On-line coupling of sequential injection extraction with restricted-access materials and post-column derivatization for sample clean-up and determination of propranolol in human plasma

The presented paper deals with a new methodology for direct determination of propranolol in human plasma. The methodology described is based on sequential injection analysis technique (SIA) coupled with solid phase extraction (SPE) column based on restricted access materials (RAM). Special RAM column containing 30 μm polymeric material—N-vinylacetamide copolymer was integrated into the sequential injection manifold. SIA–RAM system was used for selective retention of propranolol, while the plasma matrix components were eluted with two weak organic solutions to waste.

Due to the acid–basic and polarity properties of propranolol molecule and principles of reversed-phase chromatography, it was possible to retain propranolol on the N-vinylacetamide copolymer sorbent (Shodex MSpak PK-2A 30 μm (2 mm × 10 mm)). Centrifuged plasma samples were aspirated into the system and loaded onto the column using acetonitrile–water (5:95, v/v), pH 11.00, adjusted by triethylamine. The analyte was retained on the column while proteins contained in the sample were removed to waste. Interfering endogenous substances complicating detection were washed out by acetonitrile–water (15:85), pH 11.00 in the next step. The extracted analyte was eluted by means of tetrahydrofuran–water (25:75), pH 11.00 to the fluorescence detector (emission filter 385 nm). The whole procedure comprising sample pre-treatment, analyte detection and column reconditioning took about 15 min. The recoveries of propranolol from undiluted plasma were in the range 96.2–97.8% for three concentration levels of analyte. The proposed SIA–RAM method has been applied for direct determination of propranolol in human plasma.     

On-line coupling of flow injection sequential extraction to hydride generation atomic fluorescence spectrometry for fractionation of arsenic in soils

A new flow injection on-line sequential extraction procedure coupled with hydride generation atomic fluorescence spectrometry (HG-AFS) was developed for rapid and automatic fractionation of arsenic in soils. The developed methodology involved a three-step sequential extraction procedure with deionized water, KOH solution, and HCl solution. 25 mg of the soil sample packed into a microcolumn (4 mm i.d. × 3 cm long) was dynamically extracted by continuously pumping each individual extractant through the column. The extracted arsenic solution was merged with 4% (m/v) K₂S₂O₈ solution for on-line oxidation of all arsenic species into As^V. The total extracted arsenic was on-line detected by HG-AFS, and quantitated using an on-line standard addition calibration strategy. The total time for the three-step sequential extraction and on-line detection lasted only 10 min. The developed methodology offers several advantages over conventional batch sequential extraction protocols, including minimization of readsorption/redistribution problem, improvement of accuracy, high speed, less amounts of sample/reagents required, less risk of contamination and analyte loss. The developed methodology was successfully applied to the fractionation of arsenic in certified soil reference materials.     

On-line sample-pre-treatment schemes for trace-level determinations of metals by coupling flow injection or sequential injection with ICP-MS

Within the last decade, the first generation of flow injection (FI) has been supplemented by sequential injection (SI), also termed the second generation, and, recently, by the third generation, i.e., SI-Lab-on-Valve (SI-LOV). As apparent from the literature, FI and/or SI have become dominant as substitutes for labor-intensive, manual, sample-pre-treatment and/or solution-handling procedures prior to analyte detection by inductively coupled plasma mass spectrometry (ICP-MS). The present review presents and discusses the progress of the state of the art in implementing miniaturized FI/SI systems for on-line matrix separation and pre-concentration of trace levels of metals with detection by ICP-MS. It highlights some of the frequently applied on-line, sample-pre-treatment schemes, including solid phase extraction (SPE), on-wall molecular sorption and precipitate/(co)-precipitate retention using a polytetrafluoroethylene (PTFE) knotted reactor (KR), solvent extraction-back extraction and hydride/vapor generation. It also addresses a novel, robust approach, whereby the protocol of SI-LOV-bead injection (BI) on-line separation and pre-concentration of ultra-trace levels of metals by a renewable microcolumn is interfaced to ICP-MS, as conducted in the present authors' group. It discusses the future outlook in this field.     

