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Purpose: Mebos, a traditional South Africa confection consisting of dried, pulped, and sugared apricots, is rich in fibre and vitamins, but also contains salicylic acid, flavonoids, and citric acid. We report a case of postoperative surgical site bleeding in a healthy patient who consumed approximately 2 kg of mebos per day prior to his elective spinal surgery., Methods: The clinical course of a previously healthy 54-year-old male patient with cauda equina syndrome secondary to lumbar spinal stenosis who underwent surgical intervention with subsequent bleeding into the surgical site is discussed. The cause was investigated through biochemical analysis, thromboelastometry (ROTEM®) and mass and absorption spectrometry were applied to assess flavonoid, citric acid, and salicylic acid content., Results: ROTEM® revealed an abnormal clotting profile with an increased clot forming time, suggesting intrinsic coagulopathy. Mass and absorption spectrometry revealed a high total flavonoid content as well as citric acid concentration in the mebos. Salicylic acid was at detection limits of the instrument., Conclusion: Results highlighted the effect of flavonoids and citric acid and therefore explain the abnormal clotting profile in this patient. Inhibition of coagulation prior to elective surgery is a known contraindication and may pose great risks in spinal surgery. In the present report, we demonstrated an association between inhibition of coagulation and an excess of the flavonoids content and citric acid concentration in mebos consumed in the days prior to elective spinal surgery., (© 2024. The Author(s).)

Introduction: Aspalathus linearis (commonly known as rooibos) is endemic to the Cape Floristic Region of South Africa and is a popular herbal drink and skin phytotherapeutic ingredient, with health benefits derived primarily from its unique phenolic content. Several, seemingly habitat-specific ecotypes from the Cederberg (Western Cape) and Northern Cape have morphological, ecological, genetic and biochemical differences., Objectives and Methods: Despite the commercial popularity of the cultivated variety, the uncultivated ecotypes are largely understudied. To address gaps in knowledge about the biochemical constituency, ultra-performance liquid chromatography-mass spectrometry analysis of fifteen populations was performed, enabling high-throughput metabolomic fingerprinting of 50% (v/v) methanolic extracts. Antioxidant screening of selected populations was performed via three assays and antimicrobial activity on two microbial species was assessed. The metabolomic results were corroborated with total phenolic and flavonoid screening of the extracts., Results and Discussion: Site-specific chemical lineages of rooibos ecotypes were confirmed via multivariate data analyses. Important features identified via PLS-DA disclosed higher relative abundances of certain tentative metabolites (e.g., rutin, aspalathin and apiin) present in the Dobbelaarskop, Blomfontein, Welbedacht and Eselbank sites, in comparison to other locations. Several unknown novel metabolites (e.g., m/z 155.0369, 231.0513, 443.1197, 695.2883) are responsible for metabolomic separation of the populations, four of which showed higher amounts of key metabolites and were thus selected for bioactivity analysis. The Welbedacht and Eselbank site 2 populations consistently displayed higher antioxidant activities, with 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activities of 679.894 ± 3.427 µmol Trolox/g dry matter and 635.066 ± 5.140 µmol Trolox/g dry matter, respectively, in correlation with a high number of phenolic and flavonoid compounds. The contribution of the individual metabolites to the pharmacological effectiveness of rooibos remains unknown and as such, further structural elucidation and phytopharmacological testing is thus urgently needed., (© 2024. The Author(s).)

Stevens' Cure (Umckaloabo) emerged as a patent medicine claiming to treat tuberculosis in the United Kingdom at the beginning of the 20th century. However, due to its identity being shrouded in secrecy, it was never truly accepted by the medical community. It was "rediscovered" in the 1970s and subsequently developed into a very popular and successful phytopharmaceutical for the treatment of upper respiratory tract infections. Whether Stevens' Cure contained the same ingredient(s) as the modern Umckaloabo has not yet been demonstrated. We attempted to elucidate for the first time the identity of the original ingredient by comparative analysis of historical product samples. Three historical samples of Stevens' Cure were compared with Pelargonium sidoides DC. and P. reniforme Curt. root per UPLC-MS analysis. We confirm that the ingredient- P. sidoides DC.-is indeed the same as used in modern phytotherapy. We also attribute the first ethnopharmacological record of P. sidoides DC. being used for the treatment of tuberculosis to C. H. Stevens, the "creator" of Umckaloabo., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Brendler, Stander and van Wyk.)

South Africa has a rich history of medicinal plant species and their documented uses as traditional medicines, and is also home to three well-known, blue-flowered sage species of ethnobotanical importance. The Namaqualand bloublomsalie ( Salvia dentata ) has so far remained unstudied and apparently overlooked. Our study is the first to report on the essential oil chemistry of this medicinally relevant species and provide a comparison with the other two (well-studied) closely related Cape bloublomsalies ( Salvia africana and S. chamelaeagnea ). The data, generated from three geographically isolated populations comprised of 13 individual plants of S. dentata , revealed diagnostically high levels of camphor (14.37%), α-pinene (11.43%), camphene (10.18%), 1,8-cineole (eucalyptol) (9.42%) and bornyl acetate (8.56%) which provide a distinct chemical profile from the other two species.

Hot water blanching at 80 °C for 3 min can be used as a novel pre-treatment step in pomegranate peel to preserve the integrity of the phytochemical content within the peel extracts by lowering or inactivating enzymes such as polyphenol (PPO) oxidase and peroxidase (POD) that are responsible for the break-down of phytochemicals within the peel. The aim of this study was to investigate the effect of hot water blanching pre-treatment on yield, bioactive compounds, antioxidants, enzyme inactivation, and antibacterial activity of ‘Wonderful’, ‘Acco’, and ‘Herskawitz’ pomegranate peel extracts. We used a variety of spectrophotometric-based assays and liquid chromatography mass spectrometry (LC-MS)-based approach to characterize and quantify metabolites within the peel extracts. Blanching significantly (p < 0.05) reduced PPO activity in all peel extracts, with the highest PPO reduction in ‘Herskawitz’ peel extracts at 0.25 U/mL. Furthermore, higher antioxidant activity in ‘Herskawitz’ blanched peel extracts using 2,2-diphenyl-1-picryl hydrazyl (DPPH) antioxidant activity, ferric ion reducing antioxidant power (FRAP), and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging activity at 567.78 ± 9.47 µmol Trolox/g DM, 800.05 ± 1.60 µmol Trolox/g DM, and 915.27 ± 0.61 µmol Trolox/g DM, respectively, was noted. ‘Herskawitz’ blanched peel extracts were recorded with the lowest minimum inhibitory concentration (MIC) value of 80 µg/mL for Gram-positive Bacillus subtilis and Gram-negative Klebsiella pneumoniae bacteria strains. A total of 30 metabolites were present in ‘Acco’ and ‘Herskawitz’ peel extracts and were tentatively identified after LC-MS profiling. This study demonstrates that blanched peel extracts from ‘Herskawitz’ cultivar have great potential for commercial use in value-added products in the nutraceutical, cosmeceutical, and pharmacological industries.

