Automated analysis of fluvoxamine in rat plasma using a column-switching system and ion-pair high-performance liquid chromatography

We have established a robust, fully automated analytical method for the analysis of fluvoxamine in rat plasma using a column-switching ion-pair high-performance chromatography system. The plasma sample was injected onto a precolumn packed with Shim-pack MAYI-ODS (50 microm), where the drug was automatically purified and enriched by on-line solid-phase extraction. After elution of the plasma proteins, the analyte was back-flushed from the precolumn and then separated isocratically on a reversed-phase C18 column (L-column ODS) with a mobile phase (acetonitrile-0.1% phosphoric acid, 36:64, v/v) containing 2 mM sodium 1-octanesulfonate. The analyte was monitored by a UV detector at a wavelength of 254 nm. The calibration line for fluvoxamine showed good linearity in the range of 5-5000 ng/mL (r > 0.999) with the limit of quantification of 5 ng/mL (RSD = 6.51%). Accuracy ranged from -2.94 to 4.82%, and the within- and between-day precision of the assay was better than 8% across the calibration range. The analytical sensitivity and accuracy of this assay is suitable for characterization of the pharmacokinetics of orally-administered fluvoxamine in rats.     

Determination of fluoxetine enantiomers in rat plasma by pre-column fluorescence derivatization and column-switching high-performance liquid chromatography

A column-switching HPLC method employing both octadecylsilica (ODS) and chiral columns with fluorescence detection was developed for the determination of enantiomer of fluoxetine (FLX), an antidepressant drug, in rat plasma. Racemic FLX was derivatized with a fluorescent reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) or 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and the enantiomeric separation of the resultant derivatives was examined on an amylose-based chiral column (CHIRALPAK AD-RH) in the reversed-phase mode. The derivative with NBD-COCl (NBD-FLX) showed a sufficient separation factor (a) and resolution (Rs) compared with that with DBD-COCl. Thus, FLX was derivatized with NBD-COCl and the resultant NBD-FLX was first quantified on the ODS column and then introduced to the CHIRALPAK AD-RH column via a six-port switching valve to examine the enantiomeric ratio. The intra- and inter-day accuracy (97.6-112.7%) and precision (1.47-10.60%) were satisfactory in the range 10-1000 nM FLX and the limit of quantification was approximately 10 nM. The absolute recoveries of FLX with hexane from rat plasma were in the range 87.5-92.2% (n = 3). The method was applied to determine FLX enantiomers in the plasma of rats administered FLX orally, and it was shown that the R-isomer was eliminated faster than the S-isomer.     

Simultaneous quantitation of loganin and sweroside in plasma using column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of loganin and sweroside, which are the active ingredients of purified herbal extract from Lonicera japonica (SKL JI), in rat plasma using column-switching and ultraviolet (UV) absorbance detection. Plasma was simply filtrated prior to injection to the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with two six-port switching valves. Detection of loganin and sweroside was accurate and repeatable, with a limit of quantitation of 0.05 μg ml⁻¹ in plasma. The calibration curves for both loganin and sweroside were linear over the concentration ranges of 0.05-40.0 and 0.02-40.0 μg ml⁻¹ in rat plasma, respectively. The intra- and inter-day precision over the concentration range for loganin and sweroside were lower than 8.1 and 10.9% (relative standard deviation, R.S.D.), and accuracy was between 94.7 and 113.5 and 95.0 and 113.1%, respectively. This method has been successfully applied to determine the levels of loganin and sweroside in rat plasma samples from pharmacokinetics studies.     

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.     

Direct determination of benzamides in serum by column-switching high-performance liquid chromatography.

