TY - JOUR
AU - Costa, Jose Luiz
AB - Abstract The development of new sample preparation alternatives in analytical toxicology leading to quick, effective, automated and environmentally friendly procedures is growing in importance. One of these alternatives is the QuEChERS, originally developed for the analysis of pesticide residues, producing cleaner extracts than liquid–liquid extraction, and easier separation of aqueous and organic phases. However, there are few published studies on the miniaturization of this technique for forensic toxicology, especially in postmortem analysis. We developed and validated a modified micro-QuEChERS and LC–MS-MS assay to quantify 16 antidepressants, 7 antipsychotics and 3 metabolites and semi-quantify norfluoxetine and norsertraline in postmortem blood. The calibration curve was linear from 1 to 500 ng/mL, achieved an r > 0.99, with all standards quantifying within ±15% of target except ±20% at the limit of quantification of 1 ng/mL for 26 substances. The F test was applied to evaluate if the variance between replicates remained constant for all calibrators. Six weighting factors were analyzed (1/x, 1/x2, 1/x0,5, 1/y, 1/y2 and 1/y0,5), with the weighting factor with the lowest sum of residual regression errors (1/x2) selected. No endogenous or exogenous interferences were observed. Method imprecision and bias were <19.0% and 19.7%, respectively. Advantages of this method include a low sample volume of 100 µL, simple but effective sample preparation and a rapid 8.5-min run time. The validated analytical method was successfully applied to the analysis of 100 authentic postmortem samples. Introduction Identification and quantification of psychoactive substances (PAS) are crucial for postmortem forensic toxicology because these compounds are frequently involved in overdoses and deaths and drug-facilitated crimes. However, many PAS are also pharmacotherapies to treat depression and other mental disorders, such as antidepressants and mood stabilizers. Acute intoxication by antidepressants leads to excitement and delirium, seizures, dryness of the mouth and skin, inhibition of bowel and bladder activity, respiratory depression, cardiac dysrhythmias and coma. When used in association, monoaminoxidase inhibitors and selective serotonin reuptake inhibitors may cause serotonergic syndrome, characterized by hyperthermia, muscle stiffness, myoclonus and rapid changes in mental state and vital signs (1). Antipsychotics are another class of drugs prescribed for the treatment of depression, anxiety and personality disorders, with the atypical antipsychotics most commonly prescribed. The choice of the biological matrix to perform toxicological analysis depends on the purpose of the analysis and the route, dose, frequency of use and pharmacokinetics of the xenobiotic of interest. Blood (whole blood, serum or plasma) is the main matrix of choice for the quantification of toxicants because it best correlates with a drug’s pharmacodynamic effects (2, 3). Sample pretreatment is necessary to separate and concentrate analytes of interest from the matrix and for preparing an extract compatible with the instrument selected for identification and quantification of analytes (4). Currently, methods based on microextraction processes are available as alternatives to classic liquid–liquid extraction (LLE) and solid-phase extraction (SPE) techniques. LLE is simple and inexpensive, but matrix interferences may lead to poor extraction efficiencies and contamination of equipment. SPE has outstanding selectivity and eliminates interfering substances; however, it is time consuming and more expensive than LLE (5). There is an ongoing search for new sample preparation techniques that are rapid, effective, automatable and environmentally friendly. QuEChERS, the acronym for quick, easy, cheap, effective, rugged and safe, also known as dispersive SPE extraction, achieves these sample preparation goals. Anastassiades et al. (6) purposed the original QuEChERS technique in 2003 to extract pesticide residues in food samples. The method utilized acetonitrile for the extraction, which is less toxic than those used traditionally in LLE, followed by partition of analytes enhanced by the addition of salts to increase ionic strength (salting out effect). Finally, there was a clean-up step employing dispersive SPE extraction. This technique produced cleaner extracts than LLE and facilitated the isolation of the organic phase, and the acetonitrile solvent enabled direct injection into a liquid chromatograph (LC) or gas chromatograph (GC) coupled to a mass spectrometer (MS). Other solvents, such as chloroform, hexane, acetone and ethyl acetate, are not as readily amenable for direct injection (7). The QuEChERS technique was applied for the identification and quantification of pesticides, environmental contaminants and pharmaceuticals in urine and blood samples (8–13). Thus, QuEChERS is easy, reproducible, rapid, less expensive and environmentally friendly than traditional extraction techniques, including for postmortem samples (13–17). The aim of this research was to develop and validate a modified QuEChERS method for sample preparation of postmortem blood to identify and quantify 28 PAS. The micro-QuEChERS approach for drug extraction is rapid, requires a low sample volume, is suitable for compounds with different physiochemical properties and is environmentally friendly due to low solvent consumption. To the best of our knowledge, there are few published studies on the miniaturization of this technique in forensic toxicology, especially in postmortem analysis. Experimental Standards and chemicals Reference standards of bupropion, citalopram, sertraline, desipramine, desmethylcitalopram, duloxetine, fluoxetine, imipramine, mirtazapine, norsertraline, nortriptyline, o-desmethylvenlafaxine, paroxetine, trazodone, venlafaxine, amitriptyline, amitriptyline-d3 and imipramine-d3, olanzapine, risperidone-d4 and levo-mepromazine were purchased from LoGiCal Group (Teddington, Middlesex, UK). Haloperidol, hydroxybupropion, norfluoxetine, doxepin, clomipramine, trimipramine, desipramine-d3, duloxetine-d3, citalopram-d6, doxepin-d3, paroxetine-d6 and desmethylcitalopram-d3 standards were acquired from Cerilliant (Round Rock, TX, USA). Clozapine, duloxetine and ziprasidone standards were obtained from USP (Rockville, MD, USA) and bupropion-d9 and quetiapine standards were from Toronto Research Chemicals (North York, ON, Canada). The certified plasma bi-level controls for tricyclic antidepressants were purchased from Chromsystems (Gräfelfing, Bavaria, Germany). Acetonitrile, ammonium formate, methanol and formic acid (98–100%) were acquired from Merck (Darmstadt, Hesse, Germany), and all extraction solvents were high-performance liquid chromatography (HPLC) grade. Ultra-pure deionized water was supplied by a Milli-Q RG unit from Millipore (Milford, MA, USA). Calibrators, quality controls and internal standards Individual stock solutions were prepared by dilution of reference standards in methanol. Analytes were separated into three groups and combined working stock solutions formulated. Calibrators at 1, 50, 100, 200, 300 and 500 ng/mL were prepared by fortifying 100 µL blank human postmortem blood, previously documented to be negative by laboratory testing, with 10 µL of the appropriate combined working stock solution. Quality control (QC) solutions were prepared by a different analyst from the individual preparing the calibrators. Mixed analyte QC solutions were prepared in methanol. QC samples were created at 3, 150 and 400 ng/mL by fortifying 100 µL blank human postmortem blood with 10 µL of the appropriate QC solution. The internal standard (IS) mixture (citalopram-d6, desipramine-d3, desmethylcitalopram-d3, duloxetine-d3, imipramine-d3, paroxetine-d6, amitriptyline-d3, bupropion-d9, doxepin-d3 and risperidone-d4) was prepared by appropriate