Optimization of SPME-Arrow-GC/MS Method for Determination of Free and Bound Volatile Organic Compounds from Grape Skins.

(1) Background: Solid phase microextraction (SPME)-Arrow is a new extraction technology recently employed in the analysis of volatiles in food materials. Grape volatile organic compounds (VOC) have a crucial role in the winemaking industry due to their sensory characteristics of wine.; (2) Methods: Box-Behnken experimental design and response surface methodology were used to optimise SPME-Arrow conditions (extraction temperature, incubation time, exposure time, desorption time). Analyzed VOCs were free VOCs directly from grape skins and bound VOCs released from grape skins by acid hydrolysis.; (3) Results: The most significant factors were extraction temperature and exposure time for both free and bound VOCs. For both factors, an increase in their values positively affected the extraction efficiency for almost all classes of VOCs. For free VOCs, the optimum extraction conditions are: extraction temperature 60 °C, incubation time 20 min, exposure time 49 min, and desorption time 7 min, while for the bound VOCs are: extraction temperature 60 °C, incubation time 20 min, exposure time 60 min, desorption time 7 min.; (4) Conclusions: Application of the optimized method provides a powerful tool in the analysis of major classes of volatile organic compounds from grape skins, which can be applied to a large number of samples.

Optimization of HS-SPME for GC-MS Analysis and Its Application in Characterization of Volatile Compounds in Sweet Potato.

Volatile compounds are the main chemical species determining the characteristic aroma of food. A procedure based on headspace solid-phase microextraction (HP-SPME) coupled to gas chromatography-mass spectrometry (GC-MS) was developed to investigate the volatile compounds of sweet potato. The experimental conditions (fiber coating, incubation temperature and time, extraction time) were optimized for the extraction of volatile compounds from sweet potato. The samples incubated at 80 °C for 30 min and extracted at 80 °C by the fiber with a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) coating for 30 min gave the most effective extraction of the analytes. The optimized method was applied to study the volatile profile of four sweet potato cultivars (Anna, Jieshu95-16, Ayamursaki, and Shuangzai) with different aroma. In total, 68 compounds were identified and the dominants were aldehydes, followed by alcohols, ketones, and terpenes. Significant differences were observed among the volatile profile of four cultivars. Furthermore, each cultivar was characterized by different compounds with typical flavor. The results substantiated that the optimized HS-SPME GC-MS method could provide an efficient and convenient approach to study the flavor characteristics of sweet potato. This is the basis for studying the key aroma-active compounds and selecting odor-rich accessions, which will help in the targeted improvement of sweet potato flavor in breeding.

Extraction Improvement of the Bioactive Blue-Green Pigment "Marennine" from Diatom Haslea ostrearia's Blue Water: A Solid-Phase Method Based on Graphitic Matrices.

The compound "marennine" is a blue-green pigment produced by the benthic microalgae Haslea ostrearia, with pathogenicity reduction activities against some bacteria and promising potential as a natural pigment in seafood industries. After decades of research, the chemical family of this compound still remains unclear, mainly because structural studies were impaired by the presence of co-extracted compounds in marennine isolates. To improve the purity of marennine extract, we developed a novel extraction method using a graphitic stationary phase, which provides various advantages over the previous procedure using tandem ultrafiltration. Our method is faster, more versatile, provides a better crude yield (66%, compared to 57% for ultrafiltration) and is amenable to upscaling with continuous photobioreactor cultivation. Our goal was to take advantage of the modulable surface properties of the graphitic matrix by optimizing its interactions with marennine. As such, the effects of organic modifiers, pH and reducing agents were studied. With this improvement on marennine purification, we achieved altogether the isolation of a fucoidan-related, sulfated polysaccharide from blue water. Characterization of the polysaccharides fraction suggests that roughly half of UV-absorbing compounds could be isolated from the marennine crude extracts. The identification of sulfated polysaccharides could be a major breakthrough for marennine purification, providing targeted isolation techniques. Likewise, the added value of Haslea ostrearia and the role of polysaccharides in previous marennine chemical characterization and bioactivity studies remain to be determined.

