A single-laboratory validation was completed for a method to determine total terpene lactones in Ginkgo biloba products. The method determines terpene lactones on the basis of the main terpene lactones (Bilobalide, Ginkgolide A, Ginkgolide B, Ginkgolide C, and Ginkgolide J) by high-performance liquid chromatography with evaporative light-scattering detection after extraction. Nine matrixes were chosen for study, including crude leaf material, standardized dry powder extract, single- and multiple-entity finished products, and alcohol and glycerin tinctures. The sample purification with prepacked columns allows selective extraction of the terpene lactones with no interferences from any matrix under study. A Youden ruggedness trial testing 7 instrumental and preparation factors with the potential to affect quantitative results showed that 2 factors (volume of the column elution solvent and pH of the diluent) were the most important parameters to control during sample preparation. The method performed well in terms of precision; 4 matrixes tested in triplicate over a 3-day period showed an overall repeatability relative standard deviation (RSD) of about 3%. HorRat values were within the limits for performance acceptability, ranging from 0.5 to 1.0. Analysis of variance testing at a = 0.05 showed no significant differences among the within-or between-group sources of variation, although comparison of within-day, between-day, and total precision showed that most of the RSD came from within-day determinations except those for the Ginkgo dry extract (Gb-SLV-2). Accuracy testing at 4 concentration levels of terpene lactones obtained by spiking a negative control matrix at approximately 300, 750, 1500, and 2250 microg/mL gave recoveries of about 91% for the 300 microg/mL level, about 98% for the 750 microg/mL level, about 99% for the 1500 microg/mL level, and 97% for the 2250 microg/mL level with an overall recovery of 96% and an RSD of 3.2%.

OBJECTIVES: Cimicifuga racemosa (Actaea racemosa, black cohosh) is used as an anti-inflammatory, antipyretic and analgesic remedy in traditional medicines. The present study focuses on the effects of C. racemosa root extracts on inducible nitric oxide synthase (iNOS) in lipopolysaccharide-stimulated murine macrophages (RAW 264.7). METHODS: C. racemosa rhizome and phosphate-buffered saline extracts were analysed for phenolcarboxylic acids and triterpene glycosides using an HPLC photodiode array/evaporative light-scattering detector system. iNOS was characterised by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), nitric oxide production (nitrite levels) and nuclear translocation of nuclear factor-kappaB (p65 subunit) protein. KEY FINDINGS: Incubation of lipopolysaccharide-stimulated macrophages with aqueous C. racemosa extracts (0-6 mg/ml) inhibited nitrite accumulation in a concentration-dependent manner. C. racemosa extracts also reduced iNOS protein expression and iNOS mRNA levels in a dose-dependent manner. C. racemosa extracts did not significantly inhibit iNOS activity and did not affect nuclear translocation of nuclear factor-kappaB (p65 subunit) protein. Incubation with the extract was associated with a concentration-dependent reduction of interferon beta and interferon regulatory factor 1 mRNA. Among the triterpene glycosides, 23-epi-26-deoxyactein was identified as an active principle in C. racemosa extracts. CONCLUSIONS: Extracts from the roots of C. racemosa inhibit nitric oxide production by reducing iNOS expression without affecting activity of the enzyme. This might contribute to the anti-inflammatory activities of C. racemosa.

Three preparations of Hypericum perforatum L.(a hydroalcoholic extract, a lipophilic extract and an ethylacetic fraction) and the pure compounds hypericin, adhyperforin, amentoflavone, hyperoside, isoquercitrin, hyperforin dicyclohexylammonium (DHCA) salt and dicyclohexylamine were evaluated for their topical anti-inflammatory activity. H. perforatum preparations provoked a dose-dependent reduction of Croton-oil-induced ear oedema in mice, showing the following rank order of activity: lipophilic extract > ethylacetic fraction > hydroalcoholic extract (ID50 (dose that inhibited oedema by 50%) 220, 267 and >1000 mug cm(-2), respectively). Amentoflavone (ID50 0.16 mumol cm(-2)), hypericin (ID50 0.25 mumol cm(-2)), hyperforin DHCA salt (ID50 0.25 mumol cm(-2)) and adhyperofrin (ID50 0.30 mumol cm(-2)) had anti-inflammatory activity that was more potent or comparable to that of indometacin (ID50 0.26 mumol cm(-2)), whereas isoquercitrin and hyperoside were less active (ID50 about 1 mumol cm(-2)). As dicyclohexylamine alone was inactive, the effect of hyperforin DHCA salt can be attributed completely to the phloroglucinol moiety. The pharmacological activity and phytochemical profile of the tested extracts and fraction suggest that different constituents are involved in the topical antiphlogistic property of H. perforatum in-vivo.

