Identification of post-mortem product of zolpidem degradation by hemoglobin via the Fenton reaction

Abstract

Zolpidem, N,N-dimethyl-2-[6-methyl-2-(4-methylphenyl)imidazo[1,2-a]pyridin-3-yl]acetamide, is a hypnotic agent widely used in clinical practice but is detected in many clinical cases of fatal intoxication and suicide. In forensic toxicology, the precise determination of zolpidem concentration in blood is a must to provide concrete evidence of death by zolpidem poisoning. However, the concentrations of zolpidem in blood at autopsy often differ from those at the estimated time of death. In the present study, we found that zolpidem was degraded by hemoglobin (Hb) via the Fenton reaction at various temperatures. The mechanism underlying zolpidem degradation involved the oxidation of its linker moiety. The MS and MS/MS spectra obtained by liquid chromatography quadrupole-Orbitrap mass spectrometry (LC-Q-Orbitrap-MS) showed the formation of 2-hydroxy-N,N-dimethyl-2-(6-methyl-2-(p-tolyl)imidazo[1,2-a]pyridin-3-yl)acetamide (2-OH ZOL) in Hb/H₂O₂ solution incubated with zolpidem and in the blood of several individuals who died from ingestion of zolpidem. These results suggest that 2-OH ZOL is the post-mortem product of zolpidem degradation by Hb via the Fenton reaction.

INTRODUCTION

Zolpidem, N,N-dimethyl-2-[6-methyl-2-(4-methylphenyl)imidazo[1,2-a]pyridin-3-yl]acetamide, is a hypnotic agent that was initially approved by the FDA in 1992. The drug usage has dramatically increased because zolpidem is available commercially (Jang et al., 2019). Developed for use in patients with difficulty sleeping, zolpidem is a central nervous system depressant, a gamma-aminobutyric acid receptor agonist, and a sedative. It is widely believed to be relatively safer and much more tolerable than benzodiazepines because it is not toxic and does not result in dependence or tolerance. On the other hand, zolpidem is a major compound contributing to poisoning deaths, including accidents and suicide, in the world (Jang et al., 2019; Gunja, 2013; Takayasu et al., 2008). The lethal blood zolpidem concentration is 1.5−4.0 μg/mL (Schulz et al., 2020). In forensic toxicology, the precise determination of blood zolpidem concentration is a must to provide concrete evidence of death by zolpidem poisoning. However, blood zolpidem concentration is not detected or detected at concentrations much lower than the expected one (Takayasu et al., 2008). At the moment, there is no reasonable explanation for the decreased zolpidem concentration in blood after death.

Post-mortem plasma esterase activity and bacterial contamination are important factors contributing to post-mortem decreases in blood chemical concentration (Peters and Steuer, 2019). However, the plasma esterase such as human serum albumin is unlikely to be involved in the post-mortem decreases in blood zolpidem concentration because the structure of zolpidem makes it unsuitable for use as a substrate of this enzyme. On the other hand, it is reported that zolpidem is degraded by bacteria such as Aspergillus jensenii and Mucor circinelloides (Martínez-Ramírez et al., 2016). Small quantities of zolpidem metabolites produced by these bacteria have been detected in human post-mortem blood, suggesting that bacteria may not be the key factor affecting the post-mortem decreases in blood zolpidem concentration. Recently, we have reported a new mechanism of post-mortem decreases in blood chemical concentrations by hemoglobin (Hb) via the Fenton reaction (Yamagishi et al., 2021a, 2023): antianxiety agents and atypical antipsychotic agents (e.g., bromazepam and paliperidone) are decomposed post-mortem by Hb via the Fenton reaction. In the Fenton reaction, divalent iron catalyzes the dismutation of hydrogen peroxide (H₂O₂), leading to the formation of hydroxyl radical (^•OH) and hydroxide (HO^–), along with trivalent iron (Barb et al., 1949). Hb has four divalent iron ions, and H₂O₂ is produced by the dismutation of the superoxide anion (O₂^•–) generated from HbO₂ autoxidation (Shikama et al., 2001). As far as we know, there are no reports on the interaction between zolpidem and Hb via the Fenton reaction. Thus, we speculate that Hb is the major factor contributing to the post-mortem decreases in blood zolpidem concentrations via the Fenton reaction.

