TY - JOUR
AU - Szpot, Paweł
AB - Abstract This paper presents a rapid, sensitive and precise method developed and validated for the quantification of sufentanil in biological samples using ultra-performance liquid chromatography coupled with QqQ-MS-MS. Plasma samples were extracted with simple and fast liquid-liquid extraction (ethyl acetate, pH 9). Calibration curve showed linearity in the concentration range of 0.005–30 µg/L. The lower limit of quantification was 0.010 µg/L. The most important method features are low lower limit of quantification value, simple plasma extraction and small sample volume. This method is suitable not only for evaluation of the pharmacokinetics, toxicology, bioavailability and clinical pharmacology of sufentanil but also for the detection and identification of this compound in human plasma samples for forensic purposes. Introduction Sufentanil, N-[4-(methoxymethyl)-1-[2-(2-thienyl)ethyl]-4-piperidi-nyl]-N-phenylpropanamide (Figure 1) sold under the brand names Sufenta®, Dsuvia®, Zalviso® and Fentatienil®, is a synthetic, phenylpiperidine opioid drug acting as an agonist of µ1 and µ2 as well as δ and κ opioid receptors (1,2). Since 1976, sufentanil has been used as an anesthetic agent in surgical procedures in patients undergoing tracheal intubation and mechanical ventilation during the introduction and maintenance of general anesthesia. It is also used as a postoperative or obstetric analgesic drug (3–6). Sufentanil is available as tablets (15 and 30 μg), in injection (5 and 50 μg/mL) and in the form of transdermal patches (transdermal therapeutic system) (50 μg). Dosage is determined for each patient individually. When administering booster doses, the initial dose should be considered (7–11). Sufentanil shows about 5–10 times stronger activity than fentanyl (12) and 500–1,000 stronger than morphine (7,13, 14). Sufentanil is characterized by high lipophilicity, it passes easily through the skin and passes from the bloodstream through the blood–brain barrier to the central nervous system (15, 16). The duration of action of sufentanil and its concentration depend on the dose used. A single dose of the drug (15 μg/kg) given intravenously to 8 adult patients led, after 39 minutes, to an average maximum plasma sufentanil concentration of 0.15 μg/L, which then decreased with elimination half-life of 4.6 hours (17). According to other literature data, administration to a group of 14–15 adults sufentanil at a dose of 75 μg/kg (epidural bonus thoracic administration) led after 0.3 hour to the mean maximum plasma concentration of 0.26 μg/L, and the average elimination half-life was 6.3 hours (18). After giving sufentanil in the form of a sublingual tablet (15 μg) to a group of 38 young adult patients, the peak plasma level was 0.04 μg/L, and it was reached 0.8 hour after the start of sufentanil administration. The maintenance doses were administered at intervals of 20 minutes (a total of 40 maintenance doses were administered). The average peak plasma level after the last dose was 0.28 μg/L. It was observed after 0.9 hour from the time of administration. The mean elimination half-lives after a single sublingual dose were 7–12 hours (19). Figure 1. Open in new tabDownload slide Sufentanil. Figure 1. Open in new tabDownload slide Sufentanil. Table I. MRM Conditions Applied in the Analysis of Sufentanil and Sufentanil-d5 Compound . RT [min] . Precursor Ion [m/z] . Product Ion [m/z] . CE [V] . Q1 Pre Bias [V] . Q3 Pre Bias [V] . Dwell time [ms] . Sufentanil 3.07 387.1 238.2 –21.0 –17.0 –10.0 13.0 111.1 –39.0 –13.0 –10.0 13.0 355.3 –21.0 –17.0 –16.0 13.0 Sufentanil-d5 3.06 392.1 238.2 –21.0 –18.0 –10.0 13.0 111.1 –40.0 –18.0 –10.0 13.0 360.3 –21.0 –18.0 –16.0 13.0 Compound . RT [min] . Precursor Ion [m/z] . Product Ion [m/z] . CE [V] . Q1 Pre Bias [V] . Q3 Pre Bias [V] . Dwell time [ms] . Sufentanil 3.07 387.1 238.2 –21.0 –17.0 –10.0 13.0 111.1 –39.0 –13.0 –10.0 13.0 355.3 –21.0 –17.0 –16.0 13.0 Sufentanil-d5 3.06 392.1 238.2 –21.0 –18.0 –10.0 13.0 111.1 –40.0 –18.0 –10.0 13.0 360.3 –21.0 –18.0 –16.0 13.0 Open in new tab Table I. MRM Conditions Applied in the Analysis of Sufentanil and Sufentanil-d5 Compound . RT [min] . Precursor Ion [m/z] . Product Ion [m/z] . CE [V] . Q1 Pre Bias [V] . Q3 Pre Bias [V] . Dwell time [ms] . Sufentanil 3.07 387.1 238.2 –21.0 –17.0 –10.0 13.0 111.1 –39.0 –13.0 –10.0 13.0 355.3 –21.0 –17.0 –16.0 13.0 Sufentanil-d5 3.06 392.1 238.2 –21.0 –18.0 –10.0 13.0 111.1 –40.0 –18.0 –10.0 13.0 360.3 –21.0 –18.0 –16.0 13.0 Compound . RT [min] . Precursor Ion [m/z] . Product Ion [m/z] . CE [V] . Q1 Pre Bias [V] . Q3 Pre Bias [V] . Dwell time [ms] . Sufentanil 3.07 387.1 238.2 –21.0 –17.0 –10.0 13.0 111.1 –39.0 –13.0 –10.0 13.0 355.3 –21.0 –17.0 –16.0 13.0 Sufentanil-d5 3.06 392.1 238.2 –21.0 –18.0 –10.0 13.0 111.1 –40.0 –18.0 –10.0 13.0 360.3 –21.0 –18.0 –16.0 13.0 Open in new tab Sufentanil binds with blood proteins in about 93%. It is metabolized in the liver with the participation of cytochrome CYP3A4 by N-dealkylation or O-demethylation. O-desmethylsufentanil exhibits 10% of the sufentanil activity, whereas N-desalkylsufentanil is inactive. Sufentanil is excreted with urine, in approximately 80% within 24 hours (with 2% in unchanged form). O-desmethylsufentanil can accumulate in patients with renal failure after repeated or continuous administration. The kinetics of sufentanil does not change significantly in patients with liver cirrhosis (7, 13, 15, 16, 20, 21). In the literature, cases of deadly poisoning with sufentanil are reported rarely, compared with other drugs acting on central nervous system (19). The symptoms of sufentanil overdose include depression of the respiratory center, apnea, muscle stiffness (especially of the chest muscles), drop in blood pressure, bradycardia, nausea, vomiting, dizziness and euphoria. In the case of long-term use, development of tolerance can occur, as well as mental and physical addiction. Sufentanil can also severely impair psychomotor performance; therefore, it should not be used while driving or operating mechanical devices (17). So far, sufentanil has been determined in a variety of biological materials using techniques based on gas (22–24) or liquid chromatography (25–27) and radioimmunoassay (28); however, most of these methods require a time-consuming procedure for sample preparation. It is necessary to develop an analytical procedure providing reliable results and, at the same time, fast and simple. Recently, there has been a significant development of specialist scientific equipment used for identification and determination of chemical compounds in biological material. It has been possible to detect a much larger number of substances in much lower concentrations (29). However, the interpretation of results and established standards regarding, for example, validation of methods, require much more attention, and many case reports published in recent years contain detailed data on analytical problems accompanying the study of postmortem material (30–32). The aim of this work was to develop and validate