The off-line combination of high performance liquid chromatography and comprehensive two-dimensional gas chromatography-mass spectrometry: a powerful approach for highly detailed essential oil analysis.

The present contribution is focused on the off-line combination of high performance liquid chromatography (HPLC) and comprehensive two-dimensional gas chromatography-quadrupole mass spectrometry (GC×GC-quadMS), and its application to the detailed qualitative analysis of essential oils. Specifically, a silica column was exploited for the separation of the essential oil constituents in two groups, namely hydrocarbon and oxygenated compounds. After, each HPLC-fraction was reduced in volume, and then subjected to cryogenically modulated GC×GC-quadMS analysis. The volatiles were separated on a normal-phase GC×GC column set, and identified through database matching and linear retention index information. The concentrated HPLC fractions gave origin to unexpectably crowded chromatograms, due to two fundamental GC×GC characteristics, namely the enhanced separation power and sensitivity. The results attained were particularly stimulating with regards to the oxygenated compounds, namely those constituents which contribute most to the essential oil aroma, and are of more use for the evaluation of quality and genuineness. Two genuine Citrus essential oils, bergamot and sweet orange, were subjected to analysis, and compared to applications carried out with a GC-quadMS instrument.

Determination of petitgrain oils landmark parameters by using gas chromatography-combustion-isotope ratio mass spectrometry and enantioselective multidimensional gas chromatography.

Gas chromatography-combustion-isotope mass spectrometry was employed for the assessment of the Carbon isotope ratios of volatiles in Italian mandarin and lemon petitgrain oils. In addition, the composition of the whole oil and the enantiomeric distribution of selected chiral compounds were determined for all the samples by using gas chromatography and by multidimensional and conventional enantioselective gas chromatography. The composition of the oils was compared with previous studies. The enantiomeric distribution of lemon petitgrain oils is here reported for the first time. On the composition of mandarin petitgrain oil, the information available in literature, to date, is relative only to one sample from Egypt. Carbon isotope ratio of several terpene hydrocarbons and of their oxygenated derivatives contained in petitgrains was compared with the δ (13)C(VPDB) values of the same compounds present in the corresponding genuine Italian Citrus peel oil. The results prove that the isotopic values obtained for lemon and mandarin petitgrain oils are very close to those relative to the corresponding peel oils determined in previous studies.

Multidimensional enantio gas chromtography/mass spectrometry and gas chromatography-combustion-isotopic ratio mass spectrometry for the authenticity assessment of lime essential oils (C. aurantifolia Swingle and C. latifolia Tanaka).

This article focuses on the genuineness assessment of Lime oils (Citrus aurantifolia Swingle and C. latifolia Tanaka), by Multi Dimensional Gas Chromatography (MDGC) to determine the enantiomeric distribution of α-thujene, camphene, ß-pinene, sabinene, α-phellandrene, ß-phellandrene, limonene, linalool, terpinen-4-ol, α-terpineol and by gas chromatography-combustion isotope ratio mass spectrometry (GC-C-IRMS) to determine the isotopic ratios of α-pinene, ß-pinene, limonene, α-terpineol, neral, geranial, ß-caryophyllene, trans-α-bergamotene, germacrene B. To the author's knowledge this is the first attempt to assess the authenticity and differentiate Persian Lime from Key lime oils by GC-C-IRMS. The results of the two analytical approaches were compared. The simultaneous use of the two techniques provides more reliable capability to detect adulteration in Citrus essential oils. In fact, in some circumstance only one of the two techniques allows to discriminate adulterated or contaminated oils. In cases where only small anomalies are detected by the two techniques due to subtle adulterations, their synergic use allows to express judgments. The advantage of both techniques is the low number of components the analyst must evaluate, reducing the complexity of the data necessary to deal with. Moreover, the conventional analytical approach based on the evaluation of the whole volatile fraction can fail to reveal the quality of the oils, if the adulteration is extremely subtle.

