Sensitive determination of piritramide in human plasma by gas chromatography.

A selective and sensitive method for the determination of piritramide in human plasma is described. A 1-ml aliquot of plasma was extracted with 10 ml of hexane-isoamyl alcohol (99.5:0.5, v/v) (extraction efficiency 86%) after addition of 50 microliters of 2 M ammonia and 20 microliters of aqueous strychnine solution (100 ng per 10 microliters) as internal standard. Gas chromatography was performed with J&W DB-1, 30 m x 0.53 mm I.D. separation column, film thickness 1.5 microns, using an nitrogen-phosphorus-sensitive detector. The assay was linear in the concentration range 3.75-2250 ng/ml (r = 0.999), with a lower limit of detection of 1-2 ng/ml. The precision was determined using spiked plasma samples (10 and 50 ng/ml), with coefficients of variation of 3.5 and 3.1% (intra-day; n = 5) and 4.6 and 4.1% (inter-day; n = 4). In the range 3.75-150 ng/ml, the accuracy of the assay was 3.36%. The method was used for the determination of piritramide plasma concentrations in patients receiving intra- or post-operative analgesia.     

Determination of ondansetron and its hydroxy metabolites in human serum using solid-phase extraction and liquid chromatography/positive ion electrospray tandem mass spectrometry

Ondansetron and its hydroxylated metabolites were determined in human serum using solid-phase extraction (SPE) and liquid chromatography/positive ion electrospray tandem mass spectrometry. Pyrimethamine was used as the internal standard. The analytes were eluted from the SPE cartridge using 2 x 1 ml of methanol containing 0.5% triethylamine, evaporated under vacuum and the residue was reconstituted in the mobile phase. The liquid chromatographic separation was achieved on a silica column using a mobile phase of aqueous 20 mM ammonium acetate (pH 4.7)-acetonitrile (85 : 15, v/v) at a flow-rate of 0.4 ml min(-1). The method was linear over the range 1-500 ng ml(-1) for ondansetron and each of the metabolites in human serum. The intra-day accuracy was better than 9.1% and the precision was <10.3%; the inter-day accuracy was better than 9.5% and the precision was <12.6%. The limit of detection was 250 pg ml(-1) based on a signal-to-noise ratio of 3. The absolute recovery from serum for all analytes was >90%.     

Determination of the catechol-O-methyltransferase inhibitor Ro 40-7592 in human plasma by high-performance liquid chromatography with coulometric detection.

A sensitive and specific high-performance liquid chromatographic method has been developed to measure the catechol-O-methyl-transferase (COMT) inhibitor 3,4-dihydroxy-4'-methyl-5-nitrobenzophenone (Ro 40-7592) in human plasma. The compound and the internal standard were extracted from plasma at pH 2 with n-butyl chloride-ethyl acetate (95:5, v/v). The extract was chromatographed on a reversed-phase column (Hypersil ODS, 5 microns) using a mixture of phosphate buffer (0.05 M, pH 2), methanol and tetrahydrofuran (45:55:5, v/v/v) as the mobile phase. Long-retained components were removed from the system by means of a simple column-switching system. Quantification of the catechol-O-methyltransferase inhibitor was performed by means of coulometric detection (0.1 V). The limit of quantification was about 1 ng/ml, using a 1-ml specimen of plasma. The recovery from human plasma was greater than 88%. The mean inter-assay precision was 5.3% in the range 2.5-1000 ng/ml. Linearity of the standard curve was obtained in the concentration range 2.5-500 ng/ml. The catechol-O-methyltransferase inhibitor was stable in human plasma when stored for six months at -20 degrees C and for 24 h at room temperature. The practicability of the new method was demonstrated by the analysis of more than 400 plasma samples from a tolerance study performed in human volunteers.     

