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PubMed1988

Separation of proteins by reversed-phase high-performance liquid chromatography. II. Optimizing sample pretreatment and mobile phase conditions.

The effects of separation variables such as temperature, pH and composition of the mobile phase (including additives such as chaotropes, ion-pairing agents and surfactants), sample size and sample pretreatment for reversed-phase high-performance liquid chromatography (RP-HPLC) of proteins is examined. Experimental optimization of these parameters using the preferred instrumental and column conditions described previously lead to well behaved chromatographic performance for most proteins. This allowed us to achieve the required level of performance for the first dimension (RP-HPLC) separation of most protein samples by the chromatophoresis process.

Bacterial Proteins↗

PubMed2005

Ionic liquid-based aqueous two-phase system, a sample pretreatment procedure prior to high-performance liquid chromatography of opium alkaloids.

An ionic liquid, 1-butyl-3-methylimidazolium chloride ([C4 mim]Cl)/salt aqueous two-phase systems (ATPS) was presented as a simple, rapid and effective sample pretreatment technique coupled with high-performance liquid chromatography (HPLC) for analysis of the major opium alkaloids in Pericarpium papaveris. To find optimal conditions, the partition behaviors of codeine and papaverine in ionic liquid/salt aqueous two-phase systems were investigated. Various factors were considered systematically, and the results indicated that both the pH value and the salting-out ability of salt had great influence on phase separation. The recoveries of codeine and papaverine were 90.0-100.2% and 99.3-102.0%, respectively, from aqueous samples of P. papaveris by the proposed method.

Chemical Fractionation↗

PubMed1993

Determination of catecholamines in urine and plasma by on-line sample pretreatment using an internal surface boronic acid gel.

An automated method of analysis of catecholamines using a new packing material, internal surface boronic acid gel, was developed. The new support is designed with a carboxymethylcellulose-bonded external surface in order to be non-adsorptive to proteins and with a phenylboronic acid-bonded internal surface to retain only catecholamines. This packing support displayed an affinity for basic or neutral catecholic compounds with no protein adsorption and enabled on-line sample pretreatment of catecholamines in urine and deproteinized plasma. The catecholamines were selectively adsorbed on the new material and separated on a reversed-phase or a cation-exchange column. These compounds were then detected electrochemically. The limits of quantitation were 1.5-3.0 ng/ml in urine and 10-15 pg/ml in plasma, at a signal-to-noise ratio of 5.

Boronic Acids↗

PubMed2001

Influence of buffer composition and sample pretreatment on efficiency separation for monitoring neuropeptides in plasma using capillary electrophoresis.

More efficient and faster separation conditions for qualitative as well as quantitative analysis of neuropeptides in human plasma using capillary zone electrophoresis (CZE) have been developed. The analysis method for neuropeptides has been improved specifically to study thyroid hormone related neuropetides for the regulation of thyroid disease. In this study, we investigated the pretreatment methods, composition of the running buffer and rinsing procedures between runs in order to obtain more sensitive and faster separation of trace neuropeptides in plasma by CZE. The tested neuropeptides were somatostatin (SOMA), vasopressin (VP), neurotensin (NT), and thyrotropin-releasing hormone (TRH). Plasma samples were pretreated by deproteinization and solid-phase extraction method. The fraction of neuropeptides was reconstituted in 40% acetonitrile followed by ultrafiltration, and then analyzed by CZE. Resolution and sensitivity was improved using the separation buffer composition with 100 mM Tris-phosphate buffer (pH 2.0) while the sensitivity was further improved via a stacking method using the sample buffer of 40% acetonitrile. These sample pretreatment methods and buffer condition permit quantitative analysis on tested neuropeptides at the 20 ng/mL level. The rinsing procedures between runs using 90% ethanol dramatically shortened the rinsing time to 30 min.

Buffers↗

PubMed1986

Sample pretreatment to minimize interferences from whole blood in the radioimmunoassay for cyclosporine.

