Materialsand Instrumentation

The chemical standardsused for the calibration curves, the QC samples, the HPLC mobile phases,and the solutions used in the sample extraction/dilution procedurewere purchased from Eureka srl Lab Division (code LC20000). The completelist of the analytes (even with MRM transitions) is reported inTables S.1 and S.2. The liquid chromatography-tandemmass spectrometry (LC-MS/MS) instrumentation is an ABSciex API 4500QTrap interfaced with a Shimadzu Nexera X2 LC HPLC (SIL-30AC autosampler,LC-30AD pump and CTO-20AC column oven). The method was developed andvalidated at the Pharmatoxicology and Analytical Quality Laboratory(ACCREDIA n. 2274 ASLPE, accreditation n. 1822L, according to ISO/IEC17025) of the “Santo Spirito Hospital”, Pescara, Italy.All configurations and instrumental parameters are detailed inTable S.3 andFigure S.1. Specifically, the mass spectrometer operating parameters (bothfor positive and negative ionization modes) are reported inTable S.4. The chromatographic column used, HypersilGold PFP (50 mm × 2.1 mm, 1.9 μm), was thermostated at40 °C, while the analyses were carried out under gradient elutionwith binary phase M1 (H₂O, 0.1% formic acid, 10 mM ammoniumformate) and M2 (acetonitrile) with a flow rate of 400 μL/minaccording to the profile reported inFigure S.2. The analysis takes a total of 15 min including the system reconditioningstep.

Sample Preparation

The sampling phase on the seizedmaterials was conducted following the guidelines on sampling of illicitdrugs for the qualitative analysis of the European Network of ForensicScience Institutes (ENFSI),²³ dividingthe material into aliquots and weighing them accurately in order toprovide a normalized quantitative analysis. Following the principleof less sample handling and taking advantage of the high LC-MS/MSconfiguration sensitivity and selectivity, sample preparation includesan extraction/dilution protocol for solid samples and a dilution protocolfor 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 4000rpm, 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 preparingthe sample in an autosampler vial by placing 10 μL of the samplesolution in 990 μL of reagent D (H₂O, 0.1% formicacid, 10 mM ammonium formate) and 20 μL 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 beadequate for all analytes in terms of parent ion affinity and retentiontimes such as 6-MAM D6 for acetylsalicylic acid and morphine D6 foramphetamines). For all other substances, the volumetric ratios are100 μL sample, 900 μL of reagent D, and 20 μL ofreagent C. When it is necessary to analyze seizure liquid substances,the procedure involves only two steps:(i) add 10μL of sample to 990 μL of reagent B and(ii) in autosampler vials 10 μL of the sample solution in 990 μLof reagent D and 20 μL of reagent C.

Method Validation

The method was validated accordingto the U.S. Food and Drug Administration and European Medicines Agencyinternational guidelines.^24,25 and the following parameterswere considered: linearity, lower limit of detection (LLOD), lowerlimit of quantification (LLOQ), precision and trueness (intraday andinterday), and reagents and standards stabilities.

Extraction/Dilutionand LC-MS/MS Procedures

First,the chromatographic separation reported in another study¹⁷ for the simultaneous analysis of more than 739chemicals was tested. By applying the same chromatographic parameters,it was observed that the characteristics required, such as focusingthe analytes in tight peaks to maximize the signal-to-noise ratioand consequently the sensitivity, avoiding the presence of matrixeffects by resolving possible interferents through chromatography,and increasing the parameters for the correct identification consideringalso the reproducibility of the retention time, are maintained inthe analyses of both liquid and solid seized samples. In this protocol,the first extraction of selected compounds was carried out with differentsolvents, such as methanol, isopropanol, hexane, dichloromethane,ethyl acetate, acetonitrile, and solutions formed by different percentagesand combinations of the above-mentioned solvents. Different recoveryfactors were obtained, and the better performances were observed usingacetonitrile 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 evaluatethe better system to obtain the final sample ready for the analysisand to obtain better sensibility, signal-to-noise ratio, and ionizationefficiency in the MS instrumentation and peaks symmetry during thechromatographic run. Dilution with an aqueous solution proved to bethe most efficient. In addition, a percentage of formic acid was addedbecause some molecules with acid pH were found to be more stable.The final dilution was focused on injection and chromatographic resolutionin order to reach good peak shapes. It is very important that theinjected solution is as similar as possible to the initial conditionof the gradient in order to avoid peak tailings and non-Gaussian peaksshapes. The aqueous solution used for the final dilution was modifiedusing 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 ionizationand fragmentation are optimized. Electrospray ionization (ESI) temperatureswere set from 250 to 550 °C with steps of 50 and 450 °Cselected as the best one considering all molecules. Ion spray voltageis checked varying it from 500 to 5500 V, and +5400 and −4500V are the best values (considering peaks intensities and noise) inpositive and negative ionization modes, respectively. Regarding HPLCseparation, it is very important to set a good mobile phase gradient,due to the presence of several isobar molecules with the same parentions (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 mandatorythat their resolution is by HPLC gradient. The analysis was carriedout using a gradient from 5% to 75% of organic solvent in 8 min toreach the separation between all these molecules. Acetonitrile andmethanol are tried as organic solvents for mobile phase M2, and acetonitrileresults to be the best one. In chromatographic separation development,in this case implemented to focus the analytes before their detectionby MS/MS, it was also verified that no carry over (or memory effect)problems were present that could affect the batch analyzes throughthe use of an autosampler. No effects were found with the optimizedmobile phase gradient, while maintaining the analysis within 15 minincluding the system reconditioning. The optimized LC-MS/MS parametersand the analytes chromatographic profiles, using polarity switching,are shown inFigure2. More MRM transitions and instrumental parameters details are reportedinTables S.1–S.4 andFigures S.1 and S.2.