Quantitative high-throughput determination of endogenous retinoids in human plasma using triple-stage liquid chromatography/tandem mass spectrometry

A high-throughput ultrasensitive analytical method based on liquid chromatography with positive ion atmospheric pressure chemical ionization (APCI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of all-trans-4-oxo-retinoic acid (at4oxoRA), 13-cis-4-oxo-retinoic acid (13c4oxoRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (atRA) and all-trans-retinol (atROH) in human plasma. A stable isotope of atRA was used as internal standard (IS). The analytes and IS were isolated from 100 microL plasma by acetonitrile mono-phase extraction (MPE) performed in black 96-well microtiterplates. A 100 microL injection was focused on-column and chromatographed on an Agilent ZORBAX SB-C18 rapid-resolution high-throughput (RRHT) column with 1.8-microm particles (4.6 mmx50 mm) maintained at 60 degrees C. The initial mobile phase composition was acetonitrile/water/formic acid (10:90:0.1, v/v/v) delivered at 1.8 mL/min. Elution was accomplished by a fast gradient to acetonitrile/methanol/formic acid (90:10:0.1, v/v/v). The method had a chromatographic total run time of 7 min. An Applied Biosystems 4000 Q TRAP linear tandem mass spectrometer equipped with a heated nebulizer (APCI) ionization source was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 315.4-->297 (4-oxo-retinoic acids), 301.2-->205 (retinoic acids), 305.0-->209 (IS) and 269.2-->93 (retinol) used for quantification. The assay was fully validated and found to have acceptable accuracy, precision, linearity, sensitivity and selectivity. The mean extraction recoveries from spiked plasma samples were 80-105% for the various retinoids at three different levels. The intra-day accuracy of the assay was within 8% of nominal and intra-day precision was better than 8% coefficient of variance (CV) for retinoic acids. Inter-day precision results for quality control samples run over a 12-day period alongside clinical samples showed mean precision better than 12.5% CV. The limit of quantification was in the range of 0.1-0.2 ng/mL and the mass limit of detection (mLOD) was in the range 1-4 pg on column for the retinoic acids. The assay has been successfully applied to the analysis of 1700 plasma samples.     

High-performance liquid chromatographic method for the simultaneous measurement of remacemide (a novel anticonvulsant and cerebroprotectant) and an active metabolite in human plasma.

A high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of both remacemide (a novel anticonvulsant and cerebroprotectant) and an active, major metabolite in human plasma. After the addition of an internal standard, the analytes were extracted from the plasma by ion-exchange solid-phase extraction and measured by an isocratic HPLC system with ultraviolet detection at 210 nm. The recovery of the analytes was > 90%. The standard curves were linear over the range of quantitation of approximately 10-500 ng/ml for remacemide itself and 15-250 ng/ml for the metabolite. Both intra-day and inter-day accuracy and precision data were excellent. Remacemide and its metabolite were shown to be stable in human plasma for at least a year when stored at -20 degrees C.     

Development and validation of a liquid chromatographic/tandem mass spectrometric method for the determination of sertraline in human plasma

A sensitive and rapid liquid chromatographic/tandem mass spectrometric method was developed and validated for the determination of sertraline in human plasma. The analyte and internal standard (IS, diphenhydramine) were extracted with 3 mL of diethyl ether/dichloromethane (2:1, v/v) from 0.25 mL plasma, then separated on a Zorbax Eclipse XDB C18 column using methanol/water/formic acid (75:25:0.1, v/v/v) as the mobile phase. The triple quadrupole mass spectrometry was applied via an atmospheric pressure chemical ionization (APCI) source for detection. The fragmentation pattern of the protonated sertraline was elucidated with the aid of product mass spectra of isotopologous peaks. Quantification was performed using selected reaction monitoring of the transitions of m/z 306 --> 159 for sertraline and m/z 256 --> 167 for the IS. The method was linear over the concentration range of 0.10-100 ng/mL. The intra-day and inter-day precisions, expressed by relative standard deviation, were both less than 6.7%. Assay accuracies were within +/-6.9% as terms of relative error. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.10 ng/mL with a precision of 8.3% and an accuracy of 9.6%. The validated method has been successfully applied for the pharmacokinetic study and bioequivalence evaluation of sertraline in 18 healthy volunteers after a single oral administration of 50 mg sertraline hydrochloride tablets.     

Automated liquid chromatographic determination of ranitidine in microliter samples of rat plasma

An improved high-performance liquid chromatographic method with UV detection at 313 nm has been developed for quantitation of ranitidine in 100 microliter of rat plasma over the range 25 to 1000 ng/ml. To each sample were added the internal standard (metiamide) and 2 M NaOH. After dichloromethane extraction, the nitrogen-dried extracts were reconstituted in the mobile phase of 0.01 M phosphate buffer-triethylamine-methanol-water (530:5:390:75 v/v). Chromatography on mu Bondapak C18 with quantitation by peak height ratios showed an analyte recovery of 97%; a limit of detection of 10 ng/ml; a precision of 1-10% and an accuracy of 1-5%. About 90 samples can be processed in 24 h.     

