Blagoj Mitrevski

Novel hybrid comprehensive 2D-multidimensional gas chromatography for precise, high-resolution characterization of multicomponent samples.

A novel hybrid (sequential) comprehensive 2D-multidimensional gas chromatography (GC × GC-MDGC) method for complex sample manipulation and separation is described. It incorporates a separation step that approximates slow modulation GC × GC, prior to microfluidic Deans switch heart-cutting of a targeted region(s) into a third analytical column. It allows discrete single or multiple components, bands or regions, or any combination of these to be selected and excised from within the 2D GC × GC separation space. The excised individual components can be further collected and studied. Alternatively, any unresolved or poorly resolved components, or regions that require further separation, can be transferred to an additional (third) column separation step. The method is applied to separation and quantitative analysis of oxygenates in a thermally stressed algae-derived biofuel oil by using flame ionization detection (FID), without any prefractionation. This permits oxygenated compounds to be fully resolved from saturated (matrix) compounds, which are completely excluded from the third column. Improved separation was obtained between target classes (aldehydes, 2-ketones, alcohols, acids). Excellent calibration linearity, and retention time and peak area reproducibility were obtained for 14 oxy-compounds present in trace amount in the complex biofuel matrix. Accuracy of microfluidic transfer to the third column, and the profile reproducibility before and after heart-cut operations, was demonstrated by extracting single components from a complex coffee volatile sample.

Application of comprehensive two-dimensional gas chromatography to sterols analysis

The applicability of comprehensive two-dimensional gas chromatography (GCxGC) for sterol analysis was investigated by separation and identification of endogenous sterols in standards, and spiked in human urine. The modulation temperature was optimized to achieve the best separation and signal enhancement. The separation pattern of trimethylsilyl (TMS) derivatives of sterols was compared on two complementary column sets. Whilst the BPX5/BPX50 column set offers better overall separation, BPX50/BPX5 provides better peak shape and sensitivity. Comparison of the identification power of GCxGC-TOFMS against both the NIST05 MS library and a laboratory created (in-house) TOFMS library was carried out on a free sterols extract of urine, derivatised and spiked at the World Anti-Doping Agency (WADA) limit of 2 ng mL(-1). The average match quality for 19 analysed sterols on the BPX50/BPX5 column set was 950/1000 when searched against the in-house library; only four were identified against the NIST05 library, at a match threshold of 800. The match quality of GCxGC-TOFMS spectra was superior to that for analysis using 1D GC-TOFMS for sterols spiked in urine at 10 ng mL(-1). An r(2)>0.997 was obtained for the concentration range between 0.25 ng mL(-1) and 10 ng mL(-1) for three selected sterols. The lowest limit of detection (LOD) was obtained for estrone (0.1 ng mL(-1)) and the highest LOD was for 5alpha-androstan-3alpha,11beta-diol-17-one, epitestosterone and cholesteryl butyrate (1 ng mL(-1)), using a match threshold of at least 800 and signal-to-noise ratio of at least 10. TOFMS coupled to GCxGC enabled satisfactory identification of sterols in urine at their LOD. A minimum acceptable match (MAM) criterion for urinary sterols using 2D retention times and TOF mass spectra is introduced. This study shows that GCxGC-TOFMS yields high specificity for steroids derived from urine, with detection limits appropriate for use in doping control.

Fast comprehensive two-dimensional gas chromatography method for fatty acid methyl ester separation and quantification using dual ionic liquid columns.

Safflower oil is a complex mixture of C18 saturated and unsaturated fatty acids amongst other fatty acids, and achieving separation between these similar structure components using one dimensional gas chromatography (GC) may be difficult. This investigation aims to obtain improved separation of fatty acid methyl esters in safflower oil, and their quantification using comprehensive two-dimensional GC (GC×GC). Here, GC×GC separation is accomplished by the coupling of two ionic liquid (IL) column phases: the combination of SLB-IL111 with IL59 column phases was finally selected since it provided excellent separation of a FAME standard mixture, as well as fatty acids in safflower and linseed oil, compared to other tested column sets. Safflower oil FAME were well separated in a short run of 16min. FAME validation was demonstrated by method reproducibility, linearity over a range up to 500mgL(-1), and limits of detection which ranged from 1.9mgL(-1) to 5.2mgL(-1) at a split ratio of 20:1. Quantification was carried out using two dilution levels of 200-fold for major components and 20-fold for trace components. The fatty acids C15:0 and C17:0 were not reported previously in safflower oil. The SLB-IL111/IL59 column set proved to be an effective and novel configuration for separation and quantification of vegetable and animal oil fatty acids.

