A critical review of bioanalytical and clinical applications of solid phase microextraction.

Studying the functions, mechanisms, and effects of drugs and other exogenous compounds on biological systems, together with investigations performed to understand biosystems better, comprises one of the most fascinating areas of research. Although classical sample preparation techniques are dominantly used to infer the relevant information from the investigated system, they fail to meet various imperative requirements, such as being environmentally friendly, applicable in-vivo, and compatible with online analysis. As a chameleon in the analytical toolbox, solid phase microextraction (SPME) is one of the best tools available for studying biological systems in unconventional ways. In this review, SPME is spotlighted, and its capability for bioanalytical applications, including drug analysis, untargeted and targeted metabolomics, in-vivo and clinical studies, is scrutinized based on studies reported in the past five years. In addition, novel extractive phases and instrumental coupling strategies developed to serve bioanalytical research are discussed to give the perspective for state-of-the-art and future developments. The literature assessment showed that SPME could act as a critical tool to investigate in-vivo biological systems and provide information about the elusive portion of the metabolome. Moreover, recently introduced miniaturized SPME probes further improved the low-invasive nature of the sampling and enabled sampling even from a single cell. The coupling of SPME directly to mass spectrometry significantly reduced the total analytical workflow and became one of the promising tools suitable for fast diagnostic purposes and drug analysis. The numerous applications and advancements reported in bioanalysis using SPME show that it will continue to be an indispensable technique in the future.

Mesoporous carbon hollow spheres based sensitive SPME probes for in vivo sampling analysis of selected plant hormones in Chinese aloes.

Phytohormones are a class of endogenous substances that separately or synergistically regulate the growth, development, and differentiation of plants. Accurately and efficiently detecting and monitoring the concentration of plant hormones in living plants is of significant importance. Herein, a novel mesoporous carbon hollow spheres (MCHS)-based in vivo solid phase microextraction (SPME) probe was designed for in vivo sampling of plant hormones. The designed MCHS features the advantages of high surface area, porous shells, and large hollow spaces, facilitating the dynamic adsorption and enrichment of target phytohormone. In addition, a cationic polyelectrolyte, (poly (diallyl dimethyl ammonium chloride) (PDDA), was further modified onto the MCHS to expedite the extraction process by electrostatic interaction. Utilizing the MCHS@PDDA probe in combination with HPLC-MS/MS facilitated the continuous monitoring of three plant hormones (abscisic acid (ABA), indole-3-acetic acid (IAA), and gibberellin (GA3)) in Chinese aloe. The detection limit of this method was 0.016-0.090 µg/L, the linear range was 10-1000 µg/L, and both the RSD of the single probe (n = 6) and probe-to-probe test (n = 6) were less than 7.2 %. This method had excellent accuracy and good reproducibility comparable to the traditional sample pretreatment method. Ultimately, this established in-vivo SPME method was successfully adopted to quantify three selected plant hormones in living Chinese Aloes, providing a new method for the long-term monitoring of endogenous active substances in living system.

In vivo profiling of phytocannabinoids in Cannabis spp. varieties via SPME-LC-MS analysis.

BACKGROUND: In vivo solid-phase microextraction (SPME) is a minimally invasive, non-exhaustive sample-preparation technique that facilitates the direct isolation of low molecular weight compounds from biological matrices in living systems. This technique is especially useful for the analysis of phytocannabinoids (PCs) in plant material, both for forensic purposes and for monitoring the PC content in growing Cannabis spp. plants. In contrast to traditional extraction techniques, in vivo SPME enables continuous tracking of the changes in the level of PCs during plant growth without the need for plant material collection. In this study, in vivo SPME utilizing biocompatible C18 probes and liquid-chromatography coupled to quadrupole time-of flight mass spectrometry (LC-Q-TOF-MS) is proposed as a novel strategy for the extraction and analysis of the acidic forms of five PCs in growing medicinal cannabis plants. RESULTS: The SPME method was optimized by testing various parameters, including the extraction phase (coating), extraction and desorption times, and the extraction temperature. The proposed method was validated with satisfactory analytical performance regarding linearity (10-3000 ng/mL), limits of quantification, and precision (relative standard deviations below 5.5 %). The proposed method was then successfully applied for the isolation of five acidic forms of PCs, which are main components of growing medicinal cannabis plants. As a proof-of-concept, SPME probes were statically inserted into the inflorescences of two varieties of Cannabis spp. plants (i.e., CBD-dominant and Δ9-THC-dominant) cultivated under controlled conditions for 30 min extraction of tetrahydrocannabinolic acid (Δ9-THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabiviarinic acid (CBVA), and tetrahydrocannabivarinic acid (THCVA). SIGNIFICANCE AND NOVELTY: The results confirmed that the developed SPME-LC-Q-TOF-MS method is a precise and efficient tool that enables direct and rapid isolation and analysis of PCs under in vivo conditions. The proposed methodology is highly appealing option for monitoring the metabolic pathways and compositions of multiple PCs in medicinal cannabis at different stages of plant growth.