Enantiomeric distribution of key volatile components in Citrus essential oils

Citrus as many other plants present characteristic distribution of some enantiomers, thus it is often possible to use this parameter for identification, characterization, genuineness, and pharmacological activity assessment. In particular, it is possible to reveal adulteration of different nature, such as addition of synthetic compounds, or natural components of different botanical origin, with drastic changes in the biological and olfactory properties. This study is focused on the evaluation of the enantiomeric excesses of numerous samples of different Citrus species: C. deliciosa Ten., C. limon (L.) Burm., C. bergamia, C. aurantifolia (Christm.) Swing., C. latifolia Tan., C. sinensis (L.) Osbeck, and C. aurantium L. The enantiomeric distribution is determined by direct esGC and, depending on the complexity of the essential oil, by MDGC with a chiral column in the second dimension. The research is focused on the determination of fourteen chiral components which present specific distribution in the essential oils investigated. Particular attention is given to the trend of the enantiomeric distribution during the productive season, so to identify useful parameters for quality assessment also in consideration of the wide range of variability often reported in literature. The components investigated were the following: α-thujene, α-pinene, camphene, β-pinene, sabinene, α-phellandrene, β-phellandrene, limonene, linalool, camphor, citronellal, linalyl acetate, terpinen-4-ol, α-terpineol. The use of MDGC allowed the separation of the enantiomers of camphor and citronellal, otherwise not separated by conventional esGC; however for the separation of the enantiomers of α-pinene it was preferable to use conventional esGC. The MDGC system allowed to determine the enantiomeric distribution of camphene, α- and β-phellandrene in lime essential oil for the first time. The results are discussed in function of seasonal variation and, when possible, in function of the extraction technology, with particular regards to lime oils.

Evaluation of a medium-polarity ionic liquid stationary phase in the analysis of flavor and fragrance compounds.

The present research is focused on the evaluation, in terms of efficiency and polarity, of a recently introduced gas chromatography (GC) column, coated with a 1,12-di(tripropylphosphonium) dodecane bis(trifluoromethansulfonyl) amide ionic-liquid stationary phase (SLB-IL59) and its application to the analysis of a complex essential oil. The ionic liquid column demonstrated very good efficiency, in terms of plate number, and a polarity close to that of the 100% poly(ethyleneglycol) stationary phase. In this preliminary evaluation, the SLB-IL59 30 m column was subjected to bleeding measurements, by means of conventional gas chromatography/mass spectrometry (GC/MS) and, in addition, of comprehensive 2D GC. The SLB-IL59 column (30 m × 0.20 µm d(f), 0.25 mm i.d.) was then evaluated in the analysis of typical essential oil constituents, in the form of pure standard compounds. Resolution toward several analytes was measured and the results were compared to those obtained with both apolar [silphenylene polymer, equivalent to poly(5% diphenyl/95% dimethylsiloxane)] and medium-polarity [100% poly(ethyleneglycol)] stationary phases, namely, the most common columns employed in the analysis of essential oils; peak symmetry, for different essential oil constituents, was also measured and expressed through tailing factors (at 10% of peak height). The final part of the investigation was devoted to the GC/MS analysis of lemon essential oil, with GC-flame ionization detection (FID) used for quantification. Linear retention indices of all the identified compounds were determined, and the data obtained were compared to those attained on the apolar and "wax" columns. The results obtained in the present investigation reveal the great potential of this novel stationary phase, as a medium-polarity alternative, in the analysis of essential oils.

Composition of Egyptian nerolì oil.

The bitter orange flower oil (or neroli) is an essential product, largely used in perfumery. Neroli is obtained by hydrodistillation or steam distillation, from the flowers of bitter orange (Citrus aurantium L.). Since a long time neroli production is limited and its cost on the market is considerably high. The annual production in Tunisia and Morocco is ca. 1500 Kg, representing more than 90% of the worldwide production. A small amount ofneroli is also produced in Egypt, Spain and Comorros (not exceeding 150 kg totally). Due to the high cost, the producers and the users have tried to obtain less expensive products, with odor characters close to that of neroli oil to be used as substitute and sometimes as adulterants of the genuine oil. In this study are investigated five samples of Egyptian neroli oils produced in 2008 and 2009, in the same industrial plant, declared genuine by the producer. For all the samples the composition was determined by GC/FID and by GC/MS-LRI; the samples were also analyzed by esGC to determine the enantiomeric distribution of twelve volatiles and by GC-C-IRMS for the determination of the delta13C(VPDB) values of some mono and sesquiterpene hydrocarbons, alcohols and esters. The analytical procedures allowed to quantitatively determining 86 components. In particular the variation of the composition seems to be dependent on the period of production. In fact, the amount of linalool decreases from March to April while linalyl acetate presents an opposite trend, increasing in the same period. The RSD determined for the delta13C(VPDB) are very small (max. 3.89%), ensuring the authenticity of all samples. The results are also discussed in function of the limits provided by the European Pharmacopoeia (EP) (2004), AFNOR (1995) and ISO (2002) regulations for genuine neroli oils.

