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PubMed2001

Determination of alkylphenols after derivatization to ferrocenecarboxylic acid esters with gas chromatography-atomic emission detection.

A method is described for the rapid determination of alkylphenols in nonpolar matrixes. The alkylphenols are derivatized with ferrocenecarboxylic acid chloride so that every phenol molecule is labeled with one iron atom. The resulting esters are analyzed by gas chromatography with atomic emission detection (AED) in the iron-selective detection mode. This method utilizes the AED's low detection limit (0.05 pg/s) for iron and the high selectivity versus carbon (3.5 x 10(6)) for the detection of the alkylphenols. Because the derivatization is performed before the first step of sample preparation, the risk of analyte loss by adsorption or volatilization is minimized. The total recoveries in the lower ppm concentration range vary between 79 and 125%. The quantification of 20 C0-C3-alkylphenols in crude oils is demonstrated by analyzing a shale oil (SRM 1580) and a petroleum crude oil (SRM 1582). The complete workup is easily carried out in only 45 min/sample.

Journal Article↗

PubMed1997

A desalting approach for MALDI-MS using on-probe hydrophobic self-assembled monolayers.

One of the problems encountered in preparing samples for matrix-assisted laser desorption/ionization (MALDI) analysis is the presence of nonvolatile salts in the sample. This difficulty is often exacerbated by the necessity to prepare the sample in the appropriate sample-to-matrix ratio. This paper reports a probe surface derivatization method that greatly simplifies this sample preparation process. By constructing self-assembled monolayers of octadecyl mercaptan (C18) on the MALDI probe surface, we were able to generate a surface capable of reversibly binding polypeptides via hydrophobic interactions, which in turn, permits the analyte to be easily concentrated and desalted directly on the probe tip.

Chelating Agents↗

PubMed2003

Optimization and validation of a rapid method to determine citrate and inorganic phosphate in milk by capillary electrophoresis.

Quantification of phosphate and citrate compounds is very important because their distribution between soluble and colloidal phases of milk and their interactions with milk proteins influence the stability and some functional properties of dairy products. The aim of this work was to optimize and validate a capillary electrophoresis method for the rapid determination of these compounds in milk. Various parameters affecting analysis have been optimized, including type, composition, and pH of the electrolyte, and sample extraction. Ethanol, acetonitrile, sulfuric acid, water at 50 degrees C or at room temperature were tested as sample buffers (SB). Water at room temperature yielded the best overall results and was chosen for further validation. The extraction time was checked and could be shortened to less than 1 min. Also, sample preparation was simplified to pipet 12 microl of milk into 1 ml of water containing 20 ppm of tartaric acid as an internal standard. The linearity of the method was excellent (R2 > 0.999) with CV values of response factors <3%. The detection limits for phosphate and citrate were 5.1 and 2.4 nM, respectively. The accuracy of the method was calculated for each compound (103.2 and 100.3%). In addition, citrate and phosphate content of several commercial milk samples were analyzed by this method, and the results deviated less than 5% from values obtained when analyzing the samples by official methods. To study the versatility of the technique, other dairy productssuch as cream cheese, yogurt, or Cheddar cheese were analyzed and accuracy was similar to milk in all products tested. The procedure is rapid and offers a very fast and simple sample preparation. Once the sample has arrived at the laboratory, less than 5 min (including handling, preparation, running, integration, and quantification) are necessary to determine the concentration of citric acid and inorganic phosphate. Because of the speed and accuracy of this method, it is promising as an analytical quantitative testing technique.

Animals↗

PubMed1998

Detection of Cryptosporidium parvum in human feces by PCR.

