Simplified kinetic calibration of solid-phase microextraction for in vivo pharmacokinetics

Solid-phase microextraction (SPME) has been demonstrated to be useful for in vivo sampling in pharmacokinetic studies. In this study, a single time-point kinetic calibration for in vivo dynamic monitoring was developed by simplification of the laborious multiple time-point kinetic calibration, based on the independent desorption kinetics of the preloaded standards from SPME fibers with the changing analyte concentrations. The theoretical foundation and practical application conditions, such as the replicate numbers, the optimal time-point for desorption, and the sampling time, were systematically investigated. Furthermore, the feasibility of using regular standards rather than deuterated ones for the kinetic calibration was justified by comparing to the data obtained using the deuterated standards. All the methods were verified by in vitro and in vivo experiments. The results from in vivo SPME were validated by the blood drawing and chemical assay. These simplified calibration methods improved the quantitative applications of SPME for dynamic monitoring and in vivo sampling, enhance the multiplexing capability and automatic potentials for high throughput analysis, and decrease expenses on reagents and instruments.     

In vivo solid-phase microextraction for single rodent pharmacokinetics studies of carbamazepine and carbamazepine-10,11-epoxide in mice

The use of solid-phase microextraction (SPME) for in vivo sampling of drugs and metabolites in the bloodstream of freely moving animals eliminates the need for blood withdrawal in order to generate pharmacokinetics (PK) profiles in support of pharmaceutical drug discovery studies. In this study, SPME was applied for in vivo sampling in mice for the first time and enables the use of a single animal to construct the entire PK profile. In vivo SPME sampling procedure used commercial prototype single-use in vivo SPME probes with a biocompatible extractive coating and a polyurethane sampling interface designed to facilitate repeated sampling from the same animal. Pre-equilibrium in vivo SPME sampling, kinetic on-fibre standardization calibration and liquid chromatography–tandem mass spectrometry analysis (LC–MS/MS) were used to determine unbound and total circulating concentrations of carbamazepine (CBZ) and its active metabolite carbamazepine-10,11-epoxide (CBZEP) in mice (n = 7) after 2 mg/kg intravenous dosing. The method was linear in the range of 1–2000 ng/mL CBZ in whole blood with acceptable accuracy (93–97%) and precision (<17% RSD). The single dose PK results obtained using in vivo SPME sampling compare well to results obtained by serial automated blood sampling as well as by the more conventional method of terminal blood collection from multiple animals/time point. In vivo SPME offers the advantages of serial and repeated sampling from the same animal, speed, improved sample clean-up, decreased animal use and the ability to obtain both free and total drug concentrations from the same experiment.     

Direct monitoring of ochratoxin A in cheese with solid-phase microextraction coupled to liquid chromatography-tandem mass spectrometry

An in situ application of solid-phase microextraction (SPME) as a sampling and sample preparation method coupled to HPLC-MS/MS for direct monitoring of ochratoxin A (OTA) distribution at different locations in a single cheese piece is proposed. To be suited to the acidic analyte, the extraction phase (carbon-tape SPME fiber) was acidified with aqueous solution of HCl at pH 2, instead of the traditional sample pre-treatment with acids before SPME sampling. For calibration, kinetic on-fiber-standardization was used, which allowed the use of short sampling time (20 min) and accurate quantification of the OTA in the semi-solid cheese sample. In addition, the traditional kinetic calibration that used deuterated compounds as standards was extended to use a non-deuterated analogue ochratoxin B (OTB) as the standard of the analyte OTA, which was supported by both theoretical discussion and experimental verification. Finally, the miniaturized SPME fiber was adopted so that the concentration distribution of OTA in a small-sized cheese piece could be directly probed. The detection limit of the resulting SPME method in semi-solid gel was 1.5 ng/mL and the linear range was 3.5–500 ng/mL. The SPME–LC-MS/MS method showed good precision (RSD: ∼10%) and accuracy (relative recovery: 93%) in the gel model. The direct cheese analysis showed comparable accuracy and precision to the established liquid extraction. As a result, the developed in situ SPME–LC-MS/MS method was sensitive, simple, accurate and applicable for the analysis of complicated lipid-rich samples such as cheese.     

