Chemistry of Isoeugenol and Its Oxidation Products: Mechanism and Kinetics of Isoeugenol as a Skin Sensitizer.

Structurally similar phytochemical compounds may elicit markedly different skin sensitization responses. Eugenol and isoeugenol are natural phenylpropanoids found in various essential oils are frequently used as fragrance ingredients in consumer products due to their pleasing aromatic properties. Both compounds are also skin sensitizers with isoeugenol being a stronger sensitizer than eugenol. The most commonly accepted mechanisms for haptenation by eugenol involve formation of a quinone methide or an ortho-quinone intermediate. The mechanism for the increased skin response to isoeugenol remains elusive, although quinone methide intermediates have been proposed. The recent identification of diastereomeric 7,4'-oxyneolignans as electrophilic, thiol-depleting isoeugenol derivatives has revived interest in the possible role of elusive reactive intermediates associated with the isoeugenol's haptenation process. In the present work, integrated non-animal skin sensitization methods were performed to determine the ability of syn-7,4'-oxyneolignan to promote haptenation and activation of further molecular pathways in keratinocytes and dendritic cells, confirming it as a candidate skin sensitizer. Kinetic NMR spectroscopic studies using dansyl cysteamine (DCYA) confirmed the first ordered nature of the nucleophilic addition for the syn-7,4'-oxyneolignan. Computational studies reaffirmed the "syn" stereochemistry of the isolated 7,4'-oxyneolignans along with that of their corresponding DCYA adducts and provided evidence for the preferential stereoselectivity. A plausible rationale for isoeugenol's strong skin sensitization is proposed based on the formation of a hydroxy quinone methide as a reactive intermediate rather than the previously assumed quinone methide.

Computational Tools to Expedite the Identification of Potential PXR Modulators in Complex Natural Product Mixtures: A Case Study with Five Closely Related Licorice Species.

The genus Glycyrrhiza, comprising approximately 36 spp., possesses complex structural diversity and is documented to possess a wide spectrum of biological activities. Understanding and finding the mechanisms of efficacy or safety for a plant-based therapy is very challenging, yet it is crucial and necessary to understand the polypharmacology of traditional medicines. Licorice extract was shown to modulate the xenobiotic receptors, which might manifest as a potential route for natural product-induced drug interactions. However, different mechanisms could be involved in this phenomenon. Since the induced herb-drug interaction of licorice supplements via Pregnane X receptor (PXR) is understudied, we ventured out to analyze the potential modulators of PXR in complex mixtures such as whole extracts by applying computational mining tools. A total of 518 structures from five species of Glycyrrhiza: 183 (G. glabra), 180 (G. uralensis), 100 (G. inflata), 33 (G. echinata), and 22 (G. lepidota) were collected and post-processed to yield 387 unique compounds. Visual inspection of top candidates with favorable ligand-PXR interactions and the highest docking scores were identified. The in vitro testing revealed that glabridin (GG-14) is the most potent PXR activator among the tested compounds, followed by licoisoflavone A, licoisoflavanone, and glycycoumarin. A 200 ns molecular dynamics study with glabridin confirmed the stability of the glabridin-PXR complex, highlighting the importance of computational methods for rapid dereplication of potential xenobiotic modulators in a complex mixture instead of undertaking time-consuming classical biological testing of all compounds in a given botanical.

Solvents effect on dansyl cysteamine depletion and reactivity classification of skin sensitizers: Tackling the challenges using binary solvent systems.

The high throughput method using dansyl cysteamine (HTS-DCYA™) is a sensitive and rapid in chemico approach to characterize skin sensitizers' thio-reactivity. The direct quantification of fluorescent hapten-DCYA adducts facilitates the rapid testing of pure chemicals as well as mixtures. Poor solubility in acetonitrile was occasionally observed and can represent a limitation. To enable the range of solvent options compatible with the testing, the effect of binary solvent systems on thio-reactivity and the HTS-DCYA classification was explored. The method's robustness was validated using five different solvent modifiers: water, DMSO, methanol, ethanol, and tetrahydrofuran. Some modifiers, viz., water and methanol, resulted in unexpected DCYA depletion, negatively affecting the thio-reactivity and classification of potential sensitizers. This undesirable, non-specific depletion was circumvented by optimizing the original HTS-DCYA™ method's workflow, resulting in a more robust and reliable thio-reactivity and hence classification with a binary solvent system. The results were validated for both pure compounds and plant extracts as examples of complex test samples. Based on the obtained results, the modified HTS-DCYA optimal conditions in the various solvent systems were established. Concentrations of modifiers up to 10% DMSO, 40% water, 40% EtOH, 60% MeOH, or 60% THF in acetonitrile were found acceptable for the modified protocol, with results comparable to the original method. The improved workflow with binary solvent systems provides significant advantages by expanding the applicability of the HTS-DCYA to a wider array of chemicals poorly soluble in acetonitrile.

