Electrospray Ionization Mass Spectrometry Method Research Articles

Integrated Multiplatform Analysis and Separation of Thirteen Flavonoids and Anthraquinones in Seven Medicinal Cassia Species.

Cassia (Family: Fabaceae) species are a large group of flowering plants rich in bioactive anthraquinone and flavonoids used in botanical supplements and nutraceuticals. A simple and reliable high-performance liquid chromatography-photodiode array (HPLC-PDA) method was developed and validated for separating and quantifying 13 anthraquinone and flavonoids. These compounds were further confirmed using an LC-based electrospray ionization mass spectrometry (ESI-MS/MS) method in the leaves and flowers of selected Cassia species. A simple and rapid HPTLC method was developed for chemical fingerprint analysis of all Cassia species. All 13 compounds were chromatographically separated on a Zorbax TC18 (4.6 × 250, 5 μm particle size) analytical column, and 0.1% formic acid and acetonitrile as elution solvents at a flow rate of 0.8 mL/min with detection at 259 nm. For HPTLC fingerprinting, the mobile phase compositions of toluene, ethyl acetate, and formic acid (5.5:4.2:0.6, v/v/v) were optimized to separate and identify all compounds using silica gel 60F254 aluminum plates. The validation data for the developed HPLC-PDA method for 13 compounds showed good linearity (r2 >0.99) with a sensitive LOD (0.082-1.969 μg/mL), LOQ (0.250-5.967 μg/mL), and excellent recoveries (85.22-100.32%). The quantification results were found to be precise and accurate (<5.0% and relative error), -0.77-0.44 with ESI-MS/MS confirmation in the Cassia samples. The novel HPTLC method was excellent separation for 13 compounds, with Rf values ranging between 0.12 and 0.61. The developed HPLC-PDA method was simple and precise and could separate and quantify anthraquinones and flavonoids along with confirmation, using a novel LC-based ESI-MS/MS. The HPTLC method was found to be simple and precise, with excellent separation capabilities for these compounds. This novel multiplatform approach successfully identified and quantified 13 compounds simultaneously using an integration of data strategy in seven medicinally important Cassia species' leaves and flowers.

Effectiveness and Biochemical Impact of Flubendiamide and Flonicamid Insecticides against Bemisia tabaci (Hemiptera: Aleyrodidae) and Residue Dissipation in Cherry Tomato Plants and Soil under Greenhouse Conditions

Abstract We investigated the effectiveness and biochemical impact of the insecticides flubendiamide and flonicamid compared with azadirachtin and acetamiprid against the whitefly Bemisia tabaci (Grennadius) (Hemiptera: Aleyrodidae) infesting cherry tomato (Solanum lycopersicum var. cerasiforme L.) grown under greenhouse conditions. The dissipation of both insecticides in the plants and in the soil also was determined using the QuEChERS (quick, easy, cheap, effective, rugged, and safe) and liquid chromatography–electrospray ionization–mass spectrometry methods. Both insecticides were more effective for reducing B. tabaci populations than were either acetamiprid or azadirachtin. Biochemical analysis revealed that esterase may play an important role in whitefly adaptation to flubendiamide and flonicamid. The QuEChERS method was determined suitable for quickly detecting residues of flubendiamide and flonicamid in complex matrices. The recovery rates on tomato fruit samples were 92.8–106.0%, with a relative standard deviation (RSD) range of 0.46–2.65%. For soil samples, the recovery rates were 81.3–95.7% with RSDs of 1.20–3.86%. We further determined that flubendiamide had dissipation half-lives of 3.13, 3.63, and 3.68 d in tomato fruit, tomato leaves, and soil, respectively. Flonicamid had half-lives of 4.25, 3.54, and 2.60 d in fruit, leaves, and soil, respectively. These results suggest that preharvest intervals of 3 and 7 d are appropriate for flubendiamide and flonicamid, respectively, in cherry tomato production. The risk quotient was &amp;gt;1 by the day 5 after application; however, that value declined to &amp;lt;1 on day 7 after application, indicating little long-term risk to human health.

