Conventional Solid-phase Extraction Research Articles

Analysis of Biological Fluids by Direct Combination of Solid Phase Extraction and Desorption Electrospray Ionization Mass Spectrometry

A novel, solid phase extraction (SPE)-based sample preparation method was developed for desorption electrospray ionization (DESI) mass spectrometry. Conventional SPE sample preparation was followed by a custom elution procedure. The eluate was evaporated from the closing frit of the cartridge using a gas jet. Thus the analyte was concentrated on the surface of the frit, which is ideal for DESI analysis. Application of the above SPE protocol allowed the concentration of the analyte content of up to 1 L liquid sample into a 1 mm diameter circular spot. The sample preparation procedure can improve the overall sensitivity of the method by up to 6 orders of magnitude if the sample volume is sufficient. The device has been tested using aqueous solutions of Rhodamine 116; the limit of detection was comparable to the LOD of electrospray analysis. Methodology was tested for drug monitoring applications in human serum. Levels of Cyclosporine A were determined using a 0.1 mL serum sample. Dynamic range of the method exceeded 3 orders of magnitude; the detection limit was below the therapeutic serum concentration of the drug.

Validated gas chromatographic–mass spectrometric analysis of urinary cannabinoids purified with a calcium-hardened β-cyclodextrin polymer

A comprehensive solid-phase extraction (SPE) technique based on the formation of an inclusion complex between β-cyclodextrin (βCD) and cannabinoids including Δ 9-tetrahydrocannabinol (THC), 11-hydroxy-Δ 9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ 9-tetrahydrocannabinol (THC-COOH) was developed in gas chromatographic–mass spectrometric (GC–MS) analysis. A βCD/epichlorohydrin copolymer was prepared and then ‘hardened’ in aqueous solution with 0.3 M CaCl 2 to yield a stable particulate copolymer, which was used as a novel SPE sorbent. An internal standard THC-COOH- d 9 was added to urine samples containing 3 cannabinoids and then purified with the hardened βCD polymer. The cannabinoids were extracted from the hardened βCD using tetrahydrofuran. Resulting extracts were evaporated and derivatized with MSTFA/NH 4I/dithioerythritol (500:4:2, v/w/w) and analyzed by GC–MS in selected-ion monitoring (SIM) mode. Overall recoveries ranged from 85% to 102%, with a detection limit of 0.2 μg L −1 for the three cannabinoids tested. The precision (% CV) and accuracy (% bias) of the assay were 1.2–5.1% and 93–111% in 0.2–50 μg L −1 calibration range, respectively ( r 2 > 0.9997). Twenty actual samples positive by fluorescence polarization immunoassay were also quantitatively analyzed. The devised technique based on the calcium-hardened βCD sorption of cannabinoids and subsequent GC–SIM/MS resulted in better selectivity and extraction efficiency than is possible using the conventional hydrophobicity-based SPE methods.

Monolithic solid-phase extraction for the rapid on-line monitoring of microcystins in surface waters

We describe the development and optimization of a sensitive and selective screening method for the measurement of trace levels of microcystins in surface waters. Several sample preparation techniques were compared, including solid-phase microextraction (SPME), particle-based solid-phase extraction (SPE), and monolith-based SPE. A flow-injection (FI) based approach employing a reversed-phase monolithic SPE column was found to be optimal. Quantification was performed by directly interfacing the FI-based SPE system to an electrospray ionization-mass spectrometer (ESI-MS). To more safely simulate peptidyl toxins such as the microcystins, a model peptide (i.e., angiotensin II) was used for method optimization. Sample loading flow rate and volume, eluent composition, and elution flow rate were optimized. Sample throughput was six samples per hour, a detection limit of 1.31 ng angiotensin II was demonstrated for a linear dynamic range from 1–1,000 ng and 3.4% relative standard deviation (n = 4, 100 ng sample). Sample volumes up to 1,000 ml of surface water could be loaded onto the monolithic SPE disk without exceeding the sorbent’s capacity. Unlike conventional particle-based SPE methods, the monolithic SPE disk does not need to be replaced between samples and could be used indefinitely. The FI-based SPE-ESI-MS method was successfully applied to the determination of microcystin-LR, the most common of the microcystins, in environmental samples and was demonstrated for the direct monitoring of chlorinated drinking water, with trends tracked over a period of eight months.