A novel approach for lavender essential oil authentication and quality assessment.

Currently, the quality of lavender (Lavandula angustifolia Mill.) essential oil (LEO) is defined and regulated based on standards and methods established by regulatory authorities. Unfortunately, these existing standards and methods are not sufficient for LEO quality evaluation due to the complexity of LEO and adulteration encouraged by a burgeoning market. This study provides an efficient and reliable method for LEO quality assessment and adulteration detection. After a comprehensive investigation, involving a large set of LEO samples (n = 72) analyzed by multiple techniques (GC/MS, GC/Q-ToF, NMR, and chemometric analysis), a new approach named Q-Index was proposed. Fourteen marker compounds, along with trans-furano-linalool oxide acetate (an indicator of synthetic compound adulteration in LEO), were identified. These marker compounds played significant roles in discriminating the adulterated samples from the authentic LEOs. Calculation of the Q-Index value using the identified marker compounds permitted the detection of fraudulent samples. As demonstrated, all the authentic LEOs exhibited high Q-Index values (>100), whereas the adulterated or poor-quality samples displayed low Q-Index values (<100). The NMR-based chemometric analysis, which served as an independent and complementary approach to the GC/MS and Q-Index methods, was applied in order to assess the validity of the Q-Index method. Overall, the results obtained from different methods were in good agreement. Moreover, compared to the NMR method, the Q-Index approach possessed greater sensitivity in detecting LEO adulteration associated with the addition of synthetic compounds. Results of this study demonstrated that the Q-Index method could be successfully applied for LEO quality assessment and adulteration detection. This approach may have a significant potential to improve quality control for the LEO industry.

Are atranols the only skin sensitizers in oakmoss? A systematic investigation using non-animal methods.

Oakmoss and treemoss absolutes are the major natural extracts of concern as potential sources of skin sensitizers in cosmetics and personal care products (PCP). Two single constituents, atranol and chloroatranol, have been identified as primary culprits in both lichens, and industrial self-regulation has been proposed to limit their contents to less than 100 ppm. Nonetheless, evidence points to the presence of additional candidate skin sensitizers in these multicomponent extracts. These observations, along with a lack of data from non-animal alternative methods and the chemical variability of commercial absolutes, prompted further investigation of oakmoss absolute along with altranol-like compounds in these extracts. The major chemical constituents of a commercial sample were identified by two independent analytical techniques, GC-MS and HPLC-DAD-MS. The crude oakmoss extract and pure compounds were assayed with two in chemico methods (HTS-DCYA and DPRA) to gauge their chemical reactivity. Activation of inflammatory responses in vitro was also investigated by KeratinoSens™ and human cell line activation tests (h-CLAT). Based on weight of evidence, orcinol, ethyl orsellinate, and usnic acid were classified as candidate sensitizers, along with both atranols and oakmoss extract.

Discovery of a Remarkable Methyl Shift Effect in the Vanilloid Activity of Triterpene Amides.

As part of a study on triterpenoid conjugates, the dietary pentacyclic triterpenoids oleanolic (2a) and ursolic acids (3a) were coupled with vanillamine, and the resulting amides (2b and 3b, respectively) were assayed for activity on the vanilloid receptor TRPV1. Despite a structural difference limited to the location of a methyl group in their conformationally rigid pentacyclic core, oleanoloyl vanillamide dramatically outperformed ursoloyl vanillamide in terms of potency (EC50 = 35 ± 2 nM for 2b and 5.4 ± 2.3 µM for 3b). Using molecular docking and dynamics, this difference was translated into distinct accommodation modes at the TRPV1 vanillyl ligand pocket, suggesting a critical role of a C-H πphenyl interaction between the triterpenoid C-29 methyl and Phe591 of TRPV1. Because the molecular mechanisms underlying the activation process of transient receptor channels (TRPs) remain to be fully elucidated, the observation of spatially restricted structure-activity information is of significant relevance to identify the molecular detail of TRPV1 ligand gating.

