Chemicals
Water (Chromasolv® LC–MS), methanol (Chromasolv® LC–MS), ethyl acetate, formic acid and fentnyl-d5 were purchased from Sigma-Aldrich (Steinheim, Germany); ammonium formate from Sigma-Aldrich (Bangalore, India); ammonium carbonate from Fluka (Buchs, Switzerland); 4-fluoroisobutyryl fentanyl (4-FiBF) from Chiron (Trondheim, Norway). Standard solutions of 4-FiBF and internal standard (IS) fentanyl-d5, at concentration of 100 µg/mL were prepared in methanol.
Biological material
Drug-free blank blood samples used for the development and validation of the method were obtained from Regional Blood Donation Center, while blank urine and tissues (brain, kidney, liver and stomach wall) were collected during autopsies performed in Department of Forensic Medicine. Blank samples were screened prior to spiking to ensure that they were free from drugs. Forensic biological fluids (blood, urine, bile, vitreous humor, gastric content) and tissues (brain, liver, kidney, stomach wall) were sent to our laboratory, for toxicological analyses.
Working solutions, calibration curve, and quality control samples
The stock and standard solutions were stored at − 20 °C. Standard solutions were diluted with methanol to obtain working standard solutions at the following concentrations for 4-FiBF: 1, 2, 5, 10, 20, 50, 100, 200 and 500 ng/mL. Calibration points and quality control (QC) samples were prepared by mixing the appropriate 4-FiBF working solution with blank whole blood, urine, and homogenates of brain, kidney, liver and stomach wall samples. The final concentrations of the calibrators were 0.1 (lower limit of quantification; LLOQ), 0.2, 0.5, 1, 2, 5, 10, 20 and 50 (upper limit of quantification; ULOQ) ng/mL (biological fluids) or ng/g (solid tissues) for 4-FiBF. Quality control samples were prepared by spiking blank human whole blood, urine, and homogenates of brain, kidney, liver and stomach wall to yield final concentration of 0.2 (low QC), 2.0 (medium QC) and 20 (high QC) ng/mL or ng/g.
Sample preparation
A 0.5-mL volume of biological fluid (blood, urine, bile, vitreous humor, gastric content) or 500 mg of homogenized tissue sample (brain, liver, kidney, stomach wall) was transferred into 10-mL plastic vials. Next, 20 µL of methanolic IS solution (fentanyl-d5, 100 ng/mL) and 0.5 mL of 0.5 M ammonium carbonate buffer (pH 9) were added. Liquid–liquid extraction (LLE) with 2 mL of ethyl acetate was carried out for 10 min. The samples were centrifuged for 10 min (2540 × g at 4 °C). The organic phase was transferred into 2-mL Eppendorf tubes and evaporated to dryness under a stream of inert nitrogen gas at 40 °C. The dry residues were dissolved in 50 µL of methanol. The solution was then transferred into glass inserts of autosampler vials and analyzed by UHPLC–QqQ-MS/MS. Because the concentrations of 4-FiBF in most biological fluids and tissues were markedly above ULOQ (50 ng/mL), the assay was repeated. Blood, urine, vitreous humor and brain samples were diluted with water (LC–MS grade) tenfold, while bile, kidney, liver, stomach wall and gastric content samples were diluted with water tenfold twice, finally obtaining a 100-fold dilution. To homogenize the tissue samples, 1 g of solid specimen (brain, kidney, liver, stomach wall) was transferred to plastic tube (12 mL) and mixed with 1 mL of water (LC–MS grade). The tube was placed in a glass beaker containing ice cubes. The contents of the tube was homogenized using a Q55 sonicator (QSonica, Newtown, USA).
Chromatographic and spectrometric conditions
Analyses were performed using an UHPLC, Shimadzu Nexera X2 (Kyoto, Japan). The separation was done using a Kinetex XB-C18 column (150 × 2.1 mm i.d., particle size 2.6 µm; Phenomenex, Torrance, CA, USA) with the thermostat set at 40 °C. The mobile phase consisted 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient elution was carried out at a constant flow of 0.4 mL/min. The gradient applied was as follows: 0 min, 5% B; 12 min, 98% B; 14 min, 98% B; and 15 min, 5% B. Return to the initial gradient compositions (95% A/5% B) was performed for 5 min. The injection volume was 2.0 μL.
Detection of the investigated compounds was achieved using a QqQ-MS/MS, Shimadzu 8050 (Kyoto, Japan). The spectrometer was equipped with an electrospray ionization (ESI) source; determination of the investigated substances was carried out in the multiple reaction monitoring (MRM) mode. The following MS parameters were fixed: nebulizing gas flow, 3 L/min; heating gas flow, 10 L/min; interface temperature, 250 °C; desolvation line temperature, 200 °C; heat block temperature, 350 °C; and drying gas flow, 10 L/min. A summary of precursor and product ions, collision energies, dwell time, Q1–Q3 pre-bias voltages, and retention time for each compound is presented in Table 1.
Table 1 Multiple reaction monitoring (MRM) conditions used in the high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MS/MS) method for quantification of 4-fluoroisobutyryl fentanyl (4-FiBF) in biological samples Validation
Validation of the method included examination of selectivity, linearity, precision and accuracy, carryover, limit of detection and quantification, recovery and matrix effect.
Selectivity
Ten different lots of blank whole blood, and five different lots of urine, brain, kidney, liver, stomach wall samples from different origin were tested for possible endogenous interference peaks at the retention times of 4-FiBF and IS.
Linearity
Linearity was evaluated by analysis of 4-FiBF working solutions with blank whole blood, urine, brain, kidney, liver and stomach wall in final concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20 and 50 ng/mL or ng/g. Linear calibration model was applied. The coefficient of determination (R2) were determined. According to the acceptance criteria used, the coefficient of determination should meet the condition: R2 ≥ 0.995.
Precision and accuracy
The intra-day and inter-day precision and accuracy were estimated by replicating analysis (n = 5) of QC samples at three concentration levels: 0.2, 2.0 and 20 ng/mL or ng/g. Precision was defined as relative standard deviation (RSD%), while accuracy was expressed as mean relative error (RE%).
Carryover
To investigate the carryover, three samples of each matrix type without analytes were analysed after a calibration sample at the 4-FiBF concentration in highest calibration level of 50 ng/mL or 50 ng/g. Unacceptable carry over was when peak area ratio in a zero sample exceeded 20% of the area ratio observed for the LLOQ samples.
The LLOQ and the LOD
The LLOQ was defined as the concentration at which the RSD% and RE% do not exceed 20% and 15%, respectively [19]. The limit of detection (LOD) was considered to be the lowest concentration of the sample for which the signal-to-noise ratio met the condition at least: S/N ≥ 3.
Recovery and matrix effect
The recovery and matrix effect were evaluated at each of the three different concentrations: 0.2, 2.0 and 20 ng/mL or ng/g. The recovery (%, n = 5) was determined by comparing the response of extracted analyte in spiked blank matrix with the response of analyte spiked after the extraction of blank matrix. The matrix effect (%bias, n = 5) was determined by comparing the response of analyte spiked after the extraction of blank matrix with the response of analyte in neat solution [20].