Use of a novel cation-exchange restricted-access material for automated sample clean-up prior to the determination of basic drugs in plasma by liquid chromatography

A new kind of silica-based restricted-access material (RAM) has been tested in pre-columns for the on-line solid-phase extraction (SPE) of basic drugs from directly injected plasma samples before their quantitative analysis by reversed-phase liquid chromatography (LC), using the column switching technique. The outer surface of the porous RAM particlescontains hydrophilic diol groups while sulphonic acid groups are bound to the internal surface, which gives the sorbent the properties of a strong cation exchanger towards low molecular mass compounds. Macromolecules such as proteins have no access to the internal surface of the pre-column due to their exclusion from the pores and are then flushed directly out. The retention capability of this novel packing material has been tested for some hydrophilic basic drugs, such as atropine, fenoterol, ipratropium, procaine, sotalol and terbutaline, used as model compounds. The influence of the composition of the washing liquid on the retention of the analytes in the pre-column has been investigated. The elution profiles of the different compounds and the plasma matrix as well as the time needed for the transfer of the analytes from the pre-column to the analytical column were determined in order to deduce the most suitable conditions for the clean-up step and develop on-line methods for the LC determination of these compounds in plasma. The cationic exchange sorbent was also compared to another RAM, namely RP-18 ADS (alkyl diol silica) sorbent with respect to retention capability towards basic analytes.     

Optimization of a matrix solid-phase dispersion method with sequential clean-up for the determination of alkylphenol ethoxylates in biological tissues

A modified Matrix Solid-Phase Dispersion (MSPD) method with sequential clean-up has been developed to isolate and purify alkylphenol ethoxylates (APEs) and alkylphenols in biological tissues. Elution profile, sequential clean-up adsorbent and experimental set up were optimized. Octadecylsilica was used as the solid-phase for matrix dispersion. Methanol was found to be the optimal eluting solvent for APEs. Aluminium oxide was quite efficient for removing the coeluting interferences. Quantitative analysis was done by reversed-phase HPLC with fluorescence detection. The optimized procedure was applied to analyze both fish and mussel samples. Average recoveries for all spiked tissue samples were greater than 90%. Typical limits of detection amount to tens of ppbs on a wet weight basis.     

Comparison of solid-phase extraction sorbents for sample clean-up in the analysis of organic explosives

In this study, a method was developed for determination of steroid hormones (17beta-estradiol, estrone, 17alpha-ethynylestradiol) in tap and sewage water samples from Sweden. Sample preparation and analysis were performed by a hollow-fibre microporous membrane liquid-liquid extraction (HF-MMLLE) set-up combined with gas chromatography-mass spectrometry (GC-MS). In this approach, only the organic liquid in the lumen (10microL) of the hollow-fibre membrane was utilised for depleting extraction. Several parameters were studied, including: type of organic solvent, sample pH, salt and humic acid content. The optimised method allowed the determination of the analyte at the low ngL(-1) level in tap and sewage water. A linear plot gave correlation coefficients better than 0.995 and resulted in a method limit of detection of 1.6, 3 and 10ngL(-1) for 17beta-estradiol, estrone, and 17alpha-ethynylestradiol, respectively, in sewage water. Enrichment factors were over 1400 after derivatisation. The repeatabilities at 50 and 600ngL(-1) were better than 10% and 6%, respectively.     

On-line monitoring of glucose and penicillin by sequential injection analysis

A sequential injection analysis (SIA) system has been developed for on-line monitoring of glucose and penicillin during cultivations of the filamentous fungus Penicillium chrysogenum. The SIA system consists of a peristaltic pump, an injection valve, two piston pumps, two multi-position valves and a detector. The glucose analyzer is based on an enzymatic reaction using glucose oxidase, which converts glucose to glucono-lactone with formation of hydrogen peroxide and subsequent detection of H₂O₂ by a chemiluminescence reaction involving luminol. The penicillin analysis is based on formation of penicilloic acid by penicillinase. Penicilloic acid is detected either by a chemiluminescence method or by a decolorization method. In the chemiluminescence method the penicilloic acid is quantified by its quenching effect on the chemiluminescence signal obtained when luminol reacts with iodine. In the decolorization method the penicilloic acid is detected spectrophotometrically by the decrease in the absorbance of an iodine-starch complex.     

Loop-column technique: a new sample clean-up method for the determination of gallopamil from biological matrix

Summary A new sample clean-up method for the HPLC determination of gallopamil from biological matrix has been developed. In this method the loop capillary of the injection valve was replaced by a loop column filled with Nucleosil particles. This technique offers reduced extraction time (<1 min) and high sensitivity with a limit of detection as low as 0.1 ng/ml.     

