On-line Sample Preparation Research Articles

Quantification of multiple DNA adducts formed through oxidative stress using liquid chromatography and electrospray tandem mass spectrometry.

Damage to DNA can arise through covalent modification of bases by reaction with oxidants and products of lipid peroxidation derived through normal aerobic metabolism. Such premutagenic lesions, including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), pyrimido[1,2alpha]purine-10(3H)one-2'-deoxyribose (M1-dG), etheno-2'-deoxyadenosine (epsilon-dA), and etheno-2'-deoxycytidine (epsilon-dC), are believed to be important in the development of human cancers related to diet, disease states, and lifestyle. We report the development of a method for concurrent quantification of these four adducts in DNA hydrolysates of 100 microg or less using on-line sample preparation coupled with liquid chromatography and tandem mass spectrometry. The sensitive detection permitted adduct quantification at levels below one adduct in 10(8) normal nucleotides and measurement of these adducts in DNA from untreated rodent liver and normal human liver samples. This methodology should prove useful in hypothesis-driven studies of cancer etiology in laboratory animals and humans.

Determination of lonazolac and its hydroxy and O-sulfated metabolites by on-line sample preparation liquid chromatography with fluorescence detection

A reliable method, which can be used for the determination of lonazolac and its hydroxylated and O-sulfated metabolites in cell culture media with methyllonazolac as the internal standard is described. The procedure employs on-line sample enrichment using a BioTrap 500 MS™ (20×4 mm I.D.) extraction pre-column and subsequent gradient separation on an Xterra MS C 18-H™ (100×3 mm I.D., 3.5 μm particles) analytical column in the back-flush mode. Signal monitoring was done by measurement of fluorescence responses at 273 nm for excitation and 385 nm for emission. Structural identity of analyte peaks was confirmed by liquid chromatography coupled to mass spectroscopy (LC–MS–MS) using an electrospray ionization (ESI) source in the selected reaction monitoring (SRM) mode. Mean recoveries of lonazolac, hydroxylonazolac and lonazolac sulfate, respectively, from the biological matrix were 104.2±3.5, 96.7±2.2, and 100.9±3.5%. The limit of detection (LOD) for the three compounds was about 5 ng/ml using a total sample volume of only 50 μl. Linearity of signal responses versus concentration for all three analytes was accomplished in the range 10–600 ng/ml. The mean values of the coefficients of variation (C.V.) for quality control samples measured in duplicate at three different days at the 10, 40, 100, and 400 ng/ml level were 4.46±1.15, 3.94±2.13 and 4.79±2.07% for lonazolac, hydroxylonazolac and lonazolac sulfate. The target analytes were sufficiently stable at both storage and sample preparation conditions because no substantial deviations between analyte concentrations measured before and after subsequently performed freeze and thaw cycles were observed.

Microfluidic components for protein characterization

The use of microfluidic components to create an analytical toolbox for the very rapidly growing field of proteomics is described. This toolbox provides novel generic analytical solutions that are highly adaptable for analysis of various biomolecules, ranging from high to low abundant. The components are fabricated using silicon micromachining and consist of a microchip immobilised enzyme reactor (μIMER), a piezoelectric microdispenser and high-density nanovial target plates. This microtechnology based platform interfaces matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF-MS) to a wide range of upstream sample handling and/or analytical techniques. Examples of applications such as rapid on-line digestion (12 s) and sample preparation of proteins, interfacing to capillary liquid chromatography (100 attomol sensitivity), and in-vial chemistry on femtomol amounts of sample are presented.

Strategy for the development of automated methods involving dialysis and trace enrichment as on-line sample preparation for the determination of basic drugs in plasma by liquid chromatography

Among the sample preparation techniques, dialysis followed by clean-up and enrichment of the dialysate on a pre-column has proved to be a useful approach for the LC determination of drugs in plasma. By use of sample processors, like the ASTED system, such bioanalytical methods can be fully automated, the dialysis and trace enrichment steps being directly coupled to LC. In order to facilitate the development of such automated methods, a strategy based on a decision tree has been elaborated. After the selection of appropriate conditions for the LC analysis, the decision tree provides information about suggested starting conditions and guidelines for the optimisation of the most important parameters likely to influence analyte recovery and method selectivity. The plasma samples are dialysed on a cellulose acetate membrane in the static-pulsed mode and the dialysate is enriched on a trace enrichment pre-column packed with octadecyl silica or with a strong cation-exchange material. This decision tree is until now restricted to the analysis of basic drugs in plasma. In order to demonstrate the applicability of this method development strategy, an automated procedure based on the coupling of dialysis with trace enrichment has been developed for the LC determination of antifungal agents (clotrimazole, econazole and miconazole) in plasma.