In Vivo Profiling and Quantification of Chlorinated Paraffin Homologues in Living Fish.

Herein, we demonstrate the ability of a dual-purpose periodic mesoporous organosilica (PMO) probe to track the complex chlorinated paraffin (CP) composition in living animals by assembling it as an adsorbent-assisted atmospheric pressure chemical ionization Fourier-transform ion cyclotron resonance mass spectrometry (APCI-FT-ICR-MS) platform and synchronously performing it as the in vivo sampling device. First, synchronous solvent-free ionization and in-source thermal desorption of CP homologues were achieved by the introduction of the PMO adsorbent-assisted APCI module, generating exclusive adduct ions ([M - H]-) of individual CP homologues (CnClm) with enhanced ionization efficiency. Improved detection limits of short- and medium-chain CPs (0.10-24 and 0.48-5.0 pg/µL) were achieved versus those of the chloride-anion attachment APCI-MS methods. Second, the dual-purpose PMO probe was applied to extract the complex CP compositions in living animals, following APCI-FT-ICR-MS analysis. A modified pattern-deconvolution algorithm coupled with the sampling-rate calibration method was used for the quantification of CPs in living fish. In vivo quantification of a tilapia exposed to technical CPs for 7 days was successfully achieved, with ∑SCCPs and ∑MCCPs of the sampled fish calculated to be 1108 ± 289 and 831 ± 266 µg/kg, respectively. Meanwhile, 58 potential CP metabolites were identified in living fish for the first time during in vivo sampling of CPs, a capacity that could provide an important tool for future study regarding its expected risks to humans and its environmental fate.

Solid-phase microextraction of endogenous metabolites from intact tissue validated using a Biocrates standard reference method kit.

Improved analytical methods for the metabolomic profiling of tissue samples are constantly needed. Currently, conventional sample preparation methods often involve tissue biopsy and/or homogenization, which disrupts the endogenous metabolome. In this study, solid-phase microextraction (SPME) fibers were used to monitor changes in endogenous compounds in homogenized and intact ovine lung tissue. Following SPME, a Biocrates AbsoluteIDQ assay was applied to make a downstream targeted metabolomics analysis and confirm the advantages of in vivo SPME metabolomics. The AbsoluteIDQ kit enabled the targeted analysis of over 100 metabolites via solid-liquid extraction and SPME. Statistical analysis revealed significant differences between conventional liquid extractions from homogenized tissue and SPME results for both homogenized and intact tissue samples. In addition, principal component analysis revealed separated clustering among all the three sample groups, indicating changes in the metabolome due to tissue homogenization and the chosen sample preparation method. Furthermore, clear differences in free metabolites were observed when extractions were performed on the intact and homogenized tissue using identical SPME procedures. Specifically, a direct comparison showed that 47 statistically distinct metabolites were detected between the homogenized and intact lung tissue samples (P < 0.05) using mixed-mode SPME fibers. These changes were probably due to the disruptive homogenization of the tissue. This study's findings highlight both the importance of sample preparation in tissue-based metabolomics studies and SPME's unique ability to perform minimally invasive extractions without tissue biopsy or homogenization while providing broad metabolite coverage.

Trying is believing: A pilot study of in-vivo nicotine replacement therapy sampling in disadvantaged Black adults who smoke cigarettes.

