Materials and methods

Chemicals and solutions

Pipamperone dihydrochloride (99%, HPLC-grade powder) was purchased from Sigma-Aldrich® (Darmstadt, Germany) and pipamperone-d10 dihydrochloride (manufacturer Toronto Research Chemicals (TRC)), was obtained via LGC Standards Ltd. (Wesel, Germany). Water and acetonitrile (UHPLC-MS grade, Chemsolute®) were purchased from Th. Geyer (Renningen, Germany). Formic acid (99%, ULC-MS grade) was obtained from Biosolve (Valkenswaard, The Netherlands). Methanol (UHPLC-MS grade, LiChrosolv®) and di-sodium hydrogen phosphate (Na2HPO4, p.a.) were obtained from Supelco® (Darmstadt, Germany). β-glucuronidase from E. coli was purchased from Roche Diagnostics GmbH (Mannheim, Germany). QuEChERS Dispersive Solid Phase Extraction (dSPE) Kits for fatty samples (article number: 5982 − 5121) from Agilent (Santa Clara, USA) were used.

Calibrators and quality control samples

Calibrator samples were prepared in 200 µL whole blood from volunteers. The calibration ranged from 0.05 to 5.0 mg/L, using ten calibrators (equidistant distribution). Whole blood quality control samples were prepared at concentrations of 0.25, 1.5 and 4.5 mg/L, aliquoted and stored at -20 °C until use. Furthermore, commercially available serum quality control samples were used (TDMC 1/23 (samples A and B) from Arvecon GmbH (Walldorf, Germany), TDMC A: 0.13 mg/L and TDMC B: 0.32 mg/L). The internal standard (IS) solution was prepared in a concentration of 1.5 mg/L in methanol.

Selection of authentic cases and classification

Real case samples were obtained from autopsies performed in the Institute of Legal Medicine in Cologne between July 2020 and May 2023, in which toxicological tests had been commissioned by the public prosecutor’s office. The routine analyses had already been completed, and all cases were closed. The selection of cases was based on the detection of pipamperone in the ´General Unknown Screening´ (GUS) using a High performance liquid chromatography with diode array detection (HPLC-DAD, Agilent Technologies (Waldbronn, Germany), 1260 series Infinity Binary LC System), which is used for screening of medications and drugs of abuse in death cases. The analysis was performed after liquid-liquid alkaline and acidic extraction. Routine toxicological analyses were completed in all cases. Those consisted of the GUS for medications and drugs of abuse, as well as screening for volatile substances (alcohols), also including blood and, if urine was available, urine alcohol determination. Moreover, analyzer-based immunological testing was performed in each case by using a Tecan Freedom EVOlyzer (Tecan Schweiz AG and Abbott, Männedorf, Switzerland) device with microplate EIA Kits for amphetamines, benzodiazepines, cocaine, methadone, opiates and cannabinoids (Abbot Rapid Diagnostics GmbH, Cologne, Germany)). Subsequent to positive immunological testing, confirmatory chromatographic methods (gas or liquid chromatography coupled with mass spectrometry) were performed in each case. The further selection of cases that should be included in the analyses was performed stepwise. In total, 32 cases were chosen, details are shown in Table 1. An analysis of pipamperone was performed in heart and femoral blood in duplicate. Afterwards, the cases were divided in groups depending on whether the cause of death was unknown (group 1), the cause of death was determined as natural (e.g. heart attack, organ failure, tumor disease) (group 2), or non-natural (e.g. polytrauma, death by hanging, drowning) (group 3). Cases of unknown death (group 1) were selected for further analyses and controlled for completeness of fluids and organ tissue for analyses. Specimens of interests were gastric content, cerebral spinal fluid (CSF), urine or kidney tissue, liver tissue and brain tissue. In a further step, cases with pipamperone concentrations < 0.5 mg/L in femoral blood or those where pipamperone did not relevantly contribute to death, e.g. due to an intoxication with alternative substances, were excluded. Hence, 12 cases were chosen for the analysis of the exhibits mentioned above to assess organ distribution of pipamperone (see Table 1, cases 1–12).

