Assessing the Chemical Profile and Biological Potentials of Tamarix aphylla (L.) H.Karst. and Tamarix senegalensis DC. by In Vitro, In Silico, and Network Methodologies.

The present study aimed to investigate the chemical profile, antioxidant, and enzyme inhibition properties of extracts from fruits and aerial parts (leaves and twigs) of Tamarix aphylla and T. senegalensis. Hexane, dichloromethane, ethyl acetate (EtOAc), and methanol extracts were prepared sequentially by maceration. Results revealed that EtOAc extracts of T. senegalensis and T. aphylla fruits contained the highest total phenolic content (113.74 and 111.21 mg GAE/g) while that of T. senegalensis (38.47 mg RE/g) recorded the highest total flavonoids content. Among the quantified compounds; ellagic, gallic, 3-hydroxybenzoic, caffeic, syringic, p-coumaric acids, isorhamnetin, procyanidin B2, and kaempferol were the most abundant compounds in the two species. EtOAc extracts of the two organs of T. senegalensis in addition to MeOH extract of T. aphylla aerial parts displayed the highest chelating power (21.00-21.30 mg EDTAE/g, p > 0.05). The highest anti-AChE (3.11 mg GALAE/g) and anti-BChE (3.62 mg GALAE/g) activities were recorded from the hexane and EtOAc extracts of T. senegalensis aerial parts and fruits, respectively. EtOAc extracts of the fruits of the two species exerted the highest anti-tyrosinase (anti-Tyr) activity (99.44 and 98.65 mg KAE/g, p > 0.05). Also, the EtOAc extracts of the both organs of the two species exhibited highest anti-glucosidase activity (0.88-0.90 mmol ACAE/g, p > 0.05) while the best anti-α-amylase activity was recorded from the dichloromethane extract of T. senegalensis fruits (0.74 mmol ACAE/g). In this study, network pharmacology was employed to examine the connection between compounds from Tamarix and their potential effectiveness against Alzheimer's disease. The compounds demonstrated potential interactions with pivotal genes including APP, GSK3B, and CDK5, indicating a therapeutic potential. Molecular docking was carried out to understand the binding mode and interaction of the compounds with the target enzymes. Key interactions observed, such as H-bonds, promoted the binding, and weaker ones, such as van der Waals attractions, reinforced it. These findings suggest that these two Tamarix species possess bioactive properties with health-promoting effects.

An untargeted metabolomics approach to study changes of the medium during human cornea culture.

INTRODUCTION: Two main approaches (organ culture and hypothermia) for the preservation and storage of human donor corneas are globally adopted for corneal preservation before the transplant. Hypothermia is a hypothermic storage which slows down cellular metabolism while organ culture, a corneal culture performed at 28-37 °C, maintains an active corneal metabolism. Researchers, till now, have just studied the impact of organ culture on human cornea after manipulating and disrupting tissues. OBJECTIVES: The aim of the current work was to optimize an analytical procedure which can be useful for discovering biomarkers capable of predicting tissue health status. For the first time, this research proposed a preliminary metabolomics study on medium for organ culture without manipulating and disrupting the valuable human tissues which could be still used for transplantation. METHODS: In particular, the present research proposed a method for investigating changes in the medium, over a storage period of 20 days, in presence and absence of a human donor cornea. An untargeted metabolomics approach using UHPLC-QTOF was developed to deeply investigate the differences on metabolites and metabolic pathways and the influence of the presence of the cornea inside the medium. RESULTS: Differences in the expression of some compounds emerged from this preliminary metabolomics approach, in particular in medium maintained for 10 and 20 days in presence but also in the absence of cornea. A total of 173 metabolites have been annotated and 36 pathways were enriched by pathway analysis. CONCLUSION: The results revealed a valuable untargeted metabolomics approach which can be applied in organ culture metabolomics.

Microwave-assisted hydrodiffusion and gravity extraction: An efficient method to produce pomegranate juice.

Green technologies based on microwaves have been developed by the food industry to produce organoleptically acceptable fruit juices without preliminary processing. Microwave irradiation coupled with hydrodiffusion and gravity (MHG) combines microwave heating with the earth's gravity, allowing the collection of hydrophilic substances released from the irradiated matrix. To the best of our knowledge, MHG extraction has never been experimented to produce pomegranate juice. In this work, we have evaluated it as a potential alternative to the conventional squeezing. A central composite design study (CCD) allowed the selection of the best extractive conditions (irradiation power and extraction time) to obtain a pomegranate juice with higher yield, polyphenol (e.g., catechin and delphinidin-3,5-glucoside) content, and related bioactivities (antioxidant and antidiabetic) than the one obtained by squeezing while maintaining the chemical-physical properties. Thus, this technique appears to be a functional alternative to producing high value pomegranate juice.

