Determination of tramadol and metabolites by HPLC-FL and HPLC–MS/MS in urine of dogs

Tramadol is a centrally acting analgesic drug used in veterinary and human clinical practice. Its metabolism has been largely characterized in human being but is still long to be comprehended in several animal species, especially in the dog. The aim of the present study was to develop and validate a new analytical procedure to investigate HPLC the metabolization/elimination process tramadol in urine of dogs by HPLC-FL or HPLC–MS/MS. A single oral dose of tramadol (4 mg/kg) was administered to 4 male Beagle dogs and the urine was naturally collected. This matrix either hydrolyzed than un-hydrolyzed was extracted with different blends of solvents to detect the total or free form of the analytes, respectively.     

LC–MS/MS studies of ritonavir and its forced degradation products

Forced degradation of ritonavir (RTV), under the conditions of hydrolysis (acidic, basic and neutral), oxidation, photolysis and thermal stress as prescribed by ICH was studied using LC–MS/MS. Eight degradation products were formed and their separation was accomplished on Waters XTerra^® C₁₈ column (250 mm × 4.6 mm i.d., 5 μm) using water:methanol:acetonitrile as (40:20:40, v/v/v) mobile phase in an isocratic elution mode by LC. The method was extended to LC–MS/MS for characterization of the degradation products and the pathways of decomposition were proposed. No previous reports were found in the literature regarding the characterization of degradation products of ritonavir.     

Direct quantitation of glucoraphanin in dog and rat plasma by LC–MS/MS

A rapid method to quantify levels of the β-thioglycoside N-hydroxyl sulfate, glucoraphanin, in dog and rat plasma to support pre-clinical toxicological and pharmacological studies has been developed using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Glucoraphanin was extracted from plasma by protein precipitation with acetonitrile and separated via hydrophilic interaction liquid chromatography (HILIC) using a Luna 5 μm Silica (2) 100 Å column (50 mm× 2.0 mm) at a flow rate of 0.3 mL/min. Solvent A consisted of 200 mM ammonium acetate and formic acid (99:1, v/v) and Solvent B was acetonitrile. Initial conditions (90% Solvent B) were held for 0.01 min after injection, decreased to 40% in 0.5 min and held constant for 2.5 min, returning to initial conditions for 3 min (reequilibration time). Glucoraphanin was detected by MS/MS using a turbo ion spray interface as the ion source operating in negative ion mode. Acquisition was performed in multiple reaction monitoring mode at m/z 435.8 → 96.7. The method was validated for the calibration range 10–2000 ng/mL. Within- and between-run precision for the low, mid and high QC levels was 8% R.S.D. or less and accuracy ranged from 100 to 113%. The lower limit of quantification was 10 ng/mL; calibration curves encompassed the range of plasma concentrations expected to be found in bioavailability and pharmacokinetics studies with glucoraphanin. The method has successfully been applied to the determination of glucoraphanin in dog and rat plasma and should be extendable to other species as well.     

Using ambient mass spectrometry and LC–MS/MS for the rapid detection and identification of multiple illicit street drugs

In this study the recently developed technique of thermal desorption electrospray ionization/mass spectrometry (TD–ESI/MS) was applied to the rapid analysis of multiple controlled substances. With the reallocation of mass spectral resources [from a standard ESI source coupled with liquid chromatography (LC) to an ambient TD–ESI source], this direct-analysis technique allows the identification of a wider range of illicit drugs through a dual-working mode (pretreatment-free qualitative screening/conventional quantitative confirmation). Through 60-MRM (multiple reaction monitoring) analysis—in which the MS/MS process was programmed to sequentially scan 60 precursor ion/product ion transitions and, thereby, identify 30 compounds (two precursor/product ion transitions per compound)—of a four-component (drug) standard, the signal intensity ratios of each drug transition were comparable with those obtained through 8-MRM analysis, demonstrating the selectivity of TD–ESI/MS for the detection of multiple drugs. The consecutive analyses of tablets containing different active components occurred with no cross-contamination or interference from sample to sample, demonstrating the reliability of the TD–ESI/MS technique for rapid sampling (two samples min^?1). The active ingredients in seized drug materials could be detected even when they represented less than 2 mg g^?1 of the total sample weight, demonstrating the sensitivity of TD–ESI/MS. Combining the ability to rapidly identify multiple drugs with the “plug-and-play” design of the interchangeable ion source, TD–ESI/MS has great potential for use as a pretreatment-free qualitative screening tool for laboratories currently using LC–MS/MS techniques to analyze illicit drugs.     

