Characterization of lipids in complex samples using comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry

Most lipids are a complex mixture of classes of compounds such as fatty acids, fatty alcohols, diols, sterols and hydroxy acids. In this study, the suitability of comprehensive two-dimensional gas chromatography coupled to a time-of-light mass spectrometer is studied for lipid characterization in complex samples. With lanolin, a refined wool wax, as test sample, it is demonstrated that combined methylation plus silylation is the preferred derivatization procedure to achieve (i) high-quality GC x GC separation and (ii) easily recognizable ordered structures in lipid analysis. Optimization of the GC x GC column combination, the influence of the temperature programme on the quality of the separation, and the potential and limitations of automated TOF-MS-based identification are discussed. The combined power of a 2D separation, ordered structures and MS detection is illustrated by the identification of several minor sample constituents.     

全二维气相色谱-飞行时间质谱联用技术分析重馏分油中芳烃组成

通过对升温速度、二维补偿温度、调制周期等关键实验参数的优化,建立了全二维气相色谱-飞行时间质谱(GC×GC-TOF MS)分析重馏分油中芳烃组分的方法,得到了重馏分油芳烃组分按环数分布的二维点阵图。根据谱库检索、标准化合物对照及文献报道,对重馏分油芳烃组分中菲、甲基菲及芘、苯并蒽等常见多环芳烃(PAH)进行了准确定性,并将该方法应用到重馏分油加氢处理工艺研究中,对菲、芘的加氢处理产物进行了定性分析。该研究为重馏分油芳烃组分的准确定性提供了新的技术手段,为加深对油品加氢规律的认识提供了技术支持。全二维气相色谱与普通一维色谱对比,在重馏分油的芳烃组分分析上体现了极大优势。     

Identification of individual acids in a commercial sample of naphthenic acids from petroleum by two-dimensional comprehensive gas chromatography/mass spectrometry

The identification of most individual members of the complex mixtures of carboxylic acids found in petroleum ('naphthenic acids') has eluded chemists for over a century; they remain unresolved by conventional gas chromatographic methods. Recently, however, we successfully used two-dimensional comprehensive gas chromatography/mass spectrometry to identify numerous individual diamondoid acids in the naphthenic acids of oil sands process water (OSPW). We have now applied the same methods to a study of a mixture of commercially available naphthenic acids originally refined from petroleum. The results confirm that OSPW and refined petroleum contain very different distributions of acids, as noted previously, although some of the diamondoid acids recently identified in OSPW were detectable in both. Rather, two-dimensional comprehensive gas chromatography/time-of-flight mass spectrometry (GCxGC/ToF-MS) of the methyl esters of the petroleum acids and of numerous acids synthesised for comparison showed that the former comprised mainly C(8-18) straight-chain, methyl-branched, acyclic isoprenoid, cyclohexyl and isomeric octahydropentalene, perhydroindane and perhydronaphthalene (decalin) acids. Some of the latter bicyclic acids occurred as both the non-alkyl-substituted isomers and the bicyclic ethanoic and propanoic acids. Also present in minor quantities was a range of phenyl carboxylic and substituted phenyl alkanoic acids, and traces of non-acids, including trimethylnaphthalenes, again identified by comparison with the synthesised compounds. These results represent some of the first identifications of multiple individual naphthenic acids in commercial mixtures originating from petroleum and provide a basis for future studies of the petroleum geochemistry, toxicities and environmental impacts of the acids. Furthermore, characterisation of the acids will be important for improving the understanding of the role of naphthenic acids in petroleum engineering, particularly for oil pipeline deposition problems.     