The Sceletium genus has been of medicinal importance in southern Africa for millennia and Sceletium tortuosum (Aizoaceae), one of eight species in the genus has gained pharmaceutical importance as an anxiolytic and anti-depressant due to the presence of mesembrine alkaloids. S. tortuosum is used for the manufacture of herbal teas, dietary supplements and other phytopharmaceutical products. This study aimed to provide a metabolomic characterization of S. tortuosum and its sister species as these are not easy to distinguish using morphology alone. Plant samples were thus collected from various locations in the succulent Karoo (South Africa) and analyzed through liquid chromatography-mass spectrometry (LC-MS), using MS E fragmentation as a putative tool for chemical identities. Metabolomics-based analyses in combination with molecular networking were able to distinguish between the four species of Sceletium based on the presence of 4-(3,4-dimethyoxyphenyl)-4-[2-acetylmethlamino)ethyl]cyclohexanone ( m/z 334.2020; RT 6.60 min), mesembrine ( m/z 290.1757; RT 5.10 min) and 4'-O-demethylmesembrenol ( m/z 276.1597; RT 4.17 min). Metabolomic profiles varied according to the different localities and metabolites occurred at variable quantitative levels in Sceletium ecotypes. Molecular networking provided the added advantage of being able to observe mesembrine alkaloid isomers and coeluting metabolites (from the joubertiamine group) that were difficult to discern without this application. By combining high-throughput metabolomics together with global and feature based-molecular networking, a powerful metabolite profiling platform that is able to discern chemical patterns within and between populations was established. These techniques were able to reveal chemotaxonomic relationships and allowed for the discovery of chemical markers that may be used as part of monitoring protocols during the manufacture of phytopharmaceutical and dietary products based on Sceletium ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Reddy, Stander, Stafford and Makunga.)

The presence of pharmaceutical compounds in the aquatic environment is a significant environmental health concern, which is exacerbated by recent evidence of the contribution of drug metabolites to the overall pharmaceutical load. In light of a recent report of the occurrence of metabolites of antiretroviral drugs (ARVDs) in wastewater, we investigate in the present work the occurrence of further ARVD metabolites in samples obtained from a domestic wastewater treatment plant in the Western Cape, South Africa. Pharmacokinetic data indicate that ARVDs are biotransformed into several positional isomeric metabolites, only two of which have been reported wastewater samples. Given the challenges associated with the separation and identification of isomeric species in complex wastewater samples, a method based on liquid chromatography hyphenated to ion mobility spectrometry-high resolution mass spectrometry (LC-IMS-HR-MS) was implemented. Gradient LC separation was achieved on a sub-2 µm reversed phase column, while the quadrupole-time-of-flight MS was operated in data independent acquisition (DIA) mode to increase spectral coverage of detected features. A mass defect filter (MDF) template was implemented to detect ARVD metabolites with known phase I and phase II mass shifts and fractional mass differences and to filter out potential interferents. IMS proved particularly useful in filtering the MS data for co-eluting species according to arrival time to provide cleaner mass spectra. This approach allowed us to confirm the presence of two known hydroxylated efavirenz and nevirapine metabolites using authentic standards, and to tentatively identify a carboxylate metabolite of abacavir previously reported in literature. Furthermore, three hydroxylated-, two sulphated and one glucuronidated metabolite of efavirenz, two hydroxylated metabolites of nevirapine and one hydroxylated metabolite of ritonavir were tentatively or putatively identified in wastewater samples for the first time. Assignment of the metabolites is discussed in terms of high resolution fragmentation data, while collisional cross section (CCS) values measured for the detected analytes are reported to facilitate further work in this area., Competing Interests: Declaration of Competing Interest The authors have declared no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

'Wonderful' pomegranate ( Punica granatum L.) peel contains a wide range of phytochemicals including vitamins, dietary fibre, phenolic compounds, and antioxidant properties. Yet, it is often used as animal feed or discarded in landfills, which is not the best eco-friendly way to utilize this phenolic-rich bioresource. Finding novel ways of utilizing pomegranate peel waste could prove a more profitable and eco-friendlier alternative that is far more beneficial to the economy. Adding a blanching pre-treatment step at optimal conditions prior to processing of pomegranate peel aids in the inactivation of quality changing enzymes such as polyphenol oxidase (PPO) and peroxidase (POD), which are accountable for the degradation reactions that cause breakdown of nutrients and phytochemicals. This study aimed to determine the effect of blanching at 80 °C for 3 min on the yield, polyphenol content, antioxidant properties, enzyme inactivation, and antibacterial activity of 'Wonderful' pomegranate peel ethanolic extracts from three different harvest maturities (unripe, ripe, and over ripe), including a comprehensive characterization and quantification using liquid chromatography-mass spectrometry (LC-MS). The blanched unripe peel extracts exhibited the highest total phenolic content, total tannin content, 2,2-diphenyl-1-picryl hydrazyl (DPPH) antioxidant activity, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging activity and ferric ion reducing antioxidant power (FRAP) at 14.0 mg gallic acid equivalent (GAE)/g dry mass (DM), 1.0 mg GAE/g DM, 359.1 µmol Trolox/g DM, 912.2 µmol Trolox/g DM and 802.5 µmol Trolox/g DM, respectively. There was significant ( p < 0.05) decrease in PPO and POD activity of all blanched pomegranate peel extracts. The blanched unripe peel extracts had the lowest PPO activity at 0.2 U/g fresh weight (FW), with a 70% PPO inactivation compared to ripe and over ripe harvest, whereas the highest POD inactivation was recorded at 67% in over ripe peel extracts. All blanched peel extracts, irrespective of harvest maturity, had minimum inhibitory concentration (MIC) values at 160 µg/mL against all four bacteria strains tested, which included two Gram-positive bacterial strains ( Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 12600) and two Gram-negative bacteria ( Escherichia coli 11775 and Klebsiella pneumonia ATCC 13883). A total of 25 metabolites including phenolic acids (4), organic acids (1), flavonoids (4), ellagitannins (13), and other polyphenols (3) in all three pomegranate peel samples were tentatively identified after LC-MS profiling. The blanched unripe peel extracts showed significantly higher punicalin α and β, β punicalagin, catechin, epicatechin content at 414 mg/g, and 678 mg/g, 151 mg/g, 229 mg/g, respectively, compared to peel extracts from other harvest maturities. This study provides supportive information for the commercial utilization of pomegranate fruit peel as source of value-added ingredients for the development of novel food, cosmetics, and pharmacological products.

Species from the genus Xenorhabdus, endosymbiotic bacteria of Steinernema nematodes, produce several antibacterial and antifungal compounds, some of which are anti-parasitic. In this study, we report on the effect growth conditions have on the production of antimicrobial compounds produced by Xenorhabdus khoisanae J194. The strain was cultured in aerated and non-aerated broth, respectively, and on solid media. Production of antimicrobial compounds was detected after 24 h of growth in liquid media, with highest levels recorded after 96 h. Highest antimicrobial activity was obtained from cells cultured on solid media. By using ultraperformance liquid chromatography linked to mass spectrometry and HPLC, a plethora of known Xenorhabdus compounds were identified. These compounds are the PAX lipopeptides (PAX 1', PAX 3', PAX 5, and PAX 7E), xenocoumacins and xenoamicins. Differences observed in the MS-MS fractionation patterns collected in this study, when compared to previous studies indicated that this strain produces novel xenoamicins. Three novel antimicrobial compounds, khoicin, xenopep and rhabdin, were identified and structurally characterized based on MS-MS fractionation patterns, amino acid analysis and whole genome analysis. The various compounds produced under the three different conditions indicates that the secondary metabolism of X. khoisanae J194 may be regulated by oxygen, water activity or both. Based on these findings X. khoisanae J194 produce a variety of antimicrobial compounds that may have application in disease control., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Booysen, Rautenbach, Stander and Dicks.)

