[Rapid extraction and detection of five alkaloids in dried khat by solvent extraction-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry].

Khat is a common plant that grows primarily in Eastern Africa and the Arabian Peninsula. Cathinone, norpseudoephedrine, and norephedrine are the main psychoactive components of khat. Experimental studies have shown that red and green khat have similar cathinone contents, but green khat contains more norpseudoephedrine and norephedrine than red khat. Research indicates that Ethiopians believe that red khat has stronger psychoactive effects than green khat. Therefore, we speculated that other substances in red khat may enhance its psychoactive effects. Using the sampling method, we identified two other psychoactive components in khat: methcathinone and ethcathinone. At present, only a few studies on the extraction and detection of alkaloids from khat have been published in China, and no reports on the extraction and detection of methcathinone and ethcathinone from khat are available. In this study, we established an extraction and detection method for five alkaloids in dried khat using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF MS). To establish the extraction method, we optimized the extraction solvent and process. The amounts of dichloromethane and sodium hydroxide added during the purification step were also optimized. To establish the detection method, we optimized the chromatographic and MS conditions. The final extraction and detection method was as follows: Dried khat powder (0.1 g) was loaded into a polypropylene centrifuge tube, added with 1 mL of 0.05 mol/L hydrochloride aqueous solution, and vortex-oscillated for 3 min for extraction. The sample was centrifuged at 10000 r/min for 3 min. Next, 600 µL of the supernatant was placed in a centrifuge tube, added with 1 mL of dichloromethane, shaken for 1 min, and centrifuged at 10000 r/min for 3 min. Subsequently, 300 µL of the supernatant was placed in a centrifuge tube, added with 80 µL of 1 mol/L sodium hydroxide aqueous solution, shaken for 1 min, and added with 1 mL of acetonitrile. Vortex oscillation was performed for 2 min to extract the sample, after which solid sodium chloride (0.4 g) was added to the mixture, followed by shaking for 1 min to separate the acetonitrile and aqueous phases. The mixture was then centrifuged at 10000 r/min for 3 min. Finally, the supernatant was collected and diluted for further testing. The five target analytes were separated on a ZORBAX Eclipse Plus Phenyl-Hexyl column (100 mm×3.0 mm, 1.8 µm) via gradient elution using 0.1% acetic acid aqueous solution and acetonitrile as mobile phases with a flow rate of 0.3 mL/min and column temperature of 30 ℃. The analytes were identified using the targeted MS/MS method under positive electrospray ionization mode and quantified using the external standard method. The five alkaloids showed good correlations (all correlation coefficients (r2)≥0.9976) with their respective linear ranges. The limits of detection were between 0.08 and 0.75 µg/L, and the limits of quantification were between 0.25 and 2.50 µg/L. The average recoveries of the five alkaloids from two plants with different alkaloid contents were between 90.7% and 105.2%. The intra-sample precision ranged from 0.5% to 2.3%, the intra-day precision was between 1.0% and 2.5%, and the inter-day precision was between 1.3% and 3.3%. Using the developed method, we extracted and analyzed 15 khat samples, and detected five alkaloids. This method enables rapid sample pretreatment and has high sensitivity, good stability, and suitable accuracy. Based on the above results, we conclude that the proposed method meets the inspection and identification requirements for khat. Thus, it can provide a valuable reference for the physical and chemical identification of khat and support for further studies on its psychoactive components.

Desorption Electrospray Ionization Mass Spectrometry Imaging Illustrates the Quality Characters of Isatidis Radix.

For a long history, herbal medicines have made significant contributions to human health all around the world. However, the exploration of an effective approach to illustrate their inner quality remains a challenge. So, it is imperative to develop new methods and technologies to characterize and identify quality markers of herbal medicines. Taking Isatidis Radix, the dried root of Isatis indigotica as an example, desorption electrospray ionization (DESI), in combination with quadrupole-time-of-flight mass spectrometry (Q-TOF/MS), was applied in this work for the first time to reveal the comprehensive spatial distribution of metabolites and, further, to illustrate quality characters of this herbal medicine. After simple pretreatment, 102 metabolites including alkaloids, sulfur-containing compounds, phenylpropanoids, nucleosides, amino acids, organic acids, flavonoids, phenols, terpenes, saccharides, peptides, and sphingolipids were characterized, some of which were successfully localized and visualized in the transverse section of the root. Based on the ion images, samples with different quality characters were distinguished unambiguously by the pattern recognition method of orthogonal partial least squares discrimination analysis (OPLS-DA). Simultaneously, 11 major influencing components exerting higher ion intensities in superior samples were identified as the potential quality markers of Isatidis Radix. Desorption electrospray ionization (DESI) mass spectrometry imaging (MSI), together with chemometric analysis could not only improve the understanding of the plant biology of herbal medicines but also be beneficial in the identification of quality markers, so as to carry out better quality control of herbal medicines.

