Headspace Stir Bar Sorptive Extraction Research Articles

A comparative study on flavor trapping techniques from the viewpoint of odorants of hot-pressed rapeseed oil.

Rapeseed oil, as one of the three major vegetable oils in the world, its matrix effect makes the decoding flavor a challenge. Solid-phase microextraction (SPME), SPME-Arrow, headspace stir bar sorptive extraction (HSSE), direct thermal desorption (DTD), and solvent-assisted flavor evaporation (SAFE) were compared based on the odorants in hot-pressed rapeseed oil. Besides, methodological validation for 31 aroma standards was conducted to compare reliability and robustness of these approaches. DTD showed the largest proportion of acids, while the other techniques extracted a majority of nitriles. The highest number of odorants was detected by SAFE (31), followed by HSSE (30), SPME-Arrow (30), SPME (24), and DTD (14). SPME-Arrow showed the best performance in linearity, recovery, and reproducibility followed by SPME, HSSE, DTD, and SAFE. Results reveal the advantages and limitations of diverse methodologies and provide valuable insights for the selection of extraction methods in an oil matrix and flavor decoding.

Headspace stir-bar sorptive extraction combined with gas chromatography-mass spectrometry for trace analysis of volatile organic compounds in Schisandra chinensis Baillon (omija).

Analyzing volatile organic compounds (VOCs) in food is crucial but challenging. Schisandra chinensis Baillon (omija) is an herbal plant with various functional health activities. Previous VOC analyses focused on S. chinensis fruit but not its leaves. Therefore, VOCs in S. chinensis fruit and leaves were analyzed using headspace stir-bar sorptive extraction (HS-SBSE)-GC-MS, and optimal conditions were established. Various factors, such as the sample preparation method, twister stir-bar type, sample amount, extraction temperature, and extraction time, expected to affect extraction were carefully optimized. Under the optimal conditions, 35 and 40 VOCs were identified in S. chinensis fruit and leaves, respectively. This HS-SBSE method is capable of rapid analysis and a low contamination rate without requiring organic solvents. These findings provide practical guidelines for HS-SBSE applications in various food matrices by providing analytical methods for VOC detection.

A high-throughput analysis of volatile compounds with various polarities using headspace stir bar sorptive extraction

Long sample extraction time is usually necessary in the analysis of volatile flavour compounds to achieve high extraction efficiency. However, the long extraction time reduces sample throughput, which results in waste of labour and energy. Therefore, in this study, an improved headspace-stir bar sorptive extraction was developed to extract volatile compounds with varying polarities in a short time. With the aim of achieving high throughput, extraction conditions were selected and optimised based on the combinations of different extraction temperatures (80–160 °C), extraction times (1–61 min), and sample volumes (50–850 μL) through the response surface methodology with Box-Behnken design. After obtaining the preliminary optimal conditions (160 °C, 25 min, and 850 μL), the effect of cold stir bars with shorter extraction time on the extraction efficiency was evaluated. The cold stir bar improved the overall extraction efficiency with better repeatability, and the extraction time was further shortened to 1 min. Then, the effects of different ethanol concentrations and salt additions (sodium chloride or sodium sulfate) were studied, and 10% ethanol concentration with no salt addition provided the highest extraction efficiency for most compounds. Finally, it was verified that the high-throughput extraction condition was feasible for the volatile compounds spiked in a honeybush infusion.

Individual Chemical Profiles in the Leach's Storm-Petrel.

Avian chemical communication, once largely overlooked, is a growing field that has revealed the important role that olfaction plays in the social lives of some birds. Leach's storm-petrels (Oceanodroma leucorhoa) have a remarkable sense of smell and a strong, musky scent. This long-lived, monogamous seabird relies on olfaction for nest relocation and foraging, but whether they use scent for communication is less well studied. They are nocturnally active at the breeding colony and yet successfully reunite with their mate despite poor night-vision, indicating an important role for non-visual communication. We investigated the chemical profiles of Leach's storm-petrels to determine whether there is socially relevant information encoded in their plumage odor. To capture the compounds comprising their strong scent, we developed a method to study the compounds present in the air surrounding their feathers using headspace stir bar sorptive extraction coupled with gas chromatography-mass spectrometry. We collected feathers from Leach's storm-petrels breeding on Bon Portage Island in Nova Scotia, Canada in both 2015 and 2016. Our method detected 142 commonly occurring compounds. We found interannual differences in chemical profiles between the two sampling years. Males and females had similar chemical profiles, while individuals had distinct chemical signatures across the two years. These findings suggest that the scent of the Leach's storm-petrel provides sociochemical information that could facilitate olfactory recognition of individuals and may inform mate choice decisions.