In the present study, because we evaluated the interactions between zolpidem and each reaction reagent for the Fenton reaction (e.g., H₂O₂, Hb, and Hb/H₂O₂), we measured zolpidem per se and the product of zolpidem degradation by Hb via the Fenton reaction. For detection of zolpidem per se and the product of zolpidem degradation by Hb via the Fenton reaction, a liquid chromatography coupled to a quadrupole-Orbitrap mass spectrometry (LC-Q-Orbitrap-MS) was used. Because it provides accurate molecular mass values (Orbitrap-MS) and chemical structure information (Q-Orbitrap-MS; tandem MS) (Yamagishi et al., 2023), LC-Q-Orbitrap-MS effectively measures untargeted small molecules, such as zolpidem degradation products generated by post-mortem changes.

MATERIALS AND METHODS

Chemicals

Hb (Human, H7379) and zolpidem (Z103) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (ACN, LC–MS grade) and water (LC–MS grade) were supplied from Kanto Chemical (Tokyo, Japan). Diazepam-d5 was gained from Hayashi Pure Chemical Ind., Ltd. (Osaka, Japan). 0.1 M phosphate buffer solutions (pH 6.0, 6.8, and 7.4), 1 M ammonium formate solution (HPLC grade), H₂O₂ (30%, guaranteed grade), and formic acid (FA, 98–100%, LC–MS grade) were obtained from Fujifilm Wako Pure Chemical (Osaka, Japan).

Instruments

A Q-Exactive plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a Vanquish Flex Binary LC system (Thermo Fisher Scientific) was used. The conditions for liquid chromatography and mass spectrometry are summarized in Table 1.

Table 1. Liquid chromatography and mass spectrometry conditions.

LC conditions
LC system	Vanquish Flex Binary LC(Thermo Fisher Scientific, Waltham, MA, USA)
Column	CORTECS T3 column, 2.7 μm, 100 × 2.1 mm I.D., (Waters, Milford, MA, USA)
Injection volume [µL]	2
Total flow [mL/min]	0.3
Oven [ºC]	40
Elution buffer A	0.01 M ammonium formate and 0.1% Formic acid
Elution buffer B	Acetonitrile
Gradient curve [A/B]	90/10 (0 min)–40/60 (12 min)–0/100 (18–23 min)–90/10 (23–30 min)
MS conditions
MS system	Q-Exactive plus Orbitrap mass spectrometer (Thermo Fisher Scientific)
Polarity	Positive
Mode	Full-scan MS,parallel reaction monitoring (PRM; MS/MS analyses)
m/z range	100–500
Heater temperature [°C]	413
Capillary temperature [°C]	256
Auxiliary gas [%]	11
Sheath gas [%]	48
S-lens radio frequency level	50
Spray voltage [kV]	3.5
AGC target value	5e6 (Full-scan MS), 1e5 (PRM)
Resolution at m/z 200	70000 (Full-scan MS), 17,500 (PRM)
Collision energy [V]	10, 20, and 30 (PRM)

Recovery of zolpidem from the reaction mixture containing reagents for the Fenton reaction

