Fast Quantitative LC-MS/MS Determination of Illicit
Substances in Solid and Liquid Unknown Seized Samples
Narcotic and psychotropic substances are natural, synthetic, or semisynthetic compounds that are present in both solid and liquid illicit products. The alterations effects on the central nervous system related to their use can be psycholeptic, psychoanaleptic, or psychodiseptic and are able to generate tolerance, addiction, or dependence phenomena, creating social and public order problems. In this scenario, the analytical evaluations that aim to determine these analytes in seized nonbiological samples, and which assume the character of judicial evidence, must meet high analytical requirements of reliability, transparency, and procedures uniformity at a national level. For the first time in the literature, the herein validated method is able to provide the simultaneous quantitative determination of 37 of the most common narcotic substances as well as the most commonly used excipients/adulterants found in seized illicit material. Additionally, the validated method can process both solid and liquid samples maintaining the precision and trueness levels (intraday and interday) in accordance with the U.S. Food and Drug Administration and European Medicines Agency international guidelines (<14.31 and <13.41%, respectively). Furthermore, it provides a simple and fast procedure for sample preparation using the dilute and shoot approach, exploiting the sensitivity and selectivity of the LC-MS/MS instrument configuration used and the signal acquisition in multiple reaction monitoring (MRM) mode (both positive and negative polarization modes).
<!>Materials
and Instrumentation<!>Sample Preparation<!>Method Validation<!>Extraction/Dilution
and LC-MS/MS Procedures<!><!>Figure of Merits and Method Validation<!>Real Sample Analyses<!><!>Green Analytical
Procedure Index (GAPI)<!><!>Conclusions<!><!>Author Contributions<!>
<p>Narcotic and psychotropic substances
are natural, synthetic, or semisynthetic compounds that show pharmacological
activity and are able to alter the psychic and behavioral spheres
to different degrees. The effects on the central nervous system (CNS)
of these alterations can be psycholeptic, psychoanaleptic, or psychodiseptic.
Often, these compounds could generate tolerance, addiction, or dependence
phenomena. For these reasons, many substances, based on their different
chemical structures, biological activities, and social effects, were
included in national and international regulations as prohibited substances.
In this context, these compounds are collected into specific tables
which are constantly updated. As recently reviewed,1,2 the
characterization of these seized substances can be extremely difficult,
with long analysis and reporting times, especially if the laboratory
does not have suitable protocols for the whole process and data traceability.
In this framework, the analytical assessments, which aim to determine
the narcotic and psychotropic substances in seized nonbiological samples
and which assume the character of judicial evidence, must meet high
analytical requirements of reliability, transparency, and procedures
uniformity at national levels. During illicit substances analysis,
a documentation in which the entire supply chain is traceable and
compliant with current law regulations is always necessary. Alongside
these requirements, it should also be added that high investigation
levels must also be ensured through the instrumental techniques applications,
procedures, and analytical methods that are robust and widely shared
at the scientific community level in the toxicological and forensic
fields. Generally, illicit seized samples often contain a wide range
of other adulterating compounds that are added in order to increase
the product bulk, facilitate administration, or even worse, mimic
the pharmacological effects. These compounds can be both legal (caffeine,
procaine, paracetamol, sugars, creatine, benzocaine)3 but also illegal (cocaine, 3,4-methylenedioxymethamphetamine