On-line coupling of extraction with gas chromatography

A recent trend in the development of analytical methodologies is the integration of the sample preparation step directly with the chromatographic system. In integrated on-line systems, extraction, clean-up, separation and detection are connected with each other and the whole analytical procedure takes place in a closed, usually automated system. In this review, a critical overview of the principles and benefits of on-line coupling of extraction with gas chromatography is presented. Techniques suitable for the extraction and analysis of various types of compounds in both solid and liquid samples are presented. The potential of on-line techniques is discussed and illustrated with selected examples.     

On-line speciation of bromine and bromide using sequential injection analysis with spectrophotometric detection

An on-line procedure for the simultaneous determination of bromine and total bromine (bromine+bromide oxidised to bromine) is proposed, which lead to the determination of bromide by subtraction. Phenol red was used as chromogenic reagent for bromine and total bromine after bromide was oxidised to bromine by Chloramine T. The linear range found is 1-10 mg L(-1) with a detection limit of 0.6 mg L(-1) for bromine, and a linear range of 0.8-15 mg L(-1) with a detection limit of 0.4 mg L(-1) for total bromine. The calculated RSD for bromine is less than 0.8% and for total bromine less than 0.7%. The system is fully computerised and able to run 30 samples per hour with an automated rinsing step that eliminates sample carry-over. The results for both bromine and bromide from the proposed sequential injection analysis (SIA) system compare favourably with standard manual methods and statistical evaluation proves no significant difference between the results of the proposed SIA system and the standard method at the 95% confidence level. The presence of other halides was found not to interfere.     

On-line dynamic fractionation and automatic determination of inorganic phosphorus in environmental solid substrates exploiting sequential injection microcolumn extraction and flow injection analysis

Sequential injection microcolumn extraction (SI-MCE) based on the implementation of a soil-containing microcartridge as external reactor in a sequential injection network is, for the first time, proposed for dynamic fractionation of macronutrients in environmental solids, as exemplified by the partitioning of inorganic phosphorus in agricultural soils. The on-line fractionation method capitalises on the accurate metering and sequential exposure of the various extractants to the solid sample by application of programmable flow as precisely coordinated by a syringe pump.

Three different soil phase associations for phosphorus, that is, exchangeable, Al- and Fe-bound, and Ca-bound fractions, were elucidated by accommodation in the flow manifold of the three steps of the Hieltjes–Lijklema (HL) scheme involving the use of 1.0 M NH₄Cl, 0.1 M NaOH and 0.5 M HCl, respectively, as sequential leaching reagents. The precise timing and versatility of SI for tailoring various operational extraction modes were utilized for investigating the extractability and the extent of phosphorus re-distribution for variable partitioning times.

Automatic spectrophotometric determination of soluble reactive phosphorus in soil extracts was performed by a flow injection (FI) analyser based on the Molybdenum Blue (MB) chemistry. The 3σ detection limit was 0.02 mg P L⁻¹ while the linear dynamic range extended up to 20 mg P L⁻¹ regardless of the extracting media. Despite the variable chemical composition of the HL extracts, a single FI set-up was assembled with no need for either manifold re-configuration or modification of chemical composition of reagents.

The mobilization of trace elements, such as Cd, often present in grazed pastures as a result of the application of phosphate fertilizers, was also explored in the HL fractions by electrothermal atomic absorption spectrometry.     

Non-particulate (continuous bed or monolithic) restricted-access reversed-phase media for sample clean-up and separation by capillary-format liquid chromatography

