Volatiles Composition and Antimicrobial Activities of Areca Nut Extracts Obtained by Simultaneous Distillation-Extraction and Headspace Solid-Phase Microextraction.

The volatile components of areca nuts were isolated by headspace solid-phase microextraction (HS-SPME, DVB/CAR/PDMS fiber extraction) and simultaneous hydrodistillation-extraction (SHDE) and analyzed by gas chromatography/mass spectrometry. Furthermore, all SHDE fractions were tested for antimicrobial activity using the disk diffusion method on nine Gram-negative and Gram-positive bacteria (Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus canis, Streptococcus pyogenes, and Candida albicans). In total, 98 compounds (mainly alcohols, carbonyl compounds, fatty acids, esters, terpenes, terpenoids, and aliphatic hydrocarbons) were identified in SHDE fractions and by using SPME extraction Fatty acids were the main group of volatile constituents detected in all types of extracts. The microorganism most sensitive to the extract of the areca nut was Streptococcus canis. The results can provide essential information for the application of different treatments of areca nuts in the canning industry or as natural antibiotics.

Authentification of fruit spirits using HS-SPME/GC-FID and OPLS methods.

This research provides an accurate description of the origin for fruit spirits. In total, 63 samples of various kinds of fruit spirits (especially from apples, pears, plums, apricots and mirabelle) were analysed using headspace-solid phase microextraction and gas chromatography with flame-ionization detector. Obtained volatile profiles were treated and analysed by multivariate regression with a reduction of dimensionality-orthogonal projections to latent structure for the classification of fruit spirits according to their fruit of origin. Basic result of statistical analysis was the differentiation of spirits to groups with respect to fruit kind. Tested kinds of fruit spirits were strictly separated from each other. The selection was achieved with a specificity of 1.000 and a sensitivity of 1.000 for each kind of spirit. The statistical model was verified by an external validation. Hierarchical cluster analysis (calculation of distances by Ward's method) showed a similarity of volatile profiles of pome fruit spirits (apple and pear brandies) and stone fruit spirits (especially mirabelle and plum brandies).

Comparison of Chemical Composition and Biological Properties of Essential Oils Obtained by Hydrodistillation and Steam Distillation of Laurus nobilis L.

The purpose of this study was to compare the yield, chemical composition, antimicrobial and antioxidant properties of essential oils isolated from leaves of Laurus nobilis L. by two different distillation methods. The essential oils isolated by hydrodistillation (HD) and steam distillation (SD) were analyzed by gas chromatography coupled to mass spectrometry (GC-MS) and gas chromatography with flame ionization detector (GC-FID). Hydrodistillation produced a yield of 0.95 ± 0.06% which is slightly higher than yield obtained by steam distillation 0.79 ± 0.07%. Seventy three compounds in the bay leaves oil obtained by steam distillation were identified while in essential oil obtained by hydrodistillation were identified only 54 compounds. The antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical method. Antimicrobial activity of obtained essential oils was evaluated by disc diffusion method in comparison with several chosen antimicrobials. The antimicrobial activity was tested on five microorganisms - Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Candida albicans. In general, oils produced by steam distillation had higher antimicrobial and antioxidant activities than hydrodistillation extracts. It seems that hydrodistillation is better for higher yield while steam distillation is better to use for more quality oils with stronger biological properties.

Chemical Composition and Determination of the Antibacterial Activity of Essential Oils in Liquid and Vapor Phases Extracted from Two Different Southeast Asian Herbs-Houttuynia cordata (Saururaceae) and Persicaria odorata (Polygonaceae).

