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Use of herbal medicines and supplements by consumers to prevent or treat disease, particularly chronic conditions continues to grow, leading to increased awareness of the minimal regulation standards in many countries. Fraudulent, adulterated and contaminated herbal and traditional medicines and dietary supplements are a risk to consumer health, with adverse effects and events including overdose, drug-herb interactions and hospitalisation. The scope of the risk has been difficult to determine, prompting calls for new approaches, such as the combination of DNA metabarcoding and mass spectrometry used in this study. Here we show that nearly 50% of products tested had contamination issues, in terms of DNA, chemical composition or both. Two samples were clear cases of pharmaceutical adulteration, including a combination of paracetamol and chlorpheniramine in one product and trace amounts of buclizine, a drug no longer in use in Australia, in another. Other issues include the undeclared presence of stimulants such as caffeine, synephrine or ephedrine. DNA data highlighted potential allergy concerns (nuts, wheat), presence of potential toxins (Neem oil) and animal ingredients (reindeer, frog, shrew), and possible substitution of bird cartilage in place of shark. Only 21% of the tested products were able to have at least one ingredient corroborated by DNA sequencing. This study demonstrates that, despite current monitoring approaches, contaminated and adulterated products are still reaching the consumer. We suggest that a better solution is stronger pre-market evaluation, using techniques such as that outlined in this study.

Methamphetamine is an illicit psychostimulant drug that is linked to a number of diseases of the nervous system. The downstream biochemical effects of its primary mechanisms are not well understood, and the objective of this study was to investigate whether untargeted metabolomic analysis of an in vitro model could generate data relevant to what is already known about this drug. Rat B50 neuroblastoma cells were treated with 1 mM methamphetamine for 48 h, and both intracellular and extracellular metabolites were profiled using gas chromatography–mass spectrometry. Principal component analysis of the data identified 35 metabolites that contributed most to the difference in metabolite profiles. Of these metabolites, the most notable changes were in amino acids, with significant increases observed in glutamate, aspartate and methionine, and decreases in phenylalanine and serine. The data demonstrated that glutamate release and, subsequently, excitotoxicity and oxidative stress were important in the response of the neuronal cell to methamphetamine. Following this, the cells appeared to engage amino acid-based mechanisms to reduce glutamate levels. The potential of untargeted metabolomic analysis has been highlighted, as it has generated biochemically relevant data and identified pathways significantly affected by methamphetamine. This combination of technologies has clear uses as a model for the study of neuronal toxicology.

Low intensity repetitive magnetic stimulation of neural tissue modulates neuronal excitability and has promising therapeutic potential in the treatment of neurological disorders. However, the underpinning cellular and biochemical mechanisms remain poorly understood. This study investigates the behavioural effects of low intensity repetitive magnetic stimulation (LI-rMS) at a cellular and biochemical level. We delivered LI-rMS (10 mT) at 1 Hz and 10 Hz to B50 rat neuroblastoma cells in vitro for 10 minutes and measured levels of selected metabolites immediately after stimulation. LI-rMS at both frequencies depleted selected tricarboxylic acid (TCA) cycle metabolites without affecting the main energy supplies. Furthermore, LI-rMS effects were frequency-specific with 1 Hz stimulation having stronger effects than 10 Hz. The observed depletion of metabolites suggested that higher spontaneous activity may have led to an increase in GABA release. Although the absence of organised neural circuits and other cellular contributors (e.g., excitatory neurons and glia) in the B50 cell line limits the degree to which our results can be extrapolated to the human brain, the changes we describe provide novel insights into how LI-rMS modulates neural tissue.

