Method for the determination of catechin and epicatechin enantiomers in cocoa-based ingredients and products by high-performance liquid chromatography: single-laboratory validation

A single-laboratory validation study was performed for an HPLC method to identify and quantify the flavanol enantiomers (+)- and (-)-epicatechin and (+)- and (-)-catechin in cocoa-based ingredients and products. These compounds were eluted isocratically with an ammonium acetate-methanol mobile phase applied to a modified beta-cyclodextrin chiral stationary phase and detected using fluorescence. Spike recovery experiments using appropriate matrix blanks, along with cocoa extract, cocoa powder, and dark chocolate, were used to evaluate accuracy, repeatability, specificity, LOD, LOQ, and linearity of the method as performed by a single analyst on multiple days. In all samples analyzed, (-)-epicatechin was the predominant flavanol and represented 68-91% of the total monomeric flavanols detected. For the cocoa-based products, within-day (intraday) precision for (-)-epicatechin was between 1.46-3.22%, for (+)-catechin between 3.66-6.90%, and for (-)-catechin between 1.69-6.89%; (+)-epicatechin was not detected in these samples. Recoveries for the three sample types investigated ranged from 82.2 to 102.1% at the 50% spiking level, 83.7 to 102.0% at the 100% spiking level, and 80.4 to 101.1% at the 200% spiking level. Based on performance results, this method may be suitable for routine laboratory use in analysis of cocoa-based ingredients and products.     

Separation of pyrethroid enantiomers by chiral high-performance liquid chromatography

The separation of enantiomers of pyrethroid insecticides has been systematically studied using a commercially available Pirkle type 1-A chiral-phase high-performance liquid chromatography column. Useful resolution was obtained for compounds with a variety of acid and alcohol moieties, and containing one to four chiral centres. The chromatographic behaviour of the diastereomers of some of these insecticides on a cyano-bonded column was also examined.     

Direct separation of drug enantiomers by high-performance liquid chromatography with chiral stationary phases

The development of chiral stationary phases for HPLC has resulted in renewed interest in methods for the separation of drug enantiomers. This paper provides a brief overview of some of the more recent approaches to the direct resolution of drug enantiomers by HPLC with particular emphasis on their quantification in biological fluids.     

Optimized separation of pesticide enantiomers by chiral high-performance liquid chromatography

Summary A computer-assisted method is described for optimization of multi-component, mobile phase selection for separating enantiomers of four pesticides in normal-phase HPLC. The method is based on the triangle, solvent-selection concept using a statistical scanning method. The optimization of the separation over the experimental region is based on a special polynomial estimation from seven experimental runs, and resolution (R_s) is used as the selection criterion. Excellent agreement was obtained between predicted and experimental data.     

Quantitative analysis of three chiral pesticide enantiomers by high-performance column liquid chromatography

Methods for the enantiomeric quantitative determination of 3 chiral pesticides, paclobutrazol, myclobutanil, and uniconazole, and their residues in soil and water are reported. An effective chiral high-performance liquid chromatographic (HPLC)-UV method using an amylose-tris(3,5-dimethylphenylcarbamate; AD) column was developed for resolving the enantiomers and quantitative determination. The enantiomers were identified by a circular dichroism detector. Validation involved complete resolution of each of the 2 enantiomers, plus determination of linearity, precision, and limit of detection (LOD). The pesticide enantiomers were isolated by solvent extraction from soil and C18 solid-phase extraction from water. The 2 enantiomers of the 3 pesticides could be completely separated on the AD column using n-hexane isopropanol mobile phase. The linearity and precision results indicated that the method was reliable for the quantitative analysis of the enantiomers. LODs were 0.025, 0.05, and 0.05 mg/kg for each enantiomer of paclobutrazol, myclobutanil, and uniconazole, respectively. Recovery and precision data showed that the pretreatment procedures were satisfactory for enantiomer extraction and cleanup. This method can be used for optical purity determination of technical material and analysis of environmental residues.     

