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At least 253 records · Page 14Linked to original sources
PubMed2005

Integration of supercritical fluid chromatography into drug discovery as a routine support tool. Part I. fast chiral screening and purification.

Supercritical fluid chromatography (SFC) has been implemented within our group as a purity assessment and purification tool to complement high performance liquid chromatography (HPLC) for diastereomer and chiral separations. Using a novel strategy, rapid chiral screening has been implemented using short columns, high flow rates and fast gradients. A primary screen delivers a separation assessment using one solvent modifier (methanol) and four columns (Chiralpak AD-H and AS-H, and Chiralcel OD-H and OJ-H) run serially in a total of 24 min. A secondary screen then uses ethanol and isopropanol (IPA) modifiers across the same columns. The screens can be combined to run a sequence of samples overnight where each racemate is analysed over 80 min. The fast analytical screening and optimisation process enables rapid identification of the purification method. Furthermore, subsequent preparative chiral SFC has decreased the overall sample turnaround time for the Medicinal Chemist, delivering high fraction purities and acceptable recoveries, substantial operational cost savings and increased flexibility with respect to large scale purification feasibility in comparison to HPLC. SFC has been so successful it is now used as the primary method for chiral analysis and purification within our laboratory.

Chromatography, High Pressure Liquid↗
PubMed2005

On-matrix derivatisation-extraction of precursors of nitrogen- and sulfur-mustards for verification of chemical weapons convention.

Development and refinement of sample preparation protocols for retrospective detection and identification of chemical warfare agents (CWAs) and their markers is of paramount importance from verification point of view of chemical weapons convention (CWC). Precursors of nitrogen- and sulfur-mustards (NMPs and SMPs) are polar adsorptive markers of vesicant class of CWAs. Their detection in a given environmental sample may imply past contamination with mustards. For the efficient extraction of NMPs and SMPs from soil, on-matrix derivatisation-extraction (OMDEX) method was developed and optimized. The method involved trifluoroacetylation of analytes on soil itself, followed by extraction with suitable solvent. The extracted samples were analyzed by gas chromatography-mass spectrometry (GC-MS). This virtually single-step sample preparation offered better recoveries of NMPs and SMPs in comparison to conventionally used extraction, evaporation and derivatisation. The best recoveries of analytes were obtained with acetonitrile by OMDEX method. Dynamic linearity range of trifluoroacetylated (TFA) derivatives of NMPs and SMPs was 1-12 microg/L in GC-MS analysis in SIM mode. Repeatability and reproducibility of this technique containing 5 and 10 microg analytes/gm soil was <3.3% and <4.6%, respectively. OMDEX technique was finally applied for the detection of TFA derivatives of NMPs in the soil sample supplied in 16th official proficiency test conducted by OPCW in October 2004.

Calibration↗
PubMed1987

Environmental sampling and analysis for zeranol.

Due to concern over health effects associated with the use of zeranol, a sampling and analytical method to monitor zeranol in the work environment was developed. The collection strategy included air samples on FHLP filters, area samples on gauze pads, hand/dermal samples on gauze pads and bulk samples. The sampling method evaluation included choice of media, flow rate and duration for air samples, sample preparation, extraction efficiencies and sample stability. Analysis of the samples was accomplished by high performance liquid chromatography with identification and quantification by UV and fluorescence detection. The analytical method evaluation included separation of possible interfering compounds, calibration curves, limits of detection (LOD), limits of quantitation (LOQ), precision and accuracy. The tested analytical range was 0.01 to 150 micrograms/mL with a LOD of 0.007 microgram/mL and a LOQ of 0.02 microgram/mL using UV detection. For the fluorescence detector, the LOD was 0.05 microgram/mL and the LOQ was 0.15 microgram/mL. Peak confirmation was achieved by retention time and comparing the two detector responses. Use of this method provided the sensitivity and specificity to analyze environmental samples for zeranol in the workplace environment.

Air Pollutants, Occupational↗
PubMed1988

Improved high-performance liquid chromatographic method for the determination of tiotixene in human serum.

