SFE-plus-C(18) lipid cleanup and selective extraction method for GC/MS quantitation of polycyclic aromatic hydrocarbons in smoked meat

In biological matrixes lipid material often poses an interference problem for determinations of nonpolar compounds, e.g., polycyclic aromatic hydrocarbons (PAHs). A newly developed supercritical fluid extraction plus adsorbent method, "SFE-plus-C(18)", offers selective extraction of PAHs in lipid-rich biological matrixes without the need for supplementary cleanup. This method eliminates the use of large volumes of toxic solvent and lengthy lipid removal procedures. This study reports the first application of the SFE-plus-C(18) method to the analysis of a genuine food product, i.e., smoked meat (beef). The procedure employs the addition of C(18) adsorbent beads to the initial sample slurry of pureed smoked meat prior to supercritical CO(2) extraction and GC/MS quantitation. During SF extraction, indigenous lipids are preferentially retained on the beads, and PAHs are selectively extracted with supercritical CO(2). In a comparison of determinations of PAHs by SFE-plus-C(18) vs the conventional SFE method, only 11-17% of the indigenous lipids observed by the conventional SFE method were co-extracted using the SFE-plus-C(18) method. The PAHs in smoked meat could thus be determined efficiently in the presence of a reduced background of co-extracted lipids. Out of 10 targeted PAHs, seven were detected with a range of 10.0-26.0 ng/g in the smoked meat sample. The other three PAHs were not present above the detection limit of the instrument (2.5-4.1 pg). The recoveries of PAHs obtained using the conventional SFE method were 63-94% lower than those achieved by SFE-plus-C(18).     

Accelerated solvent extraction and gas chromatography/mass spectrometry for determination of polycyclic aromatic hydrocarbons in smoked food samples.

Accelerated solvent extraction (ASE) is a new sample extraction method offering a number of advantages such as low pe -extraction cost, reduced solvent and time consumption, and simplified extraction protocols. In this study, the ASE method was applied to the extraction of polycyclic aromatic hydrocarbons (PAHs) from biological samples. For recovery studies, fish tissues and ground pork were used as sample matrices. Sample aliquots fortified with 16 PAHs were extracted by ASE, and the extracts were treated with sulfuric acid and Florisil, followed by gas chromatography-mass spectrometry analysis. The PAH recoveries by the ASE method were found to be comparable with or better than those by Soxhlet extraction. The extraction and quantitation method was then applied to the determination of PAHs in several smoked meat samples obtained from a local market. Up to 12 PAHs were found to be present at concentrations ranging from 3 to 52 ng/g wet sample.     

Hydroxylysyl pyridinoline cross-link concentration affects the textural properties of fresh and smoked Atlantic salmon (Salmo salar L.) flesh

A simple HPLC method is presented to quantify the low concentration of hydroxylysyl pyridinoline (PYD) cross-links in Atlantic salmon (Salmo salar L.) muscle. The method involved the extraction of tissue with NaOH prior to hydrolysis, which greatly reduced the amount of protein to be hydrolyzed and made downstream operations easier and more reproducible. The concentration of PYD was 426 pmol g(-)(1) dry mass muscle in post-rigor muscle stored at 0 degrees C and sampled 3 d after death. Hydroxproline (HYP) concentration was determined following NaOH extraction as a measure of collagen content. In post-rigor samples, the alkaline-insoluble HYP fraction comprised 18.3% of the total HYP. Scanning electron microscopy revealed shrinkage of muscle fibers and a retraction of the connective tissue matrix in smoked salmon. PYD concentration was relatively resistant to processing to the smoked product, decreasing by around 11.7%, as compared to a 22.2% decrease in HYP. There was a positive correlation between PYD concentration and the firmness of post-rigor muscle samples as measured by an instrumental texture analyzer, explaining 25% of the total variation. A weaker but still significant correlation was found between PYD concentration and firmness in the smoked product. There was no relationship between fillet firmness and total collagen concentration, although the correlation with HYP in the alkaline-insoluble fraction was significant at the 6% level (P = 0.057). Our results indicate that only 1-3% of collagen molecules are linked by nonreducible mature cross-links in harvest size farmed Atlantic salmon and that PYD concentration is an important raw material characteristic for flesh quality.     