Dispersive micro-solid phase extraction combined with switchable hydrophilicity solvent-based homogeneous liquid-liquid microextraction for enrichment of non-steroidal anti-inflammatory drugs in environmental water samples.

Herein, a sensitive and green method combining dispersive micro-solid phase extraction (D-µ-SPE) and homogeneous liquid-liquid microextraction (HLLME) has been developed. Magnetic layered double hydroxide nanoparticals were prepared and used as adsorbents in d-µ-SPE. The fascinating dissolvable characteristic of the material can eliminate elution step without usage of toxic organic solvents. Dipropylamine was used as a pH-triggered switchable hydrophilicity solvent that can change the miscible/immiscible states reversibly, achieving fast two-phase separation. To demonstrate the applicability of proposed method, three non-steroidal anti-inflammatory drugs including ketoprofen, naproxen and tolmetin in water samples were enriched and purified prior to HPLC-UV analysis. The influencing parameters such as pH of sample solution, amount of sorbent, vortex time, type and volume of acidic solution and SHS, volume of NaOH were optimized in detail. The method exhibits good linearity (0.1-50 ng/mL), low limits of detection (0.02-0.05 ng/mL), high precision (RSDs<9.3%) and acceptable accuracy (97.2%-105.7%). Therefore, the presented procedure is fast, sensitive, simple and suitable for determination of non-steroidal anti-inflammatory drugs from aqueous matrices.

Systematic Evaluation of Different Coating Chemistries Used in Thin-Film Microextraction.

A systematic evaluation of eight different coatings made of solid phase extraction (SPE) and carbon-based sorbents immobilized with polyacrylonitrile in the thin-film microextraction (TFME) format using LC-MS/MS was described. The investigated coatings included graphene, graphene oxide, multi-walled carbon nanotubes (MWCNTs), carboxylated MWCNTs, as carbon-based coatings, and polystyrene-divinylbenzene (PS-DVB), octadecyl-silica particles (C18), hydrophilic-hydrophobic balance particles (HLB) and phenyl-boronic acid modified particles (PBA), as SPE-based coatings. A total of 24 compounds of diverse moieties and of a wide range of polarities (log P from -2.99 to 6.98) were selected as probes. The investigated coatings were characterized based on their extraction performance toward the selected probes at different pH values and at optimized desorption conditions. In the case of SPE-based coatings, PS-DVB and HLB exhibited a balanced extraction for compounds within a wide range of polarities, and C18 showed superior extraction recoveries for non-polar analytes. Carbon-based coatings showed high affinity for non-polar compounds given that their main driving force for extraction is hydrophobic interactions. Interestingly, among the studied carbon-based coatings, graphene oxide showed the best extraction capabilities toward polar compounds owing to its oxygen-containing groups. Overall, this work provided important insights about the extraction mechanisms and properties of the investigated coatings, facilitating the coating selection when developing new TFME applications.

Bioanalytical HPLC Applications of In-Tube Solid Phase Microextraction: A Two-Decade Overview.

In-tube solid phase microextraction is a cutting-edge sample treatment technique offering significant advantages in terms of miniaturization, green character, automation, and preconcentration prior to analysis. During the past years, there has been a considerable increase in the reported publications, as well as in the research groups focusing their activities on this technique. In the present review article, HPLC bioanalytical applications of in-tube SPME are discussed, covering a wide time frame of twenty years of research reports. Instrumental aspects towards the coupling of in-tube SPME and HPLC are also discussed, and detailed information on materials/coatings and applications in biological samples are provided.

Uptake of pharmaceuticals acts as an abiotic stress and triggers variation of jasmonates in Malabar spinach (Basella alba. L).