Is Isoeugenol a Prehapten? Characterization of a Thiol-Reactive Oxidative Byproduct of Isoeugenol and Potential Implications for Skin Sensitization.

Isoeugenol is widely used by the cosmetic and fragrance industries, but it also represents a known cause of skin sensitization adverse effects. Although devoid of a structural alert, isoeugenol has been classified as prehapten in virtue of the presence of a pre-Michael acceptor domain. Isoeugenol oxidation could theoretically lead to the generation of reactive toxic quinones, and photoinduced oxidative degradation of isoeugenol was reported to generate strongly thiol reactive byproducts. Nonetheless, the isoeugenol degradation product responsible for increased reactivity was found to be elusive. In the present study, an aged isoeugenol sample was subjected to reactivity-guided experiments to trap elusive thiol reactive species with a fluorescent nucleophile, viz. dansyl cysteamine (DCYA). The results herein presented demonstrate that photo-oxidation of isoeugenol led to the formation of a dimeric 7,4'-oxyneolignan with strong chemical reactivity, capable of nucleophilic substitution with thiols. The results were confirmed by isolation, structural characterization, and further NMR reactivity studies. Isoeugenol is already well-known as moderately reactive in thiol depletion assays, and was herein demonstrated to be capable of converting to more potent electrophilic species upon degradation, thus acting as a prehapten. The application of the reactivity-guided strategy described herein was shown to serve as an effective tool to investigate elusive skin sensitizers.

In chemico assessment of potential sensitizers: Stability and direct peptide reactivity of 24 fragrance ingredients.

Twenty-four pure fragrance ingredients of concern as potential skin sensitizers were previously subjected to degradation studies and evaluated using the high throughput with dansyl cysteamine (HTS-DCYA) method. The experimental results showed that two-thirds of the 24 fragrance ingredients underwent chemical degradation. In some cases, such degradation was accompanied by an increase in thio-reactivity. These results prompted us to investigate the reactivity of the same ingredients using the direct peptide reactivity assay (DPRA). In the present work, the 24 chemicals were subjected to forced degradation for 150 days, and evaluated with both DPRA and HTS-DCYA methods. At the end of the study, four and eight compounds remained non-reactive in the DPRA and DCYA assay, respectively. Coumarin, benzyl salicylate, benzyl cinnamate and hexyl cinnamal were found unreactive in both assays, while cinnamal, cinnamyl alcohol, hydroxycitronellal and lilial were found negative in the DCYA but positive in the DPRA method. The incongruity in reactivity of these four compounds was attributed to a possible role of pro-oxidants formed upon degradation, resulting in depletion of peptide without formation of apparent covalent adducts with the test chemical. To validate this hypothesis, the effect of hydrogen peroxide as model pro-oxidant on both lysine- and cysteine-heptapeptide depletion in the DPRA method was thus investigated. The obtained results showed little effect of oxidative conditions on lysine depletion, while cysteine depletion was significantly affected by concentrations above 1.1 mg/L of hydrogen peroxide. Overall, both in chemico methods confirmed chemical instability should be considered when assessing the skin sensitization potential of (un)known chemicals with alternative methods.

In chemico skin sensitization risk assessment of botanical ingredients.

Skin sensitization risk assessment of botanical ingredients is necessary for consumers' protection and occupational hazard identification. There are currently very few available alternative methods that can assist in the evaluation of complex mixtures. Chemical methods can provide essential information in a timely manner and thus help to reduce the need for in vivo testing, and they can complement and facilitate targeted in vitro assays. In the present work, the applicability of the high-throughput screening with dansyl cysteamine (DCYA) method for the systematic evaluation of skin sensitization of complex botanicals was explored. Botanical ingredients of four unrelated plant species were obtained and tested with the high-throughput fluorescence method at three concentrations. To illustrate the minimal matrix effects of the tested extracts on the developed method, the least DCYA-reactive extract (Rosa canina) was spiked with known sensitizers at different concentrations. The data obtained from the four plant extracts and the spiking experiments with known sensitizers, suggest that the high-throughput screening-DCYA method can be successfully applied for estimating the skin sensitization potential of complex botanical matrices. This is the first report of an attempt to develop a versatile in chemico method for the rapid detection of reactive skin sensitizers in complex botanical extracts, which could complement the battery of existing validated, non-animal methods.