Toxicity testing is essential for the protection of human health from exposure to toxic environmental chemicals. As traditional toxicity testing is carried out using animal models, mammalian cell culture models are becoming an increasingly attractive alternative to animal testing. Combining the use of mammalian cell culture models with screening-style molecular profiling technologies, such as metabolomics, can uncover previously unknown biochemical bases of toxicity. We have used a mass spectrometry-based untargeted metabolomics approach to characterize for the first time the changes in the metabolome of the B50 cell line, an immortalised rat neuronal cell line, following acute exposure to two known neurotoxic chemicals that are common environmental contaminants; the pyrethroid insecticide permethrin and the organophosphate insecticide malathion. B50 cells were exposed to either the dosing vehicle (methanol) or an acute dose of either permethrin or malathion for 6 and 24 hours. Intracellular metabolites were profiled by gas chromatography-mass spectrometry. Using principal components analysis, we selected the key metabolites whose abundance was altered by chemical exposure. By considering the major fold changes in abundance (>2.0 or <0.5 from control) across these metabolites, we were able to elucidate important cellular events associated with toxic exposure including disrupted energy metabolism and attempted protective mechanisms from excitotoxicity. Our findings illustrate the ability of mammalian cell culture metabolomics to detect finer metabolic effects of acute exposure to known toxic chemicals, and validate the need for further development of this process in the application of trace-level dose and chronic toxicity studies, and toxicity testing of unknown chemicals.

Metabolomics is an analytical technique that investigates the small biochemical molecules present within a biological sample isolated from a plant, animal, or cultured cells. It can be an extremely powerful tool in elucidating the specific metabolic changes within a biological system in response to an environmental challenge such as disease, infection, drugs, or toxins. A historically difficult step in the metabolomics pipeline is in data interpretation to a meaningful biological context, for such high-variability biological samples and in untargeted metabolomics studies that are hypothesis-generating by design. One way to achieve stronger biological context of metabolomic data is via the use of cultured cell models, particularly for mammalian biological systems. The benefits of in vitro metabolomics include a much greater control of external variables and no ethical concerns. The current concerns are with inconsistencies in experimental procedures and level of reporting standards between different studies. This review discusses some of these discrepancies between recent studies, such as metabolite extraction and data normalisation. The aim of this review is to highlight the importance of a standardised experimental approach to any cultured cell metabolomics study and suggests an example procedure fully inclusive of information that should be disclosed in regard to the cell type/s used and their culture conditions. Metabolomics of cultured cells has the potential to uncover previously unknown information about cell biology, functions and response mechanisms, and so the accurate biological interpretation of the data produced and its ability to be compared to other studies should be considered vitally important.

Several studies of neuropathic pain have linked abnormal adrenergic signalling to the development and maintenance of pain, although the mechanisms underlying this are not yet fully understood. Metabolomic analysis is a technique that can be used to give a snapshot of biochemical status, and can aid in the identification of the mechanisms behind pathological changes identified in cells, tissues and biological fluids. This study aimed to use gas chromatography-mass spectrometry-based metabolomic profiling in combination with reverse transcriptase-polymerase chain reaction and immunocytochemistry to identify functional α1-adrenergic receptors on cultured N1E-115 mouse neuroblastoma cells. The study was able to confirm the presence of mRNA for the α1D subtype, as well as protein expression of the α1-adrenergic receptor. Furthermore, metabolomic data revealed changes to the metabolite profile of cells when exposed to adrenergic pharmacological intervention. Agonist treatment with phenylephrine hydrochloride (10 µM) resulted in altered levels of several metabolites including myo-inositol, glucose, fructose, alanine, leucine, phenylalanine, valine, and n-acetylglutamic acid. Many of the changes observed in N1E-115 cells by agonist treatment were modulated by additional antagonist treatment (prazosin hydrochloride, 100 µM). A number of these changes reflected what is known about the biochemistry of α1-adrenergic receptor activation. This preliminary study therefore demonstrates the potential of metabolomic profiling to confirm the presence of functional receptors on cultured cells.

Herbal medicines are growing in popularity, use and commercial value; however, there remain problems with the quality and consequently safety of these products. Adulterated, contaminated and fraudulent products are often found on the market, a risk compounded by the fact that these products are available to consumers with little or no medical advice. Current regulations and quality control methods are lacking in their ability to combat these serious problems. Metabolomics is a biochemical profiling tool that may help address these issues if applied to quality control of both raw ingredients and final products. Using the example of the popular herbal medicine, ginseng, this essay offers an overview of the potential use of metabolomics for quality control in herbal medicines and also highlights where more research is needed.

Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.

Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.