Comparison of fused-core and conventional particle size columns by LC–MS/MS and UV: Application to pharmacokinetic study

The chromatographic performance of fused-core (superficially porous) HPLC packing materials was compared with conventional fully porous particle materials for LC–MS/MS analysis of two pharmaceuticals in rat plasma. Two commercially available antidepressants, imipramine and desipramine, were assayed using a conventional analytical C₁₈ column (5 μm, 2.0 mm × 30 mm) and a fused-core C₁₈ column (2.7 μm, 2.1 mm × 30 mm). Retention time, column efficiency, pressure drop, resolution, and loading capacity were compared under the same operating conditions. The fused-core column demonstrated reduced assay time by 34% and 2–3-fold increased efficiency (N). Loading capacity up to 25 μl of extract injected on column showed no peak distortion. The registered back-pressure from a flow rate of 1.0 ml/min did not exceed 3400 psi making it compatible with standard HPLC equipment (typically rated to 6000 psi). Two mobile phases were examined, and morpholine as an organic base modifier yielded a 2–5-fold increase in S/N near the limit of detection over triethylamine. The 2.7 μm fused-core column was applied to the analysis of imipramine and desipramine in extracted, protein precipitated rat plasma by LC–MS/MS. The calibration curves were linear in the concentration range of 0.5–1000 ng/ml for both imipramine and desipramine. Intra-run precisions (%CV) and accuracies (%bias) were within ±7.8% and ±7.3% at three QC levels and within 14.7% and 14.4% at the LOQ level for both analytes. Following a single method qualification run, the method was applied to the quantitation of pharmacokinetic study samples after oral administration of imipramine to male rats.     

HPLC–MS and HPLC–MS/MS analysis of seven active constituents of Xiao-Xu-Ming decoction and application to a pharmacokinetic study after oral administration to rat

Xiao-xu-ming decoction (XXMD) is a traditional Chinese medicine that has been widely used to treat theoplegia and its sequelae. This paper reports the development of three separate assays based on reversed phase high-performance liquid chromatography–mass spectrometry (HPLC–MS) and HPLC–MS/MS for the determination of seven active constituents of XXMD viz oroxylin A-7-O-glucuronide, wogonoside, liquiritigenin, cimifugin, 5-O-methylvisammiol, glycyrrhizic acid and glycyrrhetinic acid in rat plasma. All calibration curves were linear (r >0.99) with lower limits of quantitation (LLOQs)<12.4 ng/mL. Intra- and inter-day precisions (as relative standard deviation) were all <10.7% with recoveries in the range of 88.7–113%. In addition, the seven analytes were shown to be stable in rat plasma samples under relevant storage conditions. The validated methods were successfully applied to a pharmacokinetic study in rat after oral administration of XXMD.     

Separation and characterization of forced degradation products of abacavir sulphate by LC–MS/MS

Abacavir sulphate was subjected to forced degradation under the conditions of hydrolysis (acid, alkali and neutral), oxidation, photolysis and thermal stress as prescribed by ICH. Eight degradation products were formed and their separation was accomplished on Waters XTerra C₁₈ (250 mm × 4.6 mm, 5 μm) column using 20 mM ammonium acetate:acetonitrile as a mobile phase in gradient elution mode by LC. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. No previous reports were found in the literature regarding the degradation behavior of abacavir sulphate.     

