Determination of tramadol and its metabolite O-desmethyltramadol in vitreous humor. Is it helpful in forensic casework?

Abstract

In recent years, there has been increasing interest on the use of alternative biological materials in forensic toxicology. Vitreous humor is one of them, which, due to the closed cavity it is contained, has a low degree of contamination and high purity that makes it ideal for use in postmortem specimens. The aim of this study was to investigate the distribution of tramadol and its active metabolite O-desmethyltramadol in vitreous humor and the usefulness of using this alternative biological matrix in tramadol-related forensic cases. For this purpose, a gas chromatography–mass spectrometric method for the determination of the two analytes in blood and vitreous humor samples, which included solid-phase extraction and derivatization using N,O-Bis(trimethylsilyl)trifluoroacetamide with 1% trimethylsilyl chloride, was developed. The method was fully validated according to international guidelines and was applied to blood and vitreous humor samples from 12 forensic cases. Both substances were found to be readily distributed in vitreous humor, since even in cases of very low concentrations of the analytes in blood, their detection was also possible in vitreous humor. In addition, the vitreous humor to blood concentration ratios were calculated for both substances and the mean values were found to be 0.91 for tramadol and 0.94 for O-desmethyltramadol. The results of our study indicate that the information that can be extracted from the analysis of vitreous humor samples is particularly useful during the investigation of tramadol-related cases. Nevertheless, the need for further study of this alternative material to establish therapeutic and toxic limits becomes apparent.

Introduction

Tramadol is a synthetic, centrally acting analgesic with atypical opioid action. It was formulated in 1962 by Drs Kurt Flick and Ernst Frankus in the research laboratories of Grunenthal GmbH (Stolberg, Aachen, Germany) and it was first approved in Germany in 1977 [1]. In 1995, the Food and Drug Administration (FDA) in the United States approved the per os administration of tramadol for the treatment of moderate to severe pain [1, 2]. O-Desmethyltramadol, the active metabolite of tramadol derived via o-demethylation from the parent drug with CYP2D6 coenzyme, is involved in the opioidergic mechanism and exerts the analgesic activity of the drug [1]. The maximum recommended daily per os dose of tramadol is indicated at 400 mg [3, 4]. Therapeutic concentrations of tramadol normally ranged between 0.1 and 0.3 μg/mL, but concentrations up to 1 μg/mL (supratherapeutic) are considered tolerated [4, 5]. Blood levels of tramadol higher than 1 μg/mL are considered toxic, and may indicate contribution to the cause of death, while blood concentrations above 2 μg/mL are considered lethal [5]. More specific, in fatal cases of co-ingestion drugs the tramadol concentrations were significantly lower (0.15–39 μg/mL) than the ones of fatal cases involving only tramadol and similar to the concentrations due to therapeutic doses. In fatal cases where only tramadol is involved, concentrations of 1.6–61.8 μg/mL have been reported [4].

Vitreous humor is a transparent gel-like material found in the eye cavities and consists of 98% water and solids in the form of macromolecules and low molecular weight components such as proteins, sugars, urea, creatinine, and electrolytes [6–8]. Vitreous humor was first used in forensic toxicology by Naumann in 1959 [9] and is today widely used as an alternative biological fluid. It is preferred for postmortem analyses because of its easy collection even in cases where blood and urine samples are insufficient or impossible to obtain. In addition, its composition is more stable and less affected by putrefaction and postmortem redistribution compared to other biological materials such as blood [6, 10, 11].

There are a lot of published methods for the determination of tramadol and its metabolites in conventional biological fluids such as plasma [12–24], blood [25, 26], or urine [12, 25, 27–30]. To our knowledge, only a few studies have been published concerning the determination and distribution of tramadol alone in vitreous humor despite the advantages of this biological matrix [31, 32], while the distribution of the active metabolite O-desmethytramadol in this material has not been investigated yet. The aim of this study was the development and validation of a gas chromatography–mass spectrometric (GC–MS) method for the determination of both tramadol and O-desmethyltramadol in vitreous humor and blood samples, and its application to real biological samples from forensic cases in order to investigate the distribution of the two substances in this alternative biological fluid.

Methods

Chemicals and reagents

Reference standard methanolic solutions of tramadol, O-desmethyltramadol, and codeine-d₆ (Internal Standard, IS) at a concentration of 1.00 mg/mL (>99.9% pure) were purchased from LGC Promochem (Molsheim, France). All standards were stored according to the manufacturer’s instructions.

