Quantitative analysis of bilobalide and ginkgolides from Ginkgo biloba leaves and Ginkgo products using (1)H-NMR

1H-NMR spectrometry was applied to the quantitative analysis of the bilobalide, ginkgolides A, B, and C in Ginkgo biloba leaves and six kinds of commercial Ginkgo products without any chromatographic purification. The experiment was performed by the analysis of each singlet H-12, which were well separated in the range of delta 6.0-7.0 in the (1)H-NMR spectrum. However, the H-12 protons of bilobalide and ginkgolides may have overlapped with H-6 or H-8 protons of the Ginkgo flavonoids. Therefore, the optimum (1)H-NMR solvent for the analysis of the compound was selected through the evaluation of solvent effects on the resolution of these signals from the compounds. Acetone-d(6)-benzene-d(6) (50 : 50) was found to be the best one among the solvents evaluated. The quantity of the compounds was calculated by the relative ratio of the intensity of each compound to the known amount of internal standard (25 microgram), phloroglucinol. This method allows rapid and simple quantitation of underivatized bilobalide and ginkgolides in 5 min without any pre-purification steps.     

Direct Quantitation of Phytocannabinoids by One-Dimensional 1H qNMR and Two-Dimensional 1H-1H COSY qNMR in Complex Natural Mixtures

The widespread use of phytocannabinoids or cannabis extracts as ingredients in numerous types of products, in combination with the legal restrictions on THC content, has created a need for the development of new, rapid, and universal analytical methods for their quantitation that ideally could be applied without separation and standards. Based on previously described qNMR studies, we developed an expanded ¹H qNMR method and a novel 2D-COSY qNMR method for the rapid quantitation of ten major phytocannabinoids in cannabis plant extracts and cannabis-based products. The ¹H qNMR method was successfully developed for the quantitation of cannabidiol (CBD), cannabidiolic acid (CBDA), cannabinol (CBN), cannabichromene (CBC), cannabichromenic acid (CBCA), cannabigerol (CBG), cannabigerolic acid (CBGA), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabielsoin (CBE), and cannabidivarin (CBDV). Moreover, cannabidivarinic acid (CBDVA) and Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA) can be distinguished from CBDA and Δ9-THCA respectively, while cannabigerovarin (CBGV) and Δ8-tetrahydrocannabivarin (Δ8-THCV) present the same ¹H-spectra as CBG and Δ8-THC, respectively. The COSY qNMR method was applied for the quantitation of CBD, CBDA, CBN, CBG/CBGA, and THC/THCA. The two methods were applied for the analysis of hemp plants; cannabis extracts; edible cannabis medium-chain triglycerides (MCT); and hemp seed oils and cosmetic products with cannabinoids. The ¹H-NMR method does not require the use of reference compounds, and it requires only a short time for analysis. However, complex extracts in ¹H-NMR may have a lot of signals, and quantitation with this method is often hampered by peak overlap, with 2D NMR providing a solution to this obstacle. The most important advantage of the COSY NMR quantitation method was the determination of the legality of cannabis plants, extracts, and edible oils based on their THC/THCA content, particularly in the cases of some samples for which the determination of THC/THCA content by ¹H qNMR was not feasible.     

Quantitative analysis of camptothecin derivatives in Nothapodytes foetida using 1H-NMR method

A quantitative analysis using (1)H-NMR has been developed for the determination of camptothecin derivatives and trigonelline in Nothapodytes foetida root, stems and leaves. In the region of delta 9.5-5.5, the signals of H-7 of camptothecin (1), H-10 of 9-methoxycamptothecin (2), H-19 of pumiloside (3) and H-2 of trigonelline (4), were well separated from each other in DMSO-d(6). The quantity of the compounds was calculated by the ratio of the intensity of each compound to the known amount of internal standard 3,4,5-trimethoxybenzaldehyde. These results were compared with the conventional HPLC method. The advantages of the method are that no reference compounds are required for calibration curves, the quantification could be directly realized on a crude extract, an overall profile of the preparation could be directly obtained, and a very significant time-gain could be achieved, in comparison to conventional HPLC methods, for instance.     

Metabolic fingerprinting of Ephedra species using 1H-NMR spectroscopy and principal component analysis

The metabolomic analysis of Ephedra species was performed using 1H-NMR spectroscopy and multivariate data analysis. A broad range of metabolites could be detected by 1H-NMR spectroscopy without any chromatographic separation. The principal component analysis used to reduce the huge data set obtained from the 1H-NMR spectra of the plant extracts clearly discriminated three different Ephedra species. The major differences in Ephedra sinica, Ephedra intermedia and Ephedra distachya var. distachya were found to be due to benzoic acid analogues in the aqueous fraction and ephedrine-type alkaloids in the organic fraction. Based on this metabolomic recognition, one of nine commercial Ephedra materials evaluated was shown to be a mixture of Ephedra species. This method will be a useful tool for chemotaxonomic analysis and authentification of Ephedra species including quality control of plant materials.     

