Radiographic analysis of regenerated bone around endosseous implants in the canine using recombinant human bone morphogenetic protein-2

A selective and sensitive HPLC method was developed for the determination of U-39968E in rat plasma. The assay involved solid-phase extraction of the analyte and the internal standard and precolumn derivatization with cyclohexane-1,3-dione reagent before injection on to the HPLC column. The samples were chromatographed on a Spherisorb S5 CN column (25 cm x 4.6 mm i.d.) with a mobile phase containing acetonitrile-trifluoroacetic acid-water (17:0.2:83, v/v/v) at a flow rate of 1.5 ml min-1. The column eluent was monitored by flourescence detection with excitation at 272 nm and emission at 320 nm. The assay is linear over the range 4-759 ng ml-1. The relative standard deviation at the limit of quantification, 4 ng ml-1, was 7.1%. This method was successfully applied to the determination of U-89968E in rat plasma during pharmacokinetic studies.     

MR angiographic and sonographic indications for endarterectomy

This study describes a HPLC method to determine the concentrations of acetylsalicylic acid (ASA) and salicylic acid (SA) in human stratum corneum and in plasma. The stratum corneum layers for ASA/SA analysis were removed from three patients with postherpetic hyperalgesia treated with topical and oral aspirin. Blood samples were also collected from the same patients. Tape strippings were placed in acetonitrile and sonicated for 15 min. After centrifuging, aliquots of the supernatant were injected into the chromatograph. ASA and SA from plasma samples were extracted on Isolute C8 columns. Due to interfering peaks in the tape samples, HPLC conditions were slightly different for tape and plasma samples. ASA and SA were separated on a LiChrospher 100 RP-18 column at 1 ml/min using a water-phosphate buffer (pH 2.5)-acetonitrile mobile phase (35:40:25, v/v/v). A linear response to quantities of ASA from 0.1 to 100 microg/cm2 and of SA from 0.1 to 5 microg/cm2 in tape and to quantities of ASA 0.1 to 2 microg/ml and 1 to 50 microg/ml was obtained and the recovery from tape and plasma samples was over 98%. The method is sensitive (0.1 microg/cm2) and specific enough to allow the determination of the drugs in the skin not only after topical but also after oral administration. A good sensitivity was also obtained in plasma (0.1 microg/ml) allowing study of the kinetics of ASA and SA in plasma after oral administration. Concentrations of ASA after topical administration were 100-200 times higher than after oral administration. Plasma levels of ASA and SA after oral administration were similar to those previously found. No ASA or SA were detected in plasma after topical ASA administration.     

Quantitation of acetazolamide in rat plasma, brain tissue and cerebrospinal fluid by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method for the analysis of acetazolamide (AZ) in rat blood (plasma/serum, whole blood and serum ultrafiltrate), brain tissue and cerebrospinal fluid (CSF) was described. Quantitative extraction of AZ with ethyl acetate from both buffered plasma and brain tissue homogenate (pH 8.0) was achieved. Each extract was evaporated to dryness and the residue was chromatographed on a reversed-phase column. CSF was directly analysed without extraction step. The limits of detection were 0.05 microgram ml-1 for plasma, 0.02 microgram g-1 for brain tissue and 0.004 microgram ml-1 for CSF. Calibration curves were linear over the working ranges of 0.1-100 micrograms ml-1 for plasma, 0.05-50 micrograms g-1 for brain tissue and 0.025-50 micrograms ml-1 for CSF. The reproducibility of AZ assay in the rat biologic media indicated very low relative standard deviations (RSDs). The recoveries of AZ added to plasma and brain tissue were more than 96% with an RSD of less than 5%. The present method was applied to studies of plasma concentration profiles of the drug after administration and its distribution into central nervous system.     