All solvents used (methanol, isopropanol, ethyl acetate, acetonitrile, dichloromethane, and n-hexane) were of high-performance liquid chromatography (HPLC) grade and were purchased from Merck (Darmstadt, Germany). Analytical reagents were purchased as follows: N,O-bis(trimethylsilyl)-trifluoracetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) from Sigma-Aldrich (Steinheim, Germany); analytical grade ammonium hydroxide (NH₄OH), hydrochloric acid, and sodium dihydrogen phosphate dehydrate (NaH₂PO₄^.2H₂O) were obtained from Merck (Darmstadt, Germany). Bond Elut LRC C18 (Sorbent Mass 200 mg, Column Volume 10 mL) columns that were used for solid-phase extraction (SPE) were obtained from Agilent Technologies (Santa Clara, CA, USA).

Separate drug-free femoral blood and vitreous humor samples were obtained from forensic caseworks and after their verification as negative for drugs by GC–MS analysis, they were pooled and used.

Preparation of stock and working standard solutions

Stock standard methanolic solutions of tramadol, O-desmethyltramadol, and codeine-d₆ at concentration of 1.00 mg/mL were used. Mixed working standard solutions of the two analytes of interest were prepared at concentrations of 10.0 and 0.50 μg/mL, after appropriate dilutions with methanol. A working internal standard solution containing codeine-d₆ (IS) at a concentration of 1.00 μg/mL was prepared by diluting the appropriate volume of the corresponding stock solution with methanol.

Working standard solutions were prepared daily fresh in the requested amounts for the scheduled experiments.

Calibrators and quality control samples

Six calibrators containing tramadol and O-desmethyltramadol were prepared at concentrations: 5.00, 10.0, 30.0, 100.0, 300.0, and 1000 ng/mL, for the preparation of the calibration curves. Three quality control samples containing the two analytes of interest at concentrations of 15.0, 150.0, and 800 ng/mL were also prepared from different stock solutions than the ones used for calibrators. In all samples, a volume of 50 μL of the IS (1.00 μg/mL) was added.

Calibration curves (based on the peak area ratio of each analyte to the internal standard) were plotted every day and were used for the calculation of the analytes’ concentration.

GC–MS analysis and apparatus

The chromatographic analysis of tramadol and its metabolite was performed on an Agilent GC–MSD model 6890 N/5975 equipped with a DB-5 MS fused silica column (30 m × 0.25 mm i.d. × 0.25 μm film thickness) supplied by Agilent Technologies (IL, USA). Helium was used as a carrier gas at a flow rate of 1.0 mL/min. A volume of 1 μL was injected in splitless mode using an Agilent 7683B Series auto-sampler system. The optimized GC conditions were as follows: the initial column temperature of 100°C (hold time 1 min) was increased at a rate of 30°C/min to 300°C (hold time 2 min). Injector, ion source, and interface temperatures were set to 260°C, 230°C, and 280°C, respectively. The mass spectrometer was operated in electron ionization (EI) with selective ion monitoring (SIM) mode. The mass fragments used for the identification of the analytes were: m/z  335, 245 and 216 for silylated tramadol and m/z  393, 303 and 378 for silylated O-desmethyltramadol. The bold marked ions were used for the quantification of the analytes. The respective mass fragment of the internal standard was m/z  377 (codeine-d₆). The total analysis time was less than 10 min.

An MT 19 vortex (Chiltern, London, UK) was used for the mixing of standards and samples during their preparation. A 691 digital pH-meter (Metrohm, Herisau, Switzerland) with a glass electrode was used for pH adjustments. An evaporating unit connected with nitrogen (Reacti-Vap PIERCE Model 18 780, Rockford, IL, USA) was used for the evaporation of all samples. Centrifugation was performed with a Sigma 4K10 centrifuge (Osterade, Germany).