Catalytic reductions in membrane extraction as sample preparation for gas chromatographic analysis

The paper presents a preliminary study of membrane extraction combined with catalytic reaction in the membrane. Pt catalyst was impregnated into Nafion and silicone hollow fibre membranes. The membranes were then connected to a hydrogen carrier gas feed line and the Pt(IV) was reduced to Pt black in a few minutes. Ethanol and nitrobenzene were used as model compounds for extraction and simultaneous reduction during permeation through the membranes. Such a membrane reactor can be connected with a MESI system. This initial work shows the potential for introducing a catalytic reaction into a membrane extraction procedure for on-line continuous sample preparation.

Catalytic reductions in membrane extraction as sample preparation for gas chromatographic analysis

The paper presents a preliminary study of membrane extraction combined with catalytic reaction in the membrane. Pt catalyst was impregnated into Nation and silicone hollow fibre membranes. The membranes were then connected to a hydrogen carrier gas feed line and the Pt(IV) was reduced to Pt black in a few minutes. Ethanol and nitrobenzene were used as model compounds for extraction and simultaneous reduction during permeation through the membranes. Such a membrane reactor can be connected with a MESI system. This initial work shows the potential for introducing a catalytic reaction into a membrane extraction procedure for on-line continuous sample preparation.

On-line Sample Preparation for High Throughput Reversed-phase LC / MS Analysis of Combinatorial Chemistry Libraries.

An on-line sample preparation method utilizing a time-programmed autosampler is described for high throughput liquid chromatography/mass spectrometry (LC/MS). This approach is particularly helpful for the LC/MS analysis of samples which require solvents incompatible with HPLC in the sample preparation process. The on-line sample preparation approach minimizes a bottleneck in throughput and improves sample recovery under some circumstances.

COMPARISON OF TWO DIFFERENT APPROACHES OF SAMPLE PRETREATMENT FOR STEREOSELECTIVE DETERMINATION OF (R,S)-PROPRANOLOL IN HUMAN PLASMA

A column-switching chromatographic system using the teicoplanin chiral stationary phase for stereoselective determination of the β-blocker (R,S)-propranolol in human plasma has been developed and validated. A conventional, off-line liquid–liquid extraction procedure was used for pretreatment of plasma samples and compared to an on-line sample preparation method employing a special type of packing material (LiChrospher RP-8 ADS). The HPLC-integrated sample pretreatment (on-line liquid–solid extraction) allows higher precision, accuracy, and sensitivity. Detailed validation parameters are discussed.

Quantification of etheno-DNA adducts using liquid chromatography, on-line sample processing, and electrospray tandem mass spectrometry.

Etheno-DNA adducts are promutagenic lesions present in normal animal and human tissues that are believed to be important in the etiology of cancer related to diet and lifestyle. A method has been developed for the quantification of trace levels of etheno-DNA adducts using on-line sample preparation coupled with liquid chromatography and electrospray tandem mass spectrometry. The use of automated solid-phase extraction and stable labeled internal standards permitted the robust determination of ethenodeoxyadenosine contained in crude DNA hydrolysates from untreated rodent and human tissues at levels on the order of one adduct in 10(8) normal nucleotides from 100 microg of DNA. Inherent analyte response and matrix interference made sensitivity for simultaneous determination of ethenodeoxycytidine approximately 5-fold lower. The method was applied to the analysis of liver DNA from untreated and urethane-treated B6C3F1 mice, untreated rat liver, human placenta, and several commercial DNA preparations. Some sources of potential artifactual formation of etheno-DNA adducts were investigated.