Acmella oleracea extracts as green pesticides against eight arthropods attacking stored products.

Developing sustainable control tools for managing noxious pests attacking stored foodstuffs is a timely research challenge. Acmella oleracea (L.) R. K. Jansen is a crop widely cultivated for its multiple usages on an industrial level. In this study, the extracts prepared with A. oleracea aerial parts were applied on wheat kernels for the management of eight important arthropod pests attacking stored products, i.e., Cryptolestes ferrugineus, Tenebrio molitor, Oryzaephilus surinamensis, Trogoderma granarium, Tribolium castaneum, Tribolium confusum, Alphitobius diaperinus (adults/larvae), and Acarus siro (adults/nymphs). Extraction of A. oleracea was optimized on the base of the yield and content of spilanthol and other N-alkylamides which were analysed by HPLC-DAD-MS. Two concentrations of n-hexane or methanol extracts (500 ppm and 1000 ppm), obtained through Soxhlet extraction, were tested to acquire mortality data on the above-mentioned pests after 4, 8, and 16 h and 1 to 7 days of exposure. Both extracts achieved complete mortality (100.0%) of C. ferrugineus adults. In the case of A. diaperinus adults, mortalities were very low at any concentrations of both extracts. In general, the n-hexane extract was more efficient than methanol extract against almost all species and stages. Considering both extracts, the susceptibility order, from most to least susceptible species/stage, was C. ferrugineus adults > A. diaperinus larvae > C. ferrugineus larvae > T. granarium adults > T. molitor larvae > O. surinamensis adults > O. surinamensis larvae > T. confusum larvae > T. castaneum larvae > A. siro adults > T. molitor adults > A. siro nymphs > T. granarium larvae > T. castaneum adults > T. confusum adults > A. diaperinus adults. Our research provides useful knowledge on the efficacy of N-alkylamides-rich A. oleracea extracts as grain protectants, pointing out the importance of targeting the most susceptible species/ developmental stages.

Dual Anta-Inhibitors Targeting Protein Kinase CK1δ and A2A Adenosine Receptor Useful in Neurodegenerative Disorders.

Currently, the number of patients with neurodegenerative pathologies is estimated at over one million, with consequences also on the economic level. Several factors contribute to their development, including overexpression of A2A adenosine receptors (A2AAR) in microglial cells and up-regulation and post-translational alterations of some casein kinases (CK), among them, CK-1δ. The aim of the work was to study the activity of A2AAR and CK1δ in neurodegeneration using in-house synthesized A2A/CK1δ dual anta-inhibitors and to evaluate their intestinal absorption. Experiments were performed on N13 microglial cells, which were treated with a proinflammatory CK cocktail to simulate an inflammatory state typical of neurodegenerative diseases. Results showed that the dual anta-inhibitors have the ability to counteract the inflammatory state, even if compound 2 is more active than compound 1. In addition, compound 2 displayed an important antioxidant effect similar to the reference compound ZM241385. Since many known kinase inhibitors are very often unable to cross lipid bilayer membranes, the ability of A2A/CK1δ double anta-inhibitors to cross the intestinal barrier was investigated by an everted gut sac assay. HPLC analysis revealed that both compounds are able to cross the intestinal barrier, making them promising candidates for oral therapy.

Fecal Protein Profile in Eight Dogs Suffering from Acute Uncomplicated Diarrhea before and after Treatment.

Acute diarrhea is a very frequent condition affecting dogs; nevertheless, little is known about what happens in the GI tract during such conditions. Proteomics allows the study of proteins present in a specific biologic substrate, and fecal proteomic investigations have been recently implemented to study GI diseases in dogs. In the present study, the fecal protein profiles of eight dogs suffering from acute uncomplicated diarrhea at the time of inclusion was investigated for the first time, and then the same patients were followed, replicating two further evaluations at two subsequent time points (after 2 and 14 days from the first presentation), with the aim of gaining possible new insights regarding the pathologic changes in the gastrointestinal environment during such conditions. Two-dimensional gel electrophoresis (2-DE) was performed, followed by mass spectrometry. Nine spots, corresponding to four (groups of) proteins (i.e., albumin, alkaline phosphatase, chymotrypsin-C-like, and some immunoglobulins), showed significant differences at two or more of the three time points investigated, almost all behaving similarly and decreasing at T1 (2 days after the onset of the condition) and significantly increasing at T2 (14 days after the onset), mainly evidencing a reaction of the organism. Further studies including a greater number of patients and possibly different techniques are needed to confirm the present findings.

