In situ separation and visualization of isomeric auxin derivatives in Arabidopsis by ion mobility mass spectrometry imaging.

In situ separation and visualization of synthetic and naturally occurring isomers from heterogeneous plant tissues, especially when they share similar molecular structures, are a challenging task. In this study, we combined the ion mobility separation with desorption electrospray ionization mass spectrometry imaging (DESI-IM-MSI) to achieve a direct separation and visualization of two synthetic auxin derivatives, auxinole and its structural isomer 4pTb-MeIAA, as well as endogenous auxins from Arabidopsis samples. Distinct distribution of these synthetic isomers and endogenous auxins in Arabidopsis primary roots and hypocotyls was achieved in the same imaging analysis from both individually treated and cotreated samples. We also observed putative metabolites of synthetic auxin derivatives, i.e. auxinole amino acid conjugates and hydrolysed 4pTb-MeIAA product - 4pTb-IAA, based on their unique drifting ion intensity patterns. Furthermore, DESI-IM-MSI-revealed abundance of endogenous auxins and synthetic isomers was validated by liquid chromatography-mass spectrometry (LC-MS). Our results demonstrate that DESI-IM-MSI could be used as a robust technique for detecting endogenous and exogenous isomers and provide a spatiotemporal evaluation of hormonomics profiles in plants.

Synthesis and neuroprotective activity of 3-aryl-3-azetidinyl acetic acid methyl ester derivatives.

A library of 3-aryl-3-azetidinyl acetic acid methyl ester derivatives was prepared from N-Boc-3-azetidinone employing the Horner-Wadsworth-Emmons reaction, rhodium(I)-catalyzed conjugate addition of arylboronic acids, and subsequent elaborations to obtain N-unprotected hydrochlorides, N-alkylated and N-acylated azetidine derivatives. The compounds were evaluated for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity, revealing several derivatives to possess AChE inhibition comparable to that of the AChE inhibitor rivastigmine. The binding mode of the AChE inhibitor donepezil and selected active compounds 26 and 27 within the active site of AChE was studied using molecular docking. Furthermore, the neuroprotective activity of the prepared compounds was evaluated in models associated with Parkinson's disease (salsolinol-induced) and aspects of Alzheimer's disease (glutamate-induced oxidative damage). Compound 28 showed the highest neuroprotective effect in both salsolinol- and glutamate-induced neurodegeneration models, and its protective effect in the glutamate model was revealed to be driven by a reduction in oxidative stress and caspase-3/7 activity.

Synthesis and Characterization of New Pyrano[2,3-c]pyrazole Derivatives as 3-Hydroxyflavone Analogues.

In this paper, an efficient synthetic route from pyrazole-chalcones to novel 6-aryl-5-hydroxy-2-phenylpyrano[2,3-c]pyrazol-4(2H)-ones as 3-hydroxyflavone analogues is described. The methylation of 5-hydroxy-2,6-phenylpyrano[2,3-c]pyrazol-4(2H)-one with methyl iodide in the presence of a base yielded a compound containing a 5-methoxy group, while the analogous reaction of 5-hydroxy-2-phenyl-6-(pyridin-4-yl)pyrano[2,3-c]pyrazol-4(2H)-one led to the zwitterionic 6-(N-methylpyridinium)pyrano[2,3-c]pyrazol derivative. The treatment of 5-hydroxy-2,6-phenylpyrano[2,3-c]pyrazol-4(2H)-one with triflic anhydride afforded a 5-trifloylsubstituted compound, which was further used in carbon-carbon bond forming Pd-catalyzed coupling reactions to yield 5-(hetero)aryl- and 5-carbo-functionalized pyrano[2,3-c]pyrazoles. The excited-state intramolecular proton transfer (ESIPT) reaction of 5-hydroxypyrano[2,3-c]pyrazoles from the 5-hydroxy moiety to the carbonyl group in polar protic, polar aprotic, and nonpolar solvents was observed, resulting in well-resolved two-band fluorescence. The structures of the novel heterocyclic compounds were confirmed by 1H-, 13C-, 15N-, and 19F-NMR spectroscopy, HRMS, and single-crystal X-ray diffraction data.