Simultaneous determination and pharmacokinetic studies of aloe emodin and chrysophanol in rats after oral administration of Da-Cheng-Qi decoction by high-performance liquid chromatography

A validated high-performance liquid chromatography (HPLC) method was developed for simultaneous determination and pharmacokinetic study of aloe emodin and chrysophanol in rats. It was performed on a reverse-phase C(18) column and a mobile phase made up of methanol and 0.2% acetic acid (83:17, v/v). The ultraviolet detection was 254 nm. 1,8-dihydroxyanthraquinone was used as the internal standard. The assay was linear over the range 28-2800 ng/mL (r(2) = 0.9993) for aloe emodin and 25.6-2560 ng/mL (r(2) = 0.9991) for chrysophanol. The average percentage recoveries of three spiked plasmas were 98.8-104.8% and 97.7-103.2% for aloe emodin and chrysophanol, respectively. Their RSD of intra-day and inter-day precision at concentrations of 56, 280 and 1400 ng/mL for aloe emodin and 51.6, 258 and 1290 ng/mL for chrysophanol were less than 3.5%. This method was applied for the first time to simultaneously determinate aloe emodin and chrysophanol in rats following oral administration of traditional Chinese medicine of Da-Cheng-Qi decoction. The pharmacokinetic parameters showed that chrysophanol was better absorbed with higher concentrations in plasma than aloe emodin did. They both eliminated slowly in male rats. The assay is suitable for identifying the plasma and tissue levels of aloe emodin and chrysophanol in preclinical investigations.     

Determination of tramadol and O-desmethyltramadol in human plasma by high-performance liquid chromatography with mass spectrometry detection

A new simple, sensitive and selective liquid chromatography coupled with mass spectrometry (LC/MS) method for quantification of tramadol and its active metabolite O-desmethyltramadol in human plasma was validated. The tramadol and its metabolite were separated on a reversed phase column (Zorbax SB-C18, 100 mm x 3.0 mm I.D., 3.5 microm) under isocratic conditions using a mobile phase of a 10:90 (v/v) mixture of acetonitrile and 0.2% (v/v) trifluoroacetic acid in water. The flow rate was 1 ml/min at the column temperature 45 degrees C. In these chromatographic conditions, the retention times were 2.3 min for O-desmethyltramadol and 3.5 min for tramadol, respectively. The detection of both analytes was in SIM mode using an ion trap mass spectrometer with electrospray positive ionisation. The monitored ions were m/z 264 for tramadol and m/z 250 for its metabolite. The sample preparation was very simple and rapid and consisted in plasma protein precipitation from 0.2 ml plasma using 0.2 ml solution of perchloric acid 7%. Calibration curves were generated over the range of 2-300 ng/ml for both analytes with values for coefficient of correlation greater than 0.998 and by using a weighted (1/y) quadratic regression. The values of precision and accuracy for tramadol at quantification limit were less than 10.9% and 5.1, respectively, both for within- and between-run. For O-desmethyltramadol, precision and accuracy at quantification limit were 10.1% and -9.9% for within-run determinations and 6.7% and 10.4% for between-run determinations, respectively. The mean recovery for both analytes was 96%. Both tramadol and its metabolite demonstrated good short-term, long-term, post-preparative and freeze-thaw stability. This is the first reported method for analysis of tramadol and O-desmethyltramadol in human plasma that uses protein precipitation as sample processing procedure. The method is very simple and allows obtaining a very good recovery of both analytes. The validated LC/MS method has been applied to a pharmacokinetic study of 50 mg tramadol tablets on healthy volunteers.     

Pharmacokinetics of isofraxidin in rat plasma after oral administration of the extract of Acanthopanax senticosus using HPLC with solid phase extraction method

High-performance liquid chromatography coupled with solid phase extraction method was developed for determination of isofraxidin in rat plasma after oral administration of Acanthopanax senticosus extract (ASE), and pharmacokinetic parameters of isofraxidin either in ASE or pure compound were measured. The HPLC analysis was performed on a Dikma Diamonsil RP(18) column (4.6 mm x 150 mm, 5 microm) with the isocratic elution of solvent A (acetonitrile) and solvent B (0.1% aqueous phosphoric acid, v/v) (A : B = 22 : 78) and the detection wavelength was set at 343 nm. The calibration curve was linear over the range of 0.156-15.625 microg/ml. The limit of detection was 60 ng/ml. The intra-day precision was 5.8%, and the inter-day precision was 6.0%. The recovery was 87.30+/-1.73%. When the dosage of ASE is equal to pure compound caculated by the amount of isofraxidin, it has been found to have two maximum concentrations in plasma while the pure compound only showed one peak in the plasma concentration-time curve. The determined content of isofraxidin in plasma after oral administration of ASE is the total contents of free isofraxidin and its precursors in ASE in vitro. The pharmacokinetic characteristics of ASE showed the priority of the extract and the properities of traditional Chinese medicine.     

