Identification of the metabolites of n-butylidenephthalide in rat and human liver microsomes by liquid chromatography-high-resolution mass spectrometry.

n-Butylidenephthalide (NBDP) is one of the bioactive constituents originally isolated from Ligusticum chuanxiong Hort. The aim of this study was to study the metabolic profiles of NBDP in rat and human liver microsomes. NBDP was individually incubated with liver microsomes of rat and human at 37°C for 1 h and the samples incubated were analyzed by ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry. The identities of the metabolites were identified by accurate masses, product ions and retention times. Under the current conditions, a total of 14 metabolites were detected and identified. M12, M13 and M14 were biosynthesized and unambiguously characterized by nuclear magnetic resonance spectroscopy. All the metabolites can be detected in rat liver microsomes, whereas in human liver microsomes, M1, M3, M4, M5, M6 and M7 were not detected. Our results demonstrated that the metabolic pathways of NBDP included hydroxylation, hydration, hydrolysis and glutathione conjugation. This study provides an overview of the metabolic profiles of NBDP in vitro, which is helpful to understand the action of this compound.

Systematic identification of thermal degradation products of HPMCP during hot melt extrusion process.

A systematic identification of the degradation products of hydroxypropyl methylcellulose phthalate (HPMCP) during hot melt extrusion (HME) has been performed. A reverse phase HPLC method was developed for the extrudates of both hydroxypropyl methylcellulose acetate succinate (HPMCAS) and HPMCP polymers to quantify their thermal hydrolytic products: acetic acid (AA), succinic acid (SA) for HPMCAS and phthalic acid (PA) for HPMCP, without hydrolysing the polymers in strong alkaline solutions. The polymers were extruded in the temperature range of 160-190 °C at different screw rotation speeds and hydrolytic impurities were analysed. Investigation of extruded HPMCP showed an additional thermal degradation product, who is structural elucidation revealed to be phthalic anhydride (PAH). Moreover, two environmental analytical impurities, dimethyl phthalate and methyl benzoate formed in situ were recorded on GC-MS and their origin was found to be associated with PAH derivatization. Using the experimental data gathered during this study, a degradation mechanism for HPMCP is proposed.

[Study on the constituents of essential oil of Shunaoxin dropping pills by GC-MS].

OBJECTIVE: To study the constituents of essential oil from Shunaoxin dropping pills by GC-MS. METHODS: The essential oil from Shunaoxin dropping pills were extracted by absolute alcohol and analyzed by GC-MS. RESULTS: 15 components from the essential oil of Shunaoxin dropping pills were identified. CONCLUSION: The main components in the essential oil of Shunaoxin dropping pills are lactones such as Z-ligustilide, senkyunolide A,3-butylphthalide and 3-butylidenephthalide, other components are organic acids such as ethyl linoleate, 9,12-octadecadienoic acid and ethyl palmitate.

Targeted and untargeted phytochemistry of Ligusticum canbyi: indoleamines, phthalides, antioxidant potential, and use of metabolomics as a hypothesis-generating technique for compound discovery.

Ligusticum canbyi (J.M. Coult & Rose) is a medicinal understory forest species used in traditional rituals and ceremonies for spiritual enlightenment and improved mental health. Very little is known about the phytochemical complexity or diversity of L. canbyi tissues or extracts. The current study was undertaken to determine whether Ligusticum tissues and extracts contain specifically targeted biologically active phytochemicals such as: melatonin, serotonin, Z-ligustilide, E-3-butylidenephthalide, and ferulic acid and to investigate the untargeted phytochemical complexity of the entire L. canbyi metabolome. The results of these studies identified melatonin and serotonin in roots and shoots of L. canbyi and L. porteri. Z-ligustilide, E-butylidenephthalide, and ferulic acid were quantified in roots and shoots of L. canbyi. Metabolomic analysis detected approximately 34,000 compounds in each L. canbyi extract, and predictive analysis suggests the presence of more than 70 putative phthalide metabolites. The relative contribution of the known metabolites and the unknown markers to the antioxidant potential of root and shoot tissues were compared, and it was determined that the majority of the antioxidant capacity could be attributed to ferulic acid in the tissues. These data provide new understandings of the phytomedicinal composition and potential mechanisms of activity of L. canbyi extracts and tissues.