Case | Gender, Age [years] | BMI | Case history | Relevant autopsy findings | Other relevant toxicological findings in blood[mg/L] | PMI [days] | Pipamperonein femoral blood [mg/L]** | Cause of death
1 | m, 59 | 27.2 | - psychiatric disease- found dead in flat with stab wound in abdomen | - abdominal stab wound- congestion of inner organs - lung oedema- brain oedema- white substance residues in gastric content- increase in cardiac muscle mass- narrowed coronary vessels | Diazepam ↓Nordiazepam ↓Oxazepam ↓Temazepam ↓Salicylic acid, Ibuprofen*BAC: n.d. | 2–3 | 14 | Heart reinfarction, but possible influence of concomitant pipamperone intoxication
2 | m, 44 | 28.1 | - found lifeless, depression,- known cardiac arrhythmia | - congestion of inner organs- lung oedema- brain oedema | Venlafaxine: 0.18+ O-Desmethylvenlafaxine: 0.17Mirtazapine: 0.034Bisoprolol: 0.26Lidocaine*BAC: n.d. | 3 | 9.4 | Possible pipamperone intoxication or possible cardiac arrhythmia due to cardiotoxic effects of pipamperone, mirtazapine and venlafaxine
3 | f, 22 | 22.5 | -depression-posttraumatic stress disorder- suicidality | - congestion of inner organs- lung oedema- brain oedema | Mirtazapine: 0.35 ↑Quetiapine: 0.85 ↑Citalopram: 0.76 ↑↑Sertraline: 0.62 ↑Prothipendyl: 1.6 ↑↑Ibuprofen, Cetirizine *BAC: n.d. | 5 | 12 | Polydrug intoxication
4 | f, 55 | 18.8 | - schizophrenia and depression- suicide attempt in the past- found dead in bathroom | - congestion of inner organs- lung oedema- brain oedema- white residues in gastric content- artery sclerosis- superficial head injury | Lorazepam: 0.28 ↑Trimipramine: 0.39 ↑Zuclopenthixol: 0.045 Pregabalin: 4.9BAC: n.d. | 4 | 19 | Polydrug intoxication
5 | m, 40 | 19.3 | - found dead in psychiatric clinic | - congestion of inner organs- cerebral swelling- congestion of heart and heart surrounding vessels | Olanzapine: 1.3 ↑Flupentixol: 0.043 | 1 | 1.4 | Polydrug intoxication
6 | m, 41 | 36.6 | - posttraumatic stress disorder- alcohol abstinence since 3 years- found dead in flat | - increase in cardiac muscle mass- congestion of inner organs- haemorrhagic lung oedema- brain oedema- drug residues (white tablets) in gastric content and small intestine | Amphetamine: 0.25 ↑Venlafaxine: 0.49 ↑O-Desmethylvenlafaxine: 0.007Quetiapine: 0.33Olanzapine: 0.029THC ↓BAC: 0.52‰ | 3–4 | 1.9 | Polydrug intoxication
7 | m, 70 | 26.6 | - found dead in flat- known alcohol abuse | - increase in cardiac muscle mass and musculature steatosis- extended right atrium- head laceration- inflammation of respiratory passages- blood congestion of inner organs- lung oedema with bronchial mucus | Quetiapine: 0.11 ↑Citalopram: 0.08Ibuprofen*BAC: n.d. | N/A | 3.0 | Cardiac failure with possible contribution of quetiapine and pipamperone
8 | f, 76 | 25.0 | - depression- poly-neuropathy- found deceased at the foot of the bed in a nursing home, with a forward-leaning upper body and the head resting on the bed | - ventricular hypertrophy- scarred transformation of the myocardium in the posterior wall of the left ventricle(consistent with prior infarct)- arteriosclerosis- calcification of the aortic valves- early chronic hepatic congestion- cerebral softening due to hypoperfusion (cerebral infarction)- congestion of internal organs- cerebral oedema- hemorrhagic pulmonary oedema | Tilidine: 0.11Nortilidine: 0.34 ↑Naloxone (qualitative)Mirtazapine: 0.12Bisoprolol, Salicylic acid*BAC: n.d. | 9 | 1.5 | Suspected cardiac failure and possible contribution of pipamperone due to cardiotoxic effects
