High Pressure Liquid Chromatography

We previously reported a solid-phase extraction (SPE) method for determination of the neurotoxin domoic acid (DA) in both seawater and phytoplankton by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the purpose of sample desalting without DA pre-concentration. In the present study, we optimized the SPE procedure with seawater and phytoplankton samples directly acidified with aqueous formic acid without addition of organic solvents, which allowed sample desalting and also 20-fold pre-concentration of DA in seawater and phytoplankton samples. In order to reduce MS contamination, a diverter valve was installed between LC and MS to send the LC eluant to waste, except for the 6-min elution window bracketing the DA retention time, which was sent to the MS. Reduction of the MS turbo gas temperature also helped to maintain the long-term stability of MS signal. Recoveries exceeded 90% for the DA-negative seawater and the DA-positive cultured phytoplankton samples spiked with DA. The SPE method for DA extraction and sample clean-up in seawater was extended to mammalian fluids and tissues with modification in order to accommodate the fluid samples with limited available volumes and the tissue extracts in aqueous methanol. Recoveries of DA from DA-exposed laboratory mammalian samples (amniotic fluid, cerebrospinal fluid, plasma, placenta, and brain) were above 85%. Recoveries of DA from samples (urine, feces, intestinal contents, and gastric contents) collected from field stranded marine mammals showed large variations and were affected by the sample status. The optimized SPE-LC-MS method allows determination of DA at trace levels (low pg mL −1 ) in seawater with/without the presence of phytoplankton. The application of SPE clean-up to mammalian fluids and tissue extracts greatly reduced the LC column degradation and MS contamination, which allowed routine screening of marine mammalian samples for confirmation of DA exposure and determination of fluid and tissue DA concentrations in experimental laboratory animals.

Following the intravenous administration of thromboxane (TX) i~, the stable hydration product of TXA z, to human and nonhuman prim_~.t~,~ file mo~¢ abundant urinary metabolites are 2,3-dinor-TXl~ and ll-dehydro-TXl~. However, it is not known w£,.+~~' ,,+-.-::,~:~,::,~:. :+ ~,:,,~ ~,'-~+on of TXlg z to its enzymatic metabolites is an accurate representation of TXA z metabolism. Thus, we have compared the metabolic disposition of synthetic TXA z and TXB z via the fl-oxidation and ll-OH-dehydrogenase pathways in vivo in the monkey. TXA, or ~ (20 ng/kg) was intravenously administered to four cynomolgus monkeys pretreated with aspirin in order to suppress endogenous TXA z production. Urinary TXI~, 2,3-diuor-TXl~ and ll-dehydro-TXl~ were measm'ed before, during and up to 24 h after thromboxane administration by means of reversed-phase high-pedormance liquid chromatography radioimmunoassay. Aspirin treatment suppressed urinary 2,3-dinor-TXl~ and ll-dehydro-TXl~ by approx. 75%. A similar fractional conversion of TXA, and ~ into 2,3-dinor-TXl~ and ll-dehydro-TXl~ was found. These results suggest that TXA 2 is hydrolyzod to ~ prior to enzymatic degradation and that metabolites of the latter represent reliable indices of TXA 2 biosynthesis. Due to the variability in the conversion of thromboxanes into 2,3-dinor-TXi~ and II-dehydro-TXBz, the measurement of both metabolites seems to represent a more reliable index of acute changes in TXA, production.

Salicylic acid is a major hydrolytic degradation product of aspirin, responsible especially for gastric irritation during oral aspirin administration. This impurity was investigated in 12 different brands of aspirin formulation readily available in our locality. A simple, rapid and sensitive high performance liquid chromatographic method was adopted for this investigation. The mobile phase was methanol/water (20/80, v/v) adjusted to pH 2.5 with phosphoric acid and was run on a 50 mm reversed-phase column monitored at 240 nm. The limit of detection for salicylic acid was 5 ng. Only three of these formulations showed the presence of salicylic acid impurity and all these contained salicylic acid in excess of the USP 1980 limit of 0.3% salicylic acid per tablet.

Two simple, rapid, and selective analytical procedures were developed for the simultaneous determination of paracetamol (PR) and tramadol hydrochloride (TR) in a binary mixture using highperformance liquid chromatography with UV detection (HPLC-UV) and gas chromatography with mass spectrometry (GC-MS) techniques. HPLC resolved the two compounds on a Hypurity Advance column using a mobile phase consisting of phosphate buffer pH 6.3 and acetonitrile (90:10, v/v). PR and TR were detected by their UV absorption at 220 nm. GC-MS involved separation of the two compounds using 100% dimethylpolysiloxane (Rtx-1) column with temperature programming. The EI mass spectrum of PR was characterized by [M] + at 151 and a base peak at m/z 109 while TR mass spectrum was characterized by [M] + at 263 and a base peak at m/z 58. Quantification of the analytes in both methods was based on measuring the peak areas. The reliability and analytical performance of the proposed methods including linearity, ranges, precision, accuracy, detection, and quantification limits were statistically validated. Calibration curves were linear over the range 10-400 µg/mL for both PR and TR using the HPLC method and over the ranges of 75-500 and 25-350 µg/mL for PR and TR, respectively, using the GC-MS method. The proposed methods were successfully applied for the determination of the two compounds in laboratory-prepared mixtures and in commercially available tablet formulation. No interference peaks were observed from common pharmaceutical adjuvants. The results compared favorably with those obtained by a derivative spectrophotometric method.