INTRODUCTION: While nicotine replacement therapy (NRT) is a frontline tobacco treatment that doubles smoking quit rates, only about 18% of Black adults who smoke cigarettes report lifetime use of NRT. A promising approach for increasing NRT use is in-session (in-vivo) NRT sampling within cessation interventions. The present pilot study examined the effectiveness of an in-vivo NRT sampling intervention within a single-session, culturally-targeted motivational intervention trial in Black adults who smoke cigarettes. METHODS: Non-treatment-seeking disadvantaged Black adults (N = 60) were offered the choice to sample nicotine lozenge, patch, or both in-session with the counselor present. Regardless of their choice, they were offered a one-week starter kit of both products. Data were analyzed at baseline and 1-month follow-up. Primary outcomes were 1) differences in motivation to quit smoking among NRT samplers versus non-samplers, 2) in-vivo NRT sampling preferences, and 3) in-vivo sampling's association with NRT use and improved smoking outcomes at follow up. RESULTS: Almost all participants accepted a take-home NRT starter kit, and approximately half of those offered in-vivo sampling agreed to sample. Participants preferred sampling lozenges in session (75.8% lozenge only vs. 12.1% nicotine patch only or 12.1% both; p < .001). Motivation to quit smoking was not related to likelihood of in-vivo NRT sampling (p > .05). At 1-month follow-up, in-vivo samplers were more likely to use NRT (94% vs. 35%, respectively; p < .001) and report a quit attempt (81.8% vs. 53.9%, p < .05) compared to non-samplers. CONCLUSION: In-vivo NRT sampling is a promising strategy to improve NRT uptake among Black adults who smoke cigarettes, regardless of motivation to quit smoking.

Recent advances in facemask devices for in vivo sampling of human exhaled breath aerosols and inhalable environmental exposures.

Since the COVID-19 pandemic, the unprecedented use of facemasks has been requiring for wearing in daily life. By wearing facemask, human exhaled breath aerosols and inhaled environmental exposures can be efficiently filtered and thus various filtration residues can be deposited in facemask. Therefore, facemask could be a simple, wearable, in vivo, onsite and noninvasive sampler for collecting exhaled and inhalable compositions, and gain new insights into human health and environmental exposure. In this review, the recent advances in developments and applications of in vivo facemask sampling of human exhaled bacteria, viruses, proteins, and metabolites, and inhalable facemask contaminants and air pollutants, are reviewed. New features of facemask sampling are highlighted. The perspectives and challenges on further development and potential applications of facemask devices are also discussed.

In Vivo Contaminant Monitoring and Metabolomic Profiling in Plants Exposed to Carbamates via a Novel Microextraction Fiber.

In this study, a biocompatible solid-phase microextraction (SPME) fiber with high-coverage capture capacity based on a nitrogen-rich porous polyaminal was developed. The fiber was used to track the bioaccumulation and elimination of carbamates (isoprocarb, carbofuran, and carbaryl) and their metabolites (o-cumenol, carbofuran phenol, and 1-naphthalenol) in living Chinese cabbage plants (Brassica campestris L. ssp. chinensis Makino (var. communis Tsen et Lee)). A case-and-control model was applied in the hydroponically cultured plants, with the exposed plant groups contaminated under three carbamates at 5 µg mL-1. Both bio-enrichment and elimination of carbamates and their metabolites in living plants appeared to be very fast with half-lives at ∼0.39-0.79 and ∼0.56-0.69 days, respectively. Statistical differences in the endogenous plant metabolome occurred on day 3 of carbamate exposure. In the exposed group, the plant metabolic alterations were not reversed after 5 days of contaminant-free growth, although most contaminates had been eliminated. Compared with prior nutriological and toxicological studies, >50 compounds were first identified as endogenous metabolites in cabbage plants. The contents of the glucosinolate-related metabolites demonstrated significant time-dependent dysregulations that the fold changes of these key metabolites decreased from 0.78-1.07 to 0.28-0.82 during carbamate exposure. To summarize, in vivo SPME provided new and important information regarding exogenous carbamate contamination and related metabolic dysregulation in plants.

In vivo solid-phase microextraction swab-mass spectrometry for multidimensional analysis of human saliva.

Solid phase microextraction (SPME) is one of the most powerful sample preparation techniques for analyte extraction and enrichment from complex matrices. SPME fibers are commonly used to extract analytes from collected samples. Following our recent work on development of in vivo SPME swab that integrates an SPME fiber and a medical swab (Anal Chim Acta, 2020, 1124, 71-77), the multiple SPME fibers inserted into a medical swab (multiple-SPME swab) is further developed to couple with different mass spectrometry (MS) approaches for multidimensional analysis of human saliva in this work. The new features of cotton ball and SPME fiber of multiple-SPME swab are investigated. Biomarker discovery and disease diagnosis using multiple-SPME swab are also demonstrated. The present study shows that direct coupling multiple-SPME swab with different MS-based approaches could be simple and versatile in vivo method to expand the classes of analytes extracted simultaneously from human saliva.

Contactless electrospray ionization mass spectrometry for direct detection of analytes in living organisms.

