Discussion

Validation

A method for the quantitative determination of pipamperone in postmortem blood was developed. A less intense target ion (m/z 98.1) was used for quantification, since a better linearity could be achieved over the measuring range.

According to the guidelines of the GTFCh, the signal-to-noise ratio (S/N) at the LOD (limit of detection) should be at least 3:1. The determined signal-to-noise-ratios for both, the target and qualifier ions, were comparatively higher, suggesting that an even lower concentration could likely have been determined as the LOD.

The investigation of linearity demonstrated a proportional relationship between the analytical signal and the concentration. However, reliable results for the lower calibration points could only be obtained by applying a weighting factor of 1/x.

The observed matrix effects of approximately 97% at the two measured QC sample concentrations (0.25 mg/L and 1.5 mg/L) indicate only minimal ion suppression. Considering the standard deviation, the measured matrix effects may also be attributed to measurement uncertainty rather than co-eluting substances. The investigation of the recovery revealed only minor losses during sample preparation. This may, for instance, be due to analyte entrapment during protein precipitation. Based on the consistently good accuracy of all QC samples and the similar matrix effects and extraction efficiencies observed at the above-mentioned concentrations (compare respective data in Table 3), a further determination of matrix effects or extraction efficiency for the other QC samples was not considered necessary.

The method was also suitable for the determination of pipamperone in alternative matrices like urine, CSF and organ homogenates. Control samples for CSF and urine were analyzed using the whole blood calibration curve to assess the suitability of the method for those alternative matrices. A limitation of that procedure is that individual samples for CSF and urine were used as blank matrices for control samples. Since the method was specifically developed and validated for quantitative determination of pipamperone in whole blood, this approach was considered acceptable. However, for routine analyses in urine or CSF, additional measurements using matrices derived from multiple donors would be necessary. Usually, also a calibration with the same matrix as the sample matrix would be preferable, but has to be reasonable with respect to sample preparation time.

For organ homogenates, the use of serum/blood matrix calibrations for the quantification of drugs has been described before and is a commonly used method [13–15]. Experiments concerning the use of a standard addition for the quantification of pipamperone in organ homogenates revealed minor variations (bias − 2.8% to 2.6%) in comparison to the use of a whole blood calibration as reference for quantification in two cases. Accordingly, a whole blood matrix calibration was considered efficient for the quantification of pipamperone in organ homogenates as well.

Pipamperone blood concentrations and H/F ratios

Pipamperone concentrations in femoral and in heart blood were comparable in some of the cases, showing H/F ratios of 1.0 (6 / 31 cases). Heart and femoral blood ratios (H/F ratio) of > 1.0 were observed in 19 out of 31 cases, expressing higher heart than femoral blood concentration. Femoral blood concentrations were higher in 6 out of 31 cases.

The results of heart and femoral blood measurements indicate that pipamperone may exhibit PMR, since drugs that have a higher H/F ratio (> 1.0) are believed to have greater tendency for PMR [16]. In the present study, H/F ratios between 0.5 and 3.9 (mean: 1.5 ± 0.8, median: 1.3) were observed, excluding one case (case 15, outlier) with a very high ratio of 12. In the latter case, circumstances of death (found in a burning car, severe burning injury) might have contributed to the unusual ratio in comparison to the other ratios. Changes in drug pharmacokinetics of burn patients have been described in literature [17]. Thus, postmortem drug concentrations might also be affected and concentration changes might be unpredictable.

Concerning the H/F ratios of pipamperone, literature data are limited. Henning et al. [9] investigated H/F ratios of 0.8 and 1.3 in cases of assumed fatal pipamperone intoxication and also investigated organ distribution. Based on their data they suggested, that pipamperone may exhibit PMR, although the number of cases in the study was limited (n = 3). Brockbals et al. [15] observed an average H/F ratio of 0.9 in two cases (single values of each case not shown in publication).

However, a sole consideration of the H/F ratio is not suitable for reliably assuming PMR, since there are important factors influencing the ratio. These are e.g. the type of drug, its volume of distribution (VD), blood-plasma-ratio, dose, PMI and the pKa-value [18]. An incomplete absorption and distribution of the drug prior to death may be of importance as well, since concentrations of a substance are known to be higher in the blood of the arteries than in the blood of the veins, especially short after administration [19, 20]. A diffusion from high drug amounts in gastric content might contribute to higher drug concentrations in heart blood. This has been shown especially for ethanol, but was assumed to be less substantial for drugs [21].

