Chemicals and Reagents

Analytical standards involved in this study as Cocaine Multi-component Mixture solution (250 µg/mL in acetonitrile, certified reference material Cerilliant®), and Lidocaine solution (1 mg/mL in methanol, certified reference material Cerilliant®), were purchased from Sigma-Aldrich and stored at -20 °C. Internal Standard (IS) SKF 525-A (Proadifen hydrochloride, analytical standard, > 95%, 100 mg) was purchased from Sigma-Aldrich as well. Working solutions of the analytical standards and IS were prepared in acetonitrile or methanol and stored at -20 °C until use. Solvents used in the extraction processes were purchased by PanReac AppliChem ITW Reagents (methanol, phosphate buffer solution pH 9 and phosphate buffer pH 6, 2-propanol, n-hexane, n-heptane, and water).

Sample preparation

Four blank samples of whole blood were transferred to four lab test tubes where analytical Cocaine Multi-component Mixture standard solutions and lidocaine standard solutions were previously allowed to dry to obtain a final concentration of 1 µg/mL, 100 ng/mL, 10 ng/mL and 5 ng/mL for each analyte, in addition with 10 ng of our IS.

DNA extraction

Ten microliters of each blood sample were spotted on a cotton swab and extracted with a QIAGEN QIAamp® DNA Mini kit using a QIAamp buccal swab spin protocol. All centrifugation steps were carried out at room temperature with a Thermo Scientific™ Micro CL 17 microcentrifuge. The microcentrifuge was utilized in the four steps of DNA purification (from the solution obtained from lysis to elution step) at 8000 and 13000 rpm.

DNA extraction was performed according to the QIAGEN QIAamp® DNA Mini kit manual: in the first step (lysis) the swab was placed into a microcentrifuge tube with 400 µL of Buffer AL (30% of guanine hydrochloride and 1% of maleic acid) and 20 µL of proteinase K. Subsequently, the sample was mix-pulsed for 15 s on a vortex mixer and incubated for at least 1 h at 56 °C and briefly centrifugated to remove drops from inside the lid. After the lysis, no precipitate was noted at the bottom of the microcentrifuge tube and 400 µL of ethanol 99% were added to the sample, mixed with a pulse-vortex for 15 s and, finally, briefly centrifuged. In the second step, the solution obtained was loaded in a QIAamp Spin Column and centrifuged at 6000 × g (8000 rpm) for 1 min. The QIAamp Mini Spin Column was placed in a clean 2 mL collection tube (provided in the kit) to initiate the washing steps. The third step consisted in a first wash out with 500 µL of AW1 buffer (a solution at 50% guanine hydrochloride in ethanol), while the fourth step was performed with 500 µL of AW2 buffer (Tris hydroxymethyl-aminomethane) to perform a second washing of the sample in ethanol. After each wash step, the column was centrifuged at 6000 × g (8000 rpm) for one minute and the collection tube containing the filtrate was discarded. The collection tubes in which the remains of the lysed sample and the waste of the washing steps were collected for the subsequent toxicological study. The fifth and last step was DNA elution assessed with 30 µL of buffer AE (10 mM Tris hydroxymethyl-aminomethane with 0.5 mM EDTA pH 9) after 1 min of a full-speed centrifugation (Fig. 1a).

Liquid–liquid extraction

Toxicological analyses were performed on residues of DNA extraction obtained from step no. 2 and from the wash out residues of steps nos. 3 and 4 (see Fig. 1a). This procedure was applied on each blood sample involved in the study. Those three steps were selected for the analyses considering that they produce waste residues not involved in the subsequent phases of DNA extraction.

The residues were extracted with a liquid–liquid extraction technique. Thus, the samples were diluted with 5 mL of buffer phosphate pH 9 and extracted with 5 mL of chloroform/isopropanol/n-heptane (50:33:17; v/v). Samples were then positioned on a rotating wheel (Falc F205) for 30 min, and subsequently separated via centrifugation for 10 min at 3500 rpm (Thermo Scientific, Heraeus Biofuge Primo centrifuge). After the separation of the two phases obtained, the organic phase was collected and left drying under a gentle nitrogen stream. The three specimens (per blood sample analyzed) were reconstituted with 50 µL of methanol and 2 µL of the final solutions were analyzed with a Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer (Fig. 1a).

