Investigation of deep eutectic solvents as additives to ß-CD for enantiomeric separations of Zopiclone, Salbutamol, and Amlodipine by CE.

The enantioseparation of chiral drugs via CE was first investigated using ß-CD as chiral additive and deep eutectic solvents (DESs) as auxiliary additive. The results showed that improved separation of tested chiral drugs was obtained in the presence of DESs and ß-CD compared to the single ß-CD separation system. With the optimized condition, resolutions of DESs applied ß-CD separation system for rac-Zopiclone, rac-Salbutamol, and rac-Amlodipine increased 3-4.2 times as single ß-CD separation system. The resolutions reached 4.74, 6.37, and 9.67, respectively. The results demonstrate that DESs are viable additives to CD system in CE for the separation of the chiral drugs.

Microwave-assisted radiosynthesis of [(18) F]ASEM, a radiolabeled α7-nicotinic acetylcholine receptor antagonist.

An improvement of the original radiochemical synthesis of [(18) F]ASEM, an α7-nicotinic acetylcholinergic receptor radioligand, is reported. The new procedure utilizes microwave-assisted radiofluorination. In addition, a new preparative HPLC method was developed to eliminate a chemical impurity in the final product. Quality control procedures were also enhanced to improve detection of product with enhanced resolution of potential impurities. [(18) F]ASEM was produced in 20.1 ± 8.9% non-decay corrected (NDC) yield with an average synthesis time of 57 min and an average specific radioactivity of 856 ± 332 GBq/µmol (23 ± 9 Ci/µmol).

Detection and quantification of benzodiazepines and Z-drugs in human whole blood, plasma, and serum samples as part of a comprehensive multi-analyte LC-MS/MS approach.

For the first time, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) multi-analyte approach based on a simple liquid-liquid extraction was developed and validated for fast target screening and quantification of benzodiazepines and Z-drugs in case of driving ability and crime responsibility in the three most important biosamples whole blood, plasma, and serum. Whole blood, plasma, and serum (500 µL each) were extracted twice at pH 7.4 and at pH 10 with ether/ethyl acetate (1:1). Separation, detection, and quantification were performed using LC-MS/MS with electrospray ionization in positive mode. The method was validated with respect to selectivity, ion suppression/enhancement of co-eluting analytes, matrix effects, recovery, process efficiency, accuracy and precision, stabilities, and limits of detection and quantification. For accuracy and precision, full calibration was performed with ranges from subtherapeutic to toxic concentrations. The presented LC-MS/MS approach as part of a universal multi-analyte concept for over 100 drugs was applicable for selective detection as well as accurate and precise quantification in whole blood, plasma, and serum. The approach was selective, sensitive, accurate, and precise for 16 of the 19 tested drugs in whole blood, 18 in plasma, and 17 in serum. Only semiquantitative results could be obtained for zopiclone because of its instability in all tested biosamples.

Liquid chromatography-high resolution/ high accuracy (tandem) mass spectrometry-based identification of in vivo generated metabolites of the selective androgen receptor modulator ACP-105 for doping control purposes.

Selective androgen receptor modulators (SARMs) represent an emerging class of therapeutics which have been prohibited in sport as anabolic agents according to the regulations of the World Anti-Doping Agency (WADA) since 2008. Within the past three years, numerous adverse analytical findings with SARMs in routine doping control samples have been reported despite missing clinical approval of these substances. Hence, preventive doping research concerning the metabolism and elimination of new therapeutic entities of the class of SARMs are vital for efficient and timely sports drug testing programs as banned compounds are most efficiently screened when viable targets (for example, characteristic metabolites) are identified. In the present study, the metabolism of ACP-105, a novel SARM drug candidate, was studied in vivo in rats. Following oral administration, urine samples were collected over a period of seven days and analyzed for metabolic products by Liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry. Samples were subjected to enzymatic hydrolysis prior to liquid-liquid extraction and a total of seven major phase-I metabolites were detected, three of which were attributed to monohydroxylated and four to bishydroxylated ACP-105. The hydroxylation sites were assigned by means of diagnostic product ions and respective dissociation pathways of the analytes following positive or negative ionization and collisional activation as well as selective chemical derivatization. The identified metabolites were used as target compounds to investigate their traceability in a rat elimination urine samples study and monohydroxylated and bishydroxylated species were detectable for up to four and six days post-administration, respectively.