Structural motifs in the early metallation steps of Zn(II) and Cd(II) binding to apo-metallothionein 1a

Many proteins require a metal cofactor to function and these metals are often involved in the protein folding process. The protein metallothionein (MT) has a dynamic structure capable of binding to a variety of metals with different stoichiometries. The most well-understood structure is the seven-metal, two domain structure formed upon metallation using Zn(II) or Cd(II). However, the partially metallated states and the pathways to form these clusters are less well-understood, although it is known that the pathways are pH dependent. Using stopped flow methods, it is shown that the metallation rates of the less cooperative Zn(II) binding pathway is much more impacted by low pH conditions that that of the more cooperative Cd(II) binding pathway. Electrospray ionization mass spectrometry (ESI-MS) methods reveal specific mixtures of bridging and terminally bound MxSy structures form in the first few metallation steps. Using a combination of methods, the data show that the result of unfolding this intrinsically disordered apo-MT structure using guanidinium chloride is that the formation of preliminary bridging structures that form in the first few metallation steps is impeded. The data show that more terminally bound structures form. Our conclusion is that the compact conformation of the native apo-MT at physiological pH allows for rapid formation of complex metal-thiolate structures with high affinity that provides protection from oxidation, a function that is suppressed upon unfolding. Overall, these results highlight both the importance of the apo-MT structure in the metallation pathway, but also the differences in Zn(II) and Cd(II) binding under different conditions.

Complex of antamanide with the nitrate anion

Anionic complex of antamanide with the nitrate anion has been proven by electrospray ionization mass spectrometry (ESI-MS) method. Further, applying quantum chemical DFT calculations, the most probable structure of this complex was derived. The nitrate anion is embedded in the molecule of antamanide and its oxygens atoms are bonded by seven bonds to the hydrogen atoms of the ligand. Finally, the interaction energy, E(int), of the antamanide-NO3− complex was calculated as E(int) = -175.9 kJ/mol.

Peptide Oxidation Induced by Liquid-Solid Contact Electrification as Revealed in Liquid Microjunction-Surface Sampling Probe Mass Spectrometry.

Reproducible peptide oxidation was observed using a homebuilt liquid microjunction-surface sampling probe (LMJ-SSP) platform for analyzing peptide standards. Although electrochemical oxidation and corona discharges have previously been associated with analyte oxidation in electrospray ionization (ESI) and ESI-related ambient ionization mass spectrometry (MS) methods, they were unlikely the causes for the peptide oxidation observed in the LMJ-SSP studies. A systematic investigation demonstrated that analyte oxidation was induced during the droplet drying on a solid surface through liquid-solid electrification processes. To minimize unwanted analyte oxidation, the water content in the sample solution should be decreased and the use of hydroxyl-functionalized substrates, such as glass slides, should be avoided. In addition, if water is an essential solvent component, adding an antioxidant, such as ascorbic acid, to the sample solution before droplet evaporation on the solid surface could lower the percentage of analyte oxidation. The present findings apply to all the MS methods that involve drying microliters of sample solution onto a suitable substrate in their sample preparation protocols.

Discovery of 2 New Dibenzocyclooctadiene Lignans Cauliflorins A and B From Schisandra cauliflora

