The Effect of a Synthetic Scent on Cheetah Behaviour.

In cheetahs, age at first parturition correlates negatively with reproductive lifespan (asymmetric reproductive aging); therefore, breeding cheetahs at a young age is essential to maximize reproductive performance in this species. However, younger females display a significantly reduced frequency of copulatory behaviour, which negatively affects breeding. Volatile organic compounds (VOCs) are known to regulate appropriate behavioural responses in various species, including reproductive behaviour; moreover, they have proven to play a role in captive breeding methods in cheetahs, as well as mate choice. Therefore, the objective of this study was to evaluate the effect of a synthetic scent (SS) on the frequency of the five oestrous behaviour(s) (sniff, rub, roll, spray, and meow-chirp) known to be indicative of oestrus in female cheetahs. Based on the results of a previous study from our research group, five VOCs, identified in the marking fluid of male cheetahs, and known to be pheromones involved in reproductive behaviour, were used to create the SS. This was accomplished by mixing benzaldehyde, acetophenone, indole, dimethyl disulphide and phenol with (99.9%) ethanol. Seven female cheetahs were then observed for one oestrus cycle without stimulation (control) and then once again while exposed to the SS (treatment), which was sprayed on foil trays placed around the outside of each enclosure. The occurrence of the five oestrous behaviours was recorded and tallied per day of observations. Although the SS did not have a significant effect on the frequency of oestrous behaviours displayed by the females used in this study, five of the seven (71%) did show an increase in their behaviour with the SS when oestrogen concentrations were at their highest (peak oestrus), including three of the four younger females. The SS also significantly increased the sniffing behaviour in general. Although the results of this study do indicate that VOCs influence cheetahs and their behaviour, firm conclusions cannot be drawn due to the low number of animals used, as well as the significant effect the observation methods had on the results. Nonetheless, this study represents the first of this kind in cheetahs, therefore representing an important step in determining the role of VOCs in aiding breeding in captivity.

Chemical Characterization of the Marking Fluid of Breeding and Non-Breeding Male Cheetahs.

Scent is known to play an important role in the reproduction of cheetahs and other felids. In fact, the presence/odor of a male cheetah has been noted to trigger the estrous cycle in females. The objective of this study was to analyze the marking fluid (MF) of male cheetahs from different breeding groups to determine the composition of volatile organic compounds (VOCs) present, with the aim of identifying potential pheromones relating to sexual behavior/attraction in this species. Four breeding (B; age: 8.9 ± 1.3 years old) and four non-breeding (NB; age: 5.5 ± 0.8 years old) males were selected for this study. Samples were collected into a glass beaker, transferred immediately into a 20 mL glass screw-cap vial with a polytetrafluoroethylene (PTFE) coated silicone septum, and stored until analyzed by headspace solid-phase microextraction (HS-SPME) using gas chromatography-mass spectrometry. A contingency test with Fisher's exact test, using the frequency (FREQ) procedure of SAS 9.4, was conducted to determine the difference between the number of VOCs identified per breeding group; furthermore, differences in relative concentration (RC) of the identified VOCs between breeding groups were analyzed using ANOVA for repeated measures with the GLIMMIX procedure. From the 13 MF samples analyzed, 53 VOCs were identified, and 12 were identified in all the samples. Five of these (dimethyl disulfide, benzaldehyde, acetophenone, phenol, and indole) are known to be involved in attraction/sexual behavior in mammals. Between the two groups, the RC of indole was significantly higher in the NB group, whereas the RC of dodecanoic acid was significantly higher in the B group. Although not significant, the RC of benzaldehyde was higher in the B versus the NB group. The results of this study do support the hypothesis of differences in VOCs' between B and NB male cheetahs. However, the overlapping of age and breeding status and the diet differences could not be controlled. Still, the evidence of changes in MF composition in male cheetahs necessitates further studies on possible strategies to improve reproduction in captivity.

Comprehensive Three-Dimensional LC × LC × Ion Mobility Spectrometry Separation Combined with High-Resolution MS for the Analysis of Complex Samples.