Bioactivity-guided fractionation of the volatile oil of Angelica sinensis radix designed to preserve the synergistic effects of the mixture followed by identification of the active principles.

In natural product research, it is a common experience that fractionation of biologically-active crude extracts can lead to the loss of their original activity. This is attributed to synergistic effects, where two or more components are required to be present together for full activity of the sample. Our previous study showed that a volatile oil of Angelica sinensis radix (VOAS) inhibited endothelial cell proliferation in culture. Here we have used a bioactivity-guided fractionation method to preserve any synergistic effects of VOAS combining countercurrent chromatography (CCC), the MTS cell viability assay and gas chromatography (GC). Using a two-phase CCC solvent system (heptane-ethyl acetate-methanol-water at a volume ratio of 27:23:27:23%), forty-five fractions were isolated, nine of which exhibited anti-endothelial properties. GC analysis showed two bioactive alkylphthalides, Z-ligustilide and n-butylidenephthalide (BP) were the major compounds detected in the bioactive fractions, and were absent in non-bioactive fractions. Our results indicate that Z-ligustilide and BP are the main constituents responsible for the anti-endothelial properties of VOAS. This rapid and reliable approach in preserving sample activity while isolating and identifying its active compounds suggests that this protocol can be a powerful tool for drug discovery from natural products.

[Study on the dynamic variations of Z-ligustilide and n-butylidenephthalide content in essential oil of radix angelicae sinensis from different growth period].

OBJECTIVE: To study the variation of the biomass of the root and active components of Angelica sinensis during different growth periods. METHODS: 27 batches of Angelica sinensis were harvested from different growth periods, and the biomass of underground parts were determined; The Gas Chromatography-Mass Spectrometry (GC-MS) was used for determining the contents of Z-ligustilide and n-Butylidenephthalide in essential oil of Radix Angelicae Sinensis. RESULTS: The average contents of n-Butylidenephthalide and Z-ligustilide were more than 1% and 40% in the total essential oil of Radix Angelicae sinensis respectively. Their contents showed larger difference during different growth period. CONCLUSION: The contents of Z-ligustilide and n-Butylidenephthalide of Radix Angelicae Sinensis is closely related to their growth period.

[Determination of phthalic anhydride in ambient air by high performance liquid chromatography].

The paper considers the measurement of phthalic anhydride in the ambient air samples by high performance liquid chromatography. It describes conditions for air sampling and analysis of phthalic anhydride levels in the presence of concomitant components of its production (phthalic and maleic acids, maleic anhydride, etc.) on a liquid chromatograph with an UV detector. The procedure was tested, by estimating the quality of ambient air at the border of a sanitary protection zone of phthalic anhydride production and when analyzing the air in the industrial area. Field studies detected the concentrations of phthalic anhydrate in the air of an enterprise area, which were equal to 0.017-0.115 mg/dm3. Phthalic anhydride was detectable at a concentration of 0.001-0.0021 mg/dm3 at the border of the existing sanitary protection zone in single cases. The procedure has been recommended to measure the mass concentrations of phthalic anhydride aerosol and vapors in ambient air at the reference concentration.

Perfluorosulfonic acid membrane catalysts for optical sensing of anhydrides in the gas phase.

