Extraction and analysis of colourful eggshell pigments using HPLC and HPLC/electrospray ionization tandem mass spectrometry

The literature on the pigments of avian eggshells is critically reviewed. Methods using methanolic sulfuric acid or hydrochloric acid to extract eggshell pigments are unsuitable to detect the occurrence of zinc protoporphyrin or zinc biliverdin because they demetallate these compounds. Extraction methods are described here using EDTA and acetonitrile–acetic acid or acetonitrile–dimethyl sulfoxide, which do not demetallate zinc protoporphyrin. Such extracts were prepared from eggshell of the common nighthawk, Chordeiles minor, and from another six bird species. Protoporphyrin and biliverdin were identified and fully characterized by HPLC/electrospray ionization tandem mass spectrometry (HPLC/ESI‐MS/MS) in all samples, but none contained zinc protoporphyrin. The zinc complex of biliverdin, claimed to be an additional pigment responsible for eggshell background colours, was labile to EDTA and acid pH and if occurring naturally could not be extracted intact by the published or the modified protocols. An explanation is advanced for the exceptional report that all porphyrins from uroporphyrin to protoporphyrin were found in eggshells of the fowl Gallus domesticus. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of the serine palmitoyl transferase inhibitor myriocin by electrospray and Q‐trap mass spectrometry

Myriocin is a potent inhibitor of serine‐palmitoyl‐transferase, the first and rate‐determining enzyme in the sphingolipids biosynthetic pathway. This study developed, validated and applied a LC–MS/MS method to measure myriocin in minute specimens of animal tissue. The chemical analog 14‐OH–myriocin was used as the internal standard. The two molecules were extracted from the tissue homogenate by solid‐phase extraction, separated by gradient reversed‐phase liquid chromatography and measured by negative ion electrospray mass spectrometry in the triple quadrupole. Detection was accomplished by multiple reaction monitoring, employing the most representative transitions, 400@104 and 402@104 for myriocin and 14‐OH‐myriocin, respectively. The typical limit of detection and lower limit of quantitation of the optimized method were 0.9 pmol/mL (~0.016 pmol injected) and 2.3 pmol/mL, respectively, and the method was linear up to 250 pmol/mL range (r ² = 0.9996). The intra‐ and between‐day repeatability afforded a coefficient of variation ≤7.0%. Applications included quantification of myriocin in mouse lungs after 24 h from administration of ~4 nmol by intra‐tracheal delivery. Measured levels ranged from 4.11 (median; 2.3–7.4 IQR, n = 4) to 11.7 (median; 7.6–22.7 interquartile range (IQR), n = 6) pmol/lung depending on the different formulations used. Myriocin was also measured in retinas of mice treated by intravitreal injection and ranged from 0.045 (less than the limit of detection) to 0.35 pmol/retina.     

Adsorption kinetics for arsenic removal from aqueous solutions by untreated powdered eggshell

The batch removal of arsenic from aqueous solution using low-cost adsorbent (powdered eggshell) under the influences of initial arsenic ion concentrations (0.50 to 1.50 mg/L), pH (3.2 to 11.5) and particle size of eggshells (63 to 150 μm) were investigated. Eggshells were collected from Obafemi Awolowo University, Ile-Ife, washed with distilled water, air dried, ground into powder and sieved into different sieve sizes using British standard sieve. Powdered eggshells were stored in a desiccator for use. Adsorption isotherms and dynamics of arsenic onto PES were studied. The study revealed that there was a slight reduction in the rate of adsorption of arsenic ion onto the larger particle size, but adsorption capacity and parameters were unaffected. Powdered eggshell with particle size of 63 μm removed up to 99.6% of the 1.5 mg/L of arsenic ion in synthetic water within the first 6 hours but decreased to 98.4% and 97.4% when the powdered eggshell particle sizes were increased to 75 and 150 μm respectively. The pH optimum for arsenic removal was 7.2. The adsorption isotherms and adsorption dynamic kinetic studied through the use of graphical method revealed that Freundlich, activated sludge adsorption and pseudo second-order kinetic models correlate significantly with the experimental data with correlation coefficient of not less than 0.964.     