Development of Innovative Vitamin D Enrichment Designs for Two Typical Italian Fresh Cheeses: Burrata and Giuncata.

The aim of this research was to develop innovative cheeses fortified with vitamin D3 (VD3). Formulation studies and analyses of textural properties and chemicals were carried out for these developments. Two traditional Italian varieties of cheese (giuncata and burrata) were studied. For giuncata, the fortification of milk for cheese production provided a VD3 retention level of 43.9 ± 0.6% in the food matrix. For burrata, the VD3 ingredient was incorporated into the creamy inner part after mixing, maintaining the textural quality of the product (adhesiveness 4.3 ± 0.4 J × 10-3; firmness 0.7 ± 0.0 N; and cohesiveness 0.8 ± 0.2). The optimized enrichment designs allowed to obtain homogenous contents of VD3 during the production of giuncata (0.48 ± 0.01 µg/g) and burrata cheeses (0.32 ± 0.02 µg/g). Moreover, analyses revealed the high stability of VD3 during the storage of the two fortified cheese types (2 weeks, 4 °C). These fortification designs could be implemented at an industrial scale to obtain new cheese types enriched in VD3 and thus contribute to the reduction in VD deficiency prevalence.

Evaluation of the Fecal Proteome in Healthy and Diseased Cheetahs (Acinonyx jubatus) Suffering from Gastrointestinal Disorders.

Fecal proteomics allows for the identification of proteins and peptides present in stools and is useful in finding possible new biomarkers for diagnosing and/or monitoring gastrointestinal (GI) disorders. In the present study, we investigated the fecal proteome in healthy and diseased cheetahs (Acinonyx jubatus). Captive individuals of this species frequently show gastrointestinal disorders characterized by recurrent episodes of diarrhea, rare episodes of vomiting and weight loss, associated with Helicobacter spp. infection. Fecal proteomic evaluation has been performed by two-dimensional electrophoresis followed by liquid chromatography-tandem mass spectrometry. In healthy cheetahs, the results showed the presence of the following proteins: collagen alpha-1 (II) chain, transthyretin, IgG Fc-binding protein, titin, dystonin, isopentenyl-diphosphate Delta-isomerase 1, sodium/potassium-transporting ATPase subunit alpha-1 and protein disulfide-isomerase A6. The presence of albumin isoforms was found only in diseased cheetahs. The present paper reports the study of the fecal proteome in the cheetah, evidences some differences between healthy and diseased patients and confirms, once again, the potential of fecal proteomics for the study of the GI environment, with promising developments regarding the identification of new diagnostic/monitoring markers.

A shelf-life study for the evaluation of a new biopackaging to preserve the quality of organic chicken meat.

Widespread use of traditional packaging constitutes a serious ecological problem leading to a shift to biodegradable and compostable materials. The aim of this work is to study the ability of a new biopackaging (BP), based on biodegradable and compostable material, to preserve the quality of organic chicken meat for 14 days in comparison with a polyethylene terephthalate (PET) material. Results showed that the indices of Biogenic Amines (BAs) and the 18 monitored Volatile Organic Compounds (VOCs) have a similar trend in both packaged meats. For example, the total BAs concentration in meat increased from 390 to 961 mg Kg-1 in BP and from 393 to 800 mg Kg-1 in PET, as well as the microbiological counts. The new biopackaging (BP) showed similar properties of non-biodegradable material (PET) to preserve the shelf life of organic chicken meat and it could be used instead of plastic materials to promote a circular economy.

Clinicopathological and Fecal Proteome Evaluations in 16 Dogs Presenting Chronic Diarrhea Associated with Lymphangiectasia.

Canine intestinal lymphangiectasia (IL) is a condition characterized by variably severe gastrointestinal signs, frequently associated with laboratory abnormalities; the research for markers allowing a better understanding of the severity degree and/or obtaining an early diagnosis and/or monitoring is continuously progressing. In the present study, we investigated possible new diagnostic/follow-up markers in IL dogs, namely, serum C-reactive protein, serum bacterial lipopolysaccharide, serum cleaved cytokeratin 18, serum citrulline, and zonulin (in both serum and feces). A fecal proteomic study looking for possible confirmation and/or new marker candidates was also performed. All markers in both substrates, with the exception of serum citrulline, significantly differed between diseased and control dogs. Fecal proteomics allowed the retrieval of three proteins in IL dogs (Fc fragment of IgG-binding protein; transthyretin; proproteinase E) that were not previously found in clinically healthy subjects. Although further studies are needed, C-reactive protein, bacterial lipopolysaccharide, cleaved cytokeratin 18, and zonulin (in both serum and feces) resulted as promising markers for canine IL; similarly, fecal proteomics represents a road worthy of being pursued in the search for candidate biomarkers.