Identification of individual tetra- and pentacyclic naphthenic acids in oil sands process water by comprehensive two-dimensional gas chromatography/mass spectrometry

The oils sands industry of Canada produces large volumes of process water (OSPW) which is stored in large lagoons. The OSPW contains complex mixtures of somewhat toxic, water-soluble, acid-extractable organic matter sometimes called 'naphthenic acids' (NA). Concerns have been raised over the possible environmental impacts of leakage of OSPW and a need has therefore arisen for better characterisation of the NA. Recently, we reported the first identification of numerous individual tricyclic NA in OSPW by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GCxGC/ToF-MS) of the methyl esters. The acids were diamondoid adamantane acids, resulting, it was proposed, from biotransformation of the corresponding alkyladamantane hydrocarbons, which is a known process. Biotransformation of higher alkylated diamondoid hydrocarbons was, until now, unknown but here we describe the identification of numerous pentacyclic NA as diamantane and alkyldiamantane acids, using the same methods. Further, we suggest tentative structures for some of the tetracyclic acids formed, we propose, by ring-opening of alkyldiamantanes. We suggest that this is further evidence that some of the acid-extractable organic matter in the OSPW originates from extensive biodegradation of the oil, whether in-reservoir or environmental, although other oxidative routes (e.g. processing) may also be possible. The results may be important for helping to better focus reclamation and remediation strategies for NA and for facilitating the identification of the sources of NA in contaminated environmental samples.     

Qualitative drug analysis of hair extracts by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry

A technique using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC/TOFMS) is applied to a qualitative analysis of three sample extracts from hair suspected of containing various drug compounds. The samples were also subjected to a quantitative target analysis for codeine, morphine, 6-monoacetylmorphine (6-MAM), amphetamine, methamphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethylamphetamine (MDMA), methadone, and benzylpiperazine (BZP) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). GC × GC/TOFMS provided a non-specific procedure that identified various drugs, metabolites, and impurities not included in the target analysis. They included cocaine, diazepam, and methaqualone (quaalude). Comprehensive GC × GC separation was achieved using twin-stage cryo-modulation to focus eluant from a DB-5ms (5% phenyl) to a BPX50 (50% phenyl) GC column. The TOF mass spectrometer provided unit mass resolution in the mass range m/z 5–1000 and rapid spectral acquisition (≤500 spectra/s). Clean mass spectra of the individual components were obtained using mass spectral deconvolution software. The ‘unknown’ components were identified by comparison with mass spectra stored in a library database.     

Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry peak sorting algorithm

We report a novel peak sorting method for the two-dimensional gas chromatography/time-of-flight mass spectrometry (GC x GC/TOF-MS) system. The objective of peak sorting is to recognize peaks from the same metabolite occurring in different samples from thousands of peaks detected in the analytical procedure. The developed algorithm is based on the fact that the chromatographic peaks for a given analyte have similar retention times in all of the chromatograms. Raw instrument data are first processed by ChromaTOF (Leco) software to provide the peak tables. Our algorithm achieves peak sorting by utilizing the first- and second-dimension retention times in the peak tables and the mass spectra generated during the process of electron impact ionization. The algorithm searches the peak tables for the peaks generated by the same type of metabolite using several search criteria. Our software also includes options to eliminate non-target peaks from the sorting results, e.g., peaks of contaminants. The developed software package has been tested using a mixture of standard metabolites and another mixture of standard metabolites spiked into human serum. Manual validation demonstrates high accuracy of peak sorting with this algorithm.     

Analysis of special surfactants by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry

Multidimensional gas-chromatographic analyses of olesochemically based nonionic, anionic and several cationic surfactants in industrial cleaners are demonstrated. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry allows the simultaneous determination of fatty alcohols, fatty alcohol sulphates and alkyl polyglucosides. In addition, the determination of fatty alcohol ethoxylates up to C₁₀EO₈ (highest degree of ethoxylation) and C₁₈EO₅ (longest C-chain at an ethoxylation degree of five) and the analysis of fatty alcohol alkoxylates that contain ethoxy (EO) and propoxy (PO) groups could be realized. Because of decomposition in the injector and a weak EI-fragmentation, cationic surfactants such as alkyl benzyl dimethyl ammonium chloride could also be identified by their characteristic fragments. Thermogravimetric analyses confirmed that the temperature in a normal GC injector is not high enough to cause thermal decomposition of esterquats. However, we could demonstrate that a modified silylation procedure forms decomposition products of esterquats in the GC injector which are detectable by GC × GC–(TOF)MS and allows the identification of such GC-atypical analytes.     