Preparative isolation of ganoderic acid S,ganoderic acid T and ganoderol B from Ganoderma lucidum mycelia by high‐speed counter‐current chromatography

Ganoderic acid S, ganoderic acid T and ganoderal B are the main bioactive triterpenes of Ganoderma lucidum. In this study, mycelia of G. lucidum were obtained by two‐stage fermentation and then extracted by ethanol and petroleum ether sequentially to obtain crude triterpenes. The crude sample was further purified by recycling high‐speed counter‐current chromatography with n‐hexane–ethyl acetate–methanol–water (7:12:11:5, v/v/v/v) as the optimized two‐phase solvent system. A 16.4 mg aliquot of ganoderol B with a purity of 90.4% was separated from 300 mg of the crude sample in a single run. After employing the recycling elution mode of HSCCC with n‐hexane–ethyl acetate–methanol–water (6:10:8:4.5, v/v/v/v) for five cycles, 25.7 mg ganoderic acid T and 3.7 mg ganoderic acid S with purities of 97.8 and 83.0%, respectively, were obtained. The purities of three compounds were determined by high‐performance liquid chromatography and their chemical structures were identified by NMR and MS data.     

Residual determination and risk assessment of buprofezin in plum (Prunus domestica) grown in open‐field conditions following the application of three different formulations

This study was conducted to characterize the residual level and perform a risk assessment on buprofezin formulated as an emulsifiable concentrate, wettable powder, and suspension concentrate over various treatment schedules in plum (Prunus domestica). The samples were extracted with an AOAC quick, easy, cheap, effective, rugged, and safe, ‘QuEChERS’, method after major modifications. As intrinsic interferences were observed in blank plum samples following dispersive‐solid phase extraction (consisting of primary secondary amine and C₁₈ sorbents), amino cartridges were used for solid‐phase extraction. Analysis was carried out using liquid chromatography with diode array detection and confirmed by liquid chromatography–tandem mass spectrometry. The method showed excellent linearity with determination coefficient (R² = 1) and satisfactory recoveries (at two spiking levels, 0.5 and 2.5 mg/kg) between 90.98 and 94.74% with relative standard deviation (RSD) ≤8%. The limit of quantification (0.05 mg/kg) was considerably lower than the maximum residue limit (2 mg/kg) set by the Codex Alimentarius. Absolute residue levels for emulsifiable concentrates were highest, perhaps owing to the dilution rate and adjuvant. Notably, all formulation residues were lower than the maximum residue limit, and safety data proved that the fruits are safe for consumers. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantitation of plasma angiotensin II in healthy Chinese subjects by a validated liquid chromatography tandem mass spectrometry method

Quantitation of plasma angiotensin (Ang) II, the active mediator of the renin–angiotensin system, is challenging owing to its low physiological concentration. We report a validated liquid chromatography–mass spectrometry (LCMS) method to overcome this challenge. Ang II was extracted from EDTA plasma by an offline solid-phase extraction procedure with a Waters MAX μElution plate. LCMS quantitation was performed on the Waters TQS system, monitoring the 3+ ions of the peptide. The analytical performance of the LCMS method was validated. The stability of Ang II was studied with or without the presence of a protease inhibitor. Local reference intervals were established from 143 healthy normotensive subjects (57% female, 21–60 years old). The Ang II LCMS method had a measurable range of 3.3–700 pmol/L. The between-batch precision coefficient of variation was <7% over Ang II concentrations of 8.6–110 pmol/L. No significant matrix interference and carryover were observed. There was no significant difference in Ang II concentration in EDTA blood and plasma for at least 2h and 1 h at room temperature, respectively. Ang II was stable for at least 1 year when stored at −80°C, with or without the protease inhibitor. Age-dependent Ang II reference intervals were established: 4.4–17.7 pmol/L (21–30 years) and 3.9–12.8 pmol/L (31–60 years). The present LCMS method is suitable for quantitation of plasma Ang II to study the renin-angiotensin system.     

