Comprehensive Two-Dimensional Gas Chromatography as a Bioanalytical Platform for Drug Discovery and Analysis.

Over the last decades, comprehensive two-dimensional gas chromatography (GC×GC) has emerged as a significant separation tool for high-resolution analysis of disease-associated metabolites and pharmaceutically relevant molecules. This review highlights recent advances of GC×GC with different detection modalities for drug discovery and analysis, which ideally improve the screening and identification of disease biomarkers, as well as monitoring of therapeutic responses to treatment in complex biological matrixes. Selected recent GC×GC applications that focus on such biomarkers and metabolite profiling of the effects of drug administration are covered. In particular, the technical overview of recent GC×GC implementation with hyphenation to the key mass spectrometry (MS) technologies that provide the benefit of enhanced separation dimension analysis with MS domain differentiation is discussed. We conclude by highlighting the challenges in GC×GC for drug discovery and development with perspectives on future trends.

Evaluation of Cryogen-Free Thermal Modulation-Based Enantioselective Comprehensive Two-Dimensional Gas Chromatography for Stereo-Differentiation of Monoterpenes in Citrus spp. Leaf Oils.

This study evaluates the applicability of enantioselective gas chromatography (eGC) and enantioselective comprehensive two-dimensional gas chromatography (eGC×GC) coupled with flame ionization detection for the stereospecific analysis of designated chiral monoterpenes within essential oils distilled from the leaves of Citrus hystrix (CH), C. limon (CL), C. pyriformis (CP), and C. microcarpa (CM). A cryogen-free solid-state modulator with a combination of enantioselective first-dimension and polar second-dimension column arrangements was used to resolve potential interferences in Citrus spp. leaf oils that can complicate the accurate determination of enantiomeric compositions. Interestingly, considerable variations were observed for the enantiomeric fractions (EFs) of the chiral terpenes. (+)-limonene was identified as the predominant enantiomer (60.3-98.9%) in all Citrus oils, (+)-linalool was the major enantiomer in CM (95.9%), (-)-terpenin-4-ol was the major isomer in CM (66.4%) and CP (61.1%), (-)-α-pinene was the dominant antipode in CL (55.5%) and CM (92.1%). CH contained (-)-citronellal (100%) as the pure enantiomer, while CL and CP have lower proportions (9.0-34.6%), and citronellal is absent in CM. The obtained enantiomeric compositions were compared and discussed with results from eGC using the same enantioselective column. To our knowledge, this work encapsulates the first report that details the EFs of these chiral monoterpenes in Citrus spp. leaf oil.

Comprehensive two-dimensional gas chromatography with mass spectrometry: an advanced bioanalytical technique for clinical metabolomics studies.

The detection of human-derived metabolites as potential diagnostic biomarkers of genetic disorders, metabolic diseases, systemic diseases, and infectious diseases has been much studied in recent years, especially as technical capabilities improve, and statistical procedures are increasingly able to tease critical chemical attributes from complex data sets. Given the complex distribution of human biological matrices, the characterization and/or identification of these chemical entities is technically challenging, and is often confounded by incomplete chromatographic resolution or insufficient discriminatory power of the mass spectrometry (MS) domain. Recently, comprehensive two-dimensional gas chromatography (GC×GC) has evolved into a mature higher separation order technique that offers unprecedented resolving power, which in turn can greatly advantage clinical metabolomics studies via the expansion of metabolite coverage. In this contribution, the current state of knowledge in the development of GC×GC coupled to MS as a high-resolution bioanalytical technique for the analysis of clinical metabolites is reviewed. Selected recent applications (years 2012 to 2021) that emphasize improved GC×GC-MS strategies for clinical human metabolites' detection, identification, and quantitative analysis are described. In addition, we share our perspectives on current challenges and potential future directions of GC×GC in clinical applications.

Phytochemical Constituents and Antiproliferative Activities of Essential Oils from Four Varieties of Malaysian Zingiber officinale Roscoe against Human Cervical Cancer Cell Line.