Comparison of odor-active volatile compounds of fresh and smoked salmon

The odorant volatile compounds of raw salmon and smoked salmon have been investigated by two gas chromatography-olfactometry methods (frequency detection and odorant intensity) and gas chromatography-mass spectrometry. After simultaneous steam distillation-solvent extraction with diethyl ether and the recovery of the aromatic extract in ethanol, qualitative olfactometric characterization and identification followed by a quantitative assessment of the odorant volatile compounds were carried out. The origin of many odorant compounds of smoked salmon can be attributed to wood smoke. Another part of smoked salmon aroma is due either to the odorant compounds of the raw fish flesh or to an evolution of fish flesh aroma thanks to the smoking process conditions. Forty-nine odorant compounds have been identified in fresh salmon and 74 in smoked salmon. Carbonyl compounds, such as heptanal or (E,Z)-2,6-nonadienal, show a high detection frequency and odorant intensity in unsmoked fish, giving the flesh its typical fishy odor. For smoked salmon, phenolic compounds, such as cresol or guaiacol, and furanic compounds seem to be responsible for the smoked odor.     

Evaluation of a modified QuEChERS extraction of multiple classes of pesticides from a rice paddy soil by LC-APCI-MS/MS

A new method for the determination of clomazone, fipronil, tebuconazole, propiconazole, and azoxystrobin in samples of rice paddy soil is presented. The extraction of the pesticides from soil samples was performed by using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. Some extraction conditions such as salt addition, sample acidification, use of buffer, and cleanup step were evaluated. The optimized method dealt with a single extraction of the compounds under study with acidified acetonitrile, followed by the addition of MgSO(4) and NaCl prior to the final determination by liquid chromatography-atmospheric chemical pressure ionization-tandem mass spectrometry. Validation studies were carried out in soil samples. Recoveries of the spiked samples ranged between 70.3 and 120% with relative standard deviation lower than 18.2%. The limits of quantification were between 10 and 50 μg kg(-1). The method was applied to the analysis of real samples of soils where rice is cultivated.     

Multiresidue pesticide analysis of agricultural commodities using acetonitrile salt-out extraction, dispersive solid-phase sample clean-up, and high-performance liquid chromatography-tandem mass spectrometry

A multiresidue method analyzing 209 pesticides in 24 agricultural commodities has been developed and validated using the original Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) procedure and high performance liquid chromatography-positive electrospray ionization-tandem mass spectrometry (LC-MS/MS) analysis. Using solvent-only calibration standards (SOCSs) and matrix-matched calibration standards (MMCSs), it was demonstrated that a minimal concentration of 5-10 μg/kg (part per billion, ppb) of analytes in matrix is required for the consistent identification of targeted pesticides with two MRM transitions. Method performance was validated by the precision and accuracy results obtained from fortification studies at 10, 25, 100, and 500 ppb and MMCSs. The method was demonstrated to achieve an average recovery of 100 ± 20% (n = 4) for >75% of evaluated pesticides at the low fortification level (10 ppb) and improved to >84% at the higher fortification concentrations in all 24 matrices. Matrix effects in LC-MS/MS analysis were studied by evaluating the slope ratios of calibration curves (1.0-100 ng/mL) obtained from the SOCSs and MMCSs. Principal component analysis (PCA) of LC-MS/MS and method validation data confirmed that each matrix exerts its specific effect during the sample preparation and LC-MS/MS analysis. The matrix effect is primarily dependent on the matrix type, pesticide type and concentration. Some caution is warranted when using matrix matched calibration curves for the quantitation of pesticides to alleviate concerns on matrix effects. The QuEChERS method with LC-MS/MS was used to identify and quantitate pesticides residues, with concentrations ranging from 2.5 to >1000 ppb in a variety of agricultural samples, demonstrating fitness for screening and surveillance applications.     