dilution in methanol to reach a final concentration of 1 µg/mL. All solutions were stored at −20°C in amber glass vials. Sample preparation For sample preparation, 300 µL acetonitrile was transferred to a polypropylene tube containing 100 mg Q150 AOAC QuEChERS salt (mixture of magnesium sulfate and sodium acetate, 4:1 m/m, Restek, Bellefonte, PA, USA), followed by 200 µL ultrapure water, 100 µL postmortem blood and 10 µL IS solution. The tube was capped, homogenized in a multi-bead shaker (Bead Blaster D2400, Benchmark, Sayreville, NJ, USA) at 7 m/s for 20 s (three cycles) and centrifuged at 9,400 g for 10 min. The organic phase (100 µL) was diluted in mobile phase A (100 µL) and transferred to LC vials, with 2 µL injected into the liquid chromatography–tandem mass spectrometry (LC–MS-MS) instrument. Instrumentation The analysis was performed on a Nexera X2 UFLC chromatographic system coupled to a LCMS8060 triple quadrupole MS (Shimadzu, Kyoto, Japan). The chromatographic separation was performed on a Raptor Biphenyl column (100 mm × 2.1 mm ID, 2.7 μm, Restek, Bellefonte, PA, USA) maintained at 40°C. The mobile phase consisted of ultra-pure water (A) and methanol (B), both containing 0.1% formic acid and 2 mmol/L ammonium formate. The flow rate was 0.4 mL/min, and the gradient elution consisted of a linear change in %B, from 10 to 95%, holding at 95% B for 1 min and returning to 10% over 0.1 min, for a 8.5-min total run time. The MS was equipped with an electrospray ionization source, operated in positive mode. The optimized source parameters were heat block temperature 400°C; ion spray voltage 4.0 kV; nebulizer gas (N2) flow 3 L/min; desolvation line temperature 250°C; drying gas (N2) flow 10 L/min; heating gas (N2) flow 10 L/min; and collision-induced dissociation gas pressure (Ar) 270 kPa. The analyses were performed in multiple reaction monitoring (MRM) mode. For each compound, two MRM transitions were selected, one for quantification and one qualifier for confirmation/identification (Table I), using the MRM ratio as identification criteria with a ±20% maximum acceptance criteria. Data were acquired and analyzed with the LabSolutions software (version 5.96, Shimadzu, Kyoto, Japan). Table I. Mass Spectrometer Parameters and Retention Times of Psychotropic Drugs and Internal Standards, Limit of Quantification (LOQ) and Linearity for the Analysis in Postmortem Blood by Micro-QuEChERS and LC–MS-MS Analyte . MRM transitions (m/z) . EP (V) . CE (V) . CCEP (V) . Retention time (min) . IS . LOQ (ng/mL) . Linearity (ng/mL) . Olanzapine 313.0 ≥ 256.1 –15 –24 –17 2.41 Bupropion-d9 1 1–500 313.0 > 198.0 –16 –41 –20 o-Desmethylvenlafaxine 264.1 ≥ 58.1 –14 –22 –21 2.86 Desmethylcitalopram-d3 1 1–500 264.1 > 246.2 –14 –13 –25 Hydroxybupropion 256.0 ≥ 238.2 –13 –14 –15 3.21 Bupropion-d9 1 1–500 256.0 > 139.0 –28 –28 –25 Bupropion-d9 249.0 ≥ 185.1 –16 –13 –21 3.47 – 1 1–500 249.0 > 131.1 –16 –29 –28 Bupropion 240.1 ≥ 184.1 –11 –11 –20 3.49 Bupropion-d9 1 1–500 240.1 > 131.1 –11 –27 –25 Venlafaxine 278.0 ≥ 58.1 –19 –22 –24 3.68 Desmethylcitalopram-d3 1 1–500 278.0 > 260.1 –19 –11 –29 Mirtazapine 266.1 ≥ 195.1 –45 –24 –21 3.76 Desmethylcitalopram-d3 1 1–500 266.1 > 72.1 –29 –22 –29 Clozapine 326.9 ≥ 270.2 –16 –24 –18 3.92 Citalopram-d6 1 1–500 326.9 > 192.2 –16 –47 –19 Desmethylcitalopram-d3 314.2 ≥ 109.0 –25 –26 –19 3.97 – 1 1–500 314.2 > 262.1 –16 –18 –17 Citalopram-d6 331.2 ≥ 109.2 –13 –30 –20 3.98 – 1 1–500 331.2 > 262.0 –13 –22 –17 Desmethylcitalopram 311.0 ≥ 109.1 –16 –25 –10 3.98 Desmethylcitalopram-d3 1 1–500 311.0 > 262.1 –16 –18 –17 Citalopram 325.5 ≥ 109.1 –15 –27 –21 3.99 Citalopram-d6 1 1–500 325.5 > 262.1 –15 –19 –19 Haloperidol 375.9 ≥ 123.0 –26 –41 –24 4.09 Doxepin-d3 1 1–500 375.9 > 165.0 –26 –25 –18 Trazodone 372.2 ≥ 176.0 –17 –25 –17 4.11 Citalopram-d6 1 1–500 372.2 > 148.0 –17 –39 –27 Doxepin-d3 283.2 ≥ 107.2 –22 –23 –10 4.12 – 1 1–500 283.2 > 235.0 –14 –18 –24 Risperidone-d4 415.2 ≥ 195.0 –20 –35 –20 4.14 – 1 1–500 415.2 > 114.2 –10 –49 –22 Norfluoxetine 296.3 ≥ 134.1 –15 –7 –25 4.15 Risperidone-d4 50 50–500 296.3 > 30.1 –15 –14 –30 Risperidone 411.0 ≥ 191.1 –40 –29 –12 4.15 Risperidone-d4 1 1–500 411.0 > 110.1 –20 –50 –21 Fluoxetine 310.1 ≥ 44.1 –21 –13 –17 4.17 Risperidone-d4 1 1–500 310.1 > 148.1 –15 –8 –11 Doxepin 280.2 ≥ 107.1 –11 –23 –19 4.20 Doxepin-d3 1 1–500 280.2 > 115.2 –14 –47 –24 Quetiapine 348.0 ≥ 253.0 –10 –22 –14 4.35 Desipramine-d3 1 1–500 348.0 > 221.0 –10 –35 –24 Ziprasidone 413.3 ≥ 194.0 –13 –30 –20 4.36 Paroxetine-d6 1 1–500 413.3 > 159.0 –13 –44 –15 Desipramine-d3 270.4 ≥ 75.0 –14 –17 –13 4.42 – 1 1–500 270.4 > 193.1 –14 –39 –19 Desipramine 267.1 ≥ 72.1 –28 –18 –30 4.43 Desipramine-d3 1 1–500 267.1 > 208.1 –28 –22 –24 Paroxetine-d6 336.2 ≥ 76.1 –13 –30 –13 4.43 – 1 1–500 336.2 > 198.3 –13 –22 –13 Paroxetine 330.1 ≥ 70.1 –15 –33 –27 4.44 Paroxetine-d6 1 1–500 330.1 > 192.1 –15 –20 –20 Duloxetine 297.9 ≥ 44.2 –15 –13 –17 4.47 Duloxetine-d3 1 1–500 297.9 > 154.1 –18 –6 –28 Imipramine 284.1 ≥ 86.0 –28 –18 –17 4.47 Imipramine-d3 1 1–500 284.1 > 58.0 –28 –36 –25 Imipramine-d3 284.1 ≥ 89.0 –15 –19 –15 4.47 – 1 1–500 284.1 > 61.0 –15 –40 –24 Duloxetine-d3 301.2 ≥ 47.1 –12 –26 –17 4.49 – 1 1–500 301.2 > 155.2 –15 –13 –17 Nortriptyline 264.1 ≥ 233.1 –12 –14 –26 4.52 Amitriptyline-d3 1 1–500 264.1 > 91.1 –12 –24 –18 Amitriptyline 278.1 ≥ 91.0 –29 –27 –18 4.55 Amitriptyline-d3 1 1–500 278.1 > 233.1 –29 –16 –26 Amitriptyline-d3 281.4 ≥ 233.1 –15 –19 –15 4.55 – 1 1–500 281.4 > 105.1 –15 –25 –21 Trimipramine 295.2 ≥ 100.1 –40 –21 –16 4.62 Imipramine-d3 1 1–500 295.2 > 58.0 –15 –39 –22 Levomepromazine 329.1 ≥ 100.1 –36 –22 –19 4.69 Duloxetine-d3 1 1–500 329.1 > 58.1 –16 –40 –22 Clomipramine 315.1 ≥ 86.0 –12 –21 –16 4.75 Imipramine-d3 1 1–500 315.1 > 58.0 –12 –37 –24 Norsertraline 274.9 ≥ 158.9 –14 –23 –16 4.75 Amitriptyline-d3 50 50–500 274.9 > 88.9 –14 –70 –15 Sertraline 306.0 ≥ 159.0 –15 –27 –29 4.75 Amitriptyline-d3 1 1–500 306.0 > 275.0 −15 −12 −30 Analyte . MRM transitions (m/z) . EP (V) . CE (V) . CCEP (V) . Retention time (min) . IS . LOQ (ng/mL) . Linearity (ng/mL) . Olanzapine 313.0 ≥ 256.1 –15 –24 –17 2.41 Bupropion-d9 1 1–500 313.0 > 198.0 –16 –41 –20 o-Desmethylvenlafaxine 264.1 ≥ 58.1 –14 –22 –21 2.86 Desmethylcitalopram-d3 1 1–500 264.1 > 246.2 –14 –13 –25 Hydroxybupropion 256.0 ≥ 238.2 –13 –14 –15 3.21 Bupropion-d9 1 1–500 256.0 > 139.0 –28 –28 –25 Bupropion-d9 249.0 ≥ 185.1 –16 –13 –21 3.47 – 1 1–500 249.0 > 131.1 –16 –29 –28 Bupropion 240.1 ≥ 184.1 –11 –11 –20 3.49 Bupropion-d9 1 1–500 240.1 > 131.1 –11 –27 –25 Venlafaxine 278.0 ≥ 58.1 –19 –22 –24 3.68 Desmethylcitalopram-d3 1 1–500 278.0 > 260.1 –19 –11 –29 Mirtazapine 266.1 ≥ 195.1 –45 –24 –21 3.76 Desmethylcitalopram-d3 1 1–500 266.1 > 72.1 –29 –22 –29 Clozapine 326.9 ≥ 270.2 –16 –24 –18 3.92 Citalopram-d6 1 1–500 326.9 > 192.2 –16 –47 –19 Desmethylcitalopram-d3 314.2 ≥ 109.0 –25 –26 –19 3.97 – 1 1–500 314.2 > 262.1 –16 –18 –17 Citalopram-d6 331.2 ≥ 109.2 –13 –30 –20 3.98 – 1 1–500 331.2 > 262.0 –13 –22 –17 Desmethylcitalopram 311.0 ≥ 109.1 –16 –25 –10 3.98 Desmethylcitalopram-d3 1 1–500 311.0 > 262.1 –16 –18 –17 Citalopram 325.5 ≥ 109.1 –15 –27 –21 3.99 Citalopram-d6 1 1–500 325.5 > 262.1 –15 –19 –19 Haloperidol 375.9 ≥ 123.0 –26 –41 –24 4.09 Doxepin-d3 1 1–500 375.9 > 165.0 –26 –25 –18 Trazodone 372.2 ≥ 176.0 –17 –25 –17 4.11 Citalopram-d6 1 1–500 372.2 > 148.0 –17 –39 –27 Doxepin-d3 283.2 ≥ 107.2 –22 –23 –10 4.12 – 1 1–500 283.2 > 235.0 –14 –18 –24 Risperidone-d4 415.2 ≥ 195.0 –20 –35 –20 4.14 – 1 1–500 415.2 > 114.2 –10 –49 –22 Norfluoxetine 296.3 ≥ 134.1 –15 –7 –25 4.15 Risperidone-d4 50 50–500 296.3 > 30.1 –15 –14 –30 Risperidone 411.0 ≥ 191.1 –40 –29 –12 4.15 Risperidone-d4 1 1–500 411.0 > 110.1 –20 –50 –21 Fluoxetine 310.1 ≥ 44.1 –21 –13 –17 4.17 Risperidone-d4 1 1–500 310.1 > 148.1 –15 –8 –11 Doxepin 280.2 ≥ 107.1 –11 –23 –19 4.20 Doxepin-d3 1 1–500 280.2 > 115.2 –14 –47 –24 Quetiapine 348.0 ≥ 253.0 –10 –22 –14 4.35 