a highly specific, simple and fast method for the determination of sufentanil in biological material. Experimental Chemicals and reagents Water (LiChrosolv® LC–MS), acetonitrile (LiChrosolv® LC–MS), methanol (LiChrosolv® LC–MS), ethyl acetate and formic acid were purchased from Merck (Darmstadt, Germany); ammonium formate was purchased from Sigma-Aldrich (Mumbai, India); ammonium carbonate was purchased from Merck (Darmstadt, Germany); sufentanil and sufentanil-d5 were purchased from Lipomed AG (Arlesheim Switzerland). Standard solutions of sufentanil and sufentanil-d5 were prepared in methanol. The standard solutions were stored at −20°C. Instrumentation–UHPLC–QqQ-MS-MS analysis Chromatographic analysis was performed using an ultra-high performance liquid chromatograph (UHPLC, Shimadzu Nexera X2, Kyoto, Japan). The chromatographic separation was carried out with a Kinetex Biphenyl column 2.1 × 50 mm; 2.6 μm (Phenomenex, USA) with a thermostat at 40°C. A mixture of 10 mM ammonium formate/0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) were used as mobile phases. The gradient elution was carried out at constant flow 0.4 mL/min. The gradient applied was the following: 0 min–5% B, 5 min–95% B, 8 min–95% B and then 9 min 5% B. Return to initial gradient compositions (95% A and 5% B) was performed at 1 min. The injected volume was 2 μL. Detection of the investigated compounds was achieved using triple-quadrupole mass spectrometer (QqQ, Shimadzu 8050, Kyoto, Japan). The spectrometer was equipped with an 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: 2.5 L/min, heating gas flow: 10 L/min, interface temperature: 350°C, DL temperature: 200°C, heat block temperature: 400°C and drying gas flow: 10 L/min. The analytes were then quantified by MRM. 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 I. Blank material Blank samples of human plasma were taken from Regional Blood Donation Center. Blank samples were screened prior to spiking to ensure that they were free from drugs. Working solutions, calibration curve, quality control samples Standard solutions were diluted with methanol to obtain working standard solutions at the following concentrations of sufentanil: 1, 10, 100, 1,000 and 10,000 µg/L. Calibration points were prepared by adding the appropriate working solution to blank human plasma. The final concentrations of the calibrators were 0.010 (lower limit of quantification, LLOQ), 0.025, 0.50, 0.10, 0.25, 0.50, 1.0, 5.0, 10 and 30 (upper limit of quantification, ULOQ) µg/L. Quality control (QC) samples were prepared by spiking blank human plasma to yield final sufentanil concentrations of 0.010 (low QC), 1.0 (medium QC) and 10 (high QC) µg/L. Sample preparation Human plasma (500 μL) was transferred to 12-mL plastic tube adding 5 μL internal standard (IS) (sufentanil-d5 at concentration 100 µg/L) and 500 μL of buffer (0.5 M ammonium carbonate, pH 9). Liquid–liquid extraction (LLE) with ethyl acetate (2 mL) was carried out for 10 min. Samples were centrifuged at 1390 g and the organic phase (1.8 mL) was transferred to 2-mL Eppendorf tube and evaporated to dryness under a stream of nitrogen (at 40°C). The extract was dissolved in 50 μL of methanol, transferred to a glass insert and analyzed by UHPLC–QqQ-MS-MS Method validation Selectivity Ten different lots of blank plasma from different origin were tested for possible endogenous interference peaks at the retention times of the sufentanil. Linearity Linearity was evaluated by the analysis of sufentanil working solution with human plasma