Sarcopenia is having a serious impact on highly aged populations. A Japanese traditional medicinal remedy containing Schisandra fruits and Astragalus roots recently showed effective treatment of sarcopenia and thus needed to be investigated further. This current study selected Schisandra cauliflora fruits for investigation of their chemical constituents and evaluation of their antisarcopenia effects. Dried S cauliflora fruits were ultrasonically extracted with methylene chloride. This extract was purified by a combination of chromatographic techniques. The isolated compounds were characterized by nuclear magnetic resonance spectroscopy, electronic circular dichroism, and high resolution electrospray ionization mass spectrometry methods. Antisarcopenia activities of the isolated compounds were evaluated by their effects on the viability and proliferation of C2C12 skeletal muscle cells. Seven dibenzocyclooctadiene lignans, including 2 that had been previously undescribed, cauliflorins A and B (1 and 2), ananolignan C (3), 6- epi-ananolignan D (4), ananolignan F (5), ananonin H (6), and ananonin J (7) were isolated from the methylene chloride extract of the fruits of S cauliflora. The C2C12 cells treated with compounds 1-7 (5 µM) exhibited proliferation in the range of 75.7% ± 7.4% to 98.2% ± 11.7%, compared to 100.0% ± 6.0% of the untreated cells. Two previously undescribed and 5 known dibenzocyclooctadiene lignans were isolated from S cauliflora fruits. All the isolated compounds were inactive on the proliferative enhancement of C2C12 skeletal muscle cells.

Identification of the botanical origins of honey based on nanoliter electrospray ionization mass spectrometry

The botanical origins of honey are important for the quality control and commercialization of honey. In this research, we established a nanoliter electrospray ionization mass spectrometry (Nano-ESI-MS) method to identify Castanopsis honey (CH), Eurya honey (EH), Dendropanax dentiger honey (DH), and Triadica cochinchinensis honey (TH). In total, 38 compounds were identified based on the collision-induced dissociation experiments by Nano-ESI-MS with 16 differential compounds and 7 quantified as potential differential markers. These four types of honey were distinguished from each other by their mass spectrometry data combined with multivariate analysis with three out of the 7 differential markers, i.e., phenethylamine, tricoumaroyl spermidine, and (+/–)-abscisic acid, identified as potential markers for CH, EH, and DH, respectively. Both the qualitative and quantitative results derived from Nano-ESI-MS were further verified by UPLC-Q/TOF-MS. Our studies provided the significant potential of the Nano-ESI-MS method in the identification of the botanical origins of different kinds of honey.

Metabolite Fingerprinting for Identification of Panax ginseng Metabolites Using Internal Extractive Electrospray Ionization Mass Spectrometry.

Ginseng, a kind of functional food and medicine with high nutritional value, contains various pharmacological metabolites that influence human metabolic functions. Therefore, it is very important to analyze the composition and metabolites of ginseng. However, the analysis of active metabolites in ginseng samples usually involves various experimental steps, such as extraction, chromatographic separation, and characterization, which may be time-consuming and laborious. In this study, an internal extractive electrospray ionization mass spectrometry (iEESI-MS) method was developed to analyze active metabolites in ginseng samples with sequential sampling and no pretreatment. A total of 44 metabolites, with 32 ginsenosides, 6 sugars, and 6 organic acids, were identified in the ginseng samples. The orthogonal partial least-squares discriminant analysis (OPLS-DA) score plot showed a clear separation of ginseng samples from different origins, indicating that metabolic changes occurred under different growing conditions. This study demonstrated that different cultivation conditions of ginseng can be successfully discriminated when using iEESI-MS-based metabolite fingerprints, which provide an alternative solution for the quality identification of plant drugs.

Simultaneous Determination of Glibenclamide and Silymarin Released from Chitosan Microparticles by HPLC-ESI-MS Technique: Method Development and Validation.

The study aim was to develop and validate a high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC-ESI-MS) method to simultaneously determine glibenclamide (Gli) and silymarin (Sil) released from chitosan (CS) microparticles in aqueous solutions. The CS microparticles were synthesized using an ionic gelation method, and their morphology, swelling degree, encapsulation efficiency and active substance release were investigated. Gli and Sil were loaded in different concentrations, and their identification and quantification were performed using the HPLC-ESI-MS method, which was further validated. The drugs’ characteristic m/z was found in the higher intensity of retention time (Rt) (Gli, 8.909 min; Sil A, 5.41 min; and Sil B, 5.66 min). The method selectivity and precision are very good, and the blank solution proved no interference. The linearity of the answer function is very good for Sil A (R2 = 1), Sil B (R2 = 0.9998) and Gli (R2 = 0.9991). For Gli, we obtained a limit of detection (LOD) = 0.038 mg/mL and limit of quantification (LOQ) = 1.275 mg/mL; for Sil A, a LOD = 0.285 mg/mL and LOQ = 0.95 mg/mL; and for Sil B, a LOD = 0.045 mg/mL and LOQ = 0.15 mg/mL. A high-resolution HPLC-ESI-MS method was developed and validated, which allowed the simultaneous determination of Gli and Sil loaded in CS microparticles, in a concentration range of 0.025–1 mg/mL.