Application of comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry in human metabolomic studies

In this paper several characteristic features of time-of-flight mass spectrometry in coupling with gas chromatography are demonstrated and the parameters are compared to quadrupole mass analyzer. In the second part, the comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry was applied in human metabolomic field, particularly in the determination of pathological markers of Inherited Metabolic Disorders (IMDs).     

Comparison of comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry and gas chromatography-mass spectrometry for the analysis of tobacco essential oils

A sample of tobacco essential oil was analyzed using gas chromatography-mass spectrometry (GC/MS) and comprehensive two-dimensional gas chromatography coupled to a time-of-flight mass spectrometry (GC × GC/TOFMS), respectively. In the GC/MS analysis, serially coupled columns were used. By comparing the GC/MS results with GC × GC/TOFMS results, many more components in the essential oil could be found within the two-dimensional separation space of GC × GC. The quantitative determination of components in the essential oil was performed by GC × GC with flame ionization detection (FID), using a method of multiple internal standards calibration.     

Validation of automated library-based qualitative screening of pesticides by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry

A method for automated detection and reporting of pesticides in plant materials based on comprehensive two-dimensional GC/time-of-flight MS with library-based detection by software has been developed and validated. Optimum settings for detection parameters such as spectral match threshold and first and second dimension retention time tolerances were assessed with respect to occurrence of false detects and false negatives. Next the method was validated following European Union guidelines established for qualitative screening of pesticides. The validation was largely done in retrospect by using data obtained for spiked samples (235 pesticides, various crops, 0.01-0.2 mg/kg) that had been analyzed previously with routine samples over a period of 18 months. At 0.01 mg/kg, the required 95% confidence level (<5% false negatives) was met for 83 compounds. This increased to 185 compounds at the 0.2 mg/kg level. For a number of pesticides, especially at low levels, it had to be concluded that at this stage the method was not fit-for-purpose to reliably demonstrate the absence of pesticides in samples to be analyzed. On the other hand, the fact that the overall detection rate at 0.01 mg/kg was 71% clearly showed that the method does provide added value for the numerous pesticides that are not covered by quantitative methods because the infrequent occurrence does not justify inclusion in such methods.     

Odour fingerprint acquisition by means of comprehensive two-dimensional gas chromatography-olfactometry and comprehensive two-dimensional gas chromatography/mass spectrometry

The analysis of complex matrices, such as perfumes, by means of gas chromatography-olfactometry (GC-O) can be rather imprecise due to the co-elutions, leading to a possible masking of odour-active trace-level compounds by major interferences or agglomeration of olfactive impressions resulting in unreliable olfactive characterization. To overcome these limits an innovative technique, comprehensive two-dimensional gas chromatography-olfactometry (GC x GC-O), was applied, revealing several relevant co-elutions, as in the linalool and linalyl acetate zones. A total of 177 compounds, out of these 135 odour-active, were detected by GC-O, while about 481 out of 818 compounds presented odour-activity through GC x GC-O analyses. In addition, GC/mass spectrometry (GC/MS) and GC x GC/MS analyses were also performed. Peak assignment was achieved by means of different information sources, such as GC/MS, GC x GC/MS, LRI, injection of standards and olfactive impressions.     