Restricted-access reversed-phase non-particulate (continuous bed or monolithic) stationary phases of different hydrophobicity synthesized in 100 m i.d. fused silica capillaries have been evaluated. A specific property of restricted-access media (RAM) is that they interact with small analytes and exclude big molecules, e.g. proteins, from access to the active sites and adsorption on the surface. This dual property facilitates direct injection of biological fluids for drug or drug-metabolite analysis. Different RAM and RAM-precursor capillary columns were tested to assess the influence of chromatographic bed morphology on loadability. Inverse size-exclusion chromatography was used for investigation of pore structural properties of the capillary-format continuous beds. The data obtained were used to discuss the mechanism of separation of the biological samples using capillary columns and to propose a model for the topochemical architecture of the RAM investigated. Different morphology of the non-particulate reversed-phase precursors resulted in two types of RAM material shielded with hydrophilic polymer, classified as homogeneous or heterogeneous topochemistry stationary phases. Capillary columns were applied for chromatography of biological fluids. High resolution was obtained, without the need for column switching, when capillary columns operated in gradient conditions. Extensive evaluation of the chromatographic properties (hydrophobicity, efficiency, separation impedance, and loadability) of the non-particulate reversed-phase materials was performed before and after shielding with hydrophilic polymer to generate restricted-access properties. Minor changes of hydrophobicity, efficiency, or separation impedance were observed after the shielding.     

On-line dilution with sequential injection analysis: a system for monitoring sulphate in industrial effluents

 On-line dilution with sequential injection analysis has been evaluated for sulphate monitoring in industrial effluents using a dilution coil in the conduits of the manifold system and a dilution step as part of the timing sequence. The manifold of the SIA system with the dilution coil is more complex than the system including the dilution step. The former method needs more complicated programming as well. Shorter analysis time favoured the dilution with the dilution step, but the limited linear range of this method is a large drawback. The large linear range of the SIA system with dilution coil makes it very suitable to be used as a process analyser. The proposed SIA system is suitable to monitor sulphate in industrial effluents in the range 50–5000 mg/L with a dilution coil, in the range 50–1000 mg/L with a timing sequence of 6.5 s and in the range 750–5000 mg/L with a timing sequence of 15 s using a dilution step; all three ranges with a RSD <4.5%. Received: 11 March 1996/Revised: 1 July 1996/Accepted: 4 July 1996     

On-line dilution with sequential injection analysis: a system for monitoring sulphate in industrial effluents

 On-line dilution with sequential injection analysis has been evaluated for sulphate monitoring in industrial effluents using a dilution coil in the conduits of the manifold system and a dilution step as part of the timing sequence. The manifold of the SIA system with the dilution coil is more complex than the system including the dilution step. The former method needs more complicated programming as well. Shorter analysis time favoured the dilution with the dilution step, but the limited linear range of this method is a large drawback. The large linear range of the SIA system with dilution coil makes it very suitable to be used as a process analyser. The proposed SIA system is suitable to monitor sulphate in industrial effluents in the range 50–5000 mg/L with a dilution coil, in the range 50–1000 mg/L with a timing sequence of 6.5 s and in the range 750–5000 mg/L with a timing sequence of 15 s using a dilution step; all three ranges with a RSD <4.5%. Received: 11 March 1996/Revised: 1 July 1996/Accepted: 4 July 1996     

New chiral and restricted-access materials containing glycopeptides as selectors for the high-performance liquid chromatographic determination of chiral drugs in biological matrices

Two new chiral and restricted-access materials containing glycopeptide antibiotics as chiral selectors (chiro-Glyco-RAM) were designed, suitable for the direct HPLC injection of biological fluids containing chiral drugs without any sample pre-treatment or pre-columns coupling. The external surface of the porous silica support was covered with a bio-compatible hydrophilic polymeric network (polyvinyl alcohol, PVA) while the chiral phase based on either teicoplanin (TE) or teicoplanin aglycone (TAG) was exclusively confined to the internal region. The chiro-Glyco-RAM supports were synthesized by the following steps: (a) introduction of 3-aminopropyl groups on 100 A pore size silica gel; (b) activation of the aminopropylated silica with 1,6-diisocyanatohexane; (c) functionalization of the external region of the porous silica with PVA; (d) covalent linking of TE/TAG to the internal surface. The average pore diameter of the chiro-Glyco-RAM supports, calculated by inverse size-exclusion chromatography (ISEC), was about 80 A and able to exclude macromolecules heavier than about 20,000 Da (such as the most abundant serum proteins) from the pores. The recovery of bovine serum albumin (BSA) was almost quantitative. HPLC analyses of model chiral drugs were performed using hydro-organic mobile phases consisting of an organic solvent (acetonitrile or methanol) and aqueous solutions of ammonium acetate (0.020 M) or ammonium formate (0.0025-0.0050 M).     