Desipramine-d3 1 1–500 348.0 > 221.0 –10 –35 –24 Ziprasidone 413.3 ≥ 194.0 –13 –30 –20 4.36 Paroxetine-d6 1 1–500 413.3 > 159.0 –13 –44 –15 Desipramine-d3 270.4 ≥ 75.0 –14 –17 –13 4.42 – 1 1–500 270.4 > 193.1 –14 –39 –19 Desipramine 267.1 ≥ 72.1 –28 –18 –30 4.43 Desipramine-d3 1 1–500 267.1 > 208.1 –28 –22 –24 Paroxetine-d6 336.2 ≥ 76.1 –13 –30 –13 4.43 – 1 1–500 336.2 > 198.3 –13 –22 –13 Paroxetine 330.1 ≥ 70.1 –15 –33 –27 4.44 Paroxetine-d6 1 1–500 330.1 > 192.1 –15 –20 –20 Duloxetine 297.9 ≥ 44.2 –15 –13 –17 4.47 Duloxetine-d3 1 1–500 297.9 > 154.1 –18 –6 –28 Imipramine 284.1 ≥ 86.0 –28 –18 –17 4.47 Imipramine-d3 1 1–500 284.1 > 58.0 –28 –36 –25 Imipramine-d3 284.1 ≥ 89.0 –15 –19 –15 4.47 – 1 1–500 284.1 > 61.0 –15 –40 –24 Duloxetine-d3 301.2 ≥ 47.1 –12 –26 –17 4.49 – 1 1–500 301.2 > 155.2 –15 –13 –17 Nortriptyline 264.1 ≥ 233.1 –12 –14 –26 4.52 Amitriptyline-d3 1 1–500 264.1 > 91.1 –12 –24 –18 Amitriptyline 278.1 ≥ 91.0 –29 –27 –18 4.55 Amitriptyline-d3 1 1–500 278.1 > 233.1 –29 –16 –26 Amitriptyline-d3 281.4 ≥ 233.1 –15 –19 –15 4.55 – 1 1–500 281.4 > 105.1 –15 –25 –21 Trimipramine 295.2 ≥ 100.1 –40 –21 –16 4.62 Imipramine-d3 1 1–500 295.2 > 58.0 –15 –39 –22 Levomepromazine 329.1 ≥ 100.1 –36 –22 –19 4.69 Duloxetine-d3 1 1–500 329.1 > 58.1 –16 –40 –22 Clomipramine 315.1 ≥ 86.0 –12 –21 –16 4.75 Imipramine-d3 1 1–500 315.1 > 58.0 –12 –37 –24 Norsertraline 274.9 ≥ 158.9 –14 –23 –16 4.75 Amitriptyline-d3 50 50–500 274.9 > 88.9 –14 –70 –15 Sertraline 306.0 ≥ 159.0 –15 –27 –29 4.75 Amitriptyline-d3 1 1–500 306.0 > 275.0 −15 −12 −30 Quantifier transitions are underlined; CE, collision energy; CCEP, collision cell exit potential; EP, entrance potential; IS, internal standard; MRM, multiple reaction monitoring. Open in new tab Table I. Mass Spectrometer Parameters and Retention Times of Psychotropic Drugs and Internal Standards, Limit of Quantification (LOQ) and Linearity for the Analysis in Postmortem Blood by Micro-QuEChERS and LC–MS-MS Analyte . MRM transitions (m/z) . EP (V) . CE (V) . CCEP (V) . Retention time (min) . IS . LOQ (ng/mL) . Linearity (ng/mL) . Olanzapine 313.0 ≥ 256.1 –15 –24 –17 2.41 Bupropion-d9 1 1–500 313.0 > 198.0 –16 –41 –20 o-Desmethylvenlafaxine 264.1 ≥ 58.1 –14 –22 –21 2.86 Desmethylcitalopram-d3 1 1–500 264.1 > 246.2 –14 –13 –25 Hydroxybupropion 256.0 ≥ 238.2 –13 –14 –15 3.21 Bupropion-d9 1 1–500 256.0 > 139.0 –28 –28 –25 Bupropion-d9 249.0 ≥ 185.1 –16 –13 –21 3.47 – 1 1–500 249.0 > 131.1 –16 –29 –28 Bupropion 240.1 ≥ 184.1 –11 –11 –20 3.49 Bupropion-d9 1 1–500 240.1 > 131.1 –11 –27 –25 Venlafaxine 278.0 ≥ 58.1 –19 –22 –24 3.68 Desmethylcitalopram-d3 1 1–500 278.0 > 260.1 –19 –11 –29 Mirtazapine 266.1 ≥ 195.1 –45 –24 –21 3.76 Desmethylcitalopram-d3 1 1–500 266.1 > 72.1 –29 –22 –29 Clozapine 326.9 ≥ 270.2 –16 –24 –18 3.92 Citalopram-d6 1 1–500 326.9 > 192.2 –16 –47 –19 Desmethylcitalopram-d3 314.2 ≥ 109.0 –25 –26 –19 3.97 – 1 1–500 314.2 > 262.1 –16 –18 –17 Citalopram-d6 331.2 ≥ 109.2 –13 –30 –20 3.98 – 1 1–500 331.2 > 262.0 –13 –22 –17 Desmethylcitalopram 311.0 ≥ 109.1 –16 –25 –10 3.98 Desmethylcitalopram-d3 1 1–500 311.0 > 262.1 –16 –18 –17 Citalopram 325.5 ≥ 109.1 –15 –27 –21 3.99 Citalopram-d6 1 1–500 325.5 > 262.1 –15 –19 –19 Haloperidol 375.9 ≥ 123.0 –26 –41 –24 4.09 Doxepin-d3 1 1–500 375.9 > 165.0 –26 –25 –18 Trazodone 372.2 ≥ 176.0 –17 –25 –17 4.11 Citalopram-d6 1 1–500 372.2 > 148.0 –17 –39 –27 Doxepin-d3 283.2 ≥ 107.2 –22 –23 –10 4.12 – 1 1–500 283.2 > 235.0 –14 –18 –24 Risperidone-d4 415.2 ≥ 195.0 –20 –35 –20 4.14 – 1 1–500 415.2 > 114.2 –10 –49 –22 Norfluoxetine 296.3 ≥ 134.1 –15 –7 –25 4.15 Risperidone-d4 50 50–500 296.3 > 30.1 –15 –14 –30 Risperidone 411.0 ≥ 191.1 –40 –29 –12 4.15 Risperidone-d4 1 1–500 411.0 > 110.1 –20 –50 –21 Fluoxetine 310.1 ≥ 44.1 –21 –13 –17 4.17 Risperidone-d4 1 1–500 310.1 > 148.1 –15 –8 –11 Doxepin 280.2 ≥ 107.1 –11 –23 –19 4.20 Doxepin-d3 1 1–500 280.2 > 115.2 –14 –47 –24 Quetiapine 348.0 ≥ 253.0 –10 –22 –14 4.35 Desipramine-d3 1 1–500 348.0 > 221.0 –10 –35 –24 Ziprasidone 413.3 ≥ 194.0 –13 –30 –20 4.36 Paroxetine-d6 1 1–500 413.3 > 159.0 –13 –44 –15 Desipramine-d3 270.4 ≥ 75.0 –14 –17 –13 4.42 – 1 1–500 270.4 > 193.1 –14 –39 –19 Desipramine 267.1 ≥ 72.1 –28 –18 –30 4.43 Desipramine-d3 1 1–500 267.1 > 208.1 –28 –22 –24 Paroxetine-d6 336.2 ≥ 76.1 –13 –30 –13 4.43 – 1 1–500 336.2 > 198.3 –13 –22 –13 Paroxetine 330.1 ≥ 70.1 –15 –33 –27 4.44 Paroxetine-d6 1 1–500 330.1 > 192.1 –15 –20 –20 Duloxetine 297.9 ≥ 44.2 –15 –13 –17 4.47 Duloxetine-d3 1 1–500 297.9 > 154.1 –18 –6 –28 Imipramine 284.1 ≥ 86.0 –28 –18 –17 4.47 Imipramine-d3 1 1–500 284.1 > 58.0 –28 –36 –25 Imipramine-d3 284.1 ≥ 89.0 –15 –19 –15 4.47 – 1 1–500 284.1 > 61.0 –15 –40 –24 Duloxetine-d3 301.2 ≥ 47.1 –12 –26 –17 4.49 – 1 1–500 301.2 > 155.2 –15 –13 –17 Nortriptyline 264.1 ≥ 233.1 –12 –14 –26 4.52 Amitriptyline-d3 1 1–500 264.1 > 91.1 –12 –24 –18 Amitriptyline 278.1 ≥ 91.0 –29 –27 –18 4.55 Amitriptyline-d3 1 1–500 278.1 > 233.1 –29 –16 –26 Amitriptyline-d3 281.4 ≥ 233.1 –15 –19 –15 4.55 – 1 1–500 281.4 > 105.1 –15 –25 –21 Trimipramine 295.2 ≥ 100.1 –40 –21 –16 4.62 Imipramine-d3 1 1–500 295.2 > 58.0 –15 –39 –22 Levomepromazine 329.1 ≥ 100.1 –36 –22 –19 4.69 Duloxetine-d3 1 1–500 329.1 > 58.1 –16 –40 –22 Clomipramine 315.1 ≥ 86.0 –12 –21 –16 4.75 Imipramine-d3 1 1–500 315.1 > 58.0 –12 –37 –24 Norsertraline 274.9 ≥ 158.9 –14 –23 –16 4.75 Amitriptyline-d3 50 50–500 274.9 > 88.9 –14 –70 –15 Sertraline 306.0 ≥ 159.0 –15 –27 –29 4.75 Amitriptyline-d3 1 1–500 306.0 > 275.0 −15 −12 −30 Analyte . MRM transitions (m/z) . EP (V) . CE (V) . CCEP (V) . Retention time (min) . IS . LOQ (ng/mL) . Linearity (ng/mL) . Olanzapine 313.0 ≥ 256.1 –15 –24 –17 2.41 Bupropion-d9 1 1–500 313.0 > 198.0 –16 –41 –20 o-Desmethylvenlafaxine 264.1 ≥ 58.1 –14 –22 –21 2.86 Desmethylcitalopram-d3 1 1–500 264.1 > 246.2 –14 –13 –25 Hydroxybupropion 256.0 ≥ 238.2 –13 –14 –15 3.21 Bupropion-d9 1 1–500 256.0 > 139.0 –28 –28 –25 Bupropion-d9 249.0 ≥ 185.1 –16 –13 –21 3.47 – 1 1–500 249.0 > 131.1 –16 –29 –28 Bupropion 240.1 ≥ 184.1 –11 –11 –20 3.49 Bupropion-d9 1 1–500 240.1 > 131.1 –11 –27 –25 Venlafaxine 278.0 ≥ 58.1 –19 –22 –24 3.68 Desmethylcitalopram-d3 1 1–500 278.0 > 260.1 –19 –11 –29 Mirtazapine 266.1 ≥ 195.1 –45 –24 –21 3.76 Desmethylcitalopram-d3 1 1–500 266.1 > 72.1 –29 –22 –29 Clozapine 326.9 ≥ 270.2 –16 –24 –18 3.92 Citalopram-d6 1 1–500 326.9 > 192.2 –16 –47 –19 Desmethylcitalopram-d3 314.2 ≥ 109.0 –25 –26 –19 3.97 – 1 1–500 314.2 > 262.1 –16 –18 –17 Citalopram-d6 331.2 ≥ 109.2 –13 –30 –20 3.98 – 1 1–500 331.2 > 262.0 –13 –22 –17 Desmethylcitalopram 311.0 ≥ 109.1 –16 –25 –10 3.98 Desmethylcitalopram-d3 1 1–500 311.0 > 262.1 –16 –18 –17 Citalopram 325.5 ≥ 109.1 –15 –27 –21 3.99 Citalopram-d6 1 1–500 325.5 > 262.1 –15 –19 –19 Haloperidol 375.9 ≥ 123.0 –26 –41 –24 4.09 Doxepin-d3 1 1–500 375.9 > 165.0 –26 –25 –18 Trazodone 372.2 ≥ 176.0 –17 –25 –17 4.11 Citalopram-d6 1 1–500 372.2 > 148.0 –17 –39 –27 Doxepin-d3 283.2 ≥ 107.2 –22 –23 –10 4.12 – 1 1–500 283.2 > 235.0 –14 –18 –24 Risperidone-d4 415.2 ≥ 195.0 –20 –35 –20 4.14 – 1 1–500 415.2 > 114.2 –10 –49 –22 Norfluoxetine 296.3 ≥ 134.1 –15 –7 –25 4.15 Risperidone-d4 50 50–500 296.3 > 30.1 –15 –14 –30 Risperidone 411.0 ≥ 191.1 –40 –29 –12 4.15 Risperidone-d4 1 1–500 411.0 > 110.1 –20 –50 –21 Fluoxetine 310.1 ≥ 44.1 –21 –13 –17 4.17 Risperidone-d4 1 1–500 310.1 > 148.1 –15 –8 –11 Doxepin 280.2 ≥ 107.1 –11 –23 –19 4.20 Doxepin-d3 1 1–500 280.2 > 115.2 –14 –47 –24 Quetiapine 348.0 ≥ 253.0 –10 –22 –14 4.35 Desipramine-d3 1 1–500 348.0 > 221.0 –10 –35 –24 Ziprasidone 413.3 ≥ 194.0 –13 –30 –20 4.36 Paroxetine-d6 1 1–500 413.3 > 159.0 –13 –44 –15 Desipramine-d3 270.4 ≥ 75.0 –14 –17 –13 4.42 – 1 1–500 270.4 > 193.1 –14 –39 –19 Desipramine 267.1 ≥ 72.1 –28 –18 –30 4.43 Desipramine-d3 1 1–500 267.1 > 208.1 –28 –22 –24 Paroxetine-d6 336.2 ≥ 76.1 –13 –30 –13 4.43 – 1 1–500 336.2 > 198.3 –13 –22 –13 Paroxetine 330.1 ≥ 70.1 –15 –33 –27 4.44 Paroxetine-d6 1 1–500 330.1 > 192.1 –15 –20 –20 