Continuous, on-site monitoring of personal exposure levels to occupational chemical hazards in ambient air is a long-standing analytical challenge. Such monitoring is required to institute appropriate health measures but is often limited by the time delays associated with batch air sampling and the need for off-site instrumental analyses. In this work, we report on the first attempt to use the catalytic properties of perfluorosulfonic acid (PSA) membranes to obtain a rapid, selective, and highly sensitive optical response to trimellitic anhydride (TMA) in the gas phase for portable sensor device application. TMA is used as starting material for various organic products and is recognized to be an extremely toxic agent by the National Institute for Occupational Safety and Health (NIOSH). Resorcinol dye is shown to become immobilized in PSA membranes and diffusionally constrain an orange brown product that results from acid-catalyzed reaction with more rapidly diffusing TMA molecules. FTIR, UV/vis, reaction selectivity to TMA versus trimellitic acid (TMLA), and homogeneous synthesis are used to infer 5,7- dihydroxyanthraquinone-2-carboxylic acid as the acylation product of the reaction. The color response has a sensitivity to at least 3 parts per billion (ppb) TMA exposure and, in addition to TMLA, excludes maleic anhydride (MA) and phthalic anhydride (PA). Solvent extraction at long times is used to determine that the resorcinol extinction coefficient in 1100 EW PSA membrane has a value of 1210 m(2)/g at 271.01 nm versus a value of 2010 m(2)/g at 275.22 nm in 50 vol% ethanol/water solution. The hypsochromic wavelength shift and reduced extinction coefficient suggest that the polar perfluorosulfonic acid groups in the membrane provide the thermodynamic driving force for diffusion and immobilization. At a resorcinol concentration of 0.376 g/L in the membrane, a partition coefficient of nearly unity is obtained between the membrane and solution concentrations and a maximum conversion rate of one ambient TMA molecule for every two membrane-immobilized resorcinol molecules is observed in 15 min.

[Determination of butylidenephthalide in Ligusticum chuanxiong by HPLC].

OBJECTIVE: To determine butylidenephthalide in Ligusticum Chuanxiong with RP-HPLC. METHOD: The sample was extracted with methanol using sonication. The ESTD was used to quantify butylidenephthalide. HPLC separation was carried out in a Hypersil ODS columm (4.6 mm x 150 mm, 5 microm) , eluted at 1 mL x min(-1) with methanol-5% isopropyl alcohol (60: 40) at 25 degrees C. The detection wavelength was 230 nm. RESULT: The linear range was 0.07-0.7 microg for butylidenephthalide. The average recovery was 95.3%, and RSD was 2.3% (n =6). CONCLUSION: This method was simple and could be used to determine butylidenephthalide with satisfactory accuracy and reproducibility.

Allergic contact dermatitis to copolymers in cosmetics--case report and review of the literature.

Copolymers or heteropolymers are large molecules with high molecular weights (>1000 D). They have been underestimated for a long time as to their sensitizing capacities. Allergic contact dermatitis to 6 copolymers in cosmetics and 1 in a medical dressing has been described; however, the nature of the hapten is still unknown. We report a case of allergic contact dermatitis to polyvinylpyrrolidone (PVP)/hexadecene copolymer in a purple-colored lipstick and review the literature on allergic contact dermatitis to 7 copolymers: PVP/hexadecene, PVP/eicosene, PVP/1-triacontene, methoxy polyethyleneglycol (PEG)-22/dodecyl glycols, methoxy PEG-17/dodecyl glycols, phthalic anhydride/trimellitic anhydride/glycols, and polyvinyl methyl/maleic acid anhydride.

[High performance liquid chromatography-mass spectrometry analysis of radix Angelica sciensis].

AIM: To analyze the chemical components in Danggui (the roots of Angelica sinensis (Oliv.) Diel). METHODS: HPLC-MS/MS was used to identify the main components in Danggui. Furthermore, the MS fragmentation regularity of the phthalides was proposed. The mobile phase of HPLC consisted of 0.5% acetic acid in water and 0.5% acetic acid in acetonitrile, analytical column was Hypersil ODS2 (250 mm x 4.6 mm, 5 microm), flow rate 1.0 mL x min(-1), injected volume 2 microL. The ionization source was ESI in positive ion mode. RESULTS: Ferulic acid, nine known phthalides and one unknown phthalide derivative were tentatively identified in chromatograms based on their MS data and the comparison of their UV spectra with those published in the literatures. CONCLUSION: The structural information of phthalides was obtained via HPLC-MS/MS, which provides an accurate and fast method to identify the phthalides and provides more scientific information for quality control of Danggui.

Correlations between air levels of hexahydrophthalic anhydride (HHPA) and HHPA-adducted albumin tryptic peptides in nasal lavage fluid from experimentally exposed volunteers.

