method of drug transport from blood to oral fluid is passive diffusion. Typically, the parent drug is found rather than
the metabolite because it is more lipid soluble. Its lipophilicity allows the parent drug to diffuse through the capillaries
and acinar cell membranes into the oral fluid. Drugs that are weak bases are found in higher concentrations in oral
fluid than in plasma due to ion trapping. Concentrations of specific drugs in oral fluid will vary between each class and
type since they have their own unique structures that affect size, polarity, and lipophilicity. Additionally, different clas-
ses of drugs will also have varying detection windows (Drummer, 2006).
Since oral fluid may be collected utilizing commercial collection devices that include a swab that is placed into an
extraction buffer (e.g., phosphate-buffered saline; Quintela et al., 2006). When developing and validating a sample prep-
aration method, it is necessary to assess the ratio of matrix to buffer. Common ratios are 1:3 and 1:4 (oral fluid:buffer)
and may need to be considered when preparing calibrators and controls for qualitative and quantitative analysis.

2.4 | Vitreous humor

Vitreous humor or fluid is the clear, colorless, viscous fluid that is located between the lens and retina of the eye
(Lappas & Lappas, 2016). It is composed of approximately 98% water along with a mixture of collagen, proteins, salts
and sugars. Due to the lack of complexity of this matrix, it may not require extensive sample preparation prior to analy-
sis (Flanagan & Connally, 2005; Leikin & Karch, 2008). Approximately 2–3 ml of fluid can be collected from each eye.
Vitreous fluid should be collected from both eyes and placed in separate collection containers (De Martinis et al., 2006).
Vitreous humor can be helpful in postmortem cases due to its proximity away from major thoracic and abdominal
organs. Further, the fluid is enclosed in a closed space and therefore less susceptible to contamination, putrefaction,
and post-mortem redistribution (Dinis-Oliveira et al., 2016). Vitreous humor may be less susceptible to postmortem pro-
duction of ethanol and useful when putrefactive changes are suspected (Levine & Jufer, 2008). Several drug classes and
metabolites are detectable in vitreous fluid including opioids, benzodiazepines, cocaine and its metabolites (Jenkins &
Oblock, 2008; “Princ. Forensic Toxicol.,” Levine & Kerrigan, 2020; Scott & Oliver, 2001). Compounds that are highly
protein-bound are less likely to be detected in vitreous fluid due to protein binding affinities and trace amounts of pro-
teins in vitreous fluid (Jenkins & Oblock, 2008; Lappas & Lappas, 2016; Levine & Jufer, 2008; Scott & Oliver, 2001).

2.5 | Tissues

Liver, lung, kidney, spleen, muscle, brain, and heart tissue have all been utilized in postmortem toxicological analysis
(Drummer, 2007). The liver is the primary site of metabolism for the majority of xenobiotics and therefore can be a use-
ful specimen when blood and urine are not available or lack sufficient volumes. The gallbladder, a small organ that sits
under the liver, contains digestive fluid called bile that ultimately is released into the small intestine. Bile may be
obtained during autopsy and in the absence of other usable fluids can also be used for analysis (Drummer, 2005).
JONES ET AL. 5 of 13

In specific types of cases, the lung tissue can prove useful such as when volatile compounds are suspected or when
liquid biological fluids are not available (Hirsch et al., 1977). In cases of traumatic accidents, destructive fire or
advanced decomposition the availability of tissue may result in relying on other types such as kidney, spleen, muscle,
brain, and/or heart (Park et al., 2009). Depending on the physicochemical properties of compounds or their lipophilic
nature, some compounds may penetrate specific organs more readily than others (Catalano et al., 1998). Further, some
compounds undergo postmortem redistribution, however, available data on these characteristics for all classes of com-
pounds is not available and therefore interpretations of drug concentrations in tissues may be restricted to a handful of
compounds (Kringsholm & Christoffersen, 1987).