Comparison of Dynamic Headspace Trapping on Tenax TA and Headspace Stir Bar Sorptive Extraction for Analysis of Grilled Chicken (Yakitori) Volatiles

Comparison of Dynamic Headspace Trapping on Tenax TA and Headspace Stir Bar Sorptive Extraction for Analysis of Grilled Chicken (Yakitori) Volatiles

Volatile Transference and Antimicrobial Activity of Cheeses Made with Ewes' Milk Fortified with Essential Oils.

During the last decades, essential oils (EOs) have been proven to be a natural alternative to additives or pasteurization for the prevention of microbial spoilage in several food matrices. In this work, we tested the antimicrobial activity of EOs from Melissa officinalis, Ocimum basilicum, and Thymus vulgaris against three different microorganisms: Escherichia coli, Clostridium tyrobutyricum, and Penicillium verrucosum. Pressed ewes’ cheese made from milk fortified with EOs (250 mg/kg) was used as a model. The carryover effect of each oil was studied by analyzing the volatile fraction of dairy samples along the cheese-making process using headspace stir bar sorptive extraction coupled to gas chromatography/mass spectrometry. Results showed that the EOs contained in T. vulgaris effectively reduced the counts of C. tyrobutyricum and inhibited completely the growth of P. verrucosum without affecting the natural flora present in the cheese. By contrast, the inhibitory effect of M. officinalis against lactic acid bacteria starter cultures rendered this oil unsuitable for this matrix.

A comparative study of the volatile profile of wine vinegars with protected designation of origin by headspace stir bar sorptive extraction.

The characteristic volatile profile of the Spanish Vinagre de Jerez (VJ), Vinagre de Condado de Huelva (VC) and Vinagre de Montilla-Moriles (VMM) protected designation of origin (PDO) wine vinegars has been studied and compared for the first time by headspace stir bar sorptive extraction-gas chromatography-mass spectrometry (HSSE-GC-MS). The possible markers of each category and PDO were assessed. Acetates were the majority group in all vinegars, while ketones, C13-norisoprenoids and volatile phenols showed significant differences between the three PDOs. Analysis of variance (ANOVA), heatmap and partial least squares-discriminant analysis (PLS-DA) were performed. According to these results, 1-heptanol, methyl nonanoate, 2-methylbutanoic acid, 2,2,6-trimethyl-cyclohexanone, trans-2-decenal, eucalyptol and α-terpineol, were the most significant compounds for differentiating of VC, diacetyl and acetoin, ethyl 3-ethoxypropanoate, 2- and 3-heptanone, 2-methyl-1-hexadecanol, 1-octen-3-ol, p-Cresol and camphene for VMM; and β-damascenone, 5-hydroxymethylfurfural, 3-heptanol, trans-2-hexen-1-ol and trans-2-hexen-1-yl acetate for VJ. Classification results showed that 100% of PDO samples were correctly classified, reaffirming the utility of the volatile profiles for classifying and authenticating wine vinegar PDOs.

Instrumental volatile flavor analysis of omija (Schisandra chinesis Baillon) using headspace stir-bar sorptive extraction-gas chromatography-mass spectrometry and its relationship to human sensory perceptions

Omija (Schisandra chinesis Baillon) is a cluster of berries, mainly cultivated in East Asian countries. The objective of this study was to conduct an instrumental volatile flavor analysis and descriptive analysis of omija samples using headspace stir-bar sorptive extraction gas chromatography–mass spectrometry. Three omija fruits were included and pre-treated using three different methods: frozen omija (FO), frozen-blended omija (FBO), and freeze-dried omija (FDO). A total of 28 volatiles were identified, and significant differences in concentrations found among omija samples were noted, especially for previously identified aroma-active compounds such as α-pinene, α-terpinene, and (E)-β-ocimene (p < .05). In terms of sensory perception, significant differences were observed in three sensory attributes that included, ginger, pine needle, and wet grassy aromatics among the samples (p < 0.05). While similarities and dissimilarities were observed among the three samples in the instrumental flavor analysis results, a principal component analysis revealed that FBO and FDO shared similar flavor characteristics while FO had distinctive flavor characteristics.