Enantioselective analysis of zopiclone in rat brain by liquid chromatography tandem mass spectrometry.

A new high-performance liquid chromatographic method with triple quadrupole mass spectrometry detection was developed and validated for the quantification of zopiclone enantiomers in rat brain samples. Zopiclone enantiomers were resolved on a CHIRALPAK AD column with a mobile phase consisting of acetonitrile/ethanol/methanol (60:20:20, v/v/v) at a flow rate of 1.3 mL min(-1). Moclobemide was used as internal standard. The sample treatment procedure was carried out employing solid-phase extraction, yielding mean absolute recoveries of 89.6 and 91.7% for each zopiclone enantiomer. The validated method showed linearity in the range of 0.29-344.8 ng g(-1), with quantification limits of 0.29 ng g(-1) for both enantiomers. Precision and accuracy were within acceptable levels of confidence (<15%). The method was applied in a pilot study of zopiclone kinetic disposition in rats. It could be observed that the levels of (+)-(S)-zopiclone were always higher than those of (-)-(R)-zopiclone, confirming the stereoselective disposition of zopiclone.

A novel validated ultra-performance liquid chromatography Method for separation of eszopiclone impurities and its degradants in drug products.

A selective, specific, and sensitive ultra-performance LC (UPLC) method was developed for determination of eszopiclone and its degradation products. The chromatographic separation was performed with a Waters ACQUITY UPLC system and BEH C18 column using gradient elution with mobile phases A and B. Mobile phase A was 0.01 M phosphate buffer with 0.2% (w/v) 1-octane sulfonic acid sodium salt as an ion pair reagent, adjusted pH 2.2 with orthophosphoric acid-acetonitrile (85 + 15, v/v). Mobile phase B was pH 2.2 buffer-acetonitrile (20 + 80, v/v). UV detection was performed at 303 nm. Eszopiclone and its impurities were chromatographed with a total run time of 13 min. A calibration study showed that the response for each of the impurities A, B, C, and D was linear between concentrations of 0.02 and 7.2 microg/mL (r2 > or = 0.999). The method was validated over this range for precision, intermediate precision, accuracy, linearity, and specificity. For the precision study, RSD of each impurity was <5% (n = 6). The method was found to be precise, accurate, linear, and specific. The proposed method was successfully used for determination of eszopiclone impurities in pharmaceutical preparations.

Capillary electrophoretic enantioselective determination of zopiclone and its impurities.

A capillary electrophoretic enantioselective method with UV detection was developed and validated for the simultaneous quantification of zopiclone enantiomers and its impurities, zopiclone-N-oxide enantiomers, and 2-amino-5-chloropyridine, in tablets. The analytes were extracted from the tablets using ACN and were separated in an uncoated fused-silica capillary (50 µm, 42 cm effective length, 50 cm total length) using 80 mM sodium phosphate buffer pH 2.5 and 5 mM carboxymethyl-ß-cyclodextrin as running buffer. The analytes and the internal standard (trimethoprim) were detected at 305 and 200 nm, respectively. A voltage of 27 kV was applied and the capillary temperature was maintained at 25°C. All enantiomers were analyzed within 8 min and linear calibration curves over the concentration range of 0.4-0.8 mg mL⁻¹ for each zopiclone enantiomer, 0.8-1.6 µg mL⁻¹ for 2-amino-5-chloropyridine and 0.4-0.8 µg mL⁻¹ for each zopiclone-N-oxide enantiomer were obtained. The coefficients of correlation obtained for the linear curves were greater than 0.99. The intra-day and inter-day accuracy and precision were lower than 2% for all analytes. This validated method was employed to study the degradation and racemization of zopiclone under stress conditions. This application demonstrated the importance of a stability-indicating assay method for this drug.

Direct, metal-free amination of heterocyclic amides/ureas with NH-heterocycles and N-substituted anilines in POCl3.

A POCl(3)-mediated, direct amination reaction of heterocyclic amides/ureas with NH-heterocycles or N-substituted anilines is described. Compared to the existing methods, this operationally simple protocol provides unique reactivity and functional group compatibility because of the metal-free, acidic reaction conditions. The yields are generally excellent.