2.6 | Gastric contents

Stomach or gastric content has been used in toxicological analysis and in simple visual examination for tablets, cap-
sules, pill fragments, blotter paper, or other signs of drug use. Depending on the state of degradation, it may be possible
to perform a presumptive visible identification of these types of evidence if identifying marks or by analysis by typical
analytical techniques (Peres et al., 2019). The analysis of gastric contents can be useful when other specimens are not
available for analysis. Gastric contents analysis can be useful in guiding further analysis of other biological specimens.
Depending on the composition of the contents, it may be necessary to homogenize the sample prior to further sample
preparation. For interpretation considerations, quantitative analysis of gastric contents may be limited, if not impossi-
ble, based on the sample provided.

2.7 | Hair

Hair grows approximately 1 cm per month and can be used as a toxicological specimen when either too much time has
passed since exposure to detect/quantitate in typical specimens such as blood or urine (Cuypers & Flanagan, 2018). It is
composed of primarily protein (65%–95% keratin), water (15%–35%), lipids (1%–9%), and minerals (>0.25%–
0.95%). Further, in postmortem cases it has value in determining chronic exposure to drugs with the ability to detect
compounds potentially from weeks to months depending on the compound(s) and length of hair (Cuypers &
Flanagan, 2018).
When collected, hair should be from the vertex region of the head with the proximal end (section closest to the
scalp) clearly labeled (Lappas & Lappas, 2016). Drugs enter the hair by adsorption from the environment, from sweat
and sebum, or incorporation into the hair shaft from blood that supplies the hair follicle. There is variability in drugs
and metabolites that may be present in the hair compared with blood and therefore its value as a toxicological specimen
is dependent on a number of factors. Interpretation of drug identification in hair may be hindered based on the scien-
tific research on the specific drug(s), potential for contamination and decontamination measures taken prior to analysis,
as well as what can be concluded based on identification (Tsanaclis & Wicks, 2008; Wada et al., 2010).

3 | SAMPLE PREPARATION TECHNIQUES

3.1 | Liquid–Liquid

Liquid–liquid extractions (LLE) are based on the concept of immiscible solvents, typically one aqueous and one organic,
which are added to an aliquot of a biological sample (Juhascik & Jenkins, 2009; Wells, 2018; Figure 1). Sample extrac-
tion is based on the solubility of the components present in the solvents utilized. A common LLE extraction is protein
precipitation which uses inorganic acids or solvents such as acetonitrile, chloroform, or methanol to physically remove
proteins from the matrix. The principle behind LLE is the solubility of the components present. A ratio of 2:1 aqueous
to organic solvent is recommend for LLE extractions in order to avoid excess interferences from being co-extracted.
Once these solvents are added to the sample the analytes will travel into the solvent in which they are more soluble.
The solvents chosen will depend on the compounds pKa, polarity, pH, and all should be considered to determine which
solvents to be used to increase the solubility of the analytes into one of the immiscible layers. The extractions can be
acidic/neutral or basic/neutral (Wells, 2018). The potential for the two solvents to form an emulsion when mixed
6 of 13 JONES ET AL.

FIGURE 1 General flow of liquid–liquid extraction

vigorously should be considered. This can be overcome by several different means including centrifugation, adding salt
to the aqueous phase, increasing the phase ratio of organic to aqueous, or by freezing the aqueous phase (Dinis-Oliveira
et al., 2016). Depending on the method of analysis, the layer recovered can either be directly injected into the instru-
ment or dried down and reconstituted into a solvent that is more suitable for the instrument.
The extraction may be carried out in one container in which all solvents and compound(s) of interest are added,
mixed, centrifuged, and the organic layer is removed (Wells, 2018). There is the potential to repeat these steps to
increase the analyte recovery. Compared with dilute and shoot, phospholipid depletion, and protein precipitation, LLE
may result in a cleaner extract and can be more selective. However, these extractions require intensive manual labor,
they are more difficult to automate, and there is a potential for increased sample variability due to the differences in
layer removal while pipetting.