A column-switching high-performance liquid chromatographic method with fluorescence detection was developed for the simultaneous determination of four benzamide-type anti-psychotic drugs: sulpiride, tiapride, sultopride and metoclopramide in human serum. In this method, a TSKgel Super-ODS column was used as an analytical column, and a TSKgel G 2000SW was prepared as a pretreatment column. Under the optimized analytical conditions, four benzamide-type anti-psychotic drugs were eluted within 18 min. The detection limits (S/N = 3) for sulpiride, tiapride, sultopride and metoclopramide are 1 ng/ml, 4 ng/ml, 2 ng/ml and 0.5 ng/ml, respectively. Finally, the method was applied to the determination of sulpiride in human serum samples obtained after a single oral dose of sulpiride.     

Avidin protein-conjugated column for direct injection analysis of drug enantiomers in plasma by high-performance liquid chromatography.

A new concept in high-performance liquid chromatography supports is proposed for the direct injection analysis of drug enantiomers in plasma. The new supports are designed with disuccinimidyl suberate as a hydrophobic internal region, and avidin protein as a hydrophilic and bulky surface region. Plasma proteins are excluded by the avidin phase and are eluted immediately from the column, whereas low-molecular-mass analytes can penetrate the surface region and interact with disuccinimidyl suberate. Enantiomers interact differentially with avidin, and are thereby separated. This column was used in reversed-phase high-performance liquid chromatographic analysis to determine ketoprofen enantiomers in plasma by direct injection. The recovery of racemic drug from plasma was almost 100%.     

Simple determination of azasetron in rat plasma by column-switching high-performance liquid chromatography

For the quantification of azasetron in rat plasma samples, a column-switching HPLC method was developed and validated. Following dilution of plasma samples with mobile phase A (17?mM potassium phosphate buffer (pH 3.0)) and simple protein precipitation by addition of perchloric acid (60%), the mixture was directly injected onto the pre-column. After endogenous plasma substances were eluted to waste, the analyte was transferred to the trap column by switching the system. Then, the analyte was back-flushed to the analytical column for separation with mobile phase B (a 22:78 v/v mixture of acetonitrile and 17?mM potassium phosphate buffer (pH 3.0)) and detected at 250?nm using a photodiode array detector. A linear standard curve was obtained in the concentration range of 10-800?ng/mL with the correlation coefficient (r) of 0.9998. The intra- and inter-day precision and accuracy values for azasetron were in the ranges of 0.3-12.9% and 89.7-101.4%, respectively. The method was valid in terms of specificity, precision, and accuracy. In addition, this efficient analytical method was successfully applied to determine plasma concentrations of azasetron following oral administration of azasetron at a dose of 4.0?mg/kg to rats.     

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.     

Development of a column-switching high-performance liquid chromatography for kynurenine enantiomers and its application to a pharmacokinetic study in rat plasma

Kynurenine (KYN), a tryptophan metabolite, is a precursor of kynurenic acid, which is an antagonist of N-methyl-d-aspartate receptor. In this study, an enantiomeric separation of d,l-KYN derivatized with the benzofurazan fluorescence reagent 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) (DBD-d,l-KYN) was first investigated by using a high-performance liquid chromatography (HPLC) with several chiral columns. As a consequence, DBD-d,l-KYN was enantiomerically separated on a cellulose-type chiral column (CHIRALCEL OJ-RH) with a mobile phase of H₂O/CH₃CN/MeOH (40/50/10) containing 0.1% acetic acid. Under this condition, the separation factor and resolution were 1.48 and 1.28, respectively. Next, a column-switching HPLC consisting of both octadecylsilica and chiral columns was developed and used to determine both d- and l-KYN enantiomers in 10 μL of rat plasma following the intraperitoneal administration of d,l-KYN to rats (10 mg kg⁻¹). The result revealed that the concentration of l-KYN was higher than that of d-KYN, suggesting that d-KYN was eliminated faster than l-KYN.     