Chemical stability and in chemico reactivity of 24 fragrance ingredients of concern for skin sensitization risk assessment.

Twenty-four pure fragrance ingredients have been identified as potential concern for skin sensitization. Several of these compounds are chemically unstable and convert into reactive species upon exposure to air or light. In the present work, a systematic investigation of the correlation between chemical stability and reactivity has been undertaken. The compounds were subjected to forced photodegradation for three months and the chemical changes were studied with GC-MS. At the end of the stability study, two-thirds of the samples were found to be unstable. The generation of chemically reactive species was investigated using the in chemico HTS-DCYA assay. Eleven and fourteen compounds were chemically reactive before and after three months, respectively. A significant increase in reactivity upon degradation was found for isoeugenol, linalool, limonene, lyral, citronellol and geraniol; in the same conditions, the reactivity of hydroxycitronellal decreased. The non-reactive compounds α-isomethyl ionone, benzyl alcohol, amyl cinnamal and farnesol became reactive after photo-oxidative degradation. Overall, forced degradation resulted in four non-reactive fragrance compounds to display in chemico thiol reactivity, while ten out of 24 compounds remained inactive. Chemical degradation does not necessarily occur with generation of reactive species. Non-chemical activation may be involved for the 10 stable unreactive compounds.

Identification of a compound isolated from German chamomile (Matricaria chamomilla) with dermal sensitization potential.

German chamomile is one of the most popular herbal ingredients used in cosmetics and personal care products. Allergic skin reactions following topical application of German chamomile have been occasionally reported, although it is not fully understood which of the chemical constituents is responsible for this adverse effect. In the present work, three candidate sensitizers were isolated from German chamomile based on activity-guided fractionation of chamomile extracts tested using the in vitro KeratinoSens™ assay. The compounds were identified as the polyacetylene tonghaosu (1), and both trans- and cis-glucomethoxycinnamic acids (2 and 3). These three compounds were classified as non- to weakly reactive using in chemico methods; however, aged tonghaosu was found to be more reactive when compared to freshly isolated tonghaosu. The polyacetylene (1) constituent was determined to be chemically unstable, generating a small electrophilic spirolactone, 1,6-dioxaspiro[4.4]non-3-en-2-one (4), upon aging. This small lactone (4) was strongly reactive in both in chemico HTS- and NMR-DCYA methods and further confirmed as a potential skin sensitizer by Local Lymph Node Assay (LLNA).

What Happens after Activation of Ascaridole? Reactive Compounds and Their Implications for Skin Sensitization.

To replace animal testing and improve the prediction of skin sensitization, significant attention has been directed to the use of alternative methods. The direct peptide reactivity assay (DPRA), the regulatory agencies' approved alternative in chemico method, has been applied for understanding the sensitization capacity of activated ascaridole. Ascaridole, the oxidative metabolite of α-terpinene, is considered to be one of the components responsible for the contact allergy associated with essential oils derived from Chenopodium and Melaleuca species. The recently developed high-throughput screening based on the dansyl cysteamine (HTS-DCYA) method was applied to understand the reported enhanced reactivity of activated ascaridole and possibly to identify the resulting elusive radical or other reactive species. For the first time, a substituted cyclohexenone was identified as a potential electrophilic intermediate resulting in higher depletion of nucleophilic DCYA, along with several nonreactive byproducts of ascaridole via a radical degradation mechanism. Formation of electrophilic species via radical degradation is one of the possible pathways should be considered for the peptide reactivity of in aged tea tree oil or oils rich in terpinenes along with commonly believed reactants, allylic-epoxides and allylic-peroxides.

In Chemico Evaluation of Tea Tree Essential Oils as Skin Sensitizers: Impact of the Chemical Composition on Aging and Generation of Reactive Species.