9 | m, 61 | 22.1 | - found dead in psychiatric clinic | - increase in cardiac muscle mass- narrowed left coronary vessel (25% remaining lumen)- pelvic artery sclerosis- congestion of inner organs- lung oedema- brain oedema | Paliperidone↓THC↓Bisoprolol*BAC: n.d. | 2 | 1.0 | Coronary heart failure with possible contribution of pipamperone
10 | f, 51 | 25.6 | - suicidal thoughts- depression- in psychiatric clinic for alcohol withdrawal- found lifeless in bed | - congestion of inner organs- softened and brittle liver tissue- chronic congestion of liver | Promethazine: 1.2 ↑↑Mirtazapine: 0.157-Aminoclonazepam: 0.028BAC: n.d. | 3 | 1.0 | Polydrug intoxication
11 | m, 18 | 28.3 | - cannabis abuse- psychiatric disease- found dead in bed | - congestion of upper body parts- congestion of inner organs- haemorrhagic lung oedema- brain oedema- foam in nostrils and respiratory passages- filled urinary bladder | Cocaine: 5.9 ↑↑Benzoylecgonine: 5.6 ↑↑THC: 0.011Amphetamine: 0.16Risperidone: ↓Paliperidone: ↓Dipyrone ↓BAC: n.d. | 2 | 1.0 | Polydrug intoxication with mainly cocaine and additional contribution of pipamperone and amphetamine
12 | m, 60 | 27.0 | - found dead in shelter for homeless individuals | - increased myocardial mass- rounded and enlarged cardiac apex- skull-brain trauma (laceration and contusion)- blood congestionof the heart- stasis of inner organs- hemorrhagic pulmonary edema | Clozapine: 0.65 + Norclozapine: 0.45 ↑↑Risperidone ↓BAC: n.d. | 3–4 | 0.83 | Suspected death from acute cardiac decompensation due to ethanol-toxic cardiomyopathy with possible contribution of pipamperone and clozapine due to cardiotoxic effects
13 | m, 20–40 (person unknown, estimation) | 21.3 | - jumped from 5th floor, found dead on a canopy | - polytraumatic injuries | THC: 0.025Promethazine: 0.19Chlorprothixene: 0.16Quetiapine ↓Nordiazepam ↓BAC: 2.12‰ | 3 | 0.22 | Multiple trauma
14 | f, 81 | 24.9 | - jumped from 5th floor- depression and bipolar disorder | - polytraumatic injuries | Zopiclone: 0.012 Mirtazapine: 0.14 Dipyrone: 31BAC: n.d. | 3 | 0.82 | Multiple trauma
15 | f, 41 | 19.6 | - found in burning car | - drastic burning injuries- loss of epidermis- charred musculature- fencing posture- heat-related bone fractures- carbon black in respiratory passages- lung oedema- brain oedema | Sertraline: 0.51Dipyrone ↓BAC: n.d.heart blood:COHb: 8%cyanide: 0.01 mg/L | 1 | 0.17 | Drastic burning injuries and heat effects
16 | f, 66 | 17.2 | - schizophrenia- complained about breathlessness and cough- found dead in flat | - left ventricular hypertrophy- artery sclerosis- brain oedema | Olanzapine: 0.058Dipyrone*BAC: n.d. | 3 | 0.25 | Unknown
17 | m, 85 | 27.2 | - femoral neck fracture after fall- death in foster home | - increase in cardiac muscle mass- artery sclerosis- bronchopneumonia- haemorrhagic lung oedema- subdural haematoma- hepatic cirrhosis- inflammation of urinary passages | Morphine: 0.052 (free)Sertraline: 0.075Dipyrone*BAC: n.d. | 5 | 0.11 | Pneumonia with possible influence of respiratory depression from morphine
18 | f, 41 | 35.8 | - paranoid schizophrenia- substance abuse, and anxiety disorder- previous suicide attempt via medication overdose(hospitalization for 6 days) | - pneumonia following aspiration of gastric contents- intestinal ischemia with surgical removal of large portions of bowel- sepsis- effusions in body cavities- liver and spleen loosened/ disintegrated | Diazepam ↓Nordiazepam: 0.082 Oxazepam: 0.016Lorazepam: ↓Quetiapine: ↓Olanzapine: ↓Venlafaxine: ↓Zuclopenthixol ↓Opipramol ↓Ketamine, Propofol, Amlodipine*BAC: n.d. | 5 | 0.46 | Septic-toxic multiorgan failure