Enantiomeric Separation and Thermodynamic Investigation of (R)-N-Tert-Butoxy Carbonyl-Piperidine-3-Carboxylic Acid Hydrazide, a Key Starting Material of Zidebactam.

A new selective, accurate and precise chiral high-performance liquid chromatography method for the separation of (R)-N-tert-butoxy carbonyl-piperidine-3-carboxylic acid hydrazide (RE) and its enantiomer was developed. RE is a key starting material of novel ß-lactam enhancer drug Zidebactam. Chiral resolution of more than 10 was achieved on Chiralpak IA column using mobile phase consisting of n-hexane, ethanol in the ratio of 70:30, v/v. The flow rate of the mobile phase was 1.0 mL min-1 and the column oven temperature was 30°C. Detection was carried out at 225 nm. The developed method was validated as per the International Conference on Harmonization guideline. Limit of detection and limit of quantification of the enantiomeric impurity (S)-N-tert-butoxy carbonyl-piperidine-3-carboxylic acid hydrazide (SE) was 2.5 and 7.5 µg mL-1, respectively. Mean recovery of the SE was 96.83 ± 1.4%. The effect of thermodynamic parameters on the chiral separation was evaluated.

[The present study situation and application prospect of nail analysis for abused drugs].

In forensic toxicology analysis, various types of biological samples have their own special characteristics and scope of applications. In this article, the physiological structure of nails, methods for collecting and pre-processing samples, and for analyzing some poisons and drugs in the nails are reviewed with details. This paper introduces the influence factors of drug abuse of the nails. The prospects of its further applications are concluded based on the research results. Nails, as an unconventional bio-sample without general application, show great potential and advantages in forensic toxicology.

Distribution of zopiclone and main metabolites in hair following a single dose.

In forensic investigations, such as drug-facilitated crimes, reference values are useful for interpretation of hair results. The aim of this study was to establish levels of zopiclone and two main metabolites, N-desmethylzopiclone and zopiclone N-oxide, in hair after the administration of a single dose of zopiclone, as very limited data are published. A controlled study was performed, where 16 volunteers consumed either 5 or 10mg zopiclone. Hair was sampled prior to consumption and 14, 30, 60, and 120 days after intake. The deposition of drug in hair segments of all sampling time points was followed in small hair segments of 5-mm, using a validated ultra-high performance liquid chromatography-tandem mass spectrometry method. In all participants, hair segments corresponding to the time of intake were positive for zopiclone, but also with lower concentrations in the neighbouring segments. The highest zopiclone concentrations were detected in samples collected 30 or 60 days after intake. For all sampling time points maximum values for the 5-mg dose ranged from 5.0-370pg/mg for zopiclone and 5.4 to 300pg/mg for N-desmethylzopiclone, where the maximum values for the 10-mg dose ranged from 17 to 590pg/mg for zopiclone and 25-410pg/mg for N-desmethylzopiclone for all sampling time points. No significant difference in concentrations was found between the two dosing groups for either zopiclone or N-desmethylzopiclone. Almost half of the participants showed lower levels 14 days after intake than in the later sampling time points. The metabolite to parent drug ratio of N-desmethylzopiclone to zopiclone varied from 0.6 to 3.4 (median=1.2) for the maximum levels of all sampling time points. N-desmethylzopiclone are suggested to serve as an additional marker to confirm the intake of zopiclone. Traces of zopiclone N-oxide were detected in hair from only eight participants. This study showed, that it was possible to follow zopiclone and N-desmethylzopiclone in hair for 4 months even though the drugs was divided into several segments in the latest collected hair samples, and no obvious wash-out effect between the sampling time points by e.g. personal hygiene could be discerned because the cumulated amount at each sampling time point was similar. We conclude that the analysis of short segments e.g. segments of 5-mm can help determine the time of a single intake of zopiclone and that obtaining a sample 1-2 months after a drug exposure provide the best conditions to detect and interpret the results.

Screen the Effective Components of Lycopodii herba on Rheumatoid Arthritis with the Aid of Spectrum-Effect Relationship and Uncover its Potential Mechanism.

