Long-term Stability Of Samples Research Articles

Long-term stability of five atypical antipsychotics (risperidone, olanzapine, paliperidone, clozapine, quetiapine) and the antidepressant mirtazapine in human serum assessed by a validated SPE LC-MS/MS method.

Long-term sample stability of five atypical antipsychoticdrugs risperidone, paliperidone, clozapine, quetiapine and olanzapine and the antidepressant drug mirtazapine in serum was studied by use of a newly developed and validated analytical method based on solid-phase extraction and liquid chromatography-tandem mass spectrometry. Ascorbic acid was used as an antioxidative agent to stabilize olanzapine during storage and sample preparation. We assessed analyte stability on long-term storage in serum samples at 25°C, 5°C, -20°C and -80°C, and during five freeze-thaw cycles. Analytes were stable for 23 days at room temperature except for olanzapine and mirtazapine (17 days). All analytes were stable for at least 30 days at 5°C. All analytes were stable for 270 days at -20°C, except for paliperidone and mirtazapine with 60 days and 180 days, respectively. All analytes were stable for 270 days at -80°C. Furthermore, all analytes were stable for five freeze-thaw cycles. We recommend storage at -80°C when samples drawn for analysis of antipsychotic drugs are stored for more than 60 days, whereas a temperature of -20°C is sufficient for storage less than 60 days.

Microfluidic-Integrated Chip Resonators for Electron Spin Sensing in Submicromolar, Submicroliter Solutions.

Planar or chip microresonators decrease the sample volume required for magnetic resonance spectroscopies to the nanoliter scale. However, the interrogation of nanoliter-scale solution samples on planar sensors is hindered by the lack of microfluidic devices that can simultaneously provide a small total volume and long-term sample stability. Here, we report microfluidic devices that decrease the total required sample volume to the submicroliter scale and also provide long-term physical stability and storability. We also report a 3D-printed microfluidic with a self-contained actuation mechanism, which allows the sample to be retracted from the microresonator surface for storage. The microfluidic devices are fabricated easily by laser cutting or 3D printing and are integrable with a broad range of planar sensors. We use planar inverse anapole (PIA) microresonators to obtain continuous wave (CW) electron paramagnetic resonance (EPR) spectra of natural-isotopic-abundance nitroxide radicals, which are ubiquitously used as reporters of biomolecular dynamics. We provide experimental evidence for a concentration sensitivity of 330 ± 40 nmol L-1, a concentration sensitivity limit of 800 ± 100 nmol L-1/mT√Hz, and an active volume no greater than 30 nL. Together, these developments represent an advance not only in the sensitivity of EPR spectroscopy but also in the design of microfluidics for stable, dead-volume-free placement of nanoliter-scale volumes of solutions on planar sensors.

Development of a high-performance liquid chromatography–tandem mass spectrometric method for the determination of Methimazole in human blood matrices

Methimazole (MMI, 1-methyl-2-mercaptoimidazole) is widely used for the treatment of hyperthyroidisms. There are methods available for the measurement of MMI concentration in human serum or plasma from the past, but none meet the current regulatory standards for bioanalytical method validations. In this paper, we developed and validated a total MMI measurement method using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a technique that conforms to current bioanalytical method validation. To form a free sulfhydryl group on MMI, sodium bisulfite was added to 50 µl of plasma or serum samples containing MMI before the derivatization step. The internal standard (MMI-D3) was spiked into samples, then these samples were derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole. After derivatization, these samples were extracted by supported liquid extraction. Then, the organic solvent was evaporated and the residue was dissolved in 50% methanol and injected into the LC-MS/MS system. A calibration curve was plotted over the concentration range 1–1000 ng/mL (r2 = 0.999). The intra-day and inter-day precisions were less than 10.2% and 9.8%, respectively. The intra-day and inter-day accuracies were between 89.5% and 101.1%, and 96.0% and 99.7%, respectively. The long-term stability of samples showed good precision and accuracy. The validated method was successfully applied to determine serum total MMI concentration in Graves’ disease patients after oral administration of 5, 10 or 15 mg MMI. The range of circulating total MMI concentrations was found to be between 2.69 and 304.27 ng/mL in this study. It was shown that the measured serum total MMI concentrations changed in a dose-dependent manner.