Fluorescence-activated multi-organelle mapping of subcellular plant hormone distribution.

Auxins and cytokinins are two major families of phytohormones that control most aspects of plant growth, development and plasticity. Their distribution in plants has been described, but the importance of cell- and subcellular-type specific phytohormone homeostasis remains undefined. Herein, we revealed auxin and cytokinin distribution maps showing their different organelle-specific allocations within the Arabidopsis plant cell. To do so, we have developed Fluorescence-Activated multi-Organelle Sorting (FAmOS), an innovative subcellular fractionation technique based on flow cytometric principles. FAmOS allows the simultaneous sorting of four differently labelled organelles based on their individual light scatter and fluorescence parameters while ensuring hormone metabolic stability. Our data showed different subcellular distribution of auxin and cytokinins, revealing the formation of phytohormone gradients that have been suggested by the subcellular localization of auxin and cytokinin transporters, receptors and metabolic enzymes. Both hormones showed enrichment in vacuoles, while cytokinins were also accumulated in the endoplasmic reticulum.

Metabolic profiles of 2-oxindole-3-acetyl-amino acid conjugates differ in various plant species.

Auxins are a group of phytohormones that play a key role in plant growth and development, mainly presented by the major member of the family - indole-3-acetic acid (IAA). The levels of free IAA are regulated, in addition to de novo biosynthesis, by irreversible oxidative catabolism and reversible conjugation with sugars and amino acids. These conjugates, which serve as inactive storage forms of auxin and/or degradation intermediates, can also be oxidized to form 2-oxindole-3-acetyl-1-O-ß-d-glucose (oxIAA-glc) and oxIAA-amino acids (oxIAA-AAs). Until now, only oxIAA conjugates with aspartate and glutamate have been identified in plants. However, detailed information on the endogenous levels of these and other putative oxIAA-amino acid conjugates in various plant species and their spatial distribution is still not well understood but is finally getting more attention. Herein, we identified and characterized two novel naturally occurring auxin metabolites in plants, namely oxIAA-leucine (oxIAA-Leu) and oxIAA-phenylalanine (oxIAA-Phe). Subsequently, a new liquid chromatography-tandem mass spectrometry method was developed for the determination of a wide range of IAA metabolites. Using this methodology, the quantitative determination of IAA metabolites including newly characterized oxIAA conjugates in roots, shoots and cotyledons of four selected plant models - Arabidopsis thaliana, pea (Pisum sativum L.), wheat (Triticum aestivum L.) and maize (Zea mays L.) was performed to compare auxin metabolite profiles. The distribution of various groups of auxin metabolites differed notably among the studied species as well as their sections. For example, oxIAA-AA conjugates were the major metabolites found in pea, while oxIAA-glc dominated in Arabidopsis. We further compared IAA metabolite levels in plants harvested at different growth stages to monitor the dynamics of IAA metabolite profiles during early seedling development. In general, our results show a great diversity of auxin inactivation pathways among angiosperm plants. We believe that our findings will greatly contribute to a better understanding of IAA homeostasis.

Pyrazole-based lamellarin O analogues: synthesis, biological evaluation and structure-activity relationships.

A library of pyrazole-based lamellarin O analogues was synthesized from easily accessible 3(5)-aryl-1H-pyrazole-5(3)-carboxylates which were subsequently modified by bromination, N-alkylation and Pd-catalysed Suzuki cross-coupling reactions. Synthesized ethyl and methyl 3,4-diaryl-1-(2-aryl-2-oxoethyl)-1H-pyrazole-5-carboxylates were evaluated for their physicochemical property profiles and in vitro cytotoxicity against three human colorectal cancer cell lines HCT116, HT29, and SW480. The most active compounds inhibited cell proliferation in a low micromolar range. Selected ethyl 3,4-diaryl-1-(2-aryl-2-oxoethyl)-1H-pyrazole-5-carboxylates were further investigated for their mode of action. Results of combined viability staining via Calcein AM/Hoechst/PI and fluorescence-activated cell sorting data indicated that cell death was triggered in a non-necrotic manner mediated by mainly G2/M-phase arrest.

Convenient Synthesis of N-Heterocycle-Fused Tetrahydro-1,4-diazepinones.