On-line Sample Preparation System Using Column-Switching HPLC for the Structure Elucidation of Compounds in Mixtures by NMR

A new on-line sample preparation system using column-switching HPLC was developed for the structure elucidation of compounds in mixtures by NMR. This system consists of three HPLC sections, separating the compounds of interest (LC-1), trapping the separated compounds on a trapping column using H2O and then replacing H2O with D2O (LC-2), and eluting the compounds from the trapping column using a deuterated solvent for an NMR measurement (LC-3). The system allows easy separation of a trace amount of the compounds from a mixture, desalting and concentration of the separated compounds, and the use of a deuterated solvent, such as CD3OD, for an NMR measurement. It was successfully applied to the structure elucidation of (±)-propranolol hydrochloride and a mixture of vitamin E derivatives as models. This new tool should be of considerable value in various fields for rapid sample preparation for NMR measurements.

In-tube solid-phase microextraction coupled to capillary LC for carbamate analysis in water samples.

Recently, the on-line sample preparation technique, intube solid-phase microextraction (SPME), was successfully implemented with a Hewlett-Packard 1100 HPLC system for analysis of carbamates in water samples. This paper describes the coupling of in-tube SPME to capillary LC and explores its utility as a sample preparation method in that format, relative to conventional LC. The Hewlett-Packard HPLC system was upgraded to a capillary LC system using commercially available accessories from LC Packings. The combination of in-tube SPME with a capillary LC system was expected to build on the merits of both in-tube SPME and the capillary LC to generate a sensitive method with an easy, effective, and efficient sample preparation. Due to the relatively large effective injection volume of the in-tube SPME technique (30-45 microL), on-column focusing was employed in order to achieve good chromatographic efficiency. Excellent sensitivity was achieved with very good method precision. For all carbamates studied, the RSD of retention time was between 0.5 and 0.8% under 4 microL/min microgradient conditions. The RSD of peak area counts was between 1.5 and 4.6%. The detection limits for all carbamates studied were less than 0.3 microg/L and, for carbaryl, just 0.02 microg/L (20 ppt). Compared with the conventional in-tube SPME/LC method, the LODs were lowered for carbaryl, propham, methiocarb, promecarb, chlorpropham, and barban, by factors of 24, 45, 42, 81, 62, and 56, respectively. The optimized method was successfully applied to the analysis of carbamates in surface water samples.

On-line sample preparation using restricted-access media in the analysis of the soy isoflavones, genistein and daidzein, in rat serum using liquid chromatography electrospray mass spectrometry.

Soy isoflavones are the subject of many investigations in experimental animals and humans regarding possible modulation of endocrine activity and chemoprevention of carcinogenesis. Genistein and daidzein, the principal biologically active isoflavones in soy, were measured using on-line solid-phase extraction (SPE) and liquid chromatography electrospray mass spectrometry (LC/ES-MS) detection in serum of rats consuming a common open-formula (NIH 31) chow that contained approximately 30 microg each of genistein and daidzein per gram of feed and a specially designed 'soy-free' chow that contained approximately 60-fold lower isoflavones. The use of a restricted-access/reverse phase trap cartridge and automated column switching permitted rapid and robust analytical performance with many injections of plasma onto a reverse phase LC column. Enzymatic deconjugation and a single centrifugation step were the only sample preparation steps required. The limit of detection for the isoflavones, based on the MS responses observed in serum from male and female rats consuming the soy-free chow, was 0.020 microM. The method, which uses deuterated isoflavones as internal standards, was determined to be accurate using spiked control serum (102-110% of added amounts) and precise using spiked control serum and incurred serum (<6% relative standard deviation). The average genistein and daidzein levels were determined in female (0.62 and 0.25 microM, respectively) and male rats (0.35 and 0.20 microM, respectively) consuming the standard diet. The sex difference observed for serum genistein concentrations was statistically significant (p < 0.0001). These results underscore the potential impact of standard open-formula diets on the results from rodent bioassays of biological activity.

On-line D/H analysis for water, natural gas, and organic solvents by manganese reduction

A new technique for on-line sample preparation and D/H determination is described. The technique is suitable for the preparation of fresh and brine waters, as well as natural gases and organic solvents. A 5-microL sample of water or hydrogen equivalent is injected and reduced by means of hot manganese metal in a specially designed reaction tube surrounded by a tube furnace and attached directly to the mass spectrometer inlet without modification.The hydrogen gas flows directly into the MS to be analyzed by reference/sample comparison. The reproducibility varied between 0.7 and 1.8% for all liquid and gas samples. The accuracy of this technique is confirmed by analysis of IAEA standard waters V-SMOW, GISP, and SLAP, as well as NGS-3 (IAEA methane intercomparison material).