Determination of arsenic compounds in marine mammals with high-performance liquid chromatography and an inductively coupled plasma mass spectrometer as element-specific detector

Total arsenic concentrations and the concentrations of individual arsenic compounds were determined in liver samples of pinnipeds [nine ringed seals (Phoca hispida), one bearded seal (Erginathus barbatus)] and cetaceans [two pilot whales (Globicephalus melas), one beluga whale (Deliphinapterus leucus)]. Total arsenic concentrations ranged from 0.167 to 2.40 mg As kg⁻¹ wet mass. The arsenic compounds extracted from the liver samples with a methanol/water mixture (9:1, v/v) were identified and quantified by anion- and cation-exchange chromatography. An ICP–MS equipped with a hydraulic high-pressure nebulizer served as the arsenic-specific detector. Arsenobetaine (0.052–1.67 mg As kg⁻¹ wet mass) was the predominant arsenic compound in all the liver samples. Arsenocholine was present in all livers (0.005–0.044 mg As kg⁻¹ wet mass). The tetramethylarsonium cation was detected in all pinnipeds ( < 0.009 to 0.043 mg As kg⁻¹) but not in any of the cetaceans. The concentration of dimethylarsinic acid ranged from < 0.001 to 0.109 mg As kg⁻¹ wet mass. Most of the concentrations for methylarsonic acid ( < 0.001 to 0.025 mg As kg⁻¹ wet mass) were below the detection limit. Arsenous acid and arsenic acid concentrations were below the detection limit of the method (0.001 mg As kg⁻¹). An unknown arsenic compound was present in all liver samples at concentrations from 0.002–0.027 mg As kg⁻¹. © 1998 John Wiley & Sons, Ltd.     

Simultaneous determination of boscalid and fludioxonil in grape and soil under field conditions by gas chromatography/tandem triple quadrupole mass spectrometry

A gas chromatography–tandem mass spectrometry method was developed and validated to simultaneously determine boscalid and fludioxonil in grape and soil samples. These samples were extracted with 10 mL of acetonitrile and purified using a mixed primary secondary amine/octadecylsilane sorbent. The method showed good linearity (R² > 0.99) in the calibration range 0.005–2 μg/mL for both pesticides. The limits of detection and quantification for the two analytes in grape and soil were 0.006 and 0.02 mg/kg, respectively. Fungicide recoveries in grape and soil were 81.18–92.11% for boscalid and 82.73–97.67% for fludioxonil with relative standard deviations of 1.31–10.31%. The established method was successfully applied to the residual analysis of boscalid and fludioxonil in real grape and soil samples. The terminal residue concentrations of boscalid and fludioxonil in grape samples collected from Anhui and Guizhou were <5 mg/kg (the maximum residue limit set by China) 7 days after the last application and 1 mg/kg (the maximum residue limit set by USA) 14 days after the last application. These results could provide guidance for the proper and safe use of boscalid and fludioxonil in grape and help the Chinese government to establish an MRL for fludioxonil in grape.     

Derivatization following hollow‐fiber microextraction with tetramethylammonium acetate as a dual‐function reagent for the determination of benzoic acid and sorbic acid by GC

Derivatization at the injection port following hollow‐fiber‐based liquid–liquid–liquid microextraction with tetramethylammonium acetate as a dual‐function reagent, i.e. an acceptor and derivatization reagent, for the determination of benzoic acid (BA) and sorbic acid (SA) in real samples by GC was developed. BA and SA were extracted from aqueous samples to an organic phase impregnated into the pores of the hollow fiber wall, and then back‐extracted to the acceptor solution located inside the lumen of the hollow fiber. Upon injection, the extracted analytes were quantitatively derivatized to their methyl esters with tetramethylammonium acetate in the GC injection port. Several parameters related to the derivatization and extraction efficiency were optimized. The linearity was satisfactory over a concentration range of 0.1–50 mg/L with r > 0.993 for both analytes. The LODs were 2.0 μg/L for SA and 20 μg/L for BA. The recoveries (83–116%) and precisions (RSDs of 1.2–11.4% (n = 3)) were examined by analyzing real spiked samples. The enrichment factors of BA and SA were 300 and 425. The results demonstrated that this is a simple, rapid, accurate, and sensitive method for the determination of BA and SA in various samples.     