This study evaluates the volatile metabolic constituents and anticancer potential of essential oils distilled from the rhizomes of four Malaysian Zingiber officinale Roscoe (Zingiberaceae family) varieties (Bentong (BE), Cameron Highlands (CH), Sabah (SA), and Bara (BA)). The ginger essential oils were analyzed by gas chromatography coupled with quadrupole mass spectrometry (GC qMS). A total of 58 secondary compounds were tentatively identified, representing 82.6-87.4% of the total ion count. These metabolites comprise mainly of monoterpene hydrocarbons (19.7-25.5%), oxygenated monoterpenes (23.6-33.7%), sesquiterpene hydrocarbons (21.3-35.6%), oxygenated sesquiterpenes (1.5-3.9%), and other minor classes of compounds (0.7-2.7%). Principal component analysis (PCA) enabled differentiation of the analyzed ginger essential oils according to their varieties, with respect to their metabolites and relative quantities. The antiproliferative activity against the HeLa cervical cancer cell line was investigated via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The oils were found to exhibit strong antiproliferative activities with IC50 values of 23.8, 35.3, 41.3, and 42.5 µg/mL for BA, BE, SA, and CH, respectively. These findings suggest that the differences among the secondary metabolites and their abundance in different varieties of Z. officinale essential oils appear to be related to their antiproliferative potential. The strong antiproliferative effects of these oils signified their potential in the prevention and chemotherapy of cervical carcinoma treatment.

Phytochemical Composition, Antioxidant and Antiproliferative Activities of Citrus hystrix, Citrus limon, Citrus pyriformis, and Citrus microcarpa Leaf Essential Oils against Human Cervical Cancer Cell Line.

The essential oil derived from Citrus plants has long been used for medicinal purposes, due to its broad spectrum of therapeutic characteristics. To date, approximately 162 Citrus species have been identified, and many investigational studies have been conducted to explore the pharmacological potential of Citrus spp. oils. This study investigated the volatile constituents of essential oil distilled from the leaves of C. hystrix, C. limon, C. pyriformis, and C. microcarpa, using gas chromatography-quadrupole mass spectrometry. A total of 80 secondary compounds were tentatively identified, representing 84.88-97.99% of the total ion count and mainly comprising monoterpene (5.20-76.15%) and sesquiterpene (1.36-27.14%) hydrocarbons, oxygenated monoterpenes (3.91-89.52%) and sesquiterpenes (0.21-38.87%), and other minor chemical classes (0.10-0.52%). In particular, 27 compounds (1.19-39.06%) were detected across all Citrus species. Principal component analysis of the identified phytoconstituents and their relative quantities enabled differentiation of the Citrus leaf oils according to their species, with the loading variables contributing to these metabolic differences being identified. The Citrus leaf oils were tested for their antioxidant and antiproliferative activities using 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The results indicated that C. limon displayed the highest DPPH radical scavenging ability (IC50 value of 29.14 ± 1.97 mg/mL), while C. hystrix exhibited the lowest activity (IC50 value of 279.03 ± 10.37 mg/mL). On the other hand, all the Citrus oils exhibit potent antiproliferative activities against the HeLa cervical cancer cell line, with IC50 values of 11.66 µg/mL (C. limon), 20.41 µg/mL (C. microcarpa), 25.91 µg/mL (C. hystrix), and 87.17 µg/mL (C. pyriformis).

Simultaneous Quantitative Assessment of Ochratoxin A, Patulin, 5-Hydroxymethylfurfural, and Bisphenol A in Fruit Drinks Using HPLC with Diode Array-Fluorimetric Detection.

The analysis of regulated contaminants in fruit drinks often requires suitable validated and rapid analytical methods for cost-effective food control, and is of considerable interest among the fruit beverage industry. This study demonstrated a rapid and sensitive high-performance liquid chromatography approach for the simultaneous determination of ochratoxin A (OTA), patulin (PAT), 5-hydroxymethylfurfural (HMF), and bisphenol A (BPA) in various fruit drinks. The separations were achieved using a C18 core-shell column with both photo-diode array and fluorimetric detections connected in series. A gradient system consisting of methanol and 0.1% formic acid at a flow rate of 1.2 mL min-1, thermostated at 35 °C, provided fast elution with run time <9 min. Sample pretreatment was optimised to enable extraction of all analytes from fruit drink matrices. The optimised method was validated. Correlation coefficients of R > 0.99 were achieved with detection limits of 0.5 ng mL-1 (OTA), 1.1 ng mL-1 (PAT), 7.9 ng mL-1 (HMF), and 1.0 ng mL-1 (BPA). Recoveries ranged from 82% to 99%. Good relative standard deviations for intraday retention times (≤3.54%) and peak area (≤3.5%) were achieved. The developed multi-contaminants analysis method was successfully applied to determine OTA, PAT, HMF, and BPA in various fruit drinks.