Solid-Phase extraction

Ten microliters of each blood sample, previously spiked with the above-mentioned molecules of interest and diluted in 5 mL of pH 6 buffer phosphate, were extracted with Solid Phase Extraction technique using Bond Elut™ Certify 130 mg (Agilent). Cartridges were conditioned with 3 mL of methanol and 3 mL of pH 6 phosphate buffer. Samples were centrifuged and the supernatant was loaded into the cartridge. Cartridges were then washed out with 3 mL of pH 6 phosphate buffer and 0.5 mL of methanol and subsequently led to exsiccation. Eluting solution was composed by 2 mL of ethyl acetate/ammonium hydroxide (98:2, v:v) and 2 mL of methanol/ammonia solution (98:2, v:v). Eluates were exsiccated and then reconstituted with 50 μL of methanol (Fig. 1a).

Hypothetical real sample preparation

As a last step, three drops of the blood samples were dropped on a surface and were allowed to dry for 72 h. Each spot was then sampled with a different cotton swab, one for each drop. Ten microliters of Internal Standard (IS) were added to the swabs and then used for DNA extraction with QIAGEN QIAamp® DNA Mini kit following the procedure previously described. At the end, the three residues of DNA extraction obtained from passages nos. 2, 3 and 4 were collected for toxicological analyses with liquid–liquid extraction procedure as reported before (Fig. 1b).

Instrumental conditions

The Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer (Thermo Scientific, San Jose, CA, U.S.A.) was associated to a HPLC system constituted by a Surveyor MS quaternary pump with degasser, Surveyor AS auto-sampler, oven with Rheodyne valve, 20 µL loop and with a heated electrospray ionization source (HESI). The chromatographic column used was a reverse phase Thermo Scientific Zorbax Eclipse XDB-C18 4.6 × 50 mm, with particle size 1.8 μm, stabilized at 35 °C and with a constant flow rate of 0.600 mL/min. Twenty millimolar ammonium formate in water and methanol were the solvents that constituted the mobile phase for the analyses. The gradient of the mobile phase was set for solvent A at: 90% in the first minute, decreased to 15% at the fourth minute and maintained at this percentage until minute 6, at the end was brought back to starting conditions from minute 6 to minute 10. The capillary and vaporization temperature were set at 330 and 280 °C respectively. Electrospray tension (with positive mode) and positive ion spray voltage were set at 3.5 kV. Sheath gas, aux gas and sweep gas were set at 45, 20 and 10 Arb, respectively. CID gas was set at 1.5 mTorr, Q1 resolution was selected at 0.4 FWHM and Q3 resolution was 0.7 FWHM. The Resolution power of Full Size was 70.000 FWHM. The mass range was between 50 to 650 m/z. Automatic Gain Control was set at 5 × 10−4 with a maximum injection time of 100 ms. Quadrupole filtered precursor ions had an isolation range of 2 m/z.

Identification criteria for qualitative confirmation

All the samples under investigation were screened with a customized inclusion list containing our molecules of interest and the IS. The molecules under investigation were confirmed following international standard guidelines for forensic toxicology and assessed via reference material (analytical standards) [9, 10]. The parent ion, product ions, retention time and signal-to noise ratio of each molecule are reported in Tables 1 and 2.

Table 1 Identification criteria for molecules under investigationTable 2 Signal-to-noise ratio of samples analyzedQuantitative confirmation

Linear calibration curves were prepared starting from working solutions with the following ranges: 1–5-10–20-40–80 ng/mL and the coefficient of determination (r2) for linear calibration model was calculated ≥ 0.99 for each molecule.

The carryover effect was investigated by injecting in triplicates an extracted blank sample of whole blood after the highest calibration points. No carryover could be noted.

LOD (Limit Of Detection) was determined as the lowest concentration with a signal-to-noise (S/N) ratio of the peak areas ≥ 3 (0.3 ng/mL) and with all the acceptance criteria met (retention time, peak shape, mass spectral ion ratio. LLOQ was determined as the lowest non-zero calibration point in which all criteria were met (detection, identification, bias, and precision) and S/N ratio of the peak areas ≥ 10 (1 ng/mL) [9, 10].