The Use of Liquid Chromatography and Mass Spectrometry to Identify and Quantify Chemical Components in Tea Extracts

A fast, straightforward, efficient, and high throughput reverse-phase high-performance liquid chromatography and electrospray ionization mass spectrometry method has been developed to analyze tea extracts containing polyphenols. The polyphenols detected in green tea (GT), black tea (BT), and paradise tropical tea (PTT) were separated in 18 min at wavelengths 254 nm, 260 nm, and 280 nm with a gradient elution on the RP-HPLC system. Our approach detected, identified, and quantified three catechins in the tea extracts analyzed, and the response at 280 nm wavelength was the best. The ESI-MS data confirmed the fragmentation patterns of the catechins detected in the tea extract. The validation data showed that the limit of detection (LOD) and limit of quantitation (LOQ) of catechins ranged from 5.26 ± 0.02 to 36.44 ± 0.02 ppb, and 17.52 ± 0.03 to 121.45 ± 0.16 ppb, respectively, for six catechins studied. The standard addition calibration approach used to quantify the catechin content in the tea extract simultaneously showed that PTT has a higher catechin content than GT and BT. The content of polyphenols in GT, BT, and PTT are summarized. This approach holds great promise for quality control studies to quantify polyphenols in nutritional products.

Technical note: Analysis of biological substances in ink fingerprint by desorption electrospray ionization mass spectrometry

As a custom in China, a visible fingerprint pressed with red seal ink on a signature represents personal confirmation of a document. This custom makes ink fingerprints important evidence for individual identification in the forensic practice of questioned document examination. Recently, forged ink fingerprints using stamps or silica gel fingerprint models have emerged. Consequently, detecting and profiling the pattern of biological substances in visible fingerprints on questioned documents is crucial to prove that a fingerprint was imprinted by its real owner. To solve this problem, a desorption electrospray ionization mass spectrometry (DESI-MS) method was developed to detect the biological substances in ink fingerprints on paper. In the positive ion mode, more signals were detected, but the interference from the seal inks could not be ignored. In the negative ion mode, the ion detected in the sweat latent fingerprints at m/z 187 presented high chemical specificity for MS imaging. It was most likely a borate compound in human sweat. Using this biomarker, the fingerprint pattern was successfully profiled and the hand-imprinted fingerprint was clearly distinguished from the stamp-imprinted one. This method can help in determining the authenticity of ink fingerprints on questioned documents in forensic practice.

Anti-enteric efficacy and mode of action of tridecanoic acid methyl ester isolated from Monochoria hastata (L.) Solms leaf.