in final concentrations of 0.005, 0.010, 0.025, 0.050, 0.10, 0.25, 0.50, 1.0, 5.0, 10 and 30 µg/L (5 replicates per level). Linearity of calibration curve was determined by plotting the peak area ratio of sufentanil to IS in human plasma with concentration for assessment of method performance; linear calibration model was applied. The calibration range was determined as the range where all calibration levels fell within 80–120% of the theoretical concentration. The coefficient of determination (R2) was determined. According to the acceptance criteria used, the coefficient of determination should meet the condition: R2 ≥ 0.995. Precision and accuracy The precisions and accuracies of the method were estimated by replicating analysis (n = 3) of QC samples at 3 concentration levels: 0.010 (low QC), 1.0 (medium QC) and 10.0 (high QC) µg/L. Intra-day precision was evaluated by analyzing QC samples 4 times over 1 day, while inter-day precision was estimated by analyzing QC samples 5 times on 5 different days. The precision was defined as the intra-day and inter-day relative standard deviation (RSD%). The intra- and inter-day accuracy was expressed as mean relative error [MRE% = (mean of the measured concentration − theoretical concentration)/theoretical concentration × 100%]. Carryover To investigate the carryover, three samples without analytes were analyzed after a calibration sample at the ULOQ. Unacceptable carryover was when peak area ratio in a zero sample after analysis of a sample containing a high concentration of sufentanil exceeded 20% of the area ratio observed for the LLOQ samples. LLOQ and LOD LLOQ was defined as the concentration at which the RSD% does not exceed 20%. Limit of detection (LOD) was considered to be the lowest concentration of the sample for which the signal to noise ratio met the condition: S/N ≥ 3. Recovery and matrix effects The recovery (n = 5) of the sufentanil was evaluated at each of the 3 different concentrations 0.010, 1.0 and 10 µg/L. The recovery (in percent) was determined by comparing calculated concentration of sufentanil in spiked blank human plasma versus calculated concentration of standard solutions at the same concentration. Matrix effects were calculated using equation described by Chambers et al. (33): $$\begin{equation*}\%\,\,\textrm{Matrix~effects}=\left( {{\frac{Response\,\,extracted \,\,sample}{Response \,\,standard}} - 1} \right) {\rm{*}}\; 100\end{equation*}$$ A negative result indicates suppression, while a positive result indicates enhancement of the analyte signal. Results Validation No interfering ion current signals were observed at the retention times of sufentanil or IS (Figure 2A). Figure 2. Open in new tabDownload slide MRM of sufentanil in blank human plasma (A) and blank human plasma fortified with analyte solution to concentrations of 0.005 µg/L (B), 0.01 µg/L (low QC) (C) and 1.0 µg/L (medium QC) (D). Figure 2. Open in new tabDownload slide MRM of sufentanil in blank human plasma (A) and blank human plasma fortified with analyte solution to concentrations of 0.005 µg/L (B), 0.01 µg/L (low QC) (C) and 1.0 µg/L (medium QC) (D). Table II. Methods for Determination of Sufentanil in Human Samplesa Matrix . Sample preparation . Method . Recovery/IS . LLOQ . Ref. . Plasma (500 µL) LLE—ethyl acetate (pH 9) LC–MS-MS 104.52–115.53%/sufentanil-d5 0.01 µg/L Presented method Plasma (880 µL) SPE—Agela Cleanert PEP LC–MS-MS 84.08 ± 7.29%/fentanyl 0.1 µg/L (34) Plasma (1,000 µL) SPE—C18 adsorbent LC–MS-MS –/fentanyl 0.3 µg/L (25) Serum (100 