Sensitive determination of erythromycin in human plasma by LC-MS/MS

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of erythromycin in human plasma (EDTA as anticoagulant) was developed and validated. The concentration ranges were 0.5-50 and 50-5000 ng ml-1. The procedure involved alkalization of 0.5 ml of plasma, one step liquid-liquid extraction, dryness of the extract and reconstitution in 80:20 water:acetonitrile. An Inertsil ODS-2 5 microns, 3.0 x 50 mm column (Metachem) with a C8 guard column and isocratic mobile phase were used for liquid chromatography. The mobile phase consisted of 1:1 acetonitrile:water with 2 mM NH4OAc and 0.1% HOAc. A flow rate of 0.7 ml min-1 was used. The analysis time on LC-MS/MS for one sample was approximately 2 min. A Turbo-Ionspray source was interfaced between the HPLC and triple quadrupole mass spectrometer (Sciex API III Plus). MS/MS analysis used Multi-Reaction Monitoring (MRM) mode. The lowest limit of quantitation (LOQ) was 0.5 ng ml-1 with all Quality Control (QC) sample recoveries varying between 88 and 105%. Nine intraday and interday calibration curves were generated yielding correlation coefficients ranging from 0.995 to 1.000. Average recovery for erythromycin at 1 ng ml-1 was 105% (+/- 4.5%). Average recovery for the internal standard was 83-103%. Short-term and long-term stability in the freezer (-20 degrees C), bench stability, and stability after 3 freeze/thaw cycles at -20 and -80 degrees C were conducted. The samples were found to be stable under all conditions. The method developed and validated proved useful for clinical pharmacokinetic study sample analysis with high throughput due to its high sensitivity and very short analysis time.     

Isocratic, simultaneous reversed-phase high-performance liquid chromatographic estimation of six drugs for combined hypertension therapy

We report an isocratic, HPLC procedure for assay of the orally administered hypertension drugs [atenolol, amlodipine, nifedipine, nitrendipine, nimodipine and felodipine given in retention order] of which atenolol, an aryloxypropanolamine beta-blocker is administered with anyone of the other five dihydropyridine calcium channel blockers in combined hypertension therapy. The drugs were dissolved in methanol and 20 microliters of a mix of the drugs was injected onto a reverse phase JASCO-metaphase ODS (250 x 4.0 mm) 5 mu column. Any one of the six drugs could be used as the internal standard. The drugs were resolved by elution with a pH 4.5 equivolume mobile phase of acetonitrile-0.01 M KH2PO4, with pH adjustments done with H3PO4 (flow-rate 1.5 ml min-1). The column effluent was monitored at 250 nm. The detector response (peak height ratio) was linear in the dynamic range of 25-3200 ng ml-1 of these drugs, with the detection limits at approximately 15 ng ml-1. Full statistical evaluation of the data including linear regression (least-square fit) analysis was performed. The suggested procedure has the advantage that all the five dihydropyridine derivatives can be quantified alone or in formulation with atenolol.     

Validation of a high-performance liquid chromatographic assay method for pharmacokinetic evaluation of busulfan

The development and validation of a high-performance liquid chromatographic (HPLC) assay for determination of busulfan concentrations in human plasma for pharmacokinetic studies is described. Plasma samples containing busulfan and 1,6-bis(methanesulfonyloxy)hexane, and internal standard, were prepared by derivatization with sodium diethyldithiocarbamate (DDTC) followed by addition of methanol and extraction with ethyl acetate. The extract was dried under nitrogen and the samples reconstituted with 100 microl of methanol prior to HPLC determination. Chromatography was accomplished using a Waters NovaPak octadecylsilyl (ODS) (150 x 3.9 mm I.D.) analytical column, NovaPak ODS guard column, and mobile phase of methanol-water (80:20, v/v) at a flow-rate of 0.8 ml/min with UV detection at 251 nm. The limit of detection was 0.0200 microg/ml (signal-to-noise ratio of 6) with a limit of quantitation (LOQ) of 0.0600 microg/ml for busulfan in plasma. Calibration curves were linear from 0.0600 to 3.00 microg/ml in plasma (500 microl) using a 1/y weighting scheme. Precision of the assay, as represented by C.V. of the observed peak area ratio values, ranged from 4.41 to 13.5% (13.5% at LOQ). No day-to-day variability was observed in predicted concentration values and the bias was low for all concentrations evaluated (bias: 0 to 4.76%; LOQ: 2.91%). The mean derivatization and extraction yield observed for busulfan in plasma at 0.200, 1.20 and 2.00 microg/ml was 98.5% (range 93.4 to 107%). Plasma samples containing potential busulfan metabolites and co-administered drugs, which may be present in clinical samples, provided no response indicating this assay procedure is selective for busulfan. This method was used to analyze plasma concentrations following administration of a 1 mg/kg oral busulfan dose.     

