INTRODUCTION
Pregabalin (PRG) is a gabapentinoid, with great structural similarity to gabapentin. It is commonly used in the treatment of neuropathic pain, partial seizures, and generalized anxiety disorder. However, potential PRG abuse has been described.[1]
It is commonly abused due to its recreational hallucinogenic dissociative effects. PRG misuse seemed to occur orally, but intravenous and rectal self-administration were also reported.[1]
PRG achieves steady state within 24–48 h, has a short half-life (within 4–7 h postingestion), and is excreted unchanged in the urine.[2]
PRG binds to the alpha-2-delta ligands, located at presynaptic endings of neurons. This binding attenuates Ca2+ flux into neurons and blocks the release of neurotransmitters, including the excitatory neurotransmitters, resulting in drowsiness up to coma. Prolonged use can lead to tolerance, which necessitates dose escalation.[2]
PRG can be a possible cause of death, especially when combined with other illicit street drugs.[3] It was reclassified into Class C drug controlled substances from April 2019.[4]
In case of drug intoxication deaths, toxicological forensic investigations are requested to detect the postmortem drug concentration and estimate the amount of drug present at the time of death, or the number of tablets consumed, for this reasons, establishing the identity of certain drug and its ante-mortem and postmortem stability is needed.[5]
This study aimed to assess the stability of orally ingested PRG in antemortem and postmortem femoral blood samples collected from the rat model using Gas chromatographyMass spectrometry (GC-MS) and the relation between them were assessed.
MATERIALS AND METHODS
Chemicals
PRG and gabapentin (internal standard) were obtained from Pfizer® (Berlin, Germany).
Animals and experimental design
Ninety-six male Sprague Dawley rats weighing 250–300 g were used in the study. They were distributed into 32 cages (3 rats/cage) and kept under standard laboratory conditions; the temperature was 24°C ± 3°C with 40% humidity and allowed free access on food and water provided ad libitum.
The experiment was ethically approved by the Institutional Animal Care and Use Committee, King Saud University.
Experimental protocol
Rats were divided into four groups with 24 animals each. Group A was given oral single dose of PRG (4 g/kg body weight/day). Group B was given oral single dose of PRG (2.5 g/kg body weight/day). Group C was given oral 21 consecutive doses of PRG (50 mg/kg body weight/day). Group D was given oral 21 consecutive doses of PRG (20 mg/kg body weight/day). The 4 g, 2.5 g, 50 mg, and 20 mg/kg animal doses were equivalent to human doses of 600 mg, 400 mg, 8 mg, and 3 mg/kg, respectively,.[6] PRG was administered through a gastric tube once a day.
The animals in each group were divided into four subgroups with six animals each where the femoral blood samples were collected based on a previous animal study of postmortem drug detection[7] at 4 and 6 h after the last ingested dose (at the time of death), 24 and 48 h after the time of death.
At the end of the experiment, all rats were anesthetized using pentobarbital sodium intraperitoneal injection (80 mg/kg) and were sacrificed.
The samples were preserved at -80°C till the assessment time (3 days from sampling) then they were prepared according to Hložek etal.[8] A volume of 100 μL of blood sample (100 mg) was mixed with 200 μL water and 10 μL of gabapentin (Internal standard) then 1.0 ml acetonitrile was added. The resulting denatured protein precipitate was separated by centrifugation 5000 rpm for 5 min. The supernatant layer was transferred to a clean test tube. One hundred μL of 2.5 M sodium hydroxide, 1.0 ml of water, 0.5 ml of ethanol, and 50 μL of pyridine were added to the supernatant sample and mixed for 30 s. Then, 50 μL of ethyl chloroformate (derivatizing reagent) was added and vigorously mixed with the sample by vortex for 2 min. PRG in the samples were extracted with 1.5 ml ethyl acetate by mixing for 3 min and were centrifuged at 5000 rpm for 3 min. The upper organic layer of ethyl acetate was separated and dried under nitrogen. Finally, samples were reconstituted with 100 μL ethyl acetate and transferred to GC-MS vial for the analysis.
The GC-MS analysis was carried out using an Agilent Technologies, model 7890B, Santa Clara, CA, USA) that was equipped with an electronically controlled splitless injection port, Helium as a carrier gas, a full scan detector, selective ion monitoring quantification with 102-quantifier ion (m/z) and 128,142, and 172 qualifier ions (m/z).
A representative gas chromatography PRG chromatogram and calibration curve are presented in Figures 1-4].