The problem of accurate determination of tiotixene in body fluids is still challenging. Several methods have been published but most of them require a tedious, time-consuming sample preparation, are not specific enough and lack the necessary sensitivity or require highly sophisticated analytical devices. As carefully validated analytical methods represent the basis of conclusive clinical trials (e.g. evaluating bioavailability/bioequivalence), an assay was developed to fulfill these needs. The method present employs an HPLC system combined with a UV-detector and uses perazine as an internal standard. The achieved lower limit of detection in serum was 0.05 ng/ml and the calibration curves were linear in the range of 0.5-20 and 0.1-2.0 ng/ml, respectively. The chromatographic peaks were well resolved and the cis-/transisomers well separated. The imprecision and inaccuracy data typically ranged from 2 to 7%; the recovery from serum was always better than 80%. The assay has been successfully used for the determination of very low tiotixene serum levels during several clinical studies.

Calibration↗
PubMed2002

Neurotransmitter metabolites in CSF: an external quality control scheme.

We report an international external quality control scheme on neurotransmitter metabolites in cerebrospinal fluid (CSF). The neurotransmitter metabolites homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) are analysed to diagnose inborn errors of neurotransmitter metabolism. HVA is the catabolite of dopamine; 5-HIAA is the catabolite of serotonin; and MHPG is the catabolite of noradrenaline. In the first phase, 12 laboratories from six countries participated in this special quality control scheme to define the present state of the art and achieve harmonization in analytical outcome and interpretation. In the second part, recoveries, dilutions and methods for sample preparation were compared. The results of 3 of 12 laboratories were excluded because of unacceptable intralaboratory coefficients of variations (CV) for HVA and/or 5-HIAA. The inter- and intralaboratory CVs, the linearity and the recovery were acceptable for the other laboratories for both parameters. Unacceptable differences in the reference ranges between laboratories, leading to differences in interpretation of the results, became obvious. There was a significant improvement of the interlaboratory CV for HVA after standardization with a calibrator. The reproducibility of MHPG measurement appeared to be adequately established in only two laboratories and recovery was low in all five measuring this metabolite. The quality control scheme is an invaluable tool for controlling the analytical outcome and providing support to laboratories to improve their quality.

Algorithms↗
PubMed2003

[Precision of internal standard method in HPLC analysis].

The internal standard methods are known to compensate for the errors from sample preparation and injection into an analytical instrument. However, recent HPLC apparatuses have injectors of excellent repeatability and it is dubious whether the cancellation of injection error can lead to substantial improvement in the precision of analysis. This paper answers the above question experimentally and theoretically. The HPLC analysis of butylscopolamine bromide is taken as an example. The relative standard deviations (RSD) of measurements in the internal standard method and absolute calibration curve method are compared and the advantages of these methods are discussed. The measurement RSD is shown to be well estimated by the (function of mutual information) (FUMI) theory without repeating measurements. This report also demonstrates simple equations for calculating the measurement RSD at an arbitrary concentration of analyte and for selecting the better method between the internal standard method and absolute calibration curve method under specific experimental conditions.

Calibration↗
PubMed2005

Simple fluorimetric liquid chromatographic method for the analysis of undecylenic acid and zinc undecylenate in pharmaceutical preparations.

A simple and selective liquid chromatographic method is described for the analysis of undecylenic acid (UA) and zinc undecylenate (ZnUA) in pharmaceutical preparations. The method is based on the derivatization of the analytes extracted from various samples with 2-(2-naphthoxy)ethyl 2-(piperidino)ethanesulfonate. The resulting derivative was analyzed by liquid chromatography with fluorimetric detection. The quantitation of the method is in the range of 3.0-50.0 microM UA with a detection limit of about 0.3 microM (S/N = 3 with 10 microl injection). We found that acetonitrile is a selective solvent for differentially dissolving UA from coexisted ZnUA in compound formulation. This results in the specific analysis of UA in the presence of ZnUA and simply analyzing the coexisted ZnUA by the value of total UA (UA+ZnUA) minus that of UA. Application of the method to the analysis of undecylenic acid and zinc undecylenate in ointment, powder and solution preparations proved feasible.

Chromatography, High Pressure Liquid↗
PubMed2003

Liquid chromatography/electrospray ionization tandem mass spectrometric screening and confirmation methods for beta2-agonists in human or equine urine.