Evaluation of analysis of polycyclic aromatic hydrocarbons by the QuEChERS method and gas chromatography-mass spectrometry and their formation in poultry meat as affected by marinating and frying

The objectives of this research were to develop a method for the determination of 16 polycyclic aromatic hydrocarbons (PAHs) in poultry meat by combining the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method with gas chromatography-mass spectrometry (GC-MS) and study their formation during marinating and frying. The recoveries of 16 PAHs ranged from 94.5 to 104% in blank samples and from 71.2 to 104% in poultry meat samples. The quantitation limits of 16 PAHs were from 0.02 to 1 ng/mL, with the intraday variability being from 2.4 to 6.6% [percent relative standard deviation (RSD%)] and interday variability being from 3.3 to 7.1% (RSD%). Most PAHs followed a time-dependent increase over a 24 h marinating period, with naphthalene being generated in the largest amount. Among the various poultry meat, chicken gizzard produced the highest level of total PAHs after 24 h of marinating. A similar tendency was observed for most PAHs during frying of poultry meat, but a high amount of total PAHs was shown in duck drumstick after 15 min of frying.     

Polycyclic aromatic hydrocarbon profile analysis of high-protein foods, oils, and fats by gas chromatography.

A method is described for the determination of polycyclic aromatic hydrocarbons (PAHs) with 3-7 rings in (I) meat, poultry, fish, and yeast; and (II) oils and fats. The extraction of PAHs from group I is incomplete, and, therefore, group I samples must be dissolved homogeneously by saponification in 2N methanolic potassium hydroxide. The PAHs are concentrated by liquid-liquid extraction (methanol-water-cyclohexane, N,N - dimethylformamide - water-cyclohexane) and by column chromatography on Sephadex LH 20. The PAHs are separated by high-performance gas-liquid chromatography (GLC) with columns containing 5% OV-101 on Gas-Chrom Q and estimated by integration of the flame ionization detector signals in relation to an internal standard (3,6-dimethylphenanthrene and/or benzo(b)chrysene). The sensitivity is significantly higher than that obtained with ultraviolet spectroscopic methods. The reproducibility and margin of error were tested with meat samples fortified with 11 PAHs and with samples of sunflower oil. The method was further applied to meat, smoked fish, yeast, and unrefined sunflower oil. All samples investigated contained more than 100 PAHs (characterized by mass spectrometry) of which only the main components were determined: phenanthrene, anthracene, fluorene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene + benzo (j)fluoranthene + benzo(k) fluoranthene, benzo(e)pyrene, benzo(a)pyrene, perylene, dibenz(a,j)anthracene, dibenz(a,h)anthracene + indeno(1,2,3,-cd)pyrene, benzo(ghi)perylene, anthanthrene, and coronene. In contrast to other methods, the GLC profile analysis allows the recording of known and unknown PAH peaks simultaneously and also allows a compilation of all PAHs.     

Simultaneous determination of five plant growth regulators in fruits by modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction and liquid chromatography-tandem mass spectrometry

An effective method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and optimized to obtain a complete separation of five representative plant growth regulators (PGRs) [gibberellic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), thidiazuron, forchlorfenuron, and paclobutrazol] in fruits. Extraction was performed with acetonitrile containing 0.1% (v/v) acetic acid, applying modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) methodology. LC-MS/MS conditions including composition of mobile phases and mass spectrometry (MS) conditions were evaluated to achieve the highest sensitivity in MS detection. All of the data acquisition was employed in the segmented multiple-reaction monitoring mode for the selected negative and positive transition ions. The octadecylsilyl (C18) dispersive solid-phase extraction (SPE) sorbent was found to provide the more satisfied recoveries than primary secondary amine (PSA) and graphitized carbon black (GCB) for five target PGRs. The optimized method allowed for recoveries of 76-112% for the five PGRs from fruit samples with relative standard deviation (RSD) values less than 10%. Limits of quantification (0.5-16.5 μg/kg) were lower than the maximum limit of residues established for PGRs. The results demonstrated that the developed LC-MS/MS and QuEChERS extraction method is highly effective for analyzing trace amounts of target PGRs in fruit samples. Finally, the method was successfully used to detect residual PGRs in Beijing, China, in 2010. The concentrations of 2,4-D (5.1-1503 μg/kg) and paclobutrazol (1-1381 μg/kg) found in orange and peach, respectively, suggesting that the use of these PGRs in these fruits should be regulated in China in the future.     