C. parvum has a high pathologic potential also for man, especially for immununosuppressed patients. The microscopic detection of cysts in feces is neither easy nor always reliable. During the recent years, considerable progress has been achieved in establishing PCR-based approaches for i) sensitive detection of C. parvum in a variety of specimen types [3, 9-16, 18, 21, 24, 25, 27, 29-32, 34], ii) identification of individual genotypes of C. parvum [2, 4-7, 17, 19, 20, 22, 23, 26, 28, 33] and iii) viability testing of C. parvum organisms [8, 9, 13, 29, 30]. The protocols published so far include nested PCR [3, 8, 18, 34], RT-PCR [13, 29], and use of the UNG carryover prevention system [10]. The aim of this work was to establish a PCR system for the detection of C. parvum oocysts in stool samples, applying the same specimen preparation procedure as applied for immunofluorescence. In addition, we combined the UNG carryover prevention system with the use of long, PCR-generated digoxigenin-labelled probes, thus achieving a sensitivity comparable to nested PCR und circumventing the contamination risks associated with nested PCR protocols. We developed a simplified sucrose-cushion-based protocol for preparation of clinical specimens (adopted from [1]), satisfying both the needs of immunofluorescence and PCR. When tested with stool samples spiked with C. parvum oocysts, the analytical sensitivity of PCR was 3,500 oocysts/ml stool (immunofluorescence: 3,000 oocysts/ml stool), demonstrating that both methods were equivalent with respect to analytical sensitivity. However, when PCR and immnunofluorescence were applied to clinical samples (n=5) with known positivity for C. parvum, only the specimen with the shortest duration of storage (5 weeks) could be correctly identified by PCR (clinical sensitivity: 20%). Our results demonstrate, that the PCR approach presented in this work is not suited for highly sensitive detection of C. parvum in faeces. This was mainly due to the fact that sucrose-gradient purified material was used, which relies on the presence of morphologically intact oocysts in the specimens. Desintegration of oocysts by excystation and/or storage may lower the parasite yield of the protocol drastically. As a consequence, a protocol extracting the entire DNA from faeces should be used for PCR for detection of C. parvum [34].

Animals↗

PubMed1993

Urinary metanephrine and normetanephrine determined without extraction by using liquid chromatography and coulometric array detection.

We describe a procedure for the direct measurement of metanephrine (MN) and normetanephrine (NMN) in hydrolyzed urine, using HPLC with coulometric array detection. Acid-hydrolyzed samples were diluted and filtered before separation by isocratic reversed-phase ion-pair chromatography. Eight serial coulometric sensors, set at incrementally increasing anodic potentials, were used to screen lower-oxidizing interferences and provide stepwise oxidation of the metanephrines. Voltammetric behavior across three adjacent sensors was used to assess resolution and aid in peak identification. Values obtained in commercial controls were consistently within the specified target range. Variability, expressed as CV, was 5.45-9.22% between runs and 1.60-4.52% within-run for both compounds. The limit of detection was 2.6 micrograms/L for MN and 2.8 micrograms/L for NMN, with a linear response to 15.0 mg/L for both analytes. Results from patients' samples correlated well with those by a method involving dual ion-exchange extraction (r = 0.963, n = 82 for MN; r = 0.9768, n = 83 for NMN). This procedure provided high selectivity and objective peak purity information while greatly simplifying sample preparation.

Chromatography, High Pressure Liquid↗

PubMed2003

Metabolite characterization in drug discovery utilizing robotic liquid-handling, quadruple time-of-flight mass spectrometry and in-silico prediction.

An assay method for identification of metabolites from in vitro microsomal incubations was developed for use in the early stage of drug discovery. We have developed a practical approach which involves integrated sample generation, sample preparation, bioanalysis, and data handling to maximize sample throughput and speed up the process for identification of metabolites. The assay system consisted of a robotic liquid handler (Genesis workstation) to generate and process samples, PALLAS MetabolExpert software to predict possible metabolites, exact mass measurement via a tandem quadrupole time-of-flight mass spectrometer (QTOF-MS) coupled with liquid chromatography to analyze samples, MetaboLynx software to find potential metabolites and Advanced Chemistry Development/MS (ACD/MS) software to provide guidance to the most likely hypothetical metabolite chemical structures. For purposes of evaluating this new method, dextromethorphan, alprenolol, and propranolol were incubated separately for up to 60 minutes with rat and human hepatic microsomes. The incubation and sample preparation were carried out in 96-well plates using the Genesis workstation. The bioanalysis was performed by LC-MS/MS using QTOF with MetaboLynx software to find metabolites. Metabolic products formed in vitro by rat and human microsomes were separated using an analytical column C18 with gradient elution at flow rate of 250 micro l/min. The internal mass calibration was performed by continuous postcolumn infusion of Haloperidol. The mass spectra from incubations containing NADPH were compared to those without NADPH (control) using the MetaboLynx software to find potential metabolites. Finally, the MS/MS spectra were processed by the ACD/MS software to predict the chemical structure. MetaboLynx software successfully identified metabolites for each of the drugs studied by automatically discerning expected metabolites. Exact differences in masses between each metabolite and parent drug were measured from five replicate sample injections. All measured values are accurate to less than 0.001Da or 3.8 ppm with the standard deviation within 0.0015 Da, which allowed good prediction/confirmation of empirical formulae. Hypothetical chemical structures were achieved by the ACD/MS software and provided a useful tool to assist in prediction of the metabolic pathways of the drugs. The metabolites identified were in good agreement with previously published results for all three compounds. This new method will greatly enhance throughput, which in turn will facilitate our ability to rapidly provide this guidance to the synthetic chemist.

Alprenolol↗

PubMed2005

Direct determination of verapamil in rat plasma by coupled column microbore-HPLC method.

This report describes an automated coupled column microbore-high-performance liquid chromatography (HPLC) with fluorescence detection for direct determination of verapamil in small volume of rat plasma. We used HPLC system consisting of three columns such as precolumn, intermediate and analytical column and six-port switching valve and injected small volume of rat plasma to the system without sample preparation. An aliquot of sample was directly injected into Capcell Pak MF Ph precolumn for clean-up and enrichment, 35 mm Capcell Pak C18, intermediate column for concentration of compounds and 250 mm Capcell Pak C18 analytical column for separation of compounds and two mobile phases are used as mobile phase A (50mM ammonium phosphate, pH 4.5) and B (50mM ammonium phosphate:acetonitrile=70:30 v/v). Analysis of verapamil and internal standard, propranolol was performed with direct injection of 10 microl of rat plasma to the system and were eluted at 22 and 12 min, respectively, at a mobile phase flow rate of 0.5 (mobile phase A) and 0.15 ml/min (mobile phase B). The peaks of verapamil and internal standard were good shapes and well separated from any interfering endogenous peaks during a total run time of 25 min. The calibration curve for verapamil showed good linearity (r(2)=0.9997) over the concentration range of 0.01-2.50 microg/ml. The mean RSD (%) values of intra-day (n=5) and inter-day (n=5) variability of verapamil ranged from 1.96 to 9.06 and 0.62 to 3.08%, respectively. The LOD and LOQ were 0.01 and 0.025 microg/ml, respectively, for verapamil using 10 microl of rat plasma. An automated coupled column microbore-HPLC method was successfully applied to a pharmacokinetic study after intravenous injection of 3mg/kg of verapamil to the normal and dimethylnitrosamine (DMN)-induced hepatofibrotic rats.

Animals↗

PubMed1993

Automated determination of free phenytoin in human plasma with on-line equilibrium dialysis and column-switching high-performance liquid chromatography.

Free phenytoin in human plasma was automatically determined by on-line equilibrium dialysis using the automated sequential trace enrichment of dialysate (ASTED) sample preparation system and HPLC. The dialysis cell was a modification of the cell supplied with the ASTED. Total phenytoin was analysed with the same analytical set-up and plasma protein binding was determined. Free phenytoin was determined in plasma from epileptic patients and the results were compared to those obtained by ultrafiltration. Automated determination of free and total phenytoin in plasma by the ASTED-HPLC combination was shown to be an accurate and reproducible method and the results in free phenytoin analyses were in agreement with those found with ultrafiltration. The sample throughput with the automated on-line combination of dialysis and column-switching HPLC was 75 samples in 24 h when the sample was dialysed at 37 degrees C.

Autoanalysis↗

PubMed2004

A new, rapid, fully automated method for determination of fluconazole in serum by column-switching liquid chromatography.

A sensitive and rapid HPLC assay for the determination of fluconazole in serum is described. HPLC-integrated sample preparation allows direct injection of serum samples without any pretreatment. The in-line extraction technique is carried out by automatically switching from the extraction column (Lichrospher ADS C8) to the analytic column (Nucleosil C18). After 6 minutes the matrix passes the extraction column, and the retained analyte is quantitatively transferred to the analytic column, where separation by isocratic HPLC is performed. The extraction eluent is sodium dihydrogen phosphate buffer, pH 5.0 (50 mM), and the analytic eluent is acetonitrile/sodium dihydrogen phosphate buffer, pH 5.0 (50 mM) (26.8/73.2, vol/vol). Fluconazole is detected according to its absorption maximum at 210 nm. The lower limit of quantification (LLOQ) is 0.65 microg/mL, the limit of detection (LOD) is 0.2 microg/mL, and the quantification range is 0.65-23.3 microg/mL. The assay was precise with a between-run coefficient of variation of < or = 5.59%. The within-run accuracy was 99.8% and 103.4%, and the between-run accuracy was 99.2% and 99.7%, respectively, for the concentrations 23.3 microg/mL and 1.3 microg/mL. The recovery was 78%. The described procedure allows sample cleanup and determination within 20 minutes, thereby facilitating drug monitoring in clinical routine. The method was applied successfully.

Antifungal Agents↗

PubMed2005

Determination of the GABA analogue succinic semialdehyde in urine and cerebrospinal fluid by dinitrophenylhydrazine derivatization and liquid chromatography-tandem mass spectrometry: application to SSADH deficiency.

Succinic semialdehyde (SSA) accumulates in the inborn error of meta- bolism succinic semialdehyde dehydrogenase deficiency owing to impaired enzymatic conversion to succinic acid. We developed a stable-isotope dilution liquid chromato- graphy-tandem mass spectrometry method for the determination of SSA in urine and cerebrospinal fluid samples. Stable-isotope-labelled [13C4]SSA, serving as internal standard, was prepared by reaction of ninhydrin with L-[13C5]glutamic acid. SSA in body fluids was converted to its dinitrophenylhydrazine (DNPH) derivative, without sample purification prior to the derivatization procedure. The DNPH derivative of SSA was injected onto a C18 analytical column and chromatography was performed by isocratic elution. Detection was accomplished by tandem mass spectrometry operating in the negative multiple-reaction monitoring mode. The limit of detection was 10 nmol/L and the calibration curves over the range 0-500 pmol of SSA showed good linearity (r2 > 0.99). The intra-day coefficient of variation (n = 10) for urine was 2.7% and inter-day coefficient of variation (n = 5) for urine was 8.5%. The average recoveries performed on two levels by enriching urine and cerebrospinal fluid samples ranged between 85 and 115%, with coefficients of variation < 8%. The method enabled the first determination of normal values for SSA in urine and pathological values of SSA in urine and cerebrospinal fluid samples derived from patients with succinic semialdehyde dehydrogenase deficiency.

Blood Chemical Analysis↗

PubMed2000

Determination of Al, Cu, Li and Mn in spruce seeds and plant reference materials by slurry sampling graphite furnace atomic absorption spectrometry.

An ultrasonic slurry sampling graphite furnace AAS method was developed for the determination of Al, Cu, Li and Mn in spruce seeds, NBS SRM 1575 pine needles and GBW CRM 07602 bush branches and leaves. The only sample preparation was grinding in a Mixer Mill before preparing a slurry by adding 0.14 mol/L nitric acid to a small sample aliquot. Cryogenic grinding was used for the spruce seeds to solve the problem of agglomerating during grinding at room temperature. A modified sample tray was applied allowing the use of both the commercial 1.5 mL vials and home-made 15 mL vials. With optimal conditions for ultrasonic agitation the homogeneity and particle size distributions in the slurries prepared in the two different vials were similar. Several aspects of the slurry sampling approach are discussed and data of important parameters are given, including the total number of particles injected into the graphite furnace, densities of the materials and percentage of analyte extracted into the liquid phase of the slurry. The density of the materials was determined by two methods; by using a Coulter particle analyser and by using a gravimetric method. The two methods gave similar accuracy and precision. The concentration ranges of the elements (in microg g(-1)) were: 80-2100 for Al, 3-15 for Cu, 0.06-2.5 for Li and 50-700 for Mn. External calibration with aqueous standards was employed. Chemical modifiers were not found to be necessary. The relative standard deviations were in the range 1.7-7%. Analyses of the two certified plant reference materials confirmed the accuracy of the method. In addition no significant difference was found for analyses of digested and slurried spruce seeds. The detection limit was 10 ng g(-1) for Li and 170 ng g(-1) for Cu. The characteristic mass (area measurements) was 4.4 pg for Li and 11 pg for Cu. For Al and Mn less sensitive wavelengths were used.