There is much controversy as to whether the analysis of cyclosporine (CsA) should be performed by radioimmunoassay (RIA) or high-performance liquid chromatography (HPLC), and whether the specimen should be serum or whole blood. Whole-blood specimens present specific advantages, but the presence of hemoglobin (Hgb) and other endogenous compounds can produce major errors in the RIA by "quenching" the analytical signal or by interfering with the antigen-antibody binding in the assay. We have developed a simple pretreatment step to remove the Hgb and other proteins responsible for this error. Red cells in whole blood are hemolyzed with a mixture of acetonitrile and water, the protein precipitated with acetonitrile, and the supernatant assayed by RIA. In a controlled study in which CsA concentration was kept constant and the Hgb concentration varied, the errors in measurement were directly proportional (r = 0.999) to the Hgb concentration. CsA values were spuriously deflated or inflated by 22.7 micrograms/L for each gram per 100 milliliters that the Hgb deviated from the 9.2 g/100 ml Hgb in the CsA calibration standards. In a similar study in which patient samples (n = 57) were assayed with and without pretreatment, the fractional error induced by Hgb was compounded in some patients by additional interferences that also appear to be removed by sample pretreatment. Without the pretreatment, CsA values could be in error by 33% when the Hgb varied 4 g/100 ml, thus providing potentially misleading results to the clinician. An I-125-labeled CsA tracer (purported not to be affected by the "quenching" interference of Hgb) produced consistently higher results when it was substituted for the tritiated CsA tracer contained in the Sandoz kit. In summary, sample pretreatment appears to be the simplest method of effectively removing endogenous interferences and minimizing erroneous results from whole blood submitted to the Sandoz RIA for CsA analysis.

Chromatography, High Pressure Liquid↗

PubMed1982

Automated analysis of acetaminophen and caffeine in serum using the FAST-LC system: contributions to assay imprecision in procedures based on HPLC with sample pretreatment.

Two procedures are described for the fully automated analysis of several therapeutic drugs in serum, using HPLC with on-line pretreatment (solvent extraction) of the sample. The FAST-LC system (Technicon Instruments) was used for the assay of mixtures of 1) acetaminophen, theophylline, and/or caffeine, or 2) phenylethylmalonamide, primidone, phenobarbital, carbamazepine epoxide, phenyltoin, and/or carbamazepine. The rate of sample analysis was 15/hr for the theophylline group of drugs and 12/hr for the six anticonvulsants. The precision of resulting assays was about 3% (CV), and only 75 microliter of sample was required. The precision of resulting assays, in terms of a previously reported model, is also discussed.

Acetaminophen↗

PubMed2006

Molecular imprinting of natural flavonoid antioxidants: application in solid-phase extraction for the sample pretreatment of natural products prior to HPLC analysis.

As shown in the past years, SPE based on molecularly imprinted polymers (MIPs) may provide significant enhancement of selectivity in sample preparation and analyte preconcentration. The objective of this work was the fabrication of MIPs for the specific adsorption of rutin and quercetin. The two flavonoids were used as the template molecules for the preparation of MIP phases in a self-assembly (noncovalent) approach. The produced MIPs were validated with regard to the imprinting efficiency as media for LC and SPE. The retention behavior of several flavonoid compounds was studied using as stationary phases imprinted, control nonimprinted polymers, and commercial silica-based materials. MIPs were applied as materials for the selective SPE and preconcentration of the flavonoids from white and red wine, orange juice, and tea. The collected fractions were analyzed by high-pressure LC. MIP-SPE facilitated specific analyte isolation and effective sample clean-up. The results show that molecularly imprinted SPE can be a useful tool for the simple, selective, and cost-effective pretreatment of samples containing natural antioxidants.

Antioxidants↗

PubMed1981

Precision of "high-performance" liquid-chromatographic assays with sample pretreatment. Error analysis for the Technicon "FAST-LC" system.

A comprehensive theory has been presented elsewhere (Anal. Chem. 53: 877-884, 1981) for the various contributions to assay imprecision in procedures that are based on sample extraction followed by "high-performance" liquid chromatography. Experimental data (1800 assays, 8400 results) for the Technicon FAST-LC system are used with this theory in an effort to understand and control the precision of clinical-laboratory procedures, both automated and manual. This study provides specific conclusions and recommendations on matters such as: standardization procedures and protocols, physical properties requires in internal standards, the relative importance of different sources of imprecision and means for improving precision, the relative importance of temperature control in pretreatment and liquid-chromatographic analysis, and the precision obtainable with small sample volumes or with samples containing very low concentrations of analyte (e.g., in assays for free drugs). Automation evidently can lead to twofold greater assay precision, other factors being equal, for liquid-chromatographic procedures that include sample pretreatment. Similarly, internal standardization, properly applied, can decrease assay imprecision by twofold.

Anticonvulsants↗

PubMed2003

Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.

Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding <0.1 cent in material costs to the price of each tip. The system allows fast loading with low backpressure (>300 micro/min for the packed column using manual force) while eliminating the possibility of blocking. The loading capacity of C18-StageTips (column bed: 0.4 mm diameter, 0.5 mm length) is 2-4 microg of protein digest, which can be increased by using larger diameter or stacked disks. Five femtomole of tryptic BSA digest could be recovered quantitatively. We have found that the Stage system is well-suited as a universal sample preparation system for proteomics.