New method for the analysis of heterocyclic amines in meat extracts using pressurised liquid extraction and liquid chromatography–tandem mass spectrometry

Heterocyclic amines (HAs) were analysed in meat extract samples using a new method based on pressurised liquid extraction (PLE) and liquid chromatography–tandem mass spectrometry. This method combines the use of a pressurised fluid with a triple quadrupole MS/MS system, resulting in benefits from both systems: high extraction efficiency and sensitivity. The effects of solvent type and PLE operational parameters, such as temperature and extraction time, were studied to obtain maximum recovery of the analytes with minimum contamination. HA extraction was best achieved using dichloromethane/acetone (50/50, v/v) at 80 °C for 10 min. Recoveries ranged from 45% to 79% with good quality parameters: limit of detection values between 0.02 and 1 ng g −1, linearity ( r 2 > 0.997), and run-to-run and day-to-day precisions with relative standard deviations lower than 13% achieved at both low (0.20 μg g −1) and medium (1.0 μg g −1) concentrations. This method reduces sample manipulation and total extraction time by nearly four-fold compared to conventional solid phase extraction. The optimised method was validated using laboratory reference material based on a meat extract, and was successfully applied to HA analysis in several cooked beef samples.

Alternative chromatographic processes for no-carrier added 177Lu radioisotope separation

The conventional multi-column solid phase extraction (SPE) chromatography technique using di-(2-ethylhexyl)orthophosphoric acid (HDEHP) impregnated OASIS-HLB sorbent based SPE resins (OASIS-HDEHP) was developed for the separation of no-carrier added (n.c.a) 177 Lu from the bulk quantity of ytterbium target. This technique exploited the large variation of lutetium metal ion distribution coefficients in the varying acidity of the HCl solution–OASIS-HDEHP resin systems for the consecutive loading-eluting cycles performed on different columns. The production batches of several hundred mCi n.c.a 177 Lu radioisotope separated from 50 mg Yb target activated in a nuclear reactor of medium neutron flux (Φ = 5 . 10 13 n . cm –2. s –1 ) were successfully performed using the above mentioned separation technique. With the target irradiation in a reactor of thermal neutron flux Φ = 2 . 10 14 n . cm –2. s –1 or the parallel run of several separation units , many Ci-s of n.c.a 177 Lu can be profitably produced. The OASIS-HDEHP resin based multi-column SPE chromatography technique makes the separation process simple and economic and offers an automation capability for operation in highly radioactive hazardous environments.

Alternative chromatographic processes for no-carrier added 177Lu radioisotope separation

HPLC technique combined with the simple conventional column solid phase extraction (SPE) chromatography using di-(2ethylhexyl)orthophosphoric acid (HDEHP) impregnated OASIS-HLB sorbent based SPE resins (OASIS-HDEHP ) was developed for the separation of no-carrier added (n.c.a) 177 Lu from the bulk quantity of ytterbium target. This combination strategy was based on combining the advantages of the better resolution of HPLC separation of n.c.a 177 Lu from the few milligram level Yb target with the high capacity of the OASIS-HDEHP column for the separation of 177 Lu from the bulk Yb target. The production batches of several hundred mCi activity of n.c.a 177 Lu radioisotope separated from 50 mg Yb target activated in a nuclear reactor of medium neutron flux (Φ = 5·10 13 n·cm -2 ·s -1 ) were successfully performed using this combined separation technique. With the target irradiation in a reactor of higher thermal neutron flux or with the parallel run of several separation units, several Ci-s of n.c.a 177 Lu can be profitably produced on a commercial production basis.

Analysis of trace contamination of phthalate esters in ultrapure water using a modified solid-phase extraction procedure and automated thermal desorption–gas chromatography/mass spectrometry