Rapid Recovery of DNA from Dried Blood Spots: Use of a Novel Electrophoretic Method

The demand for rapid, effective point of care diagnostics has created a pressing need for new and improved methods to quantify DNA from patient samples. Dried blood spotting is a well‐established testing methodology that has gained increasing importance in in vitro diagnostics due to its low cost, low sampling requirements, and long‐term sample stability. In recent years, dried blood spots have been increasingly used for targeted genetic and metabolite profiling. Large‐scale genetic screening of neonatal dried blood spots has a great potential to lower patient mortality and morbidity through early diagnosis of primary immunodeficiencies. However, DNA extraction from the surface of the dried blood spot remains the most time consuming, costly and labor‐intensive part of DNA analysis. Thus, the objective of this study was to develop and optimize a simple and rapid methodology to extract DNA from dried blood spots.In the present study, we created a novel methodology to extract DNA from a dried blood spot using only a lysis buffer (10mM KOH/30mM Tris buffer), 50V, and heat. Briefly, during the electric field extraction spots and lysis buffer were placed in an electroporation cuvette and DNA was extracted by applying 25–50 volts across the plates of the cuvette. The spot was then removed and DNA in the supernatant was analyzed using qPCR or fluorescent nucleic acid stains. This methodology was optimized by the addition of a 70°C heating step before the electric field extraction to better retrieve episomal DNA.This electric field DNA extraction is to our knowledge, the first methodology to use an electric field to extract episomal DNA from a dried blood spot. Results of this study show that this methodology produces significant recoveries of both genomic and episomal DNA (Ct (FAM) 33.0 (50V, 60s) to Ct (FAM) 35.0 (heating alone) p <0.01). Varying recoveries of episomal and genomic DNA were also seen at different voltage conditions suggesting that this methodology may be useful for the isolation and purification of episomal DNA (FAM (25V, 300s (no amplification) and 50V, 60s (Ct 33.0)). Furthermore, preliminary qualitative and spectroscopic analyses suggest that protein removal could be achieved using this dual voltage technique. The 25‐minute electric field extraction procedure has one of the lowest times for the extraction of DNA found within the literature. This optimized procedure negates the need for costly treatment and wash steps, simplifying DNA extraction considerably. Most importantly, the electric field extraction reduces the time of proprietary procedures by more than 30 minutes, while retaining 75–80% of the yield. Thus, the results show that this novel method of electric field extraction provides a rapid and effective protocol to remove episomal and genomic DNA from dried blood spots. This optimized electric field device not only simplifies bioanalytical techniques, but provides a significant step towards point of care diagnostics and should be expanded to other sample types.Support or Funding InformationThe authors thank PerkinElmer for providing the funding for this study.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bio-inspired graphene-based coatings on Mg alloy surfaces and their integrations of anti-corrosive/wearable performances

Mg and its alloys generally exhibit the poor corrosion and wear resistances, which greatly limits their industrial applications. Inspired from an ordered layered structure within natural nacre, the homogeneous reduced graphene oxide/poly (vinyl alcohol) (RGO/PVA) coating is successfully fabricated on Mg alloy surface in the current study, through a facile spin-assisted layer-by-layer assembly (SA-LBL) technology. The hybrid hydrogen/covalent bond networks form between PVA chains and RGO sheets, resulting in a compact lamellar “bricks-and-mortar” structure. The microstructure of hybrid coating is strongly influenced by the cross-linking effect of PVA molecule, whose suitable content is beneficial to generating the densely stacked lamellar coating with excellent binding strength and reduced intrinsic defects, which can significantly improve its corrosion resistance. And the water molecules can hardly diffuse into the hybrid coating, leading to a long-term stability of samples in aqueous solution, ethanol and N, N-dimethylformamide. Besides, the high hardness and good lubricant effect of RGO sheets confer the hybrid coatings outstanding wear-resistant properties. Therefore, the bio-inspired RGO coating with a certain amount of PVA allows it to have a superior barrier effect and maintain the lubrication, which can provide an integrated protection for the corrosion and wear behaviors of Mg alloy.

Quantification of acyclovir in dermal interstitial fluid and human serum by ultra-high-performance liquid-high-resolution tandem mass spectrometry for topical bioequivalence evaluation.