Lycopodii herba (SJC), a traditional Chinese medicine, has the effect of dispelling wind and eliminating dampness (a therapeutic principle and method of traditional Chinese medicine for rheumatoid arthritis), relaxing tendon and activating collaterals. However, the major effective components and its therapeutic mechanism were unclear. In this study, different SJC samples with slightly different compositions were prepared by extracting with different concentrations of ethanol. Then, the therapeutic effects on rheumatoid arthritis (RA) of different SJC samples were evaluated. Finally, the spectrum-effect relationship between UPLC-Q-TOF/MS fingerprints and the effect of RA was explored to screen the effective components. Western blotting was used to study the potential mechanism. The volume of hind paw and the level of RF, TNF-α, and IL-1ß were lower after administrating with different SJC samples, compared with the model group. Histopathological findings also confirmed that SJC could relieve the symptoms of RA. Combined with identification of the components in plasm from SJC, lycojaponicumin C, des-N-methyl-α-obscurine, 8ß-acetoxy-12ß-hydroxy-lycopodine or 8ß-acetoxy-11α-hydroxy-lycopodine or 8ß-hydroxy-11α-acetoxylycopodine were considered to be the major effective components. The mechanism may be related to AChE/NF-κB signaling pathway. This work provides a general method to screen the potential effective components of herb medicines and would be benefit to understand the mechanism of SJC for the treatment of RA.

New otonecine-type pyrrolizidine alkaloid from Petasites japonicus.

One new otonecine-type pyrrolizidine alkaloid secopetasitenine (1), along with petasitenine (fukinotoxine, 2), neopetasitenine (3), and senkirkine (4), was isolated from the whole plant of Petasites japonicus. The structure of 1 was determined by spectroscopic analyses and chemical conversion from the known alkaloid petasitenine (2).

Detection Time of Oxazepam and Zopiclone in Urine and Oral Fluid after Experimental Oral Dosing.

Data from previous experimental studies on the detection time of oxazepam and zopiclone in biological matrices are limited. The aim of this study was to examine the detection time in urine and oral fluid after single oral doses of oxazepam and zopiclone. Ten healthy volunteers received 25 mg of oxazepam in the evening of Day 1 and 7.5 mg of zopiclone in the evening of Day 3. Urine and oral fluid samples were collected twice daily for 9 days, with an additional sampling the day after ingestion of zopiclone. A total of 19 samples of both urine and oral fluid from each participant were analyzed using fully validated chromatographic methods. The median detection time for oxazepam was 91 h (range 73-108) in urine and 67 h (range 50-98) in oral fluid. The median detection time for zopiclone in urine was 49 h (range 25-98) and 59 h (range 48-146) in oral fluid. The metabolite zopiclone N-oxide showed a detection time of 36 h (range 25-84) in urine. The area under the concentration-time curve (AUCTotal) in urine corrected for creatinine was 150 µmol/L/mmol/L*h (range 105-216) for oxazepam and 1.60 µmol/L/mmol/L*h (range 0.79-4.53) for zopiclone. In oral fluid, the AUCtotal was 673 nmol/L*h (range 339-1,316) for oxazepam and 2,150 nmol/L*h (range 493-4,240) for zopiclone. In conclusion, oxazepam can be detected longer in urine than in oral fluid, while zopiclone can be detected longer in oral fluid than in urine. The high AUCTotal for zopiclone in oral fluid shows that the transfer into oral fluid is significant. In certain individuals the detection time of zopiclone in oral fluid is long. These results can be helpful when interpreting drug testing analyzes.

Segmental Hair Analysis-Interpretation of the Time of Drug Intake in Two Patients Undergoing Drug Treatment.

The present study involved segmental testing of hair in two clinical cases with known dosage histories. Hair analysis confirmed the first patient's exposure to the prescribed sertraline and citalopram for several months. Citalopram was generally distributed along the hair shaft in accordance with the drug ingestion period. By contrast, "false" positive results were observed for sertraline in distal hair segments, corresponding to a period of no sertraline exposure, which may indicate incorporation from sweat or sebum, which transport the drugs along the hair surface. The second patient received various drugs during her treatment for brain cancer. Metoclopramide, morphine, oxazepam, paracetamol, sumatriptan, tramadol, and zopiclone, which had been part of the therapy, were all detected in the proximal hair segment. The results of these two cases indicated that results-especially concerning the time of drug intake-must be interpreted with caution and allow for the possibility of incorporation from sweat or sebum.

Zopiclone concentrations in oral fluid and blood after, administration of therapeutic doses of zopiclone.

