Simultaneous Analysis of Cimetidine and Ranitidine in Human Plasma by HPLC

Abstract

A rapid method for the simultaneous quantitation of the H₂-receptor antagonist drugs cimetidine and ranitidine in human plasma by isocratic ion-pair reverse-phase HPLC is described. The method involves a simple organic extraction step of the alkalinized plasma containing added internal standard followed by back extraction of the extract with dilute acetic acid and subsequent analysis of the aqueous acidic phase on a reverse-phase (C18) column. The eluting solvent was acetonitrile-water (20:80 v/v) containing 0.005 mole/litre octanesulphonic acid and was monitored at 229 nm. The run time for the assay was 12.5 minutes, with a detection limit for cimetidine of 50 ng/m1/(0.2 μmole/1) and that for ranitidine was 20 ng/ml (0.06 umole/1).     

A sensitive and rapid determination of ranitidine in human plasma by HPLC with fluorescence detection and its application for a pharmacokinetic study

A novel precolumn derivatization reversed-phase high-performance liquid chromatography method with fluorescence detection is described for the determination of ranitidine in human plasma. The method was based on the reaction of ranitidine with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole forming yellow colored fluorescent product. The separation was achieved on a C(18) column using methanol-water (60:40, v/v) mobile phase. Fluorescence detection was used at the excitation and emission of 458 and 521 nm, respectively. Lisinopril was utilized as an internal standard. The flow rate was 1.2 mL/min. Ranitidine and lisinopril appeared at 3.24 and 2.25 min, respectively. The method was validated for system suitability, precision, accuracy, linearity, limit of detection, limit of quantification, recovery and robustness. Intra- and inter-day precisions of the assays were in the range of 0.01-0.44%. The assay was linear over the concentration range of 50-2000 ng/mL. The mean recovery was determined to be 96.40 ± 0.02%. This method was successfully applied to a pharmacokinetic study after oral administration of a dose (150 mg) of ranitidine.     

Gas chromatographic assay for the new antitumor agent pyrazine-2-diazohydroxide (diazohydroxide) and its stability in buffer, blood and plasma

Diazohydroxide is a new antitumor agent being considered for clinical trial. A sensitive and specific assay for diazohydroxide in physiological media, plasma and blood has been developed based on conversion of diazohydroxide to 2-chloropyrazine in the presence of strong hydrochloric acid. The 2-chloropyrazine is extracted into the ethyl acetate and separated by capillary gas chromatography with nitrogen-phosphorus detection. Using 0.2 ml plasma the assay was linear up to 100 micrograms/ml diazohydroxide and had a lower limit of detectability for diazohydroxide of 50 ng/ml. The coefficient of variation of the assay at 1 micrograms/ml was 6.7%. Breakdown of diazohydroxide was rapid under mild acid conditions but slower under alkaline conditions,. The half-life of diazohydroxide in 0.1 M sodium phosphate buffer, pH 6.0, at room temperature was 5 min and at pH 8.0, 480 min. Breakdown of diazohydroxide in plasma was biphasic. In fresh mouse plasma diazohydroxide had a terminal half-life at 37 degrees C of 72 min while in fresh human plasma the terminal half-life was 23 min and in fresh blood 21 min. Diazohydroxide accumulated in red blood cells at 37 degrees C to a concentration 68% above the concentration in plasma. Diazohydroxide was 49% bound to human plasma proteins at room temperature.     

High-performance liquid chromatographic assay for the antitumor glycoside phyllanthoside and its stability in plasma of several species