Electrospray ionization (ESI) mass spectra of 19 common beta(2)-agonists were investigated in terms of fragmentation pattern and dissociation behavior of the analytes, proving the origin of fragment ions and indicating mechanisms of charge-driven and charge-remote fragmentation. Based on these data, liquid chromatographic/ESI tandem mass spectrometric (LC/ESI-MS/MS) screening and confirmation methods were developed for doping control purposes. These procedures employ established sample preparation steps including either acidic or enzymatic hydrolysis, alkaline extraction and, in the case of equine urine specimens, acidic re-extraction of the analytes. In addition, a degradation product of formoterol caused by acidic hydrolysis during sample preparation could be identified and utilized as target compound in screening and also confirmation methods. The screening procedures cover 18 or 19beta(2)-agonists, the estimated limits of detection of which for equine and human urine samples vary between 2 and 100 ng ml(-1) and between 2 and 50 ng ml(-1), respectively. A single LC/MS/MS analysis can be performed in 9 min.

Adrenergic beta-Agonists↗
PubMed1996

Plasma protein adsorption patterns on liposomes: establishment of analytical procedure.

After intravenous (i.v.) injection, colloidal drug carriers such as liposomes, emulsions, polymeric or solid lipid nanoparticles immediately interact with plasma proteins. The adsorbed plasma protein patterns depend on physico-chemical characteristics of the carriers' surface and are regarded as a key factor for the in vivo behavior of the carriers. The comprehension of the correlation between protein adsorption and in vivo organ distribution can be utilized to obtain drug targeting to different tissues. Carriers with different protein adsorption patterns will interact with different tissue-specific receptors or will be recognized by different macrophage subpopulations. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was applied to determine the protein adsorption patterns on polystyrene particles as model carriers. To transfer this analytical method to i.v. injectable colloidal carriers such as liposomes, a new sample preparation method was developed. The separation of liposomes from plasma after incubation was achieved by gel filtration using a Sepharose 2B column. This technique allowed a mild separation independent from eluent composition and only according to size differences. Possible protein desorption from the liposomes and adsorption onto the gel were minimized by using an eluent with a sufficiently high ionic strength. To estimate the efficiency of separation, the content of liposomes and plasma in each fraction being eluted was determined by ultraviolet (UV) spectroscopy. With this new separation method plasma protein adsorption patterns on liposomes could be analyzed for the first time. The sample preparation by gel filtration seemed to have no influence on liposome stability as far as size distribution is concerned.

Absorption↗
PubMed2005

Proteomics analysis of human cerebrospinal fluid.

Cerebrospinal fluid (CSF) is secreted from several different central nervous system (CNS) structures, and any changes in the CSF composition will accurately reflect pathological processes. Proteomics offers a comprehensive bird's eye view to analyze CSF proteins at a systems level. This paper reviews the variety of analytical methods that have been used for proteomics analysis of CSF, including sample preparation, two-dimensional liquid and gel electrophoresis, mass spectrometry, bioinformatics, and non-gel methods. The differentially expressed CSF proteins that have been identified by proteomics methods are discussed.

Cerebrospinal Fluid Proteins↗
PubMed2004

Steviol quantification at the picomole level by high-performance liquid chromatography.

A simple and highly sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of steviol (SV) using dihydroisosteviol (DHISV) as an internal standard (IS). SV and DHISV were derivatized by reaction of the acids with 4-(bromomethyl)-7-methoxycoumarin in an aprotic solvent (DMF or acetone). The resulting ester derivatives were separated on an ODS column (250 x 4.6 mm i.d., 5 microm particle size) using fluorescence detection with excitation at 321 nm and emission at 391 nm. The mobile phase consisted of acetonitrile/water (80:20 v/v) with a flow rate of 1 mL min(-)(1). A linear relationship was observed for concentrations between 0.5 and 50 microg/mL of SV, and the detection limit was 100 pg. For application of this method to samples of beer fortified with stevioside, a simple procedure for extraction of the beer with diethyl ether and derivatization in DMF was applied. Whereas beer samples spiked with SV gave a linear response over the range 0.1-15 microg/mL beer, no SV could be detected in beer samples enriched in stevioside that had been stored for over 3 years. The application of the method to plant samples involved preparation of an acid fraction containing the SV analyte, derivatization, and sample cleanup using small silica columns and thin-layer chromatography. A sensitive determination of 594 ng of steviol present in 100 mg of dry plant material was performed with high precision and accuracy.