Sample preparation

In all blood or vitreous humor samples (1.0 mL), 50 μL of the working internal standard solution (1.00 μg/mL) was added and the samples were vortex mixed for 15 s. Therefore, all calibrators, QC, and casework samples contained 50.0 ng/mL of codeine-d₆. Then, a volume of 4-mL of phosphate buffer pH 6.0 was added and vortexed. Centrifugation was performed for 10 min at 3000 rpm. The supernatants were then loaded on Bond Elut LRC C18 SPE cartridges that were previously conditioned with 2 mL of methanol and 2 mL of phosphate buffer pH 6.0. The samples were slowly passed through the cartridge at a rate of 1 mL/min and then the columns were washed with 2 mL of mixture of distilled water: methanol (80:20, v/v) and were vacuumed for 10 s (>10 mmHg). A volume of 100 μL of n-hexane were then added, and columns were vacuumed for 10 min (>10 mmHg). Finally, the analytes were eluted twice with 1.5 mL freshly prepared mixture solution of ethyl acetate: ammonium hydroxide (98:2, v/v). The eluates were then evaporated to dryness under continuous N₂ stream. A volume of 50 μL of acetonitrile was used to reconstitute the dried residues, and 50 μL of BSTFA with 1% TMCS was added for the derivatization of the analytes. Derivatization was performed in sealed tubes at 70°C for 30 min. After cooling the tubes, the derivatized extracts were transferred to glass vials and 1 μL was injected into the GC–MS system (splitless mode).

Results and discussion

Optimization of extraction procedure

During the development of the method for the isolation of the analytes of interest from biological materials, spiked blood samples were initially used, as a more difficult matrix compared to vitreous humor, and different tests were performed, using liquid–liquid extraction (LLE) or solid-phase extraction (SPE). The selection of the optimal extraction parameters was based on the results of the recoveries and these parameters were also verified in spiked vitreous humor samples. As far as the LLE, different values of pH (7.0, 8.0, 9.0, 10.0, 11.0) and solvent systems [ethyl acetate, ethyl acetate:hexane (1:2, v/v), dichloromethane:hexane (2:3, v/v), dichloromethane:hexane:isopropanol (2:3:0,1, v/v/v)] were checked and optimized.

Furthermore, experiments were carried out to optimize the conditions of SPE with columns Bond Elut LRC C18 (200 mg) and Bond Elut LRC Certify (130 mg). The pH of the samples was adjusted with 4 mL of phosphate buffer solution pH 6.0, followed by centrifugation (3000 rpm, 10 min). Different elution solvent systems [dichloromethane:isopropanol:ammonium hydroxide (85:15:2, v/v/v), dichloromethane:isopropanol:ammonium hydroxide (80:20:2, v/v/v), dichloromethane:isopropanol:ammonium hydroxide (75:25:2, v/v/v), ethyl acetate:ammonium hydroxide (98:2, v/v)] were tested. Moreover, different mixtures of solvents (0%, 10%, 20%, 30%, 40%, and 50% of methanol in H₂O) were tested during the washing step.

It was found that the higher absolute recovery values for tramadol (84%) and O-desmethyltramadol (90%) were achieved with the SPE technique using Bond Elut LRC C18 columns with elution solvent system of ethyl acetate: ammonium hydroxide (98:2, v/v) and by using the mixture of distilled water: methanol (80:20, v/v) during the washing step of the columns, so it was chosen as the optimal one.

Optimization of derivatization procedure

During the method development, derivatization was considered necessary, as the sensitivity especially for the metabolite O-desmethyltramadol was not adequate, while the chromatographic characteristics of the two peaks were not satisfactory. The stationary phase of the gas-chromatographic column was nonpolar, while the molecules under study are characterized by a lipophilic part of a phenyl attached to a cyclohexane and triple-substituted nitrogen with polar substituents. The O-desmethyltramadol, with the additional hydroxyl group on the phenyl part, has increased polarity compared to tramadol. Derivatization results in the alteration of polarity of the two molecules under study, resulting in better chromatography and sensitivity.

Silylation reagents such as BSTFA with 1% TMCS and MTBSTFA with 1% TBDMCS and acylation reagents using anhydrides such as PFPA, TFAΑ, and HFBA were tested. The procedures were different for each group and are represented in Table 1.

Table 1.

Derivatization procedures

Derivatization procedure for silylation	Derivatization procedure for acylation
Evaporation under a nitrogen stream of the methanolic solutions or the eluted samples to dryness   Reconstitution with 50 µL of acetonitrile Addition of 50 µL of reagent Vortex mix Incubation at 70°C for 30 min Cooling and injection of 1 µL volume into the GC–MS system	Evaporation under a nitrogen stream of the methanolic solutions or the eluted samples to dryness Addition of 50 µL of reagent Vortex mix Incubation at 70°C for 30 min Cooling and evaporation under a stream of nitrogen until dry Reconstitution with 100 μL ethyl acetate Injection of 1 µL volume into the GC–MS system

The acylation reagents showed nonsatisfactory chromatographic results for both substances, while MTBSTFA with 1% TBDMCS improved the chromatographic behavior of only O-desmethyltramadol. Finally, BSTFA reagent with 1% TMCS resulted to increased sensitivity with satisfactory chromatographic behavior for both tramadol and its metabolite, and it was chosen as the optimal reagent for derivatization.