A comprehensive UHPLC-MS/MS screening method for the analysis of 98 New Psychoactive Substances and related compounds in human hair.

In this study, a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the targeted analysis of 98 New Psychoactive Substances (NPS) from the hair matrix. The monitored compounds included various chemical classes (7 phenethylamines, 10 tryptamines, 18 cathinones, 24 synthetic opioids, and 38 synthetic cannabinoids) with emphasis given to newly emerged NPS. The method employed a direct extraction process through the incubation of hair samples (25 mg) and internal standards with M3® reagent at 100 °C for 60 min, followed by extract purification through acid and basic liquid-liquid micro-extraction (LLME). Extracted compounds were analyzed through LC-MS/MS system operating in multiple reaction monitoring mode. NPS were separated in 9.5 min with a Poroshell 120 EC-C18 column (2.7 µm, 4.6 × 50 mm) using a gradient eluting mobile phase composed of water and acetonitrile/water (95:5) both containing 0.1 % of formic acid. The developed and validated method shows a good precision (≤ 15 %), linearity (R2 between 0.993 and 0.999), selectivity, and sensitivity (LOD: 0.6-10.3 pg mg-1 and LOQ: 2.1-34.4 pg mg-1). The method showed also reduced matrix effect and acceptable recovery for most of the targeted compounds. Our results showed that this method is suitable for quantifying NPS in hair matrix and could be employed in the context of routine analyses in analytical laboratories.

Faecal proteome in clinically healthy dogs and cats: Findings in pooled faeces from 10 cats and 10 dogs.

BACKGROUND: In the scientific literature, there are only a few manuscripts available on small animal faecal proteomics. METHODS: In the present pilot study, this evaluation was performed using pooled faecal samples from 10 clinically healthy dogs and, for the first time, in 10 clinically healthy cats by mean of two-dimensional electrophoresis followed by liquid chromatography-tandem mass spectrometry. RESULTS: Our results showed the presence of nine (albumin, alkaline phosphatase, chymotrypsin-C-like, cytosol aminopeptidase, elastase-3B/proteinase E, immunoglobulins and nuclear pore membrane glycoprotein 210) and 14 (albumin, caspase recruitment domain-containing protein, chymotrypsin-like, deleted in malignant brain tumours 1 protein-like, hypothetical protein LOC107375, immunoglobulin, kallikrein-1, superoxide dismutase, transthyretin precursor, interstitial collagenase-like) different proteins in canine and feline faeces, respectively. CONCLUSION: These preliminary findings document the presence of a range of proteins in the faeces of apparently healthy dogs and cats and may serve as a basis for larger, prospective studies to establish reference proteomic data against which diseased populations can be compared.

Isofuranodiene, a Natural Sesquiterpene Isolated from Wild Celery (Smyrnium olusatrum L.), Protects Rats against Acute Ischemic Stroke.

The myrrh-like furanosesquiterpene isofuranodiene (IFD) is the main constituent of wild celery (Smyrnium olusatrum L., Apiaceae), an overlooked vegetable that was cultivated during the Roman Empire. In the present study, we investigated the protective effects of IFD pre-treatment against oxidative stress and inflammatory response in an animal model of ischemic stroke. IFD was isolated by the crystallization of Smyrnium olusatrum essential oil, and its structure and purity were confirmed by NMR and HPLC analyses. Acute pre-treatment of IFD (10 mg/kg i.p.) significantly reduced the levels of the inflammatory cytokines IL-1ß and TNF-α, the expression of pNF-κB/NF-κB, and the lipid peroxidation indicator MDA. Finally, IFD boosted a faster recovery and better scores in grid-walking and modified neurological severity scores (mNSS) tests. Taken together, these findings indicate IFD as a promising lead compound for the discovery of new treatments of brain ischemia.

Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS.