Senecio coronatus (known as izonkozonko and ubulibazi in Zulu) is commonly used in traditional medicine in South Africa as purification purgative and enemas for infants during weaning. We show for the first time that this species does not contain pyrrolizidine alkaloids and that reported cases of fatal hepatic sinusoidal obstruction syndrome in infants were caused by wrongly identified Senecio species containing large amounts of retrorsine-N-oxide. A validated ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the detection and quantitation of pyrrolizidine alkaloids is described., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ion mobility spectrometry (IMS) is a rapid separation technique capable of extracting complementary structural information to chromatography and mass spectrometry (MS). IMS, especially in combination with MS, has experienced inordinate growth in recent years as an analytical technique, and elicited intense interest in many research fields. In natural product analysis, IMS shows promise as an additional tool to enhance the performance of analytical methods used to identify promising drug candidates. Potential benefits of the incorporation of IMS into analytical workflows currently used in natural product analysis include the discrimination of structurally similar secondary metabolites, improving the quality of mass spectral data, and the use of mobility-derived collision cross-section (CCS) values as an additional identification criterion in targeted and untargeted analyses. This review aims to provide an overview of the application of IMS to natural product analysis over the last six years. Instrumental aspects and the fundamental background of IMS will be briefly covered, and recent applications of the technique for natural product analysis will be discussed to demonstrate the utility of the technique in this field., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Tryptocidine C (TpcC, cyclo[D-Phe 1 -Pro 2 -Trp 3 -D-Trp 4 -Asn 5 -Gln 6 -Trp 7 -Val 8 -Orn 9 -Leu 10 ]) is a broad-spectrum antimicrobial peptide in the tyrothricin complex produced by a soil bacterium, Brevibacillus parabrevis. Electrospray mass spectrometric studies reveal the oligomerisation of TpcC into dimers and higher oligomers, analogous to tyrocidine C (TrcC, Trp 7 replaced by Tyr 7 ). Ion mobility mass spectrometry (IMMS) further confirms the formation of stable peptide dimers and tetramers with diameters of 2.7 nm and 3.3 nm, respectively, calculated from collisional cross section (CCS). Molecular dynamic simulations and docking studies support the formation of amphipathic dimers, with a diameter of 2.5 ± 0.07 nm calculated from low energy model CCS. Circular dichroism and IMMS studies point towards dynamic hydrogen-bonded conformational changes up to 28-33 μM after which the structures become more static (or in equilibrium). Fluorescence studies indicate aromatic stacking of Trp residues with a CMC of 18 μM in aqueous solutions. The concentration and time dependent interaction of Trp in oligomers indicate cooperativity in the TpcC oligomerisation that leads to the formation of higher order microscopic structures. Scanning electron microscopy studies unequivocally shows that TpcC forms nanospheres with a mean diameter of 25 nm. Repeated smaller oligomeric units, possibly dimers and tetramers, self-assemble to form these nanospheres., Competing Interests: Declaration of competing interest We have no conflict of interest to declare., (Copyright © 2020. Published by Elsevier B.V.)

The postharvesting disorder leaf blackening is the main cause of product rejection in Protea during export. In this study, we report an investigation into metabolites associated with leaf blackening in Protea species. Methanol extracts of leaf and involucral bract tissue were analyzed by liquid chromatography hyphenated to photodiode array and high-resolution mass spectrometry (LC-PDA-HRMS), where 116 features were annotated. Analytical data obtained from 37 Protea species, selections, and hybrids were investigated using metabolomics tools, which showed that stems susceptible to leaf blackening cluster together and contained features identified as benzenetriol- and/or hydroquinone-derived metabolites. On the other hand, species, selections, and cultivars not prone to blackening were linked to metabolites with known protective properties against biotic and abiotic stressors. During the browning process, susceptible cultivars also produce these protective metabolites, yet at innately low levels, which may render these species and cultivars more vulnerable to blackening. Metabolites that were found to be correlated to the instigation of the browning process, all comprising benzenetriol- and hydroquinone-glycoside derivatives, are highlighted to provide preliminary insights to guide the development of new Protea cultivars not susceptible to leaf blackening.

The C11-oxy androgens have been implicated in the progression of many diseases and endocrine-linked disorders, such as polycystic ovarian syndrome (PCOS), congenital adrenal hyperplasia, specifically 21-hydroxylase deficiency (21OHD), castration resistant prostate cancer (CRPC), as well as premature adrenarche. While the C11-oxy C 19 steroids have been firmly established in the steroid arena, the C11-oxy C 21 steroids are now also of significance. The current study reports on a high-throughput ultra-performance convergence chromatography tandem mass spectrometry (UPC 2 -MS/MS) method for the separation and quantification of 52 steroids in peripheral serum, which include the C11-oxy C 19 and C11-oxy C 21 steroids. Fifteen deuterium-labelled steroids were included for absolute quantification, which incorporates steroid extraction efficiency, together with one steroid and four non-steroidal compounds serving as quality controls (QC). The 15 min run-time per sample (16 min injection-to-injection time with an 8-step gradient) quantifies 68 analytes in a 2 µL injection volume. A single chromatographic step simultaneously identifies steroids in the mineralocorticoid, glucocorticoid and androgen pathways in adrenal steroidogenesis, together with steroid metabolites produced in the periphery, presenting an analytical method for the application of screening in vivo clinical samples. This study highlights cross-talk between the C11-oxy steroids, and describes the optimisation of multiple reaction monitoring required to measure steroids accurately. The limit of detection for the steroid metabolites ranged from 0.002 to 20 ng/mL and the limit of quantification from 0.02 to 100 ng/mL. The calibration range for the steroids ranged from 0.002 to 1000 ng/mL and for the QC compounds from 0.075 to 750 ng/mL. The method is fully validated in terms of accuracy (%RSD, <13%), precision (including inter-day variability across a three-day period) (%RSD, <16%), recovery (average 102.42%), matrix effect (ranging from -15.25 to 14.25%) and process efficiency (average 101.79%). The dilution protocol for the steroids, internal standards and QC compounds were validated, while the ion ratios of the steroid metabolites (%RSD, <16%) and QC compounds were monitored and the accuracy bias values (%RSD, <9%) were within acceptable limits. The method was subsequently used to quantify steroid levels in a cohort of healthy women. C11-oxy steroid metabolites produced as intermediates in steroidogenic pathways, together with end-products included in the method can potentially characterise the 11β-hydroxyandrostenedione-, C 21 - and C11-oxy backdoor pathways in vivo. The identification of these C11-oxy C 19 and C11-oxy C 21 intermediates would allow insight into active pathways, while steroid metabolism could be traced in patients and reference ranges established in both normal and abnormal conditions. Furthermore, conditions currently undefined in terms of the C11-oxy steroids would benefit from the analysis provided by this method, while the C11-oxy steroids could be further explored in PCOS, 21OHD, CRPC and adrenarche., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Para-aminosalicylic acid (PAS), often the last drug remaining for treatment of drug-resistant tuberculosis, is notorious for causing gastrointestinal intolerance; however, the cause of PAS intolerance is uncertain. The objective of this study was to assess relationships between peak concentrations of PAS administered as a granular slow-release enteric coated formulation, and its metabolites acetyl-PAS and glycine-PAS, and intolerance. PAS and its metabolites were measured in 29 adult patients with drug-resistant tuberculosis at Brooklyn Hospital, Cape Town, randomized to receive granular slow-release enteric-coated PAS 4 g twice daily or 8 g once daily for 1 week, followed by the alternative regimen. Concentrations of PAS and its metabolites were determined by liquid chromatography and tandem mass spectrometry, and a visual analogue scale evaluated intolerance. Spearman's correlation test assessed the relationship between maximum plasma concentrations (C max ) and intolerance scores. A large interindividual variability was observed for the PAS C max (40.42-68.55 mg/L) following 4 g twice daily; (62.69-102.41 mg/L) for 8 g once daily and a similar wide C max range found for the metabolites acetyl-PAS and glycine-PAS. Twenty-six patients reported at least 1 intolerance episode, but most visual analogue scale scores clustered around 0. Significant inverse associations were found between acetyl-PAS C max and bloating (rho = -0.448; P = .025) and diarrhea (rho = -0.407; P = .044) for the twice-daily regimen and a similar inverse association found for glycine-PAS and diarrhea (rho = -0.412; P = .041). Plasma concentrations of the metabolites did not correlate with the occurrence of gastrointestinal symptoms, but higher metabolite concentrations correlated with lower intolerance scores; slow metabolism of PAS and its continued presence in the intestinal tract may be the main cause of intolerance., (© 2019, The American College of Clinical Pharmacology.)