In recent years, pharmaceuticals have received increasing attentions because of their potential risks to the environment, but researches focusing on their impacts on defense system of living plants are still lacking. As an important class of phytohormones, jasmonates play crucial roles in plant defense system against environmental stress. In order to investigate the effect of pharmaceuticals uptake on endogenous jasmonates, an in vivo solid phase microextraction (SPME) method was established to simultaneously detect and monitor both pharmaceuticals and jasmonates in living plants. The proposed method exhibited wide linear ranges, high sensitivity (limits of detection ranging 0.0043-0.035 ng g-1 for pharmaceuticals and 0.091-0.22 ng g-1 for jasmonates, respectively), and satisfactory reproducibility (relative standard deviation of intrafiber ranging 4.2%-8.6% and interfiber ranging 5.2%-8.2%, respectively). Subsequently, this method was successfully applied to track the concentrations of each pharmaceutical and corresponding jasmonates in living Malabar spinach plants (Basella alba. L) exposed to three common pharmaceuticals (i.e. gemfibrozil, mefenamic acid and tolfenamic acid) over 15 days. In result, all pharmaceuticals appeared to trigger intensive biosynthesis of jasmonic acid (JA) (3.1-9.4 times of control) while reduced the concentration of methyl jasmonate (MeJA) (18.3%-38.1% of control). We inferred that uptake of pharmaceuticals acted as an abiotic stress and stimulated the plant defense response because of the variation of jasmonates. To the best of our knowledge, this is the first study applying SPME to detect and track both pharmaceuticals and phytohormones in living plants, which not only provided a glimpse to the adverse effect of pharmaceuticals on plants as well as the regulation of endogenous jasmonates, but also set a promising template for future in vivo analysis of xenobiotics and plant endogenous substances.

Rapid Monitoring of Organochlorine Pesticide Residues in Various Fruit Juices and Water Samples Using Fabric Phase Sorptive Extraction and Gas Chromatography-Mass Spectrometry.

Fabric phase sorptive extraction, an innovative integration of solid phase extraction and solid phase microextraction principles, has been combined with gas chromatography-mass spectrometry for the rapid extraction and determination of nineteen organochlorine pesticides in various fruit juices and water samples. FPSE consolidates the advanced features of sol-gel derived extraction sorbents with the rich surface chemistry of cellulose fabric substrate, which could extract the target analytes directly from the complex sample matrices, substantially simplifying the sample preparation operation. Important FPSE parameters, including sorbent chemistry, extraction time, stirring speed, type and volume of back-extraction solvent, and back-extraction time have been optimized. Calibration curves were obtained in a concentration range of 0.1⁻500 ng/mL. Under optimum conditions, limits of detection were obtained in a range of 0.007⁻0.032 ng/mL with satisfactory precision (RSD < 6%). The relative recoveries obtained by spiking organochlorine pesticides in water and selected juice samples were in the range of 91.56⁻99.83%. The sorbent sol-gel poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) was applied for the extraction and preconcentration of organochlorine pesticides in aqueous and fruit juice samples prior to analysis with gas chromatography-mass spectrometry. The results demonstrated that the present method is simple, rapid, and precise for the determination of organochlorine pesticides in aqueous samples.

Strategies for Accurate Quantitation of Volatiles from Foods and Plant-Origin Materials: A Challenging Task.

The volatile fraction of foods and of plant-origin materials provides functional information on sample-related variables, and gas-phase extractions are ideal approaches for its accurate chemical characterization. However, for gas-phase sampling, the usual procedures adopted to standardize results from solvent extraction methods are not appropriate: headspace (HS) composition depends on the intrinsic physicochemical analyte properties (volatility, polarity, partition coefficient(s)) and matrix effects. Method development, design, and expression of the results are therefore challenging. This review article focuses on volatile vapor-phase quantitation methods (internal standard normalization, standard addition, stable isotope dilution assay, multiple headspace extraction) and their suitability in different applications. Because of the analyte informative role, the different ways of expressing results (normalized chromatographic area, percent normalized chromatographic areas, and absolute concentrations) are discussed and critically evaluated with examples of quality markers in chamomile, process contaminants (furan and 2-methylfuran) in roasted coffee, and key-aroma compounds from high-quality cocoa.

HPLC-MS/MS combined with membrane-protected molecularly imprinted polymer micro-solid-phase extraction for synthetic cathinones monitoring in urine.