We used the reported concentration conditions of each reagent such Hb and H₂O₂ for the Fenton reaction (Yamagishi et al., 2023). For the evaluation of the recovery of zolpidem from the reaction mixture containing reagents for the Fenton reaction, a 0.1 mL aliquot of a reaction mixture containing of Hb (final concentration, 100 mg/mL), zolpidem (final concentration, 0.01 mg/mL), and H₂O₂ (final concentration, 0.3 mg/mL) was used. The control samples contained 0.1 M phosphate buffer (pH 7.4) and zolpidem, but no Hb and H₂O₂. The reaction mixture was incubated at 37ºC for 0 and 24 hr. After incubation, 1.9 mL of ACN containing diazepam-d5 (25 ng/mL) as the internal standard (IS) was added to the reaction mixture. The mixture containing ACN was sonicated, vortexed, and centrifuged at 10,000 x g for 10 min, and the supernatant was subjected to LC-Q-Orbitrap-MS to determine the zolpidem concentration in the supernatant. The extracted ion of zolpidem was detected at m/z 308.1757 with m/z tolerance of 5 ppm.

For the evaluation of the temperature- and pH-dependent recovery of zolpidem from the reaction mixture containing reagents for the Fenton reaction, Temperature conditions were set at 4, 20, 37, and 45ºC, and pH conditions were 6.0, 6.8, and 7.4. Pretreatment condition was same as the section of “the evaluation of the recovery of zolpidem from the reaction mixture containing reagents for the Fenton reaction” mentioned above.

Detection and identification of unknown zolpidem degradation product in the presence of Hb/H₂O₂

Reaction and pretreatment conditions were same as the section of “the evaluation of the recovery of zolpidem from the reaction mixture containing reagents for the Fenton reaction” mentioned above. The samples after pretreatment were subjected to LC-Q-Orbitrap-MS to detect and identify the unknown zolpidem degradation product in the sample. A difference analysis was performed by Compound Discoverer version 3.3 software (Thermo Fisher Scientific) for detection of the unknown zolpidem degradation product.

Detection of post-mortem zolpidem degradation product in the blood of individuals who died from intentional ingestion of zolpidem

The use of autopsy samples for analysis conformed to the Code of Ethics of the World Medical Association (Declaration of Helsinki). The present study was approved by the ethics committees of Chiba University (approval no. 2819, Graduate School of Medicine; approval no. R001, Graduate School of Pharmaceutical Sciences). At the request of investigation agencies, we conducted forensic autopsies as a part of criminal investigations and were not allowed to meet or contact the families before or after the autopsies. We have included a disclaimer on our official Web site stating that we occasionally collect samples from autopsies for research purposes and that if the families do not consent to the deceased’s body being used for research, they can opt out and inform us by facsimile.

In the present study, post-mortem blood samples collected from twenty autopsy cases were used, in which zolpidem was detected by drug screening performed in our laboratory. The post-mortem intervals of blood collection were from one to ten days. Causes of death included drug poisoning, CO poisoning, drowning, fire, hypothermia, brain herniation, fat embolism, traumatic shock, choking, subdural hematoma, brain damage, and gunshot wound. Autopsies were performed in January, March, April, May, June, July, November and December. To a 0.1 mL aliquot of each blood sample, 0.4 mL of ACN containing IS (250 ng/mL) was added, and the mixture containing ACN was vortexed and centrifuged at 10,000 x g for 10 min. The supernatant was subjected to LC-Q-Orbitrap-MS. The extracted ion of the post-mortem zolpidem degradation product was detected at m/z 324.1707 with m/z tolerance of 5 ppm.

Statistics

Dunnett’s multiple comparison test and Tukey test were performed for comparisons among multiple groups in the section of “Recovery of zolpidem from the reaction mixture containing reagents for the Fenton reaction”. Significant differences are indicated by ** (p < 0.01). Data are expressed as means ± standard deviation (SD).

RESULTS

Changes in zolpidem concentrations in the presence of Hb and H₂O₂

Zolpidem concentrations in H₂O₂, Hb, and Hb/H₂O₂ solutions at 37ºC were significantly decreased after 24 hr compared with those in control (Fig. 1A). The percentages of undegraded zolpidem in H₂O₂, Hb, and Hb/H₂O₂ solutions at 24 hr relative to that at 0 hr were 91.7 ± 1.1%, 87.3 ± 2.9%, and 16.4 ± 0.5%, respectively.

