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A method has been developed for the microsequencing of protein at subnanomole levels. The protein is carboxymethylated and freed of salts and reagents by reversed-phase chromatography prior to automated Edman degradation on a gas-phase sequencer. The carboxymethylated protein can also be fragmented chemically or enzymatically for further sequence analysis. The analytical techniques used to monitor the progress of the reactions all have picomole level sensitivity.
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A sensitive and selective liquid chromatography-mass spectrometry method has been developed for the simultaneous identification and quantitation of drug substances and metabolites in rat plasma. The method combines on-line turbulent-flow chromatography, high-performance liquid chromatography and mass spectrometry. This combination is considered to be a new approach suitable for fast bio-analysis in drug discovery. Dextromethorphan, and its two metabolites, dextrorphan and 3-methoxymorphinan served as model substances. The analytes present in plasma were collected on a Cyclone column using turbulent-flow chromatography and were subsequently transferred on-line to and focused on an X-Terra MS C8 column. The analytes were eluted by a linear gradient and detected by a fast scanning mass spectrometer. The detector response was quadratic and the dynamic range was estimated to be 0.5-100 ng/ml plasma or 12.5 pg to 2.50 ng injected into the system.
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It has long been the goal of biomaterials research in the field of orthopedics to develop synthetic structures exhibiting comprehensive bioactivity. In particular, an ideal bone-biomaterial would support the activity of osteoblasts in the development of new bone, while simultaneously being resorbed by osteoclasts as part of the lifelong orderly process of bone remodelling. Such resorbable calcium phosphate-based thin films and bulk ceramics have now been created by the high-temperature processing of a fine precipitate, formed from a colloidal sol and stabilized using an additive such as silicon. The materials have two characteristic features: a phase composition which is a mixture of calcium hydroxyapatite and a silicon stabilized tricalcium phosphate, and a microporous morphology based on inter-connected particles (0.2-1 microm in diameter). X-ray diffraction, infrared spectroscopy, nuclear magnetic resonance spectroscopy, and light scattering experiments indicate that the characteristic phase composition arises during sintering through substitution reactions where silicon enters the calcium phosphate lattice under conditions of high chemical reactivity. The crystallographic features are linked through the glaserite form of the apatite structure.
Five grams of seafood products were inoculated with one to 500 viable or 10(9) heat-killed cells of Listeria monocytogenes. The presence of the pathogen was detected by the polymerase chain reaction (PCR) with primers specific for fragments of the listeriolysin O (hly) gene (two sets) and for the invasion-associated protein (iap) gene (one set). For DNA preparation, boiling, either alone or in combination with lysozyme and proteinase K treatment, was not always sufficient to lyse L. monocytogenes, while treatment with Triton X-100 produced consistently good DNA suitable for amplification. To avoid false-negative and false-positive results, 48 h incubations were necessary and a subculturing step after an initial 24 h incubation greatly improved the results. The primers that amplified regions of the listeriolysin O gene gave clearer and stronger products than primers for the invasion-associated protein gene. Using this method we were able to detect one to five L. monocytogenes cells in 5 g of product in a total of 55 h.
A simplified, sensitive screening method for organic acidemias by gas chromatography-mass spectrometry using urease/direct preparations and tert.-butyldimethylsilylation (TBDMS) instead of trimethylsilylation (TMS) is described. We compiled GC-MS data on TBDMS derivatives, including methylene unit values, and quantifying and confirming ions, for use in the automated data system we developed. Quantification using [M-57]+ ions by mass chromatography was more sensitive, and the coefficient variation was smaller, compared with TMS derivatives. We confirmed the usefulness of this system, analyzing urine specimens from 53 patients with 15 different disorders.
Quantitative analytical methods, based on high-performance liquid chromatography with electrochemical detection, were developed for fenoldopam and its metabolites in human plasma. Two extraction methods, a liquid-liquid extraction method for fenoldopam and its methoxy metabolites and a liquid-solid extraction procedure for the sulfate and glucuronide conjugates of fenoldopam were developed. The extractions can either be performed manually or by robot. The limit of detection for fenoldopam, its sulfate and methoxy metabolites was 0.025, 2 and 0.5 ng/ml, respectively, at a signal to noise ratio of 4. The intra-assay and inter-assay coefficients of variation for both manual and robotic extraction procedures were comparable. These methods were suitably selective and sensitive for pharmacokinetic and metabolic studies of fenoldopam.
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A solid-phase extraction (SPE) method has been developed for the isolation of melanotan II from rabbit urine. The proposed extraction method makes it possible to selectively isolate melanotan II without significant loss of the peptide. Standard curves obtained from high-performance liquid chromatographic (HPLC) analysis of spiked urine extracts are linear from 0.1 to 4.0 micrograms/ml. The analytical method is shown to be highly reproducible, giving a relative standard deviation of less than 5% for both between-day and same-day analyses. The accuracy of the method obtained from standard plots ranges from -3.3 to 3.1%.
A simple reversed-phase high-performance liquid chromatography (HPLC) method for the simultaneous determination of caffeine and paraxanthine in human serum is described. Serum proteins are precipitated with perchloric acid and the resulting supernatant neutralized for direct injection onto an HPLC column. The method uses a phosphate-methanol mobile phase (85:15, v/v) at pH 4.9 with a flow-rate of 1.75 ml/min and quantitation is by UV absorbance at 274 nm. Elution times are approximately 18 min for caffeine and 8 min for paraxanthine. Theobromine and theophylline have elution times of 5.4 and 9.4 min and do not interfere in the assay. The intra-assay and between-assay means for precision and accuracy for both drugs are: 4.5% C.V. and 3.3% deviation. The sensitivity of the method is 50 ng/ml for each drug.
An improved solid-phase extraction (SPE) method was developed to isolate and concentrate trace levels of selected POPs (persistent organochlorine pollutants) in human serum prior to GC-MS in SIM mode or GC-ECD quantitation. The extraction involves denaturation of serum proteins with formic acid, SPE using C18 Empore disk cartridges, followed by elimination of lipid interferences using a sulfuric acid wash of the eluate. Use of the SPE disk improved assay throughput and gave a cleaner analytical matrix compared with previously reported solid-phase and liquid-liquid extraction techniques. The extraction method provided consistent recoveries at three fortification levels using 13C12 PCB 149 as internal standard. Recoveries ranged from 48 to 140% for organochlorine pesticides (6.25, 12.5 and 25 ng/ml) and 71 to 126% for polychlorinated biphenyls (0.625, 1.25 and 2.5 ng/ml).
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