Phyllanthoside is a glycoside isolated from the roots of the Central American tree Phyllanthus acuminatus Vahl with antitumor activity against murine B-16 melanoma and P-388 leukemia. We report a reversed-phase high-performance liquid chromatographic assay for phyllanthoside in plasma using a 25-cm RP-18, 5-micron column with a linear 10-min gradient of 50% to 100% methanol in 0.3 M sodium acetate, pH 4.0, at a flow-rate of 1.5 ml/min. Eluting peaks were detected at 270 nm. The lower limit of sensitivity of the assay for phyllanthoside in 0.5 ml plasma following ethyl acetate extraction at pH 7.0 was 0.25 micrograms/ml and the coefficient of variation at 1 microgram/ml was +/- 7.4%. Phyllanthoside was very rapidly broken down by mouse and rat plasma in vitro to an unidentified less polar metabolite. Formation of this metabolite was completely inhibited by preheating mouse plasma to 100 degrees C for 10 min. When mouse plasma was diluted 1:50 with water the half-life of phyllanthoside disappearance at 37 degrees C was 2.0 min. Breakdown of phyllanthoside in plasma from other species was slower than in mouse and the initial half-life at 37 degrees C in dog plasma was 30 min, in monkey plasma 33 min and in human plasma 38 min. The same less polar metabolite as in mouse plasma was formed slowly by plasma of monkey and dog. Phyllanthoside did not accumulate in human red blood cells. Binding of phyllanthoside to human plasma protein determined by ultrafiltration at 4 degrees C was 70%.     

Determination of ranitidine and its metabolites in human urine by reversed-phase ion-pair high-performance liquid chromatography

A method using ion-pair high-performance liquid chromatography is presented for determining ranitidine, ranitidine N-oxide, ranitidine S-oxide and desmethyl ranitidine in the urine from four volunteers, given on separate occasions an intravenous and oral dose of 100 mg ranitidine. This method has been used to study the metabolism and pharmacokinetics of ranitidine by man. It was found that the elimination half-life of ranitidine ranged from 110-246 min. The mean renal clearance of ranitidine in these four volunteers was 512 ml/min.     

Determination of buprenorphine by high-performance liquid chromatography with fluorescence detection: application to human and rabbit pharmacokinetic studies.

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with fluorescence detection was developed for the determination of buprenorphine in human, rabbit, pig and dog plasma. It is comprised of only a one-step extraction procedure with hexane-isoamyl alcohol at pH 9.25 and reversed-phase chromatography on a muPorasil column. The recoveries of buprenorphine and nalbuphine (internal standard) were greater than 90%. Calibration graphs were linear over the concentration range 3-300 ng/ml with a coefficient of variation, both within-day and between-day, of less than 9% at any level. The limit of detection was 1.0 ng/ml of plasma based on a signal-to-noise ratio of 3. Eight other clinically used narcotics were investigated to check for potential interferences and their analytical conditions. The possible decomposed compounds of buprenorphine were also checked for the specificity of this assay. The method has been successfully applied to the stability and pharmacokinetic studies of buprenorphine. Buprenorphine in plasma did not decompose significantly at -20 degrees C for four weeks. Pharmacokinetic application in six rabbits and a surgical patient revealed that buprenorphine followed a linear three-compartment model with two distribution phases. The two distribution and elimination half-lives and the clearance of buprenorphine were 1.32, 24.8 and 230 min and 224 ml/min in human plasma, and 0.94, 12.5 and 232 min and 30 ml/min in rabbit plasma.     

Simultaneous determination of haloperidol and its metabolite, reduced haloperidol, in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the simultaneous determination of haloperidol and reduced haloperidol in human plasma, urine and rat tissue homogenates using bromperidol as an internal standard. The method involved extraction followed by injection of 50-80 microliters of the aqueous layer onto a C18 reversed-phase column. The mobile phase was 0.5 M phosphate buffer-acetonitrile-methanol (58:31:11, v/v/v) and the flow-rate was 0.6 ml/min. The column effluent was monitored by ultraviolet detection at 214 nm. The retention times for reduced haloperidol, haloperidol and bromperidol were 5.4, 7.2 and 8.4 min, respectively. The detection limits for haloperidol and reduced haloperidol in human plasma were both 0.5 ng/ml, and the corresponding values in human urine were both 5 ng/ml. The coefficients of variation of the assay were generally low (below 10.7%) for plasma, urine, blood and tissue homogenates. No interferences from endogenous substances or any drug tested were found.     

Rapid and Sensitive Hplc Determination of Ranitidine in Plasma. Application to Pharmacokinetics Study

Abstract

A rapid and sensitive reversed-phase high-performance liquid chrormatography (RP-HPLC) method for the separation and quantification of the H₂-receptor antagonist drug ranitidine in human plasma is described.