A high-sensitivity ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) method for screening synthetic cannabinoids and other drugs of abuse in urine

The continuing emergence of designer drugs imposes high demands on the scope and sensitivity of toxicological drug screening procedures. An ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) method was developed for screening and simultaneous confirmation of both designer drugs and other drugs of abuse in urine samples in a single run. The method covered selected synthetic cannabinoids and cathinones, amphetamines, natural cannabinoids, opioids, cocaine and other important drugs of abuse, together with their main urinary metabolites. The database consisted of 277 compounds with molecular formula and exact monoisotopic mass; retention time was included for 192 compounds, and primary and secondary qualifier ion exact mass for 191 and 95 compounds, respectively. Following a solid-phase extraction, separation was performed by UHPLC and mass analysis by HR-TOFMS. MS, and broad-band collision-induced dissociation data were acquired at m/z range 50–700. Compound identification was based on a reverse database search with acceptance criteria for retention time, precursor ion mass accuracy, isotopic pattern and abundance of qualifier ions. Mass resolving power in spiked urine samples was on average FWHM 23,500 and mass accuracy 0.3 mDa. The mean and median cut-off concentrations determined for 75 compounds were 4.2 and 1 ng/mL, respectively. The range of cut-off concentrations for synthetic cannabinoids was 0.2–60 ng/mL and for cathinones 0.7–15 ng/mL. The method proved to combine high sensitivity and a wide scope in a manner not previously reported in drugs of abuse screening. The method’s feasibility was demonstrated with 50 authentic urine samples.

二盐酸氟哌噻吨几何异构体的核磁共振研究

采用COSY、NOESY、HSQC、HMBC二维核磁共振技术，首次对二盐酸氟哌噻吨的两种几何异构体的^1H-NMR、^13C-NMR谱信号进行全归属。归属结果表明，二盐酸氟哌噻吨两种几何异构体的部分^1H-NMR、^13C-NMR谱数据差别较大，可作为区分两种异构体的一个指标。     

Identification of potential cannalis containing substance metabolites: nuclear resonance and mass spectroscopic studies on side chain hydroxylation of cannabinoids

The 100-MHz-NMR-spectra of 11 side chain hydroxylated derivatives of cannabidiol ( 1 ), Δ⁶-tetrahydrocannabinol ( 2 ) and cannabinol ( 3 ) were analysed for signals specific for the position of hydroxyl groups. The mass spectra of these compounds and their trimethylsilyl ethers were investigated at ionisation voltages between 70 and 10 eV. The mass spectra of the non-silylated compounds showed no similarities independent on the basic type of cannabinoid but in the case of their trimethylsilyl ethers it was possible to derive fragmentations specific for the site of hydroxylation. These data are presented as a general method for the identification of small quantities of side chain hydroxylated in vitro and in vivo metabolites of cannabinoids.     

Analysis of 30 synthetic cannabinoids in serum by liquid chromatography‐electrospray ionization tandem mass spectrometry after liquid‐liquid extraction

The analysis of synthetic cannabinoids in human matrices is of particular importance in the fields of forensic and clinical toxicology since cannabis users partly shift to the consumption of ‘herbal mixtures’ as a legal alternative to cannabis products in order to circumvent drug testing. However, comprehensive methods covering the majority of synthetic cannabinoids already identified on the drug market are still lacking. In this article, we present a fully validated method for the analysis of 30 synthetic cannabinoids in human serum utilizing liquid‐liquid extraction and liquid chromatography‐electrospray ionization tandem mass spectrometry. The method proved to be suitable for the quantification of 27 substances. The limits of detection ranged from 0.01 to 2.0 ng/mL, whereas the lower limits of quantification were in the range from 0.1 to 2.0 ng/mL. The presented method was successfully applied to 833 authentic serum samples during routine analysis between August 2011 and January 2012. A total of 227 (27%) samples was tested positive for at least one of the following synthetic cannabinoids: JWH‐018, JWH‐019, JWH‐073, JWH‐081, JWH‐122, JWH‐200, JWH‐203, JWH‐210, JWH‐307, AM‐2201 and RCS‐4. The most prevalent compounds in positive samples were JWH‐210 (80%), JWH‐122 (63%) as well as AM‐2201 (29%). Median serum concentrations were all below 1.0 ng/mL. These findings demonstrate a significant shift of the market of synthetic cannabinoids towards substances featuring a higher CB₁ binding affinity and clearly emphasize that the analysis of synthetic cannabinoids in serum or blood samples requires highly sensitive analytical methods covering a wide spectrum of substances. Copyright © 2012 John Wiley & Sons, Ltd.     