Metals↗

PubMed2001

Validated method for the determination of the novel organo-ruthenium anticancer drug NAMI-A in human biological fluids by Zeeman atomic absorption spectrometry.

NAMI-A is a novel ruthenium-containing experimental anticancer agent. We have developed and validated a rapid and sensitive analytical method to determine NAMI-A in human plasma, plasma ultrafiltrate and urine using atomic absorption spectrometry with Zeeman correction. The sample pretreatment procedure is straightforward, involving only dilution with an appropriate hydrochloric acid buffer-solution. Because the response signal of the spectrometer depended on the composition of the sample matrix, in particular on the amount of human plasma in the sample, all unknown samples were diluted to match the matrix composition in which the standard line was prepared (plasma-buffer 1:10 v/v). This procedure enabled the measurement of samples of different biological matrices in a single run. The validated range of determination was 1.1-220 microM NAMI-A for plasma and urine, and 0.22-44 microM for plasma ultrafiltrate. The lower limit of detection was 0.85 microM in plasma and urine and 0.17 microM in plasma ultrafiltrate. The lower limit of quantitation was 1.1 and 0.22 microM, respectively. The performance of the method, in terms of precision and accuracy was according to the generally accepted criteria for validation of analytical methodologies. The applicability of the method was demonstrated in a patient who was treated in a pharmacokinetic phase I trial with intravenous NAMI-A.

Antineoplastic Agents↗

PubMed2004

Assay of histamine by nano-liquid chromatography/tandem mass spectrometry with a packed nanoelectrospray emitter.

A nano-liquid chromatographic /tandem mass spectrometric (nanoHPLC/MS/MS) method has been developed for the sensitive determination of histamine, a vital neurotransmitter. The method involved pre-column derivatization of histamine with 7-fluoro-4-nitrobenzoxadiazole (NBD-F), thus increasing the hydrophobicity of the analyte and allowing it to be retained and stacked on a nanoelectrospay emitter packed with C18 reversed-phase particles. Sodium 1-heptanesulfonate, added as an ion-pairing reagent in the mobile phase for sample loading, was found to facilitate greatly the analyte retention. The packed nanoelectrospray emitter was easily prepared using fused-silica capillary of 75 microm i.d., and included creating an electrospray emitter tip and then slurry-packing a 5 cm long column with 5 microm silica-based particles. After sample loading, the mobile phase was changed for separation and a characteristic precursor-to-product ion transition, m/z 275 --> 258, was used for the quantification. A linear calibration curve in the range from 1.0-100 ng/mL histamine with an r2 value of 0.9995 was obtained. The detection limit was estimated to be 0.1 ng/mL histamine in water. In a preliminary validation, the method was applied to determine histamine in alcoholic beverages and rat brain tissues.

Alcoholic Beverages↗

PubMed2002

Rapid determination of 226Ra and uranium isotopes in solid samples by fusion with lithium metaborate and alpha spectrometry.

A simple and rapid method has been developed to determine 226Ra in rocks, soils, and sediments. Samples are decomposed by fusion with lithium metaborate and the melt is dissolved in a solution containing sulfates and citric acid. During the dissolution, a fine suspension of mixed barium and radium sulfates is formed. The microcrystals are collected on a membrane filter (pore size 0.1 microm) and analysed in an alpha spectrometer. Application of a 133Ba tracer enables us to assess the loss of the analyte, which only rarely exceeds 10%. All analytical operations, beginning from sample decomposition to source preparation for alpha spectrometry, can be accomplished within 1 or 2 h. With uranium determination, the filtrate is spiked with a 232U tracer and passed through a column loaded with a Dowex AG (1 x 4) anion-exchange resin in the sulfate form. Interfering elements are eluted with dilute sulfuric acid followed by concentrated hydrochloric acid. Uranium is eluted with water, electrodeposited on silver discs, and analysed in the alpha spectrometer. The method was tested on reference soil and sediment materials and was found to be accurate within the estimated uncertainties.

Borates↗

PubMed1986

Efficient HPLC separation of N-p-nitrophenylglycosylamines derived from complex oligosaccharide mixtures. Human orosomucoid as a model.