Monochoria hastata (L.) Solms (family Pontederiaceae), an ethnomedicinal aquatic herb, is used to remedy several gastrointestinal diseases by various ethnic groups in India. The present study aimed to purify and characterize the antibacterial active ingredient against gastrointestinal (GI) diseases and its mode of action using in vitro experimental models. The active lead molecule in the ethyl acetate extract (EA-Mh) fraction has been purified and characterized through high-performance liquid chromatography (HPLC), proton nuclear magnetic resonance (1H NMR), and electrospray ionization mass spectrometry (ESI-MS) methods. The anti-enteric efficacy has been evaluated against enteropathogenic Gram-positive and Gram-negative bacteria by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), lactate dehydrogenase (LDH), and scanning electron microscopy (SEM) studies. The synergistic and antagonistic studies were done on E. coli MTCC 723 using standard antibiotics (ampicillin and kanamycin, final conc. 50µg/ml) in a sterilized 96-well micro-plate, incubated at 37 ℃ for 24h. The chromatographic and spectroscopic analyses revealed the presence of tridecanoic acid methyl ester (TAME) in the bioactive fraction. The compound causes significant extracellular leakage activity by disrupting cellular morphology in the Enterococcus faecalis MCC 2041T and Salmonella enterica serovar Typhimurium MTCC 98, at a dose of 375μg/ml and 750μg/ml, respectively. The SEM study shows a significant rupturing of E. coli and E. faecalis cells due to TAME induced autolysis. It has synergistic activity with ampicillin. The in silico molecular docking through the AutoDock Vina 4.2 and GROMACS (ver. 5.1) Charmm27 force field results showed that the TAME had a strong binding affinity Escherichia coli DNA Gyrase B (PDB ID: 5l3j.pdb) protein and caused conformational changes. Thus, the manuscript reports the first time on the characterization of TAME from this plant with a detailed antibacterial mode of action studies.

Identification of novel neutrophil very long chain plasmalogen molecular species and their myeloperoxidase mediated oxidation products in human sepsis

Plasmalogens are a class of phospholipids containing vinyl ether linked aliphatic groups at the sn-1 position. Plasmalogens are known to contain 16- and 18-carbon aliphatic groups at the sn-1 position. Here, we reveal that the human neutrophil plasmenylethanolamine pool uniquely includes molecular species with very long carbon chain (VLC) aliphatic groups, including 20-, 22- and 24-carbon vinyl ether linked aliphatic groups at the sn-1 position. We identified these novel VLC plasmalogen species by electrospray ionization mass spectrometry methods. VLC plasmalogens were only found in the neutrophil plasmenylethanolamine pool. During neutrophil activation, VLC plasmenylethanolamines undergo myeloperoxidase-dependent oxidation to produce VLC 2-chlorofatty aldehyde and its oxidation product, 2-chlorofatty acid (2-ClFA). Furthermore, plasma concentrations of VLC 2-ClFA are elevated in human sepsis. These studies demonstrate for the first time VLC plasmenylethanolamine molecular species, their myeloperoxidase-mediated chlorolipid products and the presence of these chlorolipids in human sepsis.

Quantifying Carbohydrate-Active Enzyme Activity with Glycoprotein Substrates Using Electrospray Ionization Mass Spectrometry and Center-of-Mass Monitoring

Carbohydrate-active enzymes (CAZymes) play critical roles in diverse physiological and pathophysiological processes and are important for a wide range of biotechnology applications. Kinetic measurements offer insight into the activity and substrate specificity of CAZymes, information that is of fundamental interest and supports diverse applications. However, robust and versatile kinetic assays for monitoring the kinetics of intact glycoprotein and glycolipid substrates are lacking. Here, we introduce a simple but quantitative electrospray ionization mass spectrometry (ESI-MS) method for measuring the kinetics of CAZyme reactions involving glycoprotein substrates. The assay, referred to as center-of-mass (CoM) monitoring (CoMMon), relies on continuous (real-time) monitoring of the CoM of an ensemble of glycoprotein substrates and their corresponding CAZyme products. Notably, there is no requirement for calibration curves, internal standards, labeling, or mass spectrum deconvolution. To demonstrate the reliability of CoMMon, we applied the method to the neuraminidase-catalyzed cleavage of N-acetylneuraminic acid (Neu5Ac) residues from a series of glycoproteins of varying molecular weights and degrees of glycosylation. Reaction progress curves and initial rates determined with CoMMon are in good agreement (initial rates within ≤5%) with results obtained, simultaneously, using an isotopically labeled Neu5Ac internal standard, which enabled the time-dependent concentration of released Neu5Ac to be precisely measured. To illustrate the applicability of CoMMon to glycosyltransferase reactions, the assay was used to measure the kinetics of sialylation of a series of asialo-glycoproteins by a human sialyltransferase. Finally, we show how combining CoMMon and the competitive universal proxy receptor assay enables the relative reactivity of glycoprotein substrates to be quantitatively established.

