Enantiospecific Analysis of 8‐Prenylnaringenin in Biological Fluids by Liquid‐Chromatography‐Electrospray Ionization Mass Spectrometry: Application to Preclinical Pharmacokinetic Investigations

8‐Prenylnaringenin (8PN) is a naturally occurring bioactive chiral prenylflavonoid found most commonly in the female flowers of hops (Humulus lupulus L.). A stereospecific method of analysis for 8PN in biological fluids is necessary to study the pharmacokinetic disposition of each enantiomer. A novel and simple liquid chromatographic‐electrospray ionization‐mass spectrometry (LC‐ESI‐MS) method was developed for the simultaneous determination of R‐ and S‐8PN in rat serum and urine. Carbamazepine was used as the internal standard (IS). Enantiomeric resolution of 8PN was achieved on a Chiralpak^® AD‐RH column with an isocratic mobile phase consisting of 2‐propanol and 10 mM ammonium formate (pH 8.5) (40:60, v/v) and a flow rate of 0.7 mL/min. Detection was achieved using negative selective ion monitoring (SIM) of 8PN at m/z 339.15 for both enantiomers and positive SIM m/z at 237.15 for the IS. The calibration curves for urine were linear over a range of 0.01–75 µg/mL and 0.05–75 µg/mL for serum with a limit of quantification of 0.05 µg/mL in serum and 0.01 µg/mL in urine. The method was successfully validated showing that it was sensitive, reproducible, and accurate for enantiospecific quantification of 8PN in biological matrices. The assay was successfully applied to a preliminary study of 8PN enantiomers in rat. Chirality 26:419–426, 2014. © 2014 Wiley Periodicals, Inc.     

HPLC‐PDA‐ORD Bioassay of S‐(+) and R‐(−) Clopidogrel on Rat Dried Blood Spots

A simple and rapid chiral high‐performance liquid chromatography (HPLC) method was developed and validated for bioanalysis of clopidogrel enantiomers on rat dried blood spots (DBS). Clopidogrel enantiomers were extracted from DBS using ethanol: methanol (80:20, v/v) and separated on a Chiralcel OJ‐H column containing cellulose tris (4‐methly benzoate) as a polysaccharide stationary phase using n‐hexane–ethanol‐diethylamine (70:30, 0.1 v/v) as a mobile phase at a flow rate of 1.0 mL/min. The detection was carried out at 220 nm using a photodiode array (PDA) detector while the elution order of the enantiomers was determined by a polarimeter connected to PDA in series. The effect of hematocrit on extraction of clopidogrel enantiomers from DBS was evaluated and no interference from endogenous substances was noticed. The overall accuracy of (R) and (S) enantiomers of clopidogrel from DBS were 91.6 and 89.2%, respectively. The calibration curves were linear over the concentration range of 1–500 µg/mL for both enantiomers. The results show that the method is specific, precise, and reproducible (intra‐ and interday precision relative standard deviations (RSDs) <10.0%). The stability of racemic clopidogrel was performed under all storage conditions and the results were found to be well within the acceptance limits. Chirality 26:102–107, 2014.© 2014 Wiley Periodicals, Inc.     

Simultaneous Enantiomeric Determination of Propranolol,Metoprolol, Pindolol,and Atenolol in Natural Waters by HPLC on New Polysaccharide‐Based Stationary Phase using a Highly Selective Molecularly Imprinted Polymer Extraction