Resolution of enantiomers of the antiarrhythmic drug encainide and its major metabolites by chiral derivatization and high-performance liquid chromatography

Commercially available chiral columns were unable to provide adequate resolution of enantiomers of the antiarrhythmic drug encainide or its major metabolites. The homochiral derivatizing agent, (-)-menthyl chloroformate, was found to react at the tertiary piperidine nitrogen of racemic encainide providing two menthyl carbamate diastereomers. The individual diastereomers could be separated with baseline resolution on normal-phase high-performance liquid chromatography on a silica column. Structures of the derivatives were confirmed by electron impact mass spectrometry and 1H NMR spectroscopy. The method was adapted for the chiral analysis of the major metabolites of encainide. The limit of sensitivity for racemic encainide was 10 ng on column and it was possible to detect a mixture containing (+)- and (-)-encainide in a ratio of 1:99. Preliminary studies indicated that (-)-encainide was O-demethylated to a greater extent than the (+)-enantiomer by rat liver microsomes.     

Simultaneous determination of propranolol and 4-hydroxypropranolol enantiomers after chiral derivatization using reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method is described, which allows the simultaneous quantification of propranolol and 4-hydroxypropranolol enantiomers in human plasma. After extraction from plasma (pH 10.5) using ethyl acetate, the enantiomers are derivatized with R-(+)-phenylethylisocyanate as chiral derivatization reagent and triethylamine as basic catalyst in chloroform. Ascorbic acid is used to prevent 4-hydroxypropranolol from oxidation during the extraction. Chromatographic separation on ODS columns and fluorescence detection (228 nm/greater than 340 nm) allows sensitive quantitation of all derivatives. Incubation of the plasma samples with beta-glucuronidase/arylsulfatase and the use of the specific beta-glucuronidase inhibitor saccharo-1,4-lactone allows the quantitation of both the sulfate and glucuronide conjugates of the enantiomers. The method was applied to human plasma samples from a subject after administration of 60 mg racemic propranolol three times daily.     

Analysis of tofisopam in human serum by column-switching semi-micro high-performance liquid chromatography and evaluation of tofisopam bioequivalency

A rapid and sensitive column-switching semi-micro HPLC method is described for the direct analysis of tofisopam in human serum. The sample (100 microL) was directly injected onto the precolumn (Capcell Pak MF Ph-1), where unretained proteins were eluted to waste. Tofisopam was then eluted into an enrichment column using 13% acetonitrile in 50 mM phosphate buffer (pH 7.0) containing 5 mM sodium octanesulfonate and subsequently into the analytical column using 43% acetonitrile in 0.1% phosphoric acid containing 5 mM sodium octanesulfonate. The detection limit (2 ng/mL), good precision (CV < or = 4.2%) and speed (total analysis time 24 min) of the present method were sufficient for drug monitoring. This method was successfully applied to a bioequivalence test of two commercial tofisopam tablets.     

Quantitative determination of clenbuterol enantiomers in human plasma by high-performance liquid chromatography using the macrocyclic antibiotic chiral stationary phase teicoplanin

We report a method for the high-performance liquid chromatographic (HPLC) chiral separation of racemic clenbuterol in human plasma. Human plasma was spiked with stock solutions of clenbuterol hydrochloride and practolol as the internal standard. Following a liquid-liquid extraction procedure with 10% (+/-)-2-butanol/isopropyl ether under alkaline conditions, the dried samples were reconstituted in methanol and chromatographed using a macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T(trade mark) (teicoplanin). The mobile phase composition was methanol:acetonitrile (70:30, v/v), containing 0.3% (v/v) acetic acid and 0.2% (v/v) triethylamine. The resulting chromatogram achieved baseline separation for the clenbuterol enantiomers. Calibration curves (peak area ratio vs plasma concentration, n = 10) were constructed for the (-)-R-and (+)-S-clenbuterol enantiomers with a plasma concentration range of 0. 25-10 microM. The correlation coefficient (r) range was 0.99988-0. 99999 (mean = 0.99999). The lowest concentration measured was 0.25 microM. Inter- and intra-assay variation was determined for the lowest, medium and highest plasma concentration (0.25, 2 and 10 microM) by calculating the analytical recoveries with a range of 96-104%. The percentage recoveries for the clenbuterol enantiomers were 88.4-102% over the concentration range used. Detailed methodology is presented.     