Animals↗

PubMed2005

Sample pretreatment and determination of non steroidal anti-inflammatory drugs (NSAIDs) in pharmaceutical formulations and biological samples (blood, plasma, erythrocytes) by HPLC-UV-MS and micro-HPLC.

The article discusses the qualitative and quantitative determination of non-steroidal anti-inflammatory drugs like salicin, salicylic acid, tenoxicam, ketorolac, piroxicam, tolmetin, naproxen, flurbiprofen, diclofenac and ibuprofen by reversed phase high performance liquid chromatography (RP-HPLC) and micro-HPLC (micro-HPLC) hyphenated with UV-absorbance and mass spectrometric detection. Both detection methods delivered calibration plots with good linearity (r(2) > 0.9800), limits of detection in the low nanogram range and recovery rates between 94 and 104 %. For the analysis of biological samples such as blood, plasma and erythrocytes liquid-liquid extraction (LLE) and solid phase extraction (SPE) on the basis of new synthesized glycidylmethacrylate/divinylbenzene copolymer (GMA/DVB) particles and commercially available material on the basis of poly(divinylbenzene-co-N-vinylpyrrolidone) copolymer were investigated. Finally the use of a micro-HPLC system with separation columns in the range of 8 cm x 200 microm I.D. for the determination of non-steroidal anti-inflammatory drugs (NSAIDs) is presented, emphasizing on the type of column and sample amount needed.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMedDate not supplied

[A device for sample pretreatment during flow cytometry].

A device for the preliminary treatment of samples immediately prior to flow cytofluorimetric analysis is described. The device is intended for several procedures: (a) mixing of batched sample volumes with the reagent and efficient stirring of the mixture; (b) disintegration of cell aggregates; and (c) disruption of cell membranes to release the cell contents (chromosomes, micronuclei, nuclei etc.). The pretreatment is useful for studying the kinetic parameters of fast cellular processes in the flow, a more correct analysis of the cell cycle and the study of karyotypes of single mitotic cells. The device was called a magnetic microstirrer.

Cell Aggregation↗

PubMed2002

[A system for biomaterial handling and sample pretreatment in conditions of weightlessness].

To perform laboratory biochemical, immunologic and microbiological analyses and special pretreatment and stabilization of biosamples immediately on the space station, system Plasma-03 was designed and manufactured and associated procedure for sample collection and pretreatment was developed. Main elements of the system are a refrigerator/thermostat, centrifuge and returnable container, and a set of accessories and expendables. The system meets the requirements to the equipment to be employed in crew health monitoring and biochemical experiments on board the International space station.

Biochemical Phenomena↗

PubMed1989

Determination of beta-19-nortestosterone and its metabolite alpha-19-nortestosterone in biological samples at the sub parts per billion level by high-performance liquid chromatography with on-line immunoaffinity sample pretreatment.

An immunoaffinity precolumn (immuno precolumn) packed with Sepharose-immobilized polyclonal antibodies against the anabolic hormone 17 beta-19-nortestosterone (beta-19-NT) was used for the selective on-line pretreatment of raw extracts of urine, bile and tissue samples by high-performance liquid chromatography. Using UV detection (247 nm), beta-19-NT and its metabolite 17 alpha-19-nortestosterone (alpha-19-NT) can be determined in biological samples with a detection limit of 0.05 microgram/kg. Owing to the high clean-up efficiency of the immuno precolumn and the large sample volumes used, confirmation by gas chromatography-mass spectrometry is possible at this level. In urine samples from a calf treated with 19-nortestosterone 17 beta-laurate, the maximum concentrations of beta-19-NT (1.3 micrograms/l) and alpha-19-NT (3.1 micrograms/l) were found seven days after intramuscular administration. In a bile sample from this calf only alpha-19-NT (55 microgram/l) was detected. In meat samples from three treated calves, the concentration of beta-19-NT varied from 0.1 to 1.6 micrograms/kg and no alpha-19-NT could be detected. In liver samples from these calves, the concentrations of beta-19-NT and alpha-19-NT were less than 0.05-0.1 and 0.5-0.9 micrograms/kg, respectively. In the corresponding kidney samples, the concentrations of beta-19-NT and alpha-19-NT were 0.4-0.5 and 0.5-1.6 micrograms/kg, respectively. The application of the same immuno precolumn to the determination of 17 beta- and 17 alpha-trenbolone, two structurally related steroids, is also demonstrated.

Animals↗

PubMed1998

On-line microporous membrane liquid-liquid extraction for sample pretreatment combined with capillary gas chromatography applied to local anaesthetics in blood plasma.