Screening for pesticide residues in oil seeds using solid-phase dispersion extraction and comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry

In this paper, we describe the development of an oil-absorbing matrix solid-phase dispersion extraction with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry suitable for screening of 68 pesticide residues (PRs) in peanut, soybean, rape seed, sesame, and sunflower seed. The 68 PRs include 27 kinds of organophosphorus, 23 organic chlorines, 11 synthetic pyrethroids, and 7 carbamates. Heptachlor epoxide was used as the internal standard. Aminopropyl silica was chosen as the dispersion sorbent of the oil-absorbing matrix solid-phase dispersion extraction and was applied to capture hydrophobic components from high oil samples. A 35-min orthogonal separation was performed by using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry with a nonpolar-polar column set. Identification of 68 PRs in the extract was finished by using the time-of-flight mass spectrometry in the assistance of an automated peak-find and spectral deconvolution software. A screening based on control design was introduced and explained. This screening method considerably reduced the cost for the quantitative and confirmatory analyses. The quality of present screening method was evaluated by the Document No. SANCO/10684/2009. The false positive rate and false negative rate provide a useful tool for the evaluation of screening performance.     

Determination of radix ginseng volatile oils at different ages by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry

Comprehensive 2-D GC (GC x GC) coupled with TOF MS or flame ionization detector (FID) was employed to characterize and quantify the chemical composition of volatile oil in the radixes of Panax ginseng C. A. Mey. (ginseng) at different ages. Thirty-six terpenoids were tentatively identified based on the MS library search and retention index in a ginseng sample at the age of 3 years. An obvious group-type separation was obtained in the GC x GC-TOF MS chromatogram. The data collected by GC x GC-FID were processed using a principal component analysis (PCA) method to classify the samples at different ages. The compounds responsible for the significant differentiation among samples were defined. It was found that the relative abundances of alpha-cadinol, alpha-bisabolol, thujopsene, and n-hexadecanoic acid significantly rise with the increase in age.     

Analysis of Artemisia annua L. volatile oil by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry

Artemisia annua L. is an annual herb native of Asia, it has been used for many centuries for the treatment of fever and malaria. In this paper, analysis of the volatile oil of Artemisia annua L. was performed by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC x GC-TOF MS). Three hundred and three components were tentatively identified and terpene compounds are the main components of Artemisia annua L. volatile oil. Artemisinic acid is tentatively qualified.     

Study of alkyl phosphates in industrial petroleum mixtures by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry

Dialkyl phosphate esters used as gellants in some oil well fracturing processes for conventional oil production can result in contamination of the collected crude. Though the exact mechanism is unclear, such compounds form volatile phosphorus that compromises refinery processes. Our initial research involved producing a comprehensive two-dimensional gas chromatographic method (GC × GC) for the detection and quantification of alkyl phosphate esters in petroleum samples, which surpassed the current method employed in sensitivity and speciation capabilities. However, selective detection is required for such analytes in petroleum matrices. This article describes the application of GC × GC with time-of-flight mass spectrometry for selective detection to the analysis of di- and tri-alkyl phosphates in petroleum samples. Features in the electron impact mass spectra of alkyl phosphates are discussed along with the GC × GC retention characteristics of the compounds. Based on these discussions, a preliminary classification and quantification of alkyl phosphate contamination in a suite of industrial samples is then presented.     

Analysis of tensides in complex samples with comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry

Multidimensional gas-chromatographical analysis of various tensides of natural or synthetic origin in cosmetic products is demonstrated. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry allows the qualitative and quantitative determination of alkyl polyglucosides (AG), fatty alcohol ethoxylates (FAEO), fatty alcohol sulfates (FAS), fatty alcohol ether sulfates (FAES) and cocamidopropyl betaines (CAPB) in shower gel and cleaning agents. The samples were aliquoted in two parts. The first part was silylated, diluted and analysed; then, in order to detect anionic tensides (FAES, FAS) too, the second aliquot was hydrolysed before being silylated for analysis. Because of their amphoteric character, the betaines can only be analysed by gas chromatography after thermal decomposition in the injector, which leads to the corresponding amidoamines among other products.     