Background Pulsed Magnetic Fields (PMFs) are currently being used to treat a range of neurological conditions such as depression. However, little is known about the cellular mechanisms behind their therapeutic effects. Objective To investigate the influence of PMFs on neuronal biochemical processes. Method B50 rat neuroblastoma cells were seeded on to 6-well plates, grown to confluence and stimulated with PMFs (~10 mT) at 1 or 10 Hz for 10 minutes (controls were unstimulated). Cells were immediately quenched post-stimulation with ice-cold PBS and then freeze-dried. Cells were subsequently lysed, derivatised and analysed using untargeted gas chromatography-mass spectrometry (GC-MS). Key Findings Initial principal component analysis revealed 3 distinct groups (control, 1 and 10 Hz). Significant differences were found in 12 metabolites (PMF stimulated vs unstimulated controls, ANOVA, n= 4-6). PMF stimulated cells had significantly lower levels (p≤0.05) of GABA precursors (succinate, aspartate, proline and glutamate). These changes may be due to increased inhibitory neurotransmitter release during PMF stimulation. Two metabolites involved in calcium signaling (inositol and serine) were also significantly lower in PMF treated cells. In addition, PMF stimulation at 1Hz reduced metabolite levels to a greater extent than 10 Hz, an effect which reached significance for glycine (p≤0.05). Conclusion PMFs at either 1 or 10 Hz significantly reduced intracellular metabolites involved with inhibitory neurotransmission and calcium signaling in neuronal cell cultures. The changes to neuronal metabolism were frequency dependent. Our data suggest that acute PMF stimulation induce biochemical changes that may modulate inhibitory neurotransmission and calcium signaling.

Introduction: Hypoxia has been implicated in nerve cell deaths that occur in a variety of neurological disorders. Opioid receptor agonists have been shown to have some positive benefits on the nervous system. The aim of the present work was to investigate the effects of hypoxia and opioid receptor agonists' treatment on the morphology of B50 neuronal cell lines cultured in hypoxia. Materials and Methods: The B50 cells were cultured under normoxic conditions (21%O2; 5% CO2) as the control group and under hypoxic conditions (5%O2; 5% CO2) as the experimental group. Three opioid receptor agonists namely DAMGO (μ) DSLET (δ) and ICI-199,441 (κ) were administered to the cells for 48 hours as treatment against hypoxia after 48 hours of culture at doses of 10 μM, 50 μM and 100 μM respectively. Neuronal morphology, viability, proliferation and differentiation were assessed using same field morphological assessment. In addition lactate dehydrogenase (LDH) leakage, cellular proliferation and DbcAMP induced differentiation were also assessed. μ opioid receptor mRNA was assessed using RT-PCR. Results: The results showed groups of dead and degenerating B50 neuronal cells and some significant changes (P<0.05) in cellular proliferation, viability cellular differentiation. The levels of LDH leakage showed normal B50 cells (100%), hypoxic cells (587%), and treated cells with 100 μM DAMGO (μ) (143%), 50 μM DSLET (δ) (140%) and 50 μM ICI-199,441 (κ) (109%). The changes in morphology, LDH release, neuronal viability, proliferation and differentiation were shown to be dose-dependent between treated hypoxic B50 neurons in culture. Conclusion: The results indicate that opioid agonists have some potential benefits in the treatment of hypoxia-induced changes in neuronal B50 cells in culture.

Oxidative stress adversely affects cells and tissues, and neuronal cells in particular have been shown to be more susceptible to the injurious effects of oxidative stress in which the cells may die when oxygen supply is reduced or completely eliminated. The aim of the present study was to study the effect of oxidative stress using hypoxia as a bench mark on the morphology of B50 neuronal cell lines cultured in hypoxia using neuronal pattern and pattern formation as case study. The B50 cells were cultured in normal incubator (21%O2; 5% CO2) as control group and hypoxic incubator (5%O2; 5% CO2) as the experimental group. Neuronal morphology, pattern and wellbeing were assessed using same field morphological assessment of cells and lactate dehydrogenase leakage (LDH). The result showed groups of dead and degenerating B50 neuronal cells, altered neuronal pattern and pattern formation and some significant changes (P<0.05) in cellular levels of LDH leakage in normal B50 cells and hypoxic cells. The changes in morphology, neuronal pattern and LDH release indicate that oxidative stress has induced morphological and cellular changes in cortical B50 cells in culture and that the B50 neuronal cells are susceptible to damage and injurious effects of oxidative stress represented by hypoxia as most brain cells.