Rapid analysis of quinones in complex matrices by derivatization-based wooden-tip electrospray ionization mass spectrometry

Quinones are important components participating in various biological processes as well as hazardous substances to human health. Rapid determination of quinones in environmental samples and biofluids is the basis for assessing their health effect. Here, we presented a rapid, straightforward, highly sensitive and environmental-friendly wooden-tip electrospray ionization mass spectrometry (ESI-MS) method for the determination of quinones in PM2.5, urine and serum. An amine group “tag” was introduced to the quinone structure through in situ derivatization with cysteamine to improve ionization efficiency of quinones in wooden-tip ESI-MS. The toothpicks were treated by sharpening and acidification with HNO3. Experimental parameters, including sample volume, spray voltage, and spray solvent composition were optimized to be 1 μL, 3.5 kV, and ACN/CH3COOC2H5 (v/v, 9:1), respectively. The limits of detection for the determination of 1,4-benzoquinone, methyl-p-benzoquinone, 1,4-naphthoquinone and 1,4-anthraquinone in ACN under the optimal conditions were 1.00, 0.96, 0.13, 0.16 ng (1.00, 0.96, 0.13, 0.16 μg/mL, sample volume, 1 μL), respectively. This approach was successfully applied to the determination of 1,4-naphthoquinone and 1,4-anthraquinone in complex matrices, including PM2.5, urine and serum without or with minimal sample preparation (LOD range: 0.22–1.48 ng).

Hand-Held Diode Laser for On-Site Analysis Using Transportable Mass Spectrometry.

A hand-held laser diode thermal desorption electrospray ionization (LDTD-ESI) mass spectrometry (MS) method was developed for rapid screening of illegal substances in solid samples. To achieve that, a simple, inexpensive, battery-powered surgical laser diode at 940 nm was employed to ablate the solid samples. The potential of using a black polytetrafluoroethylene substrate to enhance the analytes’ desorption to the gas phase was investigated and demonstrated. Among the optimized ESI parameters, the solvent (methanol/water, 50:50, v/v) and the flow rate (50 μL h–1) were critical to obtain the best sensitivity. The applicability was demonstrated for the rapid identification of selective androgen receptor modulators (SARMs) in pills and powders based on accurate mass measurements by time-of-flight MS. Also, the hand-held LDTD-ESI was combined with a transportable single quadrupole MS. The same SARMs samples were analyzed, and identifications were based on in-source cone voltage fragmentation patterns observed. These initial results demonstrate the applicability of the developed simplified LDTD-ESI MS method for future on-site testing of organic compounds in solid samples.

LC-MS/MS method for determination of seneciphylline and its metabolite, seneciphylline N-oxide in rat plasma, and its application to a rat pharmacokinetic study.

A rapid and sensitive ultra-performance liquid chromatography-tandem electrospray ionization mass spectrometry (UPLC-ESI/MS) method was established and validated for simultaneous determination of seneciphylline and its main metabolite in rat plasma. The plasma sample was prepared by simple methanol-mediated precipitation. Chromatographic separation was achieved within 3min by gradient elution using acetonitrile and water containing 0.1% formic acid as mobile phase on a Waters ACQUITY BEH C18 column (100 × 2.1mm, i.d. 1.7 μm). Quantitation was conducted in a positive multiple reaction monitoring mode. The linearity of the method was over the range of 1-1,000 ng/mL, with the lower limit of quantification of 1ng/mL. The intra- and inter-day precision and accuracy, extraction recovery, and matrix effect of analytes were within the acceptable limit. The analytes were stable during the process of sample collection, preparation, and analysis. The validated method was further applied to a pharmacokinetic study of seneciphylline in rats after oral and intravenous administration. The results revealed that seneciphylline was quickly absorbed into plasma (Tmax , 0.23-0.32 h) and reached the maximum concentration of 0.82-1.75 μg/mL after oral administration. Both seneciphylline and seneciphylline N-oxide were eliminated from plasma quickly. The low system exposure (oral bioavailability, 5.43-10.31%) was related to the extensive metabolism in the liver and microflora.