Characterization of Kimchi Flavor with Preconcentration by Head Space Solid-Phase Microextraction and Stir Bar Sorptive Extraction and Analysis by Gas Chromatography-Mass Spectrometry

Kimchi is a traditional fermented vegetable, known for its complex flavor. Herein, we compared compounds related to the kimchi flavor, identified by gas chromatography-mass spectrometry (GC-MS) with the developed solid phase microextraction (SPME) and stir bar sorptive extraction (SBSE) techniques. Although headspace-solid phase microextraction (HS-SPME) detected more volatile compounds than nondestructive-headspace-solid-phase microextraction (ND-HS-SPME), those identified by ND-HS-SPME were considered closely related to the flavor of the intact kimchi. Furthermore, direct immersion-stir bar sorptive extraction (DI-SBSE) detected more volatile and nonvolatile compounds than headspace-stir bar sorptive extraction (HS-SBSE), while more sulfur compounds were identified by HS-SBSE. Therefore, we recommend the use of the HS-SPME method using a divinylbenzene/carboxen/polydimethylsiloxane fiber for identifying compounds related to the kimchi flavor. In addition, principal component analysis showed ND-HS-SPME and HS-SBSE to be closely clustered. Overall, we estimated that the samples obtained via the nondestructive sample preparation emits fewer polar volatile flavor compounds than those obtained using the destructive sample preparation. Considering the findings presented herein, we believe that this study contributes to optimizing the flavor analysis of kimchi and other fermented vegetables.

Identification of volatile markers for the detection of adulterants in red ginseng (Panax ginseng) juice using headspace stir-bar sorptive extraction coupled with gas chromatography and mass spectrometry.

Red ginseng (Panax ginseng) products are frequently adulterated by manufacturers with cheaper medicinal plant products including deodeok (Codonopsis lanceolata) and doraji (Platycodon grandiflorum) to increase profits. To identify possible volatile markers for the adulteration of red ginseng juices with deodeok or doraji, a headspace stir-bar sorptive extraction method was developed. Gas chromatography with mass spectrometry and untargeted metabolomics analysis revealed that 1-hexanol, cis-3-hexen-1-ol, and trans-2-hexen-1-ol are abundantly present in deodeok and doraji but not red ginseng. The peak area ratios in gas chromatograms of these compounds in red ginseng juices mixed with deodeok or doraji indicate that these volatile chemicals can be used as markers to detect the adulteration of red ginseng juice.

Sampling methods for the study of volatile profile of PDO wine vinegars. A comparison using multivariate data analysis

High-quality wine vinegars have been registered in Spain under protected designation of origin (PDO): “Vinagre de Jerez”, “Vinagre de Condado de Huelva” and “Vinagre de Montilla-Moriles”. The raw material, production and aging processes determine their quality and their aromatic composition. Vinegar volatile profile is usually analyzed by gas chromatography–mass spectrometry (GC–MS), being necessary a previous extraction step. Thus, three different sampling methods (Headspace solid phase microextraction “HS-SPME”, Headspace stir bar sorptive extraction “HSSE” and Dynamic headspace extraction “DHS”) were studied for the analysis of the volatile composition of Spanish PDO wine vinegars. Multivariate curve resolution (MCR) was used to solve chromatographic problems, improving the results obtained. Principal component analysis (PCA) showed that not all the sampling methods were equally suitable for the characterization and differentiation between PDOs and categories, being HSSE the technique that made able the best vinegar characterization.