µL) Precipitation—acetonitrile LC–MS-MS 97.8–113.5%/fentanyl 0.25 µg/L (35) Plasma (1,000 µL) Solid phase microextraction: 65-µm polydimethylsiloxane-divinylbenzene (PDMS-DVB) fiber from Supelco GC–MS 1.1%/fentanyl 6 µg/L (36) Plasma (100 µL) Precipitation—methanol and then dispersive liquid–liquid microextraction methanol/chloroform HPLC–DAD 11%/—no IS added 5,500 µg/L (37) Plasma (3,000 µL) LLE—ethyl acetate/n-heptane GC–MS Not determined/fentanyl Not determined (38) Serum (1,000 µL) LLE—n-hexane/EtOH GC–NPD 93.2%/[3H]sufentanil Not determined (39) Plasma (1,000 µL) SPE—C18 adsorbent GC–NPD 98.7%/[3H]sufentanil Not determined (39) Plasma (1,000 µL) SPE—Oasis MCX 60 mg GC–MS 94–95%/alfentanil 0.3 µg/L (40) Plasma (1,000 µL) LLE—extracted twice: Heptane/isoamyl alcohol/sulfuric acid GC–MS 86–89%/alfentanil 0.3 µg/L (40) Plasma (500 µL) SPE—ISOLUTE solid phase extraction LC–MS-MS 77–87%/sufentanil-d5 0.005 µg/L (41) Serum (1,000 µL) LLE—toluene/2-propanol LC–MS-MS 75%/fentanyl 0.01 µg/L (42) Whole blood (250 μL) SPE-CEREX® Clin II SPE cartridges LC–QTOF-MS 63.6%/fentanyl 0.1 µg/L (43) Urine (1000 µL) LLE-phosphate buffer/butyl acetate LC–MS-MS Not determined/fentanyl-d5 0.2 µg/L (44) Whole blood (1,000 µL) LLE-phosphate buffer/butyl acetate LC–MS-MS Not determined/fentanyl-d5 0.2 µg/L (44) Urine (2,000 µL) SPE C18 adsorbent LC–MS-MS 96.0%/norfentanyl-d5 Not determined (45) Urine (500 µL) SPE OASIS HLB C18 LC–MS-MS 114%/fentanyl-d5 0.009 µg/L (LOD) (46) Blood (100 µL) Precipitation—acetonitrile UHPLC–QTOFMS 81–98%/fentanyl-d5 not determined (47) Whole blood (1,000 µL) SPE—Clean Screen® DAU (C8) phase and an ion exchange (benzenesulfonic acid) LC–MS-MS 71.78–89.59%/fentanyl-d5 0.25 µg/L (48) Matrix . Sample preparation . Method . Recovery/IS . LLOQ . Ref. . Plasma (500 µL) LLE—ethyl acetate (pH 9) LC–MS-MS 104.52–115.53%/sufentanil-d5 0.01 µg/L Presented method Plasma (880 µL) SPE—Agela Cleanert PEP LC–MS-MS 84.08 ± 7.29%/fentanyl 0.1 µg/L (34) Plasma (1,000 µL) SPE—C18 adsorbent LC–MS-MS –/fentanyl 0.3 µg/L (25) Serum (100 µL) Precipitation—acetonitrile LC–MS-MS 97.8–113.5%/fentanyl 0.25 µg/L (35) Plasma (1,000 µL) Solid phase microextraction: 65-µm polydimethylsiloxane-divinylbenzene (PDMS-DVB) fiber from Supelco GC–MS 1.1%/fentanyl 6 µg/L (36) Plasma (100 µL) Precipitation—methanol and then dispersive liquid–liquid microextraction methanol/chloroform HPLC–DAD 11%/—no IS added 5,500 µg/L (37) Plasma (3,000 µL) LLE—ethyl acetate/n-heptane GC–MS Not determined/fentanyl Not determined (38) Serum (1,000 µL) LLE—n-hexane/EtOH GC–NPD 93.2%/[3H]sufentanil Not determined (39) Plasma (1,000 µL) SPE—C18 adsorbent GC–NPD 98.7%/[3H]sufentanil Not determined (39) Plasma (1,000 µL) SPE—Oasis MCX 60 mg GC–MS 94–95%/alfentanil 0.3 µg/L (40) Plasma (1,000 µL) LLE—extracted twice: Heptane/isoamyl alcohol/sulfuric acid GC–MS 86–89%/alfentanil 0.3 µg/L (40) Plasma (500 µL) SPE—ISOLUTE solid phase extraction LC–MS-MS 77–87%/sufentanil-d5 0.005 µg/L (41) Serum (1,000 µL) LLE—toluene/2-propanol LC–MS-MS 75%/fentanyl 0.01 µg/L (42) Whole blood (250 μL) SPE-CEREX® Clin II SPE cartridges LC–QTOF-MS 63.6%/fentanyl 0.1 µg/L (43) Urine (1000 µL) LLE-phosphate buffer/butyl acetate LC–MS-MS Not determined/fentanyl-d5 0.2 µg/L (44) Whole blood (1,000 µL) LLE-phosphate buffer/butyl acetate LC–MS-MS Not determined/fentanyl-d5 0.2 µg/L (44) Urine (2,000 µL) SPE C18 adsorbent LC–MS-MS 96.0%/norfentanyl-d5 Not determined (45) Urine (500 µL) SPE OASIS HLB C18 LC–MS-MS 114%/fentanyl-d5 0.009 µg/L (LOD) (46) Blood (100 µL) Precipitation—acetonitrile