Essential oils obtained via the hydrodistillation of two Asian herbs (Houttuynia cordata and Persicaria odorata) were analyzed by gas chromatography coupled to mass spectrometry (GC-MS) and gas chromatography with flame ionization detector (GC-FID). Additionally, both the liquid and vapor phase of essential oil were tested on antimicrobial activity using the broth microdilution volatilization method. Antimicrobial activity was tested on Gram-negative and Gram-positive bacteria-Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Streptococcus pyogenes, Klebsiella pneumoniae, Seratia marcescense and Bacillus subtilis. Hydrodistillation produced a yield of 0.34% (Houttuynia cordata) and 0.40% (Persicaria odorata). 41 compounds were identified in both essential oils. Essential oils contained monoterpenes and their oxidized forms, sesquiterpenes and their oxidized forms, oxidized diterpenes, derivates of phenylpropene and other groups, such as, for example, aldehydes, alcohols or fatty acids. Both essential oils were antimicrobial active in both vapor and liquid phases at least in case of one bacterium. They expressed various antimicrobial activity in the range of 128-1024 µg∙mL-1, 512-1024 µg∙mL-1 in broth and 1024 µg∙mL-1, 512-1024 µg∙mL-1 in agar, respectively. Research showed new interesting information about P. odorata and H. cordata essential oils and demonstrated that both essential oils could be possibly used in the field of natural medicine or natural food preservation.

The issue of HPLC determination of endogenous lipoic acid in human plasma.

Lipoic acid (LA) is used extensively as a therapeutic agent for the treatment of various diseases. Many methods have been reported for the determination of LA plasma levels and its metabolites after its supplementation, but available information concerning endogenous plasma levels is still scarce. Studies which directly focused on determining the endogenous plasma levels provided highly controversial results, <4.9 nmol/L or 143.7-197.0 nmol/L. The main aim of this study was to verify the levels of free LA in the plasma of 40 individuals (17 women, 23 men). This group was nonsupplemented with LA and met the conditions for incorporation into the blood donors register. We measured the levels of LA using an HPLC method with very sensitive coulometric detection after previous sample preparation including deproteination and solid-phase extraction with a Phenyl cartridge. Our limit of detection was 1.85 nmol/L and was better than the values reported in studies that directly focused on determining the endogenous plasma levels of LA: 2.4 and 4.9 nmol/L respectively. However, the levels of free LA in the plasma of nonsupplemented voluntary blood donors were not detectable in all cases. The presented results of our study show that endogenous concentrations of LA are <1.85 nmol/L.

On-line monitoring of in-vitro oral bioaccessibility tests as front-end to liquid chromatography for determination of chlorogenic acid isomers in dietary supplements.

A novel fully automated in-vitro oral dissolution test assay as a front-end to liquid chromatography has been developed and validated for on-line chemical profiling and monitoring of temporal release profiles of three caffeoylquinic acid (CQA) isomers, namely, 3-CQA,4-CQA and 5-CQA, known as chlorogenic acids, in dietary supplements. Tangential-flow filtration is harnessed as a sample processing approach for on-line handling of CQA containing extracts of hard gelatin capsules and introduction of protein-free samples into the liquid chromatograph. Oral bioaccessibility/dissolution test assays were performed at 37.0±0.5°C as per US Pharmacopeia recommendations using pepsin with activity of ca. 749,000 USP units/L in 0.1mol/L HCl as the extraction medium and a paddle apparatus stirred at 50rpm. CQA release rates and steady-state dissolution conditions were determined accurately by fitting the chromatographic datasets, namely, the average cumulative concentrations of bioaccessible pools of every individual isomer monitored during 200min, with temporal resolutions of ≥10min, to a first-order dissolution kinetic model. Distinct solid-to-liquid phase ratios in the mimicry of physiological extraction conditions were assessed. Relative standard deviations for intra-day repeatability and inter-day intermediate precision of 5-CQA within the 5-40µg/mL concentration range were <3.4% and <5.5%, respectively. Trueness of the automatic flow method for determination of 5-CQA released from dietary supplements in gastric fluid surrogate was demonstrated by spike recoveries, spanning from 91.5-104.0%, upon completion of the dissolution process. The proposed hyphenated setup was resorted for evaluating potential differences in dissolution profiles and content of the three most abundant chlorogenic acid isomers in dietary supplements from varied manufacturers.