19 | m, 66 | 27.0 | - stationary in psychiatric clinic before death- found dead in flat | - beginning putrefaction- brain oedema- haemorrhagic lung oedema- filled urinary bladder- white residues in gastric content | Amitriptyline: 1.7 ↑↑Nortriptyline: 0.311Lorazepam: 0.03Dipyrone*BAC: n.d. | 6–7 | 0.05 | Intoxication with amitriptyline
20 | f, 89 | 13.9 | - previous COVID infection- symptoms of stroke (unconscious-ness, respiratory insufficiency),- palliative treatment with morphine (but mistakenly higher dose given)- found dead in retirement home | - increase in cardiac muscle mass- congestion of inner organs- haemorrhagic lung oedema- brain oedema- white residues in gastric content- artery sclerosis | Morphine (free): 0.15 ↑Mirtazapine ↓Lorazepam ↓Dipyrone, Valsartan*BAC: n.d. | 2 | 0.16 | Most likely morphine intoxication
21 | m, 25 | 21.7 | - repeated suicide announce-ments- found hanging in a public place | - alcohol-aromatic odour during autopsy- strangulation mark at throat- congestion of inner organs- haemorrhagic lung oedema- brain oedema | Tramadol: 0.88O-Desmethyltramadol: 0.018THC: ↓BAC: 0.68‰ (femoral blood)UAC: 1.11‰ | 1 | 0.15 | Hanging
22 | f, 48 | 24.9 | - under partial hospitalization treatment for depressive disorder, with a history of diuretic abuse (following paralysis and speech- disturbances due to severe potassium deficiency- found unresponsive in the prone position at the residential address | - congestion of inner organs- cerebral oedema- haemorrhagic pulmonary oedema- foam in the respiratory passages | Trimipramine: 0.30Sertraline: 0.22Lorazepam: 0.036Furosemide: 1.0Zopiclone: ↓Ibuprofen*BAC: n.d. | 2 | 0.77 | Death following the long-term administration of furosemide, psychotropic medications may have contributed to death
23 | m, 74 | 18.4 | - condition after stroke and car accident, care-dependent- diabetes- dementia- unstable personality disorder- suffers from cardiac arrhythmias- found unresponsive in residence | - stenosis of the coronary arteries- cardioembolic infarcts- cerebellar infarct (not recent)- atrophic brain tissue with enlarged gyri and sulci- gallstones- pneumonia- haemorrhagic brain oedema | Dipyrone: 54Acetaminophen, Ibuprofen, Diclofenac*BAC: n.d. | 5 | 0.30 | Pneumonia after fall and cerebellar infarct
24 | m, 50 | 24.8 | - history of frequent hospitalizations due to alcohol intoxications and cirrhosis of the liver- possible suicidal intent- found in an unusual position in the footwell of a vehicle head-down on the passenger side, with the buttocks elevated and legs drawn up in the air | -alcohol-aromatic odour- abdominal ascites- small-nodular cirrhotic and enlarged liver- enlarged spleen | Mirtazapine: 0.041BAC: 2.91‰UAC: 4.22‰ | 2–3 | 0.36 | Positional asphyxiation, possibly favoured through alcoholisation
25 | m, 59 | 29.4 | - paranoid schizophrenia- exhibited severe pain symptoms.- COVID-19 disease and experienced physical decline- found unresponsive, lying supine on the floor in psychiatric clinic | - increase in myocardial mass- arteriosclerosis - evidence on early chronic hepatic stasis and chronic cholecystitis- indicators of pneumonia- blood statis of inner organs- cerebral oedema, - pulmonary oedema | Clozapine: 1.0 + Norclozapine 0.63 ↑↑Amisulprid: 1.3Tilidine: 29Nortilidine: 83BAC: n.d. | 3 | 0.53 | Suspected cardiac decompensation associated with COVID-19 infection with possible contribution of clozapine and pipamperone due to cardiotoxic effects