The structure of a new prenylated coumarin (E-omega-benzoyloxyferulenol, 1b) from the Sardinian giant fennel (Ferula communis) has been confirmed by synthesis. The parent compound ferulenol (1a) showed sub-micromolar antimycobacterial activity, which was partly retained in 1b and in the simplified synthetic analogue 3, but diminished in its omega-hydroxy and omega-acetoxy derivatives (1c and 1d, respectively). The outstanding activity of 1a, its low toxicity, and the evidence for definite structure-activity relationships make this prenylated 4-hydroxycoumarin an interesting antibacterial chemotype worth further investigation.

Ginsenosides are considered the main active principles of the famous Chinese traditional medicine "ginseng". For more than 30 years many researchers developed methods for the identification and quantification of ginsenosides in ginseng plant material, extracts and products. Separation of ginsenosides has been achieved using thin layer chromatography (TLC), gas chromatography (GC) and high performance liquid chromatography (HPLC). Among these techniques HPLC is by far the most employed. Ultraviolet (UV), evaporative light scattering (ELSD), fluorescence and, recently, mass spectrometry (MS) were coupled with HPLC for the detection of ginsenosides. The most recent methods are here discussed together with a critical evaluation of the published results. Furthermore new techniques such as near infrared spectroscopy (NIRS) and enzyme immunosassay (EIA) recently used for the determination of ginsenosides will be discussed.

A simple HPLC method was developed to distinguish between 'poisonous' and 'non-poisonous' chemotypes of Ferula communis. The method was performed on a C(8) reverse phase analytical column using a binary eluent (aqueous TFA 0.01%-TFA 0.01% in acetonitrile) under gradient condition. The two chemotypes showed different fingerprints. The identification of five coumarins and eleven daucane derivatives by HPLC-diode array detection (HPLC-DAD) and HPLC-MS is described. A coumarin, not yet described, was detected.

The octamine cage 5, L, incorporates Ni(II), Cu(II), and Zn(II) in aqueous solution by a fast and reversible process. Equilibrium studies indicated the formation of metal complexes of protonated forms of the ligands, i.e. M(II)(LH(2))](4+) and [M(II)(LH)](+), and of the neutral ligand,[M(II)(L)](2+). The crystal structure of a complex of the monoprotonated ligand,[Ni(II)(LH)](ClO(4))(3), has been determined by single-crystal X-ray crystallography. The complex salt (C(18)H(43)N(8)Cl(3)NiO(12).H(2)O) crystallizes in the orthorombic Pbca space group, with cell constants a = 14.173(2) Å, b = 14.383(1) Å, c = 30.622(3) Å, V = 6242(1) Å(3), and Z = 8. The Ni(II) ion is coordinated to six of the eight available nitrogen atoms, in a very distorted octahedral stereochemistry: of the two uncoordinated donor atoms, a tertiary nitrogen atom and an adjacent secondary one, it is the latter that is protonated. The easy access of protons to uncoordinated amine groups of the cage accounts for the fast demetalation of [Ni(II)(L)](2+) and [Cu(II)(L)](2+) in acidic solution, which was investigated by stopped-flow spectrophotometry. Dependence of k(obs) upon [H(+)] for [Ni(II)(L)](2+) and upon [H(+)](2) for [Cu(II)(L)](2+) indicated that the protonation of uncoordinated nitrogen atoms of the cage is the key step of the demetalation process.

Harvested longan (Dimocarpus longan Lour.) fruit are susceptible to decay caused by both bacterial and fungal infections. Ochratoxin A (OTA) is a kind of mycotoxin produced by a number of fungi. In this study, OTA was extracted from longan fruit pulp by 80% methanol and then loaded on C-18 solid-phase extraction columns. The extract solution was then analyzed by high-performance liquid chromatography - fluorescence detection (HPLC-FD) and an electron spray ionization-mass spectrometry (ESI-MS), respectively. The HPLC-FD analysis showed that a compound similar to OTA might exist in longan fruit pulp, but further analysis by the ESI-MS method demonstrated that OTA was not present in the longan pulp, indicating that the presence of OTA in longan fruit pulp detected by the HPLC-FD analysis needed to be confirmed by the ESI-MS method.

A novel high performance liquid chromatography method for the determination of dimethindene maleate in pharmaceutical gel using hydrophilic interaction liquid chromatography (HILIC) with UV detection was developed and validated. Following optimal conditions for the analysis of dimethindene maleate were used: analytical column SeQuant ZIC((R))-HILIC (50mmx2.1mm, 5mum), and mobile phase consisted of a mixture of acetonitrile and aqueous solution of acetic acid (25mM) and ammonium acetate (2.5mM)(87.5:12.5, v:v). The analysis time was less than 3min at a flow rate of 0.3mlmin(-1). UV detection was performed at 258nm. The method was validated and system suitability parameters were evaluated. The method is suitable for application for routine determination of dimethindene maleate in topical pharmaceutical preparation.