Improved procedure for the the determination of rofecoxib in human plasma involving 96-well solid-phase extraction and fluorescence detection

An improved assay for the determination of rofecoxib in human plasma samples is described. The analyte and an internal standard were extracted from the plasma matrix using solid-phase extraction in the 96-well format with an Empore C8-SD extraction plate. The analytes are chromatographed on a Waters Symmetry C18 analytical column (3.5 microm, 50x4.6 mm) with a mobile phase consisting of acetonitrile-water (35:65, v/v). Analyte detection was via fluorescence following post-column photochemical derivatization. Eight point calibration curves over the concentration range of 0.5-80 ng/ml yielded a linear response when a 1/y weighted linear regression model was employed. Based on the replicate analyses (n=5) of spiked standards, the within-day assay precision was better than 8% RSD at all points on the calibration curve, within-day accuracy was within 6% of nominal at all standard concentrations. The between-run precision and accuracy of the assay, as calculated from the results of the analysis of quality control samples, was better than 7% RSD and within 5% of nominal. Assay throughput was improved by a factor of three as compared to previously described methods. The method was partially automated using a combination of a Packard Multi-Probe liquid handling system and a TomTec Quadra 96 workstation.     

Liquid chromatographic assay for the protease inhibitor atazanavir in plasma

Atazanavir is the most recently introduced protease inhibitor for the suppression of the anti-human immunodeficiency virus. A sensitive and selective reversed-phase liquid chromatographic assay for this drug in human plasma has been developed and validated. Atazanavir was isolated from a 500 microL plasma sample using liquid-liquid extraction with dichloromethane. After evaporation and reconstitution of the extract the sample was analysed using liquid chromatography and ultraviolet detection at 280 nm. In the evaluated concentration range (44-4395 ng/mL atazanavir), intra-day precisions were < or =7% and inter-day precisions were < or =14%. Accuracies between 96 and 106% were found. The lower limit of quantification was 44 ng/mL with an intra-day precision of 7%, an inter-day precision of 14% and an accuracy of 87%. There was no interference from 32 tested potentially co-administrated drugs and metabolites. The usefulness of the assay was demonstrated for samples obtained from an HIV-infected patient treated with atazanavir.     

Determination of a novel steroidal androgen receptor antagonist (Win 49596) in human plasma using solid-phase extraction and high-performance liquid chromatography with ultraviolet detection.

A rapid, sensitive and selective method was developed for the determination of a novel steroidal androgen receptor antagonist (Win 49596, I) in human plasma. The procedure involved extraction from plasma using a solid-phase phenyl support and elution directly onto a reversed-phase C8 column using a mobile phase consisting of 0.2 mol/l sodium acetate buffer at pH 7-acetonitrile (45:55, v/v). Drug was monitored by ultraviolet detection at a wavelength of 238 nm. Linear responses were observed for standards over the range 0.01-5.0 micrograms/ml. The minimum quantifiable level was 0.02 microgram/ml, using a 0.5-ml plasma sample. The precision was 5.5% and the accuracy ranged from -9.4% to 0.23%. The analytical method has been used to quantify I in plasma from dogs and rats and is projected for use with human plasma from clinical trials.     

Liquid chromatography-tandem mass spectroscopic method for the determination of zerumbone in human plasma and its application to pharmacokinetics