Hypoxia has been implicated in nerve cell deaths in many neurological disorders and opioid receptor agonists have some positive benefits on the nervous system. The aim of the present work was to investigate the effects of hypoxia and opioid receptor agonists’ treatment on the morphology of B50 cells cultured in hypoxia using neuronal pattern and pattern formation as a case study. The B50 cells were cultured in normal incubator (21%O2; 5% CO2) as the control group and hypoxic incubator (5%O2; 5% CO2) as the experimental group and three opioid receptor agonists namely DAMGO (μ), DSLET (δ) and ICI-199,441 (κ) were administered to the cells for 48 hours as treatment against hypoxia after 48 hours of culture at 10μM, 50μM and 100μM concentrations. Neuronal morphology and wellbeing was assessed using same field morphological assessment and lactate dehydrogenase leakage (LDH). The result showed groups of dead and degenerating B50 neuronal cells, altered neuronal pattern and pattern formation and some significant changes (P<0.05) in cellular levels of LDH leakage in normal, hypoxic cells and cells treated with different agonists. The changes in morphology, neuronal pattern and LDH release indicate that hypoxia induced morphological and cellular changes in B50 cells in hypoxia and opioid agonists have some potential benefits in the treatment of hypoxia-induced changes in B50 cells in culture.

Oxidative stress adversely affects neuronal cells in which they may die when oxygen supply is reduced or eliminated and opioid receptor agonists elicit several central nervous system effects. The aim of this study was to evaluate the effect of oxidative stress on opioid receptor gene expression in cortical B50 cells. The cells were cultured in normoxia, hypoxia and treated with opioid agonists; DAMGO (μ), DSLET (δ) and ICI-199,441 (κ) for 48 hours after 48 hours of initial culture at dose of 10μM, 50μM and 100μM. The level of mu opioid receptor mRNA was assessed using RT-PCR. The results show that oxidative stress induced changes in B50 cells in hypoxia while mu opioid mRNA levels showed no change. The results show that B50 cells are susceptible to damage by oxidative stress and opioid agonist treatments showed no change in the level of mu opioid receptor gene expression in B50 cells.

The super family of G-protein-coupled receptors (GPCRs) is the main target for the actions exerted by hormones, drugs and neurotransmitters. Each GPCR shows preferential coupling to some members of the G-protein family such as Gs, Gi and Gq which in turn activates the defined second messenger pathways. The G protein-coupled receptors (GPCRs) represent 50–60% of the current drug targets and this family of membrane proteins plays a crucial role in drug discovery, health and disease conditions. The G-protein-mediated signalling system has been used to study transmembrane signalling mechanisms in eukaryotic organisms resulting in different cellular activities and effects such as cellular growth, proliferation and differentiation. The G-protein-mediated signalling systems are made up of three main components, the receptors, the heterotrimeric G-proteins and the effectors in addition to various proteins that modulate the G-protein-mediated signalling process like the regulators of G-protein signalling (RGS) proteins. Mammalian cells express many GPCRs and several types of heterotrimeric G-proteins and their effectors. A number of drugs based on GPCRs have been developed for such different indications as cardiovascular, metabolic, neurodegenerative, psychiatric, and oncologic diseases. Most neurotransmitters of the central nervous system (CNS) act on GPCRs to mediate different cellular responses in normal and disease states. The activation of receptors that interact through Gi e.g. cannabinoid receptor types convey neuronal protection against hypoxic insult and resultant excitotoxic death.