Assessment of the phytochemical profiles of novel hop (Humulus lupulus L.) cultivars: A potential route to beer crafting.

This study investigated the volatile phytochemical diversity of 30 samples obtained from experimental hybrid and commercial H. lupulus L. plants. Essential oils distilled from these samples were analysed by high resolution gas chromatography coupled with accurate mass time-of-flight mass spectrometry (GC-accTOFMS). A total of 58 secondary metabolites, mainly comprising 18 esters, 6 monoterpene hydrocarbons, 2 oxygenated monoterpenes, 20 sesquiterpene hydrocarbons, 7 oxygenated sesquiterpenes, and 4 ketones, were positively or tentatively identified. A total of 24 metabolites were detected in all samples, but commercial cultivars (selected for brewing performance) had fewer compounds identified compared to experimental genotypes. Chemometrics analyses enabled distinct differentiation of experimental hybrids from commercial cultivars, discussed in terms of the different classes of compounds present in different genotypes. Differences among the mono- and sesquiterpenoids, appear to be related to either: i) the genetic origin of the plants; or ii) the processes of bioaccumulation of the identified secondary metabolites.

Incubation of Aquilaria subintegra with Microbial Culture Supernatants Enhances Production of Volatile Compounds and Improves Quality of Agarwood Oil.

Incubation with microbial culture supernatants improved essential oil yield from Aquilaria subintegra woodchips. The harvested woodchips were incubated with de man, rogosa and sharpe (MRS) agar, yeast mold (YM) agar medium and six different microbial culture supernatants obtained from Lactobacillus bulgaricus, L. acidophilus, Streptococcus thermophilus, Lactococcus lactis, Saccharomyces carlsbergensis and S. cerevisiae prior to hydrodistillation. Incubation with lactic acid bacteria supernatants provided higher yield of agarwood oil (0.45% w/w) than that obtained from yeast (0.25% w/w), agar media (0.23% w/w) and water (0.22% w/w). The composition of agarwood oil from all media and microbial supernatant incubations was investigated by using gas chromatography-mass spectrometry. Overall, three major volatile profiles were obtained, which corresponded to water soaking (control), as well as, both YM and MRS media, lactic acid bacteria, and yeast supernatant incubations. Sesquiterpenes and their oxygenated derivatives were key components of agarwood oil. Fifty-two volatile components were tentatively identified in all samples. Beta-agarofuran, α-eudesmol, karanone, α-agarofuran and agarospirol were major components present in most of the incubated samples, while S. cerevisiae-incubated A. subintegra provided higher amount of phenyl acetaldehyde. Microbial culture supernatant incubation numerically provided the highest yield of agarwood oil compared to water soaking traditional method, possibly resulting from activity of extracellular enzymes produced by the microbes. Incubation of agarwood with lactic acid bacteria supernatant significantly enhanced oil yields without changing volatile profile/composition of agarwood essential oil, thus this is a promising method for future use.

Observation and explanation of two-dimensional interconversion of oximes with multiple heart-cutting using comprehensive multidimensional gas chromatography.

Real-time interconversion processes produce unconventional peak broadening in gas chromatography (GC), and can be used to generate kinetic and thermodynamic data. In this study, an unusual separation situation in comprehensive two dimensional GC where two dimensional interconversion (i.e. a raised plateau in both first and second dimension, 1D and 2D) was observed in analysis of oxime isomers. This resulted in a characteristic and unusual rectangular peak shape in the two dimensional result. A related theoretical approach was introduced to explain the peak shape supported by simulation results which can be varied depending on concentration profiles and kinetics of the process. The simulated results were supported by experimental results obtained by a comprehensive heart-cut multidimensional GC (H/C MDGC) approach which was developed to clearly investigate isomerisation of E/Z oxime molecules in both 1D and 2D separations under different isothermal conditions. The carrier gas flow and oven temperature were selected according to initial results for 1D interconversion on a poly(ethyleneglycol) stationary phase, which was further used in both 1D and 2D separations to result in broad zones of oxime interconversion in both dimensions. The method involved repetitive injections of oxime sample, then sampling contiguous fractions of sample into a long 2D column which is intended to promote considerable interconversion. Comprehensiveness arises from the fact that the whole sample is sampled from the 1D to the 2D column, with the long 2D column replacing the short 2D column used in classical comprehensive two-dimensional gas chromatography, where the latter will not promote sufficient interconversion. Data processing and presentation permits a 'rectangular' distribution corresponding to the separated compounds, characteristic of this experiment.