Extracts of Sutherlandia frutescens (cancer bush) exhibit considerable qualitative and quantitative chemical variability depending on their natural wild origins. The purpose of this study was thus to determine bioactivity of extracts from different regions using in vitro antioxidant and anti-cancer assays. Extracts of the species are complex and are predominantly composed of a species-specific set of triterpene saponins (cycloartanol glycosides), the sutherlandiosides, and flavonoids (quercetin and kaempferol glycosides), the sutherlandins. For the Folin-Ciocalteu phenolics test values of 93.311 to 125.330 mg GAE/g DE were obtained. The flavonoids ranged from 54.831 to 66.073 mg CE/g DE using the aluminum chloride assay. Extracts from different sites were also assayed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) radical scavenging method and ferric reducing anti-oxidant power (FRAP) methods. This was followed by an in vitro Cell Titer-Glo viability assay of various ecotypes using the DLD-1 colon cancer cell line. All test extracts displayed anti-oxidant activity through the DPPH • radical scavenging mechanism, with IC 50 values ranging from 3.171 to 7.707 µg·mL -1 . However, the degree of anti-oxidant effects differed on a chemotypic basis with coastal plants from Gansbaai and Pearly Beach (Western Cape) exhibiting superior activity whereas the Victoria West inland group from the Northern Cape, consistently showed the weakest anti-oxidant activity for both the DPPH • and FRAP methods. All extracts showed cytotoxicity on DLD-1 colon cancer cells at the test concentration of 200 µg·mL -1 but Sutherlandia plants from Colesburg (Northern Cape) exhibited the highest anti-cancer activity. These findings confirm that S. frutescens specimens display variability in their bioactive capacities based on their natural location, illustrating the importance of choosing relevant ecotypes for medicinal purposes.

In this study we report a detailed investigation of the polyphenol composition of Protea pure ( P. cynaroides and P. neriifolia ) and hybrid cultivars (Black beauty and Limelight). Aqueous methanol extracts of leaf and bract tissues were analyzed by ultrahigh pressure liquid chromatography hyphenated to photodiode array and ion mobility-high resolution mass spectrometric (UHPLC-PDA-IM-HR-MS) detection. A total of 67 metabolites were characterized based on their relative reversed phase (RP) retention, UV-vis spectra, low and high collision energy HR-MS data, and collisional cross section (CCS) values. These metabolites included 41 phenolic acid esters and 25 flavonoid derivatives, including 5 anthocyanins. In addition, an undescribed hydroxycinnamic acid-polygalatol ester, caffeoyl- O -polygalatol (1,5-anhydro-[6- O -caffeoyl]-sorbitol(glucitol)) was isolated and characterized by 1D and 2D NMR for the first time. This compound and its isomer are shown to be potential chemo-taxonomic markers.

As a contribution towards a better understanding of phenolic variation in the genus Cyclopia (honeybush tea), a collection of 82 samples from 15 of the 23 known species was analysed using liquid-chromatography-high resolution mass spectrometry (UPLC-HRMS) in electrospray ionization (ESI) negative mode. Mangiferin and isomangiferin were found to be the main compounds detected in most samples, with the exception of C. bowiena and C. buxifolia where none of these compounds were detected. These xanthones were found to be absent from the seeds and also illustrated consistent differences between species and provenances. Results for contemporary samples agreed closely with those based on analysis of a collection of ca. 30-year-old samples. The use of multivariate tools allowed for graphical visualizations of the patterns of variation as well as the levels of the main phenolic compounds. Exclusion of mangiferin and citric acid from the data was found to give better visual separation between species. The use of UPLC-HRMS generated a large dataset that allowed for comparisons between species, provenances and plant parts (leaves, pods, flowers and seeds). Phenetic analyses resulted in groupings of samples that were partly congruent with species but not with morphological groupings within the genus. Although different provenances of the same species were sometimes found to be very variable, Principle Component Analysis (PCA) indicated that a combination of compounds have some (albeit limited) potential as diagnostic characters at species level. 74 Phenolic compounds are presented, many of which were identified for the first time in Cyclopia species, with nine of these being responsible for the separation between samples in the PCAs.

The widespread implementation of antiretroviral therapy medication has made antiretroviral drugs (ARVDs) a significant pharmaceutical class in regions of high HIV infection rates. However, relatively little is known regarding the environmental occurrence of these emerging contaminants, and this is especially true for their metabolites. In this work, we report analytical methods to study the simultaneous occurrence of a range of common ARVDs and some of their known metabolites in surface water and wastewater. A novel direct injection liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is reported for the analysis of ARVDs of different therapeutic classes and their selected metabolites in wastewater samples. In addition, a solid phase extraction (SPE) procedure was developed for pre-concentration of ARVs and metabolites from surface water samples. The respective methods proved suitable for the quantitative analysis of six parent ARVDs from three ARV classes, as well as three metabolites. Method validation showed average recoveries of 86% for the direct injection method, and 64% for the SPE method. With the exception of Zidovudine and the metabolites of Zidovudine and Ritonavir, all target ARVDs were detected in wastewater samples from two wastewater treatment plants in the Western Cape, South Africa. Higher concentrations were generally measured in influent compared to effluent samples, in the dry compared to the wet season as well as in chlorinated compared to uv-irradiated effluents. This study contributes for the first time quantitative data on the environmental occurrence of the known metabolites of Nevirapine (12-hydroxy-Nevirapine) and Efavirenz (8,14-dihydroxy-Efavirenz)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 ( 2 D) 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.