or MDMA, amphetamines, mephedrone).4−6 Some examples of both
solid and liquid unknown seized materials are shown in Figure 1. Currently, the works reported
in the literature often consider only a limited quota of the possible
substance combinations that can be found in seized samples7−16 by applying different instrumental configurations.</p><p>General appearance of
some seized solid and liquid materials.</p><p>Our ongoing research aimed to improve the sampling and sample processing
tool development17−19 by applying validated procedures useful for both
clinical20 and legal21 purposes, coupled also with a more simple dilute
and shoot concept;22 this method
aims to provide a valid support in seized solid and liquid samples
quantitative analysis. In particular, this method is able to simultaneously
quantify 37 of the most common narcotic substances (cocaine, buprenorphine,
amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine or
MDMA, 3,4-methylenedioxyiamphetamine or MDA, 3,4-methylenedioxy-N-ethylphetamine or MDE, 3,4-methylenedioxy-N-methyl-α-ethylphenylethylamine or MBDB, ketamine, diacetylmorphine,
ephedrine, pseudoephedrine, methadone, methorphan, 6-monoacetylmorphine
or 6-MAM, delta-9-tetrahydrocannabinol or THC, cannabidiol or CBD,
and morphine), as well as the most commonly used excipients/adulterants
(acetylsalicylic acid, aminophenazone, benzocaine, caffeine, diltiazem,
phenacetin, hydroxyzine, levamisole, lidocaine, naloxone, nicotine,
noscapine, paracetamol, paroxetine, procaine, procainamide, trimethoprim,
sulfametoxazole, tropacocaine).</p><!><p>The chemical standards
used for the calibration curves, the QC samples, the HPLC mobile phases,
and the solutions used in the sample extraction/dilution procedure
were purchased from Eureka srl Lab Division (code LC20000). The complete
list of the analytes (even with MRM transitions) is reported in Tables S.1 and S.2. The liquid chromatography-tandem
mass spectrometry (LC-MS/MS) instrumentation is an ABSciex API 4500
QTrap interfaced with a Shimadzu Nexera X2 LC HPLC (SIL-30AC autosampler,
LC-30AD pump and CTO-20AC column oven). The method was developed and
validated at the Pharmatoxicology and Analytical Quality Laboratory
(ACCREDIA n. 2274 ASLPE, accreditation n. 1822L, according to ISO/IEC
17025) of the "Santo Spirito Hospital", Pescara, Italy.
All configurations and instrumental parameters are detailed in Table S.3 and Figure S.1. Specifically, the mass spectrometer operating parameters (both
for positive and negative ionization modes) are reported in Table S.4. The chromatographic column used, Hypersil
Gold PFP (50 mm × 2.1 mm, 1.9 μm), was thermostated at
40 °C, while the analyses were carried out under gradient elution
with binary phase M1 (H2O, 0.1% formic acid, 10 mM ammonium
formate) and M2 (acetonitrile) with a flow rate of 400 Î¼L/min
according to the profile reported in Figure S.2. The analysis takes a total of 15 min including the system reconditioning
step.</p><!><p>The sampling phase on the seized
materials was conducted following the guidelines on sampling of illicit
drugs for the qualitative analysis of the European Network of Forensic
Science Institutes (ENFSI),23 dividing
the material into aliquots and weighing them accurately in order to
provide a normalized quantitative analysis. Following the principle
of less sample handling and taking advantage of the high LC-MS/MS
configuration sensitivity and selectivity, sample preparation includes
an extraction/dilution protocol for solid samples and a dilution protocol
for liquid samples. In the case of seized solid samples: (i) weigh 20 mg of sample and add 5 mL of reagent A (acetonitrile), (ii) sonicate for 10 min and centrifuge for 10 min at 4000
rpm, and (iii) dilute 1:400 (v:v) with reagent B (H2O, 1% formic acid). At this point,
for the quantitative determination of cocaine, proceed by preparing
the sample in an autosampler vial by placing 10 Î¼L of the sample
solution in 990 Î¼L of reagent D (H2O, 0.1% formic
acid, 10 mM ammonium formate) and 20 Î¼L of reagent C (methanol).