Tea tree oil (TTO) is an essential oil obtained from the leaves of Melaleuca alternifolia, M. linariifolia, or M. dissitiflora. Because of the commercial importance of TTO, substitution or adulteration with other tea tree species (such as cajeput, niaouli, manuka, or kanuka oils) is common and may pose significant risks along with perceived health benefits. The distinctive nature, qualitative and quantitative compositional variation of these oils, is responsible for the various pharmacological as well as adverse effects. Authentic TTOs (especially aged ones) have been identified as potential skin sensitizers, while reports of adverse allergic reactions to the other tea trees essential oils are less frequent. Chemical sensitizers are usually electrophilic compounds, and in chemico methods have been developed to identify skin allergens in terms of their ability to bind to biological nucleophiles. However, little information is available on the assessment of sensitization potential of mixtures, such as essential oils, due to their complexity. In the present study, 10 "tea tree" oils and six major TTO constituents have been investigated for their sensitization potential using a fluorescence in chemico method. The reactivity of authentic TTOs was found to correlate with the age of the oils, while the majority of nonauthentic TTOs were less reactive, even after aging. Further thio-trapping experiments with DCYA and characterization by UHPLC-DAD-MS led to the identification of several possible DCYA-adducts which can be used to deduce the structure of the candidate reactive species. The major TTO components, terpinolene, α-terpinene, and terpinene-4-ol, were unstable under accelerated aging conditions, which led to the formation of several DCYA-adducts.

A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers.

Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles after incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, 'HTS-DCYA assay', is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time.

Alternative testing methods for skin sensitization: NMR spectroscopy for probing the reactivity and classification of potential skin sensitizers.

Evaluating consumer products for potentially harmful side effects of chemical ingredients is important for the protection of both the consumer and those involved in the manufacturing process. In order to assess the risk potential of chemicals, regulatory agencies have encouraged the development of several in silico, in vitro, and in chemico methods as alternatives to eliminate or minimize the use of animals. To add structural information to the existing in chemico methods, an NMR-based method is proposed for probing the reactivity and classification of the potential electrophiles (E) using a model thiol, DCYA, as a nucleophile. The major advantage of the NMR method is the quantitation of the actual adduct, DCYA-E. The degree of reaction is here provided as a direct measurement of adduct formation and/or electrophile depletion, in contrast to other in chemico assays, e.g., ADRA and DPRA, where the reactivity is inferred from the quantification of the test nucleophile depletion. Moreover, the developed NMR method should serve as a qualitative and quantitative tool in understanding the site of reaction and other structural information associated with test sensitizer. This is particularly valuable and advantageous over methods encouraged by regulatory agencies, which merely provide quantification of the reaction but lack any structural information. Several compounds with multiple reaction sites were successfully tested with the proposed NMR method. Otherwise, these compounds have proven to be a challenge to identify and classify using existing alternative methods.

Quantitative Determination of α-Arbutin, ß-Arbutin, Kojic Acid, Nicotinamide, Hydroquinone, Resorcinol, 4-Methoxyphenol, 4-Ethoxyphenol, and Ascorbic Acid from Skin Whitening Products by HPLC-UV.

An HPLC-UV method was developed for the quantitative analysis of nine skin whitening agents in a single injection. These compounds are α-arbutin, ß-arbutin, kojic acid, nicotinamide, resorcinol, ascorbic acid, hydroquinone, 4-methoxyphenol, and 4-ethoxyphenol. The separation was achieved on a reversed-phase C18 column within 30 min. The mobile phase was composed of water and methanol, both containing 0.1% acetic acid (v/v). The stability of the analytes was evaluated at different pH values between 2.3 and 7.6, and the extraction procedure was validated for different types of skin whitening product matrixes, which included two creams, a soap bar, and a capsule. The best solvent system for sample preparation was 20 mM NaH2PO4 containing 10% methanol at pH 2.3. The analytical method was validated for accuracy, precision, LOD, and LOQ. The developed HPLC-UV method was applied for the quantitation of the nine analytes in 59 skin whitening products including creams, lotions, sera, foams, gels, mask sheets, soap bars, tablets, and capsules.

In Vitro Evaluation of Reversible and Time-Dependent Inhibitory Effects of Kalanchoe crenata on CYP2C19 and CYP3A4 Activities.