A rapid reversed phase high performance liquid chromatographic method for the determination of docetaxel (Taxotere) in human plasma using a column switching technique

A rapid, simple and sensitive isocratic high performance liquid chromatography (HPLC) method was developed to measure the concentration of docetaxel in plasma samples with UV detection at 227 nm. The method uses a column switching technique with an Ultrasphere C18 column (75 x 4.6 mm ID, 3 mu, Altex, USA) as clean-up column and a CSC-nucleosil C8 column (150 x 4.6 mm ID, 5 mu, CSC, Montreal, Canada) as the analytical column. The mobile phase consisted of Phosphate buffer (30 mM, pH = 3)-acetonitrile (47:53, v/v) with the flow rates of 1.1 and 1.3 ml min-1 for clean-up and analytical columns, respectively. Paclitaxel was used as an internal standard. The plasma samples were extracted using a solid phase extraction method with Ammonium acetate (30 mM, pH = 5)-acetonitrile (50:50, v/v) as final eluent. The extraction method showed a recovery of 92% for docetaxel. In this system, the retention times of docetaxel and Paclitaxel were 7.2 and 8.5 min, respectively. The detection limit of docetaxel in plasma is 2.5 ng ml-1. This analytical method has a very good reproducibility (7.2% between-day variability at a concentration of 10 ng ml-1). It is applicable in clinical and pharmacokinetic studies.     

Sensitive determination of a new antiarrhythmic agent, trecetilide, in plasma by high-performance liquid chromatography with fluorescence detection

Two high-performance liquid chromatography (HPLC) methods were developed for the determination of trecetilide in plasma samples. Differing only in the addition of a derivatization step and different detection wavelengths, the two methods encompassed a wide concentration range. In both methods, plasma samples (0.1 ml) with added internal standard were applied to solid-phase extraction discs containing a non-polar/strong cation mixed-phase, washed and eluted with an acetone-acetonitrile triethylamine mixture. The eluate was evaporated to dryness, and either reconstituted and directly injected onto an HPLC column or first derivatized with 1-naphthyl isocyanate before HPLC analysis. In both methods, the separation was performed isocratically on a cyano analytical column utilizing a mobile phase composed of acetonitrile-pH 7.9 phosphate buffer (70:30, v/v). The column effluent was monitored by fluorescence detection at 290/345 nm (with derivatization) or 235/320 nm (without derivatization). The limits of detection and quantitation of the assay were 0.57 and 1.9 ng/ml, respectively, when derivatization was used, or 4.3 and 14 ng/ml, respectively, without derivatization.     

Stereoselective determination of unchanged and glucuroconjugated eliprodil, a new anti-ischaemic drug, in human plasma and urine by precolumn derivatization and column-switching high-performance liquid chromatography with fluorescence detection