Chemical composition of essential oils from plantago lanceolata L. leaves extracted by hydrodistillation.

Extensive traditional use of medical plants leads to research dealing with chemical composition of essential oils. The aim of this work was evaluation of quality of the essential oil and extending of the knowledge about chemical composition of essential oil from ribwort (Plantago lanceolata L.) and proportional representation of compounds. Extractions of essential oils from samples of ribwort were performed by hydrodistillation. GC-MS and GC-FID techniques were used for investigation of the qualitative and semi-quantitative content of aromatic compounds in the essential oils, respectively. Major aroma constituents of ribwort leaves were groups of fatty acids 28.0-52.1 % (the most abundant palmitic acid 15.3-32.0 %), oxidated monoterpenes 4.3-13.2 % (linalool 2.7-3.5 %), aldehydes and ketones 6.9-10.0 % (pentyl vinyl ketone 2.0-3.4 %) and alcohols 3.8-9.2 % (1-octen-3-ol 2.4-8.2 %). In relative high amount were identified apocarotenoids (1.5-2.3 %) which are important constituents because of their intense fragrant. The importance is in potential manufacture control of feedstocks before producing of food supplements.

Using headspace solid-phase microextraction for comparison of volatile sulphur compounds of fresh plants belonging to families Alliaceae and Brassicaceae.

In this study, an optimisation of extraction of sulphur volatile compounds (SVCs) has been performed using Central Composite Design. The conditions of the highest amount of eluated peaks and total peaks area have been treated. Factors such as coating of fiber for SPME (Solid Phase Microextraction), extraction temperature and extraction time have been optimised. The SVCs have shown the optimal extraction using a DVB/CAR/PDMS (divinylbenzene/carboxen/polydimethylsiloxane) fiber at 73 °C during 50 min. Furthermore, a pre-incubation step lasting 20 min at the extraction temperature has been used. In total, 12 samples have been investigated at the mentioned optimal conditions, eight from the Alliaceae and four from the Brassicaceae family. The highest number of SVCs (24) has been identified in the sample of chive. The most frequently identified compound found in 11 of 12 samples has been dimethyl trisulphide.

Comparison of various techniques for the extraction and determination of antioxidants in plants.

The following extraction techniques have been used for extracting antioxidants (apigenin, coumarin, esculetin, umbelliferone, bergapten, quercetin, rutin, scopoletin and xanthotoxin) from plant material: supercritical fluid extraction, pressurized liquid extraction, extraction by means of Soxhlet apparatus, ultrasonic extraction in ultrasonic bath, and by means of ultrasonic probe. The analytical method based on HPLC-UV detection for the determination of selected antioxidants was developed. For all extracts the antioxidant capacity based on the reduction of free 2,2-diphenyl-1-picrylhydrazyl radical was also determined. Comparing all results the ultrasonic probe method using 0.75 g of sample extracted by 50 mL of acetonitrile in water (30%, v/v) for 25 min at room temperature and with amplitude at 60% (equal to 90 W) without pulsation was evaluated as the best tool. The most significant indicator demonstrating this statement is the antioxidant capacity expressed as gallic acid equivalent where the ultrasonic probe method showed the best results in 10 of 16 samples. Also the operability of ultrasonic probe extraction method compared to other tested methods is more favorable.

Comparison of inhibitory effects between acetaminophen-glutathione conjugate and reduced glutathione in human glutathione reductase.