Sequential Hybrid Three-Dimensional Gas Chromatography with Accurate Mass Spectrometry: A Novel Tool for High-Resolution Characterization of Multicomponent Samples.

A novel sequential three-dimensional gas chromatography-high-resolution time-of-flight mass spectrometry (3D GC-accTOFMS) approach for profiling secondary metabolites in complex plant extracts is described. This integrated system incorporates a nonpolar first-dimension (1Dnp) separation step, prior to a microfluidic heart-cut (H/C) of a targeted region(s) to a cryogenic trapping device, directly followed by the rapid reinjection of a trapped solute into a polar second-dimension (2DPEG) column for multidimensional separation (GCnp-GCPEG). For additional separation, the effluent from 2DPEG can then be modulated according to a comprehensive 2D GC process (GC×GC), using an ionic liquid phase as a third-dimension (3DIL) column, to produce a sequential GCnp-GCPEG×GCIL separation. Thus, the unresolved or poorly resolved components, or regions that require further separation, can be precisely selected and rapidly transferred for additional separation on 2D or 3D columns, based on the greater separation realized by these steps. The described integrated system can be used in a number of modes, but one useful approach is to target specific classes of compounds for improved resolution. This is demonstrated through the separation and detection of the oxygenated sesquiterpenes in hop ( Humulus lupulus L.) essential oil and agarwood ( Aquilaria malaccensis) oleoresin. Improved resolution and peak capacity were illustrated through the progressive comparison of the tentatively identified components for GCnp-GCPEG and GCnp-GCPEG×GCIL methods. Relative standard deviations of intraday retentions (1 tR, 2 tR,, and 3 tR) and peak areas of ≤0.01, 0.07, 0.71, and 7.5% were achieved. This analytical approach comprising three GC column selectivities, hyphenated with high-resolution TOFMS detection, should be a valuable adjunct for the improved characterization of complex plant samples, particularly in the area of plant metabolomics.

Untargeted profiling of Glycyrrhiza glabra extract with comprehensive two-dimensional liquid chromatography-mass spectrometry using multi-segmented shift gradients in the second dimension: Expanding the metabolic coverage.

Metabolic profiling of Glycyrrhiza glabra using comprehensive two-dimensional liquid chromatography (LC × LC) coupled with photodiode array (PDA) and mass spectrometry (MS) detection is described. The separation was conducted under reversed-phase conditions, using a combination of first dimension (1 D) 150 mm microbore cyano column utilising 2.7 µm diameter (dp ) particles, and second dimension (2 D) 50 mm superficially porous octadecylsilica column with 2.7 µm dp particles. A multi-segmented shift gradient (MSG) for the 2 D separation was developed, and the orthogonality achieved was compared with other modes of gradients, such as full in-fraction, and shift gradient systems. Results demonstrated a significant expansion of metabolic coverage using MSG in 2 D, providing the highest measure of orthogonality compared to other gradient modes. Compound identifications were performed by employing complementary data from PDA and MS detection, with reference to structural group-type distribution in 2D space. A total of ca. 120 compounds were detected, and among them 37 were tentatively identified, distributed over the chemical families of glycosylated flavanones, triterpene saponins, and others. In comparison with one-dimensional LC, the total number of compounds detected was ca. 2-fold greater when LC × LC was employed. To the best of our knowledge, this is the first demonstration of the MSG mode in LC × LC, representing a powerful strategy to expand the metabolic coverage for analysis of plant-derived extracts, containing a multitude of different phytochemical classes.

Chemotyping of new hop (Humulus lupulus L.) genotypes using comprehensive two-dimensional gas chromatography with quadrupole accurate mass time-of-flight mass spectrometry.