3.2 | Solid phase

When analytes of interest have similar polarities, or acid/base properties, LLE may not be suitable for their recovery.
Therefore, solid phase extractions (SPE) may prove to be more effective (Figure 2). The concept of SPE utilizes adsorp-
tion of components onto a solid phase or sorbent to separate unwanted components and extract analytes of interest
(Berrueta et al., 1995). These extractions aid in removing interferences such as phospholipids found in blood, other
interfering drugs, and other components found in the matrix and result in cleaner extracts. These packed columns, well
plates, or bulk material consist of either silica or polymers. Solid phase extraction columns come in a variety of sorbent
weights typically ranging from 0.1 to 10 grams and tube volume sizes of 1–60 ml. Sorbent capacity is estimated to be
approximately 5%–10% of the sorbent weight. The amount of solvent, sorbent capacity, and analyte to be retained are
all taken into account when choosing the correct cartridge capacity (Aurand, 2021; Lu & Ye, 2021; Thermo
Scientific, 2014). For those sorbents composed of silica they may be limited in terms of effective pH range for extrac-
tions. Further, the particle size will be more variable, and the sorbent bed should not be allowed to dry out while being
conditioned and before the sample is loaded. Comparatively, sorbent beds made of polymers can support a larger pH
range, the packing more homogenous, with a higher sample capacity. Solid phase extractions can be used for acidic,
basic, or neutral compounds in which the sorbent bed composition is adjusted to fit the type of analyte (Berrueta
et al., 1995).
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FIGURE 2 General flow of solid phase extraction

Reverse phase SPE involves a non-polar cartridge packing (C8 or C18) and non-polar solvent or solvent mixtures for
elution. Analytes are retained through Van der Waals or dispersion forces, therefore most analytes with hydrophobic
character are likely to be retained. This method is effective at removing salts and polar matrix components
(Aurand, 2021; Lu & Ye, 2021; Thermo Scientific, 2014). Ion-exchange SPE is based on separation by electrostatic inter-
actions of the oppositely charged functional groups of the sorbent to the analyte of interest. Analytes are ionized by
adjusting pH to 2 units above (basic analytes) or below (acidic analytes) the pKa of the analyte in order to be retained
on the column. Ion exchange SPE for basic analytes is a cation exchange, while anion exchange SPE is useful for acidic
analytes. Columns are prepared with a salt solution of counter-ions than the analytes of interest. When the sample is
introduced the counter-ions are displaced by the analyte. The analytes are retained until a salt solution containing
stronger counter-ions is passed through the column (Aurand, 2021; Lu & Ye, 2021).
Traditional steps for SPE methods begin with conditioning the column (Berrueta et al., 1995). This adjusts the pH of
the sorbent bed and activates the functional groups on the particles. Next, the sample is loaded, followed by washing to
remove any matrix or other interferences present. The final step is eluting the analytes of interest. Advantages of using
SPE methods are they can produce very clean extracts due to matrix interferences, such as phospholipids, salts, or other
non-targeted components being removed during the wash steps as well as increased reproducibility. These methods can
also be very selective and amendable to automation. However, there exist disadvantages including the amount of time
involved to complete the required steps as well as the associated costs of SPE columns, ensuring all necessary equip-
ment such as vacuums or positive pressure manifolds, as well as the solvents needed to carry out the extractions
(Berrueta et al., 1995).