A simple high performance liquid chromatography method HPLC‐UV for simultaneous enantiomeric determination of propranolol, metoprolol, pindolol, and atenolol in natural water samples was developed and validated, using a molecularly imprinted polymer solid‐phase extraction. To achieve this purpose, Lux® Cellulose‐1/Sepapak‐1 (cellulose tris‐(3,5‐dymethylphenylcarbamate)) (Phenomenex, Madrid, Spain) chiral stationary phase was used in gradient elution and normal phase mode at ambient temperature. The gradient elution program optimized consisted of a progressive change of the mobile phase polarity from n‐hex/EtOH/DEA 90/10/0.5 (v/v/v) to 60/40/0.5 (v/v/v) in 13 min, delivered at a flow rate of 1.3 ml/min and a sudden change of flow rate to 2.3 ml/min in 1 min. Critical steps in any molecularly imprinted polymer extraction protocol such as the flow rate to load the water sample in the cartridges and the breakthrough volume were optimized to obtain the higher extraction recoveries for all compounds. In optimal conditions (100 ml breakthrough volume loaded at 2.0 ml/min), extraction recoveries for the four pairs of β‐blockers were near 100%. The MIP‐SPE‐HPLC‐UV method developed demonstrates good linearity (R² ≥ 0.99), precision, selectivity, and sensitivity. Method limit detection was 3.0 µg/l for propranolol and pindolol enantiomers and 20.0 and 22.0 µg/l for metoprolol and atenolol enantiomers, respectively. The proposed methodology should be suitable for routine control of these emerging pollutants in natural waters for a better understanding of the environmental impact and fate. Chirality 24:860–866, 2012. © 2012 Wiley Periodicals, Inc.     

Enantioseparation and Determination of the Chiral Fungicide Furametpyr Enantiomers in Rice,Soil, and Water by High‐Performance Liquid Chromatography

The chiral fungicide furametpyr is widely used in the rice field to control rice sheath blight; however, furametpyr enantiomers are treated as just one compound in traditional achiral analysis, which gives only partial information. An effective chiral analytical method was developed for the resolution and determination of the fungicide furametpyr enantiomers in rice, soil, and water samples. Furametpyr enantiomers were excellently separated and determined on a Chiralpak AD‐H column with n‐hexane/ethanol (90:10, v/v) as mobile phase at a flow rate of 0.8 mL min^‐1 with UV detection at 220 nm. The resolution was up to 8.85. The first eluted enantiomer was (+)‐furametpyr and the second eluted one was (?)‐furametpyr. The effects of mobile‐phase composition and column temperature on the enantioseparation were evaluated. The method was validated for linearity, repeatability, accuracy, limit of detection (LOD), and limit of quantification LOQ. LOD was 2.0 µg kg^‐1 in water, 0.02 mg kg^‐1 in soil, and 0.07 mg kg^‐1 in rice with an LOQ of 6.7 µg kg^‐1 in water, 0.07 mg kg^‐1 in soil, and 0.23 mg kg^‐1 in rice. The average recoveries of the pesticide in all matrices ranged from 73.1 to 101.8% for all fortification levels. The precision values associated with the analytical method, expressed as relative standard deviation (RSD) values, were below 14.0% in all matrices. The methodology was successfully applied for the enantioselective analysis of furametpyr enantiomers in real samples. Chirality 25:904–909, 2013. © 2013 Wiley Periodicals, Inc.     

Precolumn o‐Phthalaldehyde‐N‐acetyl‐L‐cysteine Derivatization Followed by RP‐HPLC Separation and Fluorescence Detection of Sitagliptin Enantiomers in Rat Plasma

An indirect reversed‐phase high‐performance liquid chromatographic separation and fluorescence detection of sitagliptin enantiomers in rat plasma was developed and validated. Deproteinized rat plasma containing racemic sitagliptin was derivatized with o‐phthalaldehyde and N‐acetyl‐L‐cysteine under alkaline conditions, converted to diastereomers, and separated on a Lichrospher 100 RP‐18e column using 20 mM phosphate buffer and methanol (45:55 v/v) as a mobile phase under isocratic mode of elution at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 330 and 450 nm as excitation and emission wavelengths, respectively. The method was linear in the range of 50–5000 ng/ mL for both enantiomers. The intra‐ and interday accuracy and precision were within the predefined limits of ≤15% at all concentrations. The method was successfully applied to a pharmacokinetic study of sitagliptin after 5 mg/kg oral administration to Wistar rats. Robustness of the method was evaluated using design of experiments. Chirality 25:883–889, 2013. © 2013 Wiley Periodicals, Inc.     