Determination of drugs of abuse in bovine dentin using liquid chromatography–electrospray ionization tandem mass spectrometry

Drugs deposited in human teeth are well preserved; the spectrum of toxicological investigations may therefore be supplemented by an analysis method for drugs in teeth. A liquid chromatography–electrospray ionization tandem mass spectrometry assay for the detection and quantification of basic drugs of abuse in bovine dentin samples was developed and validated. The drugs and metabolites amphetamine, methamphetamine, methylenedioxymethylamphetamine, methylenedioxyethylamphetamine, codeine, morphine, cocaine and benzoylecgonine were extracted from 50 mg ground dentin powder by ultrasonication for 60 min in methanol 3 times. The extracts were analyzed on a triple‐quadrupole mass‐spectrometer in multiple reaction monitoring mode. The method was validated and proved to be accurate, precise, selective, specific and stable with good linearity within the calibration range and a lower limit of quantification of 10 to 20 pg/mg. To artificially load bovine dentin samples with drugs, the natural process of de‐ and remineralization in the oral cavity was mimicked by a pH‐cycling experiment. The artificially drug‐loaded dentin samples showed drug concentrations of 20 to 80 pg/mg. The method can be applied in further in vitro experiments as well as in post‐mortem cases, especially where limited sample tissue is available. Copyright © 2014 John Wiley & Sons, Ltd.     

Antifungal activity of Bignoniaceae plants on Aspergillus carbonarius and Aspergillus niger

Abstract

Twenty four extracts from Bignoniaceae plants of northwest Argentina were tested for antifungal activity against Aspergillus species responsible of the grape black rot. Stems and leaves of Amphilophium cynanchoides, Macfadyena cynanchoides, Tecoma stans and Jacaranda mimosifolia were separately extracted with solvents of increasing polarity to obtain the dichloromethane (fCH₂Cl₂), ethyl acetate (fEtOAc) and methanol extracts (fMeOH). The fCH₂Cl₂ from stem of M. cynanchoides had the lowest IC₅₀ (1.0–1.2?mg/mL) and MID values (0.6–1.2?mg) and the highest ID values (5.0–6.8?mm) on A. niger and A. carbonarius. The main contributors of the antifungal activity of fCH₂Cl₂ were identified as lapachol (MIC?=?0.25–1.00?mg/ml) and 1-hydroxy-4-methylanthraquinone (MIC?=?0.0625–0.125?mg/mL). These compounds synergized the antifungal activity of sodium metabisulfite and showed an additive effect in mixtures with propiconazol. They might be used as additives of commercial antifungals to protect grapes against A. niger and A. carbonarius.     

Simultaneous determination and dissipation behaviour of thifluzamide and difenoconazole in grapes using a QuEChERS method with ultra high-performance liquid chromatography and tandem mass spectrometry

In this study, a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was used for the simultaneous determination of thifluzamide and difenoconazole in grapes by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS) using a Waters Acquity UHPLC BEH C18 column. To compare the effects of different sorbents for thifluzamide and difenoconazole in grapes, samples were extracted with acetonitrile and cleaned up using five different types of sorbents at the same concentration level (0.1 mg/kg). The method was validated at three fortification concentrations (0.05, 0.1, 0.5 mg/kg) with five replicates in each matrix using 40 mg of primary secondary amine (PSA) + 10 mg of graphitised carbon black (GCB) as clean-up sorbents. The recoveries were between 76.3 and 109.3%, and the RSDr (intra-day precision, n = 5) and RSD_R (inter-day precision, n = 15) values ranged from 3.5 to 8.1% and 5.8 to 22.2%, respectively. The limits of quantification (LOQ) for thifluzamide and difenoconazole were 0.07 and 0.04 mg/kg, respectively. The method showed excellent linearity and reliability. The results demonstrated that the method was effective in detecting the two compounds. In this study, we also investigated the dissipation behaviours of thifluzamide and difenoconazole in grapes. The dissipations of thifluzamide and difenoconazole followed the first-order kinetics with the half-lives of 3.9–16.3 days.     

