Determination of a new bisphosphonate, YM175, in plasma, urine and bone by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the determination of disodium dihydrogen(cycloheptylamino)methylenebisphosphonate monohydrate (YM175) in plasma, urine and bone is described. Plasma obtained in high-dose animal studies is pretreated by Method A, a simple method using 1 ml of plasma, which is based on deproteinization of plasma followed by coprecipitation of the drug with calcium phosphate and removal of excess calcium ions by AG 50W-X8 resin. Plasma obtained in lower-dose clinical studies is treated by Method B, a more sensitive method using 10 ml of plasma, which is based on solid-phase extraction using a Sep-Pak C₁₈ cartridge coupled with Method A. Urine and bone are treated similarly to Method B. The chromatographic system consists of a mobile phase at pH 11, an alkali-stable column and an electrochemical detector operating in the oxidation mode. The determination limit is 5 ng/ml for Method A and 0.5 ng/ml for Method B in plasma, 1 ng/ml in urine, and 25 ng/g in bone.     

Transient Photoresponses of a Phototactic Microorganism, Haematococcus Pluvialis, Revealed by Light Scattering

A new method, using incoherent light scattering, has been developed to measure the flagellar beating frequency of swimming microorganisms. By means of this method, transient changes of flagellar beating frequency in response to white light flashes have been revealed in samples of a phototactic microorganism, Haematococcus pluvialis. An increase of flagellar beating frequency occurs when the flash dose (flash intensity × flash duration) is sufficient. Reciprocity between light intensity and flash duration holds for durations not exceeding 60-80 ms. For lower doses a bimodal distribution of flagellar beating frequency is revealed. No effect is observed for very low flashes or for red stimuli, whereas green light is effective. A detailed analysis of experimental results has allowed us to determine the characteristic time of the effect and follow its evolution. The correlation of this effect with visually observed behavior is discussed and a possible underlying mechanism is suggested.     

Expanded bed protein A affinity chromatography of a recombinant humanized monoclonal antibody: process development, operation, and comparison with a packed bed method.

We show that expanded bed protein A affinity chromatography using Streamline rProtein A media is an efficient method for purifying a recombinant humanized monoclonal antibody from unclarified Chinese hamster ovary cell culture fluid and that it provides purification performance comparable to using a packed bed. We determined that the dynamic capacity of the expanded bed media is related to flow rate (measured in column volumes per hour) by a power function, which allows a high capacity at a low flow rate. At 250 cm h-1 with a 25 cm bed height (10 column volumes h-1), the dynamic capacity is 30 g l-1. The yield and purity (measured by the amount of host cell proteins, DNA, SDS-PAGE, and turbidity) of the antibody purified by expanded bed is comparable to the yield and purity obtained on a standard packed bed method using Prosep A media.     

Detection of protease inhibitors by a reverse zymography method, performed in a tris(hydroxymethyl)aminomethane-Tricine buffer system

A new detecting method for protease inhibitors, especially for low-molecular-weight inhibitors, is reported. Inhibitor samples were separated on a protein substrate-SDS-polyacrylamide gel in a Tris-Tricine buffer system that improves the separation and identification of peptides and low-molecular-weight proteins. After electrophoresis, the gel was incubated with the target proteases to hydrolyze the background protein substrate. The inhibitor bands, which were protected from proteolysis by the target proteases, were stained. Standard low-molecular-weight inhibitors, such as pepstatin A for pepsin or matrix metalloproteases inhibitor I for collagenase, as well as larger inhibitors, such as soybean trypsin inhibitor or aprotinin for tryspin and cystatin C for papain, were demonstrated by this method and showed clear blue inhibitor bands in the white background when the gels were treated with the target proteases. Some significant applications of this method are introduced. This method is an ideal system for discovering new protease inhibitors in small natural samples.     

Simple, quick and efficient site-directed antibody immobilization in a cartridge

A method is described for the simple, quick and efficient attachment of antibody within a cartridge for use as an immunoaffinity chromatography column. Antibodies are immobilized via their Fc regions through the use of periodate-oxidized carbohydrate functionalities of the immunoglobulin G. The method allows for the in situ coupling of the immunoglobulin G without prior removal of the oxidizing periodate solution. The entire procedure can be completed in 50 minutes. This method is especially useful for quick determinations of a particular monoclonal antibody's functionality or avidity towards a specific antigen. It may also be used in place of a conventional immunoaffinity column for the rapid isolation of small amounts of an antigen. This method will reduce the lengthy process of preparing an immunoaffinity column from several days to less than an hour.     