Additionally, it is stated in literature that higher drug concentrations in femoral blood compared to heart blood may be a result of resuscitation [22, 23]. Ratios of 3.8 and 0.5 were observed in two of the examined cases (cases 2 and 30) in the presented study, where resuscitation was documented. The opposing concentration ratios do not fully align with literature, although a higher number of cases where resuscitation occurred should be examined to strengthen this observation.

According to literature, basic drugs with a large volume of distribution (VD > 3 L/kg), which are present in extracellular fluid and sequestered in tissue, have a tendency for PMR [18, 24]. Those substances can show H/F ratios of up to 20. Thus, e.g. antidepressants like amitriptyline exhibit relevant PMR [18]. Conversely, for substances such as acetaminophen (VD = 0.8–1.0 L/kg) and mirtazapine (VD = 10–14 L/kg) [24] a relationship between VD and PMR has not necessarily been confirmed [19]. Only few studies address the pharmacokinetic properties of pipamperone, and the VD respectively. Kloosterbor et al. [25] calculated a mean VD of 451–456 L after a 120 mg dose in healthy volunteers (mean weight 76.8 kg), assuming a bioavailability of 100%. The reported high volume of distribution (≈ 5.9 L/kg) indicates an extensive tissue distribution, which might be another indicator for PMR.

Another factor that has to be taken into consideration is the distribution of a substance between cellular components, particularly red blood cells, and serum/plasma [18]. The blood-plasma ratio of pipamperone was estimated at 0.21 ± 0.03 by Tron et al. [26] by comparing plasma concentrations and dried blood spot concentrations of pipamperone. Postmortem changes such as autolysis and putrefaction lead to a loss of cellular integrity, a decrease in pH, and dissolution of organ structures. As a consequence, concentration gradients between tissues and blood are disrupted [27, 28]. For pipamperone, which is considered to have a low blood-to-plasma concentration ratio, no relevant sequestration in blood cells can be assumed. Hence, the loss of membrane integrity may not result in redistribution from blood cells but rather in the release of the drug from tissues into the vascular compartment.

Overall, the observed variability in H/F ratios may be explained by the rather high VD of pipamperone, together with its low blood-plasma ratio, which facilitates PMR from tissues into heart blood. Regarding higher femoral blood concentrations in comparison to heart blood, local tissue stores around the femoral vein, such as subcutaneous fat and muscle, may release pipamperone postmortem into femoral blood, slightly elevating its concentration. However, redistribution into heart blood from central organs generally exerts a stronger effect.

Organ distribution

The analysis of 12 selected cases allowed the observation of relationships in the organ distribution of pipamperone.

Brain, blood and CSF

In general, little is known regarding the correlation between concentrations in femoral blood and CSF in postmortem toxicology [19]. Hiemke et al. [29] described a rapid distribution of antipsychotics from the blood into the central nervous system, typically resulting in higher concentrations in the brain than in blood. This finding is consistent with the results of the present study. Pipamperone concentrations in brain tissue were higher than in femoral blood and CSF for all observed cases. Henning et al. [9] measured brain tissue concentrations from 41 to 71 mg/kg, which is comparable to the brain tissue concentrations of cases 1–4 in this study, where also comparable femoral blood concentrations were measured (range 28–63 mg/kg). Further information regarding the origin of examined brain tissues in the study by Henning et al. [9] is not available. A significant factor of uncertainty may be variations in concentration across different regions of postmortem organs [30]. Since pipamperone primarily binds to D4-receptors, different drug concentrations may be expected in areas with high D4-receptor density (e.g., hippocampus, frontal lobe, amygdala) compared to the medulla, which was sampled in this study.

Kidney/Urine

Pipamperone is metabolized hepatically and is primarily excreted via the kidneys. Biotransformation via N-dealkylation, keto reduction, piperidine ring hydroxylation, N-oxidation and amide hydrolysis leads to a series of pharmacologically inactive metabolites [3, 4]. For extensively metabolized drugs, the parent compound is usually not detectable in urine or only present at very low concentrations [18, 30]. Koeppel et al. [4] detected unchanged pipamperone in urine. However, data regarding the concentration ratios between the parent drug and its metabolites are not available.