A general approach towards the synthesis of tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-one, tetrahydro[1,4]diazepino[1,2-a]indol-1-one and tetrahydro-1H-benzo[4,5]imidazo[1,2-a][1,4]diazepin-1-one derivatives was introduced. A regioselective strategy was developed for synthesizing ethyl 1-(oxiran-2-ylmethyl)-1H-pyrazole-5-carboxylates from easily accessible 3(5)-aryl- or methyl-1H-pyrazole-5(3)-carboxylates. Obtained intermediates were further treated with amines resulting in oxirane ring-opening and direct cyclisation-yielding target pyrazolo[1,5-a][1,4]diazepin-4-ones. A straightforward two-step synthetic approach was applied to expand the current study and successfully functionalize ethyl 1H-indole- and ethyl 1H-benzo[d]imidazole-2-carboxylates. The structures of fused heterocyclic compounds were confirmed by 1H, 13C, and 15N-NMR spectroscopy and HRMS investigation.

High-throughput interspecies profiling of acidic plant hormones using miniaturised sample processing.

BACKGROUND: Acidic phytohormones are small molecules controlling many physiological functions in plants. A comprehensive picture of their profiles including the active forms, precursors and metabolites provides an important insight into ongoing physiological processes and is essential for many biological studies performed on plants. RESULTS: A high-throughput sample preparation method for liquid chromatography-tandem mass spectrometry determination of 25 acidic phytohormones classed as auxins, jasmonates, abscisates and salicylic acid was optimised. The method uses a small amount of plant tissue (less than 10 mg fresh weight) and acidic extraction in 1 mol/L formic acid in 10% aqueous methanol followed by miniaturised purification on reverse phase sorbent accommodated in pipette tips organised in a 3D printed 96-place interface, capable of processing 192 samples in one run. The method was evaluated in terms of process efficiency, recovery and matrix effects as well as establishing validation parameters such as accuracy and precision. The applicability of the method in relation to the amounts of sample collected from distantly related plant species was evaluated and the results for phytohormone profiles are discussed in the context of literature reports. CONCLUSION: The method developed enables high-throughput profiling of acidic phytohormones with minute amounts of plant material, and it is suitable for large scale interspecies studies.

Synthesis of N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines and their photodynamic properties in the human skin melanoma cell line G361.

A small series of N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines was synthesized from easily accessible 1-phenyl-1H-pyrazol-3-ol via 7-iodo-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine and 7-iodo-4-methyl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine intermediates and their subsequent use in palladium catalyzed Buchwald-Hartwig cross-coupling reaction with various anilines. Majority of the compounds were not significantly cytotoxic to melanoma G361 cells in the dark up to 10 µM concentration, but their activity could be increased by irradiation with visible blue light (414 nm). The most active compound 10 possessed EC50 values of 3.5, 1.6 and 0.9 µM in cells irradiated with 1, 5 and 10 J/cm2, respectively. The treatment caused generation of reactive oxygen species in cells and extensive DNA damage, documented by the comet assay and by detection of phosphorylated histone H2A.X, followed by apoptotic cell death. Our results suggest that N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines could serve as a potential source of photosensitizing compounds with anticancer activities.

Synthesis and Antiproliferative Activity of 2,4,6,7-Tetrasubstituted-2H-pyrazolo[4,3-c]pyridines.

A library of 2,4,6,7-tetrasubstituted-2H-pyrazolo[4,3-c]pyridines was prepared from easily accessible 1-phenyl-3-(2-phenylethynyl)-1H-pyrazole-4-carbaldehyde via an iodine-mediated electrophilic cyclization of intermediate 4-(azidomethyl)-1-phenyl-3-(phenylethynyl)-1H-pyrazoles to 7-iodo-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridines followed by Suzuki cross-couplings with various boronic acids and alkylation reactions. The compounds were evaluated for their antiproliferative activity against K562, MV4-11, and MCF-7 cancer cell lines. The most potent compounds displayed low micromolar GI50 values. 4-(2,6-Diphenyl-2H-pyrazolo[4,3-c]pyridin-7-yl)phenol proved to be the most active, induced poly(ADP-ribose) polymerase 1 (PARP-1) cleavage, activated the initiator enzyme of apoptotic cascade caspase 9, induced a fragmentation of microtubule-associated protein 1-light chain 3 (LC3), and reduced the expression levels of proliferating cell nuclear antigen (PCNA). The obtained results suggest a complex action of 4-(2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-yl)phenol that combines antiproliferative effects with the induction of cell death. Moreover, investigations of the fluorescence properties of the final compounds revealed 7-(4-methoxyphenyl)-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine as the most potent pH indicator that enables both fluorescence intensity-based and ratiometric pH sensing.