Essential oils have been studied for their unique ability to act as antioxidants. Antioxidant activities of 423 essential oils of 48 different botanical families were evaluated for their antioxidant activities as free radical scavenging agents using the 1,1-diphenyl-2-picrylhydrazyl method. Seventy-three oils showed 50% or more inhibition at a concentration of 1.25 mg/mL. The 73 most active oil samples were further evaluated for their scavenging activities using series of dilutions to estimate their EC 50 . The EC 50 of the 73 most active oils ranged from 4 to 2000 μg/mL. Oils having an EC 50 of less than 300 μg/mL (20 selected samples) were subjected to β-carotene bleaching antioxidant activity test and more detailed analysis including thin layer chromatography, gas chromatography/mass spectrometry, high performance liquid chromatography and bioautography. Essential oils of the botanical families Lamiaceae and Myrtaceae were the most effective antioxidants. Thymol and carvacrol were the major constituents in most of the essential oils of the family Lamiaceae and eugenol was the major terpene in all of the essential oils of the family Myrtaceae.

Buprenorphine is a strong narcotic analgesic. It is also used in the substitution therapy for opium alkaloid addicts. The aim of this paper was to develop and validate a highly sensitive highperformance liquid chromatography-electrospray ionization-mass spectrometry method for simultaneous determination of buprenorphine and norbuprenorphine in human serum. The developed methodology was then applied to real clinical cases in a clinical toxicology setting. Extraction of analytes has been done using solid-phase extraction. Chromatographic separation was achieved on a LiChroCART column with a Purospher RP-18e cartridge, and for detection an LCQ mass spectrometer with an ion trap analyzer was used. Quantitation of buprenorphine and norbuprenorphine was performed in a single ion monitoring mode (m/z 468 buprenorphine, m/z 414 for norbuprenorphine) in order to increase the sensitivity of the method. The standard curves for both compounds were linear over the range of 0.2-10 ng/mL (r 2 > 0.995). The quantitation limit was 0.2 ng/mL for both analytes. The method was used for quantitation of both buprenorphine and norbuprenorphine in the serum of 15 patients undergoing the buprenorphine substitution therapy. Serum concentrations ranged between 0.36 and 4.60 ng/mL for buprenorphine and 0.21 and 2.50 ng/mL for norbuprenorphine, with buprenorphine single dosages from 0.8 to 6.0 rag.

Peanut testae are potent sources of polyphenols. When the water extracts and acid hydrolysates of five different-colored testae were analyzed by HPLC, chromatograms monitored at 280 nm varied remarkably, whereas two major peaks in the chromatograms monitored at 530 nm were detected only in kernels having completely or partially black color. After acid hydrolysis of the extracts, cyanidin was detected in each of the hydrolysates. By respectively subjecting the black testae of raw and roasted (175°C for 20 min) kernels of a black colored cultivar to water extraction and HPLC analysis, a prominent peak was detected in both extracts. The structure of the substance under those peaks was identified by mass and NMR spectrometry as cyanidin 3-sambubioside in peanut testae for the first time. Subjection of cyanidin 3-sambubioside to antioxidation and anti-inflammation assessments revealed that it was a potent antioxidant and inhibitor of nitric oxide production.

The synthesis of a limited series of non-racemic atropisomeric 1-(2-(4methyl-2-thioxothiazol-3(2H)-yl)phenyl)-3-(hetero)aryl-(thio)ureas is described. Using NMR titration experiments monitoring the shift of the two NH of the (thio)urea and the C-5 hydrogen of the heterocycle, the binding constants for some optically pure (thio)ureas with the enantiomers of N-protected amino acid tetrabutylammonium salts were determined in CD 3 CN. The obtained enantioselectivities were modest. Contrary to what was expected on the basis of the NH acidity in thiourea versus urea group, the association constants were smaller with the thiourea than with the corresponding urea. X-ray data, DFT calculations, and NMR provided the explanation of that unexpected behavior: the urea presents a pre-organized (Z,Z) conformation suitable for a double hydrogen bond with the carboxylate anion, the thiourea presents a (Z,E) conformation, which must be reorganized in a constrained (Z,Z) conformation in the complex. An intramolecular hydrogen bond between one NH and the thiocarbonyl group of the heterocycle, which is present in the thiourea and absent in the urea, might also contribute to the smaller K ass for the thiourea. The possible implication of these observations in the field of bifunctional organocatalysis is briefly discussed.