On the redox property of Ag16Au13 clusters: One-way conversion from anionic [Au13Ag16L24]3- to charge neutral [Au13Ag16L24]0.

The redox property of metal nanoclusters plays a pivotal role and is of particular interest in catalysis and other applications, such as aerobic oxidation, hydrogenation, and singlet oxygen generation, over intact nanoclusters. In this study, we report a one-way conversion process of the anionic [Ag16Au13L24]3- nanocluster into a charge neutral nanocluster of [Ag16Au13L24]0 via oxidation in a solution phase using H2O2 as the oxidant. Three-electron loss of [Ag16Au13L24]3- occurred during the oxidation process, which was confirmed by electron paramagnetic resonance and electrospray ionization mass spectrometry methods. The one-way conversion from [Ag16Au13L24]3- to [Ag16Au13L24]0 nanoclusters is in situ monitored by UV-visible spectroscopy. A nanocluster charge effect is manifested in the UV-visible spectra of nanoclusters; an ∼10 nm redshift is observed compared with the optical absorption spectrum of [Ag16Au13L24]3-.

Purification of Four Caffeoylquinic Acid Derivatives from the Flowers of Gynura Procumbens by HSCCC.

Four caffeoylquinic acid derivatives from the Gunura procumbens flowers (GPF) were successfully isolated and purified by high-speed counter-current chromatography (HSCCC). Ethyl acetate-methanol-water (3:1:3, v/v/v) was the optimum biphasic solvent system, which was selected by high-performance liquid chromatography (HPLC) and run on a preparative scale where the lower aqueous phase was used as the mobile phase with a head-to-tail elution mode. Chlorogenic acid (3.83 mg), Isochlorogenic acid A (6.51 mg), Isochlorogenic acid B (4.38 mg) and Isochlorogenic acid C (4.47 mg) were obtained for the first time in an one-step HSCCC separation from 800 mg of the crude extracts. The purities of four compounds were determined to be >95% by HPLC. Chemical structures of each isolated compounds were identified by nuclear magnetic resonance and electrospray ionization mass spectrometry methods. It is worth noting that all the four compounds were isolated here for the first time from GPF and this work confirms the effectiveness of HSCCC for the separation of compounds contained in complex samples, and provides a foundation for further exploitation of G. procumbens.

A fast and effective approach for the discrimination of garlic origin using wooden-tip electrospray ionization mass spectrometry and multivariate classification

This paper presents the combination of wooden-tip electrospray ionization mass spectrometry (WTESI-MS) and multivariate pattern recognition methods (principal component analysis, PCA and partial least squares discriminant analysis, PLS-DA) for the rapid and reliable discrimination, via chemical fingerprints, of garlic origin. A total of 312 garlic samples grown in different countries (Brazil, China, Argentina, Spain, and Chile) were studied. The methodology was based on a direct sampling approach, which relies on loading the sample by penetrating the garlic cloves with a pre-wetted wooden tip, followed by direct prompt analysis by WTESI-MS. Thus, no sample preparation is needed, which prevents the degradation of important metabolites and increases the analytical throughput. Parameters that affects the WTESI were optimized and the best performance in terms of signal stability and intensity was achieved using the positive ion mode. Most of the ions in WTESI mass spectra were assigned to amino acids, sugars, organosulfur compounds, and lipids. The discriminative model showed good performance (accuracy rates between 81.9% and 98.6%) and enabled identifying diagnostic ions for garlic samples from different origins. The differentiation and classification of garlic origin is of major importance as this food flavoring product is widely consumed, with worldwide trade representing billions of dollars every year, and is very often the subject of fraud.