Caged Phytohormones: From Chemical Inactivation to Controlled Physiological Response.

Plant hormones, also called phytohormones, are small signaling molecules regulating a wide range of growth and developmental processes. These unique compounds respond to both external (light, temperature, water, nutrition, or pathogen attack) and internal factors (e.g., age) and mediate signal transduction leading to gene expression with the aim of allowing plants to adapt to constantly changing environmental conditions. Within the regulation of biological processes, individual groups of phytohormones act mostly through a web of interconnected responses rather than linear pathways, making elucidation of their mode of action in living organisms quite challenging. To further progress with our knowledge, the development of novel tools for phytohormone research is required. Although plenty of small molecules targeting phytohormone metabolic or signaling pathways (agonists, antagonists, and inhibitors) and labeled or tagged (fluorescently, isotopically, or biotinylated) compounds have been produced, the control over them in vivo is lost at the time of their administration. Caged compounds, on the other hand, represent a new approach to the development of small organic substances for phytohormone research. The term "caged compounds" refers to light-sensitive probes with latent biological activity, where the active molecule can be freed using a light beam in a highly spatio/temporal-, amplitude-, or frequency-defined manner. This review summarizes the up-to-date development in the field of caged plant hormones. Research progress is arranged in chronological order for each phytohormone regardless of the cage compound formulation and bacterial/plant/animal cell applications. Several known drawbacks and possible directions for future research are highlighted.

In situ characterisation of phytohormones from wounded Arabidopsis leaves using desorption electrospray ionisation mass spectrometry imaging.

Phytohormones (plant hormones) are a group of small signalling molecules that act as important endogenous regulators in plant development and stress responses. Previous research has identified the phytohormone species, jasmonates, auxins and abscisic acid, and their related compounds in stressed leaf extracts. However, in situ visualisations of endogenous phytohormones from intact plant tissues remain elusive without the usage of labels or reporters. Mass spectrometry imaging is a label-free analytical technique that has been successfully applied for the direct detection of plant proteins, lipids, carbohydrates and many other biomolecules. In this study, desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) was used for high throughput visualisation and evaluation of wound-induced phytohormones inside Arabidopsis thaliana leaves. The results showed higher levels of jasmonates, salicylic acid, abscisic acid and indole-3-acetic acid in their ion intensity maps established from wounded leaves compared to control leaves, which have been validated in the parallel liquid chromatography-mass spectrometry quantification, and the untainted distributions of the identified phytohormones in leaves were confirmed by mass spectrometry imaging of instant leaf imprinted thin-layer chromatography plate samples. Further statistical analysis has not only demonstrated a significant increase of jasmonic acid and its precursor compounds in wounded leaves/regions but also highlighted a potential correlation in different phytohormone species. Our results suggest that DESI-MSI can be used to in situ characterise multiple phytohormone compounds from intact leaves with 200 µm spatial resolution to provide insight into phytohormone distributions in wounded leaves, along with their correlated precursors and metabolites under mechanical stress.

Synthesis of 5-[(1H-indol-3-yl)methyl]-1,3,4-oxadiazole-2(3H)-thiones and their protective activity against oxidative stress.