QuEChERS提取法在农产品农药残留检测中的应用进展

食品中的农药残留检测长期以来一直是一项艰巨的挑战,其杂质干扰多,残留含量低,传统的提取方法常常因为无法将杂质分离而影响检测结果。由于食品中杂质种类众多,农药的种类繁多且理化性质各异,新型农药日益涌现,所以对样品的前处理提出了更高的要求。近年来,QuEChERS(Quick,Easy,Cheap,Effective,Rugged and Safe)作为一种新型的提取方法,由于具有快速、简单、廉价、有效、可靠、安全的特点,成为国内外广泛采用的样品前处理新技术,在多种农药、医药、兽药的气相或液相色谱分析中已经得到广泛应用。本文就国内外对QuEChERS法在各类食品以及其他动植物性农产品的农药残留检测中的应用和方法的改进进行了综述,并对QuEChERS法在今后农药残留检测的应用前景以及发展方向进行了展望。     

Extraction of polycyclic aromatic hydrocarbons from spiked soil

A homogenization method was evaluated for extracting polycyclic aromatic hydrocarbons (PAHs) from soils. Fifteen PAHs were spiked and recovered from 2 soils at concentrations ranging from 1 to 1000 micrograms/g, using the homogenization method and a Soxhlet extraction method. Each extraction method performed well in removing the 15 PAHs from both soils over a broad range of concentrations. In general, Soxhlet extraction yielded slightly but significantly (P less than 0.05) higher recoveries than did the homogenization method. The homogenization method, however, was easy to use, and the extraction step turnaround time was less than 15 min/sample. The method should be suitable for other applications requiring the extraction of hydrophobic organic compounds from soils.     

Rapid magnetic solid-phase extraction based on magnetic multiwalled carbon nanotubes for the determination of polycyclic aromatic hydrocarbons in edible oils

In this study, magnetic multiwalled carbon nanotubes were fabricated by a simple method and applied to magnetic solid-phase extraction (MSPE) of eight heavy molecular weight polycyclic aromatic hydrocarbons (PAHs) including chrysene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, dibenzo[a,h]anthracene and benzo[g,h,i]perylene from edible oil samples. Several parameters affecting the extraction efficiency were investigated, including the type and volume of desorption solvent, extraction and desorption time, washing solution and the amount of sorbent. Under the optimized conditions, a simple and effective method for the determination of PAHs in edible oils was developed by coupling with gas chromatography-mass spectrometry (GC-MS). The whole pretreatment process was rapid, and it can be accomplished within 10 min. The limits of quantitation for the target PAHs were found to be 0.34-2.9 ng/g. The recoveries in oil sample were in the range 87.8-122.3% with the RSDs less than 6.8% (intraday) and 9.6% (interday). This method was successfully applied to the analysis of PAHs in seven kinds of edible oils from local markets.     

乌鲁木齐市周边地区土壤中多环芳烃的含量及来源

在乌鲁木齐市周边,从乌拉泊到水西沟按不同距离与深度进行土壤样品采集,采用索氏提取法与层析净化法进行预处理,高效液相色谱法测定土壤中16种多环芳烃(PAHs)的含量,并对PAHs进行对比分析、污染评价和来源分析的相关研究。结果表明:总PAHs平均浓度为998.23(306.94~3 652.16)ng/g,污染程度差异不大,处中度污染水平但更接近严重污染水平;16种PAHs的最低检测限为0.20~0.80 ng/g;一些采样点的表层土壤中苯并[a]芘的含量高于土壤质量控制标准。不同层次土壤PAHs的污染程度有所不同,其顺序为表层中层下层;高分子量(4~6环)PAHs占据了总含量的84.1%,低分子量(2~3环)PAHs占据15.9%,得出在乌鲁木齐市周边土壤中PAHs的重要来源是汽车排放,同时煤燃烧排放的贡献也很大。     

LC-MS/MS analysis of neonicotinoid insecticides in honey: methodology and residue findings in Austrian honeys