A RP‐HPLC‐DAD‐APCI/MSD method for the characterisation of medicinal Ericaceae used by the Eeyou Istchee Cree First Nations

Introduction – Ericaceae medicinal plants are traditionally used by the Eeyou Istchee Cree and other northern peoples of North America to treat type 2 diabetic symptoms. Because of the importance of phenolics as potential cures for degenerative diseases including type 2 diabetes, an analytical method was developed to detect them in the leaf extracts of 14 Ericaceae plants. Objective – To develop an optimised method which is applicable to a relatively large number of Ericaceae plants using their leaf extracts. For this purpose phenolics with a wide range of polarity, including a glucosylated benzoquinone, two phenolic acids, three flavanols, a flavanone, a flavone and five flavonols, were included in this study. Methodology – Characterisation of phytochemicals in extracts was undertaken by automated matching to the UV spectra to those of an in house library of plant secondary metabolites and the authentication of their identity was achieved by reversed phase‐high‐performance chromatography–diode array detection–atmospheric pressure chemical ionisation/mass selective detection. Results – Twenty‐six phenolics were characterised within 26 min of chromatographic separation in 80% ethanol extracts of 14 Ericaceae plants. The calibration curves were linear within 0.5–880 µg/g dry mass of the plant with regression values better than 0.995. The limits of detection ranged from 0.3 for µg/mL for (+)‐catechin to 2.6 µg/mL for chlorogenic acid. This is a first study dealing with relatively large number of Ericaceae extracts and is applicable to other plants of same family. Copyright © 2010 John Wiley & Sons, Ltd.     

Validated high-performance liquid chromatographic method utilizing solid-phase extraction for the simultaneous determination of naringenin and hesperetin in human plasma

Naringenin and hesperetin, the aglycones of the flavanone glucosides naringin and hesperidin occur naturally in citrus fruits. They exert a variety of pharmacological effects such as antioxidant, blood lipid-lowering, anticarcinogenic and inhibit selected cytochrome P-450 enzymes resulting in drug interactions. A specific, sensitive, precise, and accurate solid-phase extraction high-performance liquid chromatographic (HPLC) assay for the simultaneous determination of naringenin and hesperetin in human plasma was developed and validated. After addition of 7-ethoxycoumarin as internal standard, plasma samples were incubated with beta-glucuronidase/sulphatase, and the analytes were isolated from plasma by solid-phase extraction using C(18) cartridges and separated on a C(8) reversed phase column with methanol/water/acetic acid (40:58:2, v/v/v) as the eluent at 45 degrees C. The method was linear in the 10-300 ng/ml concentration range for both naringenin and hesperetin (r>0.999). Recovery for naringenin, hesperetin and internal standard was greater than 76.7%. Intra- and inter-day precision for naringenin ranged from 1.4 to 4.2% and from 1.9 to 5.2%, respectively, and for hesperetin ranged from 1.3 to 4.1% and from 1.7 to 5.1%, respectively. Accuracy was better than 91.5 and 91.3% for naringenin and hesperetin, respectively.     

A validated HPLC method for the determination of dimethyl‐4,4′‐dimethoxy‐5,6,5′,6′‐dimethylene dioxybiphenyl‐2,2′‐dicarboxylate (DDB) with fluorescence detection in raw material and pill form: application to an in vitro dissolution test and a content uniformity test

A simple, sensitive and rapid HPLC method with fluorescence detection for the determination of dimethyl‐4,4′‐dimethoxy‐5,6,5′,6′‐dimethylene dioxybiphenyl‐2,2′‐dicarboxylate (DDB) in the raw material and pill form was developed. Liquid chromatography was performed on a C₁₈ column (250 × 4.6 mm i.d., 5 µm particle size), the mobile phase consisted of methanol and 0.05 M sodium dihydrogen phosphate buffer (80 : 20, v/v), and the apparent pH of the mobile phase was adjusted to 3. The fluorescence detector was operated at excitation/emission wavelengths of 275/400 nm. The proposed method allows the determination of DDB within concentration range 0.1–1.5 µg/mL with a limit of detection of 0.032 µg/mL, a limit of quantification of 0.097 µg/mL and a correlation coefficient of 0.9997. The proposed method has been successfully applied for the analysis of DDB in its pills with a percentage recovery of 98.45 ± 0.32. The method was fully validated according to ICH guidelines. Moreover, the high sensitivity of the method permits its use in an in vitro dissolution test for DDB under simulated intestinal conditions. In addition, the proposed method was extended to a content uniformity test according to USP guidelines. Copyright © 2014 John Wiley & Sons, Ltd.     