LC–MS/MS determination of carbamathione in microdialysis samples from rat brain and plasma

A selective liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed for the determination of S-(N, N-diethylcarbamoyl) glutathione (carbamathione) in microdialysis samples from rat brain and plasma. S-(N, N-Diethylcarbamoyl) glutathione (carbamathione) is a metabolite of disulfiram. This metabolite may be responsible for disulfiram's effectiveness in the treatment of cocaine dependence. Chromatographic separations were carried out on an Alltech Altima C-18 (50 mm long × 2.1 mm i.d., 3 μm particles) analytical column at a flow rate of 0.3 ml/min. Solvent A consisted of 10 mM ammonium formate, methanol, and formic acid (99:1:0.06, v/v/v). Solvent B consisted of methanol, 10 mM ammonium formate and formic acid (99:1:0.06, v/v/v). A 20 min linear gradient from 95% aqueous to 95% organic was used. Tandem mass spectra were acquired on a Micromass Quattro Ultima “triple” quadrupole mass spectrometer equipped with an ESI interface. Quantitative mass spectrometric analysis was conducted in positive ion mode selected reaction monitoring (SRM) mode looking at the transition of m/z 407–100 and 175 for carbamathione and m/z 392–263 for the internal standard S-hexyl glutathione. The simultaneous collection of microdialysate from blood and brain was used to monitor carbamathione concentrations centrally and peripherally. Good linearity was obtained over a concentration range of 0.25–10,000 nM. The lowest limit of quantification (LLOQ) was determined to be 1 nM and the lowest limit of detection (LLOD) was calculated to be 0.25 nM. Intra- and inter-day accuracy and precision were determined and for all the samples evaluated, the variability was less that 10% (R.S.D.).     

Development and validation of an HPLC–MS/MS method to determine clopidogrel in human plasma

A quantitative method for clopidogrel using online-SPE tandem LC–MS/MS was developed and fully validated according to the well-established FDA guidelines. The method achieves adequate sensitivity for pharmacokinetic studies, with lower limit of quantifications (LLOQs) as low as 10 pg/mL. Chromatographic separations were performed on reversed phase columns Kromasil Eternity-2.5-C18-UHPLC for both methods. Positive electrospray ionization in multiple reaction monitoring (MRM) mode was employed for signal detection and a deuterated analogue (clopidogrel-d₄) was used as internal standard (IS). Adjustments in sample preparation, including introduction of an online-SPE system proved to be the most effective method to solve the analyte back-conversion in clinical samples. Pooled clinical samples (two levels) were prepared and successfully used as real-sample quality control (QC) in the validation of back-conversion testing under different conditions. The result showed that the real samples were stable in room temperature for 24 h. Linearity, precision, extraction recovery, matrix effect on spiked QC samples and stability tests on both spiked QCs and real sample QCs stored in different conditions met the acceptance criteria. This online-SPE method was successfully applied to a bioequivalence study of 75 mg single dose clopidogrel tablets in 48 healthy male subjects.Abbreviations: ESI, electrospray ionization; IS, internal standard; LC–MS/MS, liquid chromatography tandem mass spectrometry; LLOQ, lower limit of quantification; MRM, multiple reaction monitoring; SPE, solid phase extraction; QC, quality controlKEY WORDS: Clopidogrel, Online-SPE, LC–MS/MS, Back-conversion, Bioequivalence, Deuterated analogue, Real samples stability, FDA guidelines     

A novel LC–MS/MS assay for methylprednisolone in human plasma and its pharmacokinetic application

Methylprednisolone is a synthetic glucocorticoid. In our report, the authors proposed a sensitive and selective liquid chromatography/tandem mass spectrometry (LC–MS/MS) assay for the determination of methylprednisolone applying budesonide as internal standard. Liquid–liquid extraction (LLE) having tert–butyl methyl ether (TBME) have been employed to extract methylprednisolone from the plasma samples. Immediately after reconstitution, the samples were chromatographed on a C₁₈ column using an isocratic mobile phase composed of 10 mM ammonium formate buffer and acetonitrile (35:65, v/v). A flow rate of 1.00 ml/min was used to elute the analyte form the column. Analysis was carried out with an API–4000 LC–MS/MS instrument operated in multiple reaction-monitoring (MRM) mode. The linearity has been proven within the concentration range of 10.1–804 ng/ml in plasma samples. The precision (%CV) and accuracy results in five validation batches across five concentration levels were well within the acceptance limits. The drug was stable under different conditions. The particular assay has been proficiently put on pharmacokinetic study in healthy male subjects.     