Cannabis and its derivatives have great therapeutic potential and have been used for centuries for medicinal purposes. The side effects of cannabinoids include euphoric mood changes, acute psychotic episodes, initiation and exacerbation of schizophrenic psychosis in predisposed persons, impaired cognitive and psychomotor performance, tachycardia and hypotension. The production of complex behavioural effects by cannabinoids are mediated by cannabinoid receptors (CB1 and CB2) and by interactions with other neurochemical systems. It has been shown that the therapeutic and physiological effects of cannabinoids are dependent upon whether the administration is acute or chronic and on the route of administration. The physiological effects of cannabis and its derivatives include: reduction in psychomotor coordination and performance, alterations in thermoregulation, endocrine and reproductive functions and gut motility. There is also evidence of agonist selectivity for CB1 receptors coupled to different subtypes of Gi proteins or to Gi versus Go proteins. Cannabinoid-activated receptors distinct from CB1 or CB2 exist in the central nervous system. Cannabinoids are known to inhibit GABA-mediated inhibitory postsynaptic currents in the hippocampus via a presynaptic action at CB1 receptors located on GABAergic terminals. CB1 receptors have also been implicated in the inhibition of glutamatergic excitatory postsynaptic currents. The synthetic cannabinoid, Win 55,212-2, a mixed CB1-CB2 cannabinoid receptor agonist, was found to attenuate hyperalgesia in a rat model of neuropathic pain and suppress opioid-induced emesis in ferrets.

Opioid receptors are found in the central nervous system (CNS) and are classified as mu (µ), kappa (κ), delta (δ) and sigma (σ) opioid receptors. Opioid receptors belong to the large family of G protein coupled receptors (GPCRs), and have diverse and important physiological roles. Opioid receptors are not uniformly distributed in the CNS and are found in areas concerned with pain, with the highest concentration in the cerebral cortex, followed by the amygdala, septum, thalamus, hypothalamus, midbrain and spinal cord. Activated delta opioid receptors are coupled to Gi1 while activated mu opioid receptors are coupled to Gi3 in neuroblastoma cells. Mu opioid receptors are activated by mu receptor agonists and are coupled through the Gαi1 and GαoA. Both mu and kappa opioid receptors are coupled via both Gi and Gz and opioid receptors are important targets for thousands of pharmacological agents. GPCRs typically require activation by agonists for their signalling activity to be initiated but some of the GPCRs may display basal or spontaneous signalling activity in the absence of an agonist. The stimulation of these receptors triggers analgesic effects and affects the function of the nervous system, gastrointestinal tract and other body systems. Hundreds of analogs of opioid peptides have been synthesized in an effort to make the compounds more active, selective, and resistant to biodegradation than the endogenous ligands. All these modifications resulted in obtaining very selective agonists and antagonists with high affinity at mu-, delta-, and kappa-opioid receptors, which are useful in further studies on the pharmacology of opioid receptors in a mammalian organism.

Hypoxia adversely affects cells and tissues, and neuronal cells in particular have been shown to be more susceptible to the injurious effects of hypoxia in which they may begin to die when oxygen supply is reduced or completely eliminated. Opioid receptor agonists have been shown to elicit several central nervous system effects, mediated via G protein-coupled receptors. The aim of this study was to study the effect of hypoxia on G protein coupled receptor gene expression using mu opioid receptor as a case study in cortical neuronal B50 cell lines in culture. The B50 cells were cultured in normoxia (21% O2; 5% CO2) and hypoxia (5% O2; 5% CO2), and were treated with opioid agonists to determine their effects on hypoxia-induced changes. Three opioid agonists {DAMGO(μ), DSLET(δ) and ICI--199,441(κ)}, were administered to the cells as treatment for 48 h after 48 h of initial culture for a total of 96 h of culture in hypoxic conditions at concentrations of 10, 50 and 100 μM. The levels of G-protein coupled receptor (mu opioid) mRNAs were assessed using RT-PCR. The results showed that hypoxia induced morphological changes in B50 cells in hypoxia while the mu opioid RT-PCR mRNA levels showed no appreciable changes in normal, hypoxic and treated cells. The results show that B50 neuronal cells are susceptible to damage and injurious effects of hypoxia, as are most brain cells and the opioid agonist treatments showed there were no changes in the level of mu opioid receptor gene expression due to hypoxia or agonist treatment in neuronal B50 cells in culture.