Duloxetine 297.9 ≥ 44.2 –15 –13 –17 4.47 Duloxetine-d3 1 1–500 297.9 > 154.1 –18 –6 –28 Imipramine 284.1 ≥ 86.0 –28 –18 –17 4.47 Imipramine-d3 1 1–500 284.1 > 58.0 –28 –36 –25 Imipramine-d3 284.1 ≥ 89.0 –15 –19 –15 4.47 – 1 1–500 284.1 > 61.0 –15 –40 –24 Duloxetine-d3 301.2 ≥ 47.1 –12 –26 –17 4.49 – 1 1–500 301.2 > 155.2 –15 –13 –17 Nortriptyline 264.1 ≥ 233.1 –12 –14 –26 4.52 Amitriptyline-d3 1 1–500 264.1 > 91.1 –12 –24 –18 Amitriptyline 278.1 ≥ 91.0 –29 –27 –18 4.55 Amitriptyline-d3 1 1–500 278.1 > 233.1 –29 –16 –26 Amitriptyline-d3 281.4 ≥ 233.1 –15 –19 –15 4.55 – 1 1–500 281.4 > 105.1 –15 –25 –21 Trimipramine 295.2 ≥ 100.1 –40 –21 –16 4.62 Imipramine-d3 1 1–500 295.2 > 58.0 –15 –39 –22 Levomepromazine 329.1 ≥ 100.1 –36 –22 –19 4.69 Duloxetine-d3 1 1–500 329.1 > 58.1 –16 –40 –22 Clomipramine 315.1 ≥ 86.0 –12 –21 –16 4.75 Imipramine-d3 1 1–500 315.1 > 58.0 –12 –37 –24 Norsertraline 274.9 ≥ 158.9 –14 –23 –16 4.75 Amitriptyline-d3 50 50–500 274.9 > 88.9 –14 –70 –15 Sertraline 306.0 ≥ 159.0 –15 –27 –29 4.75 Amitriptyline-d3 1 1–500 306.0 > 275.0 −15 −12 −30 Quantifier transitions are underlined; CE, collision energy; CCEP, collision cell exit potential; EP, entrance potential; IS, internal standard; MRM, multiple reaction monitoring. Open in new tab Validation of the method The method was validated according to guidelines published by the Scientific Working Group for Forensic Toxicology (SWGTOX) and the Society of Toxicological and Forensic Chemistry (GTFCh) (18, 19). Blank human postmortem blood for preparation of fortified matrix samples for validation was collected in blood collection tubes containing sodium fluoride and ethylenediaminetetraacetic tripotassium salt and confirmed negative for analytes of interest prior to use. Limit of detection, quantification and linearity Identification criteria included a symmetrical peak eluting within ±2% of the average calibrator retention time, a signal-to-noise ratio of at least 3 and an ion ratio between the quantifying and qualifier MRM within ±20% of that established by the calibrators. The limit of detection (LOD) was defined as the lowest concentration that met all identification criteria, with the limit of quantification (LOQ) being the lowest concentration fulfilling the identification criteria, a signal-to-noise ratio of at least 10 and quantifying within ±20% of each target concentration (19). Linearity of the calibration curve was assessed in five batches over 5 days, with six-point calibration curves (1, 50, 100, 200, 300 and 500 ng/mL). Calibrators were required to quantify within ±20% coefficient of variation of each target concentration. For the construction of the calibration curve, the F test was applied to evaluate if the variance between the replicates remained constant for the different concentrations of the calibration curve. Thus, six weighting factors were analyzed: 1/x, 1/x2, 1/x0,5, 1/y, 1/y2 and 1/y0,5. For each of the six conditions, the correlation coefficient (r) and sum of residual regression errors (ΣRE%) were calculated. The weighting factor with the lowest ΣRE% was selected (20). Bias Bias was evaluated at three different concentrations (low, medium and high control) in postmortem blood samples over five different batches. Four replicates of each QC concentration were run in each batch with fresh calibrators. The highest average acceptable bias from the target concentration was ±20%. Results are presented in percentages. Imprecision Intraday and interday imprecision were evaluated as the percent relative standard deviation (%RSD) of results from replicate samples (low, medium and high QC) within a day (n = 4) and over 5 days (n = 20), respectively. Imprecision values with %RSD <20% were considered acceptable. One-way analysis of variance (ANOVA) was performed on each QC concentration to assess potentially significant interday variability at P < 0.05. Interference studies Fifty-eight possible exogenous interferences (drugs of abuse and pharmaceuticals) were fortified (50 ng/mL) into the LOQ and negative postmortem blood samples and analyzed (Supplementary Table 1). In the fortified negative samples, no peak can satisfy LOD criteria for the target analytes, and when fortified into LOQ samples, the target analytes were required to quantify within ±20% RSD of the expected concentration. No interference was noted if all analytes in the low QC quantified within ±20% of target with acceptable qualifier/quantifier MRM ratios, and no peak in the negative sample satisfied LOD criteria. Matrix effect Matrix effects were evaluated by comparison of target analyte peak areas in six blank postmortem blood samples fortified with analytes after extraction compared to the average target peak areas of a set of fortified ultra-pure water samples. These 12 samples were extracted according to the method described above. Results were expressed as percentages, and a negative result is indicative of matrix suppression and a positive result of matrix enhancement. Recovery Recovery (extraction efficiency) was performed in two batches: the first using six replicates of blank postmortem blood samples fortified with all analytes and ISs at the low and high QC concentrations, extracted with the proposed procedure and injected into the LC–MS-MS, and the second using six replicates of blank postmortem blood samples extracted by the proposed procedure, and the final extract was fortified with all analytes and ISs at the low and high QC concentrations and injected into the LC–MS-MS. The average peak area of the samples fortified prior to extraction divided by the average peak area of the samples fortified after extraction is multiplied by 100 to give the extraction efficiency. Carryover Carryover is the appearance of unintended analyte signal in subsequent samples after the analysis of a highly positive sample. Carryover can produce inaccurate qualitative or quantitative results. To evaluate carryover, blank matrix samples were analyzed immediately after a fortified blood extract with all analytes at the highest calibrator concentration (500 ng/mL). If no analyte is observed (above the method’s LOD) in the blank matrix, this concentration is determined to be the concentration at which the method is free from carryover (18). Dilution integrity Dilution integrity was studied by diluting one portion of fortified postmortem blood samples (at 1 µg/mL) with three portions of blank postmortem blood (1:4). A 1:10 dilution was similarly challenged. If the measured concentration times the dilution factor is within ±20% of the target concentration, the integrity of the dilution is established. Stability All the stability studies were conducted on low and high QC samples (n = 6) in triplicate. Postmortem blood samples were fortified with working solutions on Day 0, aliquoted in 2 mL polypropylene tubes and stored at 4°C (refrigerator) and −20°C (freezer). At 7, 15 and 30 days, aliquots of each QC were fortified with IS and quantified using freshly prepared calibration curves. These drug concentrations were compared to the concentrations obtained initially from freshly prepared postmortem samples. For evaluation of autosampler stability, QC and calibrator samples were extracted and analyzed immediately. These samples were stored on the autosampler at 10°C and re-injected after 24 hours. The peak areas of these samples stored for 24 hours were compared to those obtained immediately. Stability after three freeze–thaw cycles at −20°C was evaluated by fortifying postmortem blood samples on Day 0, and after each concentration was quantified, the other triplicates were stored at −20°C. After three freeze–thaw cycles (one cycle = 24 hours), these triplicate samples were also quantified against a newly prepared calibration curve. In all stability studies, analytes were considered stable if the concentration was within ±20% of the initial concentration. Results and Discussion For optimization of