3.3 | Supported liquid

Similar to SPE, supported liquid extractions (SLE) are also robust methods of sample preparation that can remove
potential interferences and isolate a wide range of compounds and are considered modified LLE. They use the same
principles of combining water immiscible solvents to create analyte extraction (ISOLUTE® SLE+ Supported Liquid
Extraction Products, n.d.; Thermo Scientific, 2014). While LLE methods require mixing the immiscible solvents by
shaking, rocking, centrifuging, and so on, SLE utilizes a stationary phase that captures the aqueous solution while the
organic solvent flows through the stationary phase. The stationary phase is composed of diatomaceous earth which has
a high silica content. Diatomaceous earth is composed of fossilized diatoms that are high in silica content and found on
the seafloor. Diatoms are closely related to algae and have a hard shell or skeleton composed of silica, resulting in
8 of 13 JONES ET AL.

diatomaceous earth containing a rich silica content (Calvert, 2009; Thermo Scientific, 2014). The diatomaceous earth is
ground resulting in large, irregular-shaped particles with high surface area leading to greater absorption of target
analytes and therefore increasing the extraction efficiency.
There are typically three steps involved in the SLE process: (a) loading the column, (b) waiting while the matrix is
absorbed into the column, and (c) eluting the analytes of interest (Figure 3). Increased reproducibility, analyte recovery
and reduction in ion suppression have all been demonstrated with the use of SLE methods (Sauve et al., 2012). These
methods give cleaner extracts compared with LLE methods due to phospholipids and other matrix interferences bind-
ing to the column and not co-eluting. Supported liquid extractions can be more selective than LLE methods. With the
lack of wash steps, the eluent may not be as clean as those processed using SPE. Depending on the analytical focus,
there may be a loss of selectivity compared with that of SPE methods, however the amount of time consumed can be
significantly less compared with both LLE and SPE. Similar to SPE, automation is possible as well. Acidic, basic, or neu-
tral compounds can all be recovered utilizing SLE as well as applying it to typical biological matrices encountered in
forensic toxicological case work.

3.4 | Phospholipid depletion

Tissues and biological fluids, such as serum, plasma, and whole blood, contain phospholipids or lipids with a phosphate
group (Basu & Kulkarni, 2014; Burkhart et al., 2012). Phospholipids can interfere with chromatographic methods as
they can be retained on hydrophobic columns, suppress ionization in electrospray ionization sources and have an
adverse effect on reproducibility. Cartridges or plates for phospholipid removal can be designed to remove only those
matrix components or combined with a protein precipitation step to also have the added benefit of removing proteins
while not significantly adding to the number of steps in sample preparation while still providing a cleaner sample for
analysis (Aurand et al., n.d.; Aurand, 2021; Lu & Ye, 2021). Products designed for phospholipid depletion (PLD) are
amendable to automation as they require no preconditioning steps. The sample, internal standard, and precipitation
solvent can be added to the PLD plate/cartridge, mixed, and with vacuum the sample can be collected and potentially
taken directly to the instrument for analysis or dried down and reconstituted with a more suitable solvent(s) for subse-
quent analysis (Figure 4) (Aurand et al., n.d.; Aurand, 2021; Lu & Ye, 2021).

3.5 | Dilution

Direct injection of biological samples into a chromatographic and/or mass spectrometric system carries challenges and
potential risks such as matrix components or interfering substances entering the analytical system along with the
targeted analytes. However, some matrices may be amendable to dilution or “dilute and shoot” when the matrix com-
ponents and/or interferences can be diluted to levels that are acceptable (Deventer et al., 2014; Kwok et al., 2016;

FIGURE 3 General flow of supported liquid extraction

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FIGURE 4 Description of phospholipid depletion with protein precipitation

Lawson et al., 2016). The laboratory must evaluate the impact to increased sample throughput and weigh those against
potential system downtown or associated costs due to analytical column degradation, variability in chromatographic
retention times, ion suppression and/or enhancement, salt formation that can potentially decrease instrument sensitiv-
ity, or other issues that can arise from introducing contaminants into the analytical system.
Dilution can reduce some of these adverse effects, however it can also reduce the detection levels of analytes. Dilute
and shoot is typically reserved to analytes that will be present in the sample matrix (i.e., urine) at high concentrations.
Dilute and shoot has the advantage of being amendable to automation. When considering a “dilute and shoot” method,
the dilution ratio should be evaluated during method validation (ANSI/ASB, 2019).