Root barks of Chionanthus virginicus L. are used in homeopathic medicines in the treatment of icterus and hepatitis. The objective of this study is to identify novel secoiridoids and lignans and to develop a simple and reliable HPLC method for the determination of oleuropein, phillyrin, total secoiridoids and total lignans for quality control and stability studies of C. virginicus herbal drug and preparations. Secoiridoids and lignans were purified by preparative HPLC. Compounds previously described were identified by HPLC according to their retention times and UV spectra. Structures of new compounds were determined by NMR. Two compounds namely excelside B and acetoxypinoresinol-4"-O-beta-D-glucoside are described for the first time in the drug. HPLC separation was performed on Symmetry C18 (Waters) by gradient elution using acetonitrile and 0.2% aqueous phosphoric acid. The method was validated for specificity, linearity, precision, accuracy, limits of detection and quantifica...

The development and validation of an HPLC method for the quantification of eight peptide antigens from the therapeutic cancer vaccine DPX-0907 is described. The antigens were formulated in DepoVax TM , a patented liposomal vaccine delivery platform used in a phase 1 study for breast, ovarian, and prostate cancers. A gradient reversed-phase method with UV detection was optimized for separating and quantifying the peptide mixture. Several extraction methods investigated to extract the peptides from the lipids led to poor recovery of one or more of the peptides. A simple, reproducible, and high-recovery extraction procedure for the simultaneous quantification of hydrophilic and hydrophobic peptides was discovered using a liquid-liquid extraction with water-saturated n-butanol and sodium bicarbonate (0.1 M). The method was found to be specific, linear, accurate, precise, and reliable within the range of 50-150% of the nominal concentration for DPX-0907. The validated method was successfully applied to the assay of peptide content in pre-clinical and clinical batches of DPX-0907.

The construction of expression vectors encoding either the human insulin A-or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A-and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source. The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E. coli resulted in product yields of grams per litre of culture broth, e.g. 4.5 g of insulin B-chain fusion protein per litre of culture broth. This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture. Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture. The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction of the biomass yield coefficient with respect to glucose.

A method has been developed for the determination of relevant trace impurities (alkali, alkaline and transition metals) in high purity quartz by ion-chromatography. In situ reagent (HF) purification and simultaneous sample dissolution was achieved in a multichannel vapour phase digestion assembly. Twenty-one samples can be digested at a time in this vapour phase system. Significant decrease in the process blank levels for all the analytes was observed. Drastic reduction (250 times) of NH 4 + blank was achieved in the described vapour phase digestion, which enables the determination of trace concentration of sodium in high purity quartz. After volatilisation of the matrix and unreacted HF, the clear water leached solutions were injected into an ion-chromatograph equipped with conductivity detector for the determination of alkali and alkaline earth metals. In the case of transition metals, the trace residues were leached with 10 mM HCl and after separation on a mixed bed analytical column (IonPac CS5) were detected by spectrophotometry after post column derivatisation using 4-(2-pyridylazo)resorcinol (PAR). The accuracy of the result was checked by their comparison with those obtained by independent methods like inductively coupled plasma (ICP) MS and ICP atomic emission spectrometry. The achievable detection limits are between 0.4 ng/g (Li) and 22 ng/g (Mn). The application of the method to the determination of the above trace metals in two high-purity-grade quartz samples is demonstrated.

A large number of micropropagated Euphorbia millii shoots from temporary immersion bioreactor showed thick broad leaves that were translucent, wrinkled and/or curled and brittle, symptoms of hyperhydricity. The environment inside bioreactor normally used in plant micropropagation is characterised by high relative humidity, poor gaseous exchange between the internal atmosphere of the bioreactor and its surrounding environment, and the accumulation of ethylene, conditions that may induce physiological disorders. A comparison of hyperhydric shoots (HS) with normal plants shows marked increase in malondialdehyde (MDA) content in HS plants. MDA, a decomposition product of polyunsaturated fatty acids hydroperoxides, has been utilized very often as a suitable biomarker for lipid peroxidation, which is an effect of oxidative damage. This hypothesis is also confirmed by the higher lipoxygenase (LOX) activity in HS plants. The potential role of antioxidant enzymes in protecting hyperhydric shoots from oxidative injury was examined by analyzing enzyme activities and isozyme profiles of hyperhydric and non-hyperhydric leaves of E. millii. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity were significantly higher in hyperhydric tissue as compared to non-hyperhydric normal leaf tissue. After native polyacrylamide gel electrophoresis (PAGE) analysis, seven SOD isoenzymes were detected and the increase in SOD activity observed in hyperhydric tissue seemed to be mainly due to Mn-SOD and Cu/Zn-SOD. The activity of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) was proportionally increased in HS tissue compared to normal leaves indicating a crucial role in eliminating toxic H 2 O 2 from plant cells. The depletion of GSH and total glutathione in spite of higher GR activities observed in HS tissue indicates that mechanism of antioxidant defense was by enhanced oxidation of GSH to GSSG by DHAR yielding ascorbate (AA). The antioxidant metabolism has been shown to be important in determining the ability of plants to survive in hyperhydric stress and the up regulation of these enzymes would help to reduce the build up of ROS.