An HPLC method was developed and validated for the determination in human plasma and urine of the enantiomers of eliprodil, (+/-)-alpha-(4-chlorophenyl)-4[(4-fluorophenyl) methyl]piperidine-1-ethanol hydrochloride, a new anti-ischaemic agent administered as a racemate. Both enantiomers are present in human plasma in unchanged and glucuroconjugated form, whereas only the glucuroconjugated form is excreted into urine; as a consequence, such metabolites in human plasma and urine should be submitted to enzymatic deconjugation with beta-glucuronidase (Escherichia coli) before being extracted. The general method involves a liquid-liquid extraction of eliprodil and internal standard from alkalinized plasma or urine with n-hexane, evaporation of the organic phase and derivatization with (S)-(+)-naphthylethyl isocyanate to give carbamate diastereoisomeric derivatives of (S)-(+)- and (R)-(-)-eliprodil and internal standard; after evaporation of the derivatizing mixture and dissolution of the residue in a small volume of phosphate buffer-acetonitrile (60:40, v/v), an aliquot is injected into a column-switching HPLC system. The derivatized sample extract is purified on a precolumn filled with C8-bonded silica material, which is flushed with acetonitrile-water, then diastereoisomers of eliprodil and the internal standard are automatically transferred by the mobile phase to the analytical column. The analytical column is a C8 type, specially deactivated for basic compounds, the mobile phase is 0.025 M phosphate buffer (pH 2.6)-methanol-acetonitrile (42:2:56) at a flow-rate of 1.2 ml min-1 and fluorimetric detector operating at lambda ex = 275 nm and lambda em = 336 nm is used. The retention times, under these conditions, are about 16 and 17 min for (S)-(+)- and (R)-(-)-eliprodil diastereoisomers, respectively, and about 19 min for the first-eluted diastereoisomer of the internal standard. During the analysis time, the precolumn, reset in a different path from that of the analytical column, is back-flushed with different solvents, then re-equilibrated with acetonitrile-water before the next injection. Linearity in plasma, for unchanged eliprodil enantiomers, was assessed in the range 0.15-10 ng ml-1 and for total eliprodil enantiomers (unchanged + conjugated) in the range 0.75-500 ng ml-1; the limit of quantitation (LOQ) is 0.15 ng ml-1 for each unchanged enantiomer and 0.75 ng ml-1 for each total enantiomer. Linearity was also assessed in urine for total (conjugated) eliprodil enantiomers in the range 50-25 000 ng ml-1; the LOQ is 50 ng ml-1 for each enantiomer. The intra- and inter-day precision and accuracy of the method were investigated in plasma and urine and found to be satisfactory for pharmacokinetic studies. The method has been extensively used in pharamcokinetic studies in man treated with a 20-mg dose of eliprodil racemate and some results of this application are reported.     

Determination of cefixime in human plasma and urine using high performance liquid chromatography column switching technique

A double column and double pump HPLC switching system is described for the analysis of cefixime in human plasma and urine. The system used muBondapak C18 short pretreatment column for on-line sample clean-up and a Hitachi GEL 3056 (ODS) analytical column for separation. A mixed solution of 0.01 mol/L H3PO4-0.1 mol/L KH2PO4-H2O (20:1:79) was used as the pretreatment mobile phase and CH2CH-0.01 mol/L H3PO4-0.1 mol/L KH2PO4-H2O (13:20:1:66) was used as analytical mobile phase. The compound in plasma and urine is detected by ultraviolet absorption at 286 nm and 314 nm, respectively. The absolute recoveries of the method in plasma and urine were 99.1% and 98.6% respectively. The relative standard deviations of the method are 0.70-3.82% and 0.80-3.73% in plasma, 1.53-3.08% and 1.31-2.67% in urine between days and day-to-day. Linear calibration curve for cefixime was measured over the range of 0.1-3.2 micrograms/ml in plasma and 1.0-32.0 micrograms/ml in urine, and the correlation coefficients were all 0.9999. The detection limit was 0.05 micrograms/ml in plasma and 0.2 micrograms/ml in urine. The plasma and urine samples were diluted with water and injected directly onto the HPLC system. The operation is simple and the relative sensitivity is markedly increased because of higher recoveries and larger loading capacity of the sample.     

A novel implantable electromechanical ventricular assist device. First acute animal testing

A method for the simultaneous determination of eight kinds of conjunct bile acids in human bile was developed by HPLC. They were separated on a YWG-C18 (3 microns) column at 30 degrees C, with methanol/water (65/35, V/V, pH3.0) as mobile phase, and detection wavelength at UV 210 nm. The linear ranges were 50-1,000 microns.ml-1, the recoveries were 91.2%-108.6%. The biles of 30 cases with cholelithiasis cholecystolithiasis and 20 cases without gallstone were detected by HPLC. The results showed that the constitution of bile acids was different between patients with cholelithiasis cholecystolithiasis and patients without gallstone.     