Acetaminophen overdose is the most frequent cause of acute liver injury. The main mechanism of acetaminophen toxicity has been attributed to oxidation of acetaminophen. The oxidation product is very reactive and reacts with glutathione generating acetaminophen-glutathione conjugate (APAP-SG). Although this conjugate has been recognized to be generally nontoxic, we have found recently that APAP-SG could produce a toxic effect. Therefore, the aim of our study was to estimate the toxicity of purified APAP-SG by characterizing the inhibitory effect in human glutathione reductase (GR) and comparing that to the inhibitory effect of the natural inhibitor reduced glutathione. We used two types of human GR: recombinant and freshly purified from red blood cells. Our results show that GR was significantly inhibited in the presence of both APAP-SG and reduced glutathione. For example, the enzyme activity of recombinant and purified GR was reduced in the presence of 4 mm APAP-SG (with 0.5 mm glutathione disulfide) by 28% and 22%, respectively. The type of enzyme inhibition was observed to be competitive in the cases of both APAP-SG and glutathione. As glutathione inhibits GR activity in cells under physiological conditions, the rate of enzyme inhibition ought to be weaker in the case of glutathione depletion that is typical of acetaminophen overdose. Notably, however, enzyme activity likely remains inhibited due to the presence of APAP-SG, which might enhance the pro-oxidative status in the cell. We conclude that our finding could reflect some other pathological mechanism that may contribute to the toxicity of acetaminophen.

Solid-phase microextraction based method for determination of essential oils components in herbal beverages.

A method employing the direct immersion solid-phase microextraction followed by GC-MS analysis is presented for the determination of essential oils components in herbal tea infusions, i.e. their direct content in the liquid phase. The extraction performances were compared using five different microextraction fibres. Significant parameters affecting sorption process such as sample amount, sorption and desorption time and temperature, stirring speed, pH adjustment and effect of ionic strength were optimised and discussed. By optimising the key parameters, a detection limits (LOD = S/N × 3) for ten target marker compounds were obtained in the range from 5.3 to 48.2 ng/mL with recoveries ranged between 93.03 and 100.50%. Intra-day and inter-day repeatability at three concentration levels were found to be 1.1-15.3 and 7.2-15.5% RSD, respectively. Finally, the optimised procedure enabling a rapid and simple analysis of essential oils was applied for the direct determination of these compounds in ten herbal tea infusions.

Optimization of focused ultrasonic extraction of propellant components determined by gas chromatography/mass spectrometry.

A method for focused ultrasonic extraction of nitroglycerin, triphenyl amine and acetyl tributyl citrate presented in double-base propellant samples following by the gas chromatography/mass spectrometry analysis was developed. A face-centered central composite design of the experiments and response surface modeling was used for optimization of the time, amplitude and sample amount. The dichloromethane was used as the extractant solvent. The optimal extraction conditions with respect to the maximum yield of the lowest abundant compound triphenyl amine were found at the 20 min extraction time, 35% amplitude of ultrasonic waves and 2.5 g of the propellant sample. The results obtained under optimal conditions were compared with the results achieved with validated Soxhlet extraction method, which is typically used for isolation and pre-concentration of compounds from the samples of explosives. The extraction yields for acetyl tributyl citrate using both extraction methods were comparable; however, the yield of ultrasonic extraction of nitroglycerin and triphenyl amine was lower than using Soxhlet extraction. The possible sources of different extraction yields are estimated and discussed.

Microextraction by packed sorbent (MEPS) as a suitable selective method for l-ascorbic acid determination in beverages.

In this study the modern microextraction by packed sorbent (MEPS) method followed by HPLC-UV analysis was optimised and evaluated for determination of l-ascorbic acid (AA) content in beverages. In this case 300µL of sample was passed through silica BIN (barrel insert and needle assembly) and entrapped AA was eluted by 60µL of methanol-water (10%, v/v) solution. Recovery of the proposed method for three concentration levels ranged between 97.46% and 106.88% with relative standard deviation 8.7% (low conc.), 2.49% (medium conc.) and 1.98% (high). Obtained results of AA contents in real samples were in the ranges 56.5-195.4µg/mL and 130.4-1090.0µg/mL for ice-tea and fruit juice samples, respectively. Proposed method was compared with iodometric titration and DPPH methods, but microextraction method was found to be more selective for AA determination in tested samples.