The aim of the present study was to modify and validate a highperformance liquid chromatographic (HPLC) method for determining 2,3 and 2,5 dihydroxybenzoic acid (2,3-DHBA and 2,5-DHBA) from salicylic acid in human plasma. The mobile phase was a mixture of sodium acetate/citrate (pH 2.5) 30 mM-methanol (93:7, v/v). The injection volume was 10 µL. Retention time for 2,5-DHBA, and 2,3-DHBA was 4.5 ± 0.10 and 5.8 ± 0.15 min, respectively. The detection and quantification limits were 10 and 40 nM for 2,3-DHBA and 8 and 20 nM for 2,5-DHBA. Linearity was evaluated in the range of 40-1600 nM for both metabolites. Interand intra-analysis variation coefficient was below 10%. Good recoveries of more than 99% were obtained for both metabolites using this method.

Two hemolysins, Sticholysin I (St I) and Sticholysin II (St II) were puri®ed from the sea anemone Stichodactyla helianthus combining gel ®ltration and ion exchange chromatography. The amino acid composition of both cytolysins was determined revealing a high proportion of glycine, lysine, tyrosine and non-polar amino acids (alanine, leucine and valine). Cysteine was not found in either polypeptide. Molecular masses of St I and St II were 19401 and 19290 Da, respectively. N-terminal sequence analysis of St I and St II showed a high homology between them suggesting they are isoforms of the same cytolysin. Compared with other sea anemone cytolysins, St I and St II contain a 22 amino acid insertion fragment also present in Eq T II/Tn C and probably in CaT I and Hm T and absent in C III, the major hemolysin previously reported in this anemone. 7

With the aim of developing a new enzyme-linked immunosorbent assay test to detect autoantibodies in the sera of rheumatoid arthritis patients with a high sensitivity and specificity using synthetic citrullinated peptides of fibrin (which is abundant in rheumatoid synovium) as antigenic substract, peptides belonging to α- and β-fibrin chains were selected by computer-aided prediction of antigenicity and epitope mapping and synthesized in solid phase. We analysed by enzyme-linked immunosorbent assay 133 sera from patients with well-characterized rheumatic diseases, including 67 patients with rheumatoid arthritis. The results of the immunoassays reported highlight the usefulness of fibrin-related peptides in rheumatoid arthritis diagnosis and, especially, the ability and specificity of the [Cit621,627,630]α-fibrin(617–631) (αfib617) peptide sequence to recognize the autoantibodies that are present in rheumatoid arthritis patients.

The recently introduced fast protein liquid chromatography (FPLC) system of Pharmacia (Uppsala, Sweden) was employed to isolate rather large peptides derived from thermolysin by selective chemical fragmentation at methionine in positions 120 and 205 of the polypeptide chain of 316 amino acid residues. Thermolysin was cleaved under conditions of limited fragmentation in order to produce, besides fragments 1 -1 20, 12 1 -205 and 206 -3 16, the overlapping fragments 1-205 and 121 -3 16. These polypeptides were separated employing prepacked Mono Q or Mono S columns (quaternary ammonium and sulfonic acid support, respectively). The columns were equilibrated with acetate-7 M urea buffer, pH 5.0 or 6.0, and eluted with a gradient of sodium chloride or acetate. Separations were achieved in 10-20min and were carried out also at a semipreparative level (1-3 mg per run). All five protein fragments were isolated in homogeneous form, as judged by amino acid analysis and electrophoresis. Considering that protein fragmentation with cyanogen bromide is the most commonly used procedure to achieve selective chemical fragmentation of a polypeptide chain, these results indicate that FPLC with ionic exchangers can be usefully employed to isolate rather large protein fragments especially suitable for automatic sequence analysis with the sequenator. Abbreviations used: CNBr, cyanopen bromide; BDTA, ethylcnediaminotetraacetic acid; FPLC, fast protein liquid chromatography; Mono Q, anion exchange liquid chromatography (HPLC) techniques, column containing a hydrophilic polymer carrying quaternary amino groups; Mono S , cation exchange which are now finding extensive use in the column containing a hydrophilic polymer carrying sulfonic groups; SDS sodium dodecyl sulfate; TCA-isolation and characterization of peptides and thermolysin, trichloroacetic acid-inactivated thcrmo-proteins -6). Because HPLC offers advantages lysin.