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Figure 1: Total ion chromatogram of pregabalin – Ethyl chloroformate derivatives
Figure 2: Mass spectrum of pregabalin – Ethyl chloroformate derivatives
Figure 3: Selective ion monitoring spectrum of pregabalin–Ethyl chloroformate derivatives
Figure 4: Calibration curve of pregabalin from extracted spiked blood
Statistical analysis
All values are presented as the mean ± standard deviation (SD). Statistical differences between groups were detected using one-way analysis of variance (SPSS for Windows version 11.0, SPSS Inc., Chicago, IL, USA). Differences in means were considered statistically significant at P = 0.05.
RESULTS
The mean (±SD) antemortem blood PRG concentrations at 4 and 6 h after the last ingested dose are presented in Figure 5. When the oral doses were 4 g/kg, 2.5 g/kg, 50 mg/kg, and 20 mg/kg the mean blood PRG levels were 31.7 ± 1.5, 17.5±0.9, 13.5±0.7, and 4.8±.5 μL/ml, respectively (P = 0.05). It is evident that the increase in dosage was accompanied by an increase in PRG concentration in the blood [Figure 5].
Figure 5: Antemortem pregabalin blood levels (μg/ml). *and # statistically significant compared to the corresponding value in Groups A and C, respectively, (P < 0.05)
Post-mortem PRG blood concentrations at 24 and 48 h after death were decreased in comparison to antemortem concentrations. At 24 h after death and with oral doses 4 g/Kg, 2.5 g/kg, 50 mg/kg, and 20 mg/kg, the mean blood PRG levels were 31.4 ± 3.2, 17.1 ± 0.8, 13.2±0.8, and 4.8 ± 0.5 μL/ml, respectively. While at 48 h after death and with before mentioned oral doses, the mean blood PRG levels were 30.9 ± 2.9, 16.9 ± 0.7, 13 ± 0.8, and 4.6 ± 0.5, respectively. However, this decrease did not show any statistical significance difference (P > 0.05) [Table 1].
Table 1: Post-mortem blood PRG concentrations
DISCUSSION
Forensic toxicologists are frequently asked to analyze the postmortem samples, for example, blood, urine, and other materials to detect certain drug concentrations and interpret the results in the known drug pharmacology.[9]
Antemortem pharmacological assumptions and interpretations are often invalid after death.[10] For this reason, the stability of PRG in antmortem and post-mortem blood samples was investigated, and the relation between them was assessed.
At storage temperature - 80°C for 3 days after sampling, PRG was detected in antemortem and postmortem blood samples up to 48 h after death.
Blood PRG concentrations of 34.1 ± 0.9 (with range 31–37), 17.8 ± 0.8 (with range 15–20) could be considered as toxic concentrations after 4 h of acute PRG overdose; while the presence of blood PRG concentrations 13.6 ± 1.8 (with range 11–16) and 4.9 ± 1 μg/ml (with range 3.4–6.5) could be considered as toxic concentrations after chronic PRG administration.
Our results showed that with increasing the oral PRG dose, the mean blood PRG concentration significantly increased. We were in agreement with Olesen etal.[11] who reported that PRG absorption is linear and obeys the first-order absorption model, where an increase in dose can result in an increase in serum concentration. Moreover, Hong etal.[12] stated that the PRG absorption rate increased over time.
Priez-Barallon etal.,[13] who detected PRG in postmortem human samples, stated that there is no difference between cardiac and peripheral blood levels, and the blood levels of PRG in intoxicated cases showed higher PRG concentration, between 16.3 and 206.7 mg/L.
Braga and Chidley[14] found that patient's serum concentrations of PRG were 60 and 15 mg/L one and 2 days after ingesting an estimated toxic dose of 11.5 g.
Our results showed that the ante-and postmortem concentration differences increased with early postmortem interval (PMI). However, these differences did not show any statistical significance (P < 0.05). The reason of this result could be due to the stability PRG in these specimens.[15]
This result was consistent with Bockbrader etal.[16] and Zilg etal.[15] They stated that drugs with a low volume of distribution (Vd), for example, PRG with Vd 0.56 L/kg,[17] showed lower drug concentration differences compared to drugs with a high Vd.
In addition, Hilberg etal.,[18] stated that drugs with Vd <4 L/Kg did not show postmortem redistribution phenomenon.
Moreover, Zilg etal.[15] suggested that postmortem blood drug level estimation should be performed during PMI; <50 h after death and that peripheral venous blood should be selected and used for the drug analysis.
CONCLUSION
PRG is a commonly abused recreational drug and its stability significantly affects the interpretation of data to reach a reliable conclusion.
According to our findings, it would be useful to perform PRG analysis in antemortem and postmortem peripheral blood specimens within 2 days of sampling.
Recommendation
We recommend further analysis of PRG levels from different specimens, for eample, hair, cardiac blood, vitreous at different storage conditions, and longer durations to augment the results of this preliminary study before using it in a forensic context.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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