BACKGROUND: In order to understand the tryptophan catabolism in acute and chronic hepatitis B patients, we simultaneously quantitative analyzed plasma kynurenine and tryptophan by high performance liquid chromatography with programmed wavelength ultraviolet detection. METHODS: A new and specific high performance liquid chromatography method to simultaneously measure plasma kynurenine and tryptophan with programmed wavelength ultraviolet detection using 3-nitro-tyrosine as internal standard was elaborated. Thirty patients were recruited (10 patients with acute HBV, 10 with chronic HBV and 10 healthy subjects). RESULTS: The retention time of kynurenine and tryptophan were 2.9 min and 4.4 min, respectively. For kynurenine, the assay was linear from 0.442 mumol/l to 18.3 mumol/l. For tryptophan, the linearity was from 3.67 to 470 mumol/l. The detection limits were 0.014 mumol/l for Kyn and 0.122 mumol/l for Trp, respectively. Its precision and recovery are satisfactory. In this study we found that the kynurenine per tryptophan ratio of acute group is higher than control group and chronic group. CONCLUSIONS: The method is simple, fast, accurate, and suitable for applicability to clinical measurement.

In industrial and pharmaceutical processes, the study of residual products becomes essential to guarantee the quality of compounds and to eliminate or minimize toxic residual products. Knowledge about the origin of impurities (raw materials, processes, the contamination of industrial plants, etc.) is necessary in preventive treatment and in the control of a product's lifecycle. Benzyl chloride is used as raw material to synthesize several quaternary ammonium compounds, such as benzalkonium chloride, which may have pharmaceutical applications. Benzaldehyde, benzyl alcohol, toluene, chloro derivatives of toluene, and dibenzyl ether are compounds that may be found as impurities in technical benzyl chloride. We proposed a high-performance liquid chromatography method for the separation of these compounds, testing two stationary phases with different dimensions and particle sizes, with the application of photodiode array-detection. The linearity for four possible impurities (benzaldehyde, toluene, alpha,alpha-dichlorotoluene, and 2-chlorotoluene) ranged from 0.1 to 10 microg/mL, limits of detection from 11 to 34 ng/mL, and repeatability from 1% to 2.9% for a 0.3-1.2 microg/mL concentration range. The method was applied to samples of technical benzyl chloride, and alpha,alpha-dichlorotoluene and benzaldehyde were identified by spectral analysis and quantitated. The selection of benzyl chloride with lower levels of impurities is important to guarantee the reduction of residual products in further syntheses.

An extraction and analytical method was developed for determination of the content and profiles of anthocyanins in commercial dietary supplements containing blueberry extract. Dietary supplements were refluxed with hydrochloric acid-methanol solution, and spectrophotometric assay was performed to evaluate the total anthocyanidin content in extracted solutions as delphinidin, which is one of the anthocyanidins in blueberry extract. Twenty compounds (15 anthocyanins and 5 anthocyanidins) in extracted solutions were separated by HPLC analysis with gradient elution using formic acid and methanol-acetonitrile as the mobile phase, and each peak was confirmed by LC/MS analysis. The proposed method was applied to 25 kinds of commercial dietary supplements, and one supplement whose profile was different from that of the fresh bilberry extract was found.

INTRODUCTION: Although high-performance liquid chromatography with ultraviolet detection (HPLC-PAD) is widely available, it has not been used for artemisinin (1) analysis because of the lack of UV absorption reported for this lactone. Increased Artemisia annua cultivation for production of 1 requires an affordable and reliable method to analyse 1 and its precursors dihydroartemisinic acid (2) and artemisinic acid (3) simultaneously from underivatized plant extracts. OBJECTIVE: To validate HPLC-PAD for the quantification of underivatized artemisinin from A. annua and artemisinin-based drugs. METHODOLOGY: Dried A. annua leaves were extracted with petroleum ether, dried, reconstituted in acetonitrile, and analysed by HPLC-PAD at 192 nm using an isocratic mobile phase (60:40, acetonitrile:0.1% acetic acid). HPLC-PAD was evaluated through accuracy, precision, recovery and comparison with HPLC with evaporative light scattering detection (HPLC-ELSD). RESULTS: HPLC-PAD proved accurate, precise and reproducible for the direct quantification of 1 and related compounds, and was more sensitive than ELSD for most of the compounds tested. The limit of quantification of 1-3 from plants was 0.048, 0.024 and 0.008 g/100 g dry weight, respectively. Recoveries were over 98%, with good intra- and inter-day repeatability. HPLC-PAD correlated significantly (r(2 )= 0.99, p < 0.001) with HPLC-ELSD for artemisinin analysis. HPLC-PAD was also reliable for the analysis of dihydroartemisinin, artesunate and artelinic acid. CONCLUSION: HPLC with ultraviolet detection was validated for the quantification of underivatized 1, 2, and 3 from crude plant samples, and is readily applicable for the quality control of herbals and artemisinin-related pharmaceutical compounds. Copyright (c) 2008 John Wiley & Sons, Ltd.