Time-concentration curves for the topical anti-viral drug acyclovir can provide valuable information for drug development. Open flow microperfusion is used for continuous sampling of dermal interstitial fluid but it requires validated methods for subsequent sample analysis. Therefore, we developed a sensitive, selective and high-throughput ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry method to determine acyclovir in human dermal interstitial fluid and serum. We validated the method over a concentration range of 0.1-25 ng/mL for a sample volume of just 20 μL and employed cation-exchange solid-phase extraction in a fully automated sample treatment procedure. Short- and long-term sample stability data and the analysis of 5000 samples from a clinical trial demonstrate the successful application of our method.

Improved pharmacodynamic assay for dihydropyrimidine dehydrogenase activity in peripheral blood mononuclear cells.

Dihydropyrimidine dehydrogenase (DPD) activity determination in peripheral blood mononuclear cells of DPD deficient patients was hitherto inaccurate due to hemoglobin (Hb) contamination. We developed an improved method for accurate measurement of DPD activity in patients. DPD activity was determined by HPLC with online radioisotope detection using liquid scintillation counting. Hb was determined spectrophotometrically. Method accuracy and precision were significantly improved by using cumulative area of all peaks as IS. Peripheral blood mononuclear cell lysates from DPD deficient patients were highly contaminated with on average 23.3% (range 2.7-51%) of Hb resulting in up to twofold underestimated DPD activity. DPD activities were corrected for Hb contamination. The method was validated and showed good long-term sample stability. This method has increased specificity allowing accurate identification of DPD deficient patients.

Dried blood spot assay for the quantification of phenytoin using Liquid Chromatography-Mass Spectrometry

Phenytoin (PHT) is one of the most commonly used anticonvulsant drugs for the treatment of epilepsy and bipolar disorders.The large amount of plasma required by conventional methods for drug quantification makes mass spectrometry combined with dried blood spot (DBS) sampling crucial for pediatric patients where therapeutic drug monitoring or pharmacokinetic studies may be difficult to realize. DBS represents a new convenient sampling support requiring minimally invasive blood drawing and providing long-term stability of samples and less expensive shipment and storage.The aim of this study was to develop a LC-MS/MS method for the quantification of PHT on DBS.This analytical method was validated and gave good linearity (r2=0.999) in the range of 0–100mg/l. LOQ and LOD were 1.0mg/l and 0.3mg/l, respectively.The drug extraction from paper was performed in a few minutes using a mixture composed of organic solvent for 80%. The recovery ranged from 85 to 90%; PHT in DBS showed to be stable at different storage temperatures for one month. A good correlation was also obtained between PHT plasma and DBS concentrations.This method is both precise and accurate and appears to be particularly suitable to monitor treatment with a simple and convenient sample collection procedure.

Successful development and use of a thermodynamic stability screen for optimizing the yield of nucleotide binding domains

ATP sensitive potassium (KATP) channels consist of four copies of a pore-forming inward rectifying potassium channel (Kir6.1 or Kir6.2) and four copies of a sulfonylurea receptor (SUR1, SUR2A, or SUR2B). SUR proteins are members of the ATP-binding cassette superfamily of proteins. Binding of ATP to the Kir6.x subunit mediates channel inhibition, whereas MgATP binding and hydrolysis at the SUR NBDs results in channel opening. Mutations in SUR1 and SUR2A NBDs cause diseases of insulin secretion and cardiac disorders, respectively, underlying the importance of studying the NBDs. Although purification of SUR2A NBD1 in a soluble form is possible, the lack of long-term sample stability of the protein in a concentrated form has precluded detailed studies of the protein aimed at gaining a molecular-level understanding of how SUR mutations cause disease. Here we use a convenient and cost-effective thermodynamic screening method to probe stabilizing conditions for SUR2A NBD1. Results from the screen are used to alter the purification protocol to allow for significantly increased yields of the purified protein. In addition, the screen provides strategies for long-term storage of NBD1 and generating NBD1 samples at high concentrations suitable for NMR studies. NMR spectra of NBD1 with MgAMP-PNP are of higher quality compared to using MgATP, indicating that MgAMP-PNP be used as the ligand in future NMR studies. The screen presented here can be expanded to using different additives and can be employed to enhance purification yields, sample life times, and storage of other low stability nucleotide binding domains, such as GTPases.