Development of a linear dual column HPLC–MS/MS method and clinical genetic evaluation for tramadol and its phase I and II metabolites in oral fluid

Tramadol is a centrally acting synthetic opioid analgesic and has received special attention due to its abuse potential and unexpected responses induced by CYP2D6 polymorphism. Oral fluid is an advantageous biofluid for drug analysis due to non-invasive sampling and high correlation of drug concentrations with plasma. However, few studies have been performed on distribution of tramadol and its metabolites in oral fluid. In the present study, a linear dual column HPLC–MS/MS method was developed and fully validated for the simultaneous determination of tramadol and its phase I [O-desmethyltramadol (ODMT), N-desmethyltramadol (NDMT) and N,O-didesmethyltramadol (NODMT)] and II metabolites in oral fluid. Furthermore, the distribution of tramadol and its metabolites, in relation to CYP2D6 genetic variations, in oral fluid was investigated following a clinical study including 23 subjects with CYP2D6*wt/*wt, CYP2D6*10/*10 or CYP2D6*5/*5. The validation results of selectivity, matrix effect, linearity, precision and accuracy were satisfactory. Pharmacokinetic parameters, such as C_ss,max and AUC_0–τ of tramadol, NDMT and NODMT, in the CYP2D6*10/*10 group were significantly higher than those in the CYP2D6*wt/*wt group. Moreover, the ratios of ODMT/tramadol, NDMT/tramadol and NODMT/NDMT correlated well with the CYP2D6 genotypes. We demonstrated that oral fluid is a promising biofluid for pharmacokinetic evaluation in relation to genetic variations.     

LC–MS/MS method for simultaneous analysis of cladrin and equol in rat plasma and its application in pharmacokinetics study of cladrin

A rapid and selective LC–MS/MS method for simultaneous analysis of cladrin and equol in female rat plasma has been developed and validated. The chromatographic separation was carried out on RP18 column, and MS/MS analysis was performed in triple quadrupole mass spectrometer with electrospray ionization. The method was linear for the concentration range from 7.8 to 1000 ng/ml for cladrin and 3.9 to 1000 ng/ml for equol. The intra-day and inter-day accuracy and precision of the method were within the acceptable limits. The validated LC–MS/MS method was successfully applied for the pharmacokinetics study of cladrin at 10 mg/kg in female S.D. rats.     

LC–MS/MS assay for olanzapine in human plasma and its application to a bioequivalence study

This paper describes a selective and sensitive assay for the determination of olanzapine (OLZ) in human plasma based on liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and quetiapine as internal standard (IS) were extracted from 200 μL plasma via solid phase extraction on Waters Oasis HLB cartridges. Chromatographic separation was achieved on an ACE 5C18-300 column (100 mm×4.6 mm, 5 μm) under isocratic conditions in a run time of 3.5 min. Mass spectrometric detection involved electrospray ionization in the positive ion mode followed by multiple reaction monitoring (MRM) of the transitions at m/z 313/256 for OLZ and m/z 384/253 for the IS. The assay was linear in the range 0.10–40.0 ng/mL with a lower limit of quantitation and limit of detection of 0.10 and 0.012 ng/mL, respectively. Intra- and inter-day precision (as coefficient of variation) and relative recovery were <5.0% and >90%, respectively. The method was successfully applied to a bioequivalence study of 5 and 10 mg OLZ disintegrating tablets in 40 healthy Indian males with reproducibility by incurred sample reanalysis in the range ?7.43 to 8.07%.     

An accurate,rapid and sensitive determination of tramadol and its active metabolite O-desmethyltramadol in human plasma by LC–MS/MS

A rapid, sensitive and accurate liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for the simultaneous determination of tramadol and its active metabolite, O-desmethyltramadol in human plasma is developed using propranolol as internal standard (IS). The analytes and IS were extracted from 200 μL aliquots of human plasma via protein precipitation using acetonitrile. Chromatographic separation was achieved in a run time of 2.0 min on an Aquasil C18 (100 mm × 2.1 mm, 5 μm) column under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The method was fully validated for its selectivity, sensitivity, linearity, precision and accuracy, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. A linear dynamic range was established from 1.0 to 600.0 ng/mL for tramadol and 0.5–300.0 ng/mL for O-desmethyltramadol. The method was successfully applied to a bioequivalence study of 200 mg tramadol tablet formulation in 27 healthy Indian male subjects under fasting condition.     