Peak Modeling Approach to Accurate Assignment of First-Dimension Retention Times in Comprehensive Two-Dimensional Chromatography

Modeling of first-dimension retention of peaks based on modulation phase and period allows reliable prediction of the modulated peak distributions generated in the comprehensive two-dimensional chromatography experiment. By application of the inverse process, it is also possible to use the profile of the modulated peaks (their heights or areas) to predict the shape and parameters of the original input chromatographic band (retention time, standard deviation, area) for the primary column dimension. This allows an accurate derivation of the first-dimension retention time (RSD 0.02%) which is equal to that for the non-modulated experiment, rather than relying upon the retention time of the major modulated peak generated by the modulation process (RSD 0.16%). The latter metric can produce a retention time that differs by at least the modulation period employed in the experiment, which displays a discontinuity in the retention time vs modulation phase plot at the point of the 180 degrees out-of-phase modulation. In contrast, the new procedure proposed here gives a result that is essentially independent of modulation phase and period. This permits an accurate value to be assigned to the first-dimension retention. The proposed metric accounts for the time on the second-dimension, the phase of the distribution, and the hold-up time that the sampled solute is retained in the modulating interface. The approach may also be based on the largest three modulated peaks, rather than all modulated peaks. This simplifies the task of assigning the retention time with little loss of precision in band standard deviation or retention time, provided that these peaks are not all overloaded in the first or second dimension.

Evaluation of World Anti-Doping Agency criteria for anabolic agent analysis by using comprehensive two-dimensional gas chromatography–mass spectrometry

AbstractThis work presents the validation study of the comprehensive two-dimensional gas chromatography (GC×GC)–time-of-flight mass spectrometry method performance in the analysis of the key World Anti-Doping Agency (WADA) anabolic agents in doping control. The relative abundance ratio, retention time, identification and other method performance criteria have been tested in the GC×GC format to confirm that they comply with those set by WADA. Furthermore, tens of other components were identified with an average similarity of >920 (on the 0–999 scale), including 10 other endogenous sterols, and full mass spectra of 5,000+ compounds were retained. The testosterone/epitestosterone ratio was obtained from the same run. A new dimension in doping analysis has been implemented by addressing separation improvement. Instead of increasing the method sensitivity, which is accompanied by making the detector increasingly “blind” to the matrix (as represented by selected ion monitoring mode, high-resolution mass spectrometry (MS) and tandem MS), the method capabilities have been improved by adding a new “separation” dimension while retaining full mass spectral scan information. Apart from the requirement for the mass spectral domain that a minimum of three diagnostic ions with relative abundance of 5% or higher in the MS spectra, all other WADA criteria are satisfied by GC×GC operation. The minimum of three diagnostic ions arises from the need to add some degree of specificity to the acquired mass spectrometry data; however, under the proposed full MS scan method, the high MS similarity to the reference compounds offers more than the required three diagnostic ions for an unambiguous identification. This should be viewed as an extension of the present criteria to a full-scan MS method. FigureImproved separation of AAS in doping control: GC×GC format

Combined effects of gamma-irradiation and modified atmosphere packaging on quality of some spices

Thyme (Thymus vidgaris L.), rosemary (Rosmarinus officinalis L.), black pepper (Piper nigrum L.) and cumin (Cuminum cyminum L.) in ground form were packaged in either air or 100% N2 and γ-irradiated at 3 different irradiation levels (7kGy, 12kGy, 17kGy). Total viable bacterial count, yeast and mould count, colour, essential oil yield and essential oil composition were determined. Microbial load was not detectable after 12kGy irradiation of all samples. Irradiation resulted in significant changes in colour values of rosemary and black pepper. The discolouration of the irradiated black pepper was lower in modified atmosphere packaging (MAP) compared to air packaging. Essential oil yield of irradiated black pepper and cumin was lower in air packaging compared to MAP. Gamma-irradiation generally decreased monoterpenes and increased oxygenated compounds, but the effect was lower in MAP. Overall, spices should be irradiated under an O2-free atmosphere to minimise quality deterioration.