26 | m, 58 | 22.5 | - cerebral palsy and blindness- bipolar disorder (lithium medication)- was found to be disoriented and confused in nursing home- fever and elevated inflammatory markers- died in hospital after reanimation attempts(hospitalization for 7 days) | - paralytic ileus- flaccid and hypochromic organs- fragility of lung tissue- mucus accumulation in the respiratory passages- darkened lung tissue areas resembling food aspiration- septic splenomegaly- a doughy softening of the liver tissue | Morphine (free): 0.096Fluoxetine: 0.12Norfluoxetine: 0.28Lamotrigin: 6.1Dipyrone: 16Quetiapine ↓Acetaminophen*BAC: n.d.lithium: 1.36 mmol/L (day 1)lithium: 0.5 mmol/L (day 3) | 4 | 0.10 | Septic multiorgan failure
27 | m, 86 | 24.2 | - depression- found in a forest after being reported missing from psychiatric clinic | - increase in myocardial mass- left ventricular hypertrophy- arteriosclerosis- stenosis of the coronary arteries- congestion of inner organs- cerebral oedema, - filled urinary bladder- bloody contents within the colon, indicating possible gastrointestinal bleeding.- signs of hypothermia- rib fractures, cranial swelling, and subcutaneous hemorrhages on the arms, legs, and chest (injuries were considered to be not directly life-threatening) | Mirtazapine: 0.13Duloxetin: 0.11Lamotrigin: 3.0Paliperidone ↓BAC: n.d. | 4–6 | 0.05 | Consistent with cardiac decompensation and intestinal bleeding
28 | f, 83 | 14.1 | - femoral neck fracture, followed by postoperative delirium- suspected dementia- had ceased oral intake | - myocardial hypertrophy- arteriosclerosis- signs of chronic hepatic congestion- atrophy and reduction of cerebral tissue- shrinking of internal organs- overdistension and oedema of pulmonary tissue- suppurative pneumonia | Morphine (free): 0.065Fentanyl: 0.001Risperidone: 0.019 Paliperidone: 0.027Midazolam ↓Melperone ↓ | 3 | 0.07 | Multiorgan failure
29 | m, 81 | 27.4 | - Parkinson’s disease- falls- agitation- decreased responsiveness, with transfer from a senior center to a hospital- fluctuating delirium- respiratory failure (subsequently leading to circulatory failure) | - increase in myocardial mass- mild arteriosclerosis- signs of chronic hepatic congestion- renal cysts- congestion of internal organs- cerebral oedema, - pulmonary oedema (also with minimal purulent content in the left bronchi) | Levetiracetam: 2.7BAC: n.d. | 3 | 0.28 | Cardiac decompensation due to chronic pre-existing defects of the heart
30 | m, 14 | 45.6 | - Prader-Willi syndrome (intractable hyperphagia)- gastrointestinal problems and fever- initial diagnosis of gastroenteritis- septic shock- elevated lactate levels- acidosis- arterial hypotension- died in the pediatric intensive care unit | - cerebral oedema- pulmonary oedema- hemorrhagic infiltrates within the lung tissue- suspicion of myocarditis (hemorrhage within the myocardium)- septic multiorgan failure- hepatic hemorrhage- loosened spleen- bowel contained green, liquid, and blood-tinged contents | S-Amphetamine: 0.063Midazolam ↓Acetaminophen*BAC: n.d. | 1 | 1.9 | Septic multiorgan failure after Staphylococcus hominis infection
31 | f, 73 | 19.3 | - in-patient in psychiatric clinic- found in the bathroom of the ward in a hanging position (using a belt attached to the shower rod) | - minimal arteriosclerosis of the aorta- strangulation mark on throat- bilateral fractures of the superior horns of the larynx- congestion of inner organs- cerebral oedema | Mirtazapine: 0.15Zopiclone ↓lidocaine, ezetimib* | 4 | 0.24 | Hanging
32 | m, 51 | 26.3 | - found hanging in residence | - Alcoholic-aromatic odor- fractures of both cornua of the hyoid bone- bleeding on both clavicles- acute congestion of inner organs-cerebral edema | Quetiapine ↓Zopiclone ↓salicylic acid*BAC: 0.91‰ | 5 | 0.57 | Hanging