Metabolic footprinting of tumorigenic and nontumorigenic uroepithelial cells using two-dimensional gas chromatography time-of-flight mass spectrometry

In this study, gas chromatography mass spectrometry (GC-MS) and two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS) were employed for the metabolic footprinting of a pair of immortalized human uroepithelial cells namely HUC-1 (nontumorigenic) and HUC T-2 (tumorigenic). Both HUC-1 and HUC T-2 cell lines were cultivated in 1 mL of Ham’s F-12 media. Subsequent to 48 h of incubation, 200 μL of cell culture supernatant was protein-precipitated using 1.7 mL of methanol and an aliquot of 1.5 mL of the mixture was separated, dried, trimethylsilyl-derivatized, and analyzed using GC-MS and GC×GC-TOFMS. Metabolic profiles were analyzed using multivariate data analysis techniques to evaluate the changes of the metabolomes. Both GC-MS and GC×GC-TOFMS analyses showed distinct differences in metabolic phenotypes of the normal and tumorigenic human bladder cells (partial least squares-discriminant analysis (PLS-DA) of GC×GC-TOFMS data; two latent variables, R ² X = 0.418, R ² Y = 0.977 and Q ² (cumulative) = 0.852). Twenty metabolites were identified as being statistically different between the two cell types. These metabolites revealed that several key metabolic pathways were perturbed in tumorigenic urothelial cells as compared to the normal cells. Application of GC×GC-TOFMS offered several advantages compared to classical one-dimensional GC-MS which include enhanced chromatographic resolution (without increase in analytical run time), increase in sensitivity, improved identification of metabolites, and also separation of reagent artifacts from the metabolite peaks. Our results reinforced the advantages of GC×GC-TOFMS and the role of metabolomics in characterizing bladder cancer biology using in vitro cell culture models.     

全二维气相色谱-四极杆质谱法检测植物油脂中脂肪酸

建立了植物油脂中31种脂肪酸成分的全二维气相色谱-四极杆质谱(GC×GC-qMS)分析方法。样品经甲酯化衍生后,以DB-1柱(30 m×0.25 mm×0.25 μm)作为一维柱、DB-Wax柱(3.2 m×0.1 mm×0.1 μm)作为二维柱组成柱系统进行分离,在调制周期为3.5 s、四极杆质量扫描范围为m/z 40～350的条件下,植物油脂中31种脂肪酸成分可以在50 min内得到准确和灵敏的检测。将本方法应用于实际样品的分析,灵敏度较传统的气相色谱-质谱法提高了100倍以上,一些植物油中微量的脂肪酸成分也因此被检出。该研究不仅为植物油中脂肪酸成分的分析提供了新的技术手段,同时对于确保食用植物油的质量安全、消除食用植物油的掺假伪劣等均有重要意义。     

Characterisation of Stevia rebaudiana by comprehensive two-dimensional liquid chromatography time-of-flight mass spectrometry

Comprehensive two-dimensional liquid chromatography (LC x LC) connected on-line to electrospray ionisation time-of-flight mass spectrometry (ESI-TOF-MS) was employed for analysis of aqueous extract of Stevia rebaudiana. Different combinations of strong cation-exchange (SCX), amino (NH2), and octadecyl siloxane (C18) stationary phases were tested in the separation of all nine known sweet Stevia glycosides. A combination of C18 as the first-dimension column and NH2 as the second-dimension column fully separated all the glycosides from the matrix. The method proved to be quantitative and repeatable. The limit of detection (S/N=3) for stevioside, a widely used natural sweetener, was 43.4 ng/g in dry leaves. The RSD for retention times was <0.1% and that of peak areas 4.5%.     

Analysis of fresh Mentha haplocalyx volatile components by comprehensive two-dimensional gas chromatography and high-resolution time-of-flight mass spectrometry

Fresh Mentha haplocalyx is a well known traditional Chinese medicinal material (CMM) used in both China and America. This paper reports analysis of the volatile components of fresh Mentha haplocalyx by comprehensive two-dimensional gas chromatography (GCxGC) and high-resolution time-of-flight mass spectrometry (HR-TOF-MS), a combination that provides almost complete chemical separation with elemental composition determination of analytes. 163 ketones and terpenes, including menthol and menthone, were tentatively identified, including enantiomers. This study suggests that GCxGCxHR-TOF-MS is suitable for routine identification of target compounds and enantiomers in CMM.