sample preparation, two QuEChERS salts were tested: Q110 (mixture of magnesium sulfate, sodium chloride, trisodium citrate dihydrate and disodium hydrogencitrate sesquihydrate; 4:1:1:0.5 m/m, Restek) and Q150 AOAC (mixture of magnesium sulfate and sodium acetate, 4:1 m/m, Restek). The Q150 AOAC QuEChERS salt was selected as the best reagent for sample preparation because it showed cleaner extracts and higher peak intensity (evaluated by peak areas) than Q110 QuEChERS salt. Unfortunately, reference materials are very expensive and difficult to obtain for laboratories in developing countries. SWGTOX guidelines specifically permit the preparation of different stock solutions for the reference standard and QC solutions when different analysts prepare the stock solutions from different weighings. The developed method identified and quantified 26 PAS and semi-quantified two PAS metabolites of diverse structure and chemistry in 8.5 minutes, reducing the cost and time of analysis. LOD and LOQ were 1 ng/mL for all analytes, except for norsertraline and norfluoxetine. Figure 1 is the LC–MS-MS chromatogram of 28 PAS at the LOQ including 16 antidepressants, 7 antipsychotics and 5 metabolites. Although the gradient elution generates a well-defined peak for the 28 analytes, norfluoxetine and norsertraline did not achieve our predefined acceptance criteria for LOQ and, therefore, were classified as semi-quantitative. Figure 1. Open in new tabDownload slide Multiple reaction monitoring (MRM) chromatograms for postmortem blood samples containing 16 antidepressants (amitriptyline, bupropion, citalopram, clomipramine, desipramine, doxepin, duloxetine, fluoxetine, imipramine, mirtazapine, o-desmethylvenlafaxine, paroxetine, sertraline, trazodone, trimipramine and venlafaxine), 7 antipsychotics (clozapine, haloperidol, levomepromazine, olanzapine, quetiapine, risperidone and ziprasidone) and 3 metabolites (hydroxybupropion, desmethylcitalopram, and nortriptyline) at LOQ (1.0 ng/mL), except for norfluoxetine and norsertraline (50 ng/mL). Figure 1. Open in new tabDownload slide Multiple reaction monitoring (MRM) chromatograms for postmortem blood samples containing 16 antidepressants (amitriptyline, bupropion, citalopram, clomipramine, desipramine, doxepin, duloxetine, fluoxetine, imipramine, mirtazapine, o-desmethylvenlafaxine, paroxetine, sertraline, trazodone, trimipramine and venlafaxine), 7 antipsychotics (clozapine, haloperidol, levomepromazine, olanzapine, quetiapine, risperidone and ziprasidone) and 3 metabolites (hydroxybupropion, desmethylcitalopram, and nortriptyline) at LOQ (1.0 ng/mL), except for norfluoxetine and norsertraline (50 ng/mL). The most effective weighting factor for the calibration curve from 1 to 500 ng/mL with the lowest ΣRE% was 1/x2 for most analytes. Excellent performance was achieved with r > 0.99 and calculated concentrations of calibrators within ±20% of target. During method optimization, the quantitative transition for hydroxybupropion and the qualifier transitions for venlafaxine and o-desmethylvenlafaxine that involved the loss of water were selected because they were most abundant, and they were usefully employed by other authors in seven other antidepressant methods (13, 21–26). In addition, the method was carefully evaluated for endogenous or exogenous interferences, without issue. The results for imprecision and bias for each analyte are shown in Table II. The highest imprecision in this validation was 19.0% for the low olanzapine QC (3.0 ng/mL). For evaluation of imprecision, an ANOVA was performed, yielding the highest intraday imprecision of 10.8% for duloxetine and 19.0% for the interday imprecision of olanzapine, both at low QC (3.0 ng/mL). Bias was <19.7%, 14.8% and 11.4% for the low, medium and high QC, respectively. Bias was also evaluated by analysis of the certified tricyclic antidepressant controls, with deviation from established concentrations <17.3%. For all analytes, results were in good agreement with the SWGTOX guidelines that set ±20% as the acceptance criteria. Therefore, it was concluded that the method has good repeatability and reproducibility. Table II. Validation Results for Imprecision, Bias and Matrix Effects for Psychotropic Drugs in Postmortem Blood by Micro-QuEChERS and LC–MS-MS Analyte . Intraday imprecision (%RSD) (n = 4) . Interday imprecision (%RSD) (n = 20) . Matrix effect (%) (n = 10) . Bias (%) (n = 20) . Recovery (%) (n = 6) . Low . Medium . High . Low . Medium . High . Low . High . Low . Medium . High . Low . High . Amitriptyline 9.2 6.5 5.3 15.1 12.1 9.8 –4.0 0.6 –9.2 –4.6 –4.9 89.6 116 Bupropion 9.0 5.6 3.8 8.0 8.4 6.3 2.2 4.3 –1.3 –1.7 –1.7 93.2 114 Citalopram 8.3 5.8 4.2 8.7 8.0 6.4 –5.2 –4.3 –1.0 –2.8 –1.8 97.8 116 Clomipramine 6.8 7.5 6.9 11.3 13.6 9.4 3.2 9.0 –12.2 –8.7 –7.3 92.6 109 Clozapine 6.5 7.7 7.0 15.0 10.2 8.0 6.1 5.8 –7.2 –2.5 –2.0 99.3 106 Desipramine 7.5 6.1 6.2 11.3 12.9 11.4 2.5 3.3 –3.3 –5.7 –5.7 95.2 115 Desmethylcitalopram 7.7 6.2 3.7 7.3 7.9 5.1 –3.9 –3.2 3.0 0.3 –1.4 101 114 Doxepin 7.4 7.8 6.7 6.6 9.4 6.6 –10.7 1.5 –10.3 –2.4 –1.2 105 109 Duloxetine 10.8 6.2 5.0 14.0 13.9 7.7 5.1 7.8 –6.8 –9.7 –7.0 92.2 107 Fluoxetine 9.8 5.6 5.2 9.7 6.4 6.2 2.0 4.0 3.2 0.1 –0.1 92.1 114 Haloperidol 7.0 8.0 6.5 5.9 8.0 7.6 –8.7 1.8 –5.8 –4.1 –2.6 102 108 Hydroxybupropion 9.5 6.0 3.7 9.7 8.1 4.6 9.4 5.6 10.2 7.3 7.7 96.9 105 Imipramine 7.9 6.9 3.7 11.4 10.6 6.0 –1.6 0.1 –6.2 –4.1 –4.6 89.5 116 Levomepromazine 6.6 8.7 8.7 7.5 8.6 10.9 28.6 27.5 –18.0 –14.2 –3.9 102 109 Mirtazapine 6.1 6.9 5.5 8.3 15.3 7.9 3.4 3.8 –14.3 –10.7 –8.8 99.9 108 Norfluoxetine a 8.0 4.9 a 16.5 12.5 a 6.6 a –2.7 –6.3 a 112 Norsertraline a 7.5 5.0 a 13.6 11.1 a 17.6 a –4.6 –5.7 a 114 Nortriptyline 9.3 7.0 3.8 12.3 11.8 8.1 2.7 4.3 –5.0 –4.0 –5.3 90.8 116 o-Desmethylvenlafaxine 4.1 1.4 4.2 4.5 10.3 7.6 3.1 5.7 4.8 6.6 0.4 102 111 Olanzapine 8.4 7.3 9.7 19.0 16.1 15.0 24.0 37.3 –6.7 –7.2 4.0 103 106 Paroxetine 9.4 7.5 6.6 11.7 13.5 10.1 3.4 9.0 –12.5 –9.0 –7.0 102 106 Quetiapine 5.4 7.4 7.1 6.3 10.1 7.3 –4.5 0.6 –8.3 –4.4 –2.6 100 106 Risperidone 6.4 7.1 5.6 12.9 11.7 10.8 0.7 –1.9 1.0 3.6 5.2 97.0 107 Sertraline 7.4 7.5 5.2 6.4 11.6 8.8 20.0 18.2 2.7 –1.7 –4.1 85.9 114 Trazodone 6.4 7.5 6.6 7.0 9.9 7.8 0.3 –0.8 –10.3 –6.7 –5.0 105 109 Trimipramine 9.3 6.1 5.6 9.9 11.0 10.3 2.0 1.7 –6.3 –4.8 –2.4 88.8 116 Venlafaxine 9.8 5.6 6.3 11.5 13.7 14.0 –6.7 –6.0 –15.8 –12.2 –10.5 90.8 114 Ziprasidone 7.5 7.0 6.3 9.6 16.9 17.4 6.5 7.4 –19.7 –14.8 –11.4 106 104 Analyte . Intraday imprecision (%RSD) (n = 4) . Interday imprecision (%RSD) (n = 20) . Matrix effect (%) (n = 10) . Bias (%) (n = 20) . Recovery (%) (n = 6) . Low . Medium . High . Low . Medium . High . Low . High . Low . Medium . High . Low . High . Amitriptyline 9.2 6.5 5.3 15.1 12.1 9.8 –4.0 0.6 –9.2 –4.6 –4.9 89.6 116 Bupropion 9.0 5.6 3.8 8.0 8.4 6.3 2.2 4.3 –1.3 –1.7 –1.7 93.2 114 Citalopram 8.3 5.8 4.2 8.7 8.0 6.4 –5.2 –4.3 –1.0 –2.8 –1.8 97.8 116 Clomipramine 6.8 7.5 6.9 11.3 13.6 9.4 3.2 9.0 –12.2 –8.7 –7.3 92.6 109 Clozapine 6.5 7.7 7.0 15.0 10.2 8.0 6.1 5.8 –7.2 –2.5 –2.0 99.3 106 Desipramine 7.5 6.1 6.2 11.3 12.9 11.4 2.5 3.3 –3.3 –5.7 –5.7 95.2 115 Desmethylcitalopram 7.7 6.2 3.7 7.3 7.9 5.1 –3.9 –3.2 3.0 0.3 –1.4 101 114 Doxepin 7.4 7.8 6.7 6.6 9.4 6.6 –10.7 1.5 –10.3 –2.4 –1.2 105 109 Duloxetine 10.8 6.2 5.0 14.0 13.9 7.7 5.1 7.8 –6.8 –9.7 –7.0 92.2 107 Fluoxetine 9.8 5.6 5.2 9.7 6.4 6.2 2.0 4.0 3.2 0.1 –0.1 92.1 114 Haloperidol 7.0 8.0 6.5 5.9 8.0 7.6 –8.7 1.8 –5.8 –4.1 –2.6 102 108 Hydroxybupropion 9.5 6.0 3.7 9.7 8.1 4.6 9.4 5.6 10.2 7.3 7.7 96.9 105 Imipramine 7.9 6.9 3.7 11.4 10.6 6.0 –1.6 0.1 –6.2 –4.1 –4.6 89.5 116 Levomepromazine 6.6 8.7 8.7 7.5 8.6 10.9 28.6 27.5 –18.0 –14.2 –3.9 102 109 Mirtazapine 6.1 6.9 5.5 8.3 15.3 7.9 3.4 3.8 –14.3 –10.7 –8.8 99.9 108 Norfluoxetine a 8.0 4.9 a 16.5 12.5 a 6.6 a –2.7 –6.3 a 112 Norsertraline a 7.5 5.0 a 13.6 11.1 a 17.6 a –4.6 –5.7 a 114 Nortriptyline 9.3 7.0 3.8 12.3 