13C-NMR. Spectra of Natural and Semi-synthetic Cannabinoids

The ¹³C-NMR. spectra of one natural and ten semi-synthetic cannabinoids were analyzed in detail. Assignments of the signals are based on their chemical shifts, splitting patterns in ¹H-off-resonance decoupling experiments and comparison with ¹³C-NMR. data of related cannabinoids. With some compounds final assignments were made by selective ¹H-decoupling experiments and incremental calculations.     

Isolation and purification of ginkgo flavonol glycosides from Ginkgo biloba leaves by high-speed counter-current chromatography

A high-speed counter-current chromatography method was developed for the separation and purification of bioactive flavonol glycosides from a crude ethanol extract of Ginkgo biloba leaves. The separation was performed with a two-phase solvent system composed of n-hexane-butanol-ethyl acetate-methanol-0.5% acetic acid (1:0.5:3.5:1:4, v/v) and three pure compounds were eluted in high purities in a one-step separation. Their purities were determined by HPLC and identified by MS,(1)H-NMR, and(13)C-NMR.     

Synthesis of Asymmetric Curcumin Analogues from CullilawanOil using Conventional and Microwave Method

Asymmetric curcumin analogues as potential anticancer compounds. The purpose of this study was to synthesize product 5-benzo[1,3]dioxol-5-yl-1-phenyl-penta-2,4-dien-1-one uses the method conventional and microwaves. Product 5-benzo[1,3]dioxol-5-yl-1-phenyl-penta-2,4-dien-1-one can be synthesized from the cullilawan oils with several stages, among others; isolation safrole, isomerization, oxidation and condensation reactions. Isolation of safrole from cullilawanoils performed using NaOH solution and purified using distillation fractionation pressure reduction produces 19.30% safrole are tested for purity by GCMS and for the identification of the structure is done by using FTIR and ¹H-NMR. The safrole isomerization performed using KOH without solvent at a temperature of 120^oC for 8 hours resulted isosafrole (91.53%) which consists of cis-isosafrol and trans-isosafrol. Oxidation isosafrole performed using KMnO₄ in acidic conditions using a phase transfer catalyst tween 80 at a temperature of < 30^oC and separation by silica gel resulted in 65.63% piperonal were tested with the GCMS and identification using FTIR and ¹H-NMR. Products asymmetrical curcumin analogous made in the condition alkaline by conventional methods for three hours produce 99.55%, a method of microwaves in 140 watts for two minutes produce 82.82%.     

Analysis of nitrophenols and other polar nitroaromatic compounds in ammunition wastewater by high-field proton nuclear magnetic resonance (1H-NMR) spectroscopy and chromatographic methods

 It is shown that by using high-field proton nuclear magnetic resonance (¹H-NMR) spectroscopy it is possible, without prior separation, to analyse nitrophenols and other acidic nitroaromatic compounds in the “pH 2 extract” of ammunition wastewater. The ¹H-NMR chemical shifts data of a variety of reference compounds are presented. Two groundwater samples from the former ammunition plant in Elsnig (Saxony) were analysed by ¹H-NMR and also by chromatographic methods (GC/MS, HPLC). The results are compared and discussed. Received: 17 January 1996/Revised: 15 May 1996/Accepted: 17 May 1996     

Analysis of nitrophenols and other polar nitroaromatic compounds in ammunition wastewater by high-field proton nuclear magnetic resonance (1H-NMR) spectroscopy and chromatographic methods

 It is shown that by using high-field proton nuclear magnetic resonance (¹H-NMR) spectroscopy it is possible, without prior separation, to analyse nitrophenols and other acidic nitroaromatic compounds in the “pH 2 extract” of ammunition wastewater. The ¹H-NMR chemical shifts data of a variety of reference compounds are presented. Two groundwater samples from the former ammunition plant in Elsnig (Saxony) were analysed by ¹H-NMR and also by chromatographic methods (GC/MS, HPLC). The results are compared and discussed. Received: 17 January 1996/Revised: 15 May 1996/Accepted: 17 May 1996     

Optimisation and characterisation of marihuana extracts obtained by supercritical fluid extraction and focused ultrasound extraction and retention time locking GC‐MS

The optimisation of focused ultrasound extraction and supercritical fluid extraction of volatile oils and cannabinoids from marihuana has been accomplished by experimental design approach. On the one hand, the focused ultrasound extraction method of volatile compounds and cannabinoids was studied based on the optimisation of cyclohexane and isopropanol solvent mixtures, and the instrumental variables. The optimal working conditions were finally fixed at isopropanol/cyclohexane 1:1 mixture, cycles (3 s^?1), amplitude (80%) and sonication time (5 min). On the other hand, the supercritical fluid extraction method was optimised in order to obtain a deterpenation of the plant and a subsequent cannabinoid extraction. For this purpose, pressure, temperature, flow and co‐solvent percentage were optimised and the optimal working conditions were set at 100 bar, 35°C, 1 mL/min, no co‐solvent for the terpenes and 20% of ethanol for the cannabinoids. Based on the retention time locking GC‐MS analysis of the supercritical fluid extracts the classification of the samples according to the type of plant, the growing area and season was attained. Finally, three monoterpenes and three cannabinoids were quantified in the ranges of 0.006–6.2 μg/g and 0.96–324 mg/g, respectively.     