Method validation

The validation of the developed method followed international guidelines [33–36] and evaluated parameters such as selectivity, specificity, linearity, sensitivity, precision, accuracy, recovery, and dilution integrity. The results are presented in details below and were fully acceptable for the two analytes of interest.

Selectivity

The selectivity of the method was tested using six different blank blood and vitreous humor samples, which had previously been found to be free of the substances under study. No interference from endogenous components of the biological material was found for both blood and vitreous humor samples at the retention times of tramadol and O-desmethyltramadol.

Specificity

Specificity was tested by analyzing 6 different spiked blank blood and vitreous humor samples at a concentration of 10.0 µg/mL, with a mixture of 41 different compounds (cocaine, benzoylecgonine, delta9-THC, 11-nor-carboxy-delta-THC, cannabidiol, codeine, morphine, 6-acetyl-morphine, oxycodone, fentanyl, pethidine, diazepam, nordazepam, alprazolam, bromazepam, lorazepam, oxazepam, midazolam, paracetamol, lidocaine, metoprolol, amitriptyline, citalopram, duloxetine, venlafaxine, norvenlafaxine, sertraline, desmethylsertraline, mirtazapine, clomipramine, levomepromazine, biperidene, quetiapine, piracetam, valproic acid, gabapentin, pregabalin, carbamazepine, oxcarbazepine, haloperidol, olanzapine) that may co-exist in biological samples of forensic interest, in order to check exogenous interferences at the retention times of the two compounds. In the chromatograms obtained, no interferences at the retention times of both studied compounds were detected for blood and vitreous humor samples.

Limits of detection and quantification

The limits of detection (LOD) and quantification (LOQ) were determined as the concentration resulting in a peak area with a signal-to-noise ratio ≥ 3 and ≥ 10, respectively. Six spiked blood and vitreous humor samples were prepared and analyzed at consecutively reduced concentrations for tramadol and O-desmethyltramadol. The LOD and LOQ values for both analytes of interest were found to be 1.50 and 5.00 ng/mL, respectively.

Linearity

Linearity of the developed method was tested by constructing linear calibration curves with six concentration points from 5.00 ng/mL to 1000 ng/mL. Regression equations (, where was each analyte’s concentration, and the respective peak area ratio of each analyte to the IS) using a weighting factor of 1/x² were derived at 4 days for blood and vitreous humor. Furthermore, correlation coefficient (R²) was calculated for each analyte and was found to be higher than 0.991 and 0.994 for tramadol and O-desmethyltramadol, respectively. Furthermore, the % RSD of slopes was also calculated and was found to be less than 3.6% and 3.2% for tramadol and O-desmethyltramadol, respectively.

Precision and accuracy

Precision (% RSD) and accuracy (% E_r, mean relative error) of the method were determined by intraday and interday analyses and were found to be within acceptable limits (±15%). Intraday precision and accuracy were studied by analyzing six quality control (QC) vitreous humor and blood samples in three concentration levels; low (15.0 ng/mL), intermediate (150.0 ng/mL), and high (800.0 ng/mL). Interday assessment was examined by analyzing the same concentration levels of QC vitreous humor and blood samples in 4 days.

Intra-day precision was less than 8.6% and 5.4% for tramadol and O-desmethyltramadol, respectively, whereas the corresponding inter-day precision values were found to be less than 5.5% and 4.6%. Intra-day accuracy for tramadol was found to be between −7.1% and 3.8%, and for O-desmethyltramadol between −6.6% and 6.5%. The inter-day accuracy was found to be between −5.9% and 2.5% for tramadol, and between −3.6% and 3.2% for O-desmethyltramadol.

Absolute recovery

Absolute recovery was determined at the three QC concentration levels, by analyzing six spiked blood and vitreous humor samples and six mixed standard solutions of the studied substances in methanol at each concentration level. For each analyte, absolute recovery results were obtained by calculating the ratio of the peak area of the analyte after its extraction from the biological materials to the area corresponding to its respective standard in methanol multiplied by 100. For both biological samples absolute recovery was found to be higher than 85% and 92% for tramadol and O-desmethyltramadol, respectively.