Anti‐biofilm and bactericidal effects of magnolia bark‐derived magnolol and honokiol on Streptococcus mutans

Dental caries affects people of all ages and is a worldwide health concern. Streptococcus mutans is a major cariogenic bacterium because of its ability to form biofilm and induce an acidic environment. In this study, the antibacterial activities of magnolol and honokiol, the main constituents of the bark of magnolia plants, toward planktonic cell and biofilm of S. mutans were examined and compared with those of chlorhexidine. The minimal inhibitory concentrations of magnolol, honokiol and chlorhexidine for S. mutans were 10, 10 and 0.25 µg/mL, respectively. In addition, each agent showed bactericidal activity against S. mutans planktonic cells and inhibited biofilm formation in a dose‐ and time‐dependent manner. Magnolol (50 µg/mL) had greater bactericidal activity against S. mutans biofilm than honokiol (50 µg/mL) and chlorhexidine (500 µg/mL) at 5 min after exposure, while all showed scant activity against biofilm at 30 s. Furthermore; chlorhexidine (0.5–500 µg/mL) exhibited high cellular toxicity for the gingival epithelial cell line Ca9‐22 at 1 hr, whereas magnolol (50 µg/mL) and honokiol (50 µg/mL) did not. Thus; it was found that magnolol has antimicrobial activities against planktonic and biofilm cells of S. mutans. Magnolol may be a candidate for prevention and management of dental caries.     

Analysis of flumequine enantiomers in rat plasma by UFLC‐ESI‐MS/MS

In this study the analysis and confirmation of flumequine enantiomers in rat plasma by ultra‐fast liquid chromatography coupled with electron spray ionization mass spectrometry (using propranolol as an internal standard [IS]) was developed and validated. Plasma samples were prepared by liquid–liquid extraction using methyl tert‐butyl ether as the extraction solvent. Direct resolution of the R‐ and S‐isomers was performed on a CHIRALCEL OJ‐RH column (4.6 × 150 mm, 5 μm) using acetonitrile / 0.1% formic acid / 1 mM ammonium acetate as the mobile phase. Detection was operated by electron spray ionization in the selected ion monitoring and positive ion mode. The target ions at m/z 262.1 and m/z 260.1 were selected for the quantification of the enantiomers and IS, respectively. The linear range was 0.5–500 ng/mL. The precisions (coefficient of variation, CV%) and recoveries were 1.43–8.68 and 94.24–106.76%, respectively. The lowest quantitation limit for both enantiomers is 0.5 ng/mL, which is sensitive enough to be applied to sample analysis in other related studies.     

Novel Stereoselective High‐Performance Liquid Chromatographic Method for Simultaneous Determination of Guaifenesin and Ketorolac Enantiomers in Human Plasma

A novel method was developed for the simultaneous determination of guaifenesin (GUA) and ketorolac tromethamine (KET) enantiomers in plasma samples. Since GUA probably increases the absorption of coadministered drugs (e.g., KET), it would be extremely important to monitor KET plasma levels for the purpose of dose adjustment with a subsequent decrease in the side effects. Enantiomeric resolution was achieved on a polysaccharide‐based chiral stationary phase, amylose‐2, as a chiral selector under the normal phase (NP) mode and using ornidazole (ORN) as internal standard. This innovative method has the advantage of the ease and reliability of sample preparation for plasma samples. Sample clean‐up was based on simply using methanol for protein precipitation followed by direct extraction of drug residues using ethanol. Both GUA and KET enantiomers were separated using an isocratic mobile phase composed of hexane/isopropanol/trifluoroacetic acid, 85:15:0.05 v/v/v. Peak area ratios were linear over the range 0.05–20 µg/mL for the four enantiomers S (+) GUA, R (–) GUA, R (+) KET, and S (–) KET. The method was fully validated according to the International Conference on Harmonization (ICH) guidelines in terms of system suitability, specificity, accuracy, precision, robustness, and solution stability. Finally, this procedure was innovative to apply the rationale of developing a chiral high‐performance liquid chromatography (HPLC) procedure for the simultaneous quantitative analysis of drug isomers in clinical samples. Chirality 26:629–639, 2014. © 2014 Wiley Periodicals, Inc.     