Wine varietal thiols are important contributors to wine aroma. The chemical nature of thiols makes them difficult to measure due to low concentrations, high sensitivity to oxidation, and low ionization. Methods for the measurement of thiols usually consist of multiple steps of sample preparation followed by instrumental measurement. Studies have collected large datasets of thiols in white wine but not in red wine, due to the lack of availability of suitable methods. In this study, for the first time, convergence chromatography was used to measure thiols in red wine at ultratrace levels with improved sensitivity compared to previous methods. Performance parameters (selectivity, linearity, limits of detection, precision, accuracy) were tested to demonstrate the suitability of the method for the proposed application. Red wine thiols were measured in South African Pinotage, Shiraz, and Cabernet Sauvignon wines ( n = 16 each). Cultivar differentiation using the thiol profile was demonstrated.

Indigenous and non-commercial fruits can be an important source of antioxidant polyphenols; however, the identity and content of polyphenols from non-commercial fruits are often poorly described. The study aimed to extract, identify, and quantify polyphenols from the skin of the indigenous Africa fruit Ximenia caffra , using solvent extraction. Three solvents (hexane, acetone, and 70% v / v ethanol) over three extraction times (30, 60 and 120 min) were used in a 3² full factorial experimental design to determine effects on polyphenol recovery, and individual polyphenolics were characterised using liquid chromatography high-resolution mass spectrometry (LC-HRMS) . Ethanol was the most effective extraction solvent, and extracts had high levels of total phenolics and flavonoids (65 mg gallic and 40 mg catechin equivalents per gram dry sample respectively), and high antioxidant activity (18.2 mg mL -1 ascorbic acid equivalents). LC-HRMS positively identified 16 compounds, of which 14 were flavonoids including flavonoid glycosides, and indicated that concentrations of some flavonoids decreased for extraction times beyond 60 min. It was concluded that the fruit of Ximenia caffra is rich in natural polyphenolic antioxidants; the present work identified and quantified a number of these, while also establishing suitable solvent extraction conditions for the recovery of these potentially high-value compounds.

In the present study an ultra-performance convergence chromatography tandem mass spectrometry (UPC 2 -MS/MS) analytical method was developed and validated for the determination of 17 C 19 and 14 C 21 steroids, including C11-oxy C 19 and C11-oxy C 21 steroids. The limit of detection and limit of quantification ranged from 0.01 to 10 ng/mL and from 0.01 to 20 ng/mL, respectively, and the method shows the recovery, matrix effect and process efficiency of steroids isolated from a serum matrix to be within acceptable limits. Good accuracy, repeatability and reproducibility were also shown and the method provided excellent sensitivity and selectivity as stereoisomers and regioisomers were also resolved and quantified accurately. Clinical conditions such as congenital adrenal hyperplasia, polycystic ovary syndrome in females and disorders of sex development in neonates and in children, amongst others, are characterized by abnormal steroid levels. Steroid profiling is essential to accurately diagnose steroid levels in the above settings as well as in androgen excess or deficiency in adrenal-linked endocrine diseases. Our method, separating C 19 and C 21 steroids in a single chromatographic step, offers a reduced sample turnover rate in the clinical setting, while providing comprehensive steroid profiles of in vivo steroids in the nmol/L range. This is, to our knowledge, the first method reported to simultaneously separate C 19 and C 21 steroids, together with their C11-hydroxy and C11-keto metabolites -one which may hold promise in the identification of new steroid markers in steroid-linked endocrine diseases, in addition to profiling steroid metabolism and abnormal enzyme activity in patients., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Tea samples from 17 populations of "wild tea" ecotypes Aspalathus linearis (rooibos tea) and 2 populations of Aspalathus pendula were analyzed. Recent advances in column technology together with high-resolution mass spectrometry were applied to improve resolution, facilitating the identification of several new compounds as well as grouping of the wild tea ecotypes according to their chemical composition. The collisional cross-section data obtained from ion mobility-mass spectrometry is reported for the flavonoids in rooibos for the first time. Enzyme pathways for the synthesis of the unique flavonoids found in rooibos tea are also proposed. A. linearis and A. pendula produce similar combinations of main phenolic compounds, with no diagnostically different discontinuities between populations or species. Northern resprouters (Gifberg and Nieuwoudtville) contain higher phenylpropenoic acid glucoside levels while teas from Wupperthal and surrounding areas were found to contain unique dihydrochalcones (phloridzin and a sieboldin analog), which are reported here for the first time.

It was previously observed that the lipopeptide surfactants in surfactin (Srf) have an antagonistic action towards the highly potent antimicrobial cyclodecapeptide, gramicidin S (GS). This study reports on some of the molecular aspects of the antagonism as investigated through complementary electrospray ionization mass spectrometry techniques. We were able to detect stable 1:1 and 2:1 hetero-oligomers in a mixture of surfactin and gramicidin S. The noncovalent interaction between GS and Srf, with the proposed equilibrium: GS~Srf↔GS+Srf correlated to apparent K d values of 6-9 μM in gas-phase and 1 μM in aqueous solution. The apparent K d values decreased with a longer incubation time and indicated a slow oligomerization equilibrium. Furthermore, the low μM K d app values of GS~Srf↔GS+Srf fell within the biological concentration range and related to the 2- to 3-fold increase in [GS] needed for bacterial growth inhibition in the presence of Srf. Competition studies indicated that neither Na + nor Ca 2+ had a major effect on the stability of preformed heterodimers and that GS in fact out-competed Ca 2+ and Na + from Srf. Traveling wave ion mobility mass spectrometry revealed near symmetrical peaks of the heterodimers correlating to a compact dimer conformation that depend on specific interactions. Collision-induced dissociation studies indicated that the peptide interaction is most probably between one Orn residue in GS and the Asp residue, but not the Glu residue in Srf. We propose that flanking hydrophobic residues in both peptides stabilize the antagonistic and inactive peptide hetero-oligomers and shield the specific polar interactions in an aqueous environment. Graphical Abstract ᅟ.