Resolution of chiral barbiturates into enantiomers by reversed-phase high-performance liquid chromatography using methylated beta-cyclodextrins

The correlation between the capacity factors of enantiomers of chiral barbiturates and the concentrations of beta-cyclodextrin, heptakis(2,6-di-O-methyl)-beta-cyclodextrin and heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin dissolved in the mobile phase was studied using LiChrosorb RP-18 as the stationary phase. Owing to the very strong adsorption of permethylated beta-cyclodextrin on the ODS surface a chiral stationary phase is generated dynamically and forms complexes with the solutes; this mechanism has been found to be the only factor responsible for the chiral recognition of the investigated compounds at all applied concentrations. The inclusion of barbiturates in the cavities of permethylated beta-cyclodextrin involves a distinct and entirely new kind of enantioselectivity compared with that observed for beta-cyclodextrin and its dimethyl derivative. Using permethylated beta-cyclodextrin baseline resolutions have been obtained with barbiturates containing a chiral centre in the heterocyclic ring or in the aliphatic side-chain.     

薄层色谱法拆分手性药物对映体

用性能稳定的薄层色谱用β-环糊精(β-CD)、硅胶手性固定相拆分手性药物对映体.在适当比例的乙腈-1%乙酸三乙胺(TEAA)、己烷-异丙醇、乙腈-甲醇-乙酸-三乙胺、甲醇-1%TEAA及乙腈-1%TEAA-三乙胺溶剂系统中展开,8种临床常用的手性药物对映体得到有效分离,对映异构体之间的相对比移值α为1.53～4.89.     

Determination of methyldibromoglutaronitrile in cosmetic products by high-performance liquid chromatography with electrochemical detection. Method validation

An increased frequency of contact allergy to methyldibromoglutaronitrile (MDBGN), a commonly used preservative in cosmetics and other consumer products, has been reported in recent years. A high-performance liquid chromatography (HPLC) method for the determination of MDBGN in cosmetic products has been validated in the present study. The identification is performed by reductive electrochemical detection of the bromine present in the molecule. The method is suitable for compliance testing of cosmetic products as well as for the research to support clinical and epidemiological studies.     

Chiral separations of pesticide enantiomers by high-performance liquid chromatography using cellulose triphenylcarbamate chiral stationary phase

The direct chiral separations of pesticide enantiomers by high-performance liquid chromatography by applying self-prepared cellulose triphenylcarbamate chiral stationary phase are performed. The mobile phase is n-hexane modified by isopropanol as a polar modifier. Nine chiral pesticides (benalaxyl, vinclozolin, diclofop-methyl, tebuconazole, quizalofop-ethyl, hexaconazole, lactofen, isocarbophos, and paclobutrazol) show enantioselectivity on the chiral stationary phase. An online circular dichrorism detector is used for identifying the pesticide enantiomers. The influences of the volume content of isopropanol and column temperature on the separations are investigated. The thermodynamic parameters related to the chiral distinguish mechanisms are also calculated.     

Quantitative determination of amoxicillin and its decomposition products by high-performance liquid chromatography

Amoxicillin, amoxicilloates, amoxicillin oligomers and amoxicillin piperazine-2,5-dione are separated by reversed-phase (C8) high-performance liquid chromatography with gradient elution. Quantitative results are reported for a number of samples. Amoxicillin trihydrate samples mostly contain amoxicilloate as the main impurity. Samples of the sodium salt also contain the piperazine-2,5-dione and the dimer. Higher oligomers such as the trimer and tetramer were not present in significant amounts. Several samples were also analysed by a mercurimetric titration method.     

Quantitative determination by high-performance liquid chromatography of acetylsalicylic acid and related substances in tablets

High-performance liquid chromatography on a Zorbax C8 7-micron column (25 cm X 0.46 cm I.D.) with methanol-water-1 M phosphoric acid (59:36:5) as the mobile phase has been used for the analysis of several naturally aged batches of fourteen brands of acetylsalicyclic acid tablets. The extraction solvent is methanol, containing 2% v/v of formic acid. Salicylic acid is the main impurity. Acetylsalicylsalicylic acid is the second most important impurity, and the corresponding salicylsalicylic acid is rarely present. Buffered or dispersible tablets contain relatively more of the latter two impurities and eventually also the corresponding higher oligomers. Acetylsalicylic anhydride is always a minor impurity. Comparison is made with classical spectrophotometric methods, which are observed to be selective for salicylic acid.     