Automated procedure for determination of barbiturates in serum using the combined system of PrepStation and gas chromatography-mass spectrometry

A sensitive HPLC method with piroxicam as internal standard was developed for assaying amphotericin B in plasma and tissue. An Ultrabase-C18 column and a simple mobile phase consisting of an acetonitrile-acetic acid (10%)-water (41:43:16) mixture were used. The flow-rate was 1 ml/min and the effluent was monitored at 405 nm. The linearity of the assay method was up to 1000 ng/ml and 100 micrograms/g for plasma and tissue, respectively. Intra-day and inter-day RSDs were below 10% for all the sample types. This HPLC assay has been applied to determine amphotericin B in plasma and tissue samples taken during pharmacokinetic and tissue distribution studies in rats.     

An automated method for the determination of subnanogram concentrations of eprinomectin in bovine plasma

Eprinomectin is a potent anthelmintic compound that kills certain parasitic nematodes and arthropods of cattle. A sensitive and automated bioanalytical assay was developed for quantitation of eprinomectin in bovine plasma in support of clinical development of eprinomectin for use in all classes of cattle. This assay determined the concentration of eprinomectin in plasma by reversed-phase high performance liquid chromatography (HPLC) with fluorometric detection. Plasma sample preparation included liquid extraction performed by the Packard MultiPROBE robotics workstation, followed by solid phase extraction performed by the Gilson ASPEC XL automated workstation. The HPLC assay included automated pre-column derivatization with a fluorogenic reagent system which included trifluoroacetic anhydride and N-methylimidazole as the catalyst. This reversed-phase chromatographic analysis was based on the fluorescence detection of derivatized eprinomectin and an internal standard, L-648 548, which was similarly derivatized by the fluorogenic reagents. The assay was automated and validated for two concentration ranges of 0.05-10 and 0.5-200 ng ml-1. The lower limit of quantitation of eprinomectin in plasma was 0.05 ng ml-1. The %RSD of the assay was 10% or better at all concentrations. This automated analysis of eprinomectin was used for high-throughput clinical assays with acceptable accuracy and precision.     

Simultaneous determination of spironolactone and its metabolites in human plasma

This study describes a specific, precise, sensitive and accurate method for determination of unchanged spironolactone and its major active metabolites in human plasma. After one-step liquid-liquid extraction, analysis of the parent drug and its metabolites was performed in one chromatographic run, using a high performance liquid chromatography (HPLC) method with a programmed switchover of the UV wavelength. Spironolactone and 7 alpha-thiomethyl-spironolactone were detected at 245 nm, while canrenone and internal standard were detected at 280 nm. The column used was an S5 ODS2 (500 mm x 4.6 mm i.d.). The mobile phase was a mixture of acetonitrile-aqueous orthophosphoric acid (pH 3.4). Chromatographic separations were performed at 5 degrees C. The standard curves were linear over the range 10-400 ng ml-1 for spironolactone and 10-600 ng ml-1 for 7 alpha-thiomethyl-spironolactone and canrenone. The precision and accuracy of the method were confirmed by relative standard deviations below 10% for different concentrations, except for the concentration equal to the quantitation limit, where these parameters ranged from 12-15%. The recovery was above 80% for all investigated compounds and for the internal standard. The assay proved to be suitable for pharmacokinetic studies of spironolactone.     