Comprehensive two-dimensional liquid chromatography (LC × LC) and ion mobility spectrometry-mass spectrometry (IMS-MS) are increasingly being used to address challenges associated with the analysis of highly complex samples. In this work, we evaluate the potential of the combination of these techniques in the form of a comprehensive three-dimensional LC × LC × IMS separation system. As application, hydrophilic interaction chromatography (HILIC) × reversed phase LC (RP-LC) × IMS-high-resolution MS (HR-MS) was used to analyze a range of phenolic compounds, including hydrolyzable and condensed tannins, flavonoids, and phenolic acids in several natural products. A protocol for the extraction and visualization of the four-dimensional data obtained using this approach was developed. We show that the combination of HILIC, RP-LC, and IMS offers excellent separation of complex phenolic samples in three dimensions. Benefits associated with the incorporation of IMS include improved MS sensitivity and mass-spectral data quality. IMS also provided separation of trimeric procyanidin isomeric species that could not be differentiated by HILIC × RP-LC or HR-MS. On the traveling wave IMS (TWIMS) system used here, both IMS separation performance and the extent of second dimension (2D) undersampling depend on the upper mass scan limit, which might present a limitation for the analysis of larger molecular ions. The performance of the LC × LC × IMS system was characterized in terms of practical peak capacity and separation power, using established theory and taking undersampling and orthogonality into account. An average increase in separation performance by a factor of 13 was found for the samples analyzed here when IMS was incorporated into the HILIC × RP-LC-MS workflow.

Predictive kinetic optimisation of hydrophilic interaction chromatography × reversed phase liquid chromatography separations: Experimental verification and application to phenolic analysis.

Method development and optimisation for comprehensive two-dimensional liquid chromatography (LC × LC) is complex, since this involves the intricate relationships between a large number of experimental parameters with the aim of achieving three conflicting goals: maximising separation performance (peak capacity), minimising analysis time and minimising dilution. This is especially true for the on-line combination of hydrophilic interaction chromatography (HILIC) and reversed phase LC (RP-LC) due to the relative elution strengths of the mobile phases used in these modes, which has severe implications for the modulation process and dilution. In this study we report a predictive kinetic optimisation tool for on-line HILIC × RP-LC which is based on theoretical relationships between the optimisation goals, the target analyte properties and chromatographic parameters (column dimensions, flow rates, mobile phases, injection volumes, etc.), allowing all chromatographic parameters to vary simultaneously within defined ranges. Experimental restrictions, such as pressure limits, flow rates, etc., are enforced to ensure all results are practically achievable on a given instrumental configuration. A Pareto-optimality approach is then used to obtain optimal sets of experimental conditions, from which the one(s) best satisfying the requirements in terms of time, dilution and/or peak capacity can be chosen. To overcome the challenges associated with mobile phase incompatibility in the coupling of HILIC and RP-LC, splitting of the first dimension HILIC flow, dilution with an aqueous make-up, or a combination of both, were investigated to establish the best approach to minimise total dilution and maximise performance. The validity of the methodology is demonstrated by deriving optimal conditions for the HILIC × RP-LC separation of procyanidins on selected columns and subsequent experimental verification of the performance for the separation of a cocoa extract.

Detailed qualitative analysis of honeybush tea (Cyclopia spp.) volatiles by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry and relation with sensory data.

The volatile composition of honeybush (Cyclopia) species was studied by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOF-MS). Headspace-solid phase micro-extraction (HS-SPME) was used to extract the volatile compounds from tea infusions prepared from the three species C. genistoides, C. maculata and C. subternata. A total of 287 compounds were identified, 101 of which were confirmed using reference standards, while the remainder were tentatively identified using mass spectral and retention index (RI) data. The identification power of TOF-MS enabled the tentative identification of 147 compounds for the first time in honeybush tea. The majority of the compounds identified were common to all three Cyclopia species, although there were differences in their relative abundances, and some compounds were unique to each of the species. In C. genistoides, C. maculata and C. subternata 265, 257 and 238 compounds were identified, respectively. Noteworthy was the tentative identification of cinnamaldehyde in particular C. maculata samples, which points to the likely contribution of this compound to their distinct sensory profiles. This study emphasises the complexity of honeybush tea volatile composition and confirms the power of GC×GC combined with TOF-MS for the analysis of such complex samples.

Analysis of honeybush tea (Cyclopia spp.) volatiles by comprehensive two-dimensional gas chromatography using a single-stage thermal modulator.