The present study was aimed to develop a procedure modified from the conventional solid-phase extraction (SPE) method for the analysis of trace concentration of phthalate esters in industrial ultrapure water (UPW). The proposed procedure allows UPW sample to be drawn through a sampling tube containing hydrophobic sorbent (Tenax TA) to concentrate the aqueous phthalate esters. The solid trap was then demoisturized by two-stage gas drying before subjecting to thermal desorption and analysis by gas chromatography–mass spectrometry. This process removes the solvent extraction procedure necessary for the conventional SPE method, and permits automation of the analytical procedure for high-volume analyses. Several important parameters, including desorption temperature and duration, packing quantity and demoisturizing procedure, were optimized in this study based on the analytical sensitivity for a standard mixture containing five different phthalate esters. The method detection limits for the five phthalate esters were between 36 ng l −1 and 95 ng l −1 and recovery rates between 15% and 101%. Dioctyl phthalate (DOP) was not recovered adequately because the compound was both poorly adsorbed and desorbed on and off Tenax TA sorbents. Furthermore, analyses of material leaching from poly(vinyl chloride) (PVC) tubes as well as the actual water samples showed that di- n-butyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) were the common contaminants detected from PVC contaminated UPW and the actual UPW, as well as in tap water. The reduction of DEHP in the production processes of actual UPW was clearly observed, however a DEHP concentration of 0.20 μg l −1 at the point of use was still being quantified, suggesting that the contamination of phthalate esters could present a barrier to the future cleanliness requirement of UPW. The work demonstrated that the proposed modified SPE procedure provided an effective method for rapid analysis and contamination identification in UPW production lines.

Application of Porous Membrane-Protected Micro-Solid-Phase Extraction Combined with HPLC for the Analysis of Acidic Drugs in Wastewater

This report describes the use of a porous membrane-protected micro-solid-phase extraction (micro-SPE) procedure to extract acidic drugs from wastewater that are then determined by high-performance liquid chromatography with ultraviolet detection. The micro-SPE device consists of C18 sorbent held within a membrane envelope made of polypropylene. Ketoprofen and ibuprofen were selected as model compounds, and extraction parameters were optimized. Correlation coefficients of 0.9980 and 0.9953 were obtained for ketoprofen and ibuprofen, respectively, across a concentration range of 1-250 microg/L. Relative extraction recoveries were between 94 and 112%. The relative standard deviation of the analytical method ranged between 2 and 10%, respectively. The method detection limits for these target analytes in wastewater ranged from 0.03 to 0.08 microg/L. When compared to conventional solid-phase extraction (SPE), this new method showed better detection limits with good reproducibility. The results shows that this micro-SPE technique is a feasible alternative to multistep SPE for the extraction of analytes in complex samples.

Extraction of alkyl methylphosphonic acids from aqueous samples using a conventional polymeric solid-phase extraction sorbent and a molecularly imprinted polymer

The analysis of alkyl methylphosphonic acids (AMPAs) constitutes an important subject for verifying the compliance to the Chemical Weapons Convention (CWC). Indeed, alkyl methylphosphonic acids are the degradation products of V and G nerve agents such as VX, sarin or soman. Lowering the limits of detection of analytical methods for complex aqueous matrices implies the introduction of concentration and clean-up steps in the whole analytical process. Therefore a molecular imprinted polymer (MIP) has been previously developed for the selective extraction and the concentration of the alkyl methylphosphonic acids. Unfortunately, the selective retention process on this MIP has involved the development of hydrogen bonds and so does not allow the direct percolation of aqueous samples. A change of solvent is then necessary and can be performed using solid-phase extraction (SPE) with conventional non selective hydrophobic sorbents. Two polymeric sorbents, Oasis HLB and HR-P resins, were selected for their high specific surface area. The extraction recoveries obtained on both sorbents were compared and the Oasis HLB sorbent was further selected and used for the percolation of acidified aqueous samples. An optimised SPE procedure was then applied to concentrate an aqueous soil extract spiked with isobutyl methylphosphonic acid (iBMPA) and cyclopentyl methylphosphonic acid (cPMPA) that was further cleaned-up by passing through the MIP. The resulting LC–MS full scan chromatograms highlight the clean-up effect of the SPE-MIP association by the removal of the matrix substances and the preservation of 95% of the compounds of interest.

Drug Monitoring and Toxicology: A Procedure for the Monitoring of Oxcarbazepine Metabolite by HPLC-UV

This article describes a rapid high-performance liquid chromatographic (HPLC) method for the measurement of the primary metabolite of oxcarbazepine. Following a simple precipitation step, 10,11,-dihydro-10-hydroxy-5H-dibenzo(b,f)azepine-5-carboxamide is quantitated (5-60 microg/mL) by analysis on an HPLC-UV system. The instrument time is less than 5 min per injection, an improvement over most published methods. The assay's limit of quantitation, linearity, imprecision, and accuracy adequately cover the therapeutic range for appropriate patient monitoring. In comparison to other published methods, this procedure would be of interest to clinical laboratories because it employs a precipitation step for sample preparation, instead of conventional yet time-consuming solid-phase extraction.