The reagent C solution contains also the deuterated internal standards,
specifically cocaine D3, 6-MAM D6, morphine D6, buprenorphine D4,
methadone D9, and THC D3 (these internal standards were found to be
adequate for all analytes in terms of parent ion affinity and retention
times such as 6-MAM D6 for acetylsalicylic acid and morphine D6 for
amphetamines). For all other substances, the volumetric ratios are
100 Î¼L sample, 900 Î¼L of reagent D, and 20 Î¼L of
reagent C. When it is necessary to analyze seizure liquid substances,
the procedure involves only two steps: (i) add 10
Î¼L of sample to 990 Î¼L of reagent B and (ii) in autosampler vials 10 Î¼L of the sample solution in 990 Î¼L
of reagent D and 20 Î¼L of reagent C.</p><!><p>The method was validated according
to the U.S. Food and Drug Administration and European Medicines Agency
international guidelines.24,25 and the following parameters
were considered: linearity, lower limit of detection (LLOD), lower
limit of quantification (LLOQ), precision and trueness (intraday and
interday), and reagents and standards stabilities.</p><!><p>First,
the chromatographic separation reported in another study17 for the simultaneous analysis of more than 739
chemicals was tested. By applying the same chromatographic parameters,
it was observed that the characteristics required, such as focusing
the analytes in tight peaks to maximize the signal-to-noise ratio
and consequently the sensitivity, avoiding the presence of matrix
effects by resolving possible interferents through chromatography,
and increasing the parameters for the correct identification considering
also the reproducibility of the retention time, are maintained in
the analyses of both liquid and solid seized samples. In this protocol,
the first extraction of selected compounds was carried out with different
solvents, such as methanol, isopropanol, hexane, dichloromethane,
ethyl acetate, acetonitrile, and solutions formed by different percentages
and combinations of the above-mentioned solvents. Different recovery
factors were obtained, and the better performances were observed using
acetonitrile that was selected for carrying out the first extraction.
Then, this first extract was further diluted with organic solvents
(methanol, acetonitrile) or with an aqueous solution in order to evaluate
the better system to obtain the final sample ready for the analysis
and to obtain better sensibility, signal-to-noise ratio, and ionization
efficiency in the MS instrumentation and peaks symmetry during the
chromatographic run. Dilution with an aqueous solution proved to be
the most efficient. In addition, a percentage of formic acid was added
because some molecules with acid pH were found to be more stable.
The final dilution was focused on injection and chromatographic resolution
in order to reach good peak shapes. It is very important that the
injected solution is as similar as possible to the initial condition
of the gradient in order to avoid peak tailings and non-Gaussian peaks
shapes. The aqueous solution used for the final dilution was modified
using only 0.1% formic acid and adding also 10 mM ammonium formate.
The presence of 0.1% formic acid is very useful for peaks intensities.
In this protocol, the parameters related to the analytes ionization
and fragmentation are optimized. Electrospray ionization (ESI) temperatures
were set from 250 to 550 °C with steps of 50 and 450 °C
selected as the best one considering all molecules. Ion spray voltage
is checked varying it from 500 to 5500 V, and +5400 and −4500
V are the best values (considering peaks intensities and noise) in
positive and negative ionization modes, respectively. Regarding HPLC
separation, it is very important to set a good mobile phase gradient,
due to the presence of several isobar molecules with the same parent
ions (MDE/MBDB, THC/CBD, MDA/phenacetin, ephedrine/pseudoephedrine,
benzocaine), and some of them have also the same fragmentation (MDE/MBDB,
THC/CBD, ephedrine/pseudoephedrine). For this reason, it is mandatory
that their resolution is by HPLC gradient. The analysis was carried
out using a gradient from 5% to 75% of organic solvent in 8 min to
reach the separation between all these molecules. Acetonitrile and
methanol are tried as organic solvents for mobile phase M2, and acetonitrile
results to be the best one. In chromatographic separation development,
in this case implemented to focus the analytes before their detection
by MS/MS, it was also verified that no carry over (or memory effect)
problems were present that could affect the batch analyzes through
the use of an autosampler. No effects were found with the optimized
mobile phase gradient, while maintaining the analysis within 15 min
including the system reconditioning. The optimized LC-MS/MS parameters
and the analytes chromatographic profiles, using polarity switching,
are shown in Figure 2. More MRM transitions and instrumental parameters details are reported
in Tables S.1–S.4 and Figures S.1 and S.2.</p><!><p>Chromatogram obtained
under the optimized conditions for the standard
solution at 100 ng/mL for all analytes and 120 ng/mL for paracetamol,
caffeine, cocaine, methadone, phenacetin, and acetylsalicylic acid
at concentration levels. The different colors represent the different
37 analytes MRM transitions. (Top) MRM in positive ionization mode.