Dilution integrity

Dilution integrity was also evaluated by spiking six blood and six vitreous humor samples with tramadol and O-desmethyltramadol at a concentration above the upper calibration level (2000 ng/mL) and diluting each biological sample with the appropriate respective blank matrix, prior to the analysis with the developed method. Accuracy and precision were found within the set criteria (±15%).

Application of the developed method to postmortem blood and vitreous humor samples

The method was applied to 12 postmortem femoral blood and vitreous humor samples obtained from forensic cases investigated by the Department of Forensic Medicine and Toxicology, National and Kapodistrian University of Athens. In these samples, the presence of tramadol had been confirmed during general screening of blood or urine samples or the intake of tramadol had been reported in the case history. The concentration results of the two substances are presented in Table 2. SIM chromatograms of tramadol and O-desmethyltramadol from real postmortem blood and vitreous humor samples (case 12) are presented in Figs 1 and 2, respectively. In addition, the ratios of vitreous humor concentrations to the respective blood concentrations were calculated in order to study the distribution of tramadol and O-desmethyltramadol in the vitreous humor. Finally, the mean and median values of the ratios of vitreous humor to blood concentrations for the two substances were also calculated and the results are shown in Table 3.

Table 2.

Concentrations found in femoral blood and vitreous humor samples of studied forensic cases

	Femoral blood concentration (ng/mL)		Vitreous humor concentration (ng/mL)		Concentration ratio vitreous humor/femolar blood
Case	Tramadol	Ο-Desmethyltramadol	Tramadol	Ο-Desmethyltramadol	Tramadol	Ο-Desmethyltramadol	Cause of death	Age	Other detected substances
1	355.2	34.5	266.0	26.3	0.75	0.76	Lobar pneumonia	77	Codeine, Paracetamol, Citalopram
2	307.1	39.4	375.1	84.9	1.22	2.15	Myocardial infarction	55	Gabapentin, Duloxetine, Citalopram
3	1814	66.3	803.6	41.0	0.44	0.62	Αspiration of gastric contents	93	Citalopram
4	267.8	192.7	223.1	227.7	0.83	1.18	Myocardial infarction	74	—
5	984.4	239.3	722.2	251.6	0.73	1.05	Carcinomatosis	49	Codeine, Morphine, Paracetamol, Amitriptyline, Pregabalin, Lidocaine, Midazolam
6	1770	10.3	975.6	5.64	0.55	0.55	Pelvic and femoral injuries due to traffic accident	75	Midazolam, Lidocaine
7	687.7	156.4	747.6	132.6	1.09	0.85	Myocardial infarction	60	—
8	1888	419.9	1254	287.1	0.66	0.68	Drug overdose	41	Morphine, Codeine, 6-acetyl-morphine, Diazepam, Nordazepam, Oxazepam, Paracetamol
9	474.4	5.55	1085	5.18	2.29	0.93	Myocardial infarction	62	Metoprolol, Lidocaine
10	136.8	19.7	58.4	10.5	0.43	0.53	Drug overdose	58	Morphine, Codeine, 6-acetyl-morphine, 11-nor-carboxy-delta9-THC, Paracetamol
11	803.6	177.6	806.3	142.7	1.00	0.80	Αutoimmune kidney disease	48	Pethidine, Codeine, Paracetamol
12	614.6	237.2	569.8	281.5	0.93	1.19	Renal cell carcinoma	67	Paracetamol, Lidocaine, Midazolam

Figure 1.

SIM chromatograms of tramadol and O-desmethyltramadol from real postmortem blood sample (case 12).

Figure 2.

SIM chromatograms of tramadol and O-desmethyltramadol from real vitreous humor sample (case 12).

Table 3.

Mean and median values of the ratio concentrations of vitreous humor to blood

	Ratio of vitreous humor to blood concentrations
Analyte	Mean value	Standard deviation	Median value
Tramadol	0.91	0.50	0.75
Ο-desmethyltramadol	0.94	0.43	0.83

According to the results of the analysis of the forensic samples, using the developed method, it was observed that when the two substances were detected in blood, they were always found in vitreous humor, even when their concentrations in blood were low, close to the LOQ of the developed method.

These results suggest that both tramadol and its metabolite are readily distributed into vitreous humor.