Different coatings for the HS-SBSE grape volatile analysis in model solution: Preliminary results

Head space stir bar sorptive extraction (HS-SBSE) was used to evaluate the efficacy of two stir bar coatings, poly(ethylene glycol) (PEG)-modified silicone (EG-Silicone) and polyacrylate (PA), in comparison with polydimethyl siloxane (PDMS) coating, to assess volatiles in model wine solutions. In addition, two temperatures (0°C and 25°C) of the injection system (CIS) were compared. This was performed to propose an analytical procedure cheaper and faster respect to traditional extractive methods and environmental friendly, avoiding the use of large amounts of solvents and high-energy consumption for cooling. Fifteen grape volatiles with different polarities, known to have an important impact on varietal wine aroma, were tested. The affinity of each tested molecule to the new coatings was assessed. The PDMS coating showed higher relative areas for terpenes (β-citronellol, geraniol, linalool and α-terpineol) and norisoprenoids (β-ionone), especially when the CIS temperature was set at 25°C. C6 compounds such as 1-hexanol, (E)-2-hexenal, (E)-2-hexen-1-ol, (Z)-3-hexen-1-ol, and benzaldehyde and eugenol, obtained greater area response using EG-Silicone and PA coatings, regardless the temperatures. The memory effect of each coating type was studied; EG Silicone and PA coatings showed a higher memory effect for a certain compounds, probably due to the lower desorption temperature that EG Silicone and PA coatings can be submitted to, respect to PDMS (220°C the former two, 295°C the latter).

Odorant synergy effects as the cause of fishy malodors in algal marine oils.

As unsaturated lipids oxidize, they form hydroperoxides, which are susceptible to further oxidation or decomposition to secondary reaction products including aldehydes, ketones, acids, and alcohols. While oxidation reactions of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are responsible for fishy off-flavors in marine oils, gas chromatography-olfactometry (GC-O) and other types of analytical studies have failed to reveal which specific oxidation products are involved. Previous research (Marsili, R.T.; Laskonis, C. The importance of odourant synergy effects in understanding malodour problems in DHA and EPA products. Lipid Technol. 2014, 26 (2), 31-34) has indicated that fishy malodor may be caused by the presence of two lipid oxidation products, heptanal and (E,Z)-3,5-octadien-2-one. The aims of the present study are to provide experimental method details and offer further evidence that these two oxidation products are indeed the cause of fishy malodors. Initial GC-MS-O studies of marine oils with fishy malodors revealed numerous oxidation products, but none were characterized as fishy. However, when all sample volatiles were captured together and then desorbed simultaneously in GC-O experiments, the fishy malodor was evident, indicating odorant synergy effects were responsible. A simple, novel method was developed using an olfactometry detector as a fraction collector to trap various peaks in marine oil chromatograms. The nose cone of the olfactometry detector was replaced with a PDMS foam absorption tube at various times during GC analysis. Combinations of GC peaks were trapped on PDMS tubes, desorbed in a Gerstel thermal extractor (off-line), and sniffed. The combination of two analytes was found to cause fishy malodors: heptanal and (E,Z)-3,5-octadien-2-one. Purge-and-trap, solid phase microextraction (SPME), and headspace stir bar sorptive extraction (HSSE) sample preparation methods prior to GC-MS were investigated. All methods confirmed the combination of heptanal and (E,Z)-3,5-octadien-2-one as the cause of fishy odor.

Dairy matrix effect on the transference of rosemary (Rosmarinus officinalis) essential oil compounds during cheese making.

The use of aromatic plant extracts as ingredients may be compromised owing to low transference and activity lack in food matrixes compared with in vitro trials. Rosemary essential oil (REO) was added to sheep milk to study the transference of its compounds during the cheese-making process and to determine how cheese antimicrobial activity is modified. The volatile characterization of dairy samples was performed using headspace stir bar sorptive extraction coupled to gas chromatography/mass spectrometry (HS-SBSE/GC/MS) so that fat matrix interferences were reduced. This method detected a decrease in volatile recovery concentration of 19.33% when REO was added to milk. A total recovery volatile yield of 62.51% was measured from the initial quantification of milk to cheese, with hydrocarbon volatiles being transferred in a higher ratio (64.88%) than oxygenated ones (58.74%). No effects were observed for REO in fortified cheese on the counts of native flora necessary for ripening processes, but the total inhibition of Clostridium spp. was provoked The study of active compound transference during cheese elaboration was achieved. The antimicrobial results in fortified cheeses with REO showed a preventive effect in the case of clostridial species, which are responsible for late cheese blowing.