Synthetic cathinones are a type of drug belonging to group of new psychoactive substances (NPSs). The illicit market for these substances is characterized by the continuous introduction to the market of new analogs to evade legislation and to avoid detection. New screening and confirmation assays are therefore needed, mainly in forensic/clinical samples. In the current development, a porous membrane-protected, micro-solid-phase extraction (µ-SPE) has been developed for the assessment of several cathinones in urine. The µ-SPE device consisted of a cone-shaped polypropylene (PP) porous membrane containing the adsorbent (molecularly imprinted polymers, MIPs, synthesized for the first time for this class of drugs). MIPs were prepared using ethylone and 3-methylmethcathinone (3-MMC) as templates, ethylene glycol dimethacrylate (EGDMA) as a functional monomer, divinylbenzene (DVB) as a cross-linker, and 2,2´-azobisisobutyronitrile (AIBN) as an initiator. The prepared ethylone-based MIP and 3-MMC-based MIP have been fully characterized and evaluated as new selective adsorbents for µ-SPE. Cathinones separation/determination was performed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Optimum loading conditions (pH 5.0, loading for 4.0 minutes under orbital-horizontal shaking at 200 rpm) and elution conditions [2.0 mL of 75:20:5 heptane/2-propanol/ammonium hydroxide and ultrasounds assistance (37 kHz, 325 W) for 4.0 minutes] were found for ethylone-based MIP. Validation (intra-day and inter-day precision and analytical recovery) showed RSD values lower than 9 and 10% for intra-day and inter-day precision, and within the 88%-101% range for intra-day and inter-day analytical recovery.

Development of a novel solid phase microextraction calibration method for semi-solid tissue sampling.

Accurate quantitative analysis using in vivo solid phase microextraction (SPME) for semi-solid tissue can be challenging due to the complexity of the sample matrix. In this paper, a comprehensive study was carried out on the extraction kinetics of SPME in the semi-solid sample, and subsequently proposed a new theoretical model to interpret the kinetic extraction process. Theoretically derived mathematical expressions well described the experimental desorption time profiles of the SPME process. Modelling experiments were also carried out to study the effect of sample tortuosity and binding matrix on the parameters affecting the extraction kinetics. Seven polyaromatic hydrocarbons (PAHs) and eight polychlorinated biphenyls (PCBs) in agarose gel and in real fish tissue were used for these experiments. The experimental data showed excellent agreement with theoretical prediction while providing excellent interpretation of the effect of tortuosity and binding matrix. Based on the theoretical model, an on-fiber standard calibration method with fewer internal standards was developed. The newly developed calibration method was used to quantify PAHs and PCBs in agarose gel and fish tissue. By using the proposed calibration method, a large number of organic compounds can be quantified with fewer internal standards. Current study provides the theoretical foundation for in vivo SPME quantitative semi-solid tissue analysis in the future.

Analysis of volatile compounds in gluten-free bread crusts with an optimised and validated SPME-GC/QTOF methodology.

The aroma of bread crust, as one of the first characteristics perceived, is essential for bread acceptance. However, gluten-free bread crusts exhibit weak aroma. A SPME-GC/QTOF methodology was optimised with PCA and RSM and validated for the quantification of 44 volatile compounds in bread crust, extracting 0.75 g of crust at 60 °C for 51 min. LODs ranged between 3.60 and 1760 µg Kg-1, all the R2 were higher than 0.99 and %RSD for precision and %Er for accuracy were lower than 9% and 12%, respectively. A commercial wheat bread crust was quantified, and furfural was the most abundant compound. Bread crusts of wheat starch and of japonica rice, basmati rice and teff flours were also quantified. Teff flour and wheat starch crusts were very suitable for improving gluten-free bread crust aroma, due to their similar content in 2-acetyl-1-pyrroline and 4-hydroxy-2,5-dimethyl-3(2H)-furanone compared to wheat flour crust and also for their high content in pyrazines.