A simple and fast analytical method able to simultaneously identify and quantify 17 endogenous and exogenous steroidal hormones was developed in bovine and equine blood using UHPLC-MS/MS. A total amount of 500 µL of sample was deproteinized with 500 µL of a mixture of methanol and zinc sulfate and evaporated. The mixture was reconstituted with 50 µL of a solution of 25% methanol and injected in the UHPLC-MS/MS triple quadrupole. The correlation coefficients of the calibration curves of the analyzed compounds were in the range of 0.9932-0.9999, and the limits of detection and quantification were in the range of 0.023-1.833 and 0.069-5.5 ppb, respectively. The developed method showed a high sensitivity and qualitative aspects allowing the detection and quantification of all steroids in equine and bovine blood. Moreover, the detection limit of testosterone (50 ppt) is half of the threshold admitted in plasma (100 ppt). Once validated, the method was used to quantify 17 steroid hormones in both bovine and equine blood samples. The primary endogenous compounds detected were corticosterone (range 0.28-0.60 ppb) and cortisol (range 0.44-10.00 ppb), followed by androstenedione, testosterone and 11-deoxycortisol.

Studying GGDEF Domain in the Act: Minimize Conformational Frustration to Prevent Artefacts.

GGDEF-containing proteins respond to different environmental cues to finely modulate cyclic diguanylate (c-di-GMP) levels in time and space, making the allosteric control a distinctive trait of the corresponding proteins. The diguanylate cyclase mechanism is emblematic of this control: two GGDEF domains, each binding one GTP molecule, must dimerize to enter catalysis and yield c-di-GMP. The need for dimerization makes the GGDEF domain an ideal conformational switch in multidomain proteins. A re-evaluation of the kinetic profile of previously characterized GGDEF domains indicated that they are also able to convert GTP to GMP: this unexpected reactivity occurs when conformational issues hamper the cyclase activity. These results create new questions regarding the characterization and engineering of these proteins for in solution or structural studies.

Phytochemical Profile and Biological Activities of Crude and Purified Leonurus cardiaca Extracts.

Leonurus cardiaca L. (Lamiaceae) is a perennial herb distributed in Asia and Southeastern Europe and has been used in traditional medicine since antiquity for its role against cardiac and gynecological disorders. The polar extracts obtained from L. cardiaca aerial parts contain several compounds among which alkaloids, iridoids, labdane diterpenes, and phenylethanoid glycosides play a major role in conferring protection against the aforementioned diseases. On the other hand, the antioxidant activities and the enzyme inhibitory properties of these extracts have not yet been deeply studied. On the above, in the present study, crude and purified extracts were prepared from the aerial parts of L. cardiaca and have been chemically characterized by spectrophotometric assays and HPLC-DAD-MS analyses. Notably, the content of twelve secondary metabolites, namely phenolic acids (chlorogenic, caffeic, caffeoylmalic and trans-ferulic acids), flavonoids (rutin and quercetin), phenylethanoid glycosides (verbascoside and lavandulifolioside), guanidine pseudoalkaloids (leonurine), iridoids (harpagide), diterpenes (forskolin), and triterpenes (ursolic acid), has been determined. Furthermore, the extracts were tested for their antioxidant capabilities (phosphomolybdenum, DPPH, ABTS, FRAP, CUPRAC, and ferrous chelating assays) and enzyme inhibitory properties against cholinesterase, tyrosinase, amylase, and glucosidase. The purified extracts contained higher phytochemical content than the crude ones, with caffeoylmalic acid and verbascoside as the most abundant compounds. A linear correlation between total phenolics, radical scavenging activity, and reducing power of extracts has been found. Notably, quercetin, caffeic acid, lavandulifolioside, verbascoside, chlorogenic acid, rutin, and ursolic acid influenced the main variations in the bioactivities found in L. cardiaca extracts. Our findings provide further insights into the chemico-biological traits of L. cardiaca and a scientific basis for the development of nutraceuticals and food supplements.

Cigarette butts, a threat for marine environments: Lessons from benthic foraminifera (Protista).

Cigarette butts are the most common form of litter in the world and their environmental impact is related to both persistence and potential toxic effects for chemical composition. The objective of this study was to assess the acute toxicity (LC50-48 h) of human-smoked cigarette butts leachate on 3 cultured genera of benthic foraminifera: the calcareous perforate Rosalina globularis, the calcareous imperforate Quinqueloculina spp., and the agglutinated Textularia agglutinans. The specimens were exposed to 16, 8, 4, 2, and 1 cigarette butts/L concentrations that prove to be acutely toxic to all taxa. Starting from 4 cigarette butts/L, both calcareous genera showed shell decalcification, and death of almost all the individuals, except for the more resistant agglutinated species. These results suggest the potential harmfulness of cigarette butts leachate related to pH reduction and release of toxic substances, in particular nicotine, which leads to physiology alteration and in many cases cellular death.