The applicability of comprehensive two-dimensional gas chromatography (GC×GC) using a single-stage thermal modulator was explored for the analysis of honeybush tea (Cyclopia spp.) volatile compounds. Headspace solid phase micro-extraction (HS-SPME) was used in combination with GC×GC separation on a non-polar × polar column set with flame ionisation (FID) detection for the analysis of fermented Cyclopia maculata, Cyclopia subternata and Cyclopia genistoides tea infusions of a single harvest season. Method optimisation entailed evaluation of the effects of several experimental parameters on the performance of the modulator, the choice of columns in both dimensions, as well as the HS-SPME extraction fibre. Eighty-four volatile compounds were identified by co-injection of reference standards. Principal component analysis (PCA) showed clear differentiation between the species based on their volatile profiles. Due to the highly reproducible separations obtained using the single-stage thermal modulator, multivariate data analysis was simplified. The results demonstrate both the complexity of honeybush volatile profiles and the potential of GC×GC separation in combination with suitable data analysis techniques for the investigation of the relationship between sensory properties and volatile composition of these products. The developed method therefore offers a fast and inexpensive methodology for the profiling of honeybush tea volatiles. Graphical abstract Surface plot obtained for the GC×GC-FID analysis of honeybush tea volatiles.

Evaluation of capillary electrophoresis for the analysis of rooibos and honeybush tea phenolics.

Rooibos and honeybush are popular herbal teas produced from the shrubs of Aspalathus linearis and Cyclopia spp., respectively, which are indigenous to South Africa. Both herbal teas are rich in polyphenols and their consumption is associated with several health benefits, partly ascribed to their phenolic constituents. Quantification of phenolics in extracts and teas for quality control and research purposes is generally performed using HPLC, although dedicated and often species-specific methods are required. CE offers an attractive alternative to HPLC for the analysis of phenolics, with potential benefits in terms of efficiency, speed and operating costs. In this contribution, we report quantitative CZE methods for the analysis of the principal honeybush and rooibos phenolics. Optimal separation for honeybush and rooibos phenolics was achieved in 21 and 32 min, respectively, with good linearity and repeatability. Quantitative data for extracts of "unfermented" and "fermented" rooibos and two honeybush species were statistically comparable with those obtained by HPLC for the majority of compounds. The developed methods demonstrated their utility for the comparison of phenolic contents between different species and as a function of manufacturing processes, thus offering cost effective, although less sensitive and robust, alternatives to HPLC analysis.

Comprehensive Two-Dimensional Hydrophilic Interaction Chromatography (HILIC) × Reversed-Phase Liquid Chromatography Coupled to High-Resolution Mass Spectrometry (RP-LC-UV-MS) Analysis of Anthocyanins and Derived Pigments in Red Wine.

Changes in anthocyanin chemistry represent some of the most important transformations involved in red wine aging. However, accurate analysis of the derived pigments, as required to study the evolution of anthocyanins and tannins during aging, is hampered by their extreme structural diversity, low levels, and the fact that many of these compounds have identical mass spectral characteristics. In this context, chromatographic separation is critical. In this contribution, the application of online hydrophilic interaction chromatography (HILIC) × reversed-phase liquid chromatography (RP-LC) separation coupled to high-resolution mass spectrometry (MS) is described for the detailed characterization of anthocyanins and their derived pigments in aged red wine. A systematic approach was followed for the optimization of HILIC × RP-LC separation parameters using a capillary liquid chromatography (LC) system in the first dimension and an ultrahigh-pressure LC system in the second dimension to ensure maximum sensitivity and performance. Ninety four (94) anthocyanin-derived pigments were tentatively identified in one- and six-year-old Pinotage wines using accurate mass and fragmentation information obtained using quadrupole-time-of-flight mass spectrometry (Q-TOF-MS). Online HILIC × RP-LC-MS was found to offer high-resolution separation, because of the combination of two different separation modes, while the structured elution order observed improved the certainty in compound identification. Therefore, this approach shows promise for the detailed elucidation of the chemical alteration of anthocyanins during wine aging.

Comprehensive two-dimensional liquid chromatographic analysis of anthocyanins.

Anthocyanins are naturally occurring plant pigments whose accurate analysis is hampered by their complexity and unique chromatographic behaviour associated with on-column conversion reactions. This paper reports the evaluation of off-line comprehensive two-dimensional liquid chromatography (LC×LC) for the analysis of anthocyanins. Hydrophilic interaction chromatography (HILIC) was used in the first dimension in combination with reversed phase liquid chromatography (RP-LC) in the second dimension. For the selective detection of anthocyanins, diode array detection was used, while high resolution quadrupole-time-of-flight mass spectrometry (Q-TOF) was used for compound identification. As application, the HILIC×RP-LC separation of diverse anthocyanins in blueberries, red radish, black beans, red grape skins and red cabbage is demonstrated. Off-line HILIC×RP-LC revealed information which could not be obtained by one-dimensional HPLC methods, while the structured elution order for the anthocyanins simplifies compound identification and facilitates the comparison of anthocyanin content of natural products by means of contour plots.