Under chronic stress, carotenoid-based colouration has often been shown to fade. However, the ecological and physiological mechanisms that govern colouration still remain largely unknown. Colour changes may be directly induced by the stressor (for example through reduced carotenoid intake) or due to the activation of the physiological stress response (PSR, e.g. due to increased blood corticosterone concentrations). Here, we tested whether blood corticosterone concentration affected carotenoid-based colouration, and whether a trade-off between colouration and PSR existed. Using the common lizard (Lacerta vivipara), we correlatively and experimentally showed that elevated blood corticosterone levels are associated with increased redness of the lizard's belly. In this study, the effects of corticosterone did not depend on carotenoid ingestion, indicating the absence of a trade-off between colouration and PSR for carotenoids. While carotenoid ingestion increased blood carotenoid concentration, colouration was not modified. This suggests that carotenoid-based colouration of common lizards is not severely limited by dietary carotenoid intake. Together with earlier studies, these findings suggest that the common lizard's carotenoid-based colouration may be a composite trait, consisting of fixed (e.g. genetic) and environmentally elements, the latter reflecting the lizard's PSR. Citation: Fitze PS, Cote J, San-Jose LM, Meylan S, Isaksson C, et al. (2009) Carotenoid-Based Colours Reflect the Stress Response in the Common Lizard. PLoS ONE 4(4): e5111.

The pharmacokinetics, biodistribution and the metabolic pathway of roscovitine were investigated in Sprague-Dawley rats after a single intravenous dose of 25 mg/kg. Blood, lungs, kidney, liver, testis, adipose tissue, spleen and brain were removed at different time-points. Plasma and tissue samples were analyzed using high performance liquid chromatography. The metabolites were identified using liquid chromatography/tandem mass spectrometry and nuclear magnetic resonance spectroscopy.

Membrane distillation is an emerging membrane process based on evaporation of a volatile solvent. One of its often stated advantages is the low flux sensitivity toward concentration of the processed fluid, in contrast to reverse osmosis. In the present paper, we looked at 2 high-solids applications of the dairy industry: skim milk and whey. Performance was assessed under various hydrodynamic conditions to investigate the feasibility of fouling mitigation by changing the operating parameters and to compare performance to widespread membrane filtration processes. Whereas filtration processes are hydraulic pressure driven, membrane distillation uses vapor pressure from heat to drive separation and, therefore, operating parameters have a different bearing on the process. Experimental and calculated results identified factors influencing heat and mass transfer under various operating conditions using polytetrafluoroethylene flat-sheet membranes. Linear velocity was found to influence performance during skim milk processing but not during whey processing. Lower feed and higher permeate temperature was found to reduce fouling in the processing of both dairy solutions. Concentration of skim milk and whey by membrane distillation has potential, as it showed high rejection (>99%) of all dairy components and can operate using low electrical energy and pressures (<10 kPa). At higher cross-flow velocities (around 0.141 m/s), fluxes were comparable to those found with reverse osmosis, achieving a sustainable flux of approximately 12 kg/ h·m 2 for skim milk of 20% dry matter concentration and approximately 20 kg/h·m 2 after 18 h of operation with whey at 20% dry matter concentration.

Vitamin B 12 content of an algal health food, Chlorella tablets (Chlorella sp.), was determined by both Lactobacillus leichmannii ATCC 7830 microbiological and intrinsic factor-chemiluminescence methods. The values of 200.9-211.6 µg/100 g dry weight determined by the chemiluminescence method were similar to the values (201.3-285.7 µg/100 g dry weight) determined by the microbiological method. A corrinoid compound was purified to homogeneity from the Chlorella tablets and characterized. The purified corrinoid compound was identified as vitamin B 12 , on the basis of silica gel 60 TLC, C18 reversed-phase HPLC, 1 H NMR spectroscopy, and UV-Vis spectroscopy.

A simple and sensitive high-performance liquid chromatography method for the determination of four Leuzea carthamoides flavonoids, namely eriodictyol, patuletin, eriodictyol-7-β βglucopyranoside, and 6-hydroxykaempferol-7-O-(6"-O-acetyl-β β-Dglucopyranoside), is presented. Using this method, quantitative composition of flavonoids ranged from 0.011% to 0.574% in dried plant material was determined. This method could be used in the future for the quantitative evaluation of major phenolic compounds in L. carthamoides.

Lipoxygenases are a group of closely related enzymes responsible for the dioxygenation of various polyenoic fatty acids containing all-cis-methylene-interrupted double bonds. The nomenclature for lipoxygenases is based on the site of insertion of molecular oxygen on the fatty acid molecule, in this

HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Abstract: The purpose of this work was to correlate the amount of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) determined in mouse fur with the number of intestinal tumours induced by PhIP, to further evaluate incorporation of PhIP into hair as ...