A new automated procedure for analyzing complex samples has been developed utilizing microporous membrane liquid-liquid extraction (MMLLE) combined with capillary gas chromatography. Some local anaesthetics were used as model compounds in aqueous solution as well as in blood plasma. The MMLLE procedure was performed in a flow system with the sample fed to the donor side of the hydrophobic microporous membrane and with an organic solvent (hexane) in the pores and as the acceptor solution. The analytes in a small volume of sample (< 1 mL) were extracted into the organic acceptor phase which was transferred into the gas chromatographic system by utilizing a loop-type interface compatible with large-volume (300 microL) injection. High selectivity and low carry-over effects were obtained with the system. The detection limits were 0.5-1 ng/mL using 0.5 mL of human plasma, and the precision was approximately 5%. The effects of pH, flow rates, and adsorption of the analytes were evaluated.

Adsorption↗

PubMed1999

Analysis of morphine and morphine-3beta-D glucuronide in human urine by capillary zone electrophoresis with minimal sample pretreatment.

The presence of two of the metabolites of heroin, free morphine and morphine 3-beta-D-glucuronide (MO3G) in acidified urine samples was simultaneously determined by capillary zone electrophoresis (CZE). In a run buffer containing 50 mM sodium borate and 250 mM boric acid (pH 8.6), free morphine migrates before a group of neutral compounds (peak N) in urine, which move with the velocity of electro-osmotic flow. In contrast, the glucuronidated form is negatively charged and migrates behind peak N. Both analytes can be precisely identified within their respective analytical window by their migration time with respect to peak N. The on-line multi-wavelengths scanning of the peak permits further confirmation. The detection sensitivity of both analytes was increased three-four fold if the samples were introduced with electro-injection as compared with hydrodynamic injection. Limits of detection (LOD) of free and conjugated morphine using electro-injection were 200 ng/mL and 500 ng/mL, respectively, determined at a 3:1 signal to noise ratio. A dramatic increase of free morphine was observed after acid hydrolysis of the urine concomitantly with the decrease of the glucuronidated form. We conclude that CZE is a rapid, simple, sensitive and useful screening technique for detection of heroin metabolites in the urine.

Calibration↗

PubMed2001

[Application of digestion pot of HP hermetic seal in sample pretreatment in spectral analysis].

Atomic absorption spectrometry is a rapid and effective method for the quantitative determination of elements, but in complete dissolution of solid sample can greatly influence the analytical accuracy. The scale sample obtained from the power plant is hardly to digestion. In this paper, a method is proposed namely digestion pot of HP hermetic seal, it can digest the scale sample, and the digestion condition is obtained. After the sample is completely digested, the conventional elements in scale sample is determined by atomic absorption spectrometry.

Copper↗

PubMed1983

New "on-line" sample-pretreatment procedure for routine liquid-chromatographic assay of low-concentration compounds in body fluids, illustrated by triamcinolone assay.

In this fully automated technique for sample cleanup before chromatographic or other quantitation steps, analytes in body fluids are enriched and semi-purified on a first column. After their selective elution, analytes are "transformed" by admixing appropriate solvents in such a way that they are focused on the top of a second column. By backflush, they then are transferred to an analytical liquid-chromatographic column (or simply eluted for quantification by other techniques). This technique is illustrated by the liquid-chromatographic assay of triamcinolone from a 1-mL urine sample, with ultraviolet detection. Because analytical recovery is almost complete and precision high, no internal standardization is necessary. Interference is eliminated as well as or better than with manual techniques. Chief advantages of this technique are online operation, processing of samples of larger volume, low cost with respect to extraction devices, and nearly universal applicability for exogenous or endogenous compounds of clinical relevance. It potentially may be widely applied.

Body Fluids↗

PubMed2006

Headspace solvent microextraction as a simple and highly sensitive sample pretreatment technique for ultra trace determination of geosmin in aquatic media.

A headspace solvent microextraction method was developed for the trace determination of geosmin, an odorant compound, in water samples. After performing the extraction by a microdrop of an organic solvent, the microdrop was introduced directly into a GC-MS injection port. One-at-the-time optimization strategy was applied to investigate and optimize some important extraction parameters such as type of solvent, drop volume, temperature, stirring rate, ionic strength, sample volume, and extraction time. The analytical data exhibited an RSD of less than 5% (n = 5), a linear calibration range of 5-900 ng/L (r2 > 0.998), and a detection limit of 0.8 and 3.3 ng/L using two different sets of selected ions. The proposed method was successfully applied to the extraction and determination of geosmin in the spiked real water sample and reasonable recovery was achieved.

Journal Article↗