Organic acid anhydrides (OAAs) are low molecular weight, reactive compounds extensively used in industry. Exposure to these compounds may lead to allergic symptoms such as rhinitis and asthma. It is important to develop better and more informative methods for assessment of exposure to OAAs. The aim of this study was to develop a method for analysis of specific hexahydrophthalic anhydride (HHPA)-adducted tryptic peptides of human serum albumin (HSA) in nasal lavage (NAL). Furthermore, these peptides were evaluated as biomarkers of exposure. The proteins in the NAL samples were reduced, alkylated and digested with trypsin and the obtained peptides were analyzed using liquid chromatography/tandem mass spectrometry. The total amount of hydrolyzable HHPA in an HHPA-HSA conjugate was used for calibration. A deuterium-labeled HHPA-HSA conjugate was used as internal standard. Five volunteers were exposed to 10, 40 and 80 microg/m3 of HHPA in an exposure chamber and NAL samples were collected before and after exposure. Acceptable precisions of the assay at 13-14% were found for three adducted peptides. The mean levels of these three peptides for the five subjects ranged between 5-22, 15-75 and 33-125 pmol/mL NAL for the exposures at 10, 40 and 80 microg/m3, respectively. High correlations between air levels and the measured peptides were found on an individual basis but there were large inter-individual differences ranging between 63 and 110% for the three peptides. The large differences remained after protein adjustments. It was possible to detect exposures below 10 microg/m3 with the method. Thus, these adducted peptides may be used as biomarkers of exposure, which may better estimate the risk than previous biomarkers developed for OAAs.

Phthalic anhydride allergy: development and characterization of optimized hapten-carrier conjugates for improved diagnosis.

BACKGROUND: At present the diagnosis of IgE-mediated hypersensitivity to phthalic anhydride (PA) is based on conjugates that are not characterized or standardized. The aim of this study was to develop optimized and molecularly characterized PA conjugates that can be used to improve the diagnosis of PA-allergy. METHODS: The PA conjugates were synthesized and the number of haptens bound on a carrier protein was estimated by matrix-assisted laser desorption/ionization time of light (MALDI-TOF) mass spectrometry. The ability of conjugates to bind IgE and IgG antibodies was measured by enzyme-linked immunosorbent assay (ELISA). Reactivity of the conjugates in vivo was evaluated by skin prick testing. RESULTS: The most active IgE-binding conjugates had a PA : HSA molar ratio of 80 : 1. In the optimal conjugates the average numbers of PA haptens per carrier molecule of human serum albumin (HSA) were 14-16. In ELISA, all 13 patients and none of the 20 controls had IgE antibodies to optimized PA conjugate. The sensitivity and specificity of the ELISA was comparable to commercial CAP RAST. PA conjugates elicited positive test results in skin prick testing showing that conjugates are immunologically active also in vivo. CONCLUSIONS: These results indicate that optimized and molecularly characterized PA-HSA conjugates can be used both in vitro and in vivo assays to improve the diagnosis of PA allergy.

Exposure to hexahydrophthalic and methylhexahydrophthalic anhydrides--dose-response for sensitization and airway effects.

OBJECTIVES: This study clarified the exposure-response relationships for the organic acid anhydrides (OAA) hexahydrophthalic (HHPA) and methylhexahydrophthalic (MHHPA) anhydrides and the development of specific immunoglobulin (IG) E and G antibodies and work-related symptoms. METHODS: In an epoxy resin-using factory, air levels of OAA were determined by gas chromatography-mass spectrometry. Occupational, smoking, and medical histories (questionnaire) were obtained for 154 exposed workers and 57 referents. Work-related symptoms of the eyes and airways were recorded, and OAA metabolites were analyzed in urine. A skin-prick test with common allergens and conjugates of OAA were performed. Specific IgE (radioallergosorbent test) and IgG (enzyme-linked immumosorbent assay) antibodies were determined in serum, and spirometry was performed. RESULTS: Air levels of the OAA were low (HHPA < 1 to 94, MHHPA < 3 to 77 microg/m3) and associated with the concentrations of the OAA metabolites in urine. Furthermore, for the exposed workers, there were high prevalences of sensitization (IgE 22%, IgG 21%), which correlated with the exposure. Neither atopy nor smoking increased this risk significantly. Furthermore, work-related symptoms were more prevalent among the exposed workers than among the referents (eyes 23% versus 14%, nose 28% versus 16%, nose bleeding 8% versus 0%, lower airways 10% versus 4%), and they were related to the exposure (adjusted prevalence odds ratios (POR) in the highest group 7.7, 3.6 and 17, respectively) and the IgE levels (POR 4.9, 3.1 and 5.6, respectively). CONCLUSIONS: In spite of the very low OAA levels in the air and metabolites in the urine, there were high and exposure-related risks of specific IgE and IgG sensitization and of work-related symptoms for the eyes, nose (especially bleeding), and lower airways.