Determination of antihyperglycemic biguanides in serum and urine using an ion-pair solid-phase extraction technique followed by HPLC-UV on a pentafluorophenylpropyl column and on an octadecyl column

An HPLC-UV method was established for the determination of metformin and buformin in biological fluids. Metformin was not retained on particles packed in conventional solid-phase extraction cartridges; in contrast, buformin was retained too firmly and not eluted with a solvent for recovery. However, both drugs were retained on particles that had been treated with an ion-pair reagent of heptanesulfonate or dodecylsulfate and recovered almost completely. The recovered fraction was subjected to HPLC on a pentafluorophenylpropyl column which was suitable for the determination of both biguanides in serum and in urine. Limits of quantitation were low enough for clinical use, and reproducibility was high with an RSD of 0.9-2.3%. HPLC on a conventional octadecyl column was suitable only for the determination of buformin in serum since interfering peaks appeared on the chromatograms of urine samples. The method was applied to analysis of some clinical specimens.

Use of SPME extraction to determine organophosphorus pesticides adsorption phenomena in water and soil matrices

Solid-phase micro extraction (SPME) coupled with GC enables rapid and simple analysis of organophosphorus pesticides in a range of complex matrices. Investigations were made into the extraction efficiencies from water of six organophosphorus insecticides (methamidophos, omethoate, dimethoate, parathion methyl, malathion, and parathion ethyl) showing a wide range of polarities. Three SPME fibres coated with different stationary phases, polydimethylsiloxane, polyacrylate, and carbowax-divinylbenzene (CW-DVB), were investigated. Water was spiked with the pesticides at concentrations from 1 to 0.01 µg mL-1, and the solutions used for optimization of the procedure. The CW-DVB fibre, with a 65 µm coating, gave the best performance. The optimized experimental conditions were sample volume 10 mL at 20°C, equilibration time 16 min, pH 5, and presence of 10% w/v NaCl. SPME analyses were performed on solutions obtained by equilibrating aqueous pesticide solutions with six certified soils with various physico-chemical characteristics. SPME data were also assessed by comparison with analyses performed by using conventional solid-phase extraction. Results indicate the suitability of SPME for analysis of pesticides in environmental water samples.

Quantitative determination of piritramide in human plasma and urine by off‐ and on‐line solid‐phase extraction liquid chromatography coupled to tandem mass spectrometry

A method for the liquid chromatography/tandem mass spectrometric (LC/MS/MS) quantification of piritramide, a synthetic opioid, in plasma after conventional off-line solid-phase extraction (SPE) and in urine by on-line SPE-LC/MS/MS in positive electrospray mode was developed and validated. Applicability of the on-line approach for plasma samples was also tested. Deuterated piritramide served as internal standard. For the off-line SPE plasma method mixed cation-exchange SPE cartridges and a 150 x 2 mm C18 column with isocratic elution were used. For the on-line SPE method, a Waters Oasis HLB extraction column and the same C18 analytical column in a column-switching set-up with gradient elution were utilized. All assays were linear within a range of 0.5-100 ng/mL with a limit of detection of 0.05 ng/mL. The intra- and interday coefficients of variance ranged from 1.3 to 6.1% for plasma and 0.5 to 6.4% for urine, respectively. The extraction recovery for the off-line plasma assay was between 90.7 and 100.0%. Influence of matrix effects, and freeze/thaw and long-term stability were validated for both approaches; influence of urine pH additionally for quantification in urine.

Cyanobacteria and microcystins occurrence in the Guadiana River (SW Spain)

This work reports on the survey carried out to determine the presence of microcystins (MCs) by using ELISA tests and HPLC-UV for such determination in different sample sites along the Spanish course of the Guadiana River. The most important cyanobacteria species identified were, Microcystis aeruginosa and Oscillatoria spp. The highest total microcystin content recorded was 6.40 µg L−1 in 2002. The main toxins found were microcystins RR and LR, with microcystin YR present at trace levels. Improvements in sample clean up were carried out by using Immunoaffinity solid phase extraction (SPE) and its advantages regarding to conventional SPE were clearly demonstrated. The confirmation of MCs presence in the evaluated water reservoirs, underlines the necessity of monitoring programs as well as the improvement of analytical methodologies to efficiently prevent the human health risks as a consequence of MCs contamination.