Determination of alprazolam and alpha-hydroxyalprazolam in human plasma by gas chromatography/negative-ion chemical ionization mass spectrometry

A sensitive and specific method was developed for the determination of alprazolam and its major metabolite alpha-hydroxyalprazolam in plasma. After the addition of deuterium-labeled internal standards, plasma samples were buffered to pH 9 with 1 ml of saturated sodium borate buffer, extracted with toluene-methylene chloride (7:3) and evaporated to dryness. The residues were treated with N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% of trimethylchlorosilane and analyzed on a Finnigan-MAT mass spectrometer operated in the negative-ion chemical ionization mode with methane as the reagent gas. Chromatographic separation was achieved on a Restek-200 capillary column using hydrogen as the carrier gas. The assay was linear from 0.25 to 50 ng ml-1 for both compounds. The intra-assay precision for alprazolam was 16.1% at 0.5 ng ml-1 and 4.6% at 50 ng ml-1 and that for alpha-hydroxyalprazolam was 15.8% at 0.5 ng ml-1 and 4.2% at 50 ng ml-1. The method was used to determine alprazolam and alpha-hydroxyalprazolam in human plasma samples collected after a single 2 mg oral does of alprazolam. A peak concentration of 32.9 ng ml-1 of alprazolam was detected at 1 h following the dose.     

Determination of indomethacin and mefenamic acid in plasma by high-performance liquid chromatography

Indomethacin and mefenamic acid are widely used clinically as non-steroidal anti-inflammatory agents. Both drugs have also been found effective to produce closure of patent ductus arteriosus in premature neonates. A simple, rapid, sensitive and reliable HPLC method is described for the determination of indomethacin and mefenamic acid in human plasma. As these drugs are not applied together, the compounds are alternately used as analyte and internal standard. Plasma was deproteinized with acetonitrile, the supernatant fraction was evaporated to dryness and the resulting residue was reconstituted in the mobile phase and injected into the HPLC system. The chromatographic separation was performed on a C18 column (250 x 4.6 mm I.D.) using 10 mM phosphoric acid-acetonitrile (40:60, v/v) as the mobile phase and both drugs were detected at 280 nm. The calibration graphs were linear with a correlation coefficient (r) of 0.999 or better from 0.1 to 10 micrograms/ml and the detection limits were 0.06 micrograms/ml for indomethacin and 0.08 micrograms/ml for mefenamic acid, for 50-microliters plasma samples. The method was not interfered with by other plasma components and has been found particularly useful for paediatric use. The within-day precision and accuracy of the method were evaluated for three concentrations in spiked plasma samples. The coefficients of variation were less than 5% and the accuracy was nearly 100% for both drugs.     

Determination of morphine and codeine in plasma by HPLC following solid phase extraction

A cheap simple and rapid extraction procedure followed by a UV high performance liquid chromatography (HPLC) assay is described for the simultaneous determination of morphine (M) and codeine (C) in plasma. The method is based on extraction of these opiates from plasma using reversed phase (solid phase) extractions columns followed by HPLC with UV detection at 240 nm. The extraction step provides, respectively, 85 and 80% recovery for M and C. The response of the detection system is linear for both molecules in the studied range from 50 to 750 ng ml-1. No other drugs have been found to interfere with the assay. This method offers a quick, cost effective and reliable procedure for specifically determining M and C, from a small sample volume.     

Application of solid-phase extraction in the method development for determination of SEPA, an acronym for soft enhancement of percutaneous absorption, in human, rat, and rabbit serum using GC-FID method

A new nonaqueous topical minoxidil formulation containing SEPA (2-n-nonyl-1,3-dioxolane) for enhancement of percutaneous absorption was under evaluation. SEPA does not have chromophore for either ultraviolet or fluorescence detection using liquid chromatography and has no functional groups for derivatization. Therefore, a direct gas-chromatographic method with flame-ionization detection (GC-FID) was developed. Owing to the limited detection response of the FID detection, it needs a selective and concentrated extract for GC-FID analysis to improve the assay sensitivity to meet the requirement for pharmacokinetic evaluation after topical application. In addition, SEPA is a very volatile compound. Any extraction procedures involving evaporation will result in a poor recovery. The application of solid-phase extraction (SPE) makes it possible to achieve a selective and a 10-fold concentrated extract with an absolute extraction recovery of approximately 90%, which greatly improved the assay sensitivity. This method involved the extraction of SEPA and the internal standard (2-n-heptyl-1,3-dioxolane) from serum (0.1-1 ml) with 100 microliter of hexane-chloroform (1:1, v:v) using a 50 mg 1.0 ml-1 phenyl SPE column (Varian, Harbor City, CA, USA), followed by direct GC-FID analysis on a fused-silica column chemically bonded with cross-linked methyl silicone gum phase (Hewlett Packard Ultra-1, 12 m x 0.2 mm x 0.33 micron, Avondale, PA, USA). The assay demonstrated a lower limit of quantitation of 2.5 ng ml-1 and a linear range of 2.5 to 250 ng ml-1 with intra- and inter-assay precision and accuracy of < or = 10%.     