Super-Resolution Imaging of Bacteria in a Microfluidics Device

Bacteria have evolved complex, highly-coordinated, multi-component cellular engines to achieve high degrees of efficiency, accuracy, adaptability, and redundancy. Super-resolution fluorescence microscopy methods are ideally suited to investigate the internal composition, architecture, and dynamics of molecular machines and large cellular complexes. These techniques require the long-term stability of samples, high signal-to-noise-ratios, low chromatic aberrations and surface flatness, conditions difficult to meet with traditional immobilization methods. We present a method in which cells are functionalized to a microfluidics device and fluorophores are injected and imaged sequentially. This method has several advantages, as it permits the long-term immobilization of cells and proper correction of drift, avoids chromatic aberrations caused by the use of different filter sets, and allows for the flat immobilization of cells on the surface. In addition, we show that different surface chemistries can be used to image bacteria at different time-scales, and we introduce an automated cell detection and image analysis procedure that can be used to obtain cell-to-cell, single-molecule localization and dynamic heterogeneity as well as average properties at the super-resolution level.

Long-term aging characteristics of atmospheric-plasma-treated poly(ɛ-caprolactone) films and fibres

This paper reports the change in the surface properties and aging behaviours of surface-modified poly(ɛ-caprolactone) films and nanofibres after atmospheric plasma treatment with a reactive gas. The morphologies, roughness, chemical bonding states, and surface energies of pristine and atmospheric-plasma-treated nanofibres were investigated. The nanofibres subjected to O 2 atmospheric plasma treatment showed higher surface roughness (∼88%), fibre diameter (∼14%), surface energy (∼19.2 times), and hydrophilicity (O/C atomic ratio, ∼43%) than did the pristine nanofibres. Over a 6-month period, the characteristics of the nanofibres isolated from air were not significantly altered; however, in the case of the nanofibres exposed to air, there was a subsequent increase in the surface roughness (∼82%), fibre diameter (∼7%), surface energy (∼27%), and hydrophilicity (O/C atomic ratio, ∼17.6%). We confirmed that the properties of the atmospheric-plasma-treated poly(ɛ-caprolactone) nanofibres maintained for 6 months without exposure to air did not show any significant change. Consequently, these nanofibres would be well suited for biomedical applications that require long-term sample stability.

Measurement of Hemoglobin A1c from Filter Papers for Population-Based Studies

Stability and transport challenges make hemoglobin (Hb) A(1c) measurement from EDTA whole blood (WB) inconvenient and costly for large-scale population studies. This study investigated Hb A(1c) measurement from WB blotted on filter paper (FP) in a Level I National Glycohemoglobin Standardization Program (NGSP)-accredited laboratory. Three Bio-Rad Variant™ II HPLC instruments and WB and FP specimens were used. Precision, accuracy, linearity, and readable total area of the 6.5-min (β-thalassemia method) Variant II HbA(2)/HbA(1c) Dual Program were assessed. Hb A(1c) stability was measured using in-house FP QC samples. The INTERHEART (a study of the effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries) and CURE (Clopidogrel in Unstable Angina to Prevent Recurrent Events) studies provided chromatographs for morphometric analyses and interoperator variability experiments. Statistical analyses were performed to assess long-term sample stability, WB vs FP agreement, and significance of Hb A(1c) peak integration. Intra- and interassay CVs were ≤2.00%. Total area counts between 0.8 and 5.5 × 10(6) μV/s produced accurate Hb A(1c) results. The regression equation for agreement between WB(x) and FP(y) was as follows: y = 0.933x + 0.4 (n = 85). FP QC samples stored at -70 °C and tested over approximately 3 years yielded CVs of 1.72%-2.73% and regression equations with slopes of -1.08 × 10(-4) to 7.81 × 10(-4). The CURE study, with better preanalytical preparation, achieved a 97% reportable rate, and the reportable rate of the INTERHEART study was 85%. The FP collection method described provided accurate, robust, and reproducible measurement of Hb A(1c) using the Bio-Rad Variant II HPLC autoanalyzer when FP specimens were prepared according to standardized protocols, and analyses were performed in an NGSP-certified laboratory, supporting the use of FP collection cards in large multinational studies.