Figure of Merits and Method Validation

The method validationprocedure, obtained following international guidelines,^24,25 saw the evaluation of analytical parameters such as linearity, lowerlimit of detection (LLOD), lower limit of quantification (LLOQ), precisionand trueness (intra and interday), and reagents and standards stabilities.As reported inTable S.5, the method resultedlinearly in the concentration range from 5 to 100 ng/mL withr² values ≥ 0.9909, showing a lower limitof detection equal to 1.67 ng/mL. In the range, the performances ofthe methods were studied in terms of precision (CV%) and trueness(BIAS%) both intraday and interday (n = 6 for eachsample and for each parameter). In the validated method, the calibration(once verified for the absence of matrix effects thanks to thedilute and shoot process) was carried out on standard solutionsamples. To validate the precision and trueness, solid and liquidsamples (as they are and spiked) were analyzed with the procedurereported to have the background value subtracted from the instrumentalresponse of the spiked sample. The result was evaluated in terms ofprecision and trueness for the quantity added and processed on thepreviously calculated calibration, obtaining the figures of meritreported inTable S.5. In particular, theprecision was evaluated on three concentration levels and equal toLLOQ (5 ng/mL),C_m (50 ng/mL), andC_up (100 ng/mL). The performances in terms oftrueness were evaluated at two concentration levels and equal toC_i (25 ng/mL) andC_h (75 ng/mL). Repeatability expresses the precision under the sameoperating conditions over a short interval of time and is also termedintra-assay precision. The intermediate precision expresses insteadwithin-laboratory variations. As indicated by the precision and truenessvalues inTable S.5, the herein validatedmethod has shown repeatability values that fulfill the internationalguidelines, as well as the intermediate precision. The recovery couldbe reported as trueness by the assay of a known added amount of analytein the sample or as the difference between the mean and the acceptedtrue value. In this work, the repeatability of the extraction processand the extraction yield indicated as intraday and interday truenessrespects the limits for the methods validation. During the validationprocess, it was observed that all the reagents were stable up to 3years at a temperature of 2–8 °C, while the chemical calibrationstandards (and QC) and reagent C containing the internal standardsmust be stored at −20 °C. No matrix effects were observedduring the method development. This phenomenon can be ascribed tothe fact that thedilute and shoot procedure developedand implemented provides for an overall dilution of the seized materialby a factor of 1:10,000 (w:v in the case of seizedsolid materials andv:v in case of liquid). Thishigh dilution factor, thanks to the instrumental sensitivity, completelyreduces any effects on the analytes source ionization process, aswell as minimize any effects related to peak asymmetries or fluctuationsin the instrumental response.

Real Sample Analyses

In order to demonstrate the methodapplicability, solid and liquid seized samples were analyzed. Someof more interesting analyzed samples are reported inTable1, confirming the broad applicabilityof the reported method for the evaluation of unknown solid and liquidmaterials. Specifically, for the correct identification, we used thecorrespondences related to the known material, as reported inFigure S.3. In addition, in light of the proceduretraceability due to the analyses in an accredited laboratory, thequantitative determination of up to 37 analytes, the easy executionand no analyte loss (related to the “dilute and shoot”process), the high selectivity and sensitivity of the instrumentalconfiguration, together with an overall analysis time of 15 min, thismethod represents a very valid alternative to other procedures reportedin the literature. A comparative evaluation is shown inTable S.6. In fact, compared to recent literature,this method provides an analysis time comparable with others (about15–20 min), with highly sensitive and selective instrumentation(LC-MS/MS), with minimal sample handling (concept ofdiluteand shoot). However, the greatest advantage was certainlyrepresented by the capacity to provide an accurate quantitative analysis(precise and trueness) through the use of internal standards thatallow the normalization of the analyte signal. Another advantage wasrepresented by the capacity to be applied both on solid and liquidsamples without distinction while maintaining high sensitivity andreproducibility. The methods presented in the literature, in the caseof quantitative analyses, consider a limited number of analytes comparedto the present procedure (37 between narcotic substances and commonlyused excipients/adulterants) and require laborious sample handling.In support of the importance of the LC-MS/MS configuration in thetoxicological and forensic fields, it should also be emphasized thatmany recently developed devices are still based on the principlesof mass spectrometry.¹¹

Green AnalyticalProcedure Index (GAPI)

Lately, increasingimportance has been given to the development of “green”methods. In this context, the objective is to reduce the anthropogenicactivities impact on the environment, and in the case of analyticalchemistry, it reflects the attempt to replace common organic solventswith nontoxic and nonpolluting ones. Other measures that refer tothis trend can be found in the Principles of Green Chemistry.²⁶ To date, to characterize the green profile ofan analytical procedure, a reference could be made to the Green AnalyticalProcedure Index (or GAPI). For the herein reported method, as wellas for others developed in our laboratory,²⁷ we evaluated the eco-friendly profile by critically applying theprinciples that lead to the visualization of the GAPI pictogram inFigure3. In particular,the details of the color assignment according to the method parametersare reported inTable S.7 andFigure S.4 and are based on the guidelines indicatedby Płotka-Wasylka in 2018.²⁸

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Supplementary Materials