PURPOSE: Little is known about the relationship between concentrations in oral fluid (OF) and blood for the widely prescribed hypnotic drug zopiclone. The purpose of this study was to investigate the usefulness of OF zopiclone concentrations to predict blood zopiclone concentrations in order to introduce OF testing as an alternative to more cumbersome blood testing. METHODS: 16 healthy young male volunteers received capsules of either 5 or 10mg zopiclone on two different study days separated by at least one week. Blood and OF were collected simultaneously at baseline and 9 times after intake of zopiclone on each study day. In addition an OF sample was collected 24-81h after intake. Lunch was served between samples taken 2.5 and 3.5h after intake. All samples were analysed for zopiclone, and the cut-off was 10ng/ml in blood and 0.2ng/ml in OF-buffer mixture. RESULTS: Zopiclone was detected in all OF samples during the study day. After 24-81h, all subjects were also positive for zopiclone in OF, except from three subjects ingesting the 5mg dose. In a single case zopiclone was detected in a baseline OF sample 14days after intake on an earlier study day. Zopiclone was detected in both OF and blood in 231 OF/blood pairs, and a significant but weak correlation between OF and blood concentration was seen (R2 of 0.30). The median (range) zopiclone OF/blood concentration ratio (ZOBCR) for all samples were 3.3 (0.8-18). The ZOBCR decreased when the OF volume increased. After 30 of 31 given doses of zopiclone, the ZOBCR was higher in samples collected before lunch than samples collected after lunch. DISCUSSION: Vast intra- and interindividual differences in ZOBCR were found, and the correlation between OF and blood concentration was less pronounced than reported in former studies. In accordance with earlier studies we found a negative correlation between ZOBCR and OF volume. The ZOBCR decreases in relation to recent intake of a meal, probably because stimulated saliva production causes "dilution" of saliva. OF zopiclone concentration appeared unsuitable for estimation of blood zopiclone concentration. Due to long detection time, analysis of zopiclone in OF might be useful to detect non-recent, previous intake.

Enantioselective determination of (R)-zopiclone and (S)-zopiclone (eszopiclone) in human hair by micropulverized extraction and chiral liquid chromatography/high resolution mass spectrometry.

Zopiclone and its (S)-enantiomer (eszopiclone) are commonly prescribed for insomnia. Despite the high demand for enantioselective differentiation, the chiral analysis of zopiclone in hair has not been reported. In this study, a method for the enantioselective quantification of zopiclone in human hair was developed. The extraction medium and duration were optimized using real eszopiclone-positive hair samples. Specifically, micropulverized extraction with 3.0M ammonium phosphate buffer (pH 8.4) involving salting-out assisted liquid-liquid extraction with acetonitrile was utilized to minimize the degradation of zopiclone and for rapid and facile operation. On the other hand, recovery of the conventional solid-liquid extraction involved overnight soaking in 3.0M ammonium phosphate buffer (pH 8.4) was only 0.58±0.12% of the maximum recovery achieved by the present method due to the decomposition in the phosphate buffer. An excellent chiral separation (Rs=5.0) was achieved using a chiral stationary phase comprising cellulose tris(3,5-dichlorophenylcarbamate) and a volatile mobile phase of 10mM ammonium carbonate (pH 8.0)-acetonitrile (25:75, v/v). Detection was carried out using liquid chromatography/high resolution mass spectrometry (LC/HRMS) with electrospray ionization. A Q Exactive mass spectrometer equipped with a quadrupole-Orbitrap analyzer was used for detection. The concentration of 0.50pg/mg was defined as the lowest limit of quantification using 5mg of hair sample. Using the developed approach, the concentration of eszopiclone in hair after a single 2-mg dose was found to be 441pg/mg, which was higher than all the reported values regarding a single administration of zopiclone. After daily administration of racemic zopiclone (3.75mg/day), the concentrations of (R)-enantiomer and (S)-enantiomer in the black hair were 5.30-8.31ng/mg and 7.96-12.8ng/mg, respectively, and the concentration of the (S)-enantiomer was always higher than that of the (R)-enantiomer due to the enantioselective difference in the pharmacokinetics.

First derivative spectrofluorimetric determination of zopiclone and its degradation product, 2-amino-5-chloropyridine, in pharmaceutical formulations with preliminary tool in biological fluids for clinical evidence of zopiclone intake.