Simultaneous determination of some dihydroxybenzenesulphonic acid derivatives and their degradation product and main impurity (hydroquinone) by ion‐pair liquid chromatography

A simple and sensitive high‐performance liquid chromatography method was developed and validated for the determination of calcium dobesilate (DOB) or ethamsylate (ETM) in the presence of their degradation product, hydroquinone (HQ). The analyses were carried out on Promosil C₁₈ column (4.6 mm × 250 mm, 5 µm particle size) using an ion‐pair mobile phase consisting of methanol–1.5 mm tetra‐butyl ammonium bromide in 0.06 m phosphate buffer (25 : 75, v/v) at pH 6.0 with fluorescence detection at 286/333 nm. Pindolol was used as an internal standard. The proposed method was found to be rectilinear over the concentration ranges of 0.05–0.5 µg/mL for DOB, 0.1–0.8 µg/mL for ETM and 0.005–0.1 µg/mL for HQ. The method was applied for the determination of the studied drugs in different dosage forms and biological fluids. The results of the proposed method were statistically compared with those obtained by the comparison methods revealing no significance differences in the performance of the methods regarding accuracy and precision. Moreover, applying a time‐programmed fluorescence technique was valuable for the detection of trace amounts of HQ as an impurity and allowed purity testing of ETM or DOB within the BP pharmacopeial limit (0.1%). Copyright © 2013 John Wiley & Sons, Ltd.     

Trace analysis of N‐acetyl‐L‐cysteine using luminol–H2O2 chemiluminescence system catalyzed by silver nanoparticles

N‐Acetyl‐L‐cysteine (NAC) can inhibit the luminol–H₂O₂, reaction, which is catalyzed by silver nanoparticles. Based on this phenomenon a new method was developed for NAC determination. Under optimum conditions, a linear relationship between chemiluminescence intensity and NAC concentration was found in the range 0.034–0.98 µg/mL. The detection limit was 0.010 µg/mL (S/N =3), and the relative standard deviation (RSD) was <5% for 0.480 µg/mL NAC (n =5). This simple, sensitive and inexpensive method has been applied to measure the concentration of NAC in pharmaceutical tablets. Copyright © 2014 John Wiley & Sons, Ltd.     

Analysis of Oxcarbazepine and the 10‐Hydroxycarbazepine Enantiomers in Plasma by LC‐MS/MS: Application in a Pharmacokinetic Study

Oxcarbazepine is a second‐generation antiepileptic drug indicated as monotherapy or adjunctive therapy in the treatment of partial seizures or generalized tonic–clonic seizures in adults and children. It undergoes rapid presystemic reduction with formation of the active metabolite 10‐hydroxycarbazepine (MHD), which has a chiral center at position 10, with the enantiomers (S)‐(+)‐ and R‐(?)‐MHD showing similar antiepileptic effects. This study presents the development and validation of a method of sequential analysis of oxcarbazepine and MHD enantiomers in plasma using liquid chromatography with tandem mass spectrometry (LC‐MS/MS). Aliquots of 100 μL of plasma were extracted with a mixture of methyl tert‐butyl ether: dichloromethane (2:1). The separation of oxcarbazepine and the MHD enantiomers was obtained on a chiral phase Chiralcel OD‐H column, using a mixture of hexane:ethanol:isopropanol (80:15:5, v/v/v) as mobile phase at a flow rate of 1.3 mL/min with a split ratio of 1:5, and quantification was performed by LC‐MS/MS. The limit of quantification was 12.5 ng oxcarbazepine and 31.25 ng of each MHD enantiomer/mL of plasma. The method was applied in the study of kinetic disposition of oxcarbazepine and the MHD enantiomers in the steady state after oral administration of 300 mg/12 h oxcarbazepine in a healthy volunteer. The maximum plasma concentration of oxcarbazepine was 1.2 µg/mL at 0.75 h. The kinetic disposition of MHD is enantioselective, with a higher proportion of the S‐(+)‐MHD enantiomer compared to R‐(?)‐MHD and an AUC^0‐12 _{S‐(+)/R‐(?)} ratio of 5.44. Chirality 25:897–903, 2013. © 2013 Wiley Periodicals, Inc.     