A rapid, sensitive, specific and selective LC-MS/MS method for the determination of zerumbone (ZER) in human plasma using 2,4-diamino-6-(4-methoxyphenyl)-1,3,5-triazine (DMTZ) as an internal standard (IS) has been developed and validated. ZER was chromatographed on C8 column using a mobile phase of acetonitrile/water (80:20, v/v) at a flow rate of 0.25 ml min(-1) . Quantitation was achieved using ESI+ interface, employing multiple reaction monitoring (MRM) mode at m/z 219 > 81 and 218 > 134 for ZER and IS, respectively. The calibration standards were linear over a range of 5-3000 ng ml(-1) (r(2)=0.9994) with an LLOQ of 5 ng ml(-1) (RSD %; 11.4% and bias%; 9.5%). Intra- and inter-day precision of ZER assay ranged from 0.18 to 3.56% with accuracy (bias) that varied between -5.09 and 4.3%, demonstrating good precision and accuracy. Recoveries of ZER and the IS from human plasma were above 85%. The developed method was validated for the determination of ZER in rat plasma. Linearity, stability of ZER and the ME on rat plasma were discussed. The applicability of the developed method was demonstrated by measuring ZER in rat plasma samples following intravenous and intraperitoneal administration of ZER prepared in hydroxypropyl-β-cyclodextrin (HPβCD) and sodium carboxymethyl cellulose (CMC), respectively, in 20 mg kg(-1) and this study indicated a clear significant difference (p<0.05) in pharmacokinetic parameters of ZER in ZER/HPβCD complex compared with ZER in CMC preparation.     

Enantioanalysis of bisoprolol in human plasma with a macrocyclic antibiotic HPLC chiral column using fluorescence detection and solid phase extraction

A sensitive, enantioselective, high-performance liquid chromatographic (HPLC) method was developed and validated to determine S-(-)- and R-(+)-bisoprolol in human plasma. Baseline resolution was achieved using the teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with a polar ionic mobile phase (PIM) consisting of methanol-glacial acetic acid-triethylamine (100 : 0.02 : 0.025, v/v/v) at a flow rate of 1.5 ml/min and fluorescence detection set at 275 nm for excitation and 305 nm for emission. All analyses with S-(-)-atenolol as the internal standard were conducted at ambient temperature. The assay involved the use of a solid-phase extraction procedure for human plasma samples prior to HPLC analysis. The C18 cartridge gave good recovery rates for both enantiomers without any interference. The method was validated over the range of 20-200 ng/ml for each enantiomer concentration. Recovery rates for S-(-)- and R-(+)-bisoprolol enantiomers were in the range of 95-102%. The method proved to be precise (within-run precision expressed as % RSD ranged from 1.0-6.2% and between-run precision ranged from 0.9-6.7%) and accurate (within-run accuracies expressed as percentage error ranged from 0.2-4.8% and between-run accuracies ranged from 0.3-1.7%). The limit of quantitation and limit of detection for each enantiomer in human plasma were 20 and 5 ng/ml, respectively.     

High-performance liquid chromatographic assay of alpha-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazine butanol (BMY 14802), a potential antipsychotic drug, in monkey and rat plasma

A high-performance liquid chromatographic-fluorescence method was developed for the quantitative analysis of BMY-14802 (I) in monkey and rat plasma. After the addition of the internal standard (BMY-14853 I.S.), 250 microliters of plasma were made basic by the addition of 2 ml of saturated sodium carbonate buffer. Compound I and the I.S. were then extracted into 5 ml of methyl tert.-butyl ether. The organic phase was evaporated and the resulting residue was reconstituted in mobile phase. Final separation and quantitation of I was achieved on an octadecyl column with a 0.05 M potassium phosphate-acetonitrile-triethylamine-85% phosphoric acid (650:350:0.1:0.05, v/v) mobile phase. Fluorescence detection was used to monitor the eluent at an excitation wavelength of 240 nm and an emission wavelength of 400 nm. The limit of detection was 0.5 ng/ml. The standard curve was linear over the range 5.0-1000 ng/ml. Intra-assay and inter-assay precision values were less than 4.0% relative standard deviation and accuracy was within 12% of nominal values. Compound I was shown to be stable in monkey and rat plasma for at least six months when stored at -20 degrees C.     

Determination of albendazole metabolites by direct injection of bovine plasma and multidimensional achiral-chiral high performance liquid chromatography