Determination of hexafluoroisopropanol,a sevoflurane urinary metabolite,by 9-fluorenylmethyl chloroformate derivatization

A reversed-phase HPLC method with fluorescence detection for the quantification of hexafluoroisopropanol (HFIP) in urine is presented. HFIP, a metabolite of the inhalation anesthetic sevoflurane, is excreted mainly in urine as glucuronic acid conjugate. After enzymatic hydrolysis of the glucuronate, primary amino groups of interferent urinary compounds are blocked by reaction with o-phthalic dicarboxaldehyde and 3-mercaptopropionic acid, followed by labeling of HFIP with 9-fluorenylmethyl chloroformate. The derivatization reaction proceeds in a water-acetonitrile (1:1) solution at room temperature with a borate buffer of pH 12.5 as a catalyst. A stable fluorescent derivative of HFIP is formed within 5 min. The HFIP-FMOC derivative is separated by reversed-phase chromatography with isocratic elution on an octadecyl silyl column (33x4.6 mm, 3 microm) and guard column (20x4.0 mm, 40 microm), at 35 degrees C, and detected by fluorescence detection at an excitation wavelength of 265 nm and an emission wavelength of 311 nm. The method detection limit is 40 pg, per 10-microl injection volume, corresponding to 16 microg/l of HFIP in urine. The among-series relative standard deviation is <6% at 200 microg/l (n=6). As a preliminary application, the method was used to detect HFIP concentration in the urine of two volunteers exposed for 3 h to an airborne concentration of sevoflurane in the order of 2 ppm.     

Water movement within a fringing reef flat,Orpheus Island,North Queensland,Australia

A technique to examine through reef water movement by direct tracing using fluorescent dyes is described. Results from an experiment conducted on the sand dominated fringing reef flat at Pioneer Bay, Orpheus Island, North Queensland, indicate net seaward water movement velocities in the order of 40 m day^-1, and considerable vertical mixing. A conceptual model of water movement is proposed in which dispersive type water movement is predominant when the reef flat is submerged, with advection being more significant when the reef flat is exposed. The application of the method to the study of the mechanisms of diagenesis is discussed and water quality rather than water agitation is suggested as being the principal reason for most rapid lithification being reported as occurring near the sediment water interface.     

A liquid chromatography assay for a quantification of doripenem, ertapenem, imipenem, meropenem concentrations in human plasma: application to a clinical pharmacokinetic study

A simple chromatographic assay based on ultra high performance liquid chromatography with ultraviolet detection at 295 nm is proposed to determinate simultaneously human plasma concentrations of imipenem, doripenem, meropenem and ertapenem. After deproteinization by acetonitrile, carbapenems are separated on a PentaFluoroPhenyl column with a binary gradient elution. This method is specific, accurate, precise (the intra-day and inter-day imprecision and inaccuracy are lower than 15%), sensitive (the limit of quantitation is equal to 0.50 mg/L for imipenem, doripenem, ertapenem, meropenem) and not time consuming (run time=7 min). An application of this method to measure ertapenem plasma concentrations in burn patients is presented.     

Bioelectrochemical fuel cell and sensor based on a quinoprotein,alcohol dehydrogenase

A biofuel cell, yielding a stable and continuous low-power output, based on the enzymatic oxidation of methanol to formic acid has been designed and investigated. The homogeneous kinetics of the electrochemically-coupled enzymatic oxidation reaction were investigated and optimized. The biofuel cell also functioned as a sensitive method for the detection of primary alcohols. A method for medium-scale preparation of the enzyme alcohol dehydrogenase [alcohol:(acceptor) oxidoreductase, EC 1.1.99.8] is described.     

Ending a decade of deception: a valiant failure, a not-so-valiant failure, and a success story

Prior studies involving two methods, Brooks Parsimony Analysis (BPA) and TreeMap, have found BPA to be the more reliable method. Recent criticisms leveled at these studies argue that the tests were unfairly created and biased in favor of BPA. The authors of a recent critique offered new exemplars to demonstrate flaws in BPA, plus a simple fix to correct the flaws found in TreeMap. A re‐evaluation of their exemplars clearly shows that the authors' calculations are incorrect, their understanding of the methods is lacking, and that their simple fix does not work. Additional analyses using TreeMap 2.02 are run to show that TreeMap 2.02, like TreeMap 1.0, cannot adequately deal with widespread parasites, contrary to the claims of its supporters. Furthermore, the exemplars corroborate previous findings that BPA, when calculated correctly, is more reliable than TreeMap1.0 and TreeMap 2.02 and therefore the method of choice in coevolutionary and biogeographic studies.     