[Analysis of differences between unifloral honeys from different botanical origins based on non-targeted metabolomics by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry].

Different nectar plants contain various secondary metabolites. Herein, the differences in the contents of endogenous metabolites in honeys from eight botanical origins (i. e., acacia, jujube, vitex, linden, buckwheat, manuka, wolfberry, and motherwort honeys) were investigated by a non-targeted metabolomics-based method. This method involved solid-phase extraction pretreatment and ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MSE). An oasis HLB cartridge was used for the removal of many saccharides. Chromatographic experiments were performed on an HSS T3 column (100 mm×2.1 mm, 1.8 µm) using a mobile phase that consisted of 0.1% (v/v) formic acid in acetonitrile and water. Mass spectrometry was conducted in the positive and negative modes by electrospray ionization (ESI). Metabolic information about the honeys from different botanical origins was acquired using a multivariate statistical analysis model. Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) were conducted for pattern recognition and difference analysis. PCA was performed for 10557 and 2706 data variables in the positive and negative ion modes, respectively. The distribution of honeys from different botanical origins was investigated in 88 honey samples. The three principal components exhibited 48.05% and 57.88% of the total variance in positive and negative ion modes, respectively. The samples studied were divided into six different groups on the basis of their botanical origins and metabolic compounds: linden, vitex, buckwheat, manuka, jujube, and acacia honeys. A permutation test (n=200) was conducted to verify the fit of the model. The differential metabolites were screened on the basis of variable importance in project (VIP; >1), analysis of variance (ANOVA; p<0.05), and maximum fold change (>1.5) by using the PLS-DA model. The compounds were identified based on the data retrieved from the Chemspider and HMDB databases according to the quality information of precursor ions and fragment ions. Thirty-two differential metabolites were screened and primarily identified according to the characteristic fragmentation rules of specific structure types and data retrieval, including 18 flavonoids, 7 phenolic acids, 6 phenyl and terpenoid glycosides, and 1 steroid. Various flavonoids in buckwheat and manuka honeys, such as quercetin, sakuranetin, 7-hydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one, 5,7-dihydroxy-2-(3-methoxyphenyl)-4H-chromen-4-one, luteolin-7-methyl ether, and pollenitin, were found. In buckwheat honey, the contents of 3-methoxy-2-(4-methylbenzoyl)-4H-chromen-4-one, 2-hydroxy-3,4-diphenylpentanedioic acid, 3'-methoxydihydroformononetin, phenylpyruvic acid, 2-O-p-coumaroyltartronic acid, 2-(3-hydroxy-4,5-dimethoxyphenyl)-4H-chromen-4-one, 7-hydroxy-6-methoxy-3-(4-methoxyphenyl)-4H-chromen-4-one, 4-[(2E)-3-(4-hydroxyphenyl)prop-2-en-1-yl]-3-methoxyphenol, and 7-hydroxy-5-methoxyflavan were the highest; these compounds are the characteristic metabolites of buckwheat honey. In addition, manuka honey possessed the highest contents of gnaphaliin and galangin 3-methyl ether. Moreover, linden honey contained the characteristic phenyl glycosides of (S)-multifidol 2-[apiosyl-(1➝6)-glucoside], 2-phenylethyl-ß-D-glucopyranoside, benzyl O-[arabinofuranosyl-(1➝6)-glucoside], crosatoside B, and terpenoid glycosides of isopentyl gentiobioside and 6-O-oleuropeoylsucrose. Vitex honey was found to be rich in quinic acid derivatives such as caffeoyl-3-O-feruloyl-quinic acid/1-feruloyl-5-caffeoylquinic acid, 3-O-caffeoyl-4-O-methyl-quinic acid/3-feruloylquinic acid, and 3-O-caffeoyl-1-O-methyl-quinic acid, in addition to the flavonoids of vitexin, namely, 6″-(3-hydroxy-3-methylglutarate) and apigenin-7-[galactosyl-(1➝4)-mannoside]. Moreover, ponasteroside A was a characteristic marker of jujube honey, and the contents of 6-C-fucosylluteolin and kaempferol 3-(2″-rhamnosylrutinoside) were the highest in acacia honey. In conclusion, the method based on non-targeted metabolomics involving UPLC-Q-TOF-MSE for different unifloral honeys was found to be fast, effective, specific, and accurate. The differences in metabolite contents and the characteristic compounds in various unifloral honeys were preliminarily illustrated. This study provides an effective analytical strategy for honey traceability and quality analysis of unifloral honey.