Beer↗
PubMed2003

Determination of 1,4-dioxane impurity levels in Triton X-100 raw material by gas chromatography with mass spectrometric detection.

Triton X-100 (octoxynol 9) is a commercially available surfactant used as a solvent detergent in numerous pharmaceutical applications including virus inactivation. A byproduct formed during its synthesis is 1,4-dioxane, the cyclic dimer of ethylene oxide and a possible carcinogen to humans. The United States Pharmacopoeia (USP) contains a labor-intensive 1,4-dioxane test for Triton X-100. The method couples vacuum distillation to extract the 1,4-dioxane from the Triton X-100 matrix followed by gas chromatography (GC) using a packed column with flame-ionization detection. In order to provide a more automated and specific test methodology, a headspace GC-mass spectrometry (MS) method has been developed for this application. Analyte quantitation is accomplished by the method of standard additions. The automated sample preparation, coupled with the specificity inherent in high-efficiency capillary column separations together with single-ion MS detection, results in an assay that is more efficient, accurate, and precise than the USP procedure. Performance characteristics of the headspace GC-MS method are contrasted with those characteristics of the USP methodology.

Dioxanes↗
PubMed1989

A new method for the measurement of nitrosoureas in plasma: an h.p.l.c. procedure for the measurement of fotemustine kinetics.

1. An analytical method for a novel nitrosourea, fotemustine, has been developed using solid-phase extraction and h.p.l.c. with u.v. detection. As part of the development, different methods for stabilising fotemustine after sample collection have been investigated. The method has been successfully applied to pharmacokinetic studies in monkeys and man. 2. Providing plasma was separated immediately from blood and frozen within 3 min of collection, negligible degradation of fotemustine occurred. The samples could then be stored at -20 degrees C in the dark for up to six days particularly if thawing prior to analysis was accelerated using a 50 degrees C water-bath so that it was complete within 3 min. Equivalent results were also obtained with samples stabilised with 0.1 M citric acid immediately after the preparation of plasma. 3. The analytical method showed good precision with a within-day variation ranging between +/- 10.7% at the lowest concentration investigated (0.1 micrograms ml-1) to 2.0% at 50.0 micrograms ml-1. The accuracy of measurement was from 108.9% to 97.6% at 0.1 and 50.0 micrograms ml-1 respectively and the response was linear up to 50 micrograms ml-1. The minimum level of quantitation was 20 ng ml-1. 4. After a single intravenous bolus dose of [14C]fotemustine (100 mg m-2) to Cynomolgus monkeys, intact drug levels rapidly declined (t1/2 12.6 +/- 0.5 min) although the half-life of radioactivity (approx 100 h) was much longer. The plasma clearance of fotemustine was 225 +/- 63 ml min-1 with a volume of distribution based on area of 4.1 +/- 1.2 litres. 5. As with monkey, plasma levels of intact fotemustine in a patient given [14C]-drug as a 1 h constant rate intravenous infusion (approx. 100 mg m-2), declined rapidly but with a half-life of 23.2 min. Again, the half-life for total radioactivity was considerably longer (30.8 h). The plasma clearance was 1426 ml min-1 and the volume of distribution based on area was 47.71.

Animals↗
PubMed2005

In-solution digestion of proteins for mass spectrometry.

Mass spectrometry (MS) has gradually replaced classical methods as a major tool in protein sequencing and characterization. However, the sample preparation repertoire has not changed very much; it has just been adjusted to the needs of the new analytical method. In this chapter frequently used in-solution enzymatic digestions and chemical cleavages are reviewed. In addition, some practical recommendations as well as the advantages and shortcomings of the methods are discussed.

Amino Acid Sequence↗
PubMed2004

[Developments in preparation and experimental method of solid phase microextraction fibers].

Solid phase microextraction (SPME) is a simple and effective adsorption and desorption technique, which concentrates volatile or nonvolatile compounds from liquid samples or headspace of samples. SPME is compatible with analyte separation and detection by gas chromatography, high performance liquid chromatography, and other instrumental methods. It can provide many advantages, such as wide linear scale, low solvent and sample consumption, short analytical times, low detection limits, simple apparatus, and so on. The theory of SPME is introduced, which includes equilibrium theory and non-equilibrium theory. The novel development of fiber preparation methods and relative experimental techniques are discussed. In addition to commercial fiber preparation, different newly developed fabrication techniques, such as sol-gel, electronic deposition, carbon-base adsorption, high-temperature epoxy immobilization, are presented. Effects of extraction modes, selection of fiber coating, optimization of operating conditions, method sensitivity and precision, and systematical automation, are taken into considerations in the analytical process of SPME. A simple perspective of SPME is proposed at last.