Validated spectrofluorimetric method for the determination of carbamazepine in pharmaceutical dosage forms after reaction with 4‐chloro‐7‐‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl)

A sensitive and simple spectrofluorimetric method has been developed and validated for the determination of the anti‐epileptic drug carbamazepine (CBZ) in its dosage forms. The method was based on a nucleophilic substitution reaction of CBZ with 4‐chloro‐7‐nitrobenzo‐2‐ oxa‐1,3‐diazole (NBD‐Cl) in borate buffer (pH 9) to form a highly fluorescent derivative that was measured at 530 nm after excitation at 460 nm. Factors affecting the formation of the reaction product were studied and optimized, and the reaction mechanism was postulated. The fluorescence–concentration plot is rectilinear over the range of 0.6–8 µg/mL with limit of detection of 0.06 µg/mL and limit of quantitation of 0.19 µg/mL. The method was applied to the analysis of commercial tablets and the results were in good agreement with those obtained using the reference method. Validation of the analytical procedures was evaluated according to ICH guidelines. Copyright © 2015 John Wiley & Sons, Ltd.     

Estimation of Enantiomeric Impurity in Piperidin‐3‐Amine by Chiral HPLC With Precolumn Derivatization

A simple, precise, accurate, robust chiral high‐performance liquid chromatographic (chiral HPLC) method was developed for estimation of (S)‐piperidin‐3‐amine (S‐isomer) in (R)‐piperidin‐3‐amine dihydrochloride (R‐AMP). As AMP is a high‐melting solid and nonchromophoric compound, development of a suitable chiral method is a challenging task. The proposed chiral HPLC‐UV method involves a precolumn derivatization technique with para toluene sulphonyl chloride (PTSC) in the presence of a base to introduce chromophore into analytes. It utilizes chiralpak AD‐H column with a simple mobile phase of 0.1% diethyl amine in ethanol with a 0.5 mL/min flow rate. Analytes were monitored by using a UV detector at 228 nm. The resolution between the two enantiomers was more than 4.0. The developed method was validated as per current International Conference on Harmonization (ICH) guidelines. Chirality 26:775–779, 2014. © 2014 Wiley Periodicals, Inc.     

A Validated Normal Phase LC Method for Enantiomeric Separation of Rasagiline Mesylate and Its (S)‐Enantiomer on Cellulose Derivative‐Based Chiral Stationary Phase

A simple, sensitive, and robust normal‐phase isocratic HPLC‐UV method was developed and validated for the enantiomeric separation of rasagiline mesylate and its (S)‐enantiomer. The rasagiline and its (S)‐enantiomer were resolved on a Chiralcel‐OJ‐H (4‐methylbenzoate cellulose coated on silica) column using a mobile phase consisting of n‐hexane:isopropyl alcohol:ethanol:diethyl amine (96:2:2:0.01) at a flow rate of 1.0 ml/min. The column temperature was maintained at 27 °C and elution was monitored at 215 nm. The resolution (R_s) between the enantiomers was found to be more than 2.0. The limit of detection and the limit of quantification of the (S)‐enantiomer were found to be 0.35 and 1.05 µg/ml, respectively. The developed method was validated as per ICH guidelines with respect to linearity, limit of detection and quantification, accuracy, precision, and robustness—and satisfactory results were obtained. The sample solution and mobile phase were found to be stable up to 48 h. The method is useful for routine evaluation of the quality of rasagiline mesylate in bulk drug‐manufacturing units. Chirality 25:324–327, 2013. © 2013 Wiley Periodicals, Inc.     