Simultaneous determination of theophylline and caffeine by proton magnetic resonance spectroscopy using partial least squares regression techniques.

A 1H-NMR procedure based on an analysis of its data by a multivariate calibration method was conducted for the simultaneous determination of theophylline and caffeine in synthetic and real samples. Partial least squares regression (PLS) was chosen as the calibration method. The methyl signals of theophilline at 3.36 and 3.54 ppm that overlapped with those of caffeine were significant characteristics which were employed in this study for their analyses. The proposed method was successfully applied to recovery studies of theophylline and caffeine from real tablet samples.     

Purification and anticancer activity investigation of pentacyclic triterpenoids from the leaves of Sinojackia sarcocarpa L.Q. Luo by high-speed counter-current chromatography

Sinojackia sarcocarpa L.Q. Luo has high aesthetic value, which is clinically used to treat diseases such as the variations of arthritis, thromboangiitis obliterans, angina pectoris as well as many other diseases. Pentacyclic triterpenoids are the main pharmacological effective compounds. High-speed counter-current chromatography method was performed on a Midi-DE centrifuge at 25°C. The solvent system was n-hexane-ethylacetate-methanol-water (10:5:3:1, v/v). Peak fractions were collected according to the elution profile for subsequent high-performance liquid chromatography analysis. The structure identification was performed by ultraviolet, infrared, mass spectrometry, (1)H-NMR and (13)C-NMR. Compound 2α,3α,19β,23β-tetrahydroxyurs-12-en-28-oic acid and 2α,3α,23β-trihydroxyurs-12-en-28-oic acid were purified from the plant of Styracaceaen genius for the first time, whose purities were 96.57% and 97.33%, respectively. Compared with the same dose of oral 5-fluorouracil with 57.6% inhibition rate, the S(180) tumour inhibition rates of 20 mg kg(-1)d(-1) two compounds were 59.5% and 48.9%, respectively.     

Conversion Characteristics of Some Major Cannabinoids from Hemp (Cannabis sativa L.) Raw Materials by New Rapid Simultaneous Analysis Method

This study was carried out to develop a high-performance liquid chromatography method for short-time analysis of the main cannabinoids in the inflorescence of hemp (Cannabis sativa L.). We also performed decarboxylation of the raw material using our advanced analysis technique. In this study, the UV spectrum was considered to analyze each of the four common cannabinoids, solvents, and samples, where the uniform elution of acidic cannabinoids without peak tailing and acids was tested. Optimal results were obtained when readings were taken at a wavelength of 220 nm using water and methanol containing trifluoroacetic acid as mobile phases in a solvent gradient system. The established conditions were further validated by system suitability, linearity, precision, detection limit, and quantitation limit tests. The decarboxylation index (DT₅₀) confirmed that Δ9-THCA decarboxylated faster than CBDA, and both maintained a linear relationship with time and temperature. In addition, the loss of cannabidiol was better prevented during the decarboxylation process in the natural state than in the extracted state.     

以甘-谷二肽为构筑单元的扇形树状分子的合成与表征

以天然氨基酸为结构单元的树枝状化合物已有不少报道．例如，Denkewalter等首次报道了以氨基酸（L-赖氨酸）为结构单元的1～10代树枝状高分子的合成．Chapman等采用发散法合成了核心为聚氧乙烯（PEO）长链的树枝状t-Boc-聚（α，ε-L赖氨酸）．Kim等报道了两种具有光活性的由缬氨酸或亮氨酸组成的小肽树状分子．Tam等合成了一系列具有各种潜在生物医学用途的肽链结构树枝状分子．     

Extraction and analysis of different Cannabis samples by headspace solid-phase microextraction combined with gas chromatography-mass spectrometry

A headspace solid-phase microextraction combined with GC-MS method was developed for the extraction and analysis of cannabinoids from Cannabis samples. Different commercially available fibres were evaluated; polydimethylsiloxane 100 microm was selected as the most efficient one. In order to enhance sensitivity and reduce analysis time, the sampling temperature was studied and it showed that extraction should be performed at a high temperature (150 degrees C). In relation with the high lipophilicity of cannabinoids, a relatively long desorption time (3 min) was necessary to ensure a total transfer from the fibre into the injection port of the gas chromatograph. The method was finally applied to the extraction of Swiss marijuana samples from different regions. Data treatment by principal component analysis and hierarchical cluster analysis allowed a discrimination of the different batches.