UHPLC–QTOFMS 81–98%/fentanyl-d5 not determined (47) Whole blood (1,000 µL) SPE—Clean Screen® DAU (C8) phase and an ion exchange (benzenesulfonic acid) LC–MS-MS 71.78–89.59%/fentanyl-d5 0.25 µg/L (48) a Parameters of presented method are given in the first row of the table. Open in new tab Table II. Methods for Determination of Sufentanil in Human Samplesa Matrix . Sample preparation . Method . Recovery/IS . LLOQ . Ref. . Plasma (500 µL) LLE—ethyl acetate (pH 9) LC–MS-MS 104.52–115.53%/sufentanil-d5 0.01 µg/L Presented method Plasma (880 µL) SPE—Agela Cleanert PEP LC–MS-MS 84.08 ± 7.29%/fentanyl 0.1 µg/L (34) Plasma (1,000 µL) SPE—C18 adsorbent LC–MS-MS –/fentanyl 0.3 µg/L (25) Serum (100 µL) Precipitation—acetonitrile LC–MS-MS 97.8–113.5%/fentanyl 0.25 µg/L (35) Plasma (1,000 µL) Solid phase microextraction: 65-µm polydimethylsiloxane-divinylbenzene (PDMS-DVB) fiber from Supelco GC–MS 1.1%/fentanyl 6 µg/L (36) Plasma (100 µL) Precipitation—methanol and then dispersive liquid–liquid microextraction methanol/chloroform HPLC–DAD 11%/—no IS added 5,500 µg/L (37) Plasma (3,000 µL) LLE—ethyl acetate/n-heptane GC–MS Not determined/fentanyl Not determined (38) Serum (1,000 µL) LLE—n-hexane/EtOH GC–NPD 93.2%/[3H]sufentanil Not determined (39) Plasma (1,000 µL) SPE—C18 adsorbent GC–NPD 98.7%/[3H]sufentanil Not determined (39) Plasma (1,000 µL) SPE—Oasis MCX 60 mg GC–MS 94–95%/alfentanil 0.3 µg/L (40) Plasma (1,000 µL) LLE—extracted twice: Heptane/isoamyl alcohol/sulfuric acid GC–MS 86–89%/alfentanil 0.3 µg/L (40) Plasma (500 µL) SPE—ISOLUTE solid phase extraction LC–MS-MS 77–87%/sufentanil-d5 0.005 µg/L (41) Serum (1,000 µL) LLE—toluene/2-propanol LC–MS-MS 75%/fentanyl 0.01 µg/L (42) Whole blood (250 μL) SPE-CEREX® Clin II SPE cartridges LC–QTOF-MS 63.6%/fentanyl 0.1 µg/L (43) Urine (1000 µL) LLE-phosphate buffer/butyl acetate LC–MS-MS Not determined/fentanyl-d5 0.2 µg/L (44) Whole blood (1,000 µL) LLE-phosphate buffer/butyl acetate LC–MS-MS Not determined/fentanyl-d5 0.2 µg/L (44) Urine (2,000 µL) SPE C18 adsorbent LC–MS-MS 96.0%/norfentanyl-d5 Not determined (45) Urine (500 µL) SPE OASIS HLB C18 LC–MS-MS 114%/fentanyl-d5 0.009 µg/L (LOD) (46) Blood (100 µL) Precipitation—acetonitrile UHPLC–QTOFMS 81–98%/fentanyl-d5 not determined (47) Whole blood (1,000 µL) SPE—Clean Screen® DAU (C8) phase and an ion exchange (benzenesulfonic acid) LC–MS-MS 71.78–89.59%/fentanyl-d5 0.25 µg/L (48) Matrix . Sample preparation . Method . Recovery/IS . LLOQ . Ref. . Plasma (500 µL) LLE—ethyl acetate (pH 9) LC–MS-MS 104.52–115.53%/sufentanil-d5 0.01 µg/L Presented method Plasma (880 µL) SPE—Agela Cleanert PEP LC–MS-MS 84.08 ± 7.29%/fentanyl 0.1 µg/L (34) Plasma (1,000 µL) SPE—C18 adsorbent LC–MS-MS –/fentanyl 0.3 µg/L (25) Serum (100 µL) Precipitation—acetonitrile LC–MS-MS 97.8–113.5%/fentanyl 0.25 µg/L (35) Plasma (1,000 µL) Solid phase microextraction: 65-µm polydimethylsiloxane-divinylbenzene (PDMS-DVB) fiber from Supelco GC–MS 1.1%/fentanyl 6 µg/L (36) Plasma (100 µL) Precipitation—methanol and then dispersive liquid–liquid microextraction methanol/chloroform HPLC–DAD 11%/—no IS added 5,500 µg/L (37) Plasma (3,000 µL) LLE—ethyl acetate/n-heptane GC–MS Not determined/fentanyl Not determined (38) Serum (1,000 µL) LLE—n-hexane/EtOH GC–NPD 93.2%/[3H]sufentanil Not determined (39) Plasma (1,000 µL) SPE—C18 adsorbent GC–NPD 98.7%/[3H]sufentanil Not determined (39) Plasma (1,000 µL) SPE—Oasis MCX 60 mg GC–MS 94–95%/alfentanil 0.3 µg/L (40) Plasma (1,000 µL) LLE—extracted twice: Heptane/isoamyl alcohol/sulfuric acid GC–MS 