11-Oxygenated steroids such as 11-ketotestosterone and 11-ketodihydrotestosterone have recently been shown to play a putative role in the development and progression of castration resistant prostate cancer. In this study we report on the development of a high throughput ultra-performance convergence chromatography tandem mass spectrometry (UPC(2)-MS/MS) method for the analysis of thirteen 11-oxygenated and six canonical C19 steroids isolated from a cell culture matrix. Using an Acquity UPC(2) BEH 2-EP column we found that UPC(2) resulted in superior selectivity, increased chromatographic efficiency and a scattered elution order when compared to conventional reverse phase ultra-performance liquid chromatography (UPLC). Furthermore, there was a significant improvement in sensitivity (5-50 times). The lower limits of quantification ranged between 0.01-10ngmL(-1), while the upper limit of quantification was 100ngmL(-1) for all steroids. Accuracy, precision, intra-day variation, recovery, matrix effects and process efficiency were all evaluated and found to be within acceptable limits. Taken together we show that the increased power of UPC(2)-MS/MS allows the analyst to complete in vitro assays at biologically relevant concentrations for the first time and in so doing determine the routes of steroid metabolism which is vital for studies of androgen responsive cancers, such as prostate cancer, and could highlight new mechanisms of disease progression and new targets for cancer therapy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A high-performance liquid chromatographic (HPLC) method coupled with diode-array detection (DAD) was optimized for the qualitative analysis of aqueous extracts of Cyclopia genistoides. Comprehensive insight into the phenolic profile of unfermented and fermented sample extracts was achieved with the identification of ten compounds based on comparison with authentic reference standards and the tentative identification of 30 additional compounds by means of electrospray ionization mass spectrometry (ESI-MS) and tandem MS detection. Three iriflophenone-di-O,C-hexoside isomers, three xanthone-dihydrochalcone derivatives and one dihydrochalcone are herein tentatively identified for the first time in C. genistoides. Of special interest is one iriflophenone-di-O,C-hexoside present in large amounts. New compounds (tentatively) identified for the first time in this species, and also in the genus Cyclopia, include two aromatic amino acids, one flavone, an iriflophenone-di-C-hexoside, a maclurin-di-O,C-hexoside, two tetrahydroxyxanthone-C-hexoside isomers, a tetrahydroxyxanthone-di-O,C-hexoside, two symmetric tetrahydroxyxanthone-C-hexoside dimers, nine glycosylated flavanone derivatives and five glycosylated phenolic acid derivatives. The presence of new compound subclasses in Cyclopia, namely aromatic amino acids and glycosylated phenolic acids, was demonstrated. The HPLC-DAD method was successfully validated and applied to the quantitative analysis of the paired sample extracts. In-depth analysis of the chemical composition of C. genistoides hot water extracts gave a better understanding of the chemistry of this species that will guide further research into its medicinal properties and potential uses.

Hydrophilic interaction chromatography (HILIC) provides an alternative separation mode for the analysis of phenolic compounds, in which aqueous-organic mobile phases with polar stationary phases are used. This paper reports the evaluation of HILIC for the analysis of the natural pigments anthocyanins, which are of importance because of their chromophoric properties and a range of health benefits associated with their consumption. Several HILIC stationary phases (silica, diol, amine, cyanopropyl and amide) and mobile phase combinations were evaluated, with the latter proving particularly important due to the distinctive chromatographic behaviour of anthocyanins. Diode array detection was used for selective detection of anthocyanins, while high resolution quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) was used for compound identification. The potential of HILIC separation is demonstrated for a range of anthocyanins varying in glycosylation and acylation patterns found in blueberries, grape skins, black beans, red cabbage and red radish. HILIC is shown to be a complementary separation method to reversed phase liquid chromatography (RP-LC) due to the alternative retention mechanism., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A group of non-ribosomally produced antimicrobial peptides, the tyrocidines from the tyrothricin complex, have potential as antimicrobial agents in both medicine and industry. Previous work by our group illustrated that the more polar tyrocidines rich in Trp residues in their structure were more active toward Gram-positive bacteria, while the more non-polar tyrocidines rich in Phe residues had greater activity toward Plasmodium falciparum, one of the major causative pathogens of malaria in humans. Our group also found that the tyrocidines have pronounced antifungal activity, dictated by the primary sequence of the tyrocidine. By simply manipulating the Phe or Trp concentration in the culture medium of the tyrothricin producer, Bacillus aneurinolyticus ATCC 10068, we were able to modulate the production of subsets of tyrocidines, thereby tailoring the tyrothricin complex to target specific pathogens. We optimized the tailored tyrothricin production using a novel, small-scale, high-throughput deep 96-well plate culturing method followed by analyses of the peptide mixtures using ultra-performance liquid chromatography linked to mass spectrometry. We were able to gradually shift the production profile of the tyrocidines and analogues, as well as the gramicidins between two extremes in terms of peptide subsets and peptide hydrophobicity. This study demonstrated that tyrothricin peptide subsets with targeted activity can be efficiently produced by simple manipulation of the aromatic amino acid profile of the culture medium.

Despite the significant importance of tannins in viticulture and enology, relatively little is known about the detailed chemical composition of these molecules. This is due to challenges associated with the accurate analytical determination of the highly structurally diverse proanthocyanidins which comprise tannins. In this contribution, we address this limitation by demonstrating how online comprehensive two-dimensional liquid chromatography (LC × LC) coupled to high resolution mass spectrometry (HR-MS) can be exploited as a powerful analytical approach for the detailed characterization of grape seed tannins. Hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC) were employed in the two dimensions to provide complementary information in terms of separation according to hydrophilicity and hydrophobicity, respectively. Online coupling of HILIC × RP-LC with fluorescence detection and electrospray ionization MS delivered high resolution analysis in a practical analysis time, while allowing selective detection and facilitating compound identification. Time-of-flight (TOF) MS provided high acquisition rates and sensitivity coupled to accurate mass information, which allowed detection of procyanidins up to a degree of polymerization (DP) of 16 and a degree of galloylation up to 8 in a red grape seed extract. This analytical methodology promises to shed new light on these important grape constituents and potentially on their evolution during wine production.

Adulteration of fruit juices--by the addition of sugar or other less expensive fruit juices as well as preservatives, artificial sweeteners and colours--was tested for by using a developed screening method. The method employs hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) using electrospray ionisation in the negative mode and ultraviolet light detection. Different fruit juices can be differentiated by the content of marker compounds like sorbitol, certain phenolic molecules and their saccharide profile. This method was used to test 46 fruit juice samples from the retail market as well as 12 control samples. The study focused on the main types of fruit juices consumed on the South African market including apple, orange, grape and blends of these juices with other fruits like mango, pear and guava. Overall, the 46 samples tested mostly agreed with label claims. One grape juice sample was adulterated, probably with apple juice. Natamycin above the legal limits was found in two samples. In addition, two samples contained natamycin and one sample benzoate without it being indicated on the label. The method is well suited as a quick screening method for fruit juice adulteration and if used routinely would reduce fruit juice adulteration without the cost of the current array of tests needed for authenticity testing.

A novel, robust and fast ultra-high performance liquid chromatography-MS method has been developed for the simultaneous quantification of reduced glutathione (GSH) and oxidised glutathione (GSSG) in grape juice, wine and model wine solution. Sample preparation is minimal and does not require derivatisation. The method has very good performance in terms of sensitivity and selectivity. The limit of detection was 0.002 and 0.001 mg L(-1) for GSH and GSSG, respectively. The amount of GSH and GSSG released by commercial glutathione-enriched inactivated dry yeast preparations (GSH-IDYs) into a model solution was assessed. Significant differences in the amount of GSH and/or GSSG released into a model wine by different GSH-IDYs were observed, with ethanol influencing this release under certain conditions. The GSH and GSSG levels in grape juice fermentations supplemented with GSH-IDY were also assessed in relation to different addition times during fermentation. GSH-IDY addition can lead to elevated wine GSH levels, provided the supplementation is done early during alcoholic fermentation.