Comprehensive two-dimensional gas chromatography with quadrupole accurate mass time-of-flight mass spectrometry (GC×GC-Q-TOFMS) is employed to profile Humulus lupulus L. (hop) essential oils. Comparison of characterised essential oils allows discrimination among chemotypes. Experimental and commercial hop genotypes displayed distinguishable chemotypic patterns among the volatile secondary metabolites making up their essential oils. In total, 210-306 unique compounds were detected (depending on specific genotype), with 99 of these compounds either positively or tentatively identified. Identified volatile secondary metabolites were grouped into esters, monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes and ketones. Terpenoids were the dominant chemical families across all hop genotypes analysed, representing between 67% and 90% of the total ion count. The multidimensional chromatographic profiles of hop essential oils are extremely information-rich, making GC×GC-Q-TOFMS useful for fast screening of new hybrid hop genotypes, and therefore informing breeding strategies to derive new commercial hop cultivars for the development of distinctive and desirable beers.

Switchable Enantioselective Three- and Four-Dimensional Dynamic Gas Chromatography-Mass Spectrometry: Example Study of On-Column Molecular Interconversion.

A novel hybrid online enantioselective four-dimensional dynamic GC (e4D-DGC) approach to study reversible molecular interconversion through specific isolation of a diastereo and enantiopure oxime, 2-phenylpropanaldehyde oxime, from prior multidimensional separation, is described. It incorporates a pre-enantioseparation step that applies comprehensive two-dimensional GC (GC × GC), prior to multiple microfluidic (Deans) switching for selection of components of a diastereomeric (E,Z) and enantiomeric (R,S) oxime into a third reactor column where isomerization occurs. This is followed by E/Z separation in a fourth analytical column. The enantioselective first dimension (1Denant) yields enantioseparation of E(R), Z(R), E(S), and Z(S) isomers, with a characteristic interconversion zone between the E and Z isomers. However, these are contaminated with underlying stereoisomers. Selected separation regions were then modulated and separated using a second dimension (2D) column via GC × GC, resolving the interfering stereoisomers. Individual pure enantiomers were then selectively heart-cut from within the 2D separation space, cryofocused, then eluted on a 3D reactor column for E ⇌ Z isomerization under controlled oven temperature and flow. Heart-cuts taken over the resulting interconversion distribution were cryotrapped at the inlet of a 4D column, on which achiral separation allows precise quantification of each E and Z isomer of the enantiomer. From peak areas and isomerization time, the forward and backward rate constants (kE→Z and kZ→E) were determined. The described methodology is suited to other configurationally labile molecules (for instance, hydrazones and imines), which exhibit isomerization, and can be used to isolate individual compounds from multicomponent samples, without requiring pure compound synthesis, or complex mathematical models or in-silico simulations.

Qualitative analysis of Copaifera oleoresin using comprehensive two-dimensional gas chromatography and gas chromatography with classical and cold electron ionisation mass spectrometry.

Improved separation of both sesquiterpenes and diterpenic acids in Copaifera multijuga Hayne oleoresin, is demonstrated by using comprehensive two-dimensional gas chromatography (GC×GC) coupled to accurate mass time-of-flight mass spectrometry (accTOFMS). GC×GC separation employs polar phases (including ionic liquid phases) as the first dimension (1D) column, combined with a lower polarity 2D phase. Elution temperatures (Te) of diterpenic acids (in methyl ester form, DAME) increased as the 1D McReynolds' polarity value of the column phase decreased. Since Te of sesquiterpene hydrocarbons decreased with increased polarity, the very polar SLB-IL111 1D phase leads to excessive peak broadening in the 2D apolar phase due to increased second dimension retention (2tR). The combination of SLB-IL59 with a nonpolar column phase was selected, providing reasonable separation and low Te for sesquiterpenes and DAME, compared to other tested column sets, without excessive 2tR. Identities of DAME were aided by both soft (30eV) electron ionisation (EI) accurate mass TOFMS analysis and supersonic molecular beam ionisation (cold EI) TOFMS, both which providing less fragmentation and increased relative abundance of molecular ions. The inter-relation between EI energies, emission current, signal-to-noise and mass error for the accurate mass measurement of DAME are reported. These approaches can be used as a basis for conducting of GC×GC with soft EI accurate mass measurement of terpenes, particularly for unknown phytochemicals.

Evaluation of fast enantioselective multidimensional gas chromatography methods for monoterpenic compounds: Authenticity control of Australian tea tree oil.