11.8 8.1 2.7 4.3 –5.0 –4.0 –5.3 90.8 116 o-Desmethylvenlafaxine 4.1 1.4 4.2 4.5 10.3 7.6 3.1 5.7 4.8 6.6 0.4 102 111 Olanzapine 8.4 7.3 9.7 19.0 16.1 15.0 24.0 37.3 –6.7 –7.2 4.0 103 106 Paroxetine 9.4 7.5 6.6 11.7 13.5 10.1 3.4 9.0 –12.5 –9.0 –7.0 102 106 Quetiapine 5.4 7.4 7.1 6.3 10.1 7.3 –4.5 0.6 –8.3 –4.4 –2.6 100 106 Risperidone 6.4 7.1 5.6 12.9 11.7 10.8 0.7 –1.9 1.0 3.6 5.2 97.0 107 Sertraline 7.4 7.5 5.2 6.4 11.6 8.8 20.0 18.2 2.7 –1.7 –4.1 85.9 114 Trazodone 6.4 7.5 6.6 7.0 9.9 7.8 0.3 –0.8 –10.3 –6.7 –5.0 105 109 Trimipramine 9.3 6.1 5.6 9.9 11.0 10.3 2.0 1.7 –6.3 –4.8 –2.4 88.8 116 Venlafaxine 9.8 5.6 6.3 11.5 13.7 14.0 –6.7 –6.0 –15.8 –12.2 –10.5 90.8 114 Ziprasidone 7.5 7.0 6.3 9.6 16.9 17.4 6.5 7.4 –19.7 –14.8 –11.4 106 104 a Norfluoxetine and norsertraline did not achieve the acceptance criteria for the LOQ and low QC and were classified as semi-quantitative. Open in new tab Table II. Validation Results for Imprecision, Bias and Matrix Effects for Psychotropic Drugs in Postmortem Blood by Micro-QuEChERS and LC–MS-MS Analyte . Intraday imprecision (%RSD) (n = 4) . Interday imprecision (%RSD) (n = 20) . Matrix effect (%) (n = 10) . Bias (%) (n = 20) . Recovery (%) (n = 6) . Low . Medium . High . Low . Medium . High . Low . High . Low . Medium . High . Low . High . Amitriptyline 9.2 6.5 5.3 15.1 12.1 9.8 –4.0 0.6 –9.2 –4.6 –4.9 89.6 116 Bupropion 9.0 5.6 3.8 8.0 8.4 6.3 2.2 4.3 –1.3 –1.7 –1.7 93.2 114 Citalopram 8.3 5.8 4.2 8.7 8.0 6.4 –5.2 –4.3 –1.0 –2.8 –1.8 97.8 116 Clomipramine 6.8 7.5 6.9 11.3 13.6 9.4 3.2 9.0 –12.2 –8.7 –7.3 92.6 109 Clozapine 6.5 7.7 7.0 15.0 10.2 8.0 6.1 5.8 –7.2 –2.5 –2.0 99.3 106 Desipramine 7.5 6.1 6.2 11.3 12.9 11.4 2.5 3.3 –3.3 –5.7 –5.7 95.2 115 Desmethylcitalopram 7.7 6.2 3.7 7.3 7.9 5.1 –3.9 –3.2 3.0 0.3 –1.4 101 114 Doxepin 7.4 7.8 6.7 6.6 9.4 6.6 –10.7 1.5 –10.3 –2.4 –1.2 105 109 Duloxetine 10.8 6.2 5.0 14.0 13.9 7.7 5.1 7.8 –6.8 –9.7 –7.0 92.2 107 Fluoxetine 9.8 5.6 5.2 9.7 6.4 6.2 2.0 4.0 3.2 0.1 –0.1 92.1 114 Haloperidol 7.0 8.0 6.5 5.9 8.0 7.6 –8.7 1.8 –5.8 –4.1 –2.6 102 108 Hydroxybupropion 9.5 6.0 3.7 9.7 8.1 4.6 9.4 5.6 10.2 7.3 7.7 96.9 105 Imipramine 7.9 6.9 3.7 11.4 10.6 6.0 –1.6 0.1 –6.2 –4.1 –4.6 89.5 116 Levomepromazine 6.6 8.7 8.7 7.5 8.6 10.9 28.6 27.5 –18.0 –14.2 –3.9 102 109 Mirtazapine 6.1 6.9 5.5 8.3 15.3 7.9 3.4 3.8 –14.3 –10.7 –8.8 99.9 108 Norfluoxetine a 8.0 4.9 a 16.5 12.5 a 6.6 a –2.7 –6.3 a 112 Norsertraline a 7.5 5.0 a 13.6 11.1 a 17.6 a –4.6 –5.7 a 114 Nortriptyline 9.3 7.0 3.8 12.3 11.8 8.1 2.7 4.3 –5.0 –4.0 –5.3 90.8 116 o-Desmethylvenlafaxine 4.1 1.4 4.2 4.5 10.3 7.6 3.1 5.7 4.8 6.6 0.4 102 111 Olanzapine 8.4 7.3 9.7 19.0 16.1 15.0 24.0 37.3 –6.7 –7.2 4.0 103 106 Paroxetine 9.4 7.5 6.6 11.7 13.5 10.1 3.4 9.0 –12.5 –9.0 –7.0 102 106 Quetiapine 5.4 7.4 7.1 6.3 10.1 7.3 –4.5 0.6 –8.3 –4.4 –2.6 100 106 Risperidone 6.4 7.1 5.6 12.9 11.7 10.8 0.7 –1.9 1.0 3.6 5.2 97.0 107 Sertraline 7.4 7.5 5.2 6.4 11.6 8.8 20.0 18.2 2.7 –1.7 –4.1 85.9 114 Trazodone 6.4 7.5 6.6 7.0 9.9 7.8 0.3 –0.8 –10.3 –6.7 –5.0 105 109 Trimipramine 9.3 6.1 5.6 9.9 11.0 10.3 2.0 1.7 –6.3 –4.8 –2.4 88.8 116 Venlafaxine 9.8 5.6 6.3 11.5 13.7 14.0 –6.7 –6.0 –15.8 –12.2 –10.5 90.8 114 Ziprasidone 7.5 7.0 6.3 9.6 16.9 17.4 6.5 7.4 –19.7 –14.8 –11.4 106 104 Analyte . Intraday imprecision (%RSD) (n = 4) . Interday imprecision (%RSD) (n = 20) . Matrix effect (%) (n = 10) . Bias (%) (n = 20) . Recovery (%) (n = 6) . Low . Medium . High . Low . Medium . High . Low . High . Low . Medium . High . Low . High . Amitriptyline 9.2 6.5 5.3 15.1 12.1 9.8 –4.0 0.6 –9.2 –4.6 –4.9 89.6 116 Bupropion 9.0 5.6 3.8 8.0 8.4 6.3 2.2 4.3 –1.3 –1.7 –1.7 93.2 114 Citalopram 8.3 5.8 4.2 8.7 8.0 6.4 –5.2 –4.3 –1.0 –2.8 –1.8 97.8 116 Clomipramine 6.8 7.5 6.9 11.3 13.6 9.4 3.2 9.0 –12.2 –8.7 –7.3 92.6 109 Clozapine 6.5 7.7 7.0 15.0 10.2 8.0 6.1 5.8 –7.2 –2.5 –2.0 99.3 106 Desipramine 7.5 6.1 6.2 11.3 12.9 11.4 2.5 3.3 –3.3 –5.7 –5.7 95.2 115 Desmethylcitalopram 7.7 6.2 3.7 7.3 7.9 5.1 –3.9 –3.2 3.0 0.3 –1.4 101 114 Doxepin 7.4 7.8 6.7 6.6 9.4 6.6 –10.7 1.5 –10.3 –2.4 –1.2 105 109 Duloxetine 10.8 6.2 5.0 14.0 13.9 7.7 5.1 7.8 –6.8 –9.7 –7.0 92.2 107 Fluoxetine 9.8 5.6 5.2 9.7 6.4 6.2 2.0 4.0 3.2 0.1 –0.1 92.1 114 Haloperidol 7.0 8.0 6.5 5.9 8.0 7.6 –8.7 1.8 –5.8 –4.1 –2.6 102 108 Hydroxybupropion 9.5 6.0 3.7 9.7 8.1 4.6 9.4 5.6 10.2 7.3 7.7 96.9 105 Imipramine 7.9 6.9 3.7 11.4 10.6 6.0 –1.6 0.1 –6.2 –4.1 –4.6 89.5 116 Levomepromazine 6.6 8.7 8.7 7.5 8.6 10.9 28.6 27.5 –18.0 –14.2 –3.9 102 109 Mirtazapine 6.1 6.9 5.5 8.3 15.3 7.9 3.4 3.8 –14.3 –10.7 –8.8 99.9 108 Norfluoxetine a 8.0 4.9 a 16.5 12.5 a 6.6 a –2.7 –6.3 a 112 Norsertraline a 7.5 5.0 a 13.6 11.1 a 17.6 a –4.6 –5.7 a 114 Nortriptyline 9.3 7.0 3.8 12.3 11.8 8.1 2.7 4.3 –5.0 –4.0 –5.3 90.8 116 o-Desmethylvenlafaxine 4.1 1.4 4.2 4.5 10.3 7.6 3.1 5.7 4.8 6.6 0.4 102 111 Olanzapine 8.4 7.3 9.7 19.0 16.1 15.0 24.0 37.3 –6.7 –7.2 4.0 103 106 Paroxetine 9.4 7.5 6.6 11.7 13.5 10.1 3.4 9.0 –12.5 –9.0 –7.0 102 106 Quetiapine 5.4 7.4 7.1 6.3 10.1 7.3 –4.5 0.6 –8.3 –4.4 –2.6 100 106 Risperidone 6.4 7.1 5.6 12.9 11.7 10.8 0.7 –1.9 1.0 3.6 5.2 97.0 107 Sertraline 7.4 7.5 5.2 6.4 11.6 8.8 20.0 18.2 2.7 –1.7 –4.1 85.9 114 Trazodone 6.4 7.5 6.6 7.0 9.9 7.8 0.3 –0.8 –10.3 –6.7 –5.0 105 109 Trimipramine 9.3 6.1 5.6 9.9 11.0 10.3 2.0 1.7 –6.3 –4.8 –2.4 88.8 116 Venlafaxine 9.8 5.6 6.3 11.5 13.7 14.0 –6.7 –6.0 –15.8 –12.2 –10.5 90.8 114 Ziprasidone 7.5 7.0 6.3 9.6 16.9 17.4 6.5 7.4 –19.7 –14.8 –11.4 106 104 a Norfluoxetine and norsertraline did not achieve the acceptance criteria for the LOQ and low QC and were classified as semi-quantitative. Open in new tab No carryover was observed in blank samples after analysis of a 500 ng/mL sample (highest calibrator), and neither endogenous (analysis of 10 different postmortem blood samples) or exogenous interferences (Supplementary Table 1) were documented. The method’s selectivity was evaluated with 58 other pharmaceuticals and drugs of abuse, with no interference meeting the established validation acceptance criteria. The analytical column does not fully separate enantiomers of citalopram nor completely separate the isomers of doxepin; however, the high cost of a chiral column prohibited its use for this routine application. Doxepin is commercially available as a mixture of trans and cis isomers in a ratio of approximately 85:15 with cis-doxepin as a relatively minor constituent. The mixture of isomers in this ratio was present in the commercially available reference material utilized in our method development and validation. Modification of the elution gradient did not produce a single peak and adversely affected separation of the other drugs. Unfortunately, there were no positive doxepin cases in the authentic samples donated by law enforcement that were analyzed as a proof of concept for this manuscript. However, in two articles in the published literature (2, 3), the presence of the two isomers in different samples is described, with the solution proposed to sum the two peaks. The two isomers were poorly separated in the published chromatograms compared to that achieved by our method. Because our method visualized two isomers, quantification was performed on the highest intensity peak, the trans isomer, which also improved the sensitivity of the method. The results of recovery and matrix effects are described in Table II. Recovery was >85.9% (sertraline low QC) for all analytes. For most analytes, observed matrix effects were <25%, considered acceptable by SWGTOX guidelines if bias and imprecision are <20%. The highest matrix effects were for olanzapine at the high QC (37.3%) and levomepromazine 28.6% and 27.5% for the low and high QC, respectively. Both analytes achieved acceptable imprecision and bias. All analytes were stable for 15 days at 4°C, except for olanzapine, ziprasidone and bupropion. Hydroxybupropion, mirtazapine, trazodone and quetiapine were stable for 30 days at 4°C. Of all the