Chromatography, Gas↗
PubMed2006

Analysis of nonylphenol: advances and improvements in the immunochemical determination using antibodies raised against the technical mixture and hydrophilic immunoreagents.

The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of technical nonylphenol (NP) is reported. The preparation of specific antibodies has been addressed using an immunizing hapten with a four-carbon atom spacer arm placed at the ortho position that preserves both the hydroxyl group and the complexity of the branched nonyl chain mixture of the technical NP. The synthesis of the immunizing hapten 5-(2-hydroxy-5-nonylphenyl)-pentanoic acid has been accomplished through a four-step synthetic pathway using the NP commercial technical mixture as the starting material. Three types of competitor haptens have also been prepared depending on the location of the spacer arm: in ortho position to the phenol group (type A), attached to the oxygen atom (type B), and in para position, substituting the nonyl chain (type C). Drawbacks produced by the hydrophobicity of the NP or of the hapten derivatives have been circumvented by using a highly hydrophilic carrier molecule such as a high-molecular-weight aminodextran as a coating support for antigen in an indirect ELISA format. A reproducible and sensitive indirect competitive ELISA has been finally obtained, reaching a limit of detection of 2.3 +/- 0.9 microg L(-1) and an IC50 value of 29 +/- 5 microg L(-1) (both N = 16). A coefficient of variation of 11% for assays performed on different days (N = 5; IC50 = 30 +/- 3 microg L(-1)) demonstrates the assay reproducibility. The assay also recognizes the nonylphenol polyethoxylates to a different degree depending on the length of the ethoxylate chain. Recovery values in the range between 96 and 100% have been obtained using spiked blind aqueous samples although the sample preparation procedure used has been shown to have a great influence on the method accuracy. A preliminary evaluation of the analytical protocol established has been performed using real water samples.

Antibodies↗
PubMed2002

From experimental design to uncertainty estimation for the European Pharmacopoeia HPLC analysis of human insulin.

In this paper the process from experimental design (e.g. ruggedness test) to uncertainty estimation is described. The uncertainty estimate was calculated for the peak area of insulin plus A21 desamido insulin resulting from an HPLC analysis of a sample of an injectable human insulin preparation, Actrapid HM 100 IU ml(-1) (Novo Nordisk A/S). The analytical method used was the European Pharmacopoeia assay. (4) An expanded uncertainty (1) of 1.8% (of the area from the HPLC analysis) at an approximately 95% confidence level was found and confirmed by a validation study. This uncertainty refers to the peak area of the analyte from a single injection of the sample. The input parameters to the uncertainty estimate were found from a factorial experimental design (e.g. ruggedness test) consisting of 9 factors applied to the HPLC analysis. The input parameters were chosen to cover the probable contributors to the variability of the measured area of the HPLC analysis, including the sample preparation, but excluding uncertainty deriving from the reference material(s).

Chromatography, High Pressure Liquid↗
PubMed2004

Application of solvent microextraction in a single drop for the determination of new antifouling agents in waters.

A new, rapid microextraction technique termed solvent microextraction (SME) has been developed for the simultaneous determination of new generation antifouling agents, in water samples. Chlorothalonil, dichlofluanid and Sea nine 211 were employed as model compounds to asses the extraction procedure and were determined by gas chromatography with electron capture detection. Experimental parameters which control the performance of SME, such as selection of solvent, exposure time, agitation, organic drop volume, and salt concentration were optimized. The new method provided good average enrichment factors of >10.7 for all analytes, good precision (RSD < 8.5%) and good linearity (r2 > 0.9880). The limits of detection (LODs) were in the range of 0.00025-0.003 microg/L (S/N = 3). The SME was performed in different type of natural water samples and acceptable recoveries were obtained for the tested analytes. The results demonstrated that SME is a rapid, accurate and effective preparation method and could be successfully performed for the determination of antifouling agents in water samples.

Aniline Compounds↗