3.6 | Automation

Sample handling or automation has been utilized to increase laboratory throughput by incorporating liquid handlers or
systems that can collect and dispense or “handle” amendable samples, pipette tips, reagents, and associated consumable
supplies in order to prepare multiple samples simultaneously with little to no interaction by a technician or analyst.
Ultimately, the samples are prepared for analysis by liquid or gas chromatography coupled with a detector such as mass
spectrometry. There are several benefits of implementing automation such as increasing the number of samples pre-
pared for analysis within a given period of time, decreasing human error, closely controlling resources, sample tracking
with the use of barcoding, minimizing specimen usage by reducing the volume of sample needed for downstream analy-
sis, and integrating the liquid handler with analytical tools.
By its name, “liquid handler,” a sample must be in liquid form for further automated processing. Therefore, if
not already in liquid form, the sample must be transformed into such a form to be used with automation. Hair, tis-
sue samples, and gastric contents would need to undergo some form of homogenization process that involves
buffers or solvents to disrupt the sample so that target analytes are released and can be extracted into a suitable sol-
vent or proceed on to further sample cleanup which may or may not be accomplished via automation. Therefore,
samples such as blood and urine are often the most commonly explored for automation. Although variability exists
with regard to their sample composition, blood and urine are typically uniform enough to be used with liquid han-
dlers. Automated systems can be designed and adjusted to be used with different types of sample preparation tech-
niques from simple dilutions to more involved sample preparation (e.g., solid phase, supported liquid,
phospholipid depletion, etc.) as well as the associated steps needed for preparing the samples, applying the sample,
wash steps, elution, sample dry down, and reconstitution.
10 of 13 JONES ET AL.

3.7 | Method optimization and validation

Method optimization and validation is key to ensuring quality toxicological results (Eurachem, 1998; Hubert
et al., 2004; Peters & Maurer, 2002; Remane et al., 2010; Shah et al., 1992, 2000; United Nations Office on Drugs and
Crime, 2009). Many forensic toxicologists are familiar with the historic document from the Scientific Working Group
on Forensic Toxicology or SWGTOX document “Standard Practices for Method Validation in Forensic Toxicology”
(“Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation in Forensic
Toxicology,”; SWGTOX, 2013). This document was reviewed, revised and updated by the Organizational Scientific Area
Committees and then sent through the Academy Standards Board (ASB) standards development organization. This
resulted in the publication of ASB Standard 036 Standard Practices for Method Validation in Forensic Toxicology
(ANSI/ASB, 2019). Sample preparation should be assessed as part of the method validation process and validated
according to the guidelines set in American National Standards Institute (ANSI)/ASB 036 or a standard associated with
the laboratories country or government oversight. Further, ANSI/ASB 036 states “The sample preparation technique
shall be evaluated and optimized using reference materials of the analyte(s) of interest. The primary goal is to demon-
strate that the sample preparation steps allow for adequate extraction, detection, identification, and/or quantitation of
the analyte(s). Sample preparation shall be evaluated with fortified matrix samples” (ANSI/ASB, 2019).
In addition to the following parameters: calibration model, bias and precision, limit of detection and quantitation,
carryover, there are matrix specific parameters such as ionization suppression/enhancement, dilution, interference
studies [matrix], and matrix recovery that should be assessed as part of the validation process. Due to the fact that some
samples will have concentrations outside of the working range, dilution may be necessary. It is necessary to assess dilu-
tion precision. Per ANSI/ASB 036, two common dilutions (1:10 and 1:50) shall be assessed in triplicate and analyzed
five times alongside five separate calibration curves (ANSI/ASB, 2019). Interferences, matrix or other compounds, are
the presence of compounds that are not specifically targeted by the method and are important parameters to assess dur-
ing validation with regard to sample preparation (ANSI/ASB, 2019).
Commonly encountered drugs, matrix, target analyte ions, and internal standard ions can all be sources of interference
(Matuszewski et al., 2003; Van Eeckhaut et al., 2009). To evaluate the impact the matrix(s) may have on analysis, 10 different
matrix sources should be assessed to consider what components may interfere with analysis as well as evaluate the variability
that may exist with regard to different sources of biological samples (ANSI/ASB, 2019). Matrix components may suppress or
enhance the ionization of the target compound(s). Calculations to assess these effects can be accomplished using the following
equation: [1 (peak area of post-spike)/(average peak area of “n” neat blanks, where n ≥ 3)]  100 (Menasco, 2020). To eval-
uate the efficiency of the sample preparation method, matrix recovery calculations can be conducted by examining pre-spiked,
post-spiked, and neat samples run in triplicate. The equation to calculate Percent Recovery is as follows: (% recovery) = [(peak
area of pre-spike)/(average peak area of n post-spikes, where n ≥ 3)]  100 (Menasco, 2020).