High throughput µ-SPE based elution coupled with UPLC-MS/MS for determination of eluxadoline in plasma sample: Application in pharmacokinetic characterization of PLGA nanoparticle formulations in rats.

Eluxadoline is a novel µ- and κ-opioid receptor (OR) agonist and δ-OR antagonist, recently approved as a first line therapy for the treatment of irritable bowel syndrome. Due to abuse potential, poor bioavailability and high intersubject variability, a sensitive and reliable assay is prerequisite for its determination in biological samples. This work first time report the development and validation of UPLC-MS/MS assay for determination of eluxadoline in rat plasma sample using risperidone as an internal standard (IS). A high-throughput 96-well plate format µ-SPE technique was used for plasma sample extraction. The extracted samples were separated on Acquity BEH™ C18 column (100×2.1mm, 1.7µm) using mobile phase elution of acetonitrile: 20mM ammonium acetate (80:20, v/v) at a flow rate of 0.3mLmin-1. The precursor to product ion transition of m/z 570.16→118.12 (qualifier), 570.16→171.08 (quantifier) for eluxadoline, and m/z 411.18→191.07 for IS were used for MRM monitoring. The calibration curves were linear in concentration range of 0.15-50ngmL-1 with LOD and LOQ of 0.07 and 0.15ngmL-1, respectively. The validation results satisfied the criteria of USFDA and SWGTOX guidelines and were within the acceptable limit. Finally, the method was successfully applied in bioavailability enhancement study of the newly developed PLGA nanoparticles and Eudragit coated PLGA nanoparticles of eluxadoline in rats.

Development and validation of an SPME-GC method for a degradation kinetics study of propiconazole I, propiconazole II and tebuconazole in blueberries in Concordia, the main production area of Argentina.

An analytical method for the simultaneous determination of propiconazole isomers and tebuconazole residues in blueberries was developed using solid-phase microextraction (SPME) coupled to gas chromatography. Confirmation was performed by gas chromatography-mass spectrometry in selected-ion monitoring mode. The SPME fibre coating selected was CWX-DVB, and the pH was adjusted to 7 with NaOH. The method is selective with adequate precision and high accuracy and sensitivity. Recoveries ranged between 97.4% and 98.9% for all compounds; and detection and quantification limits were respectively 0.21 and 0.49 µg kg-1 for propiconazole I; 0.16 and 0.22 µg kg-1 for propiconazole II; and 0.16 and 0.48 µg kg-1 for tebuconazole. The degradation of these fungicides in blueberries followed first-order rate kinetics. The half-life times for flowering and fruit set applications were respectively 4.0 and 10.3 days for propiconazole I, 4.0 and 11.4 days for propiconazole II, and 3.5 and 12.4 days for tebuconazole.

Application of response surface methodology to optimize solid-phase microextraction procedure for chromatographic determination of aroma-active monoterpenes in berries.

Most of scientific papers concern the qualitative or semi-quantitative analysis of aroma-active terpenes in liquid food matrices. Therefore, the procedure based on solid-phase microextraction and two-dimensional gas chromatography-time-of-flight mass spectrometry for determination of monoterpenes in fresh berries was developed. The optimal extraction conditions using divinylbenzene-carboxen-polydimethylsiloxane fiber were: temperature of 50°C, extraction time of 26min, equilibrium time of 29min. The developed procedure provides a high recovery (70.8-99.2%), good repeatability (CV<10.4%), high linearity (r>0.9915) and offers practical advantages over currently used methods: reliability of compounds identification, simplicity of extraction and at least one order of magnitude lower detection limits (0.10-0.011µg/L). The method was successfully applied to determine monoterpenes in 27 berry samples of different varieties and 4 berry products. Tukey's test revealed that monoterpenes content is a reliable indicator of fruit maturity and origin. It suggests that the method may be of interest to researchers and food industry.

Optimisation and validation of a HS-SPME-GC-IT/MS method for analysis of carbonyl volatile compounds as biomarkers in human urine: Application in a pilot study to discriminate individuals with smoking habits.