(Bottom) MRM in negative ionization mode.</p><!><p>The method validation
procedure, obtained following international guidelines,24,25 saw the evaluation of analytical parameters such as linearity, lower
limit of detection (LLOD), lower limit of quantification (LLOQ), precision
and trueness (intra and interday), and reagents and standards stabilities.
As reported in Table S.5, the method resulted
linearly in the concentration range from 5 to 100 ng/mL with r2 values ≥ 0.9909, showing a lower limit
of detection equal to 1.67 ng/mL. In the range, the performances of
the methods were studied in terms of precision (CV%) and trueness
(BIAS%) both intraday and interday (n = 6 for each
sample and for each parameter). In the validated method, the calibration
(once verified for the absence of matrix effects thanks to the dilute and shoot process) was carried out on standard solution
samples. To validate the precision and trueness, solid and liquid
samples (as they are and spiked) were analyzed with the procedure
reported to have the background value subtracted from the instrumental
response of the spiked sample. The result was evaluated in terms of
precision and trueness for the quantity added and processed on the
previously calculated calibration, obtaining the figures of merit
reported in Table S.5. In particular, the
precision was evaluated on three concentration levels and equal to
LLOQ (5 ng/mL), Cm (50 ng/mL), and Cup (100 ng/mL). The performances in terms of
trueness were evaluated at two concentration levels and equal to Ci (25 ng/mL) and Ch (75 ng/mL). Repeatability expresses the precision under the same
operating conditions over a short interval of time and is also termed
intra-assay precision. The intermediate precision expresses instead
within-laboratory variations. As indicated by the precision and trueness
values in Table S.5, the herein validated
method has shown repeatability values that fulfill the international
guidelines, as well as the intermediate precision. The recovery could
be reported as trueness by the assay of a known added amount of analyte
in the sample or as the difference between the mean and the accepted
true value. In this work, the repeatability of the extraction process
and the extraction yield indicated as intraday and interday trueness
respects the limits for the methods validation. During the validation
process, it was observed that all the reagents were stable up to 3
years at a temperature of 2–8 °C, while the chemical calibration
standards (and QC) and reagent C containing the internal standards
must be stored at −20 °C. No matrix effects were observed
during the method development. This phenomenon can be ascribed to
the fact that the dilute and shoot procedure developed
and implemented provides for an overall dilution of the seized material
by a factor of 1:10,000 (w:v in the case of seized
solid materials and v:v in case of liquid). This
high dilution factor, thanks to the instrumental sensitivity, completely
reduces any effects on the analytes source ionization process, as
well as minimize any effects related to peak asymmetries or fluctuations
in the instrumental response.</p><!><p>In order to demonstrate the method
applicability, solid and liquid seized samples were analyzed. Some
of more interesting analyzed samples are reported in Table 1, confirming the broad applicability
of the reported method for the evaluation of unknown solid and liquid
materials. Specifically, for the correct identification, we used the
correspondences related to the known material, as reported in Figure S.3. In addition, in light of the procedure
traceability due to the analyses in an accredited laboratory, the
quantitative determination of up to 37 analytes, the easy execution
and no analyte loss (related to the "dilute and shoot"
process), the high selectivity and sensitivity of the instrumental
configuration, together with an overall analysis time of 15 min, this
method represents a very valid alternative to other procedures reported
in the literature. A comparative evaluation is shown in Table S.6. In fact, compared to recent literature,
this method provides an analysis time comparable with others (about
15–20 min), with highly sensitive and selective instrumentation
(LC-MS/MS), with minimal sample handling (concept of dilute
and shoot). However, the greatest advantage was certainly
represented by the capacity to provide an accurate quantitative analysis
(precise and trueness) through the use of internal standards that
allow the normalization of the analyte signal. Another advantage was
represented by the capacity to be applied both on solid and liquid
samples without distinction while maintaining high sensitivity and
reproducibility. The methods presented in the literature, in the case
of quantitative analyses, consider a limited number of analytes compared
to the present procedure (37 between narcotic substances and commonly
used excipients/adulterants) and require laborious sample handling.