Furthermore, from the vitreous humor to blood ratio concentrations of both tramadol and O-desmethyltramadol, it is obvious that both substances are distributed to an almost similar extent in the two biological materials studied, since the mean values were 0.91 and 0.94, for tramadol and O-desmethyltramadol, respectively. This observation is in agreement with two previously published studies [31, 32] dealing with the determination of tramadol, but not of its metabolite, in vitreous humor. More specifically, the study of a case of confirmed tramadol and heroin intoxication by Bogusz et al. [31] showed that for tramadol, the vitreous humor to blood ratio was 0.96 (500 ng/mL in vitreous humor: 520 ng/mL in blood), very close to the ratio found in our study results. In addition, the mean of the postmortem sample ratios for tramadol in the work of Havig et al. [32] with similar number of participants was found to be 0.98.

When analyzing the postmortem samples, it was noticed that due to the readily and almost proportional distribution of the substance in vitreous humor compared to blood, when tramadol was found in blood at therapeutic levels, its corresponding concentrations in vitreous humor were also within blood therapeutic limits (0.1-1.0 μg/mL [5]). Therefore, even in cases of absence of a blood sample, the presence of tramadol at levels within these limits suggest therapeutic concentrations in blood as well, and no association of its intake with the cause of death, as for most of the cases of this study.

For the cases where tramadol was found at toxic levels in blood, it was necessary to investigate and clarify whether this was a case of overdose, postmortem redistribution or a case of poor metabolism [37, 38]. In this study, when the concentration of tramadol in the blood was at toxic levels and at the same time the concentration of O-desmethyltramadol was very low, it was assumed that it was a case of a poor metabolizer (case 6), i.e. an accumulation of the substance in the blood due to poor metabolism that leads to toxic levels of the substance and the time from the last intake of the drug to the time of death was short. It was observed that in this case the corresponding levels in vitreous humor were similar to those in blood. This illustrates the usefulness of vitreous humor for confirming the initial observation or even using the results in vitreous humor in the absence of a blood sample. Additionally, in a corresponding case (case 9) of an alleged poor metabolizer with a very low concentration of O-desmethyltramadol in both biological materials, a very high concentration of tramadol was detected in vitreous humor while its levels in blood were within therapeutic range. The combination of these results leads to the conclusion that propably this was a case of a poor metabolizer and furthermore, the time from the last ingestion of the substance to death was long. This conclusion would have been impossible in the absence of vitreous humor and by using only the quantitative blood results. In addition, vitreous humor also appears to be useful in differentiating cases of postmortem redistribution from cases of overdose. In case 3, near-fatal concentrations of tramadol in blood were observed. The simultaneous detection of the substance in vitreous humor at levels within therapeutic blood limits argued in favor of postmortem redistribution of the substance rather than its overdose. On the other hand, in case 8, the fact that both tramadol and its metabolite are found at very high levels in both biological materials argues for an overdose of tramadol and its involvement in the cause of death. Moreover, from this case it is concluded that vitreous humor may alone confirm cases of drug overdose when very high concentrations of both tramadol and its metabolite O-desmethyltramadol are found.

Compared to the previously published studies, this work presents the first method concerning the determination of tramadol using GC–MS in vitreous humor samples. Moreover, this is the first study that illustrates the determination and distribution of the metabolite O-desmethyltramadol in vitreous humor and also the usefulness of the simultaneously determination of the metabolite along with that of tramadol in this alternative material. Despite the limitations of this study (no information regarding the dosage, the intake frequency, the time of the last tramadol intake), the results of our study indicate that the information that can be extracted from the analysis of vitreous humor samples is particularly useful for a thorough investigation concerning the cause of death in tramadol-related cases. Although it seems that the blood therapeutic and toxic levels for tramadol could be also used in vitreous humor, the need for further study toward this direction becomes apparent.

Conclusions

The study of the above postmortem samples shows the importance of using vitreous humor as an alternative biological material either in cases where blood and urine samples cannot be collected or for drawing safer conclusions since vitreous humor, due to its closed cavity, is much less affected than blood by the phenomenon of sepsis and postmortem redistribution. However, in order to establish therapeutic and toxic concentrations of tramadol and its metabolite in vitreous humor, corresponding to those in blood, further analysis of biological samples from a larger number of forensic cases, and in particular cases of overdose, seems necessary.

Funding

None declared.

Data availability

Data can be made available upon reasonable request by contacting the corresponding author.