With human orosomucoid as model compound, a new method was developed to separate neutral oligosaccharides as N-p-nitro-phenylglycosylamines. Asialo orosomucoid was prepared by treatment with neuraminidase and purified by size exclusion HPLC on TSK 2000 SW. Oligosaccharides were isolated by reversed phase HPLC on Hamilton PRP-1 after hydrazinolysis and re-N-acetylation. Glycosylamination was performed with p-nitroaniline in DMSO-formic acid-water, where the whole mixture of oligosaccharide derivatives was isolated by reversed phase HPLC on Hamilton PRP-1 and separated into single glycosylamines on Shandon Hypersil ODS. The purified glycosylamines could be methylated by a new rapid method with sodium hydroxide and methyl iodide in DMSO, isolation and purification were carried out on Hamilton PRP-1 and Spherisorb ODS 2, respectively, as described for the glycosylamines. Preparative scale HPLC separations were performed on analytical columns using repetitive collection mode and automatic sample loading by means of a peristaltic pump operated by the HPLC controller. The purified glycosylamines can be used for sugar analyses or, after permethylation, for methylation analyses or related procedures.

Amino Sugars↗

PubMed1999

A two-dimensional peptide gel electrophoresis system for phosphopeptide mapping and amino acid sequencing.

A novel two-dimensional electrophoresis system to be carried out on polyacrylamide gels under nondenaturing conditions was developed to efficiently fractionate the peptides resulting from endoproteinase digestion of 32P-labeled proteins. In particular, nondenaturing gel isoelectric focusing was combined with alkaline 40% polyacrylamide gel electrophoresis to generate phosphopeptide maps with high reproducibility, thus allowing both protein fingerprinting and comparative analysis of different samples. The potential application of this method for subsequent amino acid sequencing of the isolated phosphopeptides was further demonstrated by successful manual and automated Edman sequencing. Taken together these data show that such a simple and precise approach is suitable for both analytical and preparative aims.

Amino Acid Sequence↗

PubMed2002

Gas chromatography-high-resolution mass spectrometry based method for the simultaneous determination of nine organotin compounds in water, sediment and tissue.

A GC-HRMS based method for the accurate and sensitive determination of nine organotin compounds, tetrabutyltin (TeBT), tributyltin (TBT), dibutyltin (DBT), monobutyltin (MBT), triphenyltin (TPhT), diphenyltin (DPhT), monophenyltin (MPhT), tricyclohexyltin (TCyT), and dicyclohexyltin (DCyT) in sediment, tissue and water samples is presented and discussed. Mass spectral features of these analytes via both low resolution quadrupole and high resolution magnetic sector, GC-HRMS conditions under selective ion monitoring mode and QA/QC criteria for the positive identification of analyte are all provided. Linearity of response and minimal detectable limits are illustrated for each of the nine compounds monitored and the estimates of method limits-of-detection were 7-29 ppt for water and 0.35-1.45 ppb for tissue or sediments. Sample preparation considerations and precision are discussed for spiked water and sediment samples, whereas method accuracy was established by analysing a certified reference material (CRM) mussel sample and comparing our results to the assigned values. Good agreement was found between our results and assigned or indicative values for MBT, DBT, TBT, DPhT and TPhT (cyclohexyl-tins were not present in the CRM).

Gas Chromatography-Mass Spectrometry↗

PubMed2003

Determination of primary aromatic amines in water food simulant using solid-phase analytical derivatization followed by gas chromatography coupled with mass spectrometry.

Solid phase analytical derivatization with trifluoroacetic anhydride has been introduced as sample preparation for the determination of primary aromatic amines in water by gas chromatography coupled with mass spectrometry. Water was used as a food simulant for testing migration from laminated flexible food packaging materials. The method was evaluated for 8 primary aromatic amines in 200 ml water samples, which resulted in detection limits in the 0.1-0.4 microg/l range, relative standard deviations in the 4-17% range and acceptable linearity (R2 = 0.997-1.000). Detectable levels of 2,4-diaminotoluene, 2,6-diaminotoluene and 4,4'-methylenedianiline were found in water food simulant from some of the investigated food packaging materials.

Amines↗