As part of our commitment to empowering Indigenous youth and promoting broader appreciation of Australian Indigenous peoples' knowledges, we have developed science activities centred on antioxidant testing of customarily used plants. Conventional antioxidant assays, such as those based on 2,2 '-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power and total phenolic content, often require reagents that are hazardous, costly or difficult to obtain. By contrast, the assay developed here employs safe, low-cost and readily available materials, while providing results comparable to established methods. Based on the redox properties of Betadine (a povidone-iodine complex) and the intense blue colour formed in a starch-Betadine solution, this Blue Betadine Antioxidant Test (BBAT) demonstrated quantitative agreement with the assays listed above for a range of antioxidant compounds (e.g. r2 = 0.989 based on catechin). When applied to extracts from 10 customarily used Australian native plants, the BBAT results were in good agreement with conventional assays, with results most closely aligned with ABTS and DPPH (concordance correlation coefficients >= 0.95). The BBAT is a safe and accessible method suitable for research, education and outreach, providing a culturally relevant platform for exploring the antioxidant properties of customarily used medicinal and food plants.

Metabolic profiling of blood metabolites, particularly in plasma and serum, is vital for studying human diseases, human conditions, drug interventions and toxicology. The clinical significance of blood arises from its close ties to all human cells and facile accessibility. However, patient-specific variables such as age, sex, diet, lifestyle and health status, along with pre-analytical conditions (sample handling, storage, etc.), can significantly affect metabolomic measurements in whole blood, plasma, or serum studies. These factors, referred to as confounders, must be mitigated to reveal genuine metabolic changes due to illness or intervention onset. This review aims to aid metabolomics researchers in collecting reliable, standardized datasets for NMR-based blood (whole/serum/plasma) metabolomics. The goal is to reduce the impact of confounding factors and enhance inter-laboratory comparability, enabling more meaningful outcomes in metabolomics studies. This review outlines the main factors affecting blood metabolite levels and offers practical suggestions for what to measure and expect, how to mitigate confounding factors, how to properly prepare, handle and store blood, plasma and serum biosamples and how to report data in targeted NMR-based metabolomics studies of blood, plasma and serum.

Three Australian populations of Portulaca oleracea—Common Purslane, Omega Gold and Omega Red—were grown under identical conditions, separated into portions—leaf, bud, stem and root—and their extracts tested for total phenolic content (TPC), Trolox equivalent antioxidant capacity (TEAC), ferric-reducing antioxidant potential (FRAP), and for antioxidant activity against hydroperoxides and thiobarbituric acid reactive substances (TBARS) in a linoleic acid emulsion. Highest TPC was found in Omega Gold and Omega Red roots, with 31.1 and 36.5 mg gallic acid equivalents per gram dry weight (mg GAE/g DW), respectively, being ten times higher than for Common Purslane roots (3.1 mg GAE/g DW). Other plant portions were generally higher for Omega Gold and Omega Red, though with much less difference, i.e., <2-fold variation. Results from other antioxidant tests paralleled those of TPC. Online monitoring of antioxidant activity via post-column reaction with [2,2′-azino-bis-(3-ethyl-benzothiazoline-6-sulfonic acid)] (ABTS●+), revealed a peak with significant activity. Purification of the compound responsible yielded oleracein australis 1, and 1D and 2D NMR data are presented for the first time. The results of this study show that Australian populations of P. oleracea are high in bioactivity and may be superior to the internationally recognised medicinal plant, Common Purslane.

Acacia implexa, Eucalyptus rossii and Exocarpos cupressiformis are native plants of Australia, which were used by the First Peoples for medicinal purposes. In this study, 70% aqueous ethanol crude extracts were prepared from A. implexa bark and leaves, E. rossii leaves and E. cupressiformis leaves, and partitioned via sequential extraction with n-hexane, dichloromethane (DCM), ethyl acetate and ethanol. The crude extracts and fractions were screened for antioxidant activity using a novel, high-throughput lipid-based antioxidant assay, as well as the aqueous ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay and the Folin–Ciocalteu test for total phenols. In the lipid-based assay, non-polar n-hexane and DCM fractions showed higher antioxidant activity against the formation of peroxides and thiobarbituric acid reactive substances (TBARS) than the other fractions, whereas the non-polar fractions were not effective in aqueous assays. This illustrates that the high potential of the lipid-soluble n-hexane and DCM fractions as antioxidants would have been missed if only aqueous-based assays were used. In addition, the potent antioxidant compounds were putatively annotated using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-qTOF-MS). Gallic acid, (+)-catechin, (−)-epicatechin and tannins were found in most crude extracts.

Thin layer chromatography (TLC) has replaced paper chromatography as the most practised variant of planar chromatography. It is widely used in some niche areas but it deserves much greater recognition as a complementary technique to the more popular gas and high-performance liquid chromatography. The range of stationary phases available nowadays has expanded greatly from the still popular silica gel. Developments in automation and densitometry have made quantitative analysis by TLC a more viable option.