Determination of oxaceprol in rat plasma by LC–MS/MS and its application in a pharmacokinetic study

A sensitive method for the quantification of oxaceprol in rat plasma using high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. Sample pretreatment involved a simple protein precipitation by the addition of 60 μL of acetonitrile–methanol (1:2, v/v) to 20 μL plasma sample volume. Separation was achieved on a Dikma ODS-C18 (5 μm, 150 mm × 4.6 mm) reversed-phase column at 40 °C with acetonitrile/0.1% formic acid–4 mM ammonium acetate in water (35:65,v/v) at a flow rate of 0.6 mL/min. Detection was performed using an electrospray ionization (ESI) operating in negative ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 172 → 130 (oxaceprol) and m/z 153 → 109 (protocatechuic acid, internal standard). The calibration curve of oxaceprol in plasma showed good linearity over the concentration range of 1.25–800 ng/mL. The limit of detection and limit of quantification were 0.400 ng/mL and 1.25 ng/mL, respectively. Intra- and inter-day precisions in all samples were within 15%. There was no matrix effect. The validated method was successfully applied to a preclinical pharmacokinetic study of oxaceprol in rats. After oral administration of 20 mg/kg oxaceprol to rats, the main pharmacokinetic parameters T_max, C_max, T_1/2, V_z/F and AUC_0–t were 1.4 h, 1.2 μg/mL, 2.3 h, 19.7 L/kg and 3.4 mg h/L, respectively.     

Analysis of angiotensin II receptor antagonist and protein markers at microliter level plasma by LC–MS/MS

An analytical method based on a green approach is proposed for clinical analysis. The proposed procedure involves the reduction of the sample preparation steps, the amounts of reagents and organic solvents. This simple and sensitive method for the analysis of clinical drug and biomarkers in human plasma was developed using LC connected to tandem mass spectrometry (LC–MS/MS) with a nanospray ion source. In this study, the desired drug and proteins were separated on a 5 and 10 cm RP C18 nano-flow column. Undesired polar substances in human plasma were washed out by using ACN:1% FA = 20:80 (v/v) as the loading mobile phase for drug analysis and good linearity was attainable. Only a small volume of human plasma (10 μL) was utilized for the monitoring of drug plasma concentration and significant proteins under clinical studies. All the sample preparation procedures and the analytical scheme were at microliter level. This strategy would lower the consumption of reagents and organic solvents and make a contribution toward the goal of reduction of pollution from analytical methods in general.     

Determination of cepharanthine in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

Context: Cepharanthine (CPA) has been reported to possess a wide range of pharmacological activities.

Objective: This study investigates the pharmacokinetic characteristics after oral or intravenous administration of CPA by using a sensitive and rapid LC–MS/MS method.

Materials and methods: A sensitive and rapid LC–MS/MS method was developed for the determination of CPA in Sprague–Dawley rat plasma. Twelve rats were equally randomized into two groups, including the intravenous group (1?mg/kg) and the oral group (10?mg/kg). Blood samples (250?μL) were collected at designated time points and determined using this method. The pharmacokinetic parameters were calculated.

Results: The calibration curve was linear within the range of 0.1–200?ng/mL (r?=?0.999) with the lower limit of quantification at 0.1?ng/mL. After 1?mg/kg intravenous injection, the concentration of CPA reached a maximum of 153.17?±?16.18?ng/mL and the t_1/2 was 6.76?±?1.21?h. After oral administration of 10?mg/kg of CPA, CPA was not readily absorbed and reached C_max 46.89?±?5.25?ng/mL at approximately 2.67?h. The t_1/2 was 11.02?±?1.32?h. The absolute bioavailability of CPA by oral route was 5.65?±?0.35%, and the bioavailability was poor.