A small library of 2-[(1H-indol-3-yl)methyl]-5-(alkylthio)-1,3,4-oxadiazoles was prepared, starting from indole-3-acetic acid methyl ester and its 5-methyl-substituted derivative. The synthetic route involved the formation of intermediate hydrazides, their condensation with carbon disulfide, and intramolecular cyclization to corresponding 5-[(1H-indol-3-yl)methyl]-1,3,4-oxadiazole-2(3H)-thiones. The latter were then S-alkylated, and in case of ester derivatives, they were further hydrolyzed into corresponding carboxylic acids. All 5-[(1H-indol-3-yl)methyl]-1,3,4-oxadiazole-2(3H)-thiones and their S-alkylated derivatives were then screened for their protective effects in vitro and in vivo. Methyl substitution on the indole ring and propyl, butyl, or benzyl substitution on sulfhydryl group-possessing compounds were revealed to protect Friedreich's ataxia fibroblasts against the effects of glutathione depletion induced by the γ-glutamylcysteine synthetase inhibitor, buthionine sulfoximine. Two of the active compounds also reproducibly increased the survival of Caenorhabditis elegans exposed to juglone-induced oxidative stress.

New fluorescent auxin probes visualise tissue-specific and subcellular distributions of auxin in Arabidopsis.

In a world that will rely increasingly on efficient plant growth for sufficient food, it is important to learn about natural mechanisms of phytohormone action. In this work, the introduction of a fluorophore to an auxin molecule represents a sensitive and non-invasive method to directly visualise auxin localisation with high spatiotemporal resolution. The state-of-the-art multidisciplinary approaches of genetic and chemical biology analysis together with live cell imaging, liquid chromatography-mass spectrometry (LC-MS) and surface plasmon resonance (SPR) methods were employed for the characterisation of auxin-related biological activity, distribution and stability of the presented compounds in Arabidopsis thaliana. Despite partial metabolisation in vivo, these fluorescent auxins display an uneven and dynamic distribution leading to the formation of fluorescence maxima in tissues known to concentrate natural auxin, such as the concave side of the apical hook. Importantly, their distribution is altered in response to different exogenous stimuli in both roots and shoots. Moreover, we characterised the subcellular localisation of the fluorescent auxin analogues as being present in the endoplasmic reticulum and endosomes. Our work provides powerful tools to visualise auxin distribution within different plant tissues at cellular or subcellular levels and in response to internal and environmental stimuli during plant development.

Synthesis and anthelmintic activity of benzopyrano[2,3-c]pyrazol-4(2H)-one derivatives.

A series of benzopyrano[2,3-c]pyrazol-4(2H)-one derivatives were synthesized from readily available 1-phenyl- and 1-methyl-1H-pyrazol-3-ols by sequentially employing O-acylation, Fries rearrangement and potassium carbonate-induced cyclization. The anthelmintic properties of the obtained compounds were investigated in vivo in a model nematode, Caenorhabditis elegans. Five compounds, namely 2-phenyl[1]benzopyrano[2,3-c]pyrazol-4(2H)-one 33 and its 7-fluoro, 7-chloro-, 7-bromo- and 8-fluoro-analogues, 36, 38, 40 and 43, respectively, altered the development of C. elegans. While the activities of 33 and 43 were rather modest, compounds 36, 38 and 40 inhibited the growth of the worms at concentrations of approximately 1-3 µM. At these concentrations, the compounds did not kill the worms, but they strongly inhibited their development, with the majority of larvae never progressing past the L1 stage. Moreover, testing in non-cancer human cell lines showed that, with exception of 7-bromo derivative 40, the active compounds have favourable toxicity profiles.

New fluorescently labeled auxins exhibit promising anti-auxin activity.

The plant hormone auxin is a key player in the regulation of plant growth and development. Despite numerous studies devoted to understanding its role in a wide spectrum of physiological processes, full appreciation of its function is linked to a comprehensive determination of its spatio-temporal distribution, which plays a crucial role in its mode of action. Conjugation of fluorescent tracers to plant hormones enables sensitive and specific visualization of their subcellular and tissue-specific localization and transport in planta, which represents a powerful tool for plant physiology. However, to date, only a few fluorescently labeled auxins have been developed. We report the synthesis of four novel fluorescently labeled derivatives of indole-3-acetic acid (IAA) in the form of a conjugate with a nitrobenzoxadiazole (NBD) fluorophore together with validation of their biological activity. These compounds, unlike other previously reported auxins fluorescently labeled at N1 position (nitrogen of the indole ring), do not possess auxin activity but rather show dose-dependent inhibition of auxin-induced effects, such as primary root growth inhibition, root hair growth and the auxin reporter DR5::GUS expression. Moreover, the study demonstrates the importance of the character of the linker and optimal choice of the labeling site in the preparation of fluorescently labeled auxins as important variables influencing their biological activity and fluorescent properties.

Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis.

Auxin (indole-3-acetic acid, IAA) plays fundamental roles as a signalling molecule during numerous plant growth and development processes. The formation of local auxin gradients and auxin maxima/minima, which is very important for these processes, is regulated by auxin metabolism (biosynthesis, degradation, and conjugation) as well as transport. When studying auxin metabolism pathways it is crucial to combine data obtained from genetic investigations with the identification and quantification of individual metabolites. Thus, to facilitate efforts to elucidate auxin metabolism and its roles in plants, we have developed a high-throughput method for simultaneously quantifying IAA and its key metabolites in minute samples (<10 mg FW) of Arabidopsis thaliana tissues by in-tip micro solid-phase extraction and fast LC-tandem MS. As a proof of concept, we applied the method to a collection of Arabidopsis mutant lines and identified lines with altered IAA metabolite profiles using multivariate data analysis. Finally, we explored the correlation between IAA metabolite profiles and IAA-related phenotypes. The developed rapid analysis of large numbers of samples (>100 samples d-1) is a valuable tool to screen for novel regulators of auxin metabolism and homeostasis among large collections of genotypes.

Synthesis and anti-mitotic activity of 2,4- or 2,6-disubstituted- and 2,4,6-trisubstituted-2H-pyrazolo[4,3-c]pyridines.

An efficient synthetic route for the synthesis of 2H-pyrazolo[4,3-c]pyridines, primarily varying by the substituents at the 2-, 4- and 6-positions, is described here. A Sonogashira-type cross-coupling reaction was employed to yield 3-alkynyl-1H-pyrazole-4-carbaldehydes, ethanones and propanones from the corresponding 1H-pyrazol-3-yl trifluoromethanesulfonates. Subsequent treatment of the coupling products with dry ammonia afforded a versatile library of 2H-pyrazolo[4,3-c]pyridines, which were then evaluated for their cytotoxicity against K562 and MCF-7 cancer cell lines. The most potent of these compounds displayed low micromolar GI50 values in both cell lines. Active compounds induced dose-dependent cell-cycle arrest in mitosis, as shown by flow cytometric analysis of DNA content and phosphorylation of histone H3 at serine-10. Moreover, biochemical assays revealed increased activities of caspases-3/7 in treated cells, specific fragmentation of PARP-1, and phosphorylation of Bcl-2, collectively confirming apoptosis as the mechanism of cell death.

Stable isotope dilution ultra-high performance liquid chromatography-tandem mass spectrometry quantitative profiling of tryptophan-related neuroactive substances in human serum and cerebrospinal fluid.

Many compounds related to L-tryptophan (L-TRP) have interesting biological or pharmacological activity, and their abnormal neurotransmission seems to be linked to a wide range of neurodegenerative and psychiatric diseases. A high-throughput method based on ultra-high performance liquid chromatography connected to electrospray tandem mass spectrometry (UHPLC-ESI-MS/MS) was developed for the quantitative analysis of L-TRP and 16 of its metabolites in human serum and cerebrospinal fluid (CSF), representing both major and minor routes of L-TRP catabolism. The combination of a fast LC gradient with selective tandem mass spectrometry enabled accurate analysis of almost 100 samples in 24h. The standard isotope dilution method was used for quantitative determination. The method's lower limits of quantification for serum and cerebrospinal fluid ranged from 0.05 to 15nmol/L and 0.3 to 45nmol/L, respectively. Analytical recoveries ranged from 10.4 to 218.1% for serum and 22.1 to 370.0% for CSF. The method's accuracy ranged from 82.4 to 128.5% for serum matrix and 90.7 to 127.7% for CSF matrix. All intra- and inter-day coefficients of variation were below 15%. These results demonstrate that the new method is capable of quantifying endogenous serum and CSF levels of a heterogeneous group of compounds spanning a wide range of concentrations. The method was used to determine the physiological levels of target analytes in serum and CSF samples from 18 individuals, demonstrating its reliability and potential usefulness in large-scale epidemiological studies.