The development and validation of a fully automated achiral-chiral high performance liquid chromatography (HPLC) method for the simultaneous determination of albendazole metabolites: enantiomers of albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO(2)) and albendazole 2-aminosulphone (ABZ-SO(2)NH(2)) in bovine plasma are described. This method involves an octyl restricted access media bovine serum albumin column (C(8)-RAM-BSA) (50 mm x 4.6 mm I.D.) for sample clean-up, followed by enantioselective analysis on a column containing an amylose tris(3,5-dimethylphenylcarbamate) stationary phase (150 mm x 4.6 mm I.D.). The chromatographic separations of all target compounds were performed at 30 degrees C using a mobile phase composed of phosphate buffer (10 mmol L(-1); pH 7.5):acetonitrile (60:40, v/v), flow rate of 0.5 mL min(-1) and fluorescence detection at 290 nm and 320 nm, excitation and emission, respectively. The influence of different organic modifiers and chiral selector of the stationary phase on enantioseparation of ABZ-SO was investigated. The method developed was fully validated. The calibration curves were linear in the concentration range of 40.00-1280 ng mL(-1) for each albendazole sulphoxide enantiomer, 10.0-320 ng mL(-1) for albendazole sulphone and 20.0-320 ng mL(-1) for albendazole 2-aminosulphone. The inter- and intra-day precision ranged from 0.760% to 7.79% relative standard deviation (R.S.D.), and the accuracy ranged 101% from 114% of the nominal values while the transfer efficiency was in the range of 84.4-103%. The method showed good linearity, precision, accuracy, sensitivity and selectivity allowing it to be appropriate for further pharmacokinetics and metabolism studies of albendazole.     

LC-MS/MS method for the determination of cynandione A in rat plasma and tissues

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for the quantitative determination of cynandione A in rat plasma and tissues. The plasma samples were pretreated by liquid-liquid extraction with ethyl acetate after the internal standard (honokiol) had been spiked. The tissue samples were homogenized with physiological saline and treated further like the plasma samples. The separation was performed using a Zorbax SB-C(18) column (3.5 microm, 2.1 x 100 mm) and a C18 guard column (5 microm, 4.0 x 2.0 mm) with an isocratic mobile phase consisting of methanol-0.1% formic acid (78:22, v/v) at a flow rate of 0.2 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple-reaction monitoring mode using the electrospray ionization technique in negative mode. The nominal retention times for cynandione A and honokiol were 1.41 and 2.63 min, respectively. The method was validated within the concentration range 0.2-1000 ng/mL in plasma and homogenized tissue for cynandione A, and the calibration curves were linear with correlation coefficients >0.992. The lower limit of quantification of cynandione A was 0.2 ng/mL. The intra-day and inter-day precision and accuracy of the assay in plasma were less than 14.4%, while the intra-day and inter-day precision and accuracy of the assay in tissue homogenate were less than 14.2%. This method proved to be suitable for study of pharmacokinetics and tissue distribution of cynandione A in rat.     

Quantitation of a new cholecystokinin and gastrin receptor antagonist (L-365,260) in dog and rat plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of L-365,260 (I), a cholecystokinin and gastrin receptor antagonist, in dog and rat plasma. The method involves liquid-liquid extraction and HPLC with ultraviolet detection. Standard curves were linear over the range 7.5-2000 ng/ml for rat and dog plasma. The method is reproducible and reliable with a detection limit of 7.5 ng/ml in biological fluids. The mean coefficients of variation for concentrations within the range of the standard curve range were 3.84 and 2.56%, respectively, for intra-day analysis and 4.48 and 4.26%, respectively, for inter-day analysis. Application of the development was successfully demonstrated by quantifying the concentration of I in both dog and rat plasma samples following an intravenous or oral dose of 5 mg/kg I.     

Quantitative analysis of retinoids in biological fluids by high-performance liquid chromatography using column switching. II. Simultaneous determination of etretinate, acitretin and 13-cis-acitretin in plasma

An automated gradient high-performance liquid chromatographic method for the determination of etretinate, acitretin and 13-cis-acitretin in plasma was developed, using a column-switching technique. After protein precipitation with ethanol, 0.5 ml of the supernatant was injected onto a precolumn (17 mm x 4.6 mm I.D.), filled with 37-53 microns C18 Corasil. Polar plasma components were washed out using 1% ammonium acetate and 1% acetic acid-acetonitrile (8:2, v/v); the retained retinoids were then transferred to the analytical column (125 mm x 4 mm I.D., filled with 5-microns ODS material) in the backflush mode, separated by gradient elution and detected at 360 nm by UV detection. The limit of quantification was 2 ng/ml and the inter-assay precision in the concentration range 20-1000 ng/ml was between 0.9 and 4.0% for all three compounds. To optimize the recovery for etretinate (greater than 60%), protein was precipitated from plasma with ethanol before injection, instead of direct injection of plasma samples, and a mobile phase containing 20% acetonitrile, instead of pure water or buffer, was used.