Analytical techniques for wine analysis: an African perspective; a review.

Analytical chemistry is playing an ever-increasingly important role in the global wine industry. Chemical analysis of wine is essential in ensuring product safety and conformity to regulatory laws governing the international market, as well as understanding the fundamental aspects of grape and wine production to improve manufacturing processes. Within this field, advanced instrumental analysis methods have been exploited more extensively in recent years. Important advances in instrumental analytical techniques have also found application in the wine industry. This review aims to highlight the most important developments in the field of instrumental wine and grape analysis in the African context. The focus of this overview is specifically on the application of advanced instrumental techniques, including spectroscopic and chromatographic methods. Recent developments in wine and grape analysis and their application in the African context are highlighted, and future trends are discussed in terms of their potential contribution to the industry.

Comparative metabolic profiling to investigate the contribution of O. oeni MLF starter cultures to red wine composition.

In this research work we investigated changes in volatile aroma composition associated with four commercial Oenococcus oeni malolactic fermentation (MLF) starter cultures in South African Shiraz and Pinotage red wines. A control wine in which MLF was suppressed was included. The MLF progress was monitored by use of infrared spectroscopy. Gas chromatographic analysis and capillary electrophoresis were used to evaluate the volatile aroma composition and organic acid profiles, respectively. Significant strain-specific variations were observed in the degradation of citric acid and production of lactic acid during MLF. Subsequently, compounds directly and indirectly resulting from citric acid metabolism, namely diacetyl, acetic acid, acetoin, and ethyl lactate, were also affected depending on the bacterial strain used for MLF. Bacterial metabolic activity increased concentrations of the higher alcohols, fatty acids, and total esters, with a larger increase in ethyl esters than in acetate esters. Ethyl lactate, diethyl succinate, ethyl octanoate, ethyl 2-methylpropanoate, and ethyl propionate concentrations were increased by MLF. In contrast, levels of hexyl acetate, isoamyl acetate, 2-phenylethyl acetate, and ethyl acetate were reduced or remained unchanged, depending on the strain and cultivar evaluated. Formation of ethyl butyrate, ethyl propionate, ethyl 2-methylbutryate, and ethyl isovalerate was related to specific bacterial strains used, indicating possible differences in esterase activity. A strain-specific tendency to reduce total aldehyde concentrations was found at the completion of MLF, although further investigation is needed in this regard. This study provided insight into metabolism in O. oeni starter cultures during MLF in red wine.

Investigation of the volatile composition of pinotage wines fermented with different malolactic starter cultures using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOF-MS).

Headspace solid phase microextraction in combination with comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (HS-SPME-GC × GC-TOF-MS) was used for the detailed investigation of the impact of malolactic fermentation (MLF) using three commercial Oenococcus oeni strains on the volatile composition of Vitis vinifera cv. Pinotage wines. GC × GC allowed the identification of 115 volatile compounds, including both major constituents and trace-level compounds, in a single analysis. A number of compounds differing in mean concentration levels between the control wines and those fermented with different starter cultures were shown for the first time to be influenced by MLF and/or the bacterial strain. Principal component analysis (PCA) provided excellent separation between the wines fermented with different MLF starter cultures and the control wine. Significantly different levels for some volatile compounds in wines fermented with one of the LAB starter cultures could be indicative of metabolic differences of this strain.

Application of a headspace sorptive extraction method for the analysis of volatile components in South African wines.

A headspace sorptive extraction (HSSE) in combination with thermal desorption gas chromatography-mass spectrometry (TD-GC-MS) method for the analysis of volatile components (alcohols, esters, carbonyls, acids, phenols and lactones) in wine samples was developed. Extraction conditions such as salting-out effects, sorption time, stirring speed, phase ratio, extraction temperature, and effect of pH were thoroughly evaluated as part of method validation. The method was very sensitive with LODs and LOQs between 50 pg/L to 299 microg/L and 0.2 ng/L to 0.996 microg/L, respectively. Repeatability for all the compounds was between 3 and 22%. The intermediate repeatability was obtained within the acceptable range. Out of 39 volatile compounds selected, 37 were detected and quantitated. The method was found to be simple, cost-effective, sensitive, and use a small sample volume. The method was successfully applied for the routine analysis of 79 young red and white wine samples from various South African districts.