Automated in-tube solid-phase microextraction–high-performance liquid chromatography for carbamate pesticide analysis

In-tube solid-phase microextraction (SPME) is an automated version of SPME that can be easily coupled to a conventional HPLC autosampler for on-line sample preparation, separation and quantitation. It has been termed “in-tube” SPME because the extraction phase is coated inside a section of fused-silica tubing rather than coated on the surface of a fused-silica rod as in the conventional syringe-like SPME device. The new in-tube SPME technique has been demonstrated as a very efficient extraction method for the analysis of polar and thermally labile analytes. The in-tube SPME–HPLC method used with the FAMOS autosampler from LC Packings was developed for detecting polar carbamate pesticides in clean water samples. The main parameters relating to the extraction and desorption processes of in-tube SPME (selection of coatings, aspirate/dispense steps, selection of the desorption solvents, and the efficiency of desorption solvent, etc.) were investigated. The method was evaluated according to the reproducibility, linear range and limit of detection. This method is simple, effective, reproducible and sensitive. The relative standard deviation for all the carbamates investigated was between 1.7 and 5.3%. The method showed good linearity between 5 and 10 000 μg/l with correlation coefficients between 0.9824 and 0.9995. For the carbamates studied, the limits of detection observed are lower than or similar to that of US Environmental Protection Agency or National Pesticide Survey methods. Detection of carbaryl present in clean water samples at 1 μg/l is possible.

Spectrophotometric determination of phosphite in fertilizers in a flow injection system with online sample preparation

A flow injection system with online sample preparation is proposed for the determination of phosphite in liquid fertilizers by spectrophotometry. After loop-based injection, phosphite is oxidized by an acidic permanganate solution (1.0 10−2 mol L−1 KMnO4 + 1.0 mol L−1 H2SO4) in a heated reactor (50 °C). The phosphate generated is then determined by the molybdenum blue method. Influence of flow rates, temperature, and concentration and order of addition of reagents, sample volume, and reactor configuration for the blue complex formation on recorded signals were investigated. The flow system was applied to phosphite determination in commercial samples of liquid fertilizers. The proposed system handles about 80 samples per hour [0.05–0.40% (w/v) H3PO3; R = 0,9998), consuming about 80 μL sample, 1 mg KMnO4, 25 mg (NH4)6Mo7O24, and 10 mg ascorbic acid per determination. Results are precise [relative standard deviation ≤ 3.5% for 0.1% (w/v) H3PO3, n = 12] and in agreement with those obtained by gravimetry at 95% confidence level. © 2000 John Wiley & Sons, Inc. Lab Robotics and Automation 12:286–290, 2000

On-line coupling of in-tube solid phase microextraction (SPME) to HPLC for analysis of carbamates in water samples: Comparison of two commercially available autosamplers

In-tube solid phase microextraction (SPME) is a new on-line sample preparation technique that is easily installed on a conventional high-performance liquid chromatography (HPLC) autosampler, and has previously been used with the LC Packings FAMOS autosampler without modification of the autosampler itself. This paper describes the on-line coupling of in-tube SPME to a Hewlett Packard (HP) model 1100 HPLC autosampler, and reports on several fundamental aspects of the technology. The extraction and desorption processes with this system were thoroughly studied for a group of carbamate pesticides in water samples. The similarities and differences of the in-tube SPME technique with the two different autosamplers, FAMOS and Hewlett Packard, were compared during method development. The optimized conditions were successfully applied to extract the carbamates of interest from spiked surface, well, and tap water samples, exhibiting excellent method precision. The % relative standard deviation (RSD) for all the carbamates studied in the different environmental water samples was between 0.8 and 6.0%. There was no noticeable extraction efficiency decrease relative to pure laboratory samples when the method was applied to spiked environmental water samples. The method was found to be linear between 2 and 20,000 μg/L with correlation coefficients in the range of 0.9791 to 0.9991. With respect to the different water samples, the limits of detection (LODs) for all the carbamates studied were in the range of 0.44–17 μg/L. For carbaryl, the LODs were between 0.44 and 0.67 μg/L, which were lower than those obtained using the in-tube SPME method with the FAMOS autosampler. © 2000 John Wiley & Sons, Inc. J Micro Sep 12: 125–134, 2000