86–89%/alfentanil 0.3 µg/L (40) Plasma (500 µL) SPE—ISOLUTE solid phase extraction LC–MS-MS 77–87%/sufentanil-d5 0.005 µg/L (41) Serum (1,000 µL) LLE—toluene/2-propanol LC–MS-MS 75%/fentanyl 0.01 µg/L (42) Whole blood (250 μL) SPE-CEREX® Clin II SPE cartridges LC–QTOF-MS 63.6%/fentanyl 0.1 µg/L (43) Urine (1000 µL) LLE-phosphate buffer/butyl acetate LC–MS-MS Not determined/fentanyl-d5 0.2 µg/L (44) Whole blood (1,000 µL) LLE-phosphate buffer/butyl acetate LC–MS-MS Not determined/fentanyl-d5 0.2 µg/L (44) Urine (2,000 µL) SPE C18 adsorbent LC–MS-MS 96.0%/norfentanyl-d5 Not determined (45) Urine (500 µL) SPE OASIS HLB C18 LC–MS-MS 114%/fentanyl-d5 0.009 µg/L (LOD) (46) Blood (100 µL) Precipitation—acetonitrile UHPLC–QTOFMS 81–98%/fentanyl-d5 not determined (47) Whole blood (1,000 µL) SPE—Clean Screen® DAU (C8) phase and an ion exchange (benzenesulfonic acid) LC–MS-MS 71.78–89.59%/fentanyl-d5 0.25 µg/L (48) a Parameters of presented method are given in the first row of the table. Open in new tab The linear concentration range was from 0.010 to 30 µg/L for sufentanil. The coefficient of determination (R2) was > 0.99. The calibration line equation was y = 0.954045x + 0. An LLOQ of sufentanil in human plasma was determined to be 0.010 µg/L. The analysis of sample spiked with lower concentration (0.0050 µg/L) showed that signal from analyte was also visible, but the RSD exceeded 20% (Figure 2B). Because lower concentration samples were not analyzed, 0.0050 µ/L were also considered LOD. The intra-day RSD% data obtained from 3 plasma repetitive measurements of 3 spiked human plasma samples (0.010, 1.0 and 10 µg/L of sufentanil) ranged from 0.50% to 7.28%. The inter-day RSD% ranged from 6.35% to 15.92%. The results for intra- and inter-day accuracy at the 3 QC levels were found to be less than 20.11%. The mean recovery was from 104.52% to 115.53%. Carryover was acceptable because the peak area of a zero sample analyzed after injection of ULOQ was only 19% LOQ. In all the QC samples, positive matrix effect was observed. The developed method was subjected to an international proficiency test at the German Society of Toxicological and Forensic Chemistry (Gesellschaft für Toxikologische und Forensische Chemie) and obtained a positive result (Proficiency Test TAB 1/19—Toxicological Analysis of Diagnostics of Brain Death). Method application The developed method for the determination of sufentanil in human plasma was successfully applied by our laboratory to the investigation of forensic and clinical toxicological cases. It was verified by performing toxicological tests on a 19-year-old man who was injured in a traffic accident. After being admitted to a hospital, the patient was deeply unconscious with extensive injuries of the head, chest, abdomen, pelvis, upper and lower limbs. Due to craniocerebral trauma, he was immediately subjected to neurosurgical procedure. The patient with cardiovascular and respiratory failure underwent analgosedation: he received in infusion pump, among others, sufentanil at a dose of 4 mL/hour (solution containing 250 μg/50 mL). The patient died after 4 days of hospitalization. Toxicological analysis of biological material collected during the autopsy showed presence of thiopental (20,374.0 µg/L), pentobarbital (4,146.3 µg/L), butalbital (240.8 µg/L), fentanyl (16.0 µg/L), sufentanil (0.1 µg/L) and norfentanyl (12.8 µg/L) in the blood sample. The same compounds were also present in the vitreous humor at the following concentrations: thiopental 13,404.1 µg/L, pentobarbital 2,128.6 