Results

Validation

The validation was successfully performed according to the GTFCh guidelines [11]. The corresponding data for accuracy, matrix effects and extraction efficiency are shown in Table 3. The selectivity of the method was confirmed, as the whole blood samples spiked with antipsychotics and drugs of abuse showed no interference. Linearity of calibration was confirmed by correlation coefficient calculation (R2 > 0.99) and Mandel’s F test. Absence of outliers (Grubbs test) was demonstrated for the calibration series. Homogeneity of variance was not confirmed by the F-test, indicating heteroscedasticity. Therefore, statistical weighting was required (1/x). The LOD and LOQ were determined at 0.01 mg/L and 0.05 mg/L, respectively.

 | Trueness(Bias) [%] | Precision [%] | RSDT [%] | RSDr [%] | Extraction efficiency [%] | Matrix effects [%]
QClow [0.25 mg/L] | -5.6 | 6.4 | 6.5 | 5.3 | 88.0 ± 7.8 | 97.6 ± 3.0
QCmid [1.5 mg/L] | -0.4 | 5.7 | 5.8 | 3.3 | 91.6 ± 6.1 | 97.8 ± 2.6
QChigh [4.5 mg/L] | 4.8 | 5.9 | 6.0 | 3.5 | n.t. | n.t.
TDMC A [0.13 mg/L] | 0.6 | 5.9 | 6.0 | 4.7 | n.t. | n.t.
TDMC B [0.32 mg/L] | 4.9 | 6.2 | 6.2 | 5.6 | n.t. | n.t.

Standard addition was tested exemplarily for the quantification of pipamperone in brain and liver organ homogenates of two cases. Bias between − 2.8% and 2.6% were observed in comparison to quantification of pipamperone in the tissues via whole blood matrix calibration. Hence, whole blood matrix calibration was used for further data evaluation to minimize preparation times and simplify the quantification method. Corresponding data can be found in appendix (Table 5).

Heart and femoral blood concentrations

Pipamperone concentrations of 0.82–19 mg/L were detected in femoral blood, 1.0–36 mg/L in heart blood. Heart and femoral blood ratios (H/F ratio) ranged from 0.5 to 3.9 (mean: 1.5 ± 0.8, median 1.32; n = 32).

Raw data of measured heart and femoral blood concentrations and corresponding H/F ratios can be found in the appendix (Table 6). One case was excluded from calculation (case 15), since the ratio (H/F = 12) was assessed as an outlier via Grubbs test.

Organ distribution

The measured concentrations in all matrices of the 12 selected cases are shown in Table 4. The cases were grouped based on femoral blood concentrations of pipamperone for better comparability of the results. The concentration ranges for the groups were > 5 mg/L (group 1), 1–5 mg/L (group 2) and 0.5 – < 1 mg/L (group 3). For gastric content, measured concentrations, volume of gastric content (total volume, Vtot) and respective total amount of pipamperone are given. Urine concentrations were corrected with respect to creatinine (compare Sect. 2.8).

 | Group 1 | Group 2 | Group 3
Case 1 | Case 2 | Case 3 | Case 4 | Case 5 | Case 6 | Case 7 | Case 8 | Case 9 | Case 10 | Case 11 | Case 12
Femoral blood [mg/L] | 14 | 9.4 | 12 | 19 | 1.4 | 1.9 | 3.0 | 1.5 | 1.0 | 1.0 | 1.0 | 0.82
Heart blood [mg/L] | 31 | 36 | 13 | 27 | 1.5 | 7.6 | 1.9 | 3.0 | 2.3 | 1.0 | 1.7 | 1.3
Urine [mg/L] | 28# | 6.5# | 221# | n.a. | 18# | n.a. | n.a. | 34# | 0.84** | 0.51# | 16# | 8.7#
Kidney [mg/kg] | n.a. | n.a. | n.a. | 93 | n.a. | 9.6 | 9.4 | n.a. | n.a. | n.a. | n.a. | n.a.
Liver tissue [mg/kg] | 315 | 283 | 97 | 275 | 30 | 23 | 32 | 23 | 15.9 | 5.8 | 12 | 18
Brain tissue [mg/kg] | 49 | 28 | 32 | 63 | 1.8 | 4.7 | 8.0 | 2.7 | 1.4 | 1.2 | 1.4 | 1.1
Cerebral spinal fluid [mg/L] | 10 | 7.8 | 9.1 | 14 | 0.42 | 1.4 | 1.2 | 1.0 | 0.47 | 0.33 | 0.55 | 0.40
Gastric content [mg/L]Total volume [mL]Total amount [mg] | 585Vtot = 650380 | 346Vtot = 4013.8 | 1062Vtot = 100106 | 1492Vtot = 250373 | 4.3Vtot = 1000.43 | 70Vtot = 35024.5 | 16Vtot = 20.03 | 11Vtot = 100.11 | 2.9Vtot = 5501.5 | 5.2Vtot = 40.02 | 9.9Vtot = 250.24 | 2.0Vtot = 1500.3