This work demonstrates the potential of fast multiple heart-cut enantioselective multidimensional gas chromatography (GC-eGC) and enantioselective comprehensive two-dimensional gas chromatography (eGC×GC), to perform the stereoisomeric analysis of three key chiral monoterpenes (limonene, terpinen-4-ol and α-terpineol) present in tea tree oil (TTO). In GC-eGC, separation was conducted using a combination of mid-polar first dimension ((1)D) column and a chiral second dimension ((2)D) column, providing interference-free enantioresolution of the individual antipodes of each optically active component. A combination of (1)D chiral column and (2)D polar columns (ionic liquid and wax phases) were tested for the eGC×GC study. Quantification was proposed based on summation of two major modulated peaks for each antipode, displaying comparable results with those derived from GC-eGC. Fast chiral separations were achieved within 25min for GC-eGC and<20min for eGC×GC, while ensuring adequate interference-free enantiomer separation. The suitability of using these two enantioselective multidimensional approaches for the routine assessment of chiral monoterpenes in TTO was evaluated and discussed. Exact enantiomeric composition of chiral markers for authentic TTOs was proposed by analysing a representative number of pure TTOs sourced directly from plantations of known provenance in Australia. Consistent enantiomeric fractions of 61.6±1.5% (+):38.4±1.5% (-) for limonene, 61.7±1.6% (+):38.3±1.6% (-) for terpinen-4-ol and 79.6±1.4% (+):20.4±1.4% (-) for α-terpineol were obtained for the 57 authentic Australian TTOs. The results were compared (using principle component analysis) with commercial TTOs (declared as derived from Melaleuca alternifolia) obtained from different continents. Assessing these data to determine adulteration, or additives that affect the enantiomeric ratios, in commercially sourced TTOs is discussed. The proposed method offers distinct advantages over eGC, especially in terms of analysis times and selectivity which can serve as a reliable platform for authenticity control of TTO.

Evaluation of reversible interconversion in comprehensive two-dimensional gas chromatography using enantioselective columns in first and second dimensions.

The reversible molecular interconversion behaviour of a synthesised oxime (2-phenylpropanaldehyde oxime; (C6H5)CH(CH3)CHN(OH)) was investigated by both, single dimensional gas chromatography (1D GC) and comprehensive two-dimensional gas chromatography (GC×GC). Previous studies on small molecular weight oximes were extended to this larger aromatic oxime (molar mass 149.19gmol(-1)) with interest in the extent of interconversion, enantioselective resolution, and retention time. On a polyethylene glycol (PEG; wax-type) column, a characteristic interconversion zone between two antipodes of E and Z isomers was formed by molecules which have undergone isomerisation on the column (E⇌Z). The extent of interconversion was investigated by varying chromatographic conditions (oven temperature and carrier flow rate) to understand the nature of the behaviour observed. The extent of interconversion was negligible in both enantioselective and methyl-phenylpolysiloxane phase-columns, correlating with the low polarity of the stationary phase. In order to obtain isomerisation along with enantio-resolution, a wax-type and an enantioselective column were coupled in either enantioselective-wax or wax-enantioselective order. The most appropriate column arrangement was selected for study by using a GC×GC experiment with either a wax-phase or phenyl-methylpolysiloxane phase as (2)D column. In addition to evaluation of these fast elution columns, a long narrow-bore enantioselective column (10m) was introduced as (2)D, providing an enantioselective-PEG (coupled-column ensemble: (1)D1+(1)D2)×enantioselective ((2)D) column combination. In this instance, the (1)D1 enantioselective column provides enantiomeric separation of the corresponding enantiomers ((R) and (S)) of (E)- and (Z)-2-phenylpropanaldehyde oxime, followed by E/Z isomerisation in the coupled (1)D2 PEG (reactor) column. The resulting chromatographic interconversion region was modulated and separated into either E/Z isomers (achiral (2)D column) or into the respective (R) and (S) enantiomers of the E/Z isomers when using a (2)D enantioselective column. With this arrangement, the isomers underneath the broad interconversion plateau in 1D elution profiles, including the enantiomers, could be resolved, illuminating salient features and understanding of the molecular reversible process of the interconverting molecules during the chromatographic elution. The two-dimensional patterns (contour plots), resulting from the combination of interconversion process and chiral separation, are discussed phenomenologically.

Evaluation of comprehensive two-dimensional gas chromatography with accurate mass time-of-flight mass spectrometry for the metabolic profiling of plant-fungus interaction in Aquilaria malaccensis.