Method validation for the determination of propellant components by Soxhlet extraction and gas chromatography/mass spectrometry.

A fast, sensitive, and accurate GC/MS method for the quantification of aliphatic nitroesters (ethylene glycol dinitrate, nitroglycerin, and triethylene glycol dinitrate) and aromatic amines (diphenylamine, 2-nitrodiphenylamine, and triphenylamine) in propellants was developed and validated. This method comprises a Soxhlet extraction step with dichloromethane, followed by separation on a capillary column MDN-5. Ionization of the analytes is carried out using electron ionization. The limit of quantification of the method was 1% w/w for aliphatic nitroesters and 0.1% w/w for aromatic amines (diphenylamine and triphenylamine). Values of repeatability and reproducibility for analyzed compounds were smaller than values of the maximum allowed tolerances of the Horwitz-equation RSD(max) and 2/3 RSD(max). Values of accuracy for selected compounds were below the acceptable threshold of 15% for all tested levels in the range of calibration curve excepting the lowest concentration of calibration curve for nitroglycerin and aromatic amines. During the validation of method, temperature instability in injection port of gas chromatograph and column was observed for 2-nitrodiphenylamine. Hence, it follows worse results of accuracy and linearity and 2-nitrodiphenylamine was not validated successfully.

Determination of 2-ethylhexyl nitrate in diesel fuel.

The aim of this study was to develop and validate fast and easily applicable GC/MS assay for the quantification of the substance that increases cetane number in diesel fuel (2-ethylhexylnitrate, 2-EHN). These requirements were fulfilled best by a headspace GC-MS assay with negative chemical ionization with methane (HS-GC/MS). Chromatographic separation is achieved using a DB5-MS capillary column after the addition of known amount of internal standard (o-nitrotoluene). The limit of detection was 0.009% v/v for 2-EHN and the limit of quantification was 0.03% v/v. The HS-GC/MS method was applied for the quantification of cetane improver in spiked diesel fuel and real diesel fuel. The method is linear over the studied range (0.03-0.3%, v/v), with satisfactory intra- and inter-assay precision, and the relative standard deviations are lower than 10%. Good accuracy is achieved with bias <10% at all levels tested.

Prednisolone-α-cyclodextrin-star PEG polypseudorotaxanes with controlled drug delivery properties.

The reaction of α-amino-ω-methoxypoly(ethylene glycol) [M = 5000] or star α-amino-poly(ethylene glycol) [M = 20 000] with hemiesters of prednisolone dicarboxylic acids (succinic, glutaric, adipic, phthalic acid) has been used to prepare the corresponding conjugates. The rate of esterase catalyzed hydrolysis of the conjugates is controlled by the molecular mass of poly(ethylene glycol) and the length of the linker between prednisolone and poly(ethylene glycol) (τ(1/2)∼ 5-0.5 h). The enzymatic hydrolysis proceeds most rapidly at conjugates with linkers derived from adipic and phthalic acids. The synthesized conjugates form polypseudorotaxanes with α-cyclodextrin which were characterized by 2D NOESY NMR spectra, powder X-ray diffraction patterns and in one case also by STM microscopy. In the case of the polypseudorotaxane having the linker derived from adipic acid, the enzymatic release proceeds ca. five times slower in comparison with the rate of prednisolone release from the corresponding conjugate. The rate of prednisolone release from the carrier can be controlled by three factors: character of the linker between the polymeric carrier and prednisolone, the molecular mass of poly(ethylene glycol) and complex formation with α-cyclodextrin. The synthesized polypseudorotaxanes represent new promising transport systems intended for targeted release of prednisolone in transplanted liver.

Extraction of antioxidants from plants using ultrasonic methods and their antioxidant capacity.