The concentrations of pipamperone in urine and kidney tissue observed in this study showed a discrepancy compared to the values reported by Henning et al. [9], which ranged from 445 to 548 mg/L in urine. The highest measured concentrations in the examined cases were 257 mg/L in urine (Case 3) and 93 mg/kg in kidney tissue (Case 4), whereas the concentrations in the other cases ranged from minimum 0.15 to maximum 31 mg/L. The detected concentrations in urine or kidney homogenate were even below 10 mg/L in more than 50% of the investigated cases. These differences between the measured urine concentrations and those reported in the literature may be attributed to the efficiency of the extraction process or matrix effects. Henning et al. [9] used trimipramine-D3 as an internal standard (IS) and performed standard addition, whereas in this study, pipamperone-D10 was used as IS, and quantification was based on a blood calibration. Another factor might be a missing creatinine correction of Henning et al. in comparison to the herein presented data.

The comparability of the data might also be restricted by the smaller number of cases investigated by Henning et al. [9]. However, urine concentrations can be affected by various factors and remain a qualitative parameter for interpretation, which cannot be compared directly among cases.

Gastric content

Data for pipamperone concentrations in gastric content are not available in literature for comparison. In general, the highest drug amounts were found in gastric contents of group 1, especially in cases 1 and 4 (380 mg and 373 mg in total). The presence of substantial amounts of pipamperone in the gastric contents in those cases indicates that a portion of the administered dose remained unabsorbed at the time of death. Corresponding femoral blood concentrations (14 and 19 mg/L) reflect the fraction of the substance that has already been absorbed and systemically distributed. Heart blood concentrations (27 and 31 mg/L) are higher than femoral concentrations in both cases, which would be consistent with PMR.

Liver

The highest measured concentrations in liver tissue in the four cases of group 1 (97 to 315 mg/kg) were exceeding those reported in literature in three of the cases. Hence, Henning et al. [9] detected concentrations ranging from 91 to 190 mg/kg.

With respect to the concurrently rather high pipamperone amounts in the gastric content in three of the herein presented cases (106–380 mg), a diffusion from gastric content to liver might have occurred. Diffusion from gastric content to liver has been described in literature for other drugs [19, 21]. To the best of the authors’ knowledge, there is no evidence that pipamperone undergoes enterohepatic circulation that could account for increased liver concentrations.

Variations in sampling site (sampling site not published by Henning et al. [9]) might also account for these differences. The liver is interspersed with connective tissue and enclosed by the liver capsule. This can result in different proportions of functional hepatocytes depending on location of tissue sampling and possible drug concentration differences depending on the sampling site. A study on diffusion from gastric content with amitriptyline, acetaminophen and lithium in a human cadaver model revealed high, site-dependent, drug concentrations in the lungs and liver. Hence, e.g. amitriptyline concentrations, which has most comparable physicochemical properties to pipamperone among the investigated substance, were 9-fold higher in the left anterior lobe in comparison to the right lobe [21].

PMI

The influence of the PMI and sampling time point after death on postmortem concentrations in blood and tissue has been described in literature, especially for antidepressants and antipsychotics [15, 31, 32], whereby concentration increases and decreases can occur. Brockbals et al. [15] investigated time-dependent concentration changes for pipamperone in femoral blood in two cases. One case did not show concentration changes, while an increase of the pipamperone concentration of 21% was observed in the second case (data on PMI not shown in respective cases). The PMI was estimated between 1 and 9 days among the presented cases of this study. At a short PMI of approximately one day, H/F ratios of 0.5–1.8 (n = 3) were observed for pipamperone. At a PMI of 2–3 days, ratios ranged from 0.7 to 3.9 (n = 14). At a PMI of over 3 to 4 days, ratios ranged from 0.9 to 3.8 (n = 5). This heterogeneity of results was also observed at higher PMIs (over 4 days) among the presented cases. Thus, the PMI might contribute to the H/F ratio, but influences on the H/F ratio are rather be multifactorial.