Performance evaluation of a rapid-scanning quadrupole mass spectrometer in the comprehensive two-dimensional gas chromatography analysis of pesticides in water.

The performance of a novel rapid-scanning (20,000 amu/s) quadrupole mass spectrometer (qMS) has been evaluated in the comprehensive 2-D gas chromatography (GC×GC) analysis of pesticides contained in water. Analyte extraction was performed by using direct solid-phase microextraction (SPME). The MS system was operated using a rather wide m/z 50-450 mass range and a 33 Hz spectral production rate, a frequency which was found sufficient for reliable quantification. The qMS performance was evaluated considering: (i) number of data points per peak, (ii) mass spectral quality, (iii) extent of peak skewing, and (iv) consistency of retention times. Seven-point calibration curves (external calibration) were constructed for 28 pesticides over the limit of quantification range of 100 µg/L (1, 5, 10, 25, 50, and 100 µg/L). The solid-phase microextraction-GC×GC-qMS method was validated by calculating limits of detection and quantification, intraday peak area precision, accuracy, and intraday retention-time precision. A series of tap water samples were subjected to analysis, fortunately giving negative results.

Reliable identification and quantification of volatile components of sage essential oil using ultra HRGC.

Salvia officinalis (common sage) essential oil was chemically characterized by means of GC-MS, using 100 meter capillary columns with two different stationary phases. Identification of the analytes was carried out by means of the FFNSC mass spectrum library and the use of the Linear Retention Index (LRI) as an extra criterion of library searching. Quantitative analysis was also carried out by means of GC-FID with the internal standard method and the use of relative response factors determined for each chemical class of components. A total of 45 compounds were identified and quantified and reported with their experimental LRI values.

Comprehensive two-dimensional liquid chromatography with evaporative light-scattering detection for the analysis of triacylglycerols in Borago officinalis.

An optimized 2-D liquid chromatography (LC×LC) set-up, based on the different selectivities of a silver ion (Ag) and a non-aqueous reversed phase (NARP), employed in the first (D1) and the second dimension (D2), respectively, in combination with evaporative light-scattering detection (ELSD), has been developed for the analysis of the triacylglycerol (TAG) fraction in a Borago officinalis oil. The 2-D set-up, thanks to the complementary separation selectivity provided by the two columns, allowed to distribute 78 TAGs throughout the 2-D LC retention plane otherwise unachievable by 1-D LC.

Application of a multidimensional gas chromatography system with simultaneous mass spectrometric and flame ionization detection to the analysis of sandalwood oil.

The production and trade of Indian sandalwood oil is strictly regulated, due to the impoverishment of the plantations; for such a reason, Australian sandalwood oil has been evaluated as a possible substitute of the Indian type. International directives report, for both the genuine essential oils, specific ranges for the sesquiterpene alcohols (santalols). In the present investigation, a multidimensional gas chromatographic system (MDGC), equipped with simultaneous flame ionization and mass spectrometric detection (FID/MS), has been successfully applied to the analysis of a series of sandalwood oils of different origin. A detailed description of the system utilized is reported. Three santalol isomers, (Z)-α-trans-bergamotol, (E,E)-farnesol, (Z)-nuciferol, epi-α-bisabolol and (Z)-lanceol have been quantified. LoD (MS) and LoQ (FID) values were determined for (E,E)-farnesol, used as representative of the oxygenated sesquiterpenic group, showing levels equal to 0.002% and 0.003%, respectively. A great advantage of the instrumental configuration herein discussed, is represented by the fact that identification and quantitation of target analytes are carried out in one step, without the need to perform two separate analyses.