A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees

Collection of tissue and subsequent isolation of genomic DNA from mature tree species often proves difficult. DNA extraction from needles, leaves, or buds is recommended in many protocols. Collecting these tissues from mature trees generally requires the use of firearms or climbing if sampling is to be nondestructive. As a result, sample collection is a major expense of many tree-based projects. Tree (and plant) tissues generally contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA. Many methods aim to overcom these problems, with most involving extraction in buffers containing the nonionic detergent cetyltrimethyl-ammonium bromide (CTAB), followed by numerous steps to clean contaminants from the DNA, using organic solvents and differential salt precipitation. These steps are time-consuming, such that isolation of DNA becomes the bottleneck in many molecular studies. This paper presents a new, efficient, cambium collection method for tree species and a DNA extraction protocol based on that of Doyle and Doyle (1987), with follow-up purification using the Wizard nuclei lysis and protein precipitation solutions (Promega). Results show a significant improvement in yield and DNA purity compared with other published methods, with consistently high yields of pure genomic DNA and high sample throughput. The relatively low cost per extraction, no requirement for use of liquid nitrogen, no requirement for freezer storage, and long-term sample stability after collection are important additional benefits.

Structure and Dynamics of Micelle-Associated Human Immunodeficiency Virus gp41 Fusion Domain,

The N-terminal fusion domain of the HIV-1 gp41 envelope glycoprotein is responsible for initiating the fusion of viral and cellular membranes, leading to the subsequent infection of the host cell by HIV-1. We have investigated the backbone structure and dynamics of the 30 N-terminal residues of HIV-1 gp41 in membrane-mimicking environments using NMR spectroscopy and (15)N- and (15)N,(13)C,(2)H-labeled peptides. Similar (15)N-(1)H HSQC spectra were obtained in a variety of detergents, including SDS, DPC, mixed DPC/SDS, and LPPG micelles, indicating that the peptide structure is not strongly influenced by the type of detergent used. Detailed characterization was carried out in SDS micelles, where the long-term sample stability was found to be optimal. In addition to J-coupling and NOE restraints, a nearly complete set of backbone residual dipolar coupling restraints was recorded for the fusion domain-micelle complex aligned with respect to the magnetic field using a stretched polyacrylamide gel. Backbone amide (15)N spin relaxation and amide hydrogen exchange rates with the solvent were also measured. The ensemble of NMR structures reveals an uninterrupted alpha-helix for the least mobile residues (S(2) > 0.65), Ile-4 to Met-19, with transient helical character extending up to Ala-22. A 12-residue (Ile-4 to Ala-15) segment is fully shielded from solvent, with Gly-3 and Gly-16 found at micelle-solvent interfaces. Residues external to the micelle exhibit enhanced picosecond to nanosecond time scale dynamics relative to the residues buried in the micelle, and their mobility increases with the distance from the micelle.

Increasing spectral information with shorter acquisitions: the advantages of using F1 selection in structural determinations

AbstractThe structural elucidation of complex materials by NMR is complicated by the common occurrence of nearly coincident resonances. Congested regions necessitate increased spectral resolution, with additional time increments needed for 2D NMR spectroscopy, resulting in longer acquisition times. These demands conflict with the ever‐increasing demands for instrument time and with long‐term sample stability. Selective spectroscopy addresses both of these problems. By focusing attention only on the region of interest, fewer increments are needed. Shorter acquisitions are obtained, with a higher information content. Alternatively, the spectroscopist may choose to use these time savings to collect additional transients, allowing the study of smaller amounts of material, or low‐level impurities/isomers. Using a carbohydrate as our example, we illustrate the utility of this methodology using selHMQC, selHMBC (IMPRESS) and selHMQCTOCSY. We have found this methodology useful in the coupling of efficiency and productivity of NMR spectroscopy. Copyright © 2002 John Wiley & Sons, Ltd.

Application of a new separation analyzer for the characterization of dispersions stabilized with clay derivatives

The performance of a newly developed separation analyzer is demonstrated by characterization of the sedimentation kinetics and the swelling behavior of organoclays in binary solvent mixtures (toluene/cyclohexane) as well as by investigation of oil-in-water and water-in-oil emulsions stabilized by clay derivatives. The automatic measuring system – LUMiFuge 114 – is based on a special centrifuge with an integrated optoelectronic sensor system which allows the measuring of spatial and temporal changes of light transmission during the rotation in a variable centrifugal field (12–1200g). By using the new device a fast and reproducible characterization of settling behavior and dispersion stability was possible. Moreover, the evaluation of the long-term stability of samples with high separation stability as well as of the heterogenity of the separation process, the sample transparency and the elasticity of the sediment as a function of alternating centrifugal force could be carried out.