A new and simple analytical approach consisting of an automated headspace solid-phase microextraction (HS-SPME) sampler coupled to gas chromatography-ion trap/mass spectrometry detection (GC-IT/MS) with a prior derivatization step with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) was developed to detect volatile carbonyl metabolites with low molecular weights in human urine. A central composite design (CCD) was used to optimise the PFBHA concentration and extraction conditions that affect the efficiency of the SPME procedure. With a sample volume of 1 mL, optimal conditions were achieved by adding 300 mg/L of PFBHA and allowing the sample to equilibrate for 6 min at 62°C and then extracting the samples for 51 min at the same temperature, using a divinylbenzene/polydimethylsiloxane (DVB/PDMS) fibre. The method allowed the simultaneous identification and quantification of 44 carbonyl compounds consisting of aldehydes, dialdehydes, heterocyclic aldehydes and ketones. The method was validated with regards to the linearity, inter- and intra-day precision and accuracy. The detection limits ranged from 0.009 to 0.942 ng/mL, except for 4-hydroxy-2-nonenal (15 ng/mL), and the quantification limits varied from 0.029 to 1.66 ng/mL, except for butanal (2.78 ng/mL), 2-butanone (2.67 ng/mL), 4-heptanone (3.14 ng/mL) and 4-hydroxy-2-nonenal (50.0 ng/mL). The method accuracy was satisfactory, with recoveries ranging from 90 to 107%. The proof of applicability of the methodology was performed in a pilot target analysis of urine samples obtained from 18 healthy smokers and 18 healthy non-smokers (control group). Chemometric supervised analysis was performed using the volatile patterns acquired for these samples and clearly showed the potential of the volatile carbonyl profiles to discriminate urine from smoker and non-smoker subjects. 5-Methyl-2-furfural (p<0.0001), 2-methylpropanal, nonanal and 2-methylbutanal (p<0.05) were identified as potentially useful biomarkers to identify smoking habits.

Analysis of Volatile Markers for Virgin Olive Oil Aroma Defects by SPME-GC/FID: Possible Sources of Incorrect Data.

The need to explain virgin olive oil (VOO) aroma descriptors by means of volatiles has raised interest in applying analytical techniques for trapping and quantitating volatiles. Static headspace sampling with solid phase microextraction (SPME) as trapping material is one of the most applied solutions for analyzing volatiles. The use of an internal standard and the determination of the response factors of the main volatiles seem to guarantee the correct determination of volatile concentrations in VOOs by SPME-GC/FID. This paper, however, shows that the competition phenomena between volatiles in their adsorption to the SPME fiber, inherent in static headspace sampling, may affect the quantitation. These phenomena are more noticeable in the particular case of highly odorant matrices, such as rancid and vinegary VOOs with high intensity of defect. The competition phenomena can modify the measurement sensitivity, which can be observed in volatile quantitation as well as in the recording of internal standard areas in different matrices. This paper analyzes the bias of the peak areas and concentrations of those volatiles that are markers for each sensory defect of VOOs (rancid, vinegary, musty, and fusty) when the intensity and complexity of aroma are increased. Of the 17 volatile markers studied in this work, 10 presented some anomalies in the quantitation in highly odorant matrices due the competition phenomena. However, quantitation was not affected in the concentration ranges at which each volatile marker is typically found in the defective oils they were characteristic of, validating their use as markers.

Simultaneous analysis of 22 volatile organic compounds in cigarette smoke using gas sampling bags for high-throughput solid-phase microextraction.