Rapid determination of losartan and losartan acid in human plasma by multiplexed LC–MS/MS

A rapid LC–MS/MS method has been developed and validated for the determination of losartan (LOS) and its metabolite losartan acid (LA) (EXP‐3174) in human plasma using multiplexing technique (two HPLC units connected to one MS/MS). LOS and LA were extracted from human plasma by SPE technique using Oasis HLB® cartridge without evaporation and reconstitution steps. Hydroflumethiazide (HFTZ) was used as an internal standard (IS). The analytes were separated on Zorbax SB C‐18 column. The mass transition [M–H] ions used for detection were m/z 421.0 → 127.0 for LOS, m/z 435.0 → 157.0 for LA, and m/z 330.0 → 239.0 for HFTZ. The proposed method was validated over the concentration range of 2.5–2000 ng/mL for LOS and 5.0–3000 ng/mL for LA with correlation coefficient ?0.9993. The overall recoveries for LOS, LA, and IS were 96.53, 99.86, and 94.16%, respectively. Total MS run time was 2.0 min/sample. The validated method has been successfully used to analyze human plasma samples for applications in 100 mg fasted and fed pharmacokinetic studies.     

Determination of hydroxylated metabolites of polycyclic aromatic hydrocarbons in human hair by gas chromatography–negative chemical ionization mass spectrometry

The study describes the determination of mono-hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs), metabolites of PAHs, in human hair. Twelve selected OH-PAHs from two to four rings, generally determined in urine analysis, were investigated as markers of human exposure to PAHs. Following hydrolysis of hair specimens of 50–300 mg with 1 M NaOH, OH-PAHs were extracted using dichloromethane and submitted to an optimized derivatization with (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride. Compounds were then analyzed using gas chromatography–negative chemical ionization mass spectrometry (GC–NCIMS). The average inter-day and intra-day variability was 12% and 17%, respectively. The average recovery was 52% and the limits of detection and quantification ranged from 20 and 66 pmol/g for 1-OH-phenanthrene (i.e., 3.9 and 12.8 pg/mg) to 311 and 1030 pmol/g for 2-OH-benzo(c)phenanthrene (i.e., 75.9 and 251 pg/mg). The influence of hair washing with water as decontamination step, and enzymatic treatment (β-glucuronidase) to hydrolyze conjugated derivatives were also tested. The application of the developed method to the analysis of 30 hair specimens (17 from non-smoker and 13 from smoker volunteers) demonstrated inter-individual qualitative and quantitative variations. According to the easiness of hair sampling and based on the extended detection windows provided by hair analysis, this method is proposed as a new promising tool for the assessment of human chronic exposure to PAHs.     

Determination of alachlor residues in pepper and pepper leaf using gas chromatography and confirmed via mass spectrometry with matrix protection

Alachlor residues were determined in pepper and pepper leaf, after 49 days of manufacturer‐recommended single‐ and double‐dose application to the soil and plant. The samples were extracted with acetonitrile, partitioned with n‐hexane, and purified through solid‐phase extraction, and finally detected with a gas chromatography–microelectron capture detector. The linearity of the analytical response across the studied range of concentrations (0.05–4.0 µg/mL) was excellent, obtaining coefficients of determination (r²) of 0.999. Recovery studies were carried out on spiked pepper and pepper leaf samples, at two concentrations levels (0.2 and 1.0 mg/kg), with three replicates performed at each level. Mean recoveries of 73.1–109.0% with relative standard deviations of 1.3–2.3% were obtained. The method was successfully applied to field samples, and alachlor residue was found in pepper (0.02 mg/kg) and pepper leaf (0.03 mg/kg), at levels lower than the maximum residue limits (0.2 mg/kg) set by the Korea Food and Drug Administration. The field‐detected residues were further confirmed with gas chromatography–mass spectrometry with the help of pepper leaf matrix protection. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of 11 fluorescent whitening agents in food‐contact paper and board by ion‐pairing high‐performance liquid chromatography with fluorescence detection