Evaluation of tea tree oil quality and ascaridole: a deep study by means of chiral and multi heart-cuts multidimensional gas chromatography system coupled to mass spectrometry detection.

The natural-like assessment of essential oils is a demanding task due to the growing trend toward adulterations. Usually chiral chromatography was used for this purpose due to the capability of assessing stereospecificity which is directly related to the enzymatic pathways of each plant species. On the other hand, the quality of an essential oil involves also the evaluation of its oxidative state, mainly connected with the age and storage conditions. In fact, some modifications in the chemical profile of the oil can occur if not properly preserved. Alterations of the components due to oxidative reactions lead to the formation of peroxides, endoperoxides and epoxides, such as ascaridole and 1,2,4-trihydroxymenthane, usually present in very low amount, formed by the oxidation of terpinen-4-ol and α-terpinene, respectively. Therefore, in the present research, the quality of Australian Tea Tree oil (Melaleuca alternifolia (Maiden & Betche) Cheel, Myrtaceae) was investigated by means of a multi heart-cut multidimensional gas chromatographic system coupled to a mass spectrometer detector and by conventional enantio-GC. The MDGC system allowed the complete separation of the compounds of interest transferred from the first column to a second dimension based on a different separation mechanism. The MS detector at the end of the second column provided the identification of the peaks with high similarity values because of their high purities after the multidimensional separation. Method validation was carried out, in order to use this procedure for routine application, monitoring the repeatability of 1D retention times and 2D peak areas, LoD and LoQ. Finally, enantiomeric ratios for chiral compounds were established to support quality data obtained.

Evaluation of a rapid-scanning quadrupole mass spectrometer in an apolar × ionic-liquid comprehensive two-dimensional gas chromatography system.

Comprehensive two-dimensional gas chromatography (GC×GC) is a powerful technique which can enable a great increase in GC peak capacities. However, since secondary-column separations are very rapid, detectors with a fast acquisition rate are mandatory. Such a requirement has certainly limited the use of the quadrupole mass spectrometer in the GC×GC field. The present research is focused on the evaluation of a novel rapid-scanning quadrupole mass spectrometry (qMS) detector, characterized by a 20,000 amu/s scan speed and a 50 Hz scan frequency, using a 290 amu mass range (40-330 m/z). The performance of the MS system was assessed by analyzing mixtures of 24 allergens, as well as a perfume sample, through GC×GC/qMS. The MS parameters evaluated at different acquisition rates (50, 33, and 25 Hz), as well as in the (simultaneous) scan/selected ion monitoring (SIM) mode, were the number of data points per peak, mass spectrum quality, peak skewing, and sensitivity. Two GC×GC/qMS methods, using the 50 Hz acquisition rate and the scan/SIM mode, were validated. Both methods provided similar results in terms of repeatability, accuracy, and linearity, while a great increase in sensitivity was observed (ca. a factor of 10) under scan/SIM conditions. The validated method proved to be suitable for the analysis of perfume allergens, according to the requirements of Directive 2003/15/EC.

Accurate quadrupole MS peak reconstruction in optimized gas-flow comprehensive two-dimensional gas chromatography.

In the present research, a split-flow comprehensive 2-D GC-quadrupole MS (qMS) method was developed using: a primary apolar 30 m×0.25 mm id×0.25 µm d(f) capillary linked, via a T-union, to a secondary polar 1.0 m×0.05 mm id×0.05 µm d(f) capillary and to a 0.10 m×0.05 mm id×0.05 µm d(f) uncoated column segment. The GC×GC-qMS instrument was equipped with two GC ovens and a loop-type modulator. The polar column was connected to the MS, whereas the uncoated column directed most of the first-dimension effluent to waste and enabled the generation of optimum gas velocities in both dimensions, namely circa 20 and 80 cm/s in the first and second dimensions, respectively. The rapid-scanning qMS was operated at a scan speed of 10,000 amu/s, a 25-Hz data acquisition frequency (scan time+interscan time: 40 ms), and with a normal GC mass range (m/z 40-360). Chromatography bands at the second-dimension outlet were never less than 360 ms wide (6σ), enabling the acquisition of at least 10 spectra/peak.

