From hemolymph to in-vivo: The potential of a 1 mm microlitre flow probe with separate lock chamber for NMR metabolomics in mass limited environmental samples

•Metabolomics plays a key role in understanding environmental toxicity.•A 1 mm probe and μL flow system is necessary to study crucial mass limited samples.•D. magna hemolymph, parthenogenetic/ dormant eggs, and in-vivo neonates were studied.•In-vivo metabolic responses under high salt loads could be measured.•The 1 mm probe is ideal for metabolomics studies of mass limited samples. Metabolomics is one of the leading approaches for understanding the toxic-mode-of-action of environmental contaminants. Nuclear Magnetic Resonance (NMR) spectroscopy has been commonly used in metabolomic studies; however, its main drawback is its relatively low sensitivity, making it challenging to study mass limited but environmentally crucial samples. In this work a 1 mm microlitre probe modified with a separate lock chamber to address this challenge, provided substantial improvements in mass sensitivity relative to conventional 5 mm NMR probes. The 1 mm probe is used to analyze various components of the model organism Daphnia magna, including hemolymph, parthenogenetic eggs, dormant eggs, and neonates. A μL volume flow system is designed for the 1 mm probe to perform an in-vivo exposure of neonates to high salt concentrations. The metabolic investigation of these samples was only achieved due to the minimum sample requirements and high salt tolerance of the probe, demonstrating that the 1 mm microlitre probe modified with a separate lock chamber holds significant potential for future metabolomic studies of mass limited samples. [Display omitted]