It is rare that a sample arrives in the laboratory in a state suitable for direct chromatography. The usual situation involves receipt of a sample that must be processed in a manner that makes it amenable to chromatographic analysis. In most instances, this will involve preparing a solution of the sample, or, in the more usual case, recovery of the analytes from the sample matrix. Several processes have been developed over many decades for selective analyte recovery and elimination of matrix components. Pre-concentration of the analytes often involved an additional step prior to chromatographic measurement but is performed simultaneously with the recovery step in many modern methods of sample preparation.

High-performance liquid chromatography is an established separation technique that is widely used for the separation of non-volatile species. Instrumental components of an HPLC are outlined including notable developments and innovations, particularly with respect to pump and column technology. Detection of eluted compounds is an essential part of the process and details are provided for the most common and newly emerging detectors. Gradient elution theory and optimization are also discussed since, unlike GC separations, the mobile phase in HPLC can be varied throughout the separation. Innovations in liquid chromatography including the introduction of UHPLC and multidimensional liquid chromatography are also discussed.

Chromatography involves separation of solutes resulting from differential distribution of the solutes between two phases. Three concurrent phenomena occur during chromatography: separation of solute bands, dispersion of the solute molecules leading to broadening of solute bands, and distortion of the solute bands. Separation is desirable and depends on appropriate selection of the two phases and on operating conditions whereas solute dispersion and band distortion are unavoidable but are counterproductive. Chromatographic theory aims to understand and model these three phenomena. A thorough understanding of chromatographic theory can assist in optimizing separation while minimizing broadening and distortion. Plate theory and rate theory are the two most recognized approaches to the theory of chromatography.

Gas chromatography is the most mature of all chromatographic techniques. It can be performed with a solid or liquid stationary phase although most separations are performed with the latter. Materials used as stationary phases for both techniques are described. Mobile phases for GC are not interactive but they still play an important role in the separation process. Mobile phase selection and instrumental settings are discussed. Detection of eluted compounds is an essential part of the process and details are provided for the most common detectors. Although GC is a mature technique there are still innovative approaches that are described. The chapter concludes with a brief description of the separation mechanism in GSC and GLC plus broad aspects of applications.

There are a variety of separation mechanisms (or modes) available for use in high-performance liquid chromatography (HPLC) making HPLC a versatile analytical tool for the separation of a diverse range of solutes, including non-polar, polar, ionic, ionizable, chiral, and polymeric compounds. This chapter divides the separation modes into three broad groups based on both the nature of the solutes and the separation mechanism, and explores the separation of neutral compounds, ionogenic compounds, and specialty separations. Stationary and mobile phases for each separation mode are discussed and example applications are provided. The final section in the chapter discusses aspects of method selection.

Coupling of analytical techniques has followed two distinct but in some ways closely related approaches. Separation techniques such as gas chromatography and high-performance liquid chromatography have been coupled in order to improve separating power. Separation techniques have also been coupled with spectrometric techniques to improve identification of analytes. However, in both cases, it is the limited resolving power of the various techniques that necessitates coupling. The process follows a cycle of improved resolving power revealing greater complexity of samples followed by increasing legislative and consumer demands and expectations requiring greater resolving power.

Chromatography is simultaneously a process, a method, and a branch of science. It may be classified in different ways; one approach distinguishes planar techniques from those involving a column. It provides both qualitative and quantitative information as well as being applicable at the ultra-trace level and preparative scale. Coverage of this monograph includes all aspects of chromatography with the exception of large-scale industrial applications. This chapter serves as an introduction to chromatography and to the rest of the monograph.

Chromatography generally falls into one of two distinct categories as analytical or preparative chromatography depending on the goals. The two processes are not mutually exclusive but rather complementary, analytical chromatography providing information on what, and how much, of an analyte(s) is present and preparative chromatography providing a method of isolation and purification of analyte(s). Preparative chromatography can involve any of the approaches that are used in analytical chromatography, namely, thin layer, gas, supercritical fluid, and high-performance liquid chromatography. Preparative chromatography is introduced in this chapter.

This chapter examines the reasons for the intense interest in oxidative processes and by extension in antioxidants. Ironically, the interest in antioxidants probably exceeds that in the oxidative process itself, and yet the latter is fundamental to activity. Indeed, the ultimate answers to antioxidant activity lie in a better understanding of oxidation and its mechanisms. The chapter introduces the concepts associated with antioxidants – what is an antioxidant, how does a compound function as an antioxidant, the importance of reaction mechanisms and kinetics and thermodynamics. The chapter concludes with an examination of antioxidant literature and what can be learned from antioxidant bibliometrics.