Determination of methyltetrahydrophthalic anhydride in air using gas chromatography with electron-capture detection.

Methyltetrahydrophthalic anhydride (MTHPA) stimulates the production of specific IgE antibodies which can cause occupational allergy even at extremely low levels of exposure (15-22 micrograms/m3). Safe use in industry demands control of the levels of exposure causing allergic diseases. Thus, the air monitoring of MTHPA is very important, and sensitive methods are required to measure low air concentrations or short-time peak exposures. This paper outlines the use of silica-gel tubes for sampling airborne MTHPA vapour, followed by analysis using gas chromatography with electron-capture detection. No breakthrough was observed at 113, 217, 673 and 830 micrograms/m3 (sampling volume 30, 60, 60 and 20 l, respectively; relative humidity 40-55%). Concentrations > 1.0 microgram/m3 could be quantified at 20-min sampling with a sampling rate of 1 l/min. The present method can also be applied to measurements of exposure to hexahydrophthalic and methylhexahydrophthalic anhydride. The risk of MTHPA exposure in two condenser plants was also assessed by determining MTHPA levels in air of the workplace. In conclusion, our method was found to be reliable and sensitive, and can be applied to the evaluation of MTHPA exposure.

Quantification method of human hemoglobin adducts from hexahydrophthalic anhydride and methylhexahydrophthalic anhydride.

A method was developed for the determination of human hemoglobin (Hb) adducts from hexahydrophthalic anhydride (HHPA) and methylhexahydrophthalic anhydride (MHHPA). The procedure includes lysis of erythrocytes, dialysis of the Hb-solution followed by acid hydrolysis. The released hexahydrophthalic (HHP) acid and methylhexahydrophthalic (MHHP) acid were purified using a combined liquid-liquid and solid-phase extraction procedure followed by derivatization with pentafluorobenzyl bromide. The derivatives were analyzed using GC-MS in negative ion chemical ionization mode with ammonia as moderating gas. As internal standards, deuterium-labeled HHP and MHHP acids were used. The detection limits were 0.3 pmol/g Hb for HHP acid and 0.9 pmol/g Hb for MHHP acid. The between-day precisions for HHP acid were 18% at 2 pmol/g Hb and 10% at 13 pmol/g Hb. For MHHP acid, the precision was 27% at 2 pmol/g Hb and 14% at 22 pmol/g Hb. The method was applicable for analysis of Hb adducts from workers occupationally exposed to HHPA and MHHPA.

Risk factors for sensitisation to methyltetrahydrophthalic anhydride.

OBJECTIVES: To examine an association between specific IgE to methyltetrahydrophthalic anhydride (MTHPA) and exposure time, atopic history, smoking habits, and total IgE concentrations. METHODS: A cross sectional survey was carried out on a population of 148 workers from two condenser plants using epoxy resin with MTHPA, an acid anhydride curing agent known to cause allergy. RESULTS: Using a Pharmacia CAP system with a MTHPA human serum albumin conjugate, specific IgE antibody was detected in serum from 97 (66%) out of the 148 workers exposed to MTHPA. Stepwise multiple linear regression analysis showed a striking relation between log concentrations of specific and total IgE (P < 0.0001). Furthermore, when the workers were divided into two groups according to a cut-off point (100 IU/ml) between low and high total IgE, current smoking was significantly (P = 0.025) associated with specific IgE production only in the group with low total IgE (< 100 IU/ml). CONCLUSIONS: Smoking is the most significant risk factor for raising specific IgE to MTHPA in the group with low total IgE concentrations.