To explore the possible obligate interactions between the phytopathogenic fungus and Aquilaria malaccensis which result in generation of a complex array of secondary metabolites, we describe a comprehensive two-dimensional gas chromatography (GC × GC) method, coupled to accurate mass time-of-flight mass spectrometry (TOFMS) for the untargeted and comprehensive metabolic profiling of essential oils from naturally infected A. malaccensis trees. A polar/non-polar column configuration was employed, offering an improved separation pattern of components when compared to other column sets. Four different grades of the oils displayed quite different metabolic patterns, suggesting the evolution of a signalling relationship between the host tree (emergence of various phytoalexins) and fungi (activation of biotransformation). In total, ca. 550 peaks/metabolites were detected, of which tentative identification of 155 of these compounds was reported, representing between 20.1% and 53.0% of the total ion count. These are distributed over the chemical families of monoterpenic and sesquiterpenic hydrocarbons, oxygenated monoterpenes and sesquiterpenes (comprised of ketone, aldehyde, oxide, alcohol, lactone, keto-alcohol and diol), norterpenoids, diterpenoids, short chain glycols, carboxylic acids and others. The large number of metabolites detected, combined with the ease with which they are located in the 2D separation space, emphasises the importance of a comprehensive analytical approach for the phytochemical analysis of plant metabolomes. Furthermore, the potential of this methodology in grading agarwood oils by comparing the obtained metabolic profiles (pattern recognition for unique metabolite chemical families) is discussed. The phytocomplexity of the agarwood oils signified the production of a multitude of plant-fungus mediated secondary metabolites as chemical signals for natural ecological communication. To the best of our knowledge, this is the most complete information available so far about essential oils of A. malaccensis, which represents a valuable extension to available data for advanced studies on microbial-mediated biotransformation of terpenes, and offers promise for potential discovery of unanticipated phytochemicals, and biotechnological exploitation.

Multidimensional gas chromatography methods for bioanalytical research.

Multidimensional gas chromatography (MDGC) methods are high-resolution volatile chemical separation techniques, and comprise classical heart-cutting MDGC and its more recent incarnation, comprehensive 2D GC. Although available for a long period, MDGC approaches are still not widely practiced in the field of bioanalysis, possibly reflecting the general preference for regular GC versus MDGC approaches. With the recent introduction of '-omic' techniques that emphasize global nontargeted profiling of metabolites within living systems, it is evident that MDGC is gaining momentum as a separation tool, since it offers very high resolution. By untangling metabolites within highly complex biological matrices, and expanding the metabolic coverage, MDGC plays a frontline role in '-omics' based studies. This review highlights state-of-the-art MDGC approaches, and summarizes the recent developments in bioanalytics.

UPLC method for the determination of vitamin E homologues and derivatives in vegetable oils, margarines and supplement capsules using pentafluorophenyl column.

A sensitive and rapid reversed-phase ultra performance liquid chromatographic (UPLC) method for the simultaneous determination of tocopherols (α-, ß-, γ-, δ-), tocotrienols (α-, ß-, γ-, δ-), α-tocopherol acetate and α-tocopherol nicotinate is described. The separation was achieved using a Kinetex pentafluorophenyl (PFP) column (150 × 2.1mm, 2.6 µm) with both photodiode array (PDA) and fluorescence (FL) detectors that were connected in series. Column was thermostated at 42°C. Under a gradient system consisting of methanol and water at a constant flow rate of 0.38 mL min(-1), all the ten analytes were well separated in less than 9.5 min. The method was validated in terms of linearity, limits of detection and quantitation, precision and recoveries. Calibration curves of the ten compounds were well correlated (r(2)>0.999) within the range of 100 to 25,000 µg L(-1) for α-tocopherol acetate and α-tocopherol nicotinate, 10 to 25,000 µg L(-1) for α-tocotrienol and 5 to 25,000 µg L(-1) for the other components. The method is simple and sensitive with detection limits (S/N, 3) of 1.0 to 3.0 µg L(-1) (FL detection) and 30 to 74 µg L(-1) (PDA detection). Relative standard deviations for intra- and inter-day retention times (<1%) and peak areas (≤ 4%) were obtained. The method was successfully applied to the determination of vitamin E in vegetable oils (extra virgin olive, virgin olive, pomace olive, blended virgin and refined olive, sunflower, soybean, palm olein, carotino, crude palm, walnut, rice bran and grape seed), margarines and supplements.