In this study, we developed contactless electrospray ionization mass spectrometry (ESI-MS) for in vivo analysis of living organisms in different applications. The in vivo sampling and direct analysis processess of living organisms were integrated into an operation that only requires the organism close to MS inlet that was applied to a high voltage. Living plants and animals were directly induced to generate spray ionization. Direct detection and in vivo monitoring of metabolites and chemical residues in various living organisms were successfully demonstrated. Analysis of a single sample could be completed within 30 s. Overall, contactless ESI-MS provides an attractive in vivo method to straightforward investigation of living organisms.

Comprehensive Investigation of Metabolic Changes Occurring in the Rat Brain Hippocampus after Fluoxetine Administration Using Two Complementary In Vivo Techniques: Solid Phase Microextraction and Microdialysis.

Fluoxetine is among the most prescribed antidepressant drugs worldwide. Nevertheless, limited information is known about its definitive mechanism. Although in vivo examinations performed directly in related brain structures can provide more realistic, and therefore more insightful, knowledge regarding the mechanisms and efficacy of this drug, only a few techniques are applicable for in vivo monitoring of metabolic alterations in the brain following an inducement. Among them, solid phase microextraction (SPME) and microdialysis (MD) have emerged as ideal in vivo tools for extraction of information from biosystems. In this investigation, we scrutinized the capabilities of SPME and MD to detect ongoing changes in the brain following acute fluoxetine administration. Sequential in vivo samples were collected simultaneously from male rats' hippocampi using SPME and MD before drug administration in order to establish a baseline; then samples were collected again following fluoxetine administration for an investigation of small molecule alterations. Our results indicate that MD provides more comprehensive information for polar compounds, while SPME provides superior information with respect to lipids and other medium level polar molecules. Interestingly, in the lipidomic investigation, all dysregulated features were found to be membrane lipids and associated compounds. Moreover, in the metabolomic investigations, dysregulation of hippocampal metabolite levels associated with fatty acid transportation and purine metabolisms were among the most notable findings. Overall, our evaluation of the obtained data corroborates that, when used in tandem, SPME and MD are capable of providing comprehensive information regarding the effect of fluoxetine in targeted brain structures and further elucidating this drug's mechanisms of action in the brain.

Investigation of Early Death-Induced Changes in Rat Brain by Solid Phase Microextraction via Untargeted High Resolution Mass Spectrometry: In Vivo versus Postmortem Comparative Study.

Analysis of brain samples obtained postmortem remains a standard approach in neuroscience, despite often being suboptimal for inferring roles of small molecules in the pathophysiology of brain diseases. Sample collection and preservation further hinders conclusive interpretation of biomarker analysis in autopsy samples. We investigate purely death-induced changes affecting rat hippocampus in the first hour of postmortem interval (PMI) by means of untargeted liquid chromatography-mass spectrometry-based metabolomics. The unique possibility of sampling the same brain area of each animal both in vivo and postmortem was enabled by employing solid phase microextraction (SPME) probes. Four millimeter probes coated with mixed mode extraction phase were used to sample awake, freely roaming animals, with 2 more sampling events performed after death. Significant changes in brain neurochemistry were found to occur as soon as 30 min after death, further progressing with increasing PMI, evidenced by relative changes in levels of metabolites and lipids. These included species from several distinct groups, which can be classified as engaged in energy metabolism-related processes, signal transduction, neurotransmission, or inflammatory response. Additionally, we perform thorough analysis of interindividual variability in response to death, which provides insights into how this aspect can obscure conclusions drawn from an untargeted study at single metabolite and pathway level. The results suggest high demand for systematic studies examining the PMI time course with in vivo sampling as a starting point to eliminate artifacts in the form of neurochemical changes assumed to occur in vivo.

Investigating distributions and changes of alkaloids in living Catharanthus roseus under low-phosphorus stress using wooden-tip electrospray ionisation mass spectrometry.

