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.     

Direct injection analysis of ketoprofen enantiomers in plasma using column-switching high-performance liquid chromatography system

A high-performance liquid chromatography system was developed for the stereoselective determination of ketoprofen enantiomers in human plasma following direct sample injection. The system comprised of a pretreatment column and a chiral separation column connected in a series via a switching valve. When a 200 microliter portion of human plasma containing a therapeutic level of ketoprofen was directly applied to the system, ketoprofen was adsorbed in the pretreatment column, while plasma proteins were excluded. After the elution of proteins from the pretreatment column, the valve was switched and ketoprofen was desorbed and transferred to the chiral separation column where the enantiomers were separated and determined by ultraviolet-absorption. The mobile phase conditions for the pretreatment and chiral separation were optimized, which enabled rapid and complete recovery followed by satisfactory separation of the enantiomers. The calibration line for each enantiomer showed good linearity in the range of 0.25-5 micrograms/ml with a detection limit of 0.02 micrograms/ml (signal to noise ratio (S/N) greater than 3), which was sufficient for practical demands. The precision test indicated that the coefficient of variation for five repeated determinations of (-) ketoprofen was 5.4% at 0.1 microgram/ml and 1.4% at 1 microgram/ml.     

Characterization of an avidin-bonded column for direct injection in reversed-phase high-performance liquid chromatography

An automatic method for the determination of metabolites of Ropivacaine in urine was set up. It utilizes supported liquid membrane extraction for sample clean-up and enrichment, followed by ion-pair chromatography determination using UV detection. The extraction was very selective with no observed interfering compounds from the urine matrix, permitting simple isocratic chromatographic analysis. The detection limits for spiked urine samples were 2–18 nM for the different compounds. The repeatability was 1–3% (RSD) with an internal standard that was also extracted, and about twice without this standard. A throughput of 3.3 samples per hour was achieved and the liquid membrane was stable for more than a week.     

Determination of amoxycillin in human plasma by direct injection and coupled-column high-performance liquid chromatography

This work reports the use of multidimensional HPLC by coupling a restricted access medium (RAM) bovine serum albumin (BSA) octadecyl column (100 x 4.6 mm I.D., 10 microm particle size and 120 A pore size) to an octadecyl Hypersil column (150 x 4.6 mm I.D., 5 microm particle size and 120 A pore size) to the analysis of amoxycillin in human plasma by direct injection. Ion pairing was necessary to extract amoxycillin with good recovery from the plasma proteins. To prepare the spiked samples, aliquots (60 microl) of the appropriated standard solutions were added to each culture tube containing an 180 microl of plasma and a solution of 0.30 mM tetrabuthylammonium phosphate (60 microl). They were vortexed for 15 s and then 290 microl were transferred to autosampler vials. Aliquots (250 microl) of the spiked plasma samples were injected to a column-switching HPLC system. An analysis time of 25 min with no time spent on sample preparation was achieved. The developed method showed good selectivity, sensitivity, accuracy and precision for direct analysis of this polar low wavelength ultraviolet absorption antibiotic using only 180 microl of human plasma. The validated method proved to be reliable and sensitive for the determination of amoxycillin in plasma samples of five healthy volunteers to whom test and reference formulations were administered as an oral dose (500 mg).     

Determination of metoprolol in human plasma by column switching high-performance liquid chromatography

Summary Retention characteristics of metoprolol have been studied in reversed phase mode on RP2, RP8 and CN columns. The plots of retention time as a function of the acetonitrile content and of the ionic strength of the mobile phase permitted the choice of the best conditions to separate metoprolol from plasma components by switching of these three types of columns.Human plasma (0.5–1 ml) diluted with water is first injected on a RP2 column (25–40 m particle diameter, prepared by dry packing) and rinsed with water. The sample is then back eluted with acetonitrile-0.022 M acetate buffer (7525, v:v) and switched to a CN column (10 cm long, 5 m particle diameter). The heart cut of the eluate is selected and loaded on a RP8 analytical column (25 cm long, 5 m particle diameter) with acetonitrile-0.088 M acetate buffer (7525, v:v) as mobile phase.Auto-sampler and switching valves are actuated automatically by a computing integrator based on a fixed time schedule. The duration of one cycle is about 30 min, but the last analytical step is about 15 min and represents the time interval between two injections. Metoprolol, its alpha-hydroxy metabolite and the internal standard are detected by fluorescence (_ex= 225 nm; _em > 320 nm).Presented at the 14th International Symposium on Chromatography London, September, 1982     

Chiral analysis of methadone in plasma by high-performance liquid chromatography.

A method for the chiral high-performance liquid chromatographic analysis of methadone in plasma has been developed. The method employed organic solvent extraction, enantiomeric separation on a Chiral AGP column, and ultraviolet absorption detection at 212 nm. The intra-day variation in the quantification of methadone enantiomers was less than 9% at the 100 ng/ml level, and the values obtained correlated well with those from a gas chromatographic-mass spectrometric method. Results from patients indicate inter- and intra-individual differences in the ratio between l- and d-methadone in plasma during therapy with racemic methadone. In one patient, a higher level of d-methadone in plasma was caused by both faster elimination and lower bioavailability of l-methadone.     

Determination of oxytetracycline in blood serum by high-performance liquid chromatography with direct injection.