On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples. I. Determination of clenbuterol in urine

The potential of the direct coupling of solid-phase extraction (SPE) with mass spectrometry (MS) for the analysis of biological samples is demonstrated. For SPE a cartridge exchanger is used and the eluate is directly introduced into the mass spectrometer. This system has been investigated for the determination of clenbuterol in urine. With mixed-mode cartridges, a considerable ion suppression has been obtained. The mass spectrum at the elution time of clenbuterol is dominated by that of creatinine and adduct formation of clenbuterol and creatinine has been observed. The whole procedure including injection of 1 ml urine, washing and desorption has been developed with cartridges containing 8-microm C18-bonded silica. If only a single MS step is used, the selectivity and, therefore, the sensitivity are insufficient. The detection limit is about 100 ng/ml. However, with atmospheric pressure chemical ionisation and the tandem MS mode the detection limit has been decreased to about 2 ng/ml and the ion suppression is only about 10%. For the electrospray ionisation the detection limit is about 10-times higher and the ion suppression is less favourable. The repeatability for the SPE-MS-MS procedure was 6.5% at 10 ng/ml (n=5) and the difference between the response factors at 10 ng/ml and 100 ng/ml was only 2.5%. The MS behaviour of clenbuterol and the matrix under the present conditions is discussed.     

Development of sample preparation method for auxin analysis in plants by vacuum microwave-assisted extraction combined with molecularly imprinted clean-up procedure

A novel sample preparation method for auxin analysis in plant samples was developed by vacuum microwave-assisted extraction (VMAE) followed by molecularly imprinted clean-up procedure. The method was based on two steps. In the first one, conventional solvent extraction was replaced by VMAE for extraction of auxins from plant tissues. This step provided efficient extraction of 3-indole acetic acid (IAA) from plant with dramatically decreased extraction time, furthermore prevented auxins from degradation by creating a reduced oxygen environment under vacuum condition. In the second step, the raw extract of VMAE was further subjected to a clean-up procedure by magnetic molecularly imprinted polymer (MIP) beads. Owing to the high molecular recognition ability of the magnetic MIP beads for IAA and 3-indole-butyric acid (IBA), the two target auxins in plants can be selectively enriched and the interfering substance can be eliminated by dealing with a magnetic separation procedure. Both the VMAE and the molecularly imprinted clean-up conditions were investigated. The proposed sample preparation method was coupled with high-performance liquid chromatogram and fluorescence detection for determination of IAA and IBA in peas and rice. The detection limits obtained for IAA and IBA were 0.47 and 1.6 ng/mL and the relative standard deviation were 2.3% and 2.1%, respectively. The IAA contents in pea seeds, pea embryo, pea roots and rice seeds were determined. The recoveries were ranged from 70.0% to 85.6%. The proposed method was also applied to investigate the developmental profiles of IAA concentration in pea seeds and rice seeds during seed germination.     

On-line monitoring of phosphate in natural water and effluent streams using sequential injection analysis

Automation of the molybdenum blue method by sequential injection (SIA) for the on-line monitoring of phosphate in natural waters is presented. Although sequential injection analysis runs at one fifth the speed of conventional FIA, it presents many advantages such as simplicity of the manifold, robustness and computer compatibility. A reduction in reagent and sample consumption is also observed. Flow detector fouling does not occur in SIA manifolds as the detector is in contact with water between analysis. The proposed SIA analyser is able to monitor phosphate in the range 0–70 mg l^–1 with a standard deviation of 0.9%. The detection limit is 0.5 mg l^–1 PO ₄ ^3– .     

On-line coupling of sequential injection lab-on-valve to differential pulse anodic stripping voltammetry for determination of Pb in water samples

Sequential injection lab-on-valve (LOV) was first proposed for analyzing ultra-trace amounts of Pb using differential pulse anodic stripping voltammetry (DPASV) with a miniaturized electrochemical flow cell fabricated in the LOV unit. Deposition and stripping processes took place between the renewable mercury film carbon paste electrode and sample solution, the peak current was employed as the basis of quantification. The mercury film displayed a long-term stability and reproducibility for at least 50 cycles before next renewal, the properties of integrated miniature LOV unit not only enhanced the automation of the analysis procedure but also declined sample/reagent consumption. Potential factors that affect the present procedure were investigated in detail, i.e., deposition potential, deposition time, electrode renewable procedure and the volume of sample solution. The practical applicability of the present procedure was demonstrated by determination of Pb in environmental water samples.