Optimization of liquid scintillation counting techniques for the determination of carbon-14 in environmental samples

The goal of this work was to optimize the liquid scintillation counting techniques for the determination of14C in stack effluent gases and in environmental samples such as biological and air samples. Carbon-14 activities in most environmental samples were measured with the direct CO2 absorption method. The highest figures of merit were found through the variation of Carbosorb E and Permafluor V ratio, and measurement windows. The best condition was an 1:1 volume ratio. Average 2.35 g of CO2 was reproducibly absorbed in the 20 ml mixture within 40 minutes. The counting efficiency determined by repeated analysis of NIST oxalic acid standard and the background count rate were measured to be 58.8±1.4% and 1.88±0.06 cpm, respectively, in case of saturated solution. The correction curves of counting efficiency for partially saturated solutions and for saturated solutions with quenching were prepared, respectively. The overall uncertainty of the sample specific activity for near background levels was estimated to be about 7% for 4 hours counting at 95% confidence level. Stack effluent gas samples were measured by a gel suspension counting method. After precipitation of CO2 in the form of BaCO3, 140 mg of which was mixed with 6 ml H2O and 12 ml of Instagel XF. The counting efficiency was measured to be 71.5±1.7% and the typical sensitivity of this technique was about 510 mBq/m3 for a 100 min count at a background count rate of 4.7 cpm. For the benzene counting method measurements were performed with a mixture of 3 ml benzene and 1 ml of scintillation cocktail (5 g of butyl-PBD in 100 ml of scintillation-grade toluene) in a low potassium 7 ml borosilicate glass vial. The counting efficiency and the background count rate were measured to be 64.3±1.0% and 0.51±0.05 cpm, respectively. The long-term stability of samples has been checked for all the counting techniques over a two week period, during which no apparent change in counting efficiency and background level was found.

A Sampling and Analytical Method for the Simultaneous Determination of Multiple Organophosphorus Pesticides in Air

A sampling and analytical method for organophosphorus pesticides using a combined filter/XAD-2 sorbent sampler and gas chromatography (GC)-flame photometric detection (FPD) was developed. The method was evaluated for 19 organophosphorus pesticides based on the joint Occupational Safety and Health Administration/National Institute for Occupational Safety and Health Standards Completion Program methods evaluation protocol. The evaluation addressed analyte recovery, sampler capacity, sample stability, and precision and accuracy. Additional experiments addressed long-term sample stability (30-day storage), short-term exposure limits, limits of detection, and concentration levels down to 0.1 times an exposure limit value. Samples were stable for 30 days of storage under either ambient or refrigerated conditions. Based on this research, all 19 compounds studied can be successfully determined simultaneously using one method with an accuracy of +/- 25% of the true value 95 times out of 100.

Comparison of solid adsorbent and stainless steel canister sampling for very low ppt-concentrations of aromatic compounds (≥C6) in ambient air from remote areas

The applicability of stainless steel canister and solid adsorbent sampling has been studied for very low concentrations (> 15 ppt) of compounds ≥ C6 in ambient air samples from remote areas (Arctic). Different cleaning methods were tested to get sufficiently low blank values for the solid adsorbents Tenax TA and Carbotrap for benzene and toluene. Two techniques are described based on high-vacuum or hot air pretreatment which lowered the blank to less than 1 ppt (sampling volume 10 and 60 l, respectively). Sufficiently low blanks could be maintained for a storage period of at least 1 week. However, this required a careful selection of suitable sealing and tube materials. Furthermore, the long-term stability of samples collected with electropolished stainless steel bottles was tested. No losses were observed for benzene and toluene during 1 month storage. Quantitative analysis was carried out by combining thermodesorption or cryogenic enrichment with high resolution gas chromatography. Both sampling techniques are suitable for very low ppt-concentrations of benzene and toluene but their application range is complementary (e.g. grab sampling versus sampling over longer periods). A detailed description of all working procedures and methods is given including blanks and reproducibility data.