INTRODUCTION: Vinca alkaloids are important sources for producing anticancer drugs from Catharanthus roseus. The phosphorus of soil is one of crucial factors for planting C. roseus. OBJECTIVES: We aim to develop an in vivo sampling technique coupled with direct mass spectrometry with wooden tip for investigating distributions and changes of alkaloids in flowers, leaves, stems, veins and roots of living C. roseus under low-phosphorus stress. MATERIALS AND METHODS: Living C. roseus were prepared under low-phosphorus stress (n = 10) and control conditions (n = 10). Wooden-tip electrospray ionisation mass spectrometry and conventional liquid chromatography-mass spectrometry were applied to analyse living C. roseus and extracts of C. roseus, respectively. RESULTS: Distributions and changes of serpentine, vindoline, catharanthine, and anhydrovinblastine in living C. roseus under low-phosphorus stress and control conditions were successfully obtained. CONCLUSION: Compared to control soil conditions, low-phosphorus soil was found to induce C. roseus to generate more serpentine but less catharanthine and vindoline in leaves, veins, stems and roots, and to generate more anhydrovinblastine in flowers, leaves, stems and roots. Overall, our results showed a simple, rapid, and effective method for in vivo sampling and direct analysis of living plants.

Trace analysis of UV filters and musks in living fish by in vivo SPME-GC-MS.

A method was developed for the simultaneous determination of two groups of personal care products, namely UV filters (oxybenzone, 3-(4-methylbenzylidene)camphor, padimate-O, 2-ethylhexyl-4-methoxycinnamate, and octocrylene) and polycyclic aromatic musks (galaxolide and tonalide), in fish by in vivo solid-phase microextraction followed by gas chromatography-mass spectrometry. The in vivo method was validated by carrying out in vitro experiments; the method validation parameters were linearity (r2 > 0.98), interday precision (relative standard deviations < 35.50%), limits of detection and quantification ranging from 2 to 25 ng g-1 and 5 to 70 ng g-1, respectively. The calibrations in vivo and in vitro were determined using a pre-equilibrium sampling rate calibration method. In vivo sampling rate (Rs) was greater than that in vitro; therefore in vivo Rs was applied to the uptake and elimination tracing under controlled laboratory conditions to avoid quantitation error. All analytes were bioaccumulated in muscle tissue over the 5-day exposure in different grades depending on their molecular structure and physicochemical properties; the most absorbed compound was tonalide and the least absorbed compound was padimate-O. The elimination rate was initially high with a rapid decrease of the analyte concentrations for the first 24 h; thereafter, the rate of elimination tended to decrease which indicated that the target analytes were bioaccumulated. To our knowledge, this is the first time that UV filters have been analyzed with in vivo SPME-GC-MS. The proposed method is a simple, miniaturized, and non-lethal alternative for the determination of personal care products in living organisms. Graphical abstract.

In Vivo Sampling: A Promising Technique for Detecting and Profiling Endogenous Substances in Living Systems.

Endogenous substances, naturally occurring in living organisms, are critical components with physiological and biological functions. Discovery and quantitative measurement of endogenous substances in living biotas are important for food analysis, crop cultivation, and quality assessment. Low or non-invasive in vivo sampling techniques offer the advantages of minimal perturbation to the investigated system and potentially obtain more accurate feedback compared to in vitro sampling. In this perspective, we summarize the up-to-date progress in the development of microdialysis and solid-phase microextraction as valuable tools for in vivo sampling of endogenous substances in food and agriculture chemistry. We discuss their feasibility for on-site and real-time in vivo monitoring and highlight the prospects in searching for highly specific coatings, miniaturized sampling devices, and instruments that well meet the trend for high-efficient and high-throughput analyses.

Direct ion generation from swabs.

Medical swabs are used for biofluid and tissue sampling in clinical applications. The use of medical swabs as electrospray ionization probes for direct mass spectrometric analysis is a novel and potentially widely applicable development. Here we discuss ion generation, characterize ionization behavior via microscopic videography and describe some illustrative examples of applications.

In Vivo Selective Capture and Rapid Identification of Luteolin and Its Metabolites in Rat Livers by Molecularly Imprinted Solid-Phase Microextraction.

A method based on molecularly imprinted solid-phase microextraction (MIP-SPME) coupled with liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) was developed for the detection of luteolin and its metabolites in vivo. The MIP-SPME fibers were first fabricated by dopamine and silane, and then luteolin MIPs-coated fibers were successfully prepared using luteolin, acrylamide (AM), and ethylene glycol dimethacrylate (EGDMA) as the template, functional monomer and cross-linker, respectively. The characterizations of polymers were analyzed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and the Brunauer-Emmett-Teller method (BET). The properties involving adsorption and selective experiments were evaluated, and these results revealed that MIP fibers presented high adsorption capacity and selectivity to luteolin. Furthermore, the developed MIP-SPME coupled with the LC-QTOF-MS/MS method was adopted to capture and identify luteolin and its metabolites in rat livers in vivo, and eventually, apigenin, chrysoeriol, and diosmetin were rapidly identified as metabolites.