A simple, fast, sensitive and stability-indicating derivative spectrofluorimetric method is presented for the assay of zopiclone (ZOP), a drug with hypnotic effect, and its main degradation product and major contaminant, 2-amino-5-chloropyridine (ACP). The method is based on measuring the inherent fluorescence intensity of both drugs at λex=300nm in methanol, then differentiation using D1 (first derivative technique). The developed method was found to be rectilinear over a range of 0.2-4µg/mL of ZOP and 4-100ng/mL of ACP. The limits of detection were 0.05µg/mL of ZOP and 0.2ng/mL of ACP with the limit of quantitation of 0.17µg/mL of ZOP and 0.7ng/mL of ACP. The outcoming results of the proposed method were compared to those obtained by a reference method showing no significant statistical difference between them concerning precision and accuracy. Additionally, the developed method was applied for detecting ACP in spiked human urine and plasma specimens as a tool of clinical evidence of zopiclone intake that can be easily implemented in forensic laboratories. The proposed method was validated as per ICH guidelines.

Quantitative determination of zopiclone and its impurity by four different spectrophotometric methods.

Four simple, sensitive and selective spectrophotometric methods are presented for determination of Zopiclone (ZPC) and its impurity, one of its degradation products, namely; 2-amino-5-chloropyridine (ACP). Method A is a dual wavelength spectrophotometry; where two wavelengths (252 and 301 nm for ZPC, and 238 and 261 nm for ACP) were selected for each component in such a way that difference in absorbance is zero for the second one. Method B is isoabsorptive ratio method by combining the isoabsorptive point (259.8 nm) in the ratio spectrum using ACP as a divisor and the ratio difference for a single step determination of both components. Method C is third derivative (D(3)) spectrophotometric method which allows determination of both ZPC at 283.6 nm and ACP at 251.6 nm without interference of each other. Method D is based on measuring the peak amplitude of the first derivative of the ratio spectra (DD(1)) at 263.2 nm for ZPC and 252 nm for ACP. The suggested methods were validated according to ICH guidelines and can be applied for routine analysis in quality control laboratories. Statistical analysis of the results obtained from the proposed methods and those obtained from the reported method has been carried out revealing high accuracy and good precision.

A new and fast DLLME-CE method for the enantioselective analysis of zopiclone and its active metabolite after fungal biotransformation.

Zopiclone (ZO) is a chiral drug that undergoes extensive metabolism to N-desmethylzopiclone (N-Des-ZO) and zopiclone-N-oxide (N-Ox-ZO). Pharmacological studies have shown (S)-N-Des-ZO metabolite presents anxiolytic activity and a patent for this metabolite was requested for anxiety treatment and related disorders. In this context, biotransformation employing fungi may be a promising strategy to obtain N-Des-ZO. To perform the biotransformation study in this work, an enantioselective method based on capillary electrophoresis (CE) and dispersive liquid-liquid microextraction (DLLME) was developed. CE analyses were carried out in sodium phosphate buffer (pH 2.5; 50mmolL(-1)) containing 0.5% (w/v) carboxymethyl-ß-CD, at a constant voltage of +25kV. DLLME was conducted using 2mL of liquid culture medium pH 9.5. Chloroform (100µL) and methanol (300µL) were employed as extraction and disperser solvent, respectively. After CE and DLLME optimization, the analytical method was fully validated. The method was linear over a concentration range of 90-6000ngmL(-1) for each ZO enantiomer (r>0.999) and 50-1000ngmL(-1) for each N-Des-ZO enantiomer (r>0.998). Absolute recovery of 51 and 82% was achieved for N-Des-ZO and ZO, respectively. The accuracy and precision results agreed with the EMA (European Medicines Agency) guideline, and so did the stability study. Application of the developed method in a biotransformation study was conducted in order to investigate the ability of fungi, belonging to the genus Cunninghamella, in metabolizing ZO chiral drug. Fungi Cunninghamella elegans ATCC 10028B and Cunninghamella echinulata var elegans ATCC 8688A demonstrated to be able to enantioselectively biotransform ZO to its active metabolite, N-Des-ZO. Therefore, the proposed goals of this work, i.e. a fast DLLME-CE method and an outstanding strategy to obtain N-Des-ZO, were successfully attained.