Sampling and preparations of samples

All postmortem specimen were collected during the autopsy and then stored at − 20 °C until use. The sample from the liver was obtained centrally. For examination of the brain, the medulla was preserved – if possible, in its entirety. Blood samples were pre-treated with ultrasound for homogenization. Urine samples (2 mL) were treated with 10 µL β-glucuronidase for 24 h at room temperature. In cases where no urine was available, kidney tissue was homogenized with HPLC-grade water (1 + 2 (w/v)) and treated with β-glucuronidase accordingly. Brain and liver tissue as well as gastric contents were homogenized with HPLC-grade water (1 + 9 (w/v)).

200 µL of the respective sample were spiked with 10 µL IS solution. Subsequently, 25 µL saturated Na2HPO4 solution were added to achieve slightly alkaline conditions. Protein precipitation was carried out using 1 mL of acetonitrile. The sample was centrifuged (1000 g, 3 min) and the clear supernatant was diluted (1:100 (v/v)) in mobile phase (85% LC/MS-grade water, 15% acetonitrile, 0.1% formic acid) afterwards. For organ homogenates (brain and liver tissue), the supernatant was first purified using QuEChERS with dSPE Kit according to the manufacturer´s instruction. The purified samples were then diluted in mobile phase accordingly. Samples with concentrations above the calibrated range were diluted with UHPLC grade water and the sample preparation was carried out as described above.

LC-MS/MS analysis

Measurements were performed on a 1290 Infinity II liquid chromatograph (Agilent Technologies, Santa Clara, USA) coupled with a QTRAP® 6500 + quadrupole mass spectrometer (AB Sciex, Darmstadt, Germany), which was operated in positive electrospray ionization (ESI) mode. A packed Poroshell 120 SB-C18 column (2.1 × 75 mm, 2.7 μm particle size) (Agilent Technologies, Santa Clara, USA) and a gradient elution with water containing 0.1% formic acid (eluent A) and acetonitrile containing 0.1% formic acid (eluent B) were used. Gradient start conditions were 85%/15% (A/B, (v/v)), linear increasing in a time interval from 0 to 1.5 min to 10%/90% (A/B, (v/v)) and decreasing back to the starting conditions within 1.5–4.5 min. A subsequent re-equilibrating time of 1.5 min resulted in a total run time of 6 min. The column oven was set to 30 °C, the flow rate was 0.3 mL/min and the injection volume 2 µL.