Kalanchoe crenata popularly known as "dog's liver" is used in most African countries for the treatment of chronic diseases such as diabetes, asthma and HIV/AIDS related infections. The evaluation of K. crenata for herb-drug interactions has not been reported. This study therefore aims to evaluate the risk of K. crenata for herb-drug interaction in vitro. Crude methanol and fractions of K. crenata were incubated and preincubated with recombinant human CYP2C19 and CYP3A4. Comparative studies were conducted in both human liver microsomes and recombinant human CYP to ascertain the inhibition profile of the crude extract and the various fractions. The cocktail approach of recombinant human CYPs was conducted to confirm the inhibition potential of the fractions in the presence of other CYPs. The results showed significant time-dependent inhibition of tested samples on CYP3A4 with crude methanol (39KC), fractions 45A, 45B and 45D given IC50 fold decrease of 3.29, 2.26, 1.91 and 1.49, respective. Time dependent kinetic assessment of 39KC and 45D showed KI and kinact values for 39KC as 1.77 µg/mL and 0.091 min(-1) while that of 45D were 6.45 µg/mL and 0.024 min(-1), respectively. Determination of kinact based on IC50 calculations yielded 0.015 and 0.04 min(-1) for 39KC and 45D, respectively. Cocktail approach exhibited fold decreases in IC50 for all test fractions on CYP3A4 within the ranges of 2.10 - 4.10. At least one phytoconstituent in the crude methanol extract of Kalanchoe crenata is a reversible and time-dependent inhibitor of CYP3A4.

Echinacea purpurea up-regulates CYP1A2, CYP3A4 and MDR1 gene expression by activation of pregnane X receptor pathway.

1.This study investigated the mechanism underlying Echinacea-mediated induction of CYP1A2, CYP3A4 and MDR1 in terms of human pregnane X receptor (PXR) activation. 2.Crude extracts and fractions of Echinacea purpurea were tested for PXR activation in HepG2 cells by a reporter gene assay. Quantitative real-time PCR was carried out to determine their effects on CYP1A2 and CYP3A4 mRNA expressions. Capsules and fractions were risk ranked as high, intermediate and remote risk of drug-metabolizing enzymes induction based on EC50 values determined for respective CYPs. 3. Fractions F1, F2 and capsule (2660) strongly activated PXR with 5-, 4- and 3.5-fold increase in activity, respectively. Echinacea preparations potentiated up-regulation of CYP1A2, CYP3A4 and MDR1 via PXR activation. 4.Thus E. purpurea preparations cause herb-drug interaction by up-regulating CYP1A2, CYP3A4 and P-gp via PXR activation.

High-resolution gas chromatography/mass spectrometry method for characterization and quantitative analysis of ginkgolic acids in Ginkgo biloba plants, extracts, and dietary supplements.

A high-resolution gas chromatography/mass spectrometry (GC/MS) with selected ion monitor method focusing on the characterization and quantitative analysis of ginkgolic acids (GAs) in Ginkgo biloba L. plant materials, extracts, and commercial products was developed and validated. The method involved sample extraction with (1:1) methanol and 10% formic acid, liquid-liquid extraction with n-hexane, and derivatization with trimethylsulfonium hydroxide (TMSH). Separation of two saturated (C13:0 and C15:0) and six unsaturated ginkgolic acid methyl esters with different positional double bonds (C15:1 Δ8 and Δ10, C17:1 Δ8, Δ10, and Δ12, and C17:2) was achieved on a very polar (88% cyanopropyl) aryl-polysiloxane HP-88 capillary GC column. The double bond positions in the GAs were determined by ozonolysis. The developed GC/MS method was validated according to ICH guidelines, and the quantitation results were verified by comparison with a standard high-performance liquid chromatography method. Nineteen G. biloba authenticated and commercial plant samples and 21 dietary supplements purported to contain G. biloba leaf extracts were analyzed. Finally, the presence of the marker compounds, terpene trilactones and flavonol glycosides for Ginkgo biloba in the dietary supplements was determined by UHPLC/MS and used to confirm the presence of G. biloba leaf extracts in all of the botanical dietary supplements.

Investigating sub-2 µm particle stationary phase supercritical fluid chromatography coupled to mass spectrometry for chemical profiling of chamomile extracts.

Roman and German chamomile are widely used throughout the world. Chamomiles contain a wide variety of active constituents including sesquiterpene lactones. Various extraction techniques were performed on these two types of chamomile. A packed-column supercritical fluid chromatography-mass spectrometry method was designed for the identification of sesquiterpenes and other constituents from chamomile extracts with no derivatization step prior to analysis. Mass spectrometry detection was achieved by using electrospray ionization. All of the compounds of interest were separated within 15 min. The chamomile extracts were analyzed and compared for similarities and distinct differences. Multivariate statistical analysis including principal component analysis and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used to differentiate between the chamomile samples. German chamomile samples confirmed the presence of cis- and trans-tonghaosu, chrysosplenols, apigenin diglucoside whereas Roman chamomile samples confirmed the presence of apigenin, nobilin, 1,10-epioxynobilin, and hydroxyisonobilin.