Fluorimetric determination of proteins using 4‐chloro‐(2′‐hydroxylophenylazo)rhodanine–Ti(IV) complex as a spectral probe

A novel method for the determination of proteins was developed, based on the enhancement of fluorescence with 4‐chloro‐(2′‐hydroxylophenylazo)rhodanine–Ti(IV) [ClHARP–Ti(IV)] complex as a fluorescence probe. The excitation and emission wavelengths of the system were 335 nm and 376 nm, respectively. The presence of bis(2‐ethylhexyl)sulphosuccinate sodium salt (AOT) microemulsion greatly increased the sensitivity of the system. Under optimal conditions, four kinds of proteins, including bovine serum albumin (BSA), human serum albumin (HSA), egg albumin (Ova), and γ‐globin (γ‐G) were studied. The detection limits were 0.182 µg/mL for BSA, 0.0788 µg/mL for HSA, 0.216 µg/mL for Ova and 0.484 µg/mL for γ‐G. The linear ranges of the calibration were 0–12.0, 0–10.0, 0–18.0 and 0–18.0 µg/mL, respectively. The method possessed high sensitivity, good selectivity and was applied to the analysis of protein in milk powder and cornmeal with satisfactory results. Copyright © 2008 John Wiley & Sons, Ltd.     

A New Chiral Residue Analysis Method for Triazole Fungicides in Water Using Dispersive Liquid‐Liquid Microextraction (DLLME)

A rapid, simple, reliable, and environment‐friendly method for the residue analysis of the enantiomers of four chiral fungicides including hexaconazole, triadimefon, tebuconazole, and penconazole in water samples was developed by dispersive liquid–liquid microextraction (DLLME) pretreatment followed by chiral high‐performance liquid chromatography (HPLC)‐DAD detection. The enantiomers were separated on a Chiralpak IC column by HPLC applying n‐hexane or petroleum ether as mobile phase and ethanol or isopropanol as modifier. The influences of mobile phase composition and temperature on the resolution were investigated and most of the enantiomers could be completely separated in 20 min under optimized conditions. The thermodynamic parameters indicated that the separation was enthalpy‐driven. The elution orders were detected by both circular dichroism detector (CD) and optical rotatory dispersion detector (ORD). Parameters affecting the DLLME performance for pretreatment of the chiral fungicides residue in water samples, such as the extraction and dispersive solvents and their volume, were studied and optimized. Under the optimum microextraction condition the enrichment factors were over 121 and the linearities were 30–1500 µg L^?1 with the correlation coefficients (R²) over 0.9988 and the recoveries were between 88.7% and 103.7% at the spiking levels of 0.5, 0.25, and 0.05 mg L^?1(for each enantiomer) with relative standard deviations varying from 1.38% to 6.70% (n = 6) The limits of detection (LODs) ranged from 8.5 to 29.0 µg L^?1(S/N = 3). Chirality 25:567‐574, 2013. © 2013 Wiley Periodicals, Inc.     

An Eco‐Friendly Enantioselective Access to (R)‐Naringenin as Inhibitor of Proinflammatory Cytokine Release

(RS)‐Naringenin is a flavanone well‐known for its beneficial health‐related properties, such as its anti‐inflammatory activity. The preparative enantioselective chromatographic resolution of commercial (RS)‐naringenin was performed on a Chiralpak AD‐H column (500×50 mm i.d., d_p 20 μm) using MeOH as eluent. The developed method is in accordance with the principles of green chemistry, since the environmental impact was lowered by recycling of the eluent, and allowed the production of gram amounts of each enantiomer with high purity (chemical purity >99%, enantiomeric excess (ee) >94%). Racemic and enantiomeric naringenin were subjected to an exhaustive in vitro investigation of anti‐inflammatory activity, aimed at evaluating the relevance of chirality. The assay with cultured human peripheral blood mononuclear cells (hPBMC) activated by phytohemagglutinin A revealed that (R)‐naringenin was more effective in inhibiting T‐cell proliferation than the (S)‐enantiomer and the racemate. Moreover, (R)‐naringenin significantly reduced proinflammatory cytokine levels such as those of TNF‐α and, with less potency, IL‐6. These results evidenced the anti‐inflammatory potential of naringenin and the higher capacity of (R)‐naringenin to inhibit both in vitro hPBMC proliferation and cytokine secretion at non toxic doses. Thus, (R)‐naringenin is a promising candidate for in vivo investigation.