Semi-automated in vivo solid-phase microextraction sampling and the diffusion-based interface calibration model to determine the pharmacokinetics of methoxyfenoterol and fenoterol in rats

In vivo solid-phase microextraction (SPME) can be used to sample the circulating blood of animals without the need to withdraw a representative blood sample. In this study, in vivo SPME in combination with liquid–chromatography tandem mass spectrometry (LC–MS/MS) was used to determine the pharmacokinetics of two drug analytes, R,R-fenoterol and R,R-methoxyfenoterol, administered as 5 mg kg⁻¹i.v. bolus doses to groups of 5 rats. This research illustrates, for the first time, the feasibility of the diffusion-based calibration interface model for in vivo SPME studies. To provide a constant sampling rate as required for the diffusion-based interface model, partial automation of the SPME sampling of the analytes from the circulating blood was accomplished using an automated blood sampling system. The use of the blood sampling system allowed automation of all SPME sampling steps in vivo, except for the insertion and removal of the SPME probe from the sampling interface. The results from in vivo SPME were compared to the conventional method based on blood withdrawal and sample clean up by plasma protein precipitation. Both whole blood and plasma concentrations were determined by the conventional method. The concentrations of methoxyfenoterol and fenoterol obtained by SPME generally concur with the whole blood concentrations determined by the conventional method indicating the utility of the proposed method. The proposed diffusion-based interface model has several advantages over other kinetic calibration models for in vivo SPME sampling including (i) it does not require the addition of a standard into the sample matrix during in vivo studies, (ii) it is simple and rapid and eliminates the need to pre-load appropriate standard onto the SPME extraction phase and (iii) the calibration constant for SPME can be calculated based on the diffusion coefficient, extraction time, fiber length and radius, and size of the boundary layer. In the current study, the experimental calibration constants of 338.9 ± 30 mm⁻³ and 298.5 ± 25 mm⁻³ are in excellent agreement with the theoretical calibration constants of 307.9 mm⁻³ and 316.0 mm⁻³ for fenoterol and methoxyfenoterol respectively.     

Study of kinetic desorption rate constant in fish muscle and agarose gel model using solid phase microextraction coupled with liquid chromatography with tandem mass spectrometry

This study aims to use solid phase microextraction (SPME), a simple tool to investigate diffusion rate (time) constant of selected pharmaceuticals in gel and fish muscle by comparing desorption rate of diffusion of the drugs in both agarose gel prepared with phosphate-buffered saline (PBS; pH 7.4) and fish muscle. The gel concentration (agarose gel model) that could be used to simulate tissue matrix (fish muscle) for free diffusion of drugs under in vitro and in vivo conditions was determined to model mass transfer phenomena between fibre polymer coating and environmental matrix such that partition coefficients and desorption time constant (diffusion coefficient) can be determined. SPME procedure involves preloading the extraction phase (fibre) with the standards from spiked PBS for 1 h via direct extraction. Subsequently, the preloaded fibre is introduced to the sample such fish or agarose gel for specified time ranging from 0.5 to 60 h. Then, fibre is removed at specified time and desorbed in 100 μL of desorption solution (acetonitrile: water 1:1) for 90 min under agitation speed of 1000 rpm. The samples extract were immediately injected to the instrument and analysed using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The limit of detection of the method in gel and fish muscle was 0.01–0.07 ng mL⁻¹ and 0.07–0.34 ng g⁻¹, respectively, while the limit quantification was 0.10–0.20 ng mL⁻¹ in gel samples and 0.40–0.97 ng g⁻¹ in fish sample. The reproducibility of the method was good (5–15% RSD). The results suggest that kinetics of desorption of the compounds in fish tissue and different viscosity of gel can be determined using desorption time constant. In this study, desorption time constant which is directly related to desorption rate (diffusion kinetics) of selected drugs from the fibre to the gel matrix is faster as the viscosity of the gel matrix reduces from 2% (w/v) to 0.8% (w/v). As the concentration of gel reduces, viscosity of the gel will be reduced therefore allowing faster diffusion which invariably affect desorption time constant. Also, desorption time constant of model drugs in the fish muscle and 0.8–0.9% (w/v) gel model are similar based on free diffusion of studied compounds. In addition, in vitro and in vivo desorption time constant comparison shows that desorption time constant in an in vivo system (live fish muscle) is generally higher than an in vitro system (dead fish muscle) except for sertraline and nordiazepam. This study demonstrates SPME as a simple investigative tool to understand kinetics of desorption in an in vivo system with a goal to measure desorption rate of pharmaceuticals in fish.     

Analysis of trace levels of pesticides in rainwater by SPME and GC-tandem mass spectrometry after derivatisation with PFFBr

Solid-phase microextraction (SPME) was used for the analysis of some pesticides (bromoxynil, chlorotoluron, diuron, isoproturon, 2,4-MCPA, MCPP and 2,4-D) in rainwater after derivatisation with PFBBr and gas chromatography-ion trap mass spectrometry. The derivatisation procedure was optimized by testing different methods: direct derivatisation in the aqueous phase followed by SPME extraction, on-fibre derivatisation and derivatisation in the injector. The best result was obtained by headspace coating the PDMS/DVB fibre with PFBBr for 10 min followed by direct SPME extraction for 60 min at 68 °C (pH 2 and 75% NaCl). Good detection limits were obtained for all the compounds: these ranged between 10 and 1,000 ng L⁻¹ with a relatively high uncertainty due to the combination of derivatisation and SPME extraction steps. The optimized procedure was applied to the analysis of pesticides in rainwater and results obtained shows that this method is a fast and simple technique to assess the spatial and temporal variations of concentrations of pesticides in rainwater.     

Headspace SPME followed by GC/PFPD for the analysis of malodorous sulfur compounds in liquid industrial effluents

Headspace SPME was used to analyse malodorous sulfur compounds in liquid industrial effluents. A pulsed flame photometric detector (PFPD) was selected for a specific and sensitive analysis. Two fibres, PDMS/Dvb and PDMS/Carboxen, which are particularly convenient for extracting small and volatile molecules were tested. To compare these fibres, both sensitivity and artefact formation were considered. The PDMS/Carboxen fibre showed the lower limits of detection and moreover the least artefact formation yields. It was therefore selected and headspace SPME extraction conditions were optimised. Limits of detection of the target compounds evaluated were 12–31 ng L^–1 and repeatability was around 7%. Due to the adsorption mechanism involved, extraction is strongly influenced by the sample matrix and the low affinity compounds can suffer displacement effects. To investigate the occurrence of this phenomenon, two sampling times corresponding to non-equilibrium (5 min) and equilibrium conditions (60 min) were investigated. An external calibration was carried out by using standard solutions for both sampling times. The developed procedure was then compared to the standard addition method on a real industrial effluent. The results obtained from the two methods and for the two extraction times were in good agreement, demonstrating that even a long sampling time can be used. Therefore, the simple and timesaving external calibration was defined as relevant for an accurate quantification of sulfur compounds by headspace SPME.     

Investigation of the kinetic process of solid phase microextraction in complex sample

The presence of complex matrix in the aquatic system affects the environmental behavior of hydrophobic organic compounds (HOCs). In the current study, an automated solid-phase microextraction (SPME) desorption method was employed to study the effect of 2-hydroxypropyl-β-cyclodextrin (β-HPCD) on the kinetic process of 5 selected polyaromatic hydrocarbons (PAHs) desorbing from the fiber in aqueous sample. The results showed that the added β-HPCD facilitated the desorption rates of PAHs from SPME fiber coating, and the enhancement effect can be predicted by a proposed theoretical model. Based on this model, the kinetic parameters of organic compounds desorbing from the SPME fiber can be determined, and the calculated results showed good agreement with the experimental data. In addition, the effect of temperature on the desorption kinetic was investigated. The results found that the SPME desorption time constant increased as the sampling temperature elevated, and followed the Arrhenius equation. Also, the temperature facilitated the desorption of HOCs from the bound matrix so that increased the lability degrees of the bound compounds. Finally, a calibration method based on the proposed theoretical model was developed and applied for the analysis of unknown sample.     

Analysis of amides in the air by sampling with SPME and detection by GC-FID

This paper proposes a solid-phase microextraction (SPME) method for the passive sampling and determination of N,N-dimethylacetamide (DMA) and N,N-dimethylformamide (DMF) in air by capillary GC with flame ionization detection. The optimized conditions for SPME method were grab sampling, polydimethylsiloxane/divinylbenzene fiber, extraction for 90 min at 25 +/- 2 degrees C, desorption for 3 min at 220 degrees C, and relative humidity 45 +/- 2%. Under these conditions, the method presented good linearity (R = 0.996), repeatability (%RSD 2.79 and 9.85 for DMF and DMA, respectively), and detection limit (0.021 and 0.024 mg/m3 for DMF and DMA, respectively).     

Characterisation of absorptive sampling with SPME fibres in the EUPHORE photoreactor

The implementation of an experimental set-up for sampling, and characterisation of parameters related to absorptive sampling solid phase microextraction (SPME) in the EUPHORE photoreactor is described. Toluene was taken as probe compound. Optimisation of the sampling and calibration curves are presented. Equilibrium was achieved in just 30 s due to the good agitation in the chambers and the LOD was 0.24 ppbV. The precision was ±0.02 expressed as relative standard deviation (n = 9). The inter-fibre reproducibility was ±0.03 expressed as relative standard deviation. The effect of the temperature and the sun radiation on absorption in the fibre is also studied using a relative method to calculate the ratio K/K₀ (ratio of absorption constants at two different times during the experiment at which temperature and radiation conditions experimented important variations) calculated through known parameters, avoiding in this way the need for external calibrations in the calculation of K. The results showed a difficult to predict dependence of absorption over the combination of temperature and sun radiation and the need to develop sampling systems with absorptive SPME in which sun radiation has no influence. The stability in different conditions of compounds absorbed in the fibre was also subject to study. At room temperature half of the compound absorbed is expected to desorb in 1.21 h. Conversely, when fibres were kept at low temperatures (−86 °C) after 21 days, desorption was negligible.     

On-fibre solid-phase microextraction coupled to conventional liquid chromatography versus in-tube solid-phase microextraction coupled to capillary liquid chromatography for the screening analysis of triazines in water samples

This paper compares the advantages and disadvantages of two different configurations for the extraction of triazines from water samples: (1) on-fibre solid-phase microextraction (SPME) coupled to conventional liquid chromatography (LC); and (2) in-tube SPME coupled to capillary LC. In-tube SPME has been effected either with a packed column or with an open capillary column. A critical evaluation of the main parameters affecting the performance of each method has been carried out in order to select the most suitable approach according to the requirements of the analysis. In the on-fibre SPME configuration the fibre coating was polydimethylsiloxane (PDMS)-divinylbenzene (DVB). The limits of detection (LODs) obtained with this approach under the optimized extraction and desorption conditions were between 25 and 125 microg/L. The in-tube SPME approach with a C18 packed column (35 mm x 0.5 mm I.D., 5 microm particle size) connected to a switching micro-valve provided the best sensitivity; under such configuration the LODs were between 0.025 and 0.5 microg/L. The in-tube SPME approach with an open capillary column coated with PDMS (30 cm x 0.25 mm I.D., 0.25 microm of thickness coating) connected to the injection valve provided LODs between 0.1 and 0.5 microg/L. In all configurations UV detection at 230 nm was used. Atrazine, simazine, propazine, ametryn, prometryn and terbutryn were selected as model compounds.     

一种改进的SPME方法提取火场残留物中的促燃剂

对当前国内外用于火场残留物中促燃剂的提取方法进行研究,包括采用活性炭片法(ACS)、固相微萃取(SPME)以及对固相微萃取技术从传统的热解吸模式到溶剂脱附模式的扩展性思考。通过提取3种促燃剂(汽油,煤油和柴油)检材的效能比较与分析,优化了实验条件,确定了SPME的提取和溶剂脱附的方法,实验研究表明,SPME-溶剂脱附法是火场促燃剂残留物分析的有效方法之一。     

New approach on the alkylthiol determination in water by in situ derivatization SPME followed by GC-ECD/NPD analysis

Alkylthiols are very reactive and highly volatile compounds, and thus it is difficult to determine these in the water phase. In the present work, an in situ derivatization step prior to solid-phase microextraction (SPME) has been developed for their determination in water samples. The dinitrobenzylation reaction was selected because the high chemical stability of the corresponding thioethers formed provides a significant increase in the distribution coefficient between the SPME fibre and the aqueous phase, and a potential increase in the selectivity and sensitivity. Therefore, different derivatization reaction conditions (i.e. pH, temperature, reaction time and derivatizating reagent concentration) have been studied. Then, the main parameters affecting to the SPME process, that is coating selection, extraction time profile, extraction and desorption temperatures, have been optimized. Finally, a method based on a simple 2,4-dinitrophenylation reaction at pH 8–10, in 60?min at 75°C, coupled to direct SPME using PDMS-DVB fibres at 30°C for 45?min is proposed. The performance of the method provided a good linearity and precision data, and the detection limits were in the low ng?L^?1 level.     

Selected ion storage versus tandem MS/MS for organochlorine pesticides determination in drinking waters with SPME and GC-MS

The use of two modes for mass spectrometry (MS) detection with an ion trap instrument, selected ion storage (SIS) and tandem mass spectrometry (MS/MS), are compared for the solid-phase microextraction (SPME)–gas chromatography (GC) coupled to mass spectrometry (GC-MS) determination of 16 priority organochlorine pesticides (OCPs) in drinking water samples at the ultratrace levels (ng?L^?1) required by official guidelines in the European legislation. Experimental parameters investigated for the SPME sample preparation were: the type of coating (100?µm polydimethylsiloxane, PDMS, and 65?µm poly(dimethylsiloxane)–divinylbenzene, PDMS/DVB), SPME modality, extraction and desorption times and desorption temperature and the methanol percentage in the SPME working solution. Under the calculated optimal conditions two methodologies were developed, one for SIS and the other for MS/MS modes. The detection limits, precision and accuracy were evaluated for both alternatives and were appropriate to the official guidelines requirements. The SPME–GC-MS(SIS) methodology offered LODs from 0.2–6.6?ng?L^?1, precision below 13% and recoveries between 83 and 110%. The SPME–GC–MS/MS methodology provided limits of detection (LODs) ranging from 0.3 to 7.6 ng?L^?1, % RSD were ≤14% and recoveries of 79–108% were achieved. After the results observed within an Interlaboratory Exercise, the latest MS methodology was selected for the pursued analysis in real drinking water samples. Also, the good results in this round-robin exercise validate the proposed SPME–GC–MS/MS methodology.     

The use of solid-phase microextraction to rapidly measure dissolved PCBs in natural waters

A method to measure dissolved polychlorinated biphenyls (PCBs) in natural waters on 30?min time frames using negligible depletion non-equilibrium solid-phase microextraction (SPME) was developed with detection limits ranging from 0.6 to 5.2?ng?L^?1. SPME fibres made from optical cable were inserted into a glass tube and attached to the shaft of a motor that revolved at 130?rpm to move the SPME fibres through the sampled water at a constant rate. To test for matrix interferences, measurements were made in three solutions with the same known dissolved PCB concentration but different matrices. Dissolved PCB measurements made in the presence of 8?mg?L^?1 of DOC and 200?mg?L^?1 of suspended solids were not significantly different from measurements made in deionized water, demonstrating that neither matrix interfered with SPME measurements of dissolved PCBs. PCB concentrations measured by XAD-2 resin extraction were greater than SPME measurements, suggesting that XAD measurements included DOC-associated PCBs.     

Optimisation of the on-fibre derivatisation of volatile fatty acids in the simultaneous determination together with phenols and indoles in cow slurries

The on-fibre derivatisation of volatile fatty acids (VFAs) using N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) was optimised in the simultaneous determination of VFAs together with phenols and indoles by headspace solid-phase microextraction (SPME)–gas chromatography–mass spectrometry. Firstly, the nature of the SPME fibre was optimised and four different fibres were studied (100 μm polydimethylsiloxane, 85 μm Carboxen/polydimethylsiloxane, 5/30 μm divinylbenzene/Carboxen/polydimethylsiloxane and 85 μm polyacrylate). The optimum fibre (50/30 μm divinylbenzene/Carboxen/polydimethylsiloxane) was used to study the exposure time of the fibre to the derivatisation agent and the desorption time and temperature. Firstly, a factorial design was built but since the three variables had a significant effect, a central composite design was used to build the response surfaces. The best signals were obtained after the exposure of the fibre in the headspace of the MTBSTFA derivatisation reagent for 1 h and desorption at 300 °C for 9 min. The determination of underivatised phenols and indoles was not affected by the presence of the derivatisation reagent in the fibre.     

Flexibility of solid-phase microextraction for passive sampling of atmospheric pesticides

For low volatile pesticides, the applications of solid-phase microextraction (SPME) as an air sampler were reported with sampling time chosen in the linear stage of the sorption kinetics because of long equilibrium time. In these pre-equilibrium conditions, sampling rates (SRs) expressed as the volume of air sampled by the SPME sampler per unit of time, were used to estimate analytes concentrations in air. In the present study, to achieve good extraction performance and accurate calibration, the sorption kinetics of several pesticides with SPME were investigated in detail, with a focus on parameters influencing SRs. Linear air velocity was found to be the main parameter affecting SRs. For exposed fibers, with air velocities below 20–25 cm s⁻¹, SRs increased with increasing air velocity. When linear air velocity was equal to or greater than 25–30 cm s⁻¹, it had little effect on SRs. To improve the flexibility of SPME, different configurations of SPME were compared, i.e. different lengths of fibers exposed, retracted fibers, exposed fibers with grids. SRs were linearly proportional to exposed lengths of fibers. Using grids, lower SRs and wider calibration time range were achieved. SRs for retracted fibers were the lowest among the different experimented configurations. The accuracy of calibration was improved and more flexibility of SPME was provided.     

Solid phase microextraction using new sol–gel hybrid polydimethylsiloxane-2-hydroxymethyl-18-crown-6-coated fiber for determination of organophosphorous pesticides

A new sol–gel hybrid coating, polydimethylsiloxane–2-hydroxymethyl-18-crown-6 (PDMS–2OHMe18C6) was prepared in-house for use in solid phase microextraction (SPME). The three compositions produced were assessed for its extraction efficiency towards three selected organophosphorus pesticides (OPPs) based on peak area extracted obtained from gas chromatography with electron capture detection. All three compositions showed superior extraction efficiencies compared to commercial 100 μm PDMS fiber. The composition showing best extraction performance was used to obtain optimized SPME conditions: 75 °C extraction temperature, 10 min extraction time, 120 rpm stirring rate, desorption time 5 min, desorption temperature 250 °C and 1.5% (w/v) of NaCl salt addition. The method detection limits (S/N = 3) of the OPPs with the new sol–gel hybrid material ranged from 4.5 to 4.8 ng g⁻¹, which is well below the maximum residue limit set by Codex Alimentarius Commission and European Commission. Percentage recovery of OPPs from strawberry, green apple and grape samples with the new hybrid sol–gel SPME material ranged from 65 to 125% with good precision of the method (%RSD) ranging from 0.3 to 7.4%.     

Design of experiments and detailed uncertainty analysis to develop and validate a solid-phase microextraction/gas chromatography–mass spectrometry method for the simultaneous analysis of 16 pesticides in water

A solid-phase microextraction (SPME)/gas chromatography (GC)–mass spectrometry (MS) multiresidue analytical method was developed for 16 pesticides presenting different physicochemical properties including diphenyl ether, triazine, ureas, acetamides, benzofuran, thiocarbamate, pyridine carboxamides, chloronitrile, piperedine, and azoles. Optimization was achieved by means of the design of experiments methodology. Extraction temperature, extraction time, desorption temperature, and NaCl addition were the factors exhibiting the most significant effects on pesticide extraction. Validation was carried out through model adequacy and specificity tests, limits of quantification and detection determination, and full uncertainty assessment on the whole analytical method. Good first- and second-order model adequacy was found for pesticide calibration. LOQs were in the 0.05–0.5 μg L⁻¹ range and specificity recoveries varied from 75% to 140%. These results were considered acceptable for our research purposes on highly concentrated agricultural flows. Uncertainty calculations accounted for several steps: standard preparation, calibration model selection, and use. On average, real sample concentration uncertainties were lower than 10%, indicating that the analytical method performed very well. Its application to 61 real water samples confirmed the presence of some pesticide concentrations in relation to farmer use, whereas other molecules were usually either not detected or not quantified.     

Coupled in-tube and on-fibre solid-phase microextractions for cleanup and preconcentration of organic micropollutants from aqueous samples and analysis by gas chromatography-mass spectrometry

Matrix interference removal is an important step when large volumes of aqueous samples are required to be processed to detect trace levels of analytes. A combination of two sample extraction methods has been used in this work with the aim of cleanup and preconcentration of analytes. For first objective, mild but preferential sorption of a range of analytes has been performed with in-tube solid-phase microextraction (SPME) using polytetrafluoroethylene (PTFE) tubing, and for the second, the eluate from in-tube SPME was subjected to on-fibre SPME using DVB/Caboxen/PDMS (30/50 μm) fibre. Knitting of PTFE tubing created secondary flow pattern that enhanced radial diffusion and retention of organic analytes. Up to 2 mg L⁻¹ of a broad range of substances that are not extracted by PTFE include nitrogen containing aromatic heterocyclic compounds, anilines, phenols and certain organophosphorus pesticides, thus providing a clean extract using this method of sample preparation. The proposed combination of in-tube and on-fibre SPME produced a rectilinear calibration graph over 0.03-150 μg L⁻¹ of a range of analytes using 60 mL of aqueous sample. The overall recovery of analytes was in the range 27-78%. The detection limits were between 6.1 and 21.8 ng L⁻¹. The R.S.D. was in range 5.4-8.2% and 4.2-6.5% in the analysis of respectively 2 and 20 μg L⁻¹ of analytes.