Cyclopia subternata plants are traditionally used for the production of the South African herbal tea, honeybush, and recently as aqueous extracts for the food industry. A C. subternata aqueous extract and mangiferin (a major constituent) are known to have anti-diabetic properties. Variation in phenolic composition and antioxidant capacity is expected due to cultivation largely from seedlings, having implications for extract standardization and quality control. Aqueous extracts from 64 seedlings of the same age, cultivated under the same environmental conditions, were analyzed for individual compound content, total polyphenol (TP) content and total antioxidant capacity (TAC) in a number of assays. An HPLC method was developed and validated to allow quantification of xanthones (mangiferin, isomangiferin), flavanones (hesperidin, eriocitrin), a flavone (scolymoside), a benzophenone (iriflophenone-3-C-β-glucoside) and dihydrochalcones (phloretin-3',5'-di-C-β-glucoside, 3-hydroxyphloretin-3',5'-di-C-hexoside). Additional compounds were tentatively identified using mass spectrometric detection, with the presence of the 3-hydroxyphloretin-glycoside, an iriflophenone-di-O,C-hexoside, an eriodictyol-di-C-hexoside and vicenin-2 being demonstrated for the first time. Variability of the individual phenolic compound contents was generally higher than that of the TP content and TAC values. Among the phenolic compounds, scolymoside, hesperidin and iriflophenone-3-C-β-glucoside contents were the most variable. A combination of the measured parameters could be useful in product standardization by providing a basis for specifying minimum levels.

Fusarium ear rot of maize, caused by Fusarium verticillioides, is an important disease affecting maize production worldwide. Apart from reducing yield and grain quality, F. verticillioides produces fumonisins which have been associated with mycotoxicoses of animals and humans. Currently, no maize breeding lines are known with resistance to F. verticillioides in South Africa. The objective of this study, therefore, was to evaluate 24 genetically diverse maize inbred lines as potential sources of resistance to Fusarium ear rot and fumonisin accumulation in field trials at Potchefstroom and Vaalharts in South Africa. After artificial silk channel inoculation with F. verticillioides, Fusarium ear rot development was determined at harvest and fumonisins B 1 , B 2 , and B 3 quantified. A significant inbred line by location effect was observed for Fusarium ear rot severity (P ≤ 0.001), although certain lines proved to be consistently resistant across both locations. The individual inbred lines also differed considerably in fumonisin accumulation between Potchefstroom and Vaalharts, with differentiation between susceptible and potentially resistant inbred lines only being possible at Vaalharts. A greenhouse inoculation trial was then also performed on a subset of potentially resistant and highly susceptible lines. The inbred lines CML 390, CML 444, CML 182, VO 617Y-2, and RO 549 W consistently showed a low Fusarium ear rot (<5%) incidence at both Potchefstroom and Vaalharts and in the greenhouse. Two of these inbred lines, CML 390 and CML 444, accumulated fumonisin levels <5 mg kg -1 . These lines could potentially act as sources of resistance for use within a maize breeding program.

Anthocyanins are responsible for the colour of red grapes and wine. In addition to their contribution to the sensory properties of wine, these compounds are also of interest due to their beneficial biological properties. Wine anthocyanins exhibit a large structural diversity due to variations in glycosylation and acylation patterns, which is further exacerbated by the diverse reactions involving grape-derived anthocyanins during wine ageing. Chromatographic as well as mass spectrometric resolution of wine anthocyanins is often precluded due to the complexity of these compounds. In this paper we report a rapid, high-efficiency ultra high pressure liquid chromatography (UHPLC) procedure with tandem mass spectrometric (MS/MS) detection for the in-depth screening of wine pigments. Selective detection of wine anthocyanins and derived pigments was achieved utilizing MS/MS in neutral loss scanning mode to observe the loss of dehydrated sugar moieties. This facilitated tentative compound identification based on molar mass information as well as the structured elution order of these compounds. In a second experiment, product ion spectra were recorded to allow identification of the anthocyanidin base using characteristic fragmentation patterns. The proposed methodology therefore involves two analyses for the sensitive and accurate identification of anthocyanins and their derived products in red wines. Mass spectra of wine anthocyanins under high energy collision induced dissociation (CID) conditions are reported, some for the first time. Significantly, chemical alteration of anthocyanins during wine ageing results in an off-set of the predominant fragments for each anthocyanidin base, whilst maintaining similar relative intensities. This allows unambiguous assignment of the derived products of anthocyanidin-glycosides. Using this approach, a total of 121 anthocyanins and derived compounds were identified in wines based on their relative reversed phase elution order as well as mass spectral information., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Eight Döhne Merino rams were used to quantify apparent absorption, distribution to tissues, and excretion of dietary melamine in sheep. Two batches of concentrate pellets were made; one (CON) contained corn gluten meal with no detectable melamine and the other (MEL) contained corn gluten meal that was previously found to be highly contaminated with melamine at 15,117 mg/kg. The MEL pellets contained 1,149 mg/kg of melamine. During a 10-d adaptation period, all the animals received a forage-based diet supplemented with 600 g/d of the CON pellets. This was followed by an 8-d collection period during which 6 of the animals received MEL pellets and 2 received CON pellets. Melamine intake of sheep that received MEL pellets was 0.69 g/d. Blood samples were taken before first ingestion of MEL pellets on d 1 and again on d 3, 6, and 8 of the collection period for melamine and serum creatinine analyses. Feces and urine were collected quantitatively over the 8 d for proximate and melamine analyses. All the animals were slaughtered at the end of the trial, and samples of the LM, liver, kidneys, and abdominal fat were taken for melamine analysis. Data of the 2 sheep that received CON pellets for the duration of the trial confirmed that no melamine was detected in any of the samples, and no statistical analyses were performed on these data. The apparent digestibility or efficiency of absorption of ingested melamine was 76.7%. Melamine was detected in the urine, blood, muscle (LM), and fat tissue of all the sheep that received MEL pellets. Serum melamine concentrations reached 5.4 mg/kg on d 8 of the collection period, and the meat (LM) contained 9.6 mg/kg of melamine. Calculations on the partitioning of ingested melamine suggested that urine is the major excretion route accounting for 53.2%, whereas feces accounted for 23.3% of ingested melamine. Approximately 3.5% of the ingested melamine was detected in muscle. It was concluded that ingested melamine is highly absorbable from the small intestine and that a pathway exists for the distribution of dietary melamine to meat., (© 2011 American Society of Animal Science. All rights reserved.)