In support of the importance of the LC-MS/MS configuration in the
toxicological and forensic fields, it should also be emphasized that
many recently developed devices are still based on the principles
of mass spectrometry.11</p><!><p>L, liquid seized
sample; S, solid
seized sample.</p><!><p>Lately, increasing
importance has been given to the development of "green"
methods. In this context, the objective is to reduce the anthropogenic
activities impact on the environment, and in the case of analytical
chemistry, it reflects the attempt to replace common organic solvents
with nontoxic and nonpolluting ones. Other measures that refer to
this trend can be found in the Principles of Green Chemistry.26 To date, to characterize the green profile of
an analytical procedure, a reference could be made to the Green Analytical
Procedure Index (or GAPI). For the herein reported method, as well
as for others developed in our laboratory,27 we evaluated the eco-friendly profile by critically applying the
principles that lead to the visualization of the GAPI pictogram in Figure 3. In particular,
the details of the color assignment according to the method parameters
are reported in Table S.7 and Figure S.4 and are based on the guidelines indicated
by Płotka-Wasylka in 2018.28</p><!><p>GAPI pictogram
for the reported innovative device and procedure.</p><!><p>The major advantage of the herein reported procedure
could be represented
from the easy sample preparation process that followed the principle
of dilute and shoot, avoiding the excessive sample
manipulation. This simple procedure also included a step in which
the deuterated internal standards were added and subsequently subject
to LC-MS/MS analysis. This approach was made possible thanks to the
high instrumental selectivity and sensitivity and through the signal
acquisition in multiple reaction monitoring (MRM) mode. The herein
validated methodology showed that LC-MS/MS represents the most suitable
instrumentation to support law enforcement agencies (LEA) in terms
of methods selectivity, sensitivity, and ruggedness in quantitative
analyses, especially in the field solid and liquid seized samples
analyses. However, it should be highlighted how other alternative
methodologies (e.g., electrochemistry reported by Schram and collaborators7) can be of sure support for on-site analyses. Surely a possible limitation of the procedures in LC-MS/MS
lies in the problems related to handling this instrumentation, which
however are largely overcome by the advantages of its use for laboratory
bench analysis. Moreover, it should be highlighted that the sample
preparation process could be totally automated since, among all the
preanalytical processes, the dilution steps are immediately transferable
to automatic platforms. This step will certainly further increase
the performances reported here, especially in regard to the reproducibility
values.</p><!><p>Instrumental settings,
detailed figure of merits for
the validated procedure, chromatograms related to analyses of unknown
seized materials performed on 19 April 2021, comparative table of
this method with literature, and detailed information for the GAPI
pictogram (PDF)</p><p>ac1c03310_si_001.pdf</p><!><p># G. M. Merone and A. Tartaglia contributed equally.</p><p>G.
M. Merone:
Conceptualization, methodology, investigation, data curation, formal
analysis, and writing of original draft. A. Tartaglia: Methodology,
data curation, and writing of original draft. S. Rossi: Conceptualization
and writing of original draft. F. Santavenere: Data curation, conceptualization,
and writing of original draft. E. Bassotti: Conceptualization, methodology,
data curation, validation, and writing of original draft. C. D'Ovidio:
Writing of original draft. M. Bonelli and E. Rosato: Writing of original
draft. U. de Grazia: Writing of original draft. M. Locatelli: Methodology,
data curation, and writing of original draft. F. Savini: Data curation,
investigation, conceptualization, project administration, resources,
supervision, and writing of original draft</p><!><p>The authors
declare the following competing financial interest(s): Elisa Bassotti
is an employee of Eureka Lab Division.</p>