analytes, olanzapine, o-desmethylvenlafaxine, bupropion and venlafaxine were not stable for at least 30 days at −20°C, and levomepromazine was stable for only 7 days when frozen. Stability studies on the autosampler for 24 hours indicated that most analytes were stable for 24 hours, except for sertraline in the low QC. All analytes were stable for three freeze/thaw cycles (1 cycle = 24 hours at −20°C). The results for the stability studies are shown in Supplementary Table 2. The instabilities of olanzapine and bupropion are well known. According to Bishop-Freeman et al. (27), after 30 days, bupropion severely degraded at room temperature, but samples stored in a freezer were the most stable. Olanzapine stability varies according to sample matrix and physical storage conditions. Andreassen et al. (28) noted that UV exposure caused significant degradation, recommending that samples be protected from light. Unfortunately, sample preparation is conducted on the bench top with direct light exposure. An alternative would be to analyze active metabolites that have greater stability, as a good indicator for exposure to the parent drug. Analysis of authentic forensic toxicology cases One-hundred postmortem blood samples suspected to contain PAS were donated by law enforcement and evaluated by this newly validated analytical method. The samples were collected between 2015 and 2019 from the states of Sao Paulo (Southeast Brazil) and Sergipe (Northeast Brazil); samples were anonymized, sent to our laboratory for validation of this method and stored frozen until analysis. The most common antidepressants and antipsychotics found in these postmortem blood samples were citalopram and risperidone. Citalopram was detected in 17 of 52 positive samples, in concentrations between 1 and 789 ng/mL. Concentrations above the therapeutic level were observed in 14 samples, and in 12 samples, more than one PAS was identified. These results and the analytes’ therapeutic ranges are found in Table III. Supplementary Figure 1 depicts the LC–MS-MS results of an authentic postmortem blood sample analyzed by the new method with 225 ng/mL olanzapine, 103 ng/mL desmethylcitalopram and 789 ng/mL citalopram. Despite the described instability of olanzapine and bupropion and the age of the available cases, positive results for these two analytes or their metabolites were obtained. Dilution integrity was studied for analytes that showed results >500 ng/mL (amitriptyline, bupropion, citalopram, fluoxetine, hydroxybupropion, norfluoxetine, norsertraline, nortriptyline and sertraline), in addition to the carryover assessment in subsequent analyses. Results for these nine analytes are the measured concentration times the dilution factor. No carryover was observed, and dilution integrity was documented at the specified dilutions within ±20% of the target concentration during method validation. Table III. Authentic Postmortem Blood Sample Results (ng/mL) with the New LC–MS-MS Analytical Method for Psychoactive Substances Sample . Analyte . Therapeutic concentration (ng/mL) . Sample concentration (ng/mL) . #2 Citalopram 50–110 3.4 o-desmethylvenlafaxine 100–400 1.5 #3 Citalopram 50–110 10.8 #4 Citalopram 50–110 2.4 Mirtazapine 30–80 3.1 #5 Citalopram 50–110 1.2 Desmethylcitalopram – 1.8 Haloperidol 5–17 1.6 Hydroxybupropion – 1.4 Risperidone 2–20 2.1 Trazodone 0.7–1.0 1.1 #6 Citalopram 50–110 3.0 #12 Citalopram 50–110 1.9 #13 Citalopram 50–110 1.4 Desmethylcitalopram – 1.1 Risperidone 2–20 1.3 #14 Citalopram 50–110 16.7 #15 Citalopram 50–110 1.7 #19 Citalopram 50–110 1.9 #20 Citalopram 50–110 7.0 #38 Citalopram 50–110 1.0 #49 Amitriptyline 50–300 7.6 #54 Ziprasidone 50–200 29.5 #60 Norsertraline – 454 Sertraline 10–250 396 #61 Norsertraline – 57.3 Sertraline 10–250 27.0 #62 Norsertraline – 66.3 Sertraline 10–250 74.2 #63 Norsertraline – 7.6 Sertraline 10–250 2.3 #64 Hydroxybupropion – 262 Quetiapine 100–500 339 #65 Fluoxetine 120–500 94.9 Norfluoxetine – 910 #66 Amitriptyline 50–300 7.1 Nortriptyline 20–200 5.3 #67 Amitriptyline 50–300 7.5 Nortriptyline 20–200 12.5 #68 Fluoxetine 120–500 162 Norfluoxetine – 45.6 #69 Norsertraline – 173 Nortriptyline 20–200 21.9 Sertraline 10–250 130 #70 Paroxetine 10–120 76.4 #71 Amitriptyline 50–300 1130 Nortriptyline 20–200 718 Quetiapine 100–500 457 #72 Fluoxetine 120–500 1131 Norfluoxetine – 981 #73 Amitriptyline 50–300 68.8 Nortriptyline 20–200 121 #74 Desipramine 10–500 63.1 Olanzapine 20–80 45.9 #75 Fluoxetine 120–500 731 Norfluoxetine – 622 #76 Citalopram 50–110 63.5 Desmethylcitalopram – 19.2 #77 Amitriptyline 50–300 4503 Nortriptyline – 2072 #78 Haloperidol 5–17 8.7 #79 Bupropion 10–20 2918 Hydroxybupropion – 2118 #80 Citalopram 50–110 455 Desmethylcitalopram – 57.8 #81 Fluoxetine 120–500 499 Norfluoxetine – 2093 #82 Levomepromazine 5–200 389 #83 Citalopram 50–110 176 Desmethylcitalopram – 167 #84 Amitriptyline 50–300 4475 Nortriptyline 20–200 2057 #85 Norsertraline – 2412 Sertraline 10–250 1800 #86 Nortriptyline 20–200 28.9 Quetiapine 100–500 14.9 #87 o-desmethylvenlafaxine 100–400 152 Venlafaxine 100–400 79.0 #88 Fluoxetine 120–500 1037 Norfluoxetine – 673 Quetiapine 100–500 6.6 #89 Haloperidol 5–17 40.1 Norsertraline – 196 Sertraline 10–250 164 #90 Haloperidol 5–17 7.4 #91 Nortriptyline 20–200 10.3 #92 Norsertraline – 233 Sertraline 10–250 71.1 #93 Citalopram 50–110 789 Desmethylcitalopram – 103 Olanzapine 20–80 225 #94 Norsertraline – 70.4 Sertraline 10–250 60.6 #95 Amitriptyline 50–300 95.6 Nortriptyline 20–200 54.7 #96 Citalopram 50–110 366 Desmethylcitalopram – 150 #97 Amitriptyline 50–300 54.0 Nortriptyline 20–200 177 Sample . Analyte . Therapeutic concentration (ng/mL) . Sample concentration (ng/mL) . #2 Citalopram 50–110 3.4 o-desmethylvenlafaxine 100–400 1.5 #3 Citalopram 50–110 10.8 #4 Citalopram 50–110 2.4 Mirtazapine 30–80 3.1 #5 Citalopram 50–110 1.2 Desmethylcitalopram – 1.8 Haloperidol 5–17 1.6 Hydroxybupropion – 1.4 Risperidone 2–20 2.1 Trazodone 0.7–1.0 1.1 #6 Citalopram 50–110 3.0 #12 Citalopram 50–110 1.9 #13 Citalopram 50–110 1.4 Desmethylcitalopram – 1.1 Risperidone 2–20 1.3 #14 Citalopram 50–110 16.7 #15 Citalopram 50–110 1.7 #19 Citalopram 50–110 1.9 #20 Citalopram 50–110 7.0 #38 Citalopram 50–110 1.0 #49 Amitriptyline 50–300 7.6 #54 Ziprasidone 50–200 29.5 #60 Norsertraline – 454 Sertraline 10–250 396 #61 Norsertraline – 57.3 Sertraline 10–250 27.0 #62 Norsertraline – 66.3 Sertraline 10–250 74.2 #63 Norsertraline – 7.6 Sertraline 10–250 2.3 #64 Hydroxybupropion – 262 Quetiapine 100–500 339 #65 Fluoxetine 120–500 94.9 Norfluoxetine – 910 #66 Amitriptyline 50–300 7.1 Nortriptyline 20–200 5.3 #67 Amitriptyline 50–300 7.5 Nortriptyline 20–200 12.5 #68 Fluoxetine 120–500 162 Norfluoxetine – 45.6 #69 Norsertraline – 173 Nortriptyline 20–200 21.9 Sertraline 10–250 130 #70 Paroxetine 10–120 76.4 #71 Amitriptyline 50–300 1130 Nortriptyline 20–200 718 Quetiapine 100–500 457 #72 Fluoxetine 120–500 1131 Norfluoxetine – 981 #73 Amitriptyline 50–300 68.8 Nortriptyline 20–200 121 #74 Desipramine 10–500 63.1 Olanzapine 20–80 45.9 #75 Fluoxetine 120–500 731 Norfluoxetine – 622 #76 Citalopram 50–110 63.5 Desmethylcitalopram – 19.2 #77 Amitriptyline 50–300 4503 Nortriptyline – 2072 #78 Haloperidol 5–17 8.7 #79 Bupropion 10–20 2918 Hydroxybupropion – 2118 #80 Citalopram 50–110 455 Desmethylcitalopram – 57.8 #81 Fluoxetine 120–500 499 Norfluoxetine – 2093 #82 Levomepromazine 5–200 389 #83 Citalopram 50–110 176 Desmethylcitalopram – 167 #84 Amitriptyline 50–300 4475 Nortriptyline 20–200 2057 #85 Norsertraline – 2412 Sertraline 10–250 1800 #86 Nortriptyline 20–200 28.9 Quetiapine 100–500 14.9 #87 o-desmethylvenlafaxine 100–400 152 Venlafaxine 100–400 79.0 #88 Fluoxetine 120–500 1037 Norfluoxetine – 673 Quetiapine 100–500 6.6 #89 Haloperidol 5–17 40.1 Norsertraline – 196 Sertraline 10–250 164 #90 Haloperidol 5–17 7.4 #91 Nortriptyline 20–200 10.3 #92 Norsertraline – 233 Sertraline 10–250 71.1 #93 Citalopram 50–110 789 Desmethylcitalopram – 