Selective Quantification of Trace Palladium in Road Dusts and Roadside Soils by Displacement Solid-Phase Extraction Online Coupled with Electrothermal Atomic Absorption Spectrometry

There is a growing concern about the effect of palladium on human health because of the toxicity and increasing occurrence of palladium as a result of its extensive use in automotive catalytic converters. Development of reliable analytical methodologies for the determination of palladium in environmental materials is of great importance for critical evaluation of the possible risks for human health. In this work, a displacement solid-phase extraction technique was developed and online coupled to electrothermal atomic absorption spectrometry (ETAAS) for selective and sensitive determination of trace palladium in environmental samples without need of any special selective complexing agents, selective sorbents, and masking agents. The developed methodology involved the online formation of copper pyrrolidine dithiocarbamate (Cu-PDC), and the resultant Cu-PDC was extracted onto a microcolumn packed with the sorbent from a cigarette filter. Trace Pd(II) was selectively preconcentrated through loading the sample solution onto the microcolumn by online displacement reaction between Pd(II) and the extracted Cu-PDC on the microcolumn. The retained analyte was subsequently eluted with 40 microL of ethanol for online ETAAS determination. Interferences from coexisting heavy metal ions with lower stability of their PDC complexes relative to Cu-PDC were minimized. The tolerable concentrations of Cd-(II), Fe(III), Co(II), Mn(II), Cr(III), and Zn(II) were up to 2, 6, 40, 2, 1.5, and at least 100 mg L(-1), respectively. Compared with conventional solid-phase extraction methodology, the developed displacement solid-phase extraction protocol gave 2-4 orders of magnitude improvement in the maximum tolerable concentrations of coexisting heavy metal ions. With the consumption of only 2.8 mL of sample solution, an enhancement factor of 52 and a detection limit (3sigma) of 18 ng L(-1) were achieved at a sample throughput of 30 samples h(-1). The precision (RSD, n = 13) was 2.5% at the 1 microg L(-1) level. The present methodology was successfully applied to selective determination of trace palladium in local road dusts and roadside soils.

Determination of explosives in environmental water samples by solid-phase microextraction–liquid chromatography

When explosives are present in natural aqueous media, their concentration is usually limited to trace levels. A preconcentration step able to remove matrix interferences and to enhance sensitivity is therefore necessary. In the present study, we evaluated solid-phase microextraction (SPME) technique for the recovery of nine explosives from aqueous samples using high-performance liquid chromatography with ultraviolet detection (HPLC-UV). Several parameters, including adsorption and desorption time, coating type, rate of stirring, salt addition, and pH, were optimized to obtain reproducible data with good accuracy. Carbowax coating was the only adsorbent found capable of adsorbing all explosives including nitramines. Method detection limits (MDL) were found to range from 1 to 10 μg/L, depending on the analyte. SPME/HPLC-UV coupling was then applied to the analysis of natural ocean and groundwater samples and compared to conventional solid-phase extraction (SPE/HPLC-UV). Excellent agreement was observed between both techniques, but with an analysis time around five times shorter, SPME/HPLC-UV was considered to be applicable for quantitative analysis of explosives.

Molecularly imprinted polymers for the determination of a pharmaceutical development compound in plasma using 96-well MISPE technology

The use of molecularly imprinted polymers (MIPs) as sorbents for the solid phase extraction (SPE) of a pharmaceutical compound in development, prior to quantitative analysis was investigated. Three MIPs were synthesised using a structural analogue as the template molecule. Each polymer was prepared with different monomers and porogens. The MIPs were then tested for their performance both in organic and aqueous environments, the final aim being to load plasma directly onto the polymers. At an early development stage, there is a limited amount of compound available. Due to this limitation, reducing the amount of template required for imprinting was investigated. A MIP capable of extracting the analyte directly from plasma was produced. The specificity of the polymer allowed the method to be validated at a lower sensitivity than a more conventional SPE assay. For the first time, MIPs were packed into 96-well blocks enabling high throughput analysis. The analytical method was fully validated for imprecision and inaccuracy down to 4 ng/ml in plasma.

Feasibility of a liquid-phase microextraction sample clean-up and liquid chromatographic/mass spectrometric screening method for selected anabolic steroid glucuronides in biological samples.

Anabolic androgenic steroids (AAS) are metabolized extensively in the human body, resulting mainly in the formation of glucuronide conjugates. Current detection methods for AAS are based on gas chromatographic/mass spectrometric (GC/MS) analysis of the hydrolyzed steroid aglycones. These analyses require laborious sample preparation steps and are therefore time consuming. Our interest was to develop a rapid and straightforward method for intact steroid glucuronides in biological samples, using liquid-phase microextraction (LPME) sample clean-up and concentration method combined with liquid chromatographic/tandem mass spectrometric (LC/MS/MS) analysis. The applicability of LPME was optimized for 13 steroid glucuronides, and compared with conventional liquid-liquid extraction (LLE) and solid-phase extraction (SPE) procedures. An LC/MS/MS method was developed for the quantitative detection of AAS glucuronides, using a deuterium-labeled steroid glucuronide as the internal standard. LPME, owing to its high specificity, was shown to be better suited than conventional LLE and SPE for the clean-up of urinary AAS glucuronides. The LPME/LC/MS/MS method was fast and reliable, offering acceptable reproducibility and linearity with detection limits in the range 2-20 ng ml(-1) for most of the selected AAS glucuronides. The method was successfully applied to in vitro metabolic studies, and also tested with an authentic forensic urine sample. For a urine matrix the method still has some unsolved problems with specificity, which should be overcome before the method can be reliably used for doping analysis, but still offering additional and complementary data for current GC/MS analyses.

Fiber-in-tube solid-phase extraction using a polyetheretherketone capillary, and effective on-line coupling with microcolumn liquid chromatography

On-line coupling of fiber-in-tube solid-phase extraction (SPE), using polyetheretherketone (PEEK) tubing, with microscale liquid chromatography (LC) has been investigated. PEEK tubing and polymer filaments are introduced as new medium for extraction of phthalates—diethyl phthalate (DEP), dipropyl phthalate (DPP), and dibutyl phthalate (DBP). Approximately 300 pieces of polymer filament were packed into a PEEK capillary 100 mm long (0.25 mm i.d.) and the capillary was installed in the injection valve as the sample loop. Desorption and injection of the analytes were performed simultaneously withoutuse of the desorption solvent required by conventional SPE methods. The results obtained showed that effective on-line coupling of this sample-preparation method with liquid chromatography and other liquid-phase separation techniques can be accomplished for the high-throughput analysis of complex mixtures, without large consumption of organic solvents. The limits of detection for these phthalates, approximately 1.0 ng mL−1, and the extraction time, 10 min, indicate the future possibilities of this technique.

Gas chromatographic determination of 2-hydroxy-4-methoxybenzophenone and octyldimethyl- p-aminobenzoic acid sunscreen agents in swimming pool and bathing waters by solid-phase microextraction

A method has been developed for the trace determination of two sunscreen constituents (2-hydroxy-4-methoxybenzophenone and octyldimethyl- p-aminobenzoic acid) in water samples, which are commonly used in commercial formulations. The method employs solid-phase microextraction (SPME) and gas chromatography with flame ionization and mass spectrometric detection. The technique was developed with headspace and direct sampling in order to demonstrate the applicability of these SPME extraction modes for the identification of these two UV absorbing compounds in waters. The main parameters affecting the SPME process, such as desorption time, extraction time profile, salt additives, pH, and temperature, were investigated. The poly(dimethylsiloxane) 100-μm and polyacrylate 85-μm fiber coatings were found to be the most efficient for the extraction of these compounds from aqueous matrices. Linear calibration curves in the wide range of 10–500 μg/l were obtained for both compounds yielding typical RSD values of 5–9% for both extraction modes. The recoveries were relatively high, 82–98%, with quantitation limits below 1 μg/l. A comparison between the proposed methods and the conventional multiresidue solid-phase extraction revealed that the proposed technique(s) can be reliably used for sunscreen residue measurement in water samples with satisfactory results.