The analytical method based on the HPLC coupled with UV detection (HPLC-UV) for the determination of selected antioxidants (i.e. esculetin, scopoletin, 7-hydroxycoumarine, rutin, xanthotoxin, 5-methoxypsoralen and quercetin) in plant material was developed. Two ultrasonic extraction methods for the isolation of these compounds from the plants such as Mentha longifolia L., Mentha spicata L., Ruta graveolens L., Achyllea millefolium L., Plantago lanceolata L. and Coriandrum sativum L. were used. Both of these methods, i.e. ultrasonic probe and ultrasonic bath, were optimised and compared to each other. For the proposed HPLC-UV method LOQ values in the range from 22.7 (xanthotoxin) up to 97.2 ng/mL (rutin) were obtained. For all extracts the antioxidant capacity based on the reduction of free 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was also determined. Obtained results ranged from 10.11 up to 73.50% of DPPH radical inhibition.

Comparison of supercritical fluid and Soxhlet extractions for the isolation of nitro compounds from soils.

Supercritical fluid extraction (SFE) with CO(2), a clean and rapid alternative to conventional Soxhlet extraction, was investigated for the extraction of nitro compounds from soil samples. Quantitative extraction by SFE was accomplished at a pressure of 25 MPa and an extraction temperature of 60 degrees C, for 30 min in dynamic mode and using acetonitrile as modifier, and the results were comparable with those obtained by acetonitrile Soxhlet extraction (3 h) for all soil samples. Extracts from these two procedures were analyzed by gas chromatography coupled with mass spectrometry. Quantitative reproducibility for SFE extracts was acceptable (RSD 2-10%), and the quantity of solvent was reduced from 160 mL for Soxhlet extraction to 5 mL in the case of SFE.

Headspace single-drop microextraction of herbal essential oils.

A method employing the headspace single-drop microextraction (HS-SDME) is presented for the determination of essential oils in dried herbal leaves. By optimising the key experimental parameters, a linear response for the individual target compounds was obtained in the concentration range from LOQ to 4 mg/mL (r(2) = 0.9912-0.9998), with LODs from 3.3 up to 20.5 microg per 100 g of dried leaves, and the repeatability within the RSD of 2.1-8.9%. The HS-SDME-based procedure, enabling a rapid and simple analysis of essential oils in herbs, was applied to selected real samples (nine essential oils in four different samples) in combination with GC-FID identification and quantification of the target volatiles.

Determination of nitrate esters in water samples Comparison of efficiency of solid-phase extraction and solid-phase microextraction.

This paper deals with comparison of efficiency of extraction techniques (solid-phase extraction, SPE and solid-phase microextraction, SPME) used for extraction of nitrate esters (ethyleneglycoldinitrate, EGDN and nitroglycerin, NG), representing the first step of the method of quantitative determination of trace concentrations of nitrate esters in water samples. EGDN and NG are subsequently determined by means of high-performance liquid chromatography with ultraviolet detection (HPLC-UV). Optimization of SPE and SPME conditions was carried out using model water samples. Seven SPE cartridges were tested and the conditions were optimized (type of sorbent, type and volume of solvent to be used as eluent). For both nitrate esters the limit of detection (LOD) and the limit of quantification (LOQ) obtained using SPE/HPLC-UV were 0.23 microg mL(-1) and 0.70 microg mL(-1), respectively. Optimization of SPME conditions: type of SPME fibre (four fibres were tested), type and time of sorption/desorption, temperature of sorption. PDMS/DVB (polydimethylsiloxane/divinylbenzene) fibre coating proved to be suitable for extraction of EGDN and NG. For this fibre the LOD and the LOQ for both nitrate esters were 0.16 microg mL(-1) and 0.50 microg mL(-1), respectively. Optimized methods SPE/HPLC-UV and SPME/HPLC-UV were then used for quantitative determination of nitrate esters content in real water samples from the production of EGDN and NG.