Fig. 1

Effects of H₂O₂, Hb, and Hb/H₂O₂ (A) on zolpidem concentration and effects of temperature (B) and pH (C) on zolpidem concentration in the presence of Hb/H₂O₂. A reaction mixture of 0.01 mg/mL zolpidem and each reaction reagent, such as H₂O₂, Hb, or Hb/H₂O₂, was incubated for 0 and 24 hr. Temperatures were kept at 4, 20, 37, and 45ºC and pH values were 6.0, 6.8, and 7.4. Dunnett’s multiple comparison test (A) and Tukey test (B and C) were performed for comparisons among groups. Significant differences are indicated by ** (p < 0.01). Data are expressed as means ± standard deviation (SD).

Effects of temperature and pH on decreases in zolpidem concentration in Hb/H₂O₂ solution

Zolpidem concentrations in Hb/H₂O₂ solution were significantly decreased at 4, 20, 37, and 45ºC after 24 hr compared with those at 0 hr (Fig. 1B). The percentages of undegraded zolpidem in Hb/H₂O₂ solution at 4, 20, 37, and 45ºC after 24 hr relative to that at 0 hr were 11.4 ± 0.8%, 23.8 ± 0.5%, 16.7 ± 0.2%, and 7.0 ± 0.2%, respectively. The concentration of zolpidem in Hb solution at each temperature decreased in the following order: 45ºC > 4ºC > 37ºC > 20ºC.

Zolpidem concentrations in Hb/H₂O₂ solution at pH 6.8 were significantly decreased compared with those at pH 6.0 and 7.4 after 24 hr (Fig. 1C). The percentages of undegraded of zolpidem in Hb/H₂O₂ solution at pH 6.0, 6.8, and 7.4 after 24 hr relative to that at 0 hr were 9.4 ± 0.3%, 4.4 ± 0.1%, and 9.2 ± 0.2%, respectively. The degradability of zolpidem in Hb solution at each pH decreased in the following order: pH 6.8 > pH 6.0 and 7.4. No significant differences in the reactivity of zolpidem were observed among pH 6.0, 6.8, and 7.4, although significant differences in the reactivity of zolpidem were possible.

Detection and identification of unknown zolpidem degradation product in the presence of Hb/H₂O₂

A difference analysis was performed to compare the mass spectra of Hb/H₂O₂-treated samples at 0 and 24 hr by Compound Discoverer version 3.3 software. A specific ion detected only in the Hb/H₂O₂-treated sample at 24 hr was extracted at m/z 324.1707 (Fig. 2B), which corresponds to the unknown zolpidem degradation product eluted at the retention time of 4.5 min. We named this degradation product ukZDP1.

Fig. 2

Elution profiles of ukZDP1 at m/z 324.1707 in the presence of Hb/H₂O₂ at 0 hr (A) and 24 hr (B), MS/MS spectrum of ukZDP1 detected in the positive ion mode (C), and the assignment of ukZDP1 precursor and product ions (D). The retention time of ukZDP1 was 4.5 min. The ukZDP1 peak in Fig. 2B was subjected to further LC-Q-Orbitrap-MS analysis (MS/MS analyses). The assignment of ukZDP1 precursor and product ions is summarized in Fig. 2D and Table 2.

Figure 2C shows the results of MS/MS analyses of ukZDP1 in the positive ion mode. One precursor ion (ukZDP1-1) and four product ions (ukZDP1-2–ukZDP1-5) with m/z values of 324.1692, 251.1166, 235.1222, 223.1222, and 74.0604, respectively, were detected by Orbitrap-MS. The assignment of ukZDP1 precursor and product ions is shown in Fig. 2D and Table 2. Notably, the Δm/z values for the precursor and product ions of ukZDP1 were less than ± 6 ppm. UkZDP1 was reasonably assigned to 2-hydroxy-N,N-dimethyl-2-(6-methyl-2-(p-tolyl)imidazo[1,2-a]pyridin-3-yl)acetamide (hereinafter “2-OH ZOL”).