An analytical method for the simultaneous determination of residues of eight neonicotinoid insecticides and two metabolites in honey using LC-MS/MS was developed and validated. Two approaches of sample preparation were investigated, with the final method involving acetonitrile extraction and subsequent cleanup by dispersive solid-phase extraction (QuEChERS type). Validation was based on quintuplicate analysis at three fortification levels and showed satisfactory recoveries (60-114%) and high precision (RSDs between 2.7 and 12.8%). Low limits of detection and quantification could be achieved for all analytes ranging from 0.6 to 5 μg/kg and from 2 to 10 μg/kg, respectively. Investigations of Austrian honey samples revealed the presence of acetamiprid, thiacloprid, and thiamethoxam residues in honey; however, no sample exceeded the maximum residue limits. On average, flower honey samples contained neonicotinoid residues in higher quantities compared to forest honey samples.     

Determination of bentazone, dichlorprop, and MCPA in different soils by sodium hydroxide extraction in combination with solid-phase preconcentration

A method for the extraction of bentazone, dichlorprop, and MCPA in three selected Norwegian soils of different textures is described. Initially three different extraction methods were tested on one soil type. All methods gave recoveries >80% for the pesticide mixture, but extraction with sodium hydroxide in combination with solid-phase preconcentration was used for further recovery tests with soils of different properties spiked at four herbicide concentration levels (0.001-10 microg/g of wet soil). The method was rapid and easy and required a minimum of organic solvents. The recoveries were in the range of 82-109, 80-123, and 45-91% for the soils containing 1.4 (Hole), 2.5 (Kroer), and 37.8% (Froland) organic carbon, respectively. Limits of quantification using GC-MS were 0.0003 microg/g of wet soil for bentazone and 0.0001 microg/g of wet soil for both dichlorprop and MCPA.     

Rapid method for quantitative analysis of the aroma impact compound, 2-acetyl-1-pyrroline, in fragrant rice using automated headspace gas chromatography

A rapid method employing static headspace gas chromatography (HS-GC) has been developed and validated for quantitative analysis of the impact aroma compound, 2-acetyl-1-pyrroline (2AP), in grains of fragrant rice. This developed method excludes wet extraction, and the rice headspace volatiles are brought directly and automatically to GC analysis. The conditions of the static HS autosampler were optimized to achieve high recovery and sensitivity. The most effective amount of rice sample used was 1 g, which provided 51% recovery and a linear multiple headspace extraction (MHE) plot of the peak area of 2AP. The sensitivity of the method was enhanced by utilizing a megabore fused silica capillary column in conjunction with a nitrogen-phosphorus detector (NPD). Method validations performed for both static HS-GC-FID and HS-GC-NPD demonstrated linear calibration ranges of 20-10 000 (r(2) = 0.9997) and 5-8000 (r(2) = 0.9998) ng of 2AP/g of rice sample, respectively. The limits of detection for both systems were 20 and 5 ng of 2AP, and the limits of quantitation were 0.30 and 0.01 g of brown rice sample, respectively. Reproducibility calculated as intraday and interday coefficients of variation were 3.25% RSD (n = 15) and 3.92% RSD (n = 35), respectively, for SHS-GC-FID and 1.87% RSD (n = 15) and 2.85% RSD (n = 35), respectively, for SHS-GC-NPD. The method was found to be effective when applied to the evaluation of aroma quality, based on 2AP concentrations, of some fragrant rice samples.     

Analysis of patulin in pear- and apple-based foodstuffs by liquid chromatography electrospray ionization tandem mass spectrometry

A liquid chromatography electrospray ionization tandem mass spectrometry method for the determination of patulin in apple- and pear-based foodstuffs was developed. The sample preparation is based on the QuEChERS procedure involving an initial extraction step with water and acetonitrile, followed by a partitioning step after the addition of magnesium sulfate and sodium chloride. The cleanup was performed by using dispersive solid-phase extraction with a mixture of magnesium sulfate, primary secondary amine sorbent, and n-octadecylsiloxane sorbent added together to the extract. The cleaned extract was finally evaporated and reconstituted in water prior to injection. Quantitation was performed by isotope dilution using ((13)C(7))-patulin as internal standard. The method was first fully validated in three different baby food products including apple-pear juice, apple-pear puree, and infant cereals. Then the scope of application of the method was extended to pear concentrate, raw apples, apple flakes (naturally contaminated), dried apples, and yogurt. The sensitivity achieved by the method in all matrices gave limits of detection (LOD) and quantitation (LOQ) of ≤0.5 and ≤10 μg/kg, respectively, which was compliant with maximum levels settled in Commission Regulation (EC) No. 1881/2006. Method performances for all matrices also fulfilled the criteria established in the CEN/TR 16059:2010 document. Indeed, recoveries were within the 94-104% range; relative standard deviations of repeatability (RSD(r)) and intermediate reproducibility (RSD(IR)) were ≤7.5 and ≤13.0%, respectively, and trueness in an infant apple drink (FAPAS 1642) was measured at 99%.     