G-proteins are important mediators of cellular and tissue functions and are characterised by a recognition site for Guanine Triphosphate (GTP), Guanine Diphosphate (GDP) and possess intrinsic GTPase activity. They play important roles in signal transduction responsible for cytoskeletal remodelling, cellular differentiation and vesicular transport. They are made up of three types namely, the small G-proteins, the sensors and the heterotrimeric G-proteins. The G-protein heterotrimers consist of G-alpha (G"), G-beta (G$) and G-gamma (G() subunits. Each heterotrimeric G-protein have different subunits and the combination of these subunits define the specific role of each G -protein. The activation of G" subunits regulates the activity of effector enzymes and ion channels while G$( subunits function in the regulation of mitogen-activated protein kinase (MAP-kinase) pathway. The G-protein-mediated signal transduction is important in the regulation of a cells morphological and physiological response to external stimuli. MAPKs are involved in the phosphorylation of transcription factors that stimulate gene transcription. G"s stimulates adenylate cyclase, thereby increasing cyclic adenosine monophosphate (cAMP) leading to the phosphorylation and subsequent activation of Ca2+ channels. G proteins are involved in disease pathology through several mechanisms which interfere with the G protein activity. Other disease pathologies associated with abnormal mutations in G proteins can interfere with signal transduction pathways which may involve signal transmission that is either excessive, by augmentation o f G protein function, or insufficient, via inactivation of G proteins.

Hypoxia adversely affects cells and tissues, and neuronal cells in particular have been shown to be more susceptible to the injurious effects of hypoxia in which they may begin to die when oxygen supply is reduced or completely eliminated. Cannabinoid (CB1) receptor agonists have been shown to elicit several Central Nervous System (CNS) effects, mediated via G protein-coupled receptors. The aim of this study was to examine the effect of hypoxia on G protein coupled receptor (CB1) gene expression in cortical neuronal B50 cell lines in culture. The B50 cells were cultured in normoxia (21% O2; 5% CO2) and hypoxia (5% O2; 5% CO2), and were treated with cannabinoid agonists to determine their effects on hypoxia-induced changes. Three cannabinoid agonists [Win55,212-2 mesylate (Win), arachidonoylethanolamide (AEA) and 2- arachidonylglycerol (2-AG)], were administered to the cells as treatment for 48 hours after 48hours of initial culture for a total of 96hours of culture in hypoxic conditions at concentrations of 10, 50 and 100 nM . The levels of G-protein coupled receptor (CB1) mRNAs were assessed using RT-PCR. The results showed that hypoxia induced morphological changes in B5 0 cells in hypoxia while the CB1 RT-PCR mRNA levels showed no appreciable changes in normal, hypoxic and treated cells. The results show that B50 neuronal cells are susceptible to damage and injurious effects of hypoxia, as are most brain cells and the cannabinoid agonist treatments showed there were no changes in the level of CB1 receptor gene expression due to hypoxia or agonist treatment in neuronal B50 cells in culture.

Hepcidin has been identified as the principle regulatory hormone essential for iron homeostasis. Quantitative analysis of hepcidin in bodily fluids provides an insight into the pathogenesis of disorders of iron metabolism such as hereditary hemochromatosis and anemia of chronic disease. This study describes the use of solid phase extraction (SPE) as a preparative step followed by matrix assisted laser desorption/ionization-orthogonal-time-of-flight mass spectrometry (MALDI-TOF MS) with internal standard for the quantitative analysis of unlabelled urinary hepcidin. More than 70% extraction recovery of hepcidin (hepcidin-25) with monoisotopic resolution was achieved. Urinary creatinine was analyzed using HPLC-UV/Vis with hepcidin-25 levels of 2.2 to 2.7 nmol/mmol of creatinine observed in healthy controls. Spot-to-spot variation of hepcidin standard additions was less than 3.5%. Intra- and inter-day precision assay of less than 9.5% relative standard deviation was achieved with less than 0.5% variation between the intra-day assay data. In summary, a validated non-invasive method has been developed for the quantification of unlabelled urinary hepcidin-25 which can be used to screen for iron-related disorders such as hemochromatosis in iron-overloaded patients.