Table 2. Assignment of ukZDP1 precursor and product ions.

Peakno.	Elemental composition	Theoreticalm/z	Measuredm/z	Δ m/z[ppm]
ukZDP1-1	C19H22N3O2	324.1707	324.1692	-4.6
ukZDP1-2	C16H15N2O	251.1179	251.1166	-5.2
ukZDP1-3	C16H15N2	235.1230	235.1222	-3.4
ukZDP1-4	C15H15N2	223.1230	223.1222	-3.6
ukZDP1-5	C3H8NO	74.0600	74.0604	5.4

Detection of post-mortem zolpidem degradation product (2-OH ZOL) in the blood of individuals who died from ingestion of zolpidem

Figure 3 shows the topical elution profile of 2-OH ZOL at m/z 324.1707 in the blood of individuals who died from ingestion of zolpidem. 2-OH ZOL was detected in seven blood samples collected from autopsied individuals who died from ingestion of zolpidem (Fig. S1). The causes of death were drowning (four cases), drug poisoning (one case), traumatic shock (one case), and fire (one case). The main cause of death for the 2-OH ZOL detection case was drowning. 2-OH ZOL was not detected in 13 blood samples collected from autopsied individuals who died from ingestion of zolpidem. The causes of death for these cases were CO poisoning, fire, hypothermia, brain herniation, fat embolism, traumatic shock, choking, subdural hematoma, brain damage, and gunshot wound.

Fig. 3

Topical elution profile of 2-OH ZOL at m/z 324.1707 in the blood of individuals who died by drowning from ingestion of zolpidem.

DISCUSSION

Recently, we have reported a new mechanism underlying post-mortem changes in ingested chemicals such as pesticides and drugs by Hb in post-mortem blood (Yamagishi et al., 2021a, 2021b, 2022, 2023). Hb-mediated post-mortem changes of ingested chemicals are characterized by two mechanisms: one is the covalent binding of oxime-type carbamate pesticides such as methomyl, and aldicarb to Hb (Yamagishi et al., 2021b, 2022), and the other is the oxidative decomposition of drugs such as bromazepam and paliperidone by Hb via the Fenton reaction (Yamagishi et al., 2021a, 2023). The results in Fig. 1 suggested that zolpidem was also degraded by Hb via the Fenton reaction, indicating that its linker moiety of zolpidem was the target of oxidation by the Fenton reaction (Fig. 2D). Hb is the most abundant protein found in red blood cells and is released from them by hemolysis after death (Hirabayashi, 1953).

It is said that temperature and pH are main factors influencing post-mortem changes. First, a dead body is exposed to different temperatures depending on the circumstances of fatal poisoning (e.g., 4–45ºC in Japan). Many dead bodies are usually stored in a refrigerator at 4ºC for a few days prior to autopsy in Japan (Kennedy, 2015). Furthermore, the body's internal temperature can rise to above 40ºC depending on the cause of death of hyperthermia and stimulant intoxication (Lifschultz and Donoghue, 1998). It is reported that temperatures between 15 and 40ºC have little influence on the Fenton reaction (Shaobin, 2008). We corroborate this finding with our results in Fig. 1B, where zolpidem in Hb/H₂O₂ solution was degraded at all temperatures examined. Thus, temperature is not a key factor affecting post-mortem decreases in zolpidem concentration by Hb via the Fenton reaction. Second, it has been reported that pH is one of the factors influencing post-mortem changes in a dead body (Donaldson and Lamont, 2013). Blood pH in human ranges from 7.35 to 7.45 before death. On the other hand, pH of cardiac blood is decreased to 6.0 with elapsed time after death (Sawyer et al., 1988). It is reported that pH in the range of 6.0 to 7.0 has little influence on the Fenton reaction (Yamagishi et al., 2023; Yang and Xue, 2023). As shown in Fig. 1C, zolpidem in Hb/H₂O₂ solution was similarly degraded in the pH range of 6.0 to 7.4. Thus, pH in the range of 6.0 to 7.4 is not a key factor influencing post-mortem decreases in zolpidem concentration by Hb via the Fenton reaction. To summarize, our findings indicate that zolpidem is degraded in various environments to which dead bodies are exposed, but the degradation is not affected by temperature or pH.

We speculated that the mechanism underlying zolpidem degradation involved the oxidation of its linker moiety by Hb via the Fenton reaction, resulting in the formation of 2-OH ZOL, the post-mortem degradation product. Because 2-OH ZOL was detected only in the presence of Hb/H₂O₂ by a difference analysis, 2-OH ZOL seemed to be the only degradation product produced from zolpidem by post-mortem changes induced by Hb via the Fenton reaction. However, in the present study, we were unable to detect 2-OH ZOL in some post-mortem blood samples of individuals who died after ingestion of zolpidem. Because temperature and pH were not the key factors affecting post-mortem decreases in zolpidem concentration in a dead body as mentioned above, we looked into other factors affecting zolpidem degradation by Hb via the Fenton reaction. The Fenton reaction requires divalent iron ions and H₂O₂ (Barb et al., 1949). Ferrous ions seem to originate from Hb in a dead body, and H₂O₂ may be produced by the dismutation of O₂^•– generated from HbO₂ autoxidation (Shikama et al., 2001). One study speculated that H₂O₂ concentration in the blood of a dead body was one of the key factors driving the Fenton reaction (Shaobin, 2008). Indeed, it is reported that H₂O₂ concentration in blood varies with the cause of death (Hanifi et al., 2016). In the present study, we detected 2-OH ZOL in seven post-mortem blood samples, the cause of death being mainly drowning (Fig. 3 and Fig. S1). It is known that drowning continuously induces oxidative stress, producing H₂O₂ in the process until death (Legaz et al., 2023). Thus, 2-OH ZOL may be produced from zolpidem degradation by Hb via the Fenton reaction in the event of oxidative-stress-induced death such as drowning.

Figure 4 shows a schematic diagram of zolpidem metabolism before and after death. Before death, i.e., in an alive body, zolpidem is metabolized into diverse compounds by several isoforms of hepatic cytochrome P450s such as CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 (Von Moltke et al., 1999). Indeed, zolpidem and its metabolites named M-1 (4-COOH ZOL), M-2 (6-COOH ZOL), and M-4 (6-OH ZOL) are detected in urine before death. In addition to these compounds, M-3 (4-OH ZOL) is detected in plasma before death (Ascalone et al., 1992). As far as we know, there are no reports of 2-OH ZOL detection in blood samples before death. 2-OH ZOL was detected in post-mortem blood samples collected in January, March, April, May, June, and July in the present study. Future studies are needed to clarify the relationship between zolpidem concentration at the time of death and the production of 2-OH ZOL in blood after death.

Fig. 4

Schematic diagram of zolpidem metabolic and degradation pathways before and after death.

In conclusion, zolpidem was degraded in dead bodies exposed to ordinary temperatures on the main island of Japan. The mechanism underlying zolpidem degradation involved the oxidation of the free form by Hb via the Fenton reaction, forming 2-OH ZOL, the post-mortem degradation product of zolpidem. Estimating zolpidem concentration at the time of death from the concentration of 2-OH ZOL remains a daunting task. Further studies are needed to evaluate the relationship between zolpidem concentration at the time of death and the amount of 2-OH ZOL in post-mortem blood.

Funding information

This study was supported by a grant from The Research Foundation for Pharmaceutical Sciences and JSPS KAKENHI Grants Numbers 20K18978, 22K19664 and 24H00749.

Conflict of interest

The authors declare that there is no conflict of interest.

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