In vivo solid-phase microextraction liquid chromatography-tandem mass spectrometry for monitoring blood eicosanoids time profile after lipopolysaccharide-induced inflammation in Sprague-Dawley rats.

A fast and non-lethal in vivo solid-phase microextraction (SPME) sampling method for rat blood coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) was developed for monitoring rapid changes in concentrations of eicosanoids - lipid mediators involved in the development of inflammatory conditions - using diffusion-based calibration. Sampling rates of target eicosanoids were pre-determined under laboratory conditions with a precision of ≤10%, and directly used for quantification of analyte concentrations in blood after lipopolysaccharide-induced inflammation in Sprague-Dawley rats. Results showed significant changes in unbound plasma concentrations of arachidonic acid (AA) and 12-hydroxyeicosatetraenoic acid (12-HETE) in response to the treatment. Next, performance of the proposed method was compared with protein precipitation (PP) of plasma, a conventional sample preparation technique. Finally, percentages of plasma protein binding (PPB) of specific eicosanoids were determined. PPB of target eicosanoids was in agreement with literature values, ranging from 99.3 to 99.9% for 12-HETE and DHA, respectively. We envision that the proposed method is a particularly suitable alternative to lethal sampling and current methods based on sample depletion in animal studies for accurate monitoring of rapid changes in blood concentrations of small molecules.

Study of complex matrix effect on solid phase microextraction for biological sample analysis.

Solid phase microextraction (SPME) has become a useful tool for in vivo monitoring the behavior of environmental organic pollutants in biological species due to its simplicity, relatively non-invasive, and cost-effective manner. However, the complex matrices in biological samples could significantly influence the extraction kinetic, and bias the quantification result. In this study, we investigated the effect of complex matrix on the extraction kinetic of SPME for biological sample analysis. Two sample matrices, phosphate-buffered saline (PBS) with bovine serum albumin (BSA) and agarose gel with BSA were used to simulate the biological fluid and tissue. Results showed that the addition of BSA significantly enhanced the mass transfer of organic compounds onto SPME fiber in both PBS buffer and gel sample. Enhancement factors ranging from 1.3 to 27, and 2.0 to 80 were found for all selected polyaromatic hydrocarbons (PAHs) in PBS buffer and agarose gel with BSA concentration of 0.1-5%, respectively. Then, an improved theoretical model was applied to quantify the observed enhancement effect, and the result showed that the predicted sampling time constant agreed well with the experimental one in complex matrix. Furthermore, a simplified equation was proposed for the real biological sample analysis.

Contact solid-phase microextraction with uncoated glass and polydimethylsiloxane-coated fibers versus solvent sampling for the determination of hydrocarbons in adhesion secretions of Madagascar hissing cockroaches Gromphadorrhina portentosa (Blattodea) by gas chromatography-mass spectrometry.

Molecular profiles of adhesion secretions of Gromphadorrhina portentosa (Madagascar hissing cockroach, Blattodea) were investigated by gas chromatography mass spectrometry with particular focus on a comprehensive analysis of linear and branched hydrocarbons. For this purpose, secretions from the tarsi (feet), possibly contributing to adhesion on smooth surfaces, and control samples taken from the tibiae (lower legs), which contain general cuticular hydrocarbons that are supposed to be not involved in the biological adhesion function, were analyzed and their molecular fingerprints compared. A major analytical difficulty in such a study constitutes the representative, spatially controlled, precise and reproducible sampling from a living insect as well as the minute quantities of insect secretions on both tarsi and tibiae. Thus, three different in vivo sampling methods were compared in terms of sampling reproducibility and extraction efficiency by replicate measurement of samples from tarsi and tibiae. While contact solid-phase microextraction (SPME) with a polydimethylsiloxane (PDMS) fiber showed higher peak intensities, a self-made uncoated glass fiber had the best repeatability in contact-SPME sampling. Chromatographic profiles of these two contact-SPME sampling methods were statistically not significantly different. Inter-individual variances were larger than potentially existing minor differences in molecular patterns of distinct sampling methods. Sampling by solvent extraction was time consuming, showed lower sensitivities and was less reproducible. In general, sampling by contact-SPME with a cheap glass fiber turned out to be a viable alternative to PDMS-SPME sampling. Hydrocarbon patterns of the tarsal adhesion secretions were qualitatively similar to those of epicuticular hydrocarbon profiles of the tibiae. However, hydrocarbons were in general less abundant in tarsal secretions than secretions from tibiae.