Discussion and conclusions: The results indicate that the bioavailability of CPA was poor in rats, and further research should be conducted to investigate the reason for its poor bioavailability and address this problem.     

Simultaneous Determination of Atorvastatin and Aspirin in Human Plasma by LC–MS/MS: Its Pharmacokinetic Application

A simple, rapid, and sensitive liquid chromatography tandem mass spectro-metric (LC–MS/MS) assay method has been developed and fully validated for the simultaneous quantification of atorvastatin and aspirin in human plasma using a polarity switch. Proguanil and furosemide were used as the internal standards for the quantification of atorvastatin and aspirin, respectively. The analytes were extracted from human plasma by the liquid–liquid extraction technique using methyl tert-butyl ether. The reconstituted samples were chromatographed on a Zorbax XDB Phenyl column by using a mixture of 0.2% acetic acid buffer, methanol, and acetonitrile (20:16:64, v/v) as the mobile phase at a flow rate of 0.8 mL/min. Prior to detection, atorvastatin and aspirin were ionized using an ESI source in the multiple reaction monitoring (MRM) mode. The ions were monitored at the positive m/z 559.2→440.0 transition for atorvastatin and the negative m/z 179.0→136.6 transition for aspirin. The calibration curve obtained was linear (r² ≥ 0.99) over the concentration range of 0.20–151 ng/mL for atorvastatin and 15.0–3000 ng/mL for aspirin. The method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 3.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The proposed method was found to be applicable to clinical studies.     

Development and validation of an LC–MS/MS method for quantification of NC-8 in rat plasma and its application to pharmacokinetic studies

ent-16-Oxobeyeran-19-N-methylureido (NC-8) is a recently synthesized derivative of isosteviol that showed anti-hepatitis B virus (HBV) activity by disturbing replication and gene expression of the HBV and by inhibiting the host toll-like receptor 2/nuclear factor-κB signaling pathway. To study its pharmacokinetics as a part of the drug development process, a highly sensitive, rapid, and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for determining NC-8 in rat plasma. After protein precipitation extraction, the chromatographic separation of the analyte and internal standard (IS; diclofenac sodium) was performed on a reverse-phase Luna C₁₈ column coupled with a Quattro Ultima triple quadruple mass spectrometer in the multiple-reaction monitoring mode using the transitions, m/z 347.31 → 75.09 for NC-8 and m/z 295.89 → 214.06 for the IS. The lower limit of quantitation was 0.5 ng/mL. The linear scope of the standard curve was between 0.5 and 500 ng/mL. Both the precision (coefficient of variation; %) and accuracy (relative error; %) were within acceptable criteria of <15%. Recoveries ranged from 104% to 113.4%, and the matrix effects (absolute) were non-significant (CV ≤ 6%). The validated method was successfully applied to investigate the pharmacokinetics of NC-8 in male Sprague–Dawley rats. The present methodology provides an analytical means to better understand the preliminary pharmacokinetics of NC-8 for investigations on further drug development.     

Identification and characterization of degradation products of irbesartan using LC–MS/TOF,MS, on-line H/D exchange and LC–NMR

Irbesartan was subjected to hydrolytic, oxidative, photolytic and thermal stress, according to ICH guideline Q1A (R2). The drug showed degradation only in acidic, basic and photoacidic conditions, while it was stable to other stress conditions. A total of three degradation products were formed, which were separated on a C-8 column employing a gradient HPLC method. Initially, a complete mass fragmentation pathway of the drug was established with the help of MS/TOF, MSⁿ and H/D exchange studies. Subsequently, the degradation products were subjected to LC–MS/TOF and on-line H/D exchange mass studies to obtain their accurate mass, fragment pattern and number of labile hydrogens. The MS results helped to assign tentative structures to degradation products, which were verified through ¹H and 2D COSY LC–NMR experiments. The products were identified as (2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methanamine, 1-(1-((2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methylamino)pentylideneamino)cyclopentane carboxylic acid and 2-butyl-3-(tetrazolo[1,5-f]phenanthridin-6-ylmethyl)-1,3-diazaspiro[4.4]non-1-en-4-one. The structures were justified by mechanisms of their formation.