103 Olanzapine 20–80 225 #94 Norsertraline – 70.4 Sertraline 10–250 60.6 #95 Amitriptyline 50–300 95.6 Nortriptyline 20–200 54.7 #96 Citalopram 50–110 366 Desmethylcitalopram – 150 #97 Amitriptyline 50–300 54.0 Nortriptyline 20–200 177 Open in new tab Table III. Authentic Postmortem Blood Sample Results (ng/mL) with the New LC–MS-MS Analytical Method for Psychoactive Substances Sample . Analyte . Therapeutic concentration (ng/mL) . Sample concentration (ng/mL) . #2 Citalopram 50–110 3.4 o-desmethylvenlafaxine 100–400 1.5 #3 Citalopram 50–110 10.8 #4 Citalopram 50–110 2.4 Mirtazapine 30–80 3.1 #5 Citalopram 50–110 1.2 Desmethylcitalopram – 1.8 Haloperidol 5–17 1.6 Hydroxybupropion – 1.4 Risperidone 2–20 2.1 Trazodone 0.7–1.0 1.1 #6 Citalopram 50–110 3.0 #12 Citalopram 50–110 1.9 #13 Citalopram 50–110 1.4 Desmethylcitalopram – 1.1 Risperidone 2–20 1.3 #14 Citalopram 50–110 16.7 #15 Citalopram 50–110 1.7 #19 Citalopram 50–110 1.9 #20 Citalopram 50–110 7.0 #38 Citalopram 50–110 1.0 #49 Amitriptyline 50–300 7.6 #54 Ziprasidone 50–200 29.5 #60 Norsertraline – 454 Sertraline 10–250 396 #61 Norsertraline – 57.3 Sertraline 10–250 27.0 #62 Norsertraline – 66.3 Sertraline 10–250 74.2 #63 Norsertraline – 7.6 Sertraline 10–250 2.3 #64 Hydroxybupropion – 262 Quetiapine 100–500 339 #65 Fluoxetine 120–500 94.9 Norfluoxetine – 910 #66 Amitriptyline 50–300 7.1 Nortriptyline 20–200 5.3 #67 Amitriptyline 50–300 7.5 Nortriptyline 20–200 12.5 #68 Fluoxetine 120–500 162 Norfluoxetine – 45.6 #69 Norsertraline – 173 Nortriptyline 20–200 21.9 Sertraline 10–250 130 #70 Paroxetine 10–120 76.4 #71 Amitriptyline 50–300 1130 Nortriptyline 20–200 718 Quetiapine 100–500 457 #72 Fluoxetine 120–500 1131 Norfluoxetine – 981 #73 Amitriptyline 50–300 68.8 Nortriptyline 20–200 121 #74 Desipramine 10–500 63.1 Olanzapine 20–80 45.9 #75 Fluoxetine 120–500 731 Norfluoxetine – 622 #76 Citalopram 50–110 63.5 Desmethylcitalopram – 19.2 #77 Amitriptyline 50–300 4503 Nortriptyline – 2072 #78 Haloperidol 5–17 8.7 #79 Bupropion 10–20 2918 Hydroxybupropion – 2118 #80 Citalopram 50–110 455 Desmethylcitalopram – 57.8 #81 Fluoxetine 120–500 499 Norfluoxetine – 2093 #82 Levomepromazine 5–200 389 #83 Citalopram 50–110 176 Desmethylcitalopram – 167 #84 Amitriptyline 50–300 4475 Nortriptyline 20–200 2057 #85 Norsertraline – 2412 Sertraline 10–250 1800 #86 Nortriptyline 20–200 28.9 Quetiapine 100–500 14.9 #87 o-desmethylvenlafaxine 100–400 152 Venlafaxine 100–400 79.0 #88 Fluoxetine 120–500 1037 Norfluoxetine – 673 Quetiapine 100–500 6.6 #89 Haloperidol 5–17 40.1 Norsertraline – 196 Sertraline 10–250 164 #90 Haloperidol 5–17 7.4 #91 Nortriptyline 20–200 10.3 #92 Norsertraline – 233 Sertraline 10–250 71.1 #93 Citalopram 50–110 789 Desmethylcitalopram – 103 Olanzapine 20–80 225 #94 Norsertraline – 70.4 Sertraline 10–250 60.6 #95 Amitriptyline 50–300 95.6 Nortriptyline 20–200 54.7 #96 Citalopram 50–110 366 Desmethylcitalopram – 150 #97 Amitriptyline 50–300 54.0 Nortriptyline 20–200 177 Sample . Analyte . Therapeutic concentration (ng/mL) . Sample concentration (ng/mL) . #2 Citalopram 50–110 3.4 o-desmethylvenlafaxine 100–400 1.5 #3 Citalopram 50–110 10.8 #4 Citalopram 50–110 2.4 Mirtazapine 30–80 3.1 #5 Citalopram 50–110 1.2 Desmethylcitalopram – 1.8 Haloperidol 5–17 1.6 Hydroxybupropion – 1.4 Risperidone 2–20 2.1 Trazodone 0.7–1.0 1.1 #6 Citalopram 50–110 3.0 #12 Citalopram 50–110 1.9 #13 Citalopram 50–110 1.4 Desmethylcitalopram – 1.1 Risperidone 2–20 1.3 #14 Citalopram 50–110 16.7 #15 Citalopram 50–110 1.7 #19 Citalopram 50–110 1.9 #20 Citalopram 50–110 7.0 #38 Citalopram 50–110 1.0 #49 Amitriptyline 50–300 7.6 #54 Ziprasidone 50–200 29.5 #60 Norsertraline – 454 Sertraline 10–250 396 #61 Norsertraline – 57.3 Sertraline 10–250 27.0 #62 Norsertraline – 66.3 Sertraline 10–250 74.2 #63 Norsertraline – 7.6 Sertraline 10–250 2.3 #64 Hydroxybupropion – 262 Quetiapine 100–500 339 #65 Fluoxetine 120–500 94.9 Norfluoxetine – 910 #66 Amitriptyline 50–300 7.1 Nortriptyline 20–200 5.3 #67 Amitriptyline 50–300 7.5 Nortriptyline 20–200 12.5 #68 Fluoxetine 120–500 162 Norfluoxetine – 45.6 #69 Norsertraline – 173 Nortriptyline 20–200 21.9 Sertraline 10–250 130 #70 Paroxetine 10–120 76.4 #71 Amitriptyline 50–300 1130 Nortriptyline 20–200 718 Quetiapine 100–500 457 #72 Fluoxetine 120–500 1131 Norfluoxetine – 981 #73 Amitriptyline 50–300 68.8 Nortriptyline 20–200 121 #74 Desipramine 10–500 63.1 Olanzapine 20–80 45.9 #75 Fluoxetine 120–500 731 Norfluoxetine – 622 #76 Citalopram 50–110 63.5 Desmethylcitalopram – 19.2 #77 Amitriptyline 50–300 4503 Nortriptyline – 2072 #78 Haloperidol 5–17 8.7 #79 Bupropion 10–20 2918 Hydroxybupropion – 2118 #80 Citalopram 50–110 455 Desmethylcitalopram – 57.8 #81 Fluoxetine 120–500 499 Norfluoxetine – 2093 #82 Levomepromazine 5–200 389 #83 Citalopram 50–110 176 Desmethylcitalopram – 167 #84 Amitriptyline 50–300 4475 Nortriptyline 20–200 2057 #85 Norsertraline – 2412 Sertraline 10–250 1800 #86 Nortriptyline 20–200 28.9 Quetiapine 100–500 14.9 #87 o-desmethylvenlafaxine 100–400 152 Venlafaxine 100–400 79.0 #88 Fluoxetine 120–500 1037 Norfluoxetine – 673 Quetiapine 100–500 6.6 #89 Haloperidol 5–17 40.1 Norsertraline – 196 Sertraline 10–250 164 #90 Haloperidol 5–17 7.4 #91 Nortriptyline 20–200 10.3 #92 Norsertraline – 233 Sertraline 10–250 71.1 #93 Citalopram 50–110 789 Desmethylcitalopram – 103 Olanzapine 20–80 225 #94 Norsertraline – 70.4 Sertraline 10–250 60.6 #95 Amitriptyline 50–300 95.6 Nortriptyline 20–200 54.7 #96 Citalopram 50–110 366 Desmethylcitalopram – 150 #97 Amitriptyline 50–300 54.0 Nortriptyline 20–200 177 Open in new tab The micro-QuEChERS procedure requires a low sample volume of 100 μL and made our laboratory more efficient due to the 15-min sample preparation time, which is more rapid than LLE and SPE techniques. Moreover, this sample preparation requires less frequent instrument maintenance due to cleaner extracts as compared to simple protein precipitation. This is especially true for postmortem blood samples that may be clotted or putrefied. The current method provides fast and reliable determinations of multiple drugs in a single analysis and is fit for purpose for a forensic toxicology laboratory workload. Conclusions A sensitive method based on micro-QuEChERS and LC–MS-MS was developed to quantify 16 antidepressants, 7 antipsychotics and 3 metabolites and semi-quantify two additional metabolites. The method was fully validated, requires a low sample volume of 100 µL and combines a single sample preparation step and fast instrumental analysis. When applied to authentic postmortem samples, the efficiency of the protocol was demonstrated. Acknowledgements The authors thank Institute of Legal Medicine-Sao Paulo State Police and the Institute of Analysis and Forensic Research of Sergipe State Police for kind donation of authentic samples. Funding This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (Process Number 2018/00432-1), Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq (Process Number 425814/2018-1) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—CAPES (Process Number 88882.435302/2019-01). Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the University of Campinas committee (Comitê de Ética em Pesquisa da UNICAMP—CEP, CAAE 87316318.0.0000.5404) and Superintendence of the Technical-Scientific of Sao Paulo State Police (No. 766/2015/ATS/SPTC-SSP). Supplementary data Supplementary data is available at Journal of Analytical Toxicology online. References 1. 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TI - Development and Validation of a Method for Quantification of 28 Psychotropic Drugs in Postmortem Blood Samples by Modified Micro-QuEChERS and LC–MS-MS
JF - Journal of Analytical Toxicology
DO - 10.1093/jat/bkaa138
DA - 2020-09-30
UR - https://www.deepdyve.com/lp/oxford-university-press/development-and-validation-of-a-method-for-quantification-of-28-UkSPxBTs8q
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -