Sensitive High Pressure Liquid Chromatography Research Articles

CSF dopamine is elevated in first-episode psychosis and associates to symptom severity and cognitive performance.

The hypothesis of dopamine dysfunction in psychosis has evolved since the mid-twentieth century. However, clinical support from biochemical analysis of the transmitter in patients is still missing. The present study assessed dopamine and related metabolites in the cerebrospinal fluid (CSF) of first-episode psychosis (FEP) subjects. Forty first-episode psychosis subjects and twenty healthy age-matched volunteers were recruited via the Karolinska Schizophrenia Project, a multidisciplinary research consortium that investigates the pathophysiology of schizophrenia. Psychopathology, disease severity, and cognitive performance were rated as well as cerebrospinal fluid concentrations of dopamine and related metabolites were measured using a sensitive high-pressure liquid chromatography assay. CSF dopamine was reliably detected in 50% of healthy controls and in 65% of first-episode psychosis subjects and significantly higher in first-episode psychosis subjects compared to age-matched healthy controls. No difference in CSF dopamine levels was observed between drug-naive subjects and subjects with short exposure to antipsychotics. The dopamine concentrations were positively associated with illness severity and deficits in executive functioning. Dopamine dysfunction has long been considered a cornerstone of the pathophysiology of schizophrenia, although biochemical support for elevated brain dopamine levels has been lacking. The results of the present study, showing that FEP subjects have increased CSF dopamine levels that correlate to disease symptoms, should fill the knowledge gap in this regard.

Validation of the HPLC Analytical Method for the Determination of Chemical and Radiochemical Purity of Ga-68-DOTATATE.

Ga-68-DOTA-peptides targeting somatostatin receptors have been assessed as a valuable tool in neuroendocrine tumors imaging using positron emission tomography (PET). A new selective and sensitive high-pressure liquid chromatography (HPLC) method was developed for determining chemical and radiochemical purity of Ga-68-DOTATATE (PET) tracer. The identification of peaks was achieved on a symmetry C18 column 3 μm 120Å (3.0 mm × 150 mm spherical particles) using (A) water + 0.1% trifluoroacetic acid (TFA) and (B) acetonitrile + 0.1% TFA, as the mobile phases at a flow rate of 0.600 mL/min and monitored at 220 nm. The run time was 16 min. The method was validated to fulfill International Conference on Harmonization requirements and EDQM guidelines, and it included specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision. The calibration curve was linear over the concentration range from 0.5 to 3 μg/ml, with a correlation coefficient (r2) equal to 0.999, average coefficient of variation (CV%) 2%, and average bias% did not deviate more than 5% for all concentrations. The LOD and LOQ for DOTATATE were 0.5 and 0.1 μg/mL, respectively. The method was considered precise, obtaining coefficients of variation between 0.22% and 0.52% for intraday and 0.20% and 0.61% for interday precision. Accuracy of method was confirmed with average bias% that did not deviate more than 5% for all concentrations. All results were acceptable and this confirmed that the method is suitable for its intended use in routine quality control of Ga-68-DOTATATE to guarantee the high quality of the finished product before release.

SIMULTANEOUS SEPARATION OF GALLIC ACID, PROTOCATECHUIC ACID, RUTIN, MANGIFERIN AND CHROMATOGRAPHIC PROFILING OF SOME INDIAN MEDICINAL PLANTS

Background: Plants and plant-based extracts have been used across the globe for management of various ailments and disorders. The biological and pharmacological activities of plants and their extracts are primarily attributed to polyphenols, avonoids, and other secondary metabolites. High Pressure Liquid Chromatography (HPLC) is a reliable, sensitive, and quick method to identify bioactive ingredients in complex plant extracts. In the present study, we have developed a quick and sensitive HPLC method for separation of four polyphenols, namely, gallicMethods: acid, protocatechuic acid, rutin, and mangiferin. Further, we used the developed HPLC method for the analysis of plant extracts obtained from Asparagus racemosus, Azadirachta indica, Capsicum annuum, Nigella Sativa, Phyllanthus niruri, Rosmarinus ofcinalis, Rheum palmatum, Withania somnifera, Withania somnifera, and Zingiber ofcinale. The HPLC method developed in the present study was highly effectiveResults: in separating gallic acid, protocatechuic acid, rutin, mangiferin. The method also provides excellent phytoconstituent ngerprinting of complex plant extracts of medicinal plants. The gradient mode HPLC method developed in the present study is highly useful for identicationConclusion: of plant polyphenols in complex plant-based extracts and generating HPLC chromatograms for complex plant extracts. The present method, thus, can be used for phytoconstituent proling, quality control of herbal drugs, and assessing adulteration of plant extract or nutraceuticals.

Dietary Geranylgeranyl Pyrophosphate Counteracts the Benefits of Statin Therapy in Experimental Pulmonary Hypertension.

The mevalonate pathway generates endogenous cholesterol and intermediates including geranylgeranyl pyrophosphate (GGPP). By reducing GGPP production, statins exert pleiotropic or cholesterol-independent effects. The potential regulation of GGPP homeostasis through dietary intake and the interaction with concomitant statin therapy is unknown. We developed a sensitive high-pressure liquid chromatography technique to quantify dietary GGPP and conducted proteomics, qualitative real-time polymerase chain reaction screening, and Western blot to determine signaling cascades, gene expression, protein-protein interaction, and protein membrane trafficking in wild-type and transgenic rats. GGPP contents were highly variable depending on food source that differentially regulated blood GGPP levels in rats. Diets containing intermediate and high GGPP reduced or abolished the effects of statins in rats with hypoxia- and monocrotaline-induced pulmonary hypertension: this was rescuable by methyl-allylthiosulfinate and methyl-allylthiosulfinate-rich garlic extracts. In human pulmonary artery smooth muscle cells treated with statins, hypoxia activated RhoA in an extracellular GGPP-dependent manner. Hypoxia-induced ROCK2 (Rho associated coiled-coil containing protein kinase 2)/Rab10 (Ras-related protein rab-10) signaling was prevented by statin and recovered by exogenous GGPP. The hypoxia-activated RhoA/ROCK2 pathway in rat and human pulmonary artery smooth muscle cells upregulated the expression of Ca2+-sensing receptor (CaSR) and HIMF (hypoxia-induced mitogenic factor), a mechanism attenuated by statin treatment and regained with exogenous GGPP. Rab10 knockdown almost abrogated hypoxia-promoted CaSR membrane trafficking, a process diminished by statin and resumed by exogenous GGPP. Hypoxia-induced pulmonary hypertension was reduced in rats with CaSR mutated at the binding motif of HIMF and the interaction between dietary GGPP and statin efficiency was abolished. In humans fed a high GGPP diet, blood GGPP levels were increased. This abolished statin-lowering effects on plasma GGPP, and also on hypoxia-enhanced RhoA activity of blood monocytes that was rescued by garlic extracts. There is important dietary regulation of GGPP levels that interferes with the effects of statin therapy in experimental pulmonary hypertension. These observations rely on a key and central role of RhoA-ROCK2 cascade activation and Rab10-faciliated CaSR membrane trafficking with subsequent overexpression and binding of HIMF to CaSR. These findings warrant clinical investigation for the treatment of pulmonary hypertension and perhaps other diseases by combining statin with garlic-derived methyl-allylthiosulfinate or garlic extracts and thus circumventing dietary GGPP variations.

DEVELOPMENT AND VALIDATION OF SEVEN PHENYL HYDRAZINE CHLORO ESTER ISOMERS (PGIs) BY RP-HPLC-UV METHOD IN ANTICOAGULANT DRUG SUBSTANCE; APIXABAN

Objective: The objective of this work was to develop and validate a simple and sensitive reverse-phase high-pressure liquid chromatography method for the determination of seven potential genotoxic impurities in Apixaban drug substance.
 Methods: The optimized separation was achieved by using ACE 3 C18 PFP (150 mm×4.6 mm, 3 µm) HPLC column. The mobile phase-A was a degassed mixture of 0.01M Ammonium acetate buffer(PH adjusted 4.9±0.05 with diluted glacial acetic acid) and mobile phase-B was a degassed mixture of Acetonitrile, Isopropyl alcohol and Buffer PH 4.9 in the ratio of 60:20:20 v/v/v. The gradient program was operated at a flow rate of 1.0 ml/min and UV detection was at 330 nm.
 Results: The method was superior at linearity for seven impurities and correlation coefficient values were larger than 0.999, moreover, in the separation point of view, this method further achieved no matrix interference through chromatography by better resolution of the other impurities from the Apixaban drug substance and its related impurities for the accurate analysis of seven potential genotoxic impurities. The established limits of detection (LOD), limits of quantification (LOQ) values for the seven mutagenic impurities were each of 5 ppm (0.015µg/ml) and15 ppm (0.045µg/ml) respectively. The developed method was validated as per ICH guidelines and applied as a generic method to determine these seven potential genotoxic impurities for the pharmaceutical process control and drug material release.
 Conclusion: Validation of this analytical method was carried out including stability, selectivity, linearity, accuracy, system precision, method precision and intermediate precision thus proving that the described RP-HPLC method could be employed for fast and simple analysis of sevenphenyl hydrazine chloro ester isomers in Apixaban drug substance.

Development of a Simple Chromatographic Method for the Determination of Piracetam in Human Plasma and Its Pharmacokinetic Evaluation

The objective of study was to develop an accurate and reproducible HPLC method for determination of piracetam in human plasma and to evaluate pharmacokinetic parameters of 800 mg piracetam. A simple, rapid, accurate, precise and sensitive high pressure liquid chromatography method has been developed and subsequently validated for determination of piracetam. This study represents the results of a randomized, single-dose and single-period in 18 healthy male volunteers to assess pharmacokinetic parameters of 800 mg piracetam tablets. Various pharmacokinetic parameters were determined from plasma for piracetam and found to be in good agreement with previous reported values. The data was analyzed by using Kinetica® version 4.4 according to non-compartment model of pharmacokinetic analysis and after comparison with previous studies, no significant differences were found in present study of tested product. The major pharmacokinetic parameters for piracetam were as follows: t1/2 was (4.40 ± 0.179) h; Tmax value was (2.33 ± 0.105) h; Cmax was (14.53 ± 0.282) µg/mL; the AUC(0-∞) was (59.19 ± 4.402) µg · h/mL. AUMC(0-∞) was (367.23 ± 38.96) µg. (h)(2)/mL; Ke was (0.16 ± 0.006) h; MRT was (5.80 ± 0.227) h; Vd was (96.36 ± 8.917 L). A rapid, accurate and precise high pressure liquid chromatography method was developed and validated before the study. It is concluded that this method is very useful for the analysis of pharmacokinetic parameters, in human plasma and assured the safety and efficacy of piracetam, can be effectively used in medical practice.

Effect of strain and cultural conditions on the production of cytochalasin B by the potential mycoherbicide Pyrenophora semeniperda (Pleosporaceae, Pleosporales)

The seed pathogen Pyrenophora semeniperda has demonstrated potential as a mycoherbicidal biocontrol for eliminating persistent seed banks of annual bromes on western North American rangelands. This pathogen exhibits variation in virulence that is related to mycelial growth rate, but direct laboratory tests of virulence on seeds often have low repeatability. We developed a rapid and sensitive high pressure liquid chromatography method for quantification of the phytotoxin cytochalasin B in complex mixtures in order to evaluate its use in virulence screening. All 10 strains tested produced large quantities of this metabolite in solid wheat seed culture, with production varying over a fourfold range (535–2256 mg kg−1). No cytochalasin B was produced in liquid potato dextrose broth culture, showing that its synthesis is strongly dependent on cultural conditions. In a Bromus tectorum coleoptile bioassay, solid culture extracts showed mild toxicity similar to the cytochalasin B standard at a concentration equivalent to 10−4 M cytochalasin B (72–95% of control), whereas at 10−3 M equivalent, the extracts exhibited significantly higher toxicity (8–18% of control) than the cytochalasin B standard (34% of control). This suggests the possible presence of other phytotoxic metabolites. Cytochalasin B production in solid wheat seed culture exhibited the predicted significant negative correlation with mycelial growth rate on potato dextrose agar, but the correlation was not very strong, possibly because cytochalasin B production and growth rate were measured under different cultural conditions.

SELECTIVE REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR THE SIMULTANEOUS DETERMINATION OF SOME PHARMACEUTICAL BINARY MIXTURES CONTAINING NSAIDS

A simple, rapid, sensitive, and economic reversed-phase High Pressure Liquid Chromatography method was developed for the simultaneous evaluation of five binary mixtures in their pharmaceutical preparations. The investigated mixtures were Ibuprofen(IP)-paracetamol(PC), Ibuprofen(IP)-Chlorzoxazone(CZ), Ibuprofen(IP)- Methocarbamol(MC), Ketoprofen(KP)-Chlorzoxazone(CZ), and Diclofenac sodium(DS)-Lidocaine hydrochloride(LC). The chromatographic separation was performed using isochratic mode at a flow rate 1 mL/ min, ODS C18 column, and UV detection at 254 nm. The mobile phase consisted of acetonitrile:phosphate buffer, pH 5 (60:40). The parameters affecting separation of the investigated drugs were studied. Under the optimum assay conditions, the method gave linear calibration curves with good correlation coefficients. The limits of detection (LOD) and limits of quantitation (LOQ) ranged from 0.34–5.33 µg/mL and 1.03–16 µg/mL, respectively, indicating high sensitivity of the method. The assay of the investigated binary mixtures is completed in less than 5 min. The proposed method was successfully applied to the simultaneous analysis of the drugs in their pharmaceutical binary mixtures in dosage forms with good recoveries and no significant differences between proposed and reported method. This method can be routinely applied to the quality control of the investigated binary mixtures in their pharmaceutical formulations.

Stressed Kinetics of Nanoemulsion Formulation Encapsulated Ropinirole with a Validated Ultra High Performance Liquid Chromatography–Synapt Mass Spectrometry (UPLC‐MS/MS ESI‐Q‐TOF)

AbstractA highly sensitive ultra high pressure liquid chromatography (UHPLC‐MSMS) method for estimation of ropinirole in rat brain homogenate and plasma has been validated. The method was successfully used for the degradation kinetics in different stress condition and regulated temperature. The chromatographic separation was achieved using isocratic mobile phase, consisting of acetonitrile–2mM ammoniumacetate (28:72 v/v; 0.25 mL min−1). The mass spectrometer was operated in synapt mass spectrometry mode via positive electrospray ionization using the transitions m/z 260 → m/z 261 for ropinirole, and m/z 324.39 → m/z 262.161 as a parent ion of escitalopram (IS). The assay for ropinirole was linear over the range of 0.5–100 ng mL−1 (r2; 0.999). The intra‐ and inter day precisions were less than 11.2% in terms of relative standard deviation (R.S.D.), and the accuracy was within ±6.4% in terms of relative error (RE). The mean extraction‐efficiency of QC samples (MQC, 8 ng/mL) was ≥80%. The lower limit of quantification (LLOQ) was 0.049 ng/mL where as lower limit of detection (LLOD) was 0.016 ng/mL. All the peaks of degradation were well resolved. The degradation kinetics of ropinirole, showed highest stability (t1/2 256.66/h; t0.9, 39.11/h) in acidic medium, lower stability in alkaline environment (t1/2, 103.43/h; t0.9, 15.76/h) and highly susceptible in oxidative environment (t1/2, 21.58/h; t0.9, 3.28/h). The applicability of this assay was demonstrated and successfully applied for pharmacokinetic profiling of ropinirole in Wister rat brain homogenate after intranasal administration.

Enhancing Fluorescence LC Analysis of Biogenic Amines in Fish Tissues by Precolumn Derivatization with Naphthalene-2,3-dicarboxaldehyde

This work reports a sensitive high-pressure liquid chromatography method for the simultaneous determination of biogenic mono- and diamines in fish tissues. Highly fluorescent derivatives of the amines were obtained by precolumn derivatization with naphthalene-2,3-dicarboxaldehyde, in the presence of cyanide ion as the nucleophile and of heptylamine as the internal standard. The chromatographic separation was performed using a mobile phase of acetonitrile/methanol/10 % (v/v) acetone in water, delivered in gradient mode on an Inertsil ODS-3 column (250 × 4 mm i.d., 5 μm). The method was successfully applied to the analysis of fresh and canned tuna fish tissues, subjected to ultrasound-assisted liquid–liquid extraction. The variables affecting the derivatization yield as well as the extraction recovery of the analytes from the sample matrix were investigated. Results were quantified against the internal standard, according to the matrix-matched approach. Limits of detection between 2.5 and 330 mg kg−1 sample were achieved. The precision and accuracy of assay in samples was satisfactory, yielding relative standard deviation and recovery values between 0.3 and 4.2 % and 81.0 and 102.5 %, respectively.

Pharmacokinetics, Pharmacodynamics, and Comparative Bioavailability of Single, Oral 2-mg Doses of Dexamethasone Liquid and Tablet Formulations: A Randomized, Controlled, Crossover Study in Healthy Adult Volunteers

Background Dexamethasone is a glucocorticoid used widely worldwide for immunosuppressive treatment, allergies, bronchiolitis, and croup, among others. For children, liquid formulations are especially suitable because, compared with other dosage forms, both exact dosing and proper intake are facilitated. Objective The objective was to evaluate the pharmacokinetics, pharmacodynamics, and comparative bioavailability of a commercial liquid oral dexamethasone formulation intended for pediatric use relative to those of a tablet. Methods In a randomized, controlled, crossover study in 24 healthy adult volunteers, we administered single doses of the liquid and tablet formulation, containing 2 mg of dexamethasone each. Blood samples were taken up to 24 hours postdose. Quantification was carried out using a validated specific and sensitive high-pressure liquid chromatography with UV detector method. Noncompartmental pharmacokinetic parameters were compared between treatments according to European Medicines Agency (EMA) bioequivalence guidelines. For AUC 0–t and C max, the 90% CI for the ratio of the test and reference products should be contained within the predetermined acceptance interval of 80% to 125%. As a pharmacodynamic variable, we measured suppression of endogenous cortisol (predose and postdose). Results Both preparations showed similar pharmacokinetic and pharmacodynamic profiles but high between-subject variability of pharmacokinetic key parameters and endogenous cortisol concentrations (>30%). Mean AUC 0–t, AUC 0–∞, and C max were 37.8 ng/mL/h, 46.0 ng/mL/h, and 9.35 ng/mL, respectively, for the liquid and 41.3 ng/mL/h, 48.1 ng/mL/h, and 9.17 ng/mL, respectively, for the tablet formulation. T max was 0.89 hour (liquid) and 0.97 hour (tablet). The point estimates and 90% CIs for AUC 0–t, AUC 0–∞, and C max ratios (liquid vs tablet) were 91.42% (82.05%–101.86%), 95.72% (84.46%–108.5%), and 102.04% (86.94%–119.76%), respectively. Thus, point estimates and 90% CIs were within the bioequivalence range of 80% to 125% for all relevant parameters, including the pharmacodynamic parameter AUEC (area under the effect curve). Conclusions This single-dose study suggests that the test and reference products met the EMA regulatory criteria to assume bioequivalence in these fasting healthy male and female volunteers. German Register of Clinical Trials registration number: DRKS00000785.

Pharmacokinetics of CPU0213, a novel endothelin receptor antagonist, after intravenous administration in mice1

To determine the pharmacokinetics associated with acute toxic doses of CPU0213, a novel endothelin receptor antagonist in mice after a single intravenous administration. Concentrations in serum and the pharmacokinetic parameters of CPU0213 were assayed by high pressure liquid chromatography (HPLC) following a single intravenous bolus of CPU0213 at concentrations of 25, 50, and 100 mg/kg in mice. The intravenous acute toxicity of CPU0213 was also assessed in mice. A simple, sensitive and selective HPLC method was developed for quantitative determination of CPU0213 in mouse serum. The concentration-time data conform to a 2-compartment model after iv administration of CPU0213 at concentrations of 25, 50, 100 mg/kg. The corresponding distribution half-lives (T1/2 alpha ) were 3.6, 4.2, 1.1 min and the elimination half-lives (T1/2beta) were 39.4, 70.3, 61.9 min. There was a linear increase in C0 proportional to dose, and the same as AUC(0-t) and AUC(0-infinity);. AUC(0-t) and AUC(0-infinity) were 4.511, 13.070, 23.666 g x min x L(-1 ) and 4.596, 13.679, 24.115 g x min x L(-1), respectively. The intravenous LD50 was 315.5 mg/kg. First order rate pharmacokinetics were observed for CPU0213 within the range of doses used, and the acute toxicity of CPU0213 is mild.

Pharmacokinetics of amphotericin B in serum and tissues in mice treated with amphotericin B-Intralipid.

In our previous research we studied the efficacy of amphotericin B (AMB)-Intralipid (AMB-IL) admixtures in the treatment of experimental systemic candidosis in naïve and compromised mice. In this study we evaluated the levels of AMB in blood and several organs (kidneys, liver, spleen, lungs and heart) at different times after injection (from 5 min to 14 days). Comparisons were made with levels occurring in animals treated with Fungizone or AmBisome. A sensitive high-pressure liquid chromatography (HPLC) method was adapted for assaying AMB in blood and tissues. We used reversed-phased column and a simple mobile phase consisting of acetonitrile and EDTA. We found that blood levels of AMB in mice injected with AMB-IL were higher than those obtained in animals treated with Fungizone, but similar to those obtained in mice administered AmBisome. Compared with conventional Fungizone therapy, administration of lipid formulations of AMB (both AMB-IL and AmBisome) resulted in higher concentrations of AMB in the liver and spleen, but lower concentrations in the kidneys and lungs.

High-Pressure Liquid Chromatographic Determination of Ascorbic Acid in Cooked Sausages

The purpose of this paper was to study and optimize both extraction and high-pressure liquid chromatography (HPLC)–UV detection procedures to develop a proper method for the determination of ascorbic acid content in cooked sausages. A simple and sensitive reversed-phase HPLC method for the NH2-bonded phase has been described for the determination of ascorbic acid content in cooked sausages. Various extracting agents were tested to solubilize the vitamin, with 5% (wt/vol) metaphosphoric acid giving the best results. Samples were chromatographed with UV detection at 248nm on a 25-cm Spherisorb NH2 cartridge with a 0.4-cm inside diameter with a mixture of 0.02M potassium phosphate buffer solution (pH 3.6) and acetonitrile (40:60, vol/vol) for the mobile phase. The method's precision within a day was 1.2%, and its precision between days was 3.8%. The detection limit was 1.6 mg/100 g. Recovery ranged from 91.4 to 96.2% for ascorbic acid added to meat samples. Twenty samples of six different products were analyzed in duplicate. For the samples analyzed, the mean value for ascorbic acid ranged between 21.555 and 24.899 mg/100 g of fresh weight.

Chemical Analysis of the Developmental Pattern of Polysialylation in Chicken Brain: EXPRESSION OF ONLY AN EXTENDED FORM OF POLYSIALYL CHAINS DURING EMBRYOGENESIS AND THE PRESENCE OF DISIALYL RESIDUES IN BOTH EMBRYONIC AND ADULT CHICKEN BRAINS

Recent studies have demonstrated the involvement of two polysialyltransferases in neural cell adhesion molecule (N-CAM) polysialylation. The availability of cDNAs encoding these enzymes facilitated studies on polysialylation of N-CAM. However, there is a dearth of detailed structural information on the degree of polymerization (DP), DP ranges, and the influence of embryogenesis on the DP. It is also unclear how many polysialic acid (polySia) chains are attached to a single core N-glycan. In this paper we applied new, efficient, and sensitive high pressure liquid chromatography methods to qualitatively and quantitatively analyze the polySia structures expressed on embryonic and adult chicken brain N-CAM. Our studies resulted in the following new findings. 1) The DP of the polySia chains was invariably 40-50 throughout developmental stages from embryonic day 5 to 21 after fertilization. In contrast, glycopeptides containing polySia with shorter DPs, ranging from 15 to 35, were isolated from adult brain. 2) Chemical evidence showed glycan chains abundant in Neu5Acalpha2,8Neu5Ac were expressed during all developmental stages including adult. 3) Levels of both di- and polySia were found to show distinctive changes during embryonic development.

Quantitation of the Anti-HIV Alkaloids Michellamines A and B by High Pressure Liquid Chromatography

Michellamines A and B, naphthylisoquinoline dimeric alkaloids from the leaf of a previously undescribed species of the genus Ancistrocladus, show anti-human immunodeficiency virus (HIV) activity in an in vitro screen. In order to determine the distribution of these alkaloids in the genus, the level of production in the new species, and to gain an understanding of the horticultural aspects of production, a sensitive high pressure liquid chromatography analytical method requiring a small biomass was developed. This method resolved michellamines A and B and congeners and enabled quantitation of each. The production of the michellamines was found to be limited to the new species, A. korupensis. © 1997 John Wiley & Sons, Ltd.

A Sensitive HPLC Method for Determination of Mandelic Acid in Urine, and Its Application to Biological Monitoring of Ethylbenzene-exposed Chinese Workers

AbstractA highly sensitive high-pressure liquid chromatography (HPLC) method was developed for determination of mandelic acid (MA) in urine as a marker of exposure to ethylbenzene at low concentrations. This was achieved by the use of a mobile phase (acetonitrile: 60% perchloric acid: water = 50 ml: 100 μl: 950 ml) that has no absorption at 200 nm, the wavelength at which MA shows intense absorption. The detection limit was 0.07 mg/L urine, and the recovery rate was 101% (CV: 2.7%) when MA was added at 250 mg/L to ten urine samples from non exposed subjects. The method was applied for MA determination, in parallel to a method previously developed, to 641 urine samples (from 360 ethylbenzene-exposed workers and 281 nonexposed subjects), and the results were analyzed by regression analysis to examine the relationship between ethylbenzene exposure concentrations and urinary MA concentrations. A smaller intercept on the axis (essentially zero) and a larger correlaiion coefficient (about 0.7) indicate that the...

Pharmacokinetics of Hypericin and Pseudohypericin after Oral Intake of the Hypericum Perforatum Extract LI 160 in Healthy Volunteers

The single- and multiple-dose pharmacokinetics of the naphthodianthrones hypericin and pseudohypericin derived from St. John's wort (Hypericum perforatum, LI 160, Lichtwer Pharma GmbH, Berlin) were studied in 12 healthy male subjects. After a single oral dose of 300, 900, or 1800 mg of dried hypericum extract (250, 750, or 1500 μg hypericin and 526, 1578, or 3156 μg pseudohypericin), plasma levels were measured with a modified highly sensitive high-pressure liquid chromatography (HPLC) method (lower detection limit 0.1 ng/mL) up to 3 days. The median maximal plasma levels were 1.5, 4.1, and 14.2 ng/mL for hypericin and 2.7, 11.7, and 30.6 ng/mL for Pseudohypericin, respectively, for the three doses given above (interim evaluation of four volunteers). The median elimination half-life times of hypericin were 24.8 to 26.5 hours, and varied for pseudohypericin from 16.3 to 36.0 hours. Ranging between 2.0 to 2.6 hours, the median lag-time of absorption was remarkably prolonged for hypericin when compared to pseudohypericin (0.3 to 1.1 hours). The areas under the curves (AUC) showed a nonlinear increase with raising dose; this effect was statistically significant for hypericin. During long-term dosing (3 x 300 mg/day), a steady-state was reached after 4 days. Mean maximal plasma level during the steady-state treatment was 8.5 ng/mL for hypericin and 5.8 ng/mL for pseudohypericin, while mean trough levels were 5.3 ng/mL for hypericin and 3.7 ng/mL for pseudohypericin. In spite of their structural similarities there are substantial Pharmacokinetic differences between hypericin and pseudohypericin.

A Narrow-Bore HPLC Method for the Identification and Quantitation of Free, Conjugated, and Bound Polyamines

A modified sensitive high-pressure liquid chromatography (HPLC) method, using a narrow-bore column (2.1 × 200 mm, C-18, 5-μm particle size) with a methanol:water gradient (55-84%, v/v), is described for direct analysis of benzoylated free (S), soluble-conjugated (SH), and insoluble-bound (PH) polyamines in plant tissues. Regression curves for each fraction of polyamines allow a precise quantitative determination of putrescine, cadaverine, spermidine, spermine, and agmatine in all fractions (S, SH, PH) of a crude extract. A comparison of the results to the corresponding values dansylated polyamines separated by thin-layer chromatography and quantitated by fluorometry is also presented. S, SH, and PH polyamines as benzoylated derivatives from leaves of Vitis vinifera L. cv Dogridge were characterized and quantitatively determined with this improved sensitive HPLC method.

DETERMINATION OF CYTOSINE‐CYTOSINE PHOTODIMERS IN THE DNA OF CLOUDMAN S91 MELANOMA CELLS USING HIGH PRESSURE LIQUID CHROMATOGRAPHY

Abstract— I measured the induction of cytosine‐cytosine dimer (C‐C) densities after UV‐C (< 290 nm) and UV‐B irradiation (290–320 nm) in the 2′‐deoxy‐[3H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV‐B exposure resulted in 0.000034% C‐C/J m‐2 of the total cytosine radioactivity which is 10 times less than the rate during UV‐C irradiation. Previous work with these melanoma cells showed a 4‐fold lower rate of induction of thymine‐containing pyrimidine dimers by UV‐B than UV‐C light (Niggli Photochem. Photobiol. 52, 519–524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV‐C and UV‐B exposure. However, UV‐C and UV‐B light induce 10–20 times more thymine‐containing pyrimidine dimers than C‐C in the DNA of S91 cells.