Analytes were detected by multiple reaction monitoring (MRM) using the mass spectrometry (MS) conditions shown in Table 2. Nitrogen was used as nebulizer gas (40 psi), auxiliary gas (70 psi), curtain gas (40 psi) and collision gas (9 psi), source temperature was 500 °C and ion spray voltage was 5500 V.

Analyte | Precursor ion m/z [M + H]+ | Product ions m/z | Retention time [min] | DP [V] | EP [V] | CE [V] | CXP [V]
Pipamperone | 376.2 | 165.0123.198.174.9 | 1.4 | 46 | 10 | 396733137 | 1681210
Pipamperone-d10 | 386.2 | 291.1165.0 | 1.3 | 46 | 10 | 2939 | 1812

Creatinine measurement

Quantitative creatinine measurements were conducted on an AU 480 Analyzer (Beckmann Coulter, Krefeld, Germany) by a DRI® Creatinine-Detect® test. The method is based on the Jaffe-reaction.

Method validation

The method was validated according to the guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh) [4]. Statistical data were evaluated using the Excel-based Valistat® software version 2.04 from Arvecon GmbH (Walldorf, Germany). In brief, validation covered linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy with respect to precision and trueness, as well as recovery and matrix effects.

The selectivity was tested in four postmortem as well as two ante-mortem blood samples. Moreover, selectivity regarding different analytes was tested. Therefore, commercially available quality control samples were used (MassCheck® Neuroleptics 1/Extended (lyophilized serum sample, ChromSystems®, Gräfelfing, Germany) and STUP 05/22-A WH (lyophilized whole blood sample, ACQ Science, Rottenburg am Neckar, Germany), containing the most important drugs of abuse, as well as antipsychotics and respective metabolites. For accuracy, whole blood controls (QClow−high) and commercially available serum controls (TDMC 1/23 (samples A and B)) were used. LOQ was determined according to the guidelines of the GTFCh by sixfold measurement of the lowest calibrator (alternative method 2). LOD was determined based on signal-to-noise (SN) ratio [11].

Data evaluation of whole blood and alternative matrices

Quantification of pipamperone in femoral and heart blood was performed using the established whole blood calibration curves. Whole blood and serum quality control samples were analyzed in each run (according to GTFCh guidelines [11, 12]).

Other specimen like urine or CSF were also evaluated using the whole blood calibration curve. Control samples for urine and CSF were prepared in a respective blank matrix, which was obtained from a volunteer (urine) and a resolved death case without pipamperone detection (CSF).

The application of a whole blood matrix calibration for the quantification of pipamperone in brain and liver tissue homogenates was tested in two of the cases, additionally using a standard addition procedure to compare with (data shown in appendix, Table 5). After evaluation (see results section), whole blood matrix calibration was considered suitable for the quantification of pipamperone in organ homogenates.

The total amount of the substance in the gastric contents was calculated by multiplying the measured concentration (mg/L) by the volume of gastric content, which was determined during autopsy.

H/F ratio calculation and creatinine correction

Heart and femoral blood ratios were calculated for each of the 32 cases, based on respective mean values from duplicate measurements.

Urine concentrations were corrected based on creatinine. Standardization was achieved by using the following formula (1). Reference creatinine value was set as 100 mg/dL:

1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{l}Creatinine\:corrected\:concentration\left[\frac{mg}{L}\right]=\\Substance\:concentration\:\left[\frac{mg}{L}\right]\cdot\:\:\frac{Reference\:creatinine\:\left[\frac{mg}{dL}\right]}{Sample\:creatinine\:\left[\frac{mg}{dL}\right]}\end{array}$$\end{document}

Creatinine correction was not possible in three cases (cases 4, 6 and 7), where kidney tissue was examined instead of urine and in one case (case 9), where sample material was limited.