Ethyl carbamate (EC) is a known genotoxic carcinogen that is frequently present in alcoholic beverages and is therefore a public health concern. As a consequence, maximum concentration levels for EC in these commodities are legislated in several countries. Quantitative analytical methods are therefore essential to monitor EC levels in beverages. Most published analytical methods for the determination of EC in alcoholic beverages utilise elaborate sample pre-treatment procedures to obtain injectable samples, or yield low sensitivity, for example where direct injection is used. In addition, these procedures often require large volumes of toxic solvents and are not generally applicable to diverse alcoholic beverages. This paper describes a novel procedure for the determination of EC in wines, fortified wines and spirits. The procedure is based on reversed-phase solid-phase extraction (SPE) sample clean-up combined with normal-phase liquid chromatography-atmospheric pressure chemical ionisation tandem mass spectrometric (NP-LC-APCI-MS/MS) analysis. This method provides a rapid, robust and simple analytical procedure suitable for the analysis of a diverse range of alcoholic beverages. The accuracy of the method (expressed as average recovery from diverse matrices) is 94.5%, with limits of detection (LODs) ranging between 0.25 and 0.63 µg l(-1) for different matrices. Benefits such as simplified sample preparation, low detection limits, low solvent consumption and good selectivity render the methodology ideally suited to study the occurrence of EC in diverse commodities. The method was applied to study the occurrence of EC in South African wines, fortified wines and spirits. South African wines, aged 1-9 years, contained 1.8-31 µg l(-1) EC (RSD = 69%, n = 106), fortified wines aged 2-34 years contained 2.8-79 µg l(-1) EC (RSD = 89%, n = 21), and brandies aged 3-20 years contained 4.4-95 µg l(-1) EC (RSD = 105%, n = 26). Factors affecting the formation of EC in these commodities were investigated and storage temperature, alcohol content and pH were found to affect the rate of EC formation. Of these variables storage temperature has by far the greatest effect.

Environmental stresses can significantly alter the synthesis of both primary and secondary metabolites, resulting in medicinal plants with unpredictable biological activity. Here, in vitro shoot cultures of the medicinal plant Sutherlandia frutescens were used to study the impact of three abiotic stresses (nitrogen availability, drought and salinity), primarily on l-canavanine synthesis. This compound, a non-protein amino acid, is amongst those metabolites linked to the health benefits of Sutherlandia extracts. Nitrogen supplied to microplants positively correlated with canavanine levels, exhibited by a fourfold reduction when nitrates provided were halved. Although the biomass generated was lowered under these conditions, a higher capacity for rooting (52%) in comparison to the controls (37%) became evident. Only a small increase of the canavanine content in microplants growing on 100mM NaCl medium was detected, indicating that salinity stress was not a major limitation on cavanine production, but that it played more of a role in vitro on plantlet morphogenesis. Similarly, PEG as a supplement had little to no effect on canavanine synthesis. We conclude that a deeper understanding of the nutritional requirements for the agricultural crop management of S. frutescens, which serves the herbal products industry, is needed., (Copyright © 2010. Published by Elsevier GmbH.)

An LC-MS/MS method for the trace-level determination of 3-alkyl-2-methoxypyrazines in Sauvignon blanc wines is described. 3-Isobutyl-2-methoxypyrazine (IBMP), 3-isopropyl-2-methoxypyrazine (IPMP) and 3-sec-butyl-2-methoxypyrazine (SBMP) were analyzed by reversed phase liquid chromatography coupled to atmospheric pressure chemical ionization, as electrospray ionization was found to suffer from matrix quenching effects. A sample preparation method involving distillation of wine followed by solvent extraction and sufficient preconcentration was developed. Limits of detection and quantification for all three analytes were 0.03 ng/L and 0.10 ng/L, respectively, making the method more sensitive than gas chromatographic methods. IBMP was found to be the most abundant congener in South African Sauvignon blanc wines, with concentrations varying between 0.40 and 44 ng/L in 575 samples. IPMP and SBMP levels varied from <0.03 to 3.9 and <0.03 to 3.2 ng/L, respectively. Statistical investigation indicated no clear correlation between methoxypyrazine content and either geographical origin or vintage. The method was also successfully applied for the quantitation of IBMP in five additional South African wine varieties, including three red wine cultivars. The developed method represents a powerful new tool for the in-depth investigation of these important wine aroma constituents.

Eight lactating Holstein cows were randomly allotted to 2 groups in a trial to establish whether a pathway exists for the transmission of melamine from feed to milk. All cows received oat hay ad libitum and 15 kg of concentrate pellets per cow daily. The concentrate pellets contained either melamine-contaminated corn gluten meal of Chinese origin (melamine treatment) or locally produced melamine-free corn gluten meal (control treatment). Cows in the melamine treatment ingested 17.1 g of melamine per day. Cows were milked twice daily, and milk samples were taken once daily during the afternoon milking for melamine and milk component analyses. Melamine appeared in the milk within 8 h after first ingestion of the melamine containing pellets. Melamine concentration reached a maximum of 15.7 mg/kg within 56 h after first ingestion, with an excretion efficiency of approximately 2%. Milk solids and milk urea nitrogen were not affected by treatment. The melamine concentration dropped rapidly after changing all cows back to the control pellets, but melamine only declined to undetectable levels in the milk more than 6 d (152 h) after last ingestion of melamine. Results from the current trial are important to the feed and dairy industries because, until now, any melamine found in milk and milk products was attributed only to the deliberate external addition of melamine to these products, not to adulterated ingredients in animal feeds.

The interaction between a common soil yeast, Cryptococcus laurentii, and a slow-growing medicinal plant adapted to low-nutrient soils, Agathosma betulina (Berg.) Pillans, was studied. C. laurentii CAB 578 was isolated from the rhizosphere of wild A. betulina, and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis revealed that the yeast was capable of producing polyamines, such as cadaverine and spermine, while growing in vitro in a chemically defined medium. Since the exogenous application of polyamines are known to impact on root growth, these findings supported the results obtained when axenic cultures of A. betulina seedlings were inoculated with C. laurentii CAB 578 and cultivated for 5 months under glasshouse conditions. The presence of the yeast increased root growth by 51%. Using soil dilution plates, it was demonstrated that yeast numbers were greater in the vicinity of the roots than in the bulk soil. In addition, fluoromicroscopy, in combination with the fluorescent probes Fungolight and Calcofluor white, revealed the presence of metabolic active yeast colonies on the rhizoplane 5 months after initiation of the experimentation. The study provided evidence for a symbiosis between C. laurentii and A. betulina.

Gas chromatographic and gas chromatographic-mass spectrometric techniques were employed to identify 83 compounds, including alkanes, alkenes, aldehydes, 2-methylalkanes, carboxylic acids, 1-alkyl formates and alken-1-yl formates, benzoic acid, and cholesterol, in the preorbital secretion of the male suni, Neotragus moschatus. Dimethyl disulfide derivatization and lithium aluminum hydride reduction were used to determine the position of double bonds and to confirm the identity of the functional groups in some of the constituents of the secretion.

The toxicokinetics of ochratoxin A were investigated in vervet monkeys (Cercopithecus aethiops). Three female monkeys were treated intravenously with ochratoxin A at doses, respectively, of 0.8, 1.5 and 2 mg/ kg body weight (BW). Blood and urine samples were collected over a period of 21 days. Plasma and urine extracts were analysed by high-performance liquid chromatography (HPLC) with either fluorescence or negative ion electrospray ionization mass spectrometric detection. The clearance of ochratoxin A from plasma followed a two-compartment model. The elimination half-life of ochratoxin A in the monkeys was determined to be 19-21 days and the average total body clearance was 0.22 +/- 0.07 ml/h per kg and the average apparent distribution volume of the central compartment was 59 +/- 9 ml/kg and the peripheral compartment was 59 +/- 20 ml/kg. No evidence was found for any metabolic conversion of ochratoxin A.