Simultaneous analysis of free and conjugated estrogens, sulfonamides, and tetracyclines in runoff water and soils using solid-phase extraction and liquid chromatography-tandem mass spectrometry

The ability to monitor multiple analytes from various classes of compounds in a single analysis can increase throughput and reduce cost when compared to traditional methods of analyses. This method for analyzing free (parent estrogen) and conjugated estrogens (metabolites) along with sulfonamides and tetracyclines utilizes a high pH (10.4) mobile phase with an ammonium hydroxide buffer for both positive- and negative-mode electrospray ionization. A single-step sample preparation by solid-phase extraction (SPE) was used to isolate and concentrate all analytes simultaneously. The analytical method was developed and validated for recoveries at 3 concentration levels for water and soil and produced recoveries of 42-123% and 21-105% respectively. Method detection limits ranged from 0.3 to 1.0 ng/L for water samples and 0.01 to 0.1 ng/g for soils. The method quantification limit ranged from 0.9 to 3.3 ng/L for water samples and 0.06 to 0.7 ng/g for soils. The single-point standard addition calibration procedure was validated across a linear range of MQL to 100 ng/L with ≥82% accuracy against a matrix matched standard curve. Furthermore, sorption of tetracyclines onto glassware was investigated and minimized by 10% using nitric acid-rinsed glassware, while separation parameters were further optimized based on retention time and signal responses. This method has been used for the quantification of estrogens, tetracyclines, and sulfonamides in soil and runoff waters with multiple compounds detected simultaneously in a single analysis.     

Simplified extraction of ginsenosides from American ginseng (Panax quinquefolius L.) for high-performance liquid chromatography-ultraviolet analysis

Four methods were tested for extraction and recovery of six major ginsenosides (Rb1, Rb2, Rc, Rd, Re, and Rg1) found in roots of American ginseng (Panax quinquefolius): method A, sonication in 100% methanol (MeOH) at room temperature (rt); method B, sonication in 70% aqueous MeOH at rt; method C, water extraction (90 degrees C) with gentle agitation; and method D, refluxing (60 degrees C) in 100% MeOH. After 0.5-1 h, the samples were filtered and analyzed by high-performance liquid chromatography (HPLC)-UV. A second extraction by methods C and D was done, but 85-90% of ginsenosides were obtained during the first extraction. Lyophilization of extracts did not influence ginsenoside recovery. Method D resulted in the highest significant recoveries of all ginsenosides, except Rg1. Method C was the next most effective method, while method A resulted in the lowest ginsenoside recoveries. Method B led to similar recoveries as method C. All methods used one filtration step, omitted time-consuming cleanup, but maintained clear peak resolution by HPLC, and can be used for quantitative screening of ginsenosides from roots and commercial ginseng preparations.     

Assessment of polycyclic aromatic hydrocarbon content of smoked fish by means of a fast HPLC/HPLC method

Polycyclic aromatic hydrocarbon (PAH) concentrations in smoked food can reach levels hazardous for the human health, especially when the smoking procedure is carried out under uncontrolled conditions. In this work a simple and rapid method for the determination of PAHs in smoked fish samples is described. PAHs were extracted from the insoluble samples (together with fat substances) by homogenization with acidified chloroform. The fat extract was then submitted to a first LC sample preparation step (performed on a large silica column) to isolate PAHs from triglycerides. After reconcentration, the PAH fraction was finally injected into the reverse-phase analytical column. The proposed method, which presents good characteristics of recoveries and repeatability, was also used to analyze PAH content of some smoked trout samples packed under vacuum.