Direct analysis of clomipramine in human plasma by microbore high performance liquid chromatography with column-switching

Summary An automated microbore, liquid chromatographic method with column-switching was developed for the determination of clomipramine from human plasma samples. After direct injection of samples (60 μL), plasma proteins and clomipramine were separated in size-exclusion mode using 20% acetonitrile in 20 mM phosphate buffer (pH 7.0) on Capcell Pak MF Ph-1 precolumn (10×4 mm I.D.). By valve switching, a fraction containing clomipramine was directed to an intermediate column for subsequent main separation on a microbore C₁₈ column (250×1.5 mm I.D.) using 50% acetonitrile in 20 mM phosphate buffer (pH 2.5) at 0.1 mL min⁻¹. The method was advantageous for rapidity (total analysis time: 15 min), reproducibility (C.V.<4.8%), and increased sensitivity (1 ng mL⁻¹). The linearity of response was good (r ²≥0.999) over the concentration range 1–250 ng mL⁻¹.     

Determination of ketoprofen in plasma using high-performance liquid chromatography. Comparison with gas--liquid chromatography (author's transl)

A new method of determination of ketoprofen 2-(3-benzoyl phenyl) propionic acid in plasma using high-performance liquid chromatography (HPLC) is described. After extraction by diethyl either in acidic medium, ketoprofen and the internal standard, 2-(4-benzoyl phenyl) butyric acid, are methylated with gaseous diazomethane and their concentrations measured by HPLC using in LiChrosorb Si 60 (5 micrometer) column and dichloromethane-hexane (60:40) as the mobile phase. The absolute retention times of the internal standard and ketoprofen are 11.6 and 12.8 min, respectively. The precision of the methods is +/- 4% and the lower detection limit ranges from 0.06 to 0.10 microgram/ml. The results obtained by HPLC show a very good correlation with those obtained by gas--liquid chromatography. The proposed method is sensitive, reproducible and rapid and very suitable for ketoprofen determination in pharmacokinetic studies.     

Determination of renin activity in human plasma by column-switching high-performance liquid chromatography with fluorescence detection

A column-switching high-performance liquid chromatographic (HPLC) method with fluorescence detection is described for the determination of the renin activity in human plasma. The method is based on the quantification of the enzymatically produced angiotensin I. Angiotensin I liberated from a synthetic substrate (tridecapeptide of human angiotensinogen) and [Val5]-angiotensin I as an internal standard are converted into fluorescent derivatives by reaction with benzoin. The derivatives are separated from various interfering substances by column-switching HPLC using three reversed-phase columns. The limit of detection (signal-to-noise ratio = 3) of the renin activity is 2.7 pmol of angiotensin I formed per h per ml of plasma, which corresponds to approximately 820 fmol of angiotensin I injected. The column-switching method in combination with pre-column derivatization for the fluorimetric detection permits the sensitive and selective determination of the enzymatically formed angiotensin I. Hence low activities of renin in normal human plasma are readily measured.     

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 a new non-benzodiazepine anxiolytic and its O-demethyl metabolite in plasma by high-performance liquid chromatography using automated column-switching

An highly sensitive and fully automated high-performance liquid chromatographic assay was developed for the determination of a novel non-benzodiazepine anxiolytic (I) [(R)-2-(methoxymethyl)-1-[(7-oxo-8-phenyl-7H-thieno[2,3-a]quinolizin+ ++- 10-yl)carbonyl]pyrrolidine] and its O-demethyl metabolite (II) in plasma, using column-switching for direct injection of plasma samples. After dilution in internal standard solution, the sample was injected onto a pre-column (17 mm x 4.6 mm) dry-packed with pellicular C18 reversed-phase material. Polar plasma components were removed by flushing the pre-column with water-acetonitrile (90:10, v/v). Retained substances, including I and II, were backflushed onto an analytical column, separated by gradient elution and detected by means of fluorescence detection (excitation, 304 nm; emission, 475 nm). After washing the analytical column and re-equilibrating the pre-column, the system was ready for the next injection. The limit of quantification for I and II was 0.25 and 0.5 ng/ml, respectively, using a 350-microliter specimen of plasma. The practicability of the new method was demonstrated by analysis of more than 300 plasma samples from a tolerance study performed with human volunteers. Owing to its high sensitivity, the method can be used to calculate pharmacokinetic parameters of compounds I and II in man after a single oral dose of about 1 mg of I.