4,4′‐Diaminostilbene‐2,2′‐disulfonic acid based fluorescent whitening agents (DSD‐FWAs) are prohibited in food‐contact paper and board in many countries. In this work, a reliable high‐performance liquid chromatography method was developed for the simultaneous determination of 11 common DSD‐FWAs in paper material. Sample preparation and extraction as well as chromatographic separation of multicomponent DSD‐FWAs were successfully optimized. DSD‐FWAs in prepared samples were ultrasonically extracted with acetonitrile/water/triethylamine (40:60:1, v/v/v), separated on the C₁₈ column with the mobile phase containing tetrabutylammonium bromide, and then detected by a fluorescence detector. The limits of detection were 0.12–0.24 mg/kg, and the calibration curves showed the linear correlation (R² ≥ 0.9994) within the range of 8.0–100 ng/mL, which was equivalent to the range of 0.80–10 mg/kg in the sample. The average recoveries and the RSDs were 81–106% and 2–9% at two fortification levels (1.0 and 5.0 mg/kg) in paper bowls, respectively. The successful determination of 11 DSD‐FWAs in food‐contact paper and board obtained from local markets indicated that the newly developed method was rapid, accurate, and highly selective.     

Determination of Sudan dyes in chili pepper powder by online solid‐phase extraction with a butyl methacrylate monolithic column coupled to liquid chromatography with tandem mass spectrometry

A poly(butyl methacrylate‐co‐ethylene dimethacrylate) monolithic column was fabricated and used as a novel sorbent for online solid‐phase extraction coupled to liquid chromatography with tandem mass spectrometry for the simultaneous determination of Sudan I–IV in chili pepper powder. The prepared columns were characterized by scanning electron microscopy, nitrogen adsorption‐desorption, and pressure drop measurements. Online solid‐phase extraction was performed on the synthesized monolithic column using 10 mM ammonium acetate solution as the loading solution with the aid of an online cleanup chromatography system. The desorption of Sudan I–IV was achieved with acetonitrile as the eluting solution at the flow rate of 0.5 mL/min. The extracted analytes were subsequently eluted into a C₁₈ analytical column for chromatographic separation using a mixture of 10% acetonitrile/90% formic acid (0.5%) solution as the mobile phase. Under the optimized conditions, the developed method had linear range of 1.0–50 μg/kg, a detection limit of 0.3 μg/kg, and a quantification limit of 1.0 μg/kg for each analyte. The intraday and interday recoveries of Sudan I–IV in chili pepper powder samples ranged from 94.8 to 100.9% and 94.9 to 99.4%, respectively. The intraday and interday precision were between 3.37–7.01% and 5.01–7.68%, respectively.     

Simultaneous quantification of total eicosapentaenoic acid,docosahexaenoic acid and arachidonic acid in plasma by high‐performance liquid chromatography–tandem mass spectrometry