Herbimycin A inhibits protein kinase C in vascular smooth muscle cells

A sensitive enantioselective high performance liquid chromatographic assay for determination of the dihydropyridine-type calcium antagonist amlodipine in human plasma samples is described. Chiral chromatography is performed on an alpha 1-acid glycoprotein column (i.e. Chiral-AGP) and the eluted enantiomers are trapped and compressed on two short columns before final achiral chromatography on a narrow bore column (i.e. Zorbax SB-Ph) using electrochemical detection. Both stereoselective quantitative analysis and enantiomeric ratio analysis, for samples with a known total concentration of amlodipine are described. The quantitative assay shows linearity over the range 0.5-10 ng ml-1 for the two enantiomers and the limit of detection is about 0.2 ng ml-1. The method has been applied to a pharmacokinetic study of the enantiomers of amlodipine in human subjects.     

Development and validation of a chiral high-performance liquid chromatography assay for rogletimide and rogletimide-N-oxide isomers in plasma

The purpose of the present study was to develop and validate a stereo-specific high-performance liquid chromatography (HPLC) assay for rogletimide (Rog) and rogletimide-N-oxide (Nox) isomers in plasma. The assay was performed with a chiral cellulose-[4-methylbenzoate]ester column (Chiracel OJ). Optimal separation was achieved isocratically with a mobile phase consisting of n-hexane/anhydrous ethanol (65/35, v/v) at a flow rate of 0.9 ml/min, with the column being thermostated at +35 degrees C (UV detection at 257 nm). Under these conditions, retention times were approximately 17, 28, 31 and 76 min for R-Rog, S-Rog, R-Nox and S-Nox, respectively. S-aminoglutethimide (S-Ag) served as the internal standard (retention time 70 min). An extraction procedure from plasma samples was developed on Bond Elut RP8 500-mg cartridges; conditioning was performed with 5 ml methanol and 5 ml water, after which 1 ml plasma that had previously been spiked with 5 microM S-Ag was applied. Washing was done with 6 ml water and elution, with 4 ml methanol. After evaporation to dryness, residues were dissolved in 400 microliters anhydrous ethanol and 12-48 microliters was injected onto the HPLC system. Blank plasma from healthy donors showed the random presence of a small interference eluting at the retention time of R-Rog, precluding the accurate quantification of R-Rog concentrations below 2.5 microM. Reproducibility assays demonstrated the need to use an internal standard. Taking into account the internal standard, at 2.5 microM the intra- and inter-assay coefficients of variation were 10.5% and 21.0% for R-Rog 5.5% and 8.7% for S-Rog, 7.6% and 20.8% for R-Nox and 11.7% and 6.4% for S-Nox, respectively. The detection limit was 2.5 microM for R-Rog, 0.5 microM for S-Rog, 0.25 microM for R-Nox and 0.5 microM for S-Nox. Linearity was satisfactory at concentrations ranging from 2.5 to 10 microM for R-Rog, from 0.5 to 10 microM for S-Rog, from 0.25 to 2.5 microM for R-Nox and from 0.50 to 2.5 microM for S-Nox. This assay was applied to plasma obtained from rog-letimide-treated breast cancer patients receiving conventional oral doses and demonstrated its feasibility with regard to sensitivity. The preliminary pharmacokinetic results reported herein suggest for the first time that both the R-Rog and S-Rog isomers are metabolized into rogletimide-N-oxide.