Proteomic characterization of kefir milk by two-dimensional electrophoresis followed by mass spectrometry.

Kefir is a type of fermented milk obtained thanks to the introduction of "kefir grains" in mammalian milk. Kefir grains consist of lactic and acetic acid bacteria and yeasts in alternative proportions that are held together by a matrix of complex sugars known as "kefiran." Thanks to the fermentative process, the kefir milk is rich in nutraceutical substances such as amino acids, vitamins, and mineral salts. The most valuable compounds of kefir fermentation are mainly lactic acid, exopolysaccharides, and bioactive peptides, the resulting products of proteolytic release from milk proteins (caseins and whey proteins). Among the nutraceutical properties of kefir are antimicrobial and antitumor activity, immunomodulating effect, and cholesterol-lowering effect. Therefore, in light of these intriguing properties of kefir milk, in this work, a proteomic analysis, by two-dimensional electrophoresis followed by mass spectrometry, has been performed. As a result, milk-derived polypeptides were identified in commercial kefir milk from organic farming. In particular, polypeptides deriving from κ-, αs1 -, and αs2 -caseins that may have potentially beneficial effects on human health have been detected.

Determination of coeluted isomers in wine samples by application of MS/MS deconvolution analysis.

Two organic acids isomers, 3-isopropylmalic acid (3-IPMA) and 2-isopropylmalic acid (2-IPMA), were identified and quantified in wine samples by using an LC-MS/MS method without any chromatographic separation, but processing the MS/MS data with a recently developed deconvolution algorithm (LEDA: linear equations deconvolution analysis), thus decreasing the time necessary for the process. In particular, the LEDA tool processes the MS/MS signals and assigns the relative concentrations (abundances) of the isomers in the sample, at the mg L-1 level. The efficiency of MS/MS signal assignment was improved by introducing five linear equations to define the LEDA matrix. Then, as a novel approach, an overdetermined system of linear equations was applied for the deconvolution of isomers. The use of LEDA to identify and quantify the isomers in wine samples, together with the choice of a short LC column and a fast elution gradient, simplifies the process and shortens the time needed. Furthermore, it was evaluated the quantitative determination of the IPMA isomers by using the calibration curve provided by the precursor ion MRM transition data. The calculated values of accuracy (recovery between 82.6% and 99.8%) and precision (RSD between 0.4% and 4.0%) confirm the validity of this quantitative approach and the ability of LEDA to establish the correct percentage of the MS/MS signal for each isomer. Finally, to compare the conventional LC-MS/MS method and our proposed method of LC-MS/MS coupled with LEDA post-processing elaboration, a series of real wine samples were analysed by both methods, and the results were compared.

Fiber-Sample Distance, An Important Parameter To Be Considered in Headspace Solid-Phase Microextraction Applications.

To define and control the parameters which impact headspace solid-phase microextraction (HS-SPME), it is important to reach the highest level of reproducibility. The present study aims to assess, for the first time, the effect of fiber-sample distance during HS-SPME in pre-equilibrium conditions. Analyses were primarily performed on mixtures of standard volatiles compounds (alkanes, alcohols, organic acids) designed in our lab and then on various food matrices (wine, chicken, cheese, tea), repeating already published experiments. Extractions were performed varying fiber penetration depths (10-60 mm) at different times (10-60 min) and temperatures of extraction (30-80 °C). The study revealed that variation of the distance between the fiber and the sample into the vial clearly impacts the results obtained during HS-SPME when conditions are such that no equilibrium is reached in HS. For example, in wine analysis, the percentage of octanoic acid at 80 °C was higher at 40 mm (7.5 ± 0.2%) than that at 20 mm (4.4 ± 0.3%). Moreover, regardless of the extraction temperature, the lower the time of extraction, the stronger the dependence on the fiber-sample distance. Indeed, at 60 °C, the obtained response factors for octadecane at 20 and 40 mm of fiber penetration were 21.8 and 44.5, respectively, after 10 min of extraction, 54.1 and 71.0 after 30 min, and 79.4 and 82.4 after 60 min of extraction. The analyses have been here corroborated by a theoretical model based on the diffusion equation. Therefore, to improve the method robustness during HS-SPME studies, we suggest specifying the fiber penetration depth or the fiber-sample distance with the other parameters of extraction.