3.8 | Limitations

Although it is the intent of the authors to be as comprehensive as possible to cover as many matrices and sample prepa-
ration techniques available, those included herein were deemed as the most commonly encountered in forensic toxicol-
ogy laboratories. There are certainly other matrices such as sweat, bone, meconium, dried blood spots, cerebrospinal
fluid, amniotic fluid, and breast milk that have been used to identify and quantitate xenobiotics. Each of these have
their uses and research has been conducted to investigate their utility in understanding drug distribution, drug use,
and/or drug exposure. However, in routine analysis their submission for investigation is limited and are not considered
routine specimens in forensic toxicological casework. In addition, there are other sample preparation techniques not
covered in this work, such as solid phase microextraction or disposable pipette extraction, which have been investi-
gated. Again, it is not the author(s) intent to overlook these potentially valuable techniques but to provide the reader
with information on routine or up and coming alternatives to routine matrices and sample preparation techniques.

4 | C ON C L U S I ON S

Sample preparation is an important step in forensic toxicological analysis. With the technological advancements and
the availability of mass spectrometers with increasing sensitivities, the need to remove potential interferences, such as
matrix components or non-relevant analytes to the analysis, is essential to optimizing an analytical method. Forensic
JONES ET AL. 11 of 13

toxicological analysis involves diverse analytes with a variety of chemical properties that need to be considered when
selecting an appropriate sample preparation technique. This work was written to provide the forensic toxicology com-
munity a high-level overview of biological matrices and their relevant components that should be considered during
sample pre-treatment as well as traditional and non-traditional sample preparation techniques.

A C K N O WL E D G M E N T S
The authors wish to express their gratitude to Cassandra Swart, Michael Moretto, Mikayla Caldwell, Kelsie Jenquine,
Nichole Bynum, Katherine Bollinger, Andrew Zeigler, Megan Grabenauer, Lynn Jordan, Jillian Neifeld, Claude Mallet,
Frank Kero, Karen Scott, Jamie Foss, Joseph Jones, and Marc LeBeau. Your knowledge and expertise, which you gra-
ciously shared, has impacted this work and for your time and friendship we are grateful.

CONFLICT OF INTEREST
The authors have declared no conflicts of interest for this article.

A U T H O R C ON T R I B U T I O NS
Sabra Botch-Jones: Conceptualization; project administration; resources; writing-original draft. Courtney
McGowan: Data curation; investigation; writing-review & editing. Sarah Boyle: Methodology; writing-review &
editing. Yiling Ke: Methodology; writing-review & editing. Chi Hin Chan: Investigation; writing-review & editing.
Hajin Hwang: Investigation; writing-review & editing.

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

ORCID
Sabra Jones https://orcid.org/0000-0002-9813-4124

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How to cite this article: Jones, S., McGowan, C., Boyle, S., Ke, Y., Chan, C. H. M., & Hwang, H. (2021). An
overview of sample preparation in forensic toxicology. Wiley Interdisciplinary Reviews: Forensic Science, e1436.
https://doi.org/10.1002/wfs2.1436