We describe an isocratic, reversed-phase high-performance liquid chromatographic method for the simultaneous measurement of fully oxidised, dihydro- and tetrahydropterins in cerebrospinal fluid. Tetrahydrobiopterin is detected electrochemically using an ESA Coulochem ...

A method for determining all of the six natural vitamin B 6 compounds and pyridoxine-␤-glucoside in urine from humans consuming their usual diet was developed. These compounds were specifically converted with 5 enzymes into a high fluorescent 4-pyridoxolactone which was supersensitively determined by an isocratic HPLC. All of the compounds in urine from humans consuming their usual diets were for the first time determined together. The preparation procedure for urine samples was easy without HClhydrolysis, and the enzyme reactions took only 2 or 3 h. It required only 5 L of the urine sample for analysis of one of the compounds. Urine samples from five young Japanese males consuming their usual diet contained pyridoxal, pyridoxamine, and pyridoxine-␤-glucoside but not pyridoxine or phosphoester forms. The contents of 4-pyridoxic acid and pyridoxal correlate well with a correlation coefficient of 0.98. On the other hand, the content of pyridoxamine did not correlate with that of 4-pyridoxic acid.

A method is described for the simultaneous profiling of sample lipophilicity, integrity, and purity. The method is rapid and is applicable to high throughput profiling of pharmaceutical properties in drug discovery. A short Polaris C column is used 18 with a rapid, wide-polarity mobile phase gradient, UV detection, and MS analysis. The lipophilicity of each component is estimated from a calibration curve using six drug or organic compounds and plotting their respective measured retention time versus Log D (literature). The correlation of Log D (literature) to Log D (HPLC) for 60 structurally diverse drugs has 7.4 7.4 7.4 2 a correlation coefficient r of 0.89. The method is applicable to compounds with MW.200 and retention time.1.5 min for rapid, initial pharmaceutical profiling in drug discovery.

The mechanism of human tumor resistance to the antineoplastic drug bleomycin (BLM) is not known. We now provide evidence implicating metabolic inactivation in the resistance of human Burkitt's (Daudi) lymphoma to BLM. Daudi lymphoma and human head and neck squamous cell carcinoma (A-253) cells grown (s.c.) in nude mice were found to be resistant and sensitive to BLM treatment, respectively. Within l h of s.c. injection of (3H]BLM A2,Daudi xenografts accumulated less BLM and metabolized this drug to a much greater extent than did A-253 xenografts. The BLM-resistant Daudi xenografts metabolized BLM A2 to at least 6 metabolites and only a small proportion of the drug remained as unmetabolized BLM A2. In the BLM-sensitive A-253 xenografts, however, BLM A2 remained the major component. Incubation of BLM \; with Daudi cytosolic fractions resulted in a complex mixture of metabolites similar to that formed by Daudi xenografts in nude mice. This BLM metabolite mixture was biologically inactive in plasmili DNA degradation assays. Treatment of mice bearing Daudi xenografts with an inhibitor of BLM hydrolase, L-fra/w-epoxysuccinyl-leucylamido-(4guanidino)butane (E-64), prior to | 'I I|HI M A2 treatment did not affect the amount of BLM accumulated but inhibited BLM A2 metabolism in the xenografts. Furthermore, although E-64 alone did not inhibit the growth of Daudi xenografts, it potentiated the antitumor activity of BLM. These results indicate that Daudi tumors resist BLM by metabolically inactivating it and that inhibition of BLM metabolism in vivo enhances the antitumor activity of BLM and hence overcomes resistance.

An analytical method using a liquid chromatograph combined with a simple online column switching sample pre-treatment system was developed for the determination of 1-hydroxypyrene (1-HP) in urine. This compound is the metabolite of pyrene and is used to assess the exposure of workers to polycyclic aromatic hydrocarbons (PAHs). After enzymatic hydrolysis, a urine sample was directly injected into a high-performance liquid chromatograph (HPLC) where it automatically underwent a sample cleanup using a column switching device. The procedure is simpler than previous methods because it uses only one switching valve, one extraction column and one HPLC pump. The analyte was retained on a short extraction column and after interferences were eluted to waste, was subsequently switched onto the analytical column. This allowed a short analysis time of 15 min. The calibration graph was found to be linear within the concentration range of 0.5 to 20 mg/L with a coefficient of determination exceeding r 2 5 0.99. Recoveries were found to be greater than 96% in the range 1 to 10 mg/L with intermediate precision of 2.5 to 5.8% relative standard deviation. This online method was verified by a comparison with an existing manual method by the analysis of 81 urine samples from workers exposed to PAHs and showed that the test results from both methods were in agreement with a probability obtained from the paired Student's t-test of P > 0.76. The proposed online method was found to be simple, fast and suited to routine analyses of 1-HP in urine for the assessment of occupational exposure to PAHs.

The isoflavone content and profile in processed soy-based products consumed in Brazil were determined by high-performance liquid chromatography and photodiode array detection of the intact isoflavones (naturally occurring aglycons, malonyl, acetyl, and-glycosides derivatives). Total isoflavone content varied significantly among products, from 2 to 100 mg/100 g (wet basis, expressed as aglycons), with the lowest content being found for soy-based enteral/oral diets and the highest found for textured soy proteins. For soy beverages isoflavone content varied from 12 to 83 mg/L. Soy sauce, miso, and tofu had isoflavone contents of 5.7 mg/L, 20 mg/100 g, and 7 mg/100 g, respectively. The-glycosides were the predominant form of the isoflavones in the products analyzed, except for miso, shoyu, and "Diet Shake" in which the aglycons were present in the highest proportions. On the basis of these data, the daily intake of isoflavone from soy products was estimated: the highest values were found for infants fed soy-based formulas, from 1.6 to 6.6 mg/kg of body weight.

Hydrophobic interaction chromatography (HIC) was examined as an alternative to reversed-phase chromatography (RPC) for peptide separations by highperformance liquid chromatography. With small peptides, selectivity was similar in both modes. This was the case with commercially available standards and with a set of synthetic peptides having the same amino acid composition but different sequences. Column efficiency was higher in RPC. HIC possesses several other disadvantages, including significant baseline changes during gradient elution and a requirement for non-volatile mobile phases, which complicates peptide isolation. Thus, RPC is still the method of choice for most small peptides. Marked differences in selectivity were noted with small proteins and polypeptides large enough to possess tertiary structure. Good results were also obtained by HIC in the case of some peptides that could not be purified at all by RPC, due to aggregation or poor binding or recovery. Thus, in these cases, HIC is a useful alternative to RPC for peptide purification.

The composition and concentration of anthocyanins of grape berry skins were analyzed in order to assess phenotypic variation between four grape wine varieties belonging to 4 different species: Vitis vinifera, Vitis amurensis, Vitis cinerea and Vitis X champinii. High-performance liquid chromatography coupled to mass spectrometry (LC-MS) and NMR spectroscopy (LC-NMR) were used to separate and identify the structure of anthocyanins present in these species. Combination of LC-MS and LC-NMR data resulted in the identification of 33 anthocyanins. In particular, newly reported cis isomers of p-coumaric-derivatives were identified (petunidin-, peonidin-and malvidin-3-(6-p-coumaroyl)-5-diglucoside). In V. cinerea and V. vinifera, anthocyanins were monoglucoside derivatives whereas in V. amurensis and V. X champinii, both mono-and diglucoside derivatives were identified. Malvidin-, delphinidin-and petunidin-derivatives were, respectively, the most abundant components in V. cinerea and V. vinifera, V. amurensis and V. X champinii.

Background: 1E10 monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH) 3 , in several clinical trials for melanoma, breast, and lung cancer. During early clinical development this mAb was obtained in vivo from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the in vivo to a bioreactor-based method.

A sensitive and specific HPLC–ESI-MS/MS method for the direct determination of glucosamine in human plasma has been developed and validated. Plasma samples were analyzed after a simple, one-step protein precipitation clean-up with trichloroacetic acid using a polymer-based amino high-performance liquid chromatography (HPLC) column and a water/acetonitrile mobile phase elution gradient, with d-[1-13C]glucosamine as the internal standard. Detection was performed by mass spectrometry, using an electrospray source and employing multiple reaction monitoring to separately monitor glucosamine and the internal standard. The limit of quantification of the method was 10 ng/ml of glucosamine and the calibration curve showed a good linearity up to 1000 ng/ml. The precision (R.S.D.) and the accuracy (bias) of the method at the limit of quantification were 13.8 and 4.0%, respectively, and the mean recovery of glucosamine at three concentration levels was 101.6 ± 5.7%. The method was applied for the determination of glucosamine concentrations in human plasma samples collected from untreated healthy volunteers and, in a separate bioavailability study, to evaluate plasma glucosamine pharmacokinetics profiles after oral administration of crystalline glucosamine sulfate.

Bisphenol A (BPA) is a toxic industrial chemical that affects the endocrine system even at low concentrations. A new method, based on capillary high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) analysis, has been developed to determine BPA in atmospheric samples. The method involves collection of air samples (typically 2 m3) on glass fiber filters, with ultrasonic extraction and sample concentration under vacuum before analysis. HPLC analysis was performed isocratically at a flow rate of 10 µL min−1 using a capillary reversed-phase column and MS/MS analysis in negative ion multiple reaction monitoring (MRM) mode, using BPA-d16 as internal standard. The present method provides linear response in the range 0.007–3.5 µg/filter (R2 > 0.999) and is characterized by high accuracy (mean bias 2%) and good reproducibility (mean RSD 5%). High sensitivity (LOD = 2 ng/m3 based on 2 m3 of air collected), specificity, and speed of the analysis make the present method suitable for routine determination of BPA in the atmosphere, both for ambient and personnel monitoring. Copyright © 2005 John Wiley & Sons, Ltd.

Centaurea solstitialis (yellow star thistle) has been proven to cause equine nigropallidal encephalomalacia in horses. Over the last fifty years, nigropallidal encephalomalacia has been of interest to human medicine due to the possible connection with Parkinson's disease. Previous studies indicated the presence of neurotoxic nitrogenous compounds in polar extracts of the plant. In order to give a more detailed description of the nitrogen-containing fraction of C. solstitialis, various samples were collected at different development stages. Different aliquots of the same aqueous extract were directly derivatized with ophthaldialdehyde and dansyl chloride and analyzed separately by reversed-phase HPLC. A complete profile of the free nitrogenous fraction of C. solstitialis was given and results obtained with the two derivatization procedures were compared. No particularly high level of free aspartic and glutamic acids, two potent neuroexcitotoxic amino acids, were found in polar extracts of the plant. Tyramine resulted to be the most important biologically active amine present in C. solstitialis (with a mean concentration of 2.0 mg/100 g of dry weight). q

The fungicides vinclozolin and iprodione are widely used in agriculture. These pesticides are dicarboximide fungicides containing the common moiety 3,5-dichloroaniline (3,5-DCA). It has been suggested that low-level exposures to such compounds may be associated with adverse health effects such as endocrine disruption. In this study a method using liquid chromatography/triple quadrupole mass spectrometry (LC/MS/MS) was developed for the analysis of 3,5-DCA as a biomarker of exposure to these fungicides in human urine. The urine samples were treated by basic hydrolysis to degrade the fungicides, their metabolites and conjugates to 3,5-DCA. The 3,5-DCA was then extracted using toluene and derivatized using pentafluoropropionic anhydride (PFPA). Analysis of the derivative was carried out using selected reaction monitoring (SRM) in the negative ion mode. Quantification of the derivative was performed using [ 13 C 6 ]-labeled 3,4-DCA as an internal standard with good precision and linearity in the range 0.1-200 ng/mL urine. The limit of detection was determined to be 0.1 ng/mL. The metabolites in urine were found to be stable during storage at S208C. To validate 3,5-DCA as a biomarker the method was applied in a human experimental exposure to iprodione and vinclozolin. Two healthy volunteers received 200 mg single oral doses of each pesticide followed by urine sampling during 72-120 h post-exposure. Between 78-107% of the dose was recovered as 3,5-DCA in the urine after exposure.

Benzalkonium salts are widely used as disinfectants, biocides and detergents, among a variety of other applications. The cationic surface-activity of these salts determines their potential to act as a biocide on both target and non-target organisms. In this study, a quick synthesis of a complete set of the benzalkonium salts differing in the length of an alkylating chain (from C 2 to C 20 ) is described. Moreover, their 1 H-NMR, HPLC-MS, TLC and HPLC analysis were recorded.

Racemic RS-4-(4-hydroxyphenyl)-2-butanol (rhododendrol, RD) was used as a topical skin-whitening agent until it was recently reported to induce leukoderma. We then showed that oxidation of RD with mushroom tyrosinase rapidly produces RD-quinone, which is quickly converted to RD-cyclic quinone and RD-hydroxy-p-quinone. In this study, we examined whether either or both of the enantiomers of RD can be oxidized by human tyrosinase. Using a chiral HPLC column, racemic RD was resolved optically to R(-)-RD and S(+)-RD enantiomers. In the presence of a catalytic amount of l-dopa, human tyrosinase, which can oxidize l-tyrosine but not d-tyrosine, was found to oxidize both R(-)- and S(+)-RD to give RD-catechol and its oxidation products. S(+)-RD was more effectively oxidized than l-tyrosine, while R(-)-RD was less effective. These results support the notion that the melanocyte toxicity of RD depends on its tyrosinase-catalyzed conversion to toxic quinones and the concomitant production of rea...

Molecular chaperones have been used for the improved expression of target proteins within heterologous systems; however, the chaperone and target protein have seldom been matched in terms of origin. We have developed a heterologous co-expression system that allows independent expression of the plasmodial chaperone, PfHsp70, and a plasmodial target protein. In this study, the target was Plasmodium falciparum GTP cyclohydrolase I (PfGCHI), the first enzyme in the plasmodial folate pathway. The sequential expression of the molecular chaperone followed by the target protein increased the expression of soluble functional PfGCHI. His-tagged PfGCHI was successfully purified using nickel affinity chromatography, and the specific activity was determined by high performance liquid chromatography with spectrofluorometeric detection to be 5.93 nmol/hr/mg. This is the first report of a heterologous co-expression system in which a plasmodial chaperone is harnessed for the improved production and purification of a plasmodial target protein.