Headspace stir bar sorptive extraction followed by thermal desorption and gas chromatography with mass spectrometry to determine musk fragrances in sludge samples without sample pretreatment

A direct, simple and solvent-free method based on headspace stir bar sorptive extraction and thermal desorption gas chromatography with mass spectroscopy was developed to determine 13 musk fragrances (six polycyclic musks, three nitro musks and four macrocyclic musks) in sludge without sample treatment. The optimal headspace stir bar sorptive extraction conditions were achieved when a polydimethylsiloxane stir bar was exposed for 45 min in the headspace of a 10 mL vial filled with 100 mg of sludge mixed with 0.2 mL of water stirred at 750 rpm at 80°C. The stir bar was then desorbed in the thermal desorption gas chromatography and mass spectrometry system, obtaining limits of detection between 5 and 30 ng/g. The method applicability was tested with sewage sludge from two urban wastewater treatment plants and from a potable water treatment plant. Results showed galaxolide and tonalide to be the most abundant musk fragrances found in wastewater treatment plants with maximal concentrations of 9240 and 7500 ng/g, respectively. Maximum concentration levels between 35 and 635 ng/g were found for musk ketone, musk moskene, traseolide, phantolide and celestolide in this kind of samples. Concentrations below the limits of quantitation of phantolide, galaxolide, tonalide and musk ketone were found in sludge from a potable water treatment plant.

Non-Destructive Assessment of Aroma Volatiles from a Climacteric Near-Isogenic Line of Melon Obtained by Headspace Stir-Bar Sorptive Extraction.

A climacteric aromatic near-isogenic line (NIL) of melon (Cucumis melo L.) SC3-5-1 contained an introgression of the non-climacteric Korean cultivar “Shongwan Charmi” accession PI 161375 (SC) in the genetic background of the non-climacteric cultivar “Piel de Sapo” (PS). The aroma production was monitored during ripening at 21 °C in intact fruit using headspace sorptive bar extraction (HSSE). Bars were composed of polydimethylsiloxane (PDMS) and aromas were desorbed and analyzed by gas-chromatography mass-spectrometry. The aromatic profile was composed of 70 aromatic compounds plus 21 alkanes with a predominance of esters, particularly acetate (2-methylbutyl acetate, 2-methylpropyl acetate, hexyl acetate, and phenylmethyl acetate). Some compounds were severely affected by postharvest time. The acetate esters (3-methylbutyl acetate, butan-2-yl acetate and phenylmethyl acetate) decreased with ripening and sulfur-derived compounds (S-methyl butanethioate and S-methyl 3-methylbutanethioate) increased gradually with ripening. A few compounds increased at the senescence phase (propyl ethanoate). Other compounds such as hexadecanoic acid showed a marked decrease after harvest, some decreasing from a relative maximum at harvest (2-methylpropyl hexanoate; n-hexanoic acid; nonanoic acid).

Effect of eugenol and guaiacol application on tomato aroma composition determined by headspace stir bar sorptive extraction

The present work was carried out because there is only a small amount of literature on how the volatile composition of tomatoes can be modified by the effect of exogenous substances in contact with tomato plants. This work studies how eugenol and guaiacol, either by foliar application and/or in the surrounding atmosphere, can affect the volatile composition of this fruit. An important work of this study was also conduced to validate the analytical method [headspace stir bar sorptive extraction-gas chromatography-mass spectrometry (HS-SBSE-GC-MS)] to determine the composition of the volatiles in tomato. Analytical method validation parameters such as linearity, limit of detection, limit of quantification, and recovery proved that this method is suitable for the analysis of tomato volatiles. Their eugenol and guaiacol content changed, with an increase of 200 and 35 times, respectively, when foliar treatment was used, and an increase of 10, in both cases, when plants were in contact with the contaminated atmosphere. As consequence of the treatments other volatile compounds changed considerably. For first time, a HS-SBSE-GC-MS method was successfully validated for the study of volatiles in tomatos. Results suggests that exogenous compounds in contact with the plants, such as eugenol and guaiacol, can be absorbed changing the global volatile composition of fruits, which could produce a negative or positive effect in their aroma.