Triacylglycerols and Fatty Acid Compositions of Cucumber, Tomato, Pumpkin, and Carrot Seed Oils by Ultra-Performance Convergence Chromatography Combined with Quadrupole Time-of-Flight Mass Spectrometry.

The triacylglycerol (TAG) compositions of cucumber, tomato, pumpkin, and carrot seed oils were analyzed using ultra-performance convergence chromatography (UPC2) combined with quadrupole time-of-flight mass spectrometry (Q-TOF MS). A total of 36, 42, 39, and 27 different TAGs were characterized based on their Q-TOF MS accurate molecular weight and MS2 fragment ion profiles in the cucumber, tomato, pumpkin, and carrot seed oils, respectively. Generally, different vegetable seed oils had different TAGs compositions. Among the identified fatty acids, linoleic acid was the most abundant fatty acid in cucumber, tomato, and pumpkin seed oils and the second most abundant in carrot seed oil with relative concentrations of 54.48, 48.69, 45.10, and 15.92 g/100 g total fatty acids, respectively. Oleic acid has the highest concentration in carrot seed oil and the second highest in cucumber, tomato, and pumpkin seed oils, with relative concentrations of 78.97, 18.57, 27.16, and 33.39 g/100 g total fatty acids, respectively. The chemical compositions of TAGs and fatty acids could promote understanding about the chemical profiles of certain vegetable seed oils, thus improving the potential ability to select appropriate oils with specific functions and a high nutritional value and then develop functional foods in the future.

Qualitative Analysis of Fried Semen Strychni Seeds by Ultra-high Performance Liquid Chromatographycombined with Quadrupole Time-of-flight Mass Spectrometry / 中国药学杂志

OBJECTIVE: To identify the main chemical constituents in the extract of deep fried strychnas by UPLC-Q-TOF-MS. METHODS: Agilent SB-C18(2.1 mm×100 mm, 1.8 μm) column was used. Gradient elution was conducted with mobile phase consisting of 0.1% formic acid solution (A) and acetonitrile (B)at a flow rate of 0.2 mL•min-1. The column temperature was maitained at 35 ℃. MS analysis was based on information associated mode (IDA) that positive and negative ions were respectively collected. RESULTS: A total of 31 compounds were identified in deep fried strychnas, of which eight had not been reported as Semen Strychni,and four new compounds were found in the positive and negative ion mode. The main chemical constituents included alkaloids, organic acids, glycosides, etc. CONCLUSION: The method is accurate, reliable, and efficient, and is suitable for rapid identification of the ingredients in deep fried strychnas, which provides a reference for the development and utilization of processed products of Semen Strychni and clarification of its efficacy and material basis.

Triacylglycerol compositions of sunflower, corn and soybean oils examined with supercritical CO2 ultra-performance convergence chromatography combined with quadrupole time-of-flight mass spectrometry.

A supercritical CO2 ultra-performance convergence chromatography (UPC2) system was utilized with a quadrupole time-of-flight mass spectrometry (Q-TOF MS) to examine the triacylglycerol compositions of sunflower, corn and soybean oils. UPC2 provided an excellent resolution and separation for the triacylglycerols, while the high performance Q-TOF MS system was able to provide the molecular weight and fragment ions information for triacylglycerol compound characterization. A total of 33 triacylglycerols were identified based on their elementary compositions and MS2 fragment ion profiles, and their levels in the three oils were estimated. The combination of UPC2 and Q-TOF MS may determine triacylglycerol compositions for oils and fats, and provide sn-position information for fatty acids, which may be important for food nutritional value and stability.

Hydrophilic interaction chromatographic analysis of anthocyanins.

Hydrophilic interaction chromatography (HILIC) provides an alternative separation mode for the analysis of phenolic compounds, in which aqueous-organic mobile phases with polar stationary phases are used. This paper reports the evaluation of HILIC for the analysis of the natural pigments anthocyanins, which are of importance because of their chromophoric properties and a range of health benefits associated with their consumption. Several HILIC stationary phases (silica, diol, amine, cyanopropyl and amide) and mobile phase combinations were evaluated, with the latter proving particularly important due to the distinctive chromatographic behaviour of anthocyanins. Diode array detection was used for selective detection of anthocyanins, while high resolution quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) was used for compound identification. The potential of HILIC separation is demonstrated for a range of anthocyanins varying in glycosylation and acylation patterns found in blueberries, grape skins, black beans, red cabbage and red radish. HILIC is shown to be a complementary separation method to reversed phase liquid chromatography (RP-LC) due to the alternative retention mechanism.