Analysis of fresh and aged tea tree essential oils by using GCxGC-qMS.

The present research is focused on the qualitative elucidation of the chemical profile of fresh tea tree essential oil and an oxidized, aged (circa 1984) counterpart by using the most powerful analytical tool available today for volatile analysis, namely comprehensive two-dimensional gas chromatography (GCxGC) in combination with a mass spectrometer (MS). The rapid-scanning quadrupole (q) MS system employed generated a sufficient number of spectra/s (20/s) for the reliable identification of the high-speed GCxGC peaks. The total ion current GCxGC-qMS chromatogram of the fresh product was characterized by the presence of approximately 130 unknowns. Among these, 61 peaks were assigned with spectral similarities > or = 90%, while 28 components presented MS library matches in the 80-89% range. With regards to the oxidized essential oil, about 180 volatiles were visible on the 2D plane with 63 of these characterized by library "hits" > or = 90% and 45 presenting similarities within the 80-89% range. The use of the enhanced-resolution 3D methodology enabled the full separation of the samples analyzed and, hence, a clear distinction between the essential oils.

Optimized use of a 50mum ID secondary column in comprehensive two-dimensional gas chromatography-mass spectrometry.

The objective of the present research is directed towards the optimized use of a 50microm ID secondary column, in a comprehensive two-dimensional gas chromatography-quadrupole mass spectrometry (GCxGC-qMS) system. The analytical aim was achieved by exploiting a split-flow GCxGC approach, and a rapid-scanning qMS instrument. The stationary phase combination consisted of an apolar (silphenylene polymer) 30mx0.25mm ID column, linked by means of a Y-union, to an MS-connected 1mx0.05mm ID polar one [poly(ethyleneglycol)], and to a 0.20mx0.05mm ID uncoated capillary segment; the latter was connected to a manually operated split-valve. It will be herein demonstrated that the split-flow GCxGC approach, successfully employed in previous H(2)-based, flame ionization detection experiments, provides equally satisfactory results using mass spectrometric detection and helium as carrier gas. An optimized split-flow GCxGC-qMS method was developed and exploited for the analysis of a perfume sample. The results attained were compared with those observed using the same analytical column combination, but with no flow-splitting. It was found that it is not convenient to employ a 50microm ID secondary column in a conventional GCxGC-MS instrument. On the contrary, the use a 50microm ID secondary column, in a split-flow, twin-oven system, provided a good performance. A recently developed comprehensive chromatography software was used for data processing.

Genuineness assessment of mandarin essential oils employing gas chromatography-combustion-isotope ratio MS (GC-C-IRMS).

Cold-pressed mandarin essential oils are products of great economic importance in many parts of the world and are used in perfumery, as well as in food products. Reconstituted mandarin oils are easy to find on the market; useful information on essential oil authenticity, quality, extraction technique, geographic origin and biogenesis can be attained through high-resolution GC of the volatile fraction, or enantioselective GC, using different chiral stationary phases. Stable isotope ratio analysis has gained considerable interest for the unveiling of citrus oil adulteration, detecting small differences in the isotopic carbon composition and providing plenty of information concerning the discrimination among products of different geographical origin and the adulteration of natural essential oils with synthetic or natural compounds. In the present research, the authenticity of several mandarin essential oils was assessed through the employment of GC hyphenated to isotope ratio MS, conventional GC flame ionization detector, enantioselective GC and HPLC. Commercial mandarin oils and industrial natural (declared as such) mandarin essential oils, characterized by different harvest periods and geographic origins, were subjected to analysis. The results attained were compared with those of genuine cold-pressed Italian mandarin oils, obtained during the 2008-2009 harvest season.

Thorough evaluation of the validity of conventional enantio-gas chromatography in the analysis of volatile chiral compounds in mandarin essential oil: A comparative investigation with multidimensional gas chromatography.