The methodological aspects of the use of HPLC in determination of metals in the form of their chelates are discussed. Rational schemes for the analysis of complex environmental and industrial samples are presented, including choice of chelating reagent, chelate preparation, metal concentration, separation method and detection method. Examples of the application of HPLC of metal chelates to environmental, production and quality control are presented.

A practical, sensitive and rapid analytical method was established and validated for chemical impurity tests of 2-deoxy-2-fluoro-d-glucose (FDG), 2-deoxy-2-chloro-d-glucose (ClDG) and Kryptofix 2.2.2 (K-222) in [(18)F]FDG. This method was based on precolumn derivatization with ultraviolet (UV) detection. FDG and ClDG were rapidly derivatized with 1-phenyl-3-methyl-5-pyrazolone in the presence of borate buffer at 40 degrees C, and the labeled derivatives and K-222 were separated by reversed-phase high-performance liquid chromatography and monitored by UV absorbance at 210 nm. After optimization of the conditions, FDG, ClDG and K-222 could be determined within 15 min and showed good performance in terms of sensitivity, linearity and reproducibility. This method could be successfully applied to the quality control test of [(18)F]FDG produced by a commercially available apparatus.

A study of single-laboratory validation (SLV) of a reversed-phase liquid chromatography (RP-LC) method was conducted for the determination of diester-diterpene Aconitum alkaloids, viz., aconitine, mesaconitine, and hypaconitine, in a variety of dietary supplements, including single- and multiple-ingredient dry powder extracts, pills, capsules, and raw materials. The Aconitum alkaloids in the samples were extracted by diethyl ether in the presence of ammonia. After cleanup with solid-phase extraction to remove the matrix interferences, the alkaloids were determined by RP-LC with UV detection at 235 nm, and the results were confirmed by tandem mass spectrometry. The linear responses for aconitine, mesaconitine, and hypaconitine based on the present LC system ranged from 0.5 to 200 microg/mL. Relative standard deviations of 2.0 to 6.9% were obtained from duplicate analysis of 6 test materials of different matrixes for the 3 Aconitum alkaloids performed by 2 analysts on 5 different days. The recoveries determined for supplements and raw materials spiked with 3 Aconitum alkaloids at levels of 2.5-10 microg/g were in the range of 86-99%. In view of the attainment of satisfactory results for accuracy, precision, and recovery in the SLV study, it is recommended that the method validation process proceed to a collaborative study.

Validation of an analytical method for impurities and degradation products in an active pharmaceutical ingredient is important to assessment of quality and safety in a new pharmaceutical product. In the present study, a high-performance liquid chromatographic method was validated to evaluate purity of loteprednol etabonate (LE). LE and its four related substances, major process impurities and degradation products (PJ-90, PJ-91, LE-11-keto and LE-methyl ester) were well resolved using a phenyl-stationary phase under isocratic conditions. Two photo-degradation products were identified as chloromethyl 17alpha-ethoxycarbonyloxy-11beta-hydroxy-5alpha-methyl-2-oxo-19-norandrosta-1(10),3-diene-17beta-carboxylate and chloromethyl 17alpha-ethoxycarbonyloxy-11beta-hydroxy-1-methyl-3-oxo-6(5-->10alpha)-abeo-19-norandrosta-1,4-diene-17beta-carboxylate. A photo-degradation product, chloromethyl 1beta,11beta-epoxy-17alpha-ethoxycarbonyloxy-2-oxo-10alpha-androsta-4-ene-17beta-carboxylate, was not abundant by ultraviolet detector. The risk depending on only ultraviolet detection should be noted. Calibration curves for PJ-90, PJ-91, LE-11-keto and LE-methyl ester showed linearity over the range of 0.05-2.0% levels in LE with correlation coefficient of 0.999. Accuracy (n = 3) at the concentration of 0.5% level in LE for PJ-90, PJ-91, LE-11-keto and LE-methyl ester were 2.0, 2.0, 2.3 and 2.0%, respectively. Intra-day repeatability (n = 6) at the concentration of 0.5% level in LE for PJ-90, PJ-91, LE-11-keto and LE-methyl ester were 1.4, 1.4, 1.8 and 1.4%, respectively. The lower limits of detection for PJ-90, PJ-91, LE-11-keto and LE-methyl ester were 0.002, 0.001, 0.004 and 0.003% levels in LE, respectively.