Multidimensional gas chromatography of oxidative degradation products in algae-derived fuel oil samples using narrow heartcuts and rapid cycle times

To characterize a fuels thermal and storage stability an understanding of the process of oxidation and oxidation pathways is essential. Oxidation pathways commence with hydroperoxides which quickly decompose to form a range of alcohols, acids and other oxygen-containing species. In the presence of significant levels of hydrocarbon-based matrix, analysis of these heteroatomic species is difficult. Applying multidimensional gas chromatography with very narrow heart-cut windows (0.20 min) minimizes the number of compounds transferred to the second dimension (2D) column during each heart-cut. Successive heart-cuts every 2.00 min are taken throughout the analytical run, since each heart-cut has a maximum retention on 2D of <2.00 min on the fast elution 2D column. Subsequent analyses involve incrementing or offsetting the heart-cut windows by 0.20 min, so after 10 analyses, a complete coverage of the sample components can be obtained. On the polar 1D and non-polar 2D phase column arrangement, non-polar matrix compounds elute last on the 2D column, and this determines the largest (2t)R; i.e., (2t)R < P(M) to ensure retained components on 2D will not overlap with subsequent heart-cuts. Heartcutting is supported by cryotrapping at the start of the 2D column in order to provide significantly better resolution. Good quality MS library match data generally demonstrate the high resolution separation of oxygenates achieved. Whilst 1D GC-MS was unsuccessful in identifying any of the oxygen-containing compounds reported here, good correlation of MS data (with average MS library similarity data) for acids (903), alcohols (909), ketones (941) and aldehydes (938) in the sample is obtained. The method requires ten sequential runs, and this can be accomplished automatically once the events table is set up. However if fewer target compounds are to be transferred, a reduced number of sequential runs can be implemented.

Evaluation of comprehensive two-dimensional gas chromatography with flame photometric detection: Potential application for sulfur speciation in shale oil

Flame photometric detection in the sulfur channel has been evaluated for sulfur speciation and quantification in comprehensive two-dimensional gas chromatography [GC × GC-FPD(S)] for S-compound speciation in shale extracts. Signal non-linearity and potential quenching effects were reportedly major limitations of this detector for analysis of sulfur in complex matrices. However, reliable linear relationships with correlation coefficient >0.99 can be obtained if the sum of the square root of each modulation slice in GC × GC is plotted vs. sulfur concentration. Furthermore, the quenching effects are reduced due to essentially complete separation of S-containing components from the hydrocarbon matrix. An increase of S/N of up to 150 times has been recorded for benzothiophene and dibenzothiophene in GC × GC-FPD when compared to GC-FPD due to the modulation process. As a consequence, 10 times lower detection limits were observed in the former mode. The applicability of the method was demonstrated using shale oil sample extracts. Three sulfur classes were completely separated and the target class (thiophenes) was successfully quantified after the rest of the sample was diverted to the second detector by using a heart-cut strategy. Based on the proposed method, 70% of the sulfur in the shale oil was assigned to the thiophenes, 24% to benzothiophenes, and 5% to dibenzothiophene compounds.

Accelerating analysis for metabolomics, drugs and their metabolites in biological samples using multidimensional gas chromatography

Gas chromatography (GC) with mass spectrometry (MS) is one of the great enabling analytical tools available to the chemical and biochemical analyst for the measurement of volatile and semi-volatile compounds. From the analysis result, it is possible to assess progress in chemical reactions, to monitor environmental pollutants in a wide range of soil, water or air samples, to determine if an athlete or horse trainer has contravened doping laws, or if crude oil has migrated through subsurface rock to a reservoir. Each of these scenarios and samples has an associated implementation method for GC-MS. However, few samples and the associated interpretation of data is as complex or important as biochemical sample analysis for trace drugs or metabolites. Improving the analysis in both the GC and MS domains is a continual search for better separation, selectivity and sensitivity. Multidimensional methods are playing important roles in providing quality data to address the needs of analysts.

Quantitative preparative gas chromatography of caffeine with nuclear magnetic resonance spectroscopy

Caffeine test solute was employed in combination with an internal standard (IS), 1,4-dimethoxybenzene, in preparative-gas chromatography (prep-GC), with nuclear magnetic resonance (NMR) experiments. The IS served to: (i) quantify the trapping efficiency of an external trapping assembly, consisting of a capillary column cryotrap at the end of the analytical column; (ii) quantify the solute response in different NMR samples; and (iii) permit correlation of expected level of response of a compound in the NMR experiment, based on relative responses of the IS and solute in the GC result. The recovery rate of caffeine from multiple injections of sample (1×, 2×, 5× and 10×) was 69.6 ± 1.3%, which correlated well (R(2) = 0.999) with the number of injections of compound. The (1)H-NMR spectrum was sufficient to enable structural characterisation of the reference caffeine compound, and was achieved with recovery of amounts of ≤10 μg from a single aliquot. Less than 400 μg of collected caffeine (40 replicate injections) was sufficient for structural characterisation by (13)C-NMR spectral analysis. The method allows development of approaches to separate unknown compounds in complex samples, and to separately use MS and NMR for their characterisation.