The present research is focused on the determination of the enantiomeric distribution of chiral compounds, contained in mandarin essential oils, by means of conventional chiral gas chromatography with flame ionization detection (enantio-GC-FID); the results attained were compared with those derived from heart-cutting multidimensional GC-mass spectrometry (MDGC/MS), to evaluate the reliability of the monodimensional technique as a tool for quality control. The Deans-switch MDGC system was equipped with two GC ovens, which were connected via a heated transfer line, a flame ionization detector (FID1) in the first dimension and a quadrupole MS as second-dimension detector. The a priori knowledge of potential co-elutions concerning target compounds (an enantiomer and an interfering compound), when using enantio-GC-FID, could enable the use of corrected enantiomer excess values. Correction factors could be calculated through a preliminary GC-FID analysis (using an apolar column), considering the peak areas of the known interferences. The method used for the calculation of a so-called "coelution correction factor" is described, along with some examples.

Characterization of mandarin (Citrus deliciosa Ten.) essential oil. Determination of volatiles, non-volatiles, physico-chemical indices and enantiomeric ratios.

An investigation of 27 samples of mandarin essential oils (Citrus deliciosa Tenore), industrially produced in Sicily during the 2007-2008 season, was performed to determine the composition of the volatile fraction by GC/FID and GC/MS-LRI, the enantiomeric distribution of some monoterpene hydrocarbons and linalol by Es-GC, the non-volatile oxygen heterocyclic components by RP-HPLC/PDA and the physico-chemical indices (relative density, refractive index, optical rotation, residue on evaporation, and UV spectroscopic CD value). This study up-dates the information available in the literature on Sicilian mandarin (C. deliciosa Ten.) essential oils, and provides information on the composition and quality parameters for the evaluation of this product.

Characterization of the yerba mate (Ilex paraguariensis) volatile fraction using solid-phase microextraction-comprehensive 2-D GC-MS.

The present research is focused on the use of a solid-phase microextraction-comprehensive 2-D GC methodology, in the analysis of the volatile fraction of yerba mate. Yerba mate is used for the generation of a tea-like beverage, widely consumed in South America. A rapid-scanning quadrupole mass spectrometer (qMS), employed as a detection system and operated at a 25 Hz scanning frequency, supplied high-quality mass spectra. The effectiveness of the 3-D comprehensive 2-D GC-qMS experiment was compared to that of GC-qMS analysis on the same sample. Peak identification, in both applications, was achieved through MS library matching, with the support of linear retention index data. Apart from a great increase in the number of analytes separated (approx. by a factor of 5) and identified (approx. by a factor of 3.5), the comprehensive 2-D GC-qMS approach enabled the determination of a high number of hazardous contaminants (aliphatic hydrocarbons, polycyclic aromatic hydrocarbons, and plasticizers), barely visible in the GC-qMS analysis.

Optimized use of a 50 microm internal diameter secondary column in a comprehensive two-dimensional gas chromatography system.

The focus of the present research is directed toward the development of a comprehensive two-dimensional gas chromatography (GC x GC) method, characterized by a greatly increased separation power, if compared with GC x GC approaches using classical column combinations. The analytical objective was achieved by using a 0.05 mm internal diameter (i.d.) capillary as second dimension, a split-flow approach reported in previous research (Tranchida, P. Q., Casilli, A., Dugo, P., Dugo, G. and Mondello, L. Anal. Chem. 2007, 79, 2266-2275), and a twin-oven GC x GC instrument. The column combination employed was an orthogonal one: an apolar 30 m x 0.25 mm i.d. column was linked, by means of a Y-union, to a flame ionization detector (FID)-connected high-resolution 1 m x 0.05 mm i.d. polar one and to a 0.20 m x 0.05 mm i.d. uncoated capillary segment; the latter was connected to a manually operated split valve, located on top of the second GC. As previously shown, the generation of optimum gas linear velocities in both dimensions can be attained by splitting gas flows at the outlet of the first dimension (Tranchida, P. Q., Casilli, A., Dugo, P., Dugo, G. and Mondello, L. Anal. Chem. 2007, 79, 2266-2275). An optimized GC x GC method was developed and exploited for the analysis of a complex petrochemical sample. The satisfactory results attained were directly compared with those observed using the same instrumentation, equipped with what can be defined as a classical GC x GC split-flow column set: the same primary column was connected to an FID-linked 1 m x 0.10 mm i.d. polar one and to a 0.30 m x 0.10 mm i.d. uncoated capillary. It will be herein illustrated that there is still room for significant progress in the GC x GC field.