Development of hardware and software for on-line sample preparation using sequential-injection analysis

This work describes the software and hardware developed for implementation of on-line microwave-assisted sample digestion in an automated sequential-injection analysis (SIA) system. The system was completely developed with conventional equipment available in analytical laboratories. The software and hardware allows for the acquisition and manipulation of analytical signals and also for the synchronization of the actuation of each component. The assembled system is composed of a mixing valve, three solenoid valves, a commercial microwave oven, a peristaltic pump, and an ultrasonic bath. Additionally, it is possible to program 60 different events that can be controlled by the operator according to the requirement of analysis. The operator only needs to inject the samples, and the other steps are carried out by the automated SIA system. This system was applied for direct determination of Fe in white wines and presented a sampling frequency of 10 h−1. The limit of detection was 0.6 mg L−1, and an addition-recovery experiment led to recoveries in the 98–101% range. © 2000 John Wiley & Sons, Inc. Lab Robotics and Automation 12:246–252, 2000

Integrated microanalytical technology enabling rapid and automated protein identification.

Protein identification through peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a standard technique, used in many laboratories around the world. The traditional methodology often includes long incubations (6-24 h) and extensive manual steps. In an effort to address this, an integrated microanalytical platform has been developed for automated identification of proteins. The silicon micromachined analytical tools, i.e., the microchip immobilized enzyme reactor (mu-chip IMER), the piezoelectric microdispenser, and the high-density nanovial target plates, are the cornerstones in the system. The mu-chip IMER provides on-line enzymatic digestion of protein samples (1 microL) within 1-3 min, and the microdispenser enables subsequent on-line picoliter sample preparation in a high-density format. Interfaced to automated MALDI-TOF MS, these tools compose a highly efficient platform that can analyze 100 protein samples in 3.5 h. Kinetic studies on the microreactors are reported as well as the operation of this microanalytical platform for protein identification, wherein lysozyme, myoglobin, ribonuclease A, and cytochrome c have been identified with a high sequence coverage (50-100%).

Determination of Ifosfamide by HPLC Using On-Line Sample Preparation

The Authors have developed a novel and easily applicable HPLC method for ifosfamide (IF) determination. This method involves on-line sample processing and its solid-phase extraction by means of an automatic preparator integrated with the chromatographic system. The calibration graph of the method is linear in the concentration range 6-200 μg/ml; minimum detectable concentration is 6 μg/ml. This highly accurate and easily reproducible method was used by the Authors in the treatment of osteosarcoma with slow infusion of ifosfamide.

On-line cation exchange for suppression of adduct formation in negative-ion electrospray mass spectrometry of nucleic acids.

One major difficulty in the analysis of nucleic acids by electrospray mass spectrometry is represented by the affinity of the polyanionic sugar-phosphate backbone for nonvolatile cations, especially ubiquitous sodium and potassium ions. A simple on-line sample preparation system comprising a microflow pumping system and 45 x 0.8-mm-i.d. microcolumns packed with weak or strong cation-exchange resins is described for the efficient removal of cations from nucleic acid samples. Samples were analyzed by flow injection analysis at a 3-5 microL/min flow of 10 mM triethylamine in 50% water-50% acetonitrile. After on-line desalting, mass spectra of oligonucleotides revealed no significant sodium adduct peaks. Moreover, signal-to-noise ratios were greatly enhanced compared to direct injection of the samples. Electrospray mass spectrometry with on-line sample preparation allowed accurate molecular mass determinations of picomole amounts of crude oligonucleotide preparations ranging in size from 8 to 80 nucleotides within a few minutes. The good linearity of the calibration plot (R2 = 0.9988) over at least 2 orders of magnitude and a relative standard deviation in peak areas of less than 9% permitted the sensitive quantitative measurement of oligonucleotides in a concentration range of 0.2-20 microM with selected-ion monitoring. Finally, the on-line sample preparation system was evaluated for the mass spectrometric analysis of complex oligonucleotide mixtures.