ng/mL, butalbital 223.4 µg/L, fentanyl 35.5 µg/L, sufentanil 0.4 µg/L and norfentanyl 15.9 µg/L. This method has also been applied for the determination of sufentanil in the plasma of 20 patients for whom it was necessary to use sufentanil in the form of transdermal patches because of severe burnings. In tested plasma samples, sufentanil was found at concentrations below LLOQ. Discussion Determination of drugs in biological material in very low concentrations is a challenge for forensic and clinical toxicologists. In developing a new method of analysis, it is important, in addition to achieving the lowest LLOQ possible, to make it easy and quick to use. Recent papers describing the determination of sufentanil in biological samples are listed in Table II. Their authors mainly used liquid or gas chromatography combined with mass spectrometry. The sample preparation methods described in cited works also varied. Different LLE variants as well as solid phase extraction (SPE) or sample precipitation were used. In three of the above-mentioned works, the sensitivity of picograms per milliliter has been achieved (41, 42, 46). Saari et al. obtained LLOQ = 0.005 µg/L (41), whereas Wang and Bernert described a method that is characterized by LOD = 0.009 µg/L; an LLOQ was not given (46). In both cases, 500 μL of biological material were used for extraction, as well as in the presented method. The important difference between the methods described in these works and the protocol we propose lies in the use of a different method of sample preparation. Both Saari et al. and Wang and Bernert used SPE (ISOLUTE and OASIS, respectively), which is a more time-consuming method than the simple liquid–liquid extraction we applied. Martens-Lobenhoffer managed to achieve an LLOQ of 0.010 µg/L (42). The procedure he developed required a double extraction with a mixture of toluene and 2-propanol. It was necessary to use 1 mL of biological material. The method we present allows to achieve the same LLOQ; however, it is enough to extract only 500 μL—half of the sample volume Martens-Lobenhoffer used—without having to carry out the extraction in two steps. The undoubted advantages of the presented method are LLOQ of 0.010 µg/L and a small volume of biological material needed for extraction. These are the features that, combined with a very simple and fast method of sample preparation, determine the wide application possibilities of the presented method, including both clinical toxicology and forensic analysis. Conclusions A rapid, sensitive and reliable method for the determination of sufentanil in human plasma was developed and fully validated. This method is suitable not only for evaluation of the pharmacokinetics, toxicology, bioavailability and clinical pharmacology of sufentanil but also for the detection and identification of this compound in human plasma samples for forensic purposes. Acknowledgements The authors thank the SHIM-POL A. M. Borzymowski Company for the opportunity to carry out the analysis with the use of the UHPLC–QqQ-MS-MS 8050 system. Conflict of interest The authors declare no conflict of interest. References 1. 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TI - Rapid Determination of Sufentanil in Human Plasma by UHPLC–QqQ-MS-MS
JF - Journal of Analytical Toxicology
DO - 10.1093/jat/bkaa123
DA - 2020-09-09
UR - https://www.deepdyve.com/lp/oxford-university-press/rapid-determination-of-sufentanil-in-human-plasma-by-uhplc-qqq-ms-ms-lDERNQYCvo
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -