Regular Article
Elucidation of Degradation Behavior of Nitrazepam, a Benzodiazepine Drug, under Basic Conditions: Study on Degradability of Drugs in Stomach (IV)
2024 Volume 72 Issue 1 Pages 11-15
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2024 Volume 72 Issue 1 Pages 11-15
This study investigates the stability of nitrazepam (NZP), a benzodiazepine drug, under basic conditions, since alkaline putrefactive amines and ammonia are produced once bodies are left to decompose for a long period postmortem after a murder involving NZP or an accidental overdose of NZP. The degradation of NZP in an aqueous alkaline solution was investigated by LC/photodiode array detector (PDA) where the NZP degradation product was isolated and purified by solid-phase extraction using Oasis® MCX, and its chemical structure was determined by LC/time-of-flight (TOF)-MS, NMR spectroscopy, and single-crystal X-ray crystallography. The results revealed that NZP was immediately degraded under basic conditions with 2-amino-5-nitrobenzophenone being an intermediate which further degraded to provide 2-hydroxy-5-nitrobenzophenone as the final degradation product. These results are expected to be useful in clinical chemistry and forensic science, such as the detection of drugs during postmortem examination and suspected addiction.
Benzodiazepines (BZPs) were developed in the 1960s as safe alternatives to the sedative-hypnotic medications, barbiturates. Although BZPs are frequently used as psychotropic drugs because of their anticonvulsant, anxiolytic, and sedative properties,1) they can facilitate the commission of robbery, rape, and murder by mixing them with beverages.2,3) Consequently, the detection of drugs employed in such crimes is of utmost importance. However, the detection of drugs that do not have long-term stability in the body is difficult, since these are often appraised after the crime.4–6) It is well known that postmortem specimens provide less stability for a number of drugs than other types of specimens, which is particularly problematic for the detection of cocaine, heroin, antidepressants, antipsychotics, and benzodiazepines.7) In fact, Bolte et al. reported the detection of drugs in the brain, liver, and other tissues of 116 donated bodies exhumed 9.5–11.5 years after burial.8) Although several BZPs were detected, nitrazepam (NZP), flunitrazepam, oxazepam, temazepam, and lorazepam were not.
Our group has investigated the degradation behavior of psychotropic drugs such as BZPs and tricyclic antidepressants in artificial gastric juice.9–11) The results revealed the degradation pathway of amoxapine,9) nitrazepam,10) and lormetazepam,11) and we have also determined some degradation products of such drugs. In addition, the long-term degradation behavior of NZP under acidic condition was observed and the results revealed the presence of a degradation intermediate (2-amino-N-(2-benzoyl-4-nirophenyl)-acetamide) and a final degradation product (2-amino-5-nitrobenzophenone).10) However, if the body is left for a long period and decomposition progresses, as in the case reported by Bolte et al.,8) basic polyamines and ammonia, known as putrefactive amines such as putrescine, cadaverine, spermidine, and spermine, are expected to be generated inside the body. Consequently, to detect BZPs in a decomposed corpse, it is essential to understand the degradation behavior of BZPs under both acidic (caused by gastric juice) and basic (caused by putrefaction) conditions. However, the degradation behavior of only a limited number of BZPs including flunitrazepam,12) oxazepam,13) and NZP,14) has been investigated under basic conditions.
Following our previous investigation on the degradation behavior of NZP under acidic conditions,10) this study investigates the behavior under basic conditions by determining the degradation pathway using an LC/photodiode array detector (PDA), and products where the results revealed that the obtained degradation products produced were different from those reported by Davidson et al.14)
NZP (biochemistry grade) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), and was dissolved in methanol to make a 10 mg/mL standard stock solution. Working standard solutions were then prepared from each standard stock solution by dilution with methanol. Acetonitrile and methanol (both HPLC grade); diethyl ether, formic acid, phosphoric acid, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium hydroxide (NaOH, all special grade (> 97%)); hydrochloric acid (HCl, precision analysis grade); and leucine-enkephalin (biochemical grade) were purchased from FUJIFILM Wako Pure Chemical Corporation. Methanol-d4 (special grade) was from Kanto Chemical Co., Inc. (Tokyo, Japan). Water was purified with a Milli-Q Gradient A10 system equipped with an EDS-PAK® polisher (Merck Ltd., Darmstadt, Germany). All other chemicals were of special grade. An Oasis® MCX 6 cc Vac cartridge (150 mg, particle size 30 µm; Waters Co., Milford, MA, U.S.A.) was used. The cartridge was conditioned with methanol, and water prior to use.
Apparatus and Operating ConditionsLC/PDAAn L-6300 Intelligent pump (Hitachi, Ltd., Tokyo, Japan) system equipped with an MD-910 PDA (JASCO Corporation, Tokyo, Japan) was used. LC separation was performed using a Poroshell 120 EC-C18 column (100 × 4.6 mm I.D., 2.7 µm; Agilent Technologies, Santa Clara, CA, U.S.A.). The column temperature was maintained at 40 °C. The mobile phase was a mixture of 50 mmol/L potassium phosphate buffer solution (pH 3) and acetonitrile at a ratio of 60 : 40 (v/v), and was delivered in the isocratic elution mode at a flow rate of 0.5 mL/min. The UV monitoring wavelength was set at 220 nm. A 10 µL aliquot of the sample solution was injected into the system.
LC/Time-of-Flight Mass Spectrometry (LC/TOF-MS)An Alliance HT 2795 HPLC system equipped with an LCT Premier XE TOF-MS (Waters Corporation) was used. LC separation was performed using an L-column2 ODS (150 × 2.1 mm I.D., 3.0 µm; CERI, Tokyo, Japan). The column temperature was maintained at 40 °C. The mobile phase was a mixture of 10 mmol/L ammonium formate buffer aqueous solution (pH 6.4) and acetonitrile in the ratio of 50 : 50 (v/v), and was delivered in the isocratic elution mode at a flow rate of 0.2 mL/min. A 10 µL aliquot of the sample solution was injected into the system. The optimum working parameters for TOF-MS were as follows: electrospray ionization (ESI), negative mode; capillary voltage, 2000 V; cone voltage, 50 V; aperture #1 voltage, 15 V; desolvation temperature, 350 °C; source temperature, 120 °C; desolvation gas flow (N2), 800 L/h; and cone gas flow (N2), 50 L/h. Leucine–enkephalin solution (0.25 µL/mL) was delivered at a flow rate of 5 µL/min. Mass accuracy was maintained using lock–spray with the leucine-enkephalin [M–H]− ion, m/z = 554.2615, as the lock mass. The resolution was at least 10000 as calculated by using the full-width-at-half-maximum method.
NMRAn ECA-600II NMR spectrometer (JEOL, Tokyo, Japan) was used for the NMR analysis. The frequency for 1H-NMR measurements was set at 600.1723 MHz, and that for 13C-NMR measurements was set at 150.9134 MHz. As the reference peak, solvent peaks of methanol-d4 (1H: 3.30 ppm, 13C: 49.0 ppm) were used.
X-Ray Crystallography (XRC)A Super Nova (Rigaku, Tokyo, Japan) was used as the X-ray source and the operating conditions were as follows: X-ray was generated by Kα radiation (λ = 1.54184 Å) of Cu. X-ray crystallographic data were collected at 102 K.
Degradation Behavior of NZP under Basic ConditionThe degradation experiment of NZP was carried out according to the flunitrazepam degradation test.10) To 900 µL of 1 mol/L NaOH aqueous solution, 100 µL of NZP standard solution (1000 µg/mL) was added. The solutions were then stored in an incubator set to 80 °C. After each test solution was neutralized with 1 mol/L HCl aqueous solution, time-dependent degradation over a 24 h (15, 30, and 45 min, 1, 2, 3, 4, and 24 h) was determined by LC/PDA measurements.
Isolation and Purification of NZP Degradation ProductForty milligrams of NZP were dissolved in 4 mL of methanol, and then 1 mol/L NaOH aqueous solution was added to obtain 40 mL solution and was stored at 80 °C for 3 d. To the resulting solution, purified water was added to bring the total volume to 80 mL, which was then purified using the solid-phase extraction of Oasis® MCX cartridge (4 sticks). Twenty milliliters of aliquot were processed in one Oasis® MCX cartridge. After loading the aliquot and washing with 5 mL of purified water, 5 mL of methanol was used for elution from the solid phase. All the eluted solutions were combined, and were evaporated in vacuo with an evaporator. The residual solvent was further nitrogen purged to isolate degradation product as a yellow liquid.
Structural Analysis of NZP Degradation ProductThe degradation product dissolved in methanol-d4 was subjected to NMR analyses (1H-NMR, 13C-NMR, correlation spectroscopy (COSY), heteronuclear multiple bond connectivity (HMBC), and nuclear Overhauser effect spectroscopy (NOESY)) for structural determination. The aliquot of the methanol-d4 solution of the degradation product used in the NMR measurements was further diluted with methanol to a definite concentration for the recording of UV spectra using LC/PDA and accurate molecular weight determination using LC/TOF-MS. The remaining methanol-d4 solution of the degradation product was again evaporated and the residue was dissolved in an HCl/diethyl ether solution (3 mL, 1 mol/L) to crystallize. The solution was dried by purging with nitrogen. The residue was re-dissolved in a small amount of hot water and methanol and allowed to stand in a refrigerator for recrystallization. The crystal structure was determined by XRC.
Davidson et al.14) has previously reported the NZP degradation behavior under acidic and basic conditions. Since our group has also reported the degradation behavior of NZP under acidic conditions,10) we decided to investigate this behavior under basic conditions. The preliminary experimental results revealed that the degradation behavior pathway was different than that reported by Davidson et al.14) (Fig. 1_(I)→(II)→(IV)). Therefore, we re-examined the degradation behavior of NZP under basic conditions.
The chemical reaction pathway from (I) to (IV) passing by (III) is the previously reported definite degradation pathway under acidic condition.10)
When NZP was stored in a sodium hydroxide solution (1 mol/L), a large peak (IV) of benzophenone (2-amino-5-nitrobenzophenone) appeared, while the peak of NZP decreased after 15 min, and a slightly smaller peak (V) appeared which was assumed to be another basic degradation product of NZP (Figs. 2, 3). As time progressed, the NZP basic degradation product peak (V) continued to gradually increase, while the peak (IV) reached its maximum after 30 min; however, the peak (IV) decreased and almost disappeared after 24 h (Figs. 2, 3). These results indicated that NZP degrades immediately in an aqueous sodium hydroxide solution, producing (IV) initially, which is then degraded to obtain (V) as the final degradation product.
NZP: nitrazepam, Peak (IV): a degradation product (2-amino-5-nitrobenzophenone) shown in Fig. 1, Peak (V): a degradation product ((2-hydroxy-5-nitrophenyl)-phenylmethanone) shown in Fig. 1.
(I): Nitrazepam, (IV): Intermediate degradation product (2-amino-5-nitrobenzophenone), (V): Final degradation product ((2-hydroxy-5-nitrophenyl)-phenylmethanone).
The NZP basic degradation production was obtained as a yellow liquid (35.8 mg; yield; 89.5%). The UV spectra of NZP (I) and the degradation product (V) were recorded using an LC/PDA. The NZP exhibited UV λmax peaks at 219, 255, and 311 nm (Supplementary Fig. S1), whereas the degradation product (V) showed UV λmax peaks at 255 and 311 nm (Supplementary Fig. S1). The λmax of the degradation product (V) was slightly different than that of NZP; however, their spectral patterns were nearly identical. Therefore, the basic skeleton of the degradation product (V), that is, the resonance structure derived from the double bond, thought to be similar to that of NZP.
The deprotonated molecules ([M–H]−) m/z 242.039 was determined from the high-resolution mass spectrum of the NZP degradation product (V) (Supplementary Fig. S2-(B)) measured using LC/TOF-MS. The chemical formula was estimated to be C13H9NO4, based on the nitrogen rule and index of hydrogen deficiency. Although the accurate mass (m/z 242.039) measured by LC/TOF-MS deviated from the exact mass (m/z 242.0459) calculated from the chemical formula by about m/z 0.0071 unit, this seemed to be within the error range due to instrumental performance. The identification itself was determined comprehensively by use of NMR and XRC as shown below, in addition to the mass data.
The 1H-NMR spectrum of isolated NZP degradation product (V) indicated the presence of a monosubstituted benzene ring [δ 7.78 (2H, d, J = 7.5 Hz), δ 7.66 (1H, t, J = 7.5 Hz), and δ 7.54 (2H, t, J = 6.9 Hz)], a 1,2,4-trisubstituted benzene ring [δ 8.35 (1H, s), δ 8.32 (1H, d, J = 9.0 Hz), and δ 7.08 (1H, d, J = 9.0 Hz)] (Supplementary Table S1). The number of protons was predicted to be 9 based on the high-resolution mass spectrum. Therefore, we inferred that the one-deficient hydrogen was derived from a hydroxy group. From the 13C-NMR spectrum of NZP degradation product, 13 SP2 carbons including a carbonyl carbon (δ 199.4 ppm) were assigned as shown in Supplementary Table S1. The COSY, HMBC, and NOESY correlations are presented in Supplementary Table S1. As a result of structural analyses, NZP degradation product (V) was confirmed to be 2-hydroxy-5-nitrobenzophenone (Fig. 4). The raw NMR spectra of the NZP degradation product (V) are shown in Supplementary Figs. S3–7.
Only the main correlations are indicated by arrows.
Furthermore, the result of XRC also supported the structure of (V) (Fig. 5). The crystallographic data were deposited with the Cambridge Crystallographic Data Center as supplementary publication No. CCDC 2288479.
We then investigated the degradation product (V) obtained in this study and that (II) of Davidson et al.14) The degradation pathway was also inferred. Although both degradations occurred under alkaline conditions, the basic environments were slightly different. In this study, a 1 mol/L NaOH solution was employed according to the conditions used for the alkaline degradation of flunitrazepam described in the Interviewform,12) whereas that employed in the study of Davidson et al. was 0.1 mol/L. Although both solutions are basic, the concentration employed here is much higher. Therefore, the degradation product (II) reported by Davidson et al. was considered an intermediate degradation product, and further degradation resulted in (IV) to obtain 2-hydroxy-5-nitrobenzophenone (V) as the final degradation product.
The influence of the treatment method of the solution after degradation was then investigated. Davidson et al. may have assumed at the outset that the degradation products were “water soluble,” since they employed prior to the GC/MS analysis, a liquid–liquid partition extraction adding chloroform to the degradation solution (alkaline solution) to remove coexisting compounds, such as NZP (I) and lipophilic 2-amino-5-nitrobenzophenone (IV). Therefore, if another degradation product that is easily soluble in chloroform was present in this alkaline degradation solution, it cannot be ruled out that this degradation product may have been removed by the liquid–liquid partitioning extraction. In fact, our degradation product (V) has a predicted Log P of 3.66 and is highly lipophilic. Consequently, a similar degradation product might have been obtained in the degradation experiments of Davidson et al., but was removed by the liquid–liquid partitioning and extraction procedure performed as a pretreatment step.
Considering these results, we proposed the following pathway for NZP degradation under basic conditions: NZP degrades immediately under basic conditions to form the intermediate degradation products (II) and 2-amino-5-nitrobenzophenone (IV), followed by further aromatic nucleophilic substitution reactions to form the final degradation product (V) (Fig. 6). A common aromatic nucleophilic substitution reaction occurs when the desorbing group is often a halogen and there is an electron-withdrawing group at the ortho or para position of the halogen. In structure (IV), there is a strong electron-withdrawing nitro group at the para position of the amino group and an electron-withdrawing carbonyl group at the ortho position, which suggests that an aromatic nucleophilic substitution reaction occurred, and the amino group was desorbed.
The final degradation product (V) was first reported by the authors as a substance obtained from the degradation of NZP, and there have been no reports of the compound (V) actually being detected in human postmortems.
In conclusion, this study investigated the degradation of NZP under alkaline conditions, where the structural analysis revealed that 2-hydroxy-5-nitrobenzophenone was the degradation product. The present results revise the findings of Davidson et al.,14) who reported that the alkali-catalyzed hydrolysis product of NZP is a ring-opened compound resulting from an amide bond scission. It was thus assumed that the basic degradation product (II) obtained by Davidson et al. was a degradation intermediate and not the final degradation product, 2-hydroxy-5-nitrobenzophenone, which we isolated and purified. When NZP is detected in the human stomach contents during autopsies in cases where NZP use is suspected, acidic degradation intermediates such as (2-amino-N-(2-benzoyl-4-nitrophenyl)-acetamide) and 2-amino-5-nitrobenzophenone might be detected. However, if bodies are left to decompose for a long period postmortem, the NZP basic degradation product 2-hydroxy-5-nitrobenzophenone, which was identified in this study, may be also a useful analyte in forensic analysis. Although in vitro experiments were employed in this study due to research ethics restrictions, we believe that in the future it will be necessary to conduct in vivo experiments (animal experiments) in order to carry out more reliable appraisals when detecting drugs from putrefied postmortem specimens.
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant No. 18K06609).
The authors declare no conflict of interest.
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