Quantifying volatile organic compounds (VOCs) in cigarette smoke is necessary to establish smoke-related exposure estimates and evaluate emerging products and potential reduced-exposure products. In response to this need, we developed an automated, multi-VOC quantification method for machine-generated, mainstream cigarette smoke using solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS). This method was developed to simultaneously quantify a broad range of smoke VOCs (i.e., carbonyls and volatiles, which historically have been measured by separate assays) for large exposure assessment studies. Our approach collects and maintains vapor-phase smoke in a gas sampling bag, where it is homogenized with isotopically labeled analogue internal standards and sampled using gas-phase SPME. High throughput is achieved by SPME automation using a CTC Analytics platform and custom bag tray. This method has successfully quantified 22 structurally diverse VOCs (e.g., benzene and associated monoaromatics, aldehydes and ketones, furans, acrylonitrile, 1,3-butadiene, vinyl chloride, and nitromethane) in the microgram range in mainstream smoke from 1R5F and 3R4F research cigarettes smoked under ISO (Cambridge Filter or FTC) and Intense (Health Canada or Canadian Intense) conditions. Our results are comparable to previous studies with few exceptions. Method accuracy was evaluated with third-party reference samples (≤15% error). Short-term diffusion losses from the gas sampling bag were minimal, with a 10% decrease in absolute response after 24 h. For most analytes, research cigarette inter- and intrarun precisions were ≤20% relative standard deviation (RSD). This method provides an accurate and robust means to quantify VOCs in cigarette smoke spanning a range of yields that is sufficient to characterize smoke exposure estimates.

Qualification and application of a liquid chromatography-tandem mass spectrometric method for the determination of human Aß1-40 and Aß1-42 peptides in transgenic mouse plasma using micro-elution solid phase extraction.

A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed and applied for the determination of human Aß1-40 and Aß1-42 peptides in transgenic mouse plasma to support preclinical pharmacodynamics studies. The method consisted of micro-elution solid phase extraction for sample preparation and LC-MS/MS analysis in the negative ion mode using electrospray ionization for analysis. (15)N53-Aß1-40 and (15)N55-Aß1-42 peptides were used as internal standards. A quadratic regression (weighted 1/concentrations), with an equation y = ax(2) + bx + c, was used to fit calibration curves over the concentration range of 0.500-100 ng/mL for both Aß1-40 and Aß1-42 peptides. For quality control samples at 6.00, 40.0 and 80.0 ng/mL from the qualification experiment, the within-run accuracy ranged from -2.69 to 0.583 % with precision values ≤8.23 % for Aß1-40. Within-run accuracy ranged from -4.83 to 10.1 % with precision values ≤8.87 % for Aß1-42. Samples from a pharmacodynamics study using Tg2576 transgenic mice were analyzed by this qualified LC-MS/MS method and concentrations were compared to those generated by ELISA. The two methods were shown to be comparable for Aß1-40 quantification of samples from the Tg2576 amyloid precursor protein transgenic mouse model, but varied slightly for Aß1-42.

The approach to sample acquisition and its impact on the derived human fecal microbiome and VOC metabolome.

Recent studies have illustrated the importance of the microbiota in maintaining a healthy state, as well as promoting disease states. The intestinal microbiota exerts its effects primarily through its metabolites, and metabolomics investigations have begun to evaluate the diagnostic and health implications of volatile organic compounds (VOCs) isolated from human feces, enabled by specialized sampling methods such as headspace solid-phase microextraction (hSPME). The approach to stool sample collection is an important consideration that could potentially introduce bias and affect the outcome of a fecal metagenomic and metabolomic investigation. To address this concern, a comparison of endoscopically collected (in vivo) and home collected (ex vivo) fecal samples was performed, revealing slight variability in the derived microbiomes. In contrast, the VOC metabolomes differ widely between the home collected and endoscopy collected samples. Additionally, as the VOC extraction profile is hyperbolic, with short extraction durations more vulnerable to variation than extractions continued to equilibrium, a second goal of our investigation was to ascertain if hSPME-based fecal metabolomics studies might be biased by the extraction duration employed. As anticipated, prolonged extraction (18 hours) results in the identification of considerably more metabolites than short (20 minute) extractions. A comparison of the metabolomes reveals several analytes deemed unique to a cohort with the 20 minute extraction, but found common to both cohorts when the VOC extraction was performed for 18 hours. Moreover, numerous analytes perceived to have significant fold change with a 20 minute extraction were found insignificant in fold change with the prolonged extraction, underscoring the potential for bias associated with a 20 minute hSPME.