Headspace stir bar sorptive extraction–gas chromatography/mass spectrometry characterization of the diluted vapor phase of cigarette smoke delivered to an in vitro cell exposure chamber

Advanced smoke generation systems, such as the Borgwaldt RM20S ® smoking machine used in combination with the BAT exposure chamber, allow for the generation, dilution and delivery of fresh cigarette smoke to cell or tissue cultures for in vitro cell culture analyses. Recently, our group confirmed that the Borgwaldt RM20S ® is a reliable tool to generate and deliver repeatable and reproducible exposure concentrations of whole smoke to in vitro cultures [1]. However, the relationship between dose and diluted smoke components found within the exposure chamber has not been characterized. The current study focused on the development of a headspace stir bar sorptive extraction (HSSE) method to chemically characterize some of the vapor phase components of cigarette smoke generated by the Borgwaldt RM20S ® and collected within a cell culture exposure chamber. The method was based on passive sampling within the chamber by HSSE using a Twister™ stir bar. Following exposure, sorbed analytes were recovered using a thermal desorption unit and a cooled injection system coupled to gas chromatograph/mass spectrometry for identification and quantification. Using the HSSE method, sixteen compounds were identified. The desorption parameters were assessed using ten reference compounds and the following conditions led to the maximal response: desorption temperature of 200 °C for 2 min with cryofocussing temperature of −75 °C. During transfer of the stir bars to the thermal desorption system, significant losses of analytes were observed as a function of time; therefore, the exposure-to-desorption time interval was kept at the minimum of 10 ± 0.5 min. Repeatability of the HSSE method was assessed by monitoring five reference compounds present in the vapor phase (10.1–12.9% RSD) and n-butyl acetate, the internal standard (18.5% RSD). The smoke dilution precision was found to be 17.2, 6.2 and 11.7% RSD for exposure concentrations of 1, 2 and 5% (v/v) cigarette vapor phase in air, respectively. A linear response of analyte abundance was observed as a function of dilution. Extrapolation to 100% (v/v) cigarette vapor phase, i.e., undiluted smoke, gave yields for the five compounds ranging from 6 to 450 ng for 10 min exposure.

Headspace stir bar sorptive extraction combined with GC-ICP-MS for the speciation of dimethylselenide and dimethyldiselenide in biological samples

A new method for the determination of volatile organo seleno compounds (dimethylselenide (DMSe) and dimethyldiselenide (DMDSe)) has been developed using a headspace sorptive extraction combined with capillary gas chromatography-inductively coupled plasma mass spectrometry (GC-ICP-MS). The DMSe and DMDSe retained on the sorptive bar were desorbed by methanol and introduced into GC-ICP-MS for separation and determination. The extraction performance of various sorptive bar coatings, including poly(dimethylsiloxane) (PDMS), PDMS-poly(vinyl alcohol) (PVA), carbowax (CW)-PDMS-PVA, PDMS-β-cyclodextrin (β-CD) has been studied, and the best extraction efficiencies for DMSe and DMDSe were obtained by PDMS-PVA sorptive bar coating. The factors affecting extraction and desorption of DMSe and DMDSe were studied and the optimized operation conditions were established. The limits of detection were 33 and 7.1 ng L−1 for DMSe and DMDSe, respectively, and the correlation coefficients of 0.9986 for DMSe and 0.9998 for DMDSe were obtained in the concentration range 0.5–500 ng mL−1 and 0.025–50 ng mL−1, respectively. Repeatability, based on seven replicate determinations, expressed as relative standard deviations (RSDs), were 8.0% (CDMSe = 5 ng mL−1) and 6.1% (CDMDSe = 0.5 ng mL−1) for DMSe and DMDSe, respectively. The proposed method was applied for the analysis of DMSe and DMDSe in biological samples (garlic, onion and their juice) and a good agreement was obtained between the analytical results obtained by standard addition and external calibration methods.

A novel HS‐SBSE system coupled with gas chromatography and mass spectrometry for the analysis of organochlorine pesticides in water samples

A methodology to analyze organochlorine pesticides (OCPs) in water samples has been accomplished by using headspace stir bar sorptive extraction (HS-SBSE). The bars were in house coated with a thick film of PDMS in order to properly work in the headspace mode. Sampling was done by a novel HS-SBSE system whereas the analysis was performed by capillary GC coupled mass spectrometric detection (HS-SBSE-GC-MS). The extraction optimization, using different experimental parameters has been established by a standard equilibrium time of 120 min at 85 degrees C. A mixture of ACN/toluene as back extraction solvent promoted a good performance to remove the OCPs sorbed in the bar. Reproducibility between 2.1 and 14.8% and linearity between 0.96 and 1.0 were obtained for pesticides spiked in a linear range between 5 and 17 ng/g in water samples during the bar evaluation.