Simultaneous determination of ketoprofen enantiomers and probenecid in plasma and urine by high-performance liquid chromatography.

A rapid, sensitive, stereospecific reversed-phase high-performance liquid chromatographic method was developed for simultaneous quantitation of ketoprofen enantiomers, probenecid and their conjugates in biological fluids. Following addition of the internal standard, indoprofen, the constituents were extracted into isooctane-isopropanol (95:5), water-washed, extracted with chloroform, then evaporated and the residue sequentially derivatized with ethyl chloroformate and L-leucinamide hydrochloride. The formed diastereomers were chromatographed on a reversed-phase column with a mobile phase of 0.06 M KH2PO4-acetonitrile-triethylamine (65:35:0.1) at a flow-rate of 1 ml/min and a detection wavelength of 275 nm. The minimum quantifiable concentration was 0.5 micrograms/ml in 100 microliters of rat plasma and urine samples. The intra- and inter-day coefficients of variation for this method are less than 10%. The assay is successfully applied to a pharmacokinetic study. The simultaneous analysis of probenecid with several other non-steroidal anti-inflammatory drugs was also successful.     

Method development and validation for the determination of indiquinoline tartrate, a novel kappa opioid agonist, and its related substances by high-performance liquid chromatography

A stability-indicating reversed-phase high-performance liquid chromatography method has been developed and validated for the assay of indiquinoline tartrate and its related substances. The method was established by forced degradation experiments and system suitability experiments. The chromatographic separation was achieved with a Hedera ODS-3 column (5 μm, 250 mm × 4.6 mm) and the mobile phase was constituted (flow rate 1.0 mL/min) of eluant A, aqueous acetate buffer and eluant B, CH(3)OH using a gradient elution. A photodiode array detector set at 254 nm was used for detection. The investigated validation elements showed that the method has acceptable specificity, accuracy, linearity, precision, robustness and high sensitivity with limit of detection and limit of quantitation. The method can be used for routine quality control analysis and stability testing of indiquinoline tartrate drug substance.     

Determination of the enantiomers of fenoldopam in human plasma by reversed-phase high-performance liquid chromatography after chiral derivatization.

Fenoldopam, a selective agonist at peripheral dopaminergic (DA-1) receptors, is administered as a racemic mixture and, consequently, an indirect stereospecific high-performance liquid chromatographic assay was developed to study the disposition of the individual enantiomers in human subjects. Fenoldopam enantiomers were extracted from alkalinized plasma into ethyl acetate prior to precolumn derivatization with the chiral reagent 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC). The resulting diastereomers were separated on a reversed-phase butylsilica column and determined using triple-electrode coulometric detection. The limits of determination and detection for the S- and R-enantiomers of fenoldopam were 0.5 and 0.25 ng/ml, respectively. A linear response was observed for (S)- and (R)-fenoldopam concentrations ranging from 0.5 to 50 ng/ml in plasma. The intra-day relative standard deviations (R.S.D.s) for the plasma assay at nominal concentrations of 0.5, 5 and 50 ng/ml were 17.4, 5.2 and 6.9%, respectively, for (S)-fenoldopam and 9.9, 6.2 and 7.4%, respectively, for (R)-fenoldopam. The inter-day R.S.D.s of the method at these concentrations were 9.3, 7.7 and 7.4%, respectively, for (S)-fenoldopam and 9.5, 1.9 and 7.3%, respectively, for (R)-fenoldopam. The mean accuracy of the method at concentrations of 0.5, 5 and 50 ng/ml in plasma was found to be 106.4, 111.8 and 108.9%, respectively, for (S)-fenoldopam and 116.2, 104.2 and 111.2%, respectively, for (R)-fenoldopam. The assay developed was sufficiently sensitive, accurate and precise to support pharmacokinetic studies in human subjects.