Direct measurement of hexahydrophthalic anhydride in workplace air with a transportable Fourier transform infrared spectrometer.

A method for direct measurement of hexahydrophthalic anhydride (HHPA) in workplace air by use of a Fourier transform infrared (FTIR) spectrometer was developed. Two visits were made to a plant manufacturing capacitors where HHPA was used. On the first visit a calibration method was developed according to what was expected to give the best calibration. This was performed by collection of 82 FTIR spectra from the air while simultaneously taking samples with a reference method using Amberlite XAD-2 sorbent tubes. On the second visit, two weeks later, the calibration method was used for prediction of HHPA concentrations (n = 52) in air; these were compared with XAD-2 determinations. The predicted FTIR values as a function of the XAD-2 determinations were used to evaluate some parameters regarding the FTIR method. The limit of detection was 120 micrograms HHPA/m3, and the precision at 150 micrograms/m3 was 22% and at 400 micrograms/m3 8%. When sampling from a pure HHPA atmosphere the obtained concentration by the FTIR was 103% of that of the XAD-2 tubes. The selection of different analytical parameters for the determinations are also discussed. The method is a useful tool in fast mappings of exposure levels.

Exposure-response relationships in the formation of specific antibodies to hexahydrophthalic anhydride in exposed workers.

OBJECTIVES: Exposure-response relationships in the formation of specific antibodies to hexahydrophthalic anhydride (HHPA) was studied in exposed workers. METHODS: The relation between exposure to HHPA and the levels of specific immunoglobin E [(radioallergosorbent test (RAST)] and immunoglobin G (enzyme-linked immunosorbent assay) antibodies was investigated in a cross-sectional study on 95 workers from two plants using epoxy resin with HHPA as a hardener; the mean time of exposure was 7 (range 0.1-25) years. RESULTS: The specific immunoglobin E and immunoglobin G was significantly increased in exposed workers when they were compared with unexposed workers or external referents. There was no significant difference in the number of RAST positives [N = 23 (24%)] between the groups of workers exposed to < 10 micrograms.m-3, 10--< 50 micrograms.m-3, or > or = 50 micrograms.m-3. No effects were found of atopy or smoking habits on the prevalence of RAST positives. Five out of seven workers positive for immunoglobulin E in the group with the lowest exposures reported frequent short-time (minutes per day) exposures exceeding 50 micrograms.m-3. A correlation was seen between specific immunoglobulin E and G antibodies (rs = 0.5). CONCLUSIONS: The results indicate that HHPA is a sensitizing compound even at low exposure levels and that short-time peak exposures may have an impact on immunoglobulin E sensitization.

Immunologic specificity of IgG against trimellityl-human serum albumin in serum samples of workers exposed to trimellitic anhydride.

The specificity of immunoglobulin G (IgG) against trimellityl-human serum albumin (TM-HSA) in serum samples from 11 workers exposed to trimellitic anhydride (TMA) was characterized in this study. Levels of IgG against TM-HSA and HSA-conjugates of other acid anhydrides, phthalic anhydride (PA), maleic anhydride (MA), hexahydrophthalic anhydride (HHPA) and tetrachlorophthalic anhydride (TCPA) were estimated by enzyme-linked immunosorbent assay index. Inhibition studies using each of 5 HSA acid anhydride conjugates were performed on all 11 serum samples with IgG against TM-HSA, and on the three serum samples with highest IgG binding to P-HSA and M-HSA. The only conjugate capable of inhibiting TM-HSA was TM-HSA. Both P-HSA and TM-HSA were able to inhibit IgG bound to P-HSA, and all other anhydride conjugates were able to inhibit IgG bound to M-HSA to some degree. When using TM-HSA and the other anhydride-HSA conjugates to inhibit IgG against TM-HSA, cross-reactivity was not apparent. However, when using those same conjugates to inhibit IgG against P-HSA or M-HSA, cross-reactivity could be demonstrated in some serum samples. Thus TMA workers may have antibody that has some affinity for other anhydride-HSA conjugates, but this antibody cannot be demonstrated by inhibition studies of IgG against TM-HSA when using other acid anhydride-HSA conjugates. Further studies are needed to define the biologic relevance of these immunologic observations.