Rapid,high-yield purification of rat liver 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase

A rapid method for the purification of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase from the livers of cholestyramine-fed rats is reported. The procedure involves a sequence of separations on affinity chromatography columns consisting of Blue Dextran-Sepharose, agarose-CoA, and agarose-HMG-CoA. The advantage of this method is its flexibility in scavenging enzyme that might be lost during purification, resulting in a yield of homogeneous reductase (specific activity approximately 10,000 nmol/min/mg protein) as high as 50%, which is at least twice that previously reported.     

Crystallization and preliminary characterization of mitogillin, a ribosomal ribonuclease from Aspergillus restrictus.

Mitogillin is a ribonuclease secreted by the fungus Aspergillus restrictus. The substrate for mitogillin is a short, universally conserved, sequence in ribosomal RNA. Cleavage of this sequence inactivates protein synthesis. Mitogillin was crystallized by a two-chamber vapor/liquid diffusion method using ethanol as the precipitant. This method has wider potential in the use of volatile organic solvents as precipitants. Crystals of mitogillin diffract X-rays to lattice d-spacings of at least 1.6 A, and belong to the monoclinic space group P2(1), with a = 50.4 A, b = 82.4 A, c = 38.2 A and beta = 99.8 degrees.     

A rapid microdetermination of phosphoserine, phosphothreonine, and phosphotyrosine in proteins by automatic cation exchange on a conventional amino acid analyzer

A cation-exchange chromatographic method for the separation and determination of phosphoserine, phosphothreonine, and phosphotyrosine in proteins after partial acid hydrolysis is described. The short column (0.6 X 8 cm) of an automatic amino acid analyzer was used and elution was carried out isocratically with 10 mM trifluoroacetic acid. The method is highly sensitive and each of the three O-phosphoamino acids can be accurately determined down to the 50-pmol level. Higher sensitivity may be obtained by the use of [32P]phosphate-labeled proteins. A correction factor for the decomposition of phosphoserine or phosphothreonine during acid hydrolysis can be deduced from the amount of inorganic phosphate recovered at the column void volume. The method is sensitive enough to be used for 32P-labeled proteins isolated by two-dimensional gel electrophoresis.     

Purification, Axenic Cultivation, and Description of a Soil Amoeba, Acanthamoeba sp.

SUMMARY. A small soil amoeba was purified by an agar-surface migration method. The amoeba was grown axenicly in a liquid medium composed of 1% proteose peptone, 1% glucose, and inorganic salts. The amoeba was identified as a species of Acanthamoeba. A hypothesis on the mechanism of agar-surface purification of amoebæ is proposed. The nutritional requirements of this isolate are discussed; it is concluded that this organism metabolizes glucose.     

Quantification of (+)-calanolide A, a novel and naturally occurring anti-HIV agent, by high-performance liquid chromatography in plasma from rat, dog and human

A HPLC method was validated for quantification of (+)-calanolide A (1), a novel anti-HIV agent, in rat, dog and human plasma. The synthetic intermediate (±)-12-oxocalanolide A (2) was found to be a suitable internal standard. Compounds were extracted from plasma using a solid-phase C₁₈ cartridge and quantified over the assay range of 12.5 to 800 ng/ml. The method was utilized to determine (+)-calanolide A pharmacokinetics in rats, dogs and humans. This is the first report of a validated HPLC assay for determination of (+)-calanolide A concentrations in rat and dog plasma as well as human plasma obtained from clinical trials. There was no evidence of in vivo epimerization of (+)-calanolide A to its inactive epimer (+)-calanolide B (3).     

A liquid chromatographic-electrospray ionization-tandem mass spectrometric method for determination of buprenorphine,its metabolite,norbuprenorphine, and a coformulant,naloxone, that is suitable for in vivo and in vitro metabolism studies

A liquid chromatographic-electrospray ionization-tandem mass spectrometric method has been developed and validated for determination of the antiabuse medication, buprenorphine, its primary metabolite, norbuprenorphine, and a proposed coformulant, naloxone. The method uses deuterated internal standards and a simple liquid-liquid extraction. Mass spectrometry employed selected reaction monitoring of the transitions of m/z 468 to 396 for buprenorphine, 472 to 400 for [2H4]buprenorphine, 414 to 101 for norbuprenorphine, 423 to 110 for [2H9]norbuprenorphine, 328 to 310 for naloxone, and 345 to 327 for its internal standard, [2H3]naltrexone. The method was accurate and precise across the dynamic range of 0.1 to 10 ng/ml. All analytes were stable in human plasma stored at room temperature for up to 24 h and after three freeze-thaw cycles. Reconstituted extracts were stable at -20 degrees C for up to 3 days. In human subjects receiving a sublingual tablet of 8 mg buprenorphine and 2 mg naloxone, buprenorphine and norbuprenorphine were detected for up to 24 h with respective maximum concentrations at 1 and 1.5 h. Maximal concentrations ranged from 2.2 to 2.8 and 1.5 to 2.4 ng/ml for buprenorphine and norbuprenorphine, respectively (i.e., approximately 6 nM). The method detected norbuprenorphine formation in human liver microsomes incubated with 5-82 nM buprenorphine, which encompasses the therapeutic plasma concentration range. When cDNA-expressed P450s were incubated with 21 nM buprenorphine, norbuprenorphine formation was detected for P450s 3A4, as previously described, but also for 3A5, 3A7, and 2C8. Buprenorphine utilization generally exceeded norbuprenorphine formation, suggesting that P450s 2C18, 2C19, 2D6, and 2E1 may also be involved in buprenorphine metabolism to other products. These results suggest this method is suitable for both in vivo and in vitro studies of buprenorphine metabolism to norbuprenorphine.     