A direct injection analysis by high-performance liquid chromatography has been developed for oxytetracycline in serum of animals and fish. A Hisep shielded hydrophobic phase column (15 cm x 4.6 mm I.D.) and a mobile phase of methanol-0.2 M oxalic acid (10:90, v/v, pH 7.0) with ultraviolet detection at 360 nm were used. The standard calibration curves in serum of chicken, hog, cattle and rainbow trout were linear over the range 0.1-20 micrograms/ml. The recoveries of oxytetracycline from all serum samples determined at two different concentrations (0.5 and 2.0 micrograms/ml) were 88-103%. The detection limit was 0.05 micrograms/ml for every serum sample.     

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.     

Determination of drug enantiomers in biological samples by coupled column liquid chromatography and liquid chromatography-mass spectrometry

Liquid chromatography-mass spectrometry (LC-MS) and coupled column chromatography can be used to overcome problems likely to occur in direct separation and determination of drug enantiomers in biological samples. This is exemplified here with the direct separation and determination of terbutaline in human plasma at the nmol/l level. A beta-cyclodextrin column with an aqueous mobile phase was used for chiral separation. For coupled column chromatography, the concentration of each enantiomer was calculated from the enantiomeric area ratio and the racemate concentration. A deuterium-labelled internal standard was used in the LC-MS experiments.     

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.     

Determination of zopiclone enantiomers in plasma by liquid chromatography using a chiral cellulose carbamate column.

The enantiomers of zopiclone were determined in human plasma using a sequential achiral-chiral liquid chromatographic method. Zopiclone was separated from the biological matrix and quantified on an achiral silica column. The limit of detection was 5 ng/ml. The eluent fraction containing zopiclone was collected, evaporated, reconstituted with the mobile phase and injected onto a chiral cellulose carbamate column where the enantiomeric ratio was calculated. This validated method, applied to a pilot study, suggests that pharmacokinetics of zopiclone is stereoselective.     

Stereoselective determination of the enantiomers of methadone in plasma using high-performance liquid chromatography.

An enantioselective high-performance liquid chromatographic assay for the quantification of methadone in human and beagle plasma is described. The procedure involves extraction of methadone from alkalized plasma into hexane-isoamyl alcohol (99:1, v/v). Stereoselective separation was achieved with a silica column with covalently bound alpha 1-acid glycoprotein (Chiral-AGP) without any derivatization procedure. The detection wavelength was set at 215 nm. Using an internal standard provided reliable control of the extraction procedure as well as quantification of the enantiomers of methadone. The limit of quantification was found to be 2.5 ng/ml. The method was demonstrated to be sufficiently sensitive for stereoselective pharmacokinetic studies of methadone.     

Determination of tertatolol enantiomers in biological fluids by high-performance liquid chromatography.

A stereospecific high-performance liquid chromatographic method for the quantification of (-)- and (+)-tertatolol in plasma and urine is described. The method involves solid-phase extraction followed by derivatization with S(+)-naphthylethylisocyanate to form the urea derivative, which is more sensitive to fluorescence detection. The separation of the diastereomeric derivatives was performed by reversed-phase high-performance liquid chromatography. Fluorimetric detection (lambda excitation = 220 nm, lambda emission = 320 nm) allows the quantification of tertatolol enantiomers down to 6 ng/ml. The assay was used to study the pharmacokinetic profile of tertatolol enantiomers following oral administration of racemic tertatolol; preliminary results suggest enantioselective absorption and/or disposition of tertatolol.     

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.     

Influence of injection parameters on column performance in nanoscale high-performance liquid chromatography

Summary The influence of the injection volume and the sample solvent on column efficiency has been evaluated in packed nano liquid chromatography using columns 150μ i.d. Evaluation of column performance was by means of reduced plate height (h) versus reduced velocity (v) for four polyaromatic hydrocarbon test compounds (PAHs). When compounds are dissolved in a weak solvent (such as MeCN: H₂O, 30∶70), and whatever the injection volume −60 or 200 nL-a gain in efficiency can be observed due to the well-known on-column focusing phenomenon, but keeping constant solute retention factors. Under optimized conditions (flow rate: 150 nLmin⁻¹, solvent sample MeCN: H₂O, 30∶70, injection volume 200 nL), a reduced plate height of 1.83 has been obtained for a 15 μm C₁₈ packing corresponding to 36000 plates m⁻¹, which illustrates the absence of any extracolumn band broadening under nano LC conditions.     

Solid-phase extraction and direct high-performance liquid chromatographic determination of metoprolol enantiomers in plasma

A method is described by which underivatized metoprolol enantiomers in plasma can be quantitated by high-performance liquid chromatography with fluorescence detection. Samples are prepared for injection using a simple solid-phase extraction procedure which is essentially 100% efficient for all analytes. The metoprolol enantiomers are resolved using a cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase and a hexane-ethanol-diethylamine mobile phase. Samples were tested for adaptability to autoinjection and found to be stable for at least 16 h after reconstitution in mobile phase. The automated method was determined to be precise and accurate for enantiomer concentrations as low as 10 ng base per ml and was successfully employed in a pharmacokinetic investigation.     

Determination of melagatran in rabbit plasma by high-performance liquid chromatography with automated column switching

Melagatran is an active thrombin inhibitor showing oral and parenteral bioavailability for antithrombotic therapy. A simple and convenient liquid chromatographic method has been developed and applied to the analysis of melagatran in rabbit plasma. The clean-up and separation of the sample solutions were performed by automated on-line column switching HPLC. The method validation shows the suitability of the column switching liquid chromatographic system for the quantitation of melagatran in biological fluids.     

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.     

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.