A method for the simultaneous quantification of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) in human plasma by HPLC–tandem mass spectrometry (HPLC‐MS/MS) was developed and validated. Free and esterified forms of fatty acids were hydrolysed from plasma samples in the presence of an internal standard and subjected to liquid–liquid extraction. The chromatographic run time was 3.5 min per sample. The assay was linear from 0.5 to 300 mg/L (r² > 0.997, n = 18). Based on matrix addition, accuracy deviation was <15%, except for AA at 10 mg/L (30–90%), whereas precision was <8% for all fatty acids studied. The method was applied to the measurement of these omega‐3 fatty acids in a fish oil supplement study with healthy volunteers. Healthy males (n = 4) were administered a supplement containing 465 mg EPA and 375 mg DHA per capsule (Omacor®). A dose of two capsules was given daily over a 4 week period. Pre‐treatment concentrations varied between subjects for EPA (17–68 mg/L), DHA (36–63 mg/L) and AA (121–248 mg/L). During the dosing period EPA increased 460–480% from the baseline concentration, while DHA increased 150–160%. The EPA–AA ratio increased from 0.07–0.56 to 0.3–3.1 after 4 weeks of dosing. In conclusion, the method described could be suitable for monitoring EPA, DHA and AA in clinical studies that may aid in achieving optimal concentrations of these fatty acids in patients who could be at risk of sudden cardiac death. Copyright © 2010 John Wiley & Sons, Ltd.     

Single‐step modified QuEChERS for determination of chlorothalonil in shallot (Allium ascalonicum) using GC‐μECD and confirmation via mass spectrometry

A single extraction method was developed for chlorothalonil in shallot using gas chromatography with an electron capture detector (GC‐μECD). Samples were extracted with single‐step modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method using ethyl acetate as an extraction solvent. Significant matrix effects were observed, and the calibration curve was constructed from the matrix. The linearity of the analytical response across the studied range of concentrations (0.01–1.00 mg/L) was excellent, obtaining a correlation coefficient (r²) of 0.996. >0.996. Recovery studies were carried out on spiked shallot blank samples, at two concentration levels (0.4 and 2.0 mg/kg) with three replicates performed at each level. Mean recoveries of 97.2–104.9% with RSDs of 1.3–2.7% were obtained. The method is demonstrated to be suitable for the determination of chlorothalonil in shallot. The dissipation rates of chlorothalonil were described using first‐order kinetics, and its half‐life was 2.8 days. Based on the dissipation pattern of the pesticide residues, the pre‐harvest residue limit (PHRL) was also calculated. Residues were confirmed via mass spectrometry. Copyright © 2012 John Wiley & Sons, Ltd.     

Preparative separation of six terpenoids from Wedelia prostrata Hemsl. by two-step high-speed counter-current chromatography

Terpenoids are principal chemical compounds of Wedelia prostrata Hemsl. and have different biological activities, thus the study on separation and purification of terpenoids from W. prostrata Hemsl. is necessary. In this paper, high-speed counter-current chromatography (HSCCC) was successfully established for preparative isolation and purification of terpenoids from extracts of petroleum ether fraction which extracted from whole herbs of W. prostrata Hemsl. In the process, a total of 750?mg of sample was prepared for HSCCC isolation. Terpenoids were separated and purified with the two-phase solvent system n-hexane–ethyl acetate–methanol–water (9:1:9:1, 19:1:19:1, v/v/v/v). Therefore, 5α-hydroxy-2-oxo-p-menth-6(1)-ene (4.4?mg), 3α-angeloyloxy-ent-kaur-16-en-oic acid (5.6?mg), 3α-tigloyloxy-ent-kaur-16-en-oic acid (5.7?mg), 3α-phenylpropionoyloxy-ent-kaur-16-en-oic acid (7.3?mg), 3α-senecioyloxy-ent-kaur-16-en-oic acid (11.4?mg), and kaurenoic acid (12.3?mg) were obtained from W. prostrata Hemsl. and their purities reached standard determined by HPLC. Among them, 3α-phenylpropionoyloxy-ent-kaur-16-en-oic acid and 5α-hydroxy-2-oxo-p-menth-6(1)-ene were first isolated with high quantity as a useful chemical resource. The structures of these compounds were identified by ESI-MS, ¹H-NMR, and ¹³C-NMR. The present results demonstrated that high-speed counter-current chromatography was a fast and efficient technique for preparative separation of six terpenoids from W. prostrata Hemsl. which provided a useful reference to solve the problem of their sample availability for drug development.