Development of a novel,bioluminescence-based,fungal bioassay for toxicity testing

Naturally bioluminescent fungi, Armillaria mellea and Mycena citricolor, were used to develop a novel, bioluminescence-based bioassay for toxicity testing. Bioassays were carried out to assess the toxicity of 3,5-dichlorophenol (3,5-DCP), pentachlorophenol (PCP), copper and zinc. The results suggested that 60 min was a suitable exposure time for the bioassay. Light reduction was observed in response to 3,5-DCP, PCP and Cu for both A. mellea and M. citricolor, but to Zn only for A. mellea. Armillaria mellea was significantly less sensitive to 3,5-DCP and PCP than M. citricolor. The EC50 values for A. mellea and M. citricolor were similar to EC50 values for 3,5-DCP, PCP and Cu (but not Zn) of bioluminescence-based bacterial biosensors. They were also similar to EC50 values for Cu and Zn of a bioluminescence-based yeast biosensor. The results highlighted the importance of using both prokaryotic and eukaryotic biosensors. The novel bioassay provides a rapid and sensitive method to assess bioavailability of pollutants as well as a method to determine their toxicity to filamentous fungi. It also expands the range of organisms that can be used for bioluminescence-based toxicity testing by complementing existing biosensors.     

Determination of kynurenine by a simple gas—liquid chromatographic method applicable to urine,plasma, brain and cerebrospinal fluid

A simple, sensitive and specific method for the determination of kynurenine is described. This is based on alkaline cleavage of kynurenine, followed by solvent extraction, trifluoroacetylation and gas—liquid chromatography with electron capture detection. Using this method kynurenine has been determined in urine and plasma, and for the first time in brain and cerebrospinal fluid. Increases in kynurenine in brain, plasma and urine are demonstrated following tryptophan administration to man and rat.     

Studies of UDP-glucuronosyltransferase activity toward eugenol, using a gas chromatographic method of measurement

A method for the assay of uridine diphosphate (UDP)-glucuronosyltransferase activities toward some phenolic compounds and monoterpenoid alcohols is described. The method is based on the disappearance of the free substrate after incubation with microsomes and UDP-glucuronate. This disappearance is recorded using a gas chromatographic process. This method has been used, for example, to characterize the glucuronidation process of eugenol (4-allyl-2-methoxyphenol). The method could be extended to other substrates. Analytical conditions are given for some of them, especially monoterpenoid alcohols since the studies of their conjugations are a growing field of interest in evaluation of heterogeneity of UDP-glucuronosyltransferase. The method could also be used with other biological materials including cell suspension and crude liver biopsies.     

Affinity ligand selection from a library of small molecules: assay development, screening, and application

A facile and cost-effective process for screening synthetic libraries for an affinity ligand is described. A high throughput 96-well plate filtration method was designed to screen both discrete compounds and mixtures of compounds attached to a solid support. Human serum albumin (HSA) was used as a target protein to demonstrate the proof of concept. Detection and quantitation by fluorescence was accomplished with the use of fluorescamine to conjugate the protein in the filtrate. It is found that mixtures demonstrating low average binding reflect an overall lower hit rate of the components, whereas deconvolution of mixtures with high protein binding consistently provides a high hit rate. This differs from many of the previous experiences screening solid-phase mixtures in which high false positive rates are noted to occur. A total of 100K compounds were tested: 25K as discrete samples and 75K as mixtures. An overall hit rate of 8% was observed. Secondary screening of compounds measured specificity, recovery, and dynamic binding capacity. The effectiveness of the method is illustrated using an affinity column made with a representative lead compound. A similar purity was achieved in a single-step purification of HSA from serum as compared to that obtained by two steps of ion-exchange chromatography. The process for primary screening of a large number of compounds is simple, inexpensive, and applicable to any soluble target protein of known or unknown function from crude mixtures and may have additional utility as a generic chemical affinity tool for the functional characterization of novel proteins emerging from proteomics work.