The extraction of ranitidine from plasma by an organic solvent was eliminated in this method. Instead, the pre-chromatography isolation of the drug was done by adding approximately 50 mg of zinc sulfate and 200 μL of acetonitrile in 1.0 mL of plasma. A short column packed with pH-stable (1–13) reversed phase PLRP-S^TM particles was used with an isocratic elution of 5.0mM dibasic potassium phosphate plus 0.50mM tetraethyl ammonium hydroxide/ acetonitrile, 80:20 (v/v). The ranitidine was monitored at 315 nm and 0.20 to 0.002 absorption units full scale (AUFS). The completion time of the assay was less than 15 minutes and had a limit of detection of 1.0 ng/mL for a 100-μL injection volume.

After an oral dose of 150 mg of ranitidine, plasma samples were collected at several time points and were analyzed by using this method to determine various pharmacokinetic parameters.     

Rapid determination of succinylcholine in human plasma by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic method with fluorometric detection has been developed for the determination of succinylcholine in human plasma. Succinylcholine shows fluorescence at 282 nm with an excitation at 257 nm. The assay is sensitive, reproducible and linear for concentrations ranging from 100 ng/ml to 100 micrograms/ml of succinylcholine. In a pilot study the plasma concentration-time curve showed a triphasic elimination, with half-lives of 0.4, 1.2 and 8 min, respectively. In a clinical setting, drugs commonly administered during anaesthesia did not interfere with the assay. This method provides a simple and time-saving alternative to existing methods.     

Highly sensitive assay for alkaline and acid phosphatase activities by high-performance liquid chromatography with electrochemical detection

A highly sensitive and specific assay for alkaline and acid phosphatases in biological materials, such as plasma and saliva, has been established. Phenol, formed enzymatically from the substrate phenylphosphate, was determined by high-performance liquid chromatography with electrochemical detection. The retention time of phenol was 7 min and no other peaks were observed. The method is rapid and sensitive with a detection limit for phenol of as little as 5 pmol. Thus, as little as 0.5 microliter of rat plasma or 10 microliters of human saliva is required for both alkaline and acid phosphatase assays. The assay is accurate and reproducible. Using this assay, alkaline and acid phosphatase activities in saliva were found to be 1.12 +/- 0.12 nmol/min/ml and 9.79 +/- 1.23 nmol/min/ml, respectively. This new assay method should be applicable to extremely small biological samples.     

A Sensitive High Performance Liquid Chromatographic Determination of Clopamide in Human Plasma

Abstract

A simple and sensitive high-performance liquid chromatographic method for quantitation of clopamide in human plasma has been developed. the assay uses a reversed-phase C18 microbore column (2 mm I.D. × 100 mm) packed with 5 μm ODS Hypersil. the chromatographic separation was achieved by using an isocratic mobile phase comprising acetonitrile-10 mM phosphate buffer pH 4 (17:83, v/v) at a flow rate of 0.5 ml/min. the eluant was monitored by a UV detector operating at 241 nm. the assay was based on an organic extraction before chromatographic separation. to 1 ml plasma sample, 100 μl of the internal standard, methylparaben (300 ng/ml), and 8 ml of diethyl ether were added. the samples were shaken and centrifuged, the organic layer was then transferred to a tapered centrifuge tube and evaporated to dryness. the residue was reconstituted and injected onto the HPLC column. the inter-and intra-assay coefficients of variation were found to be less than 10%. the lowest limit of detection for clopamide in plasma was 5 ng/ml. the method is sensitive, specific and allows for routine analysis in the pharmacokinetic studies.     

Determination of exifone in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection was developed for the determination of exifone in human plasma and urine. Exifone was extracted from acidified plasma or neutralized urine with diethyl ether and the evaporated extracts were analysed on a C18 reversed-phase column. The compound was eluted in about 8 min with acetonitrile-0.3 M orthophosphoric acid (15:85, v/v) at a flow-rate of 0.9 ml/min. This method gave accurate and reproducible results; the calibration graphs were linear (r greater than 0.99) over the range of 2.8-360 nmol/l for plasma and 0.18-36 mumol/l for urine, and concentrations as low as 1 nmol/l in plasma could be quantified. These results allowed this assay to be used for determinations in single-dose pharmacokinetic studies.     

Direct analysis of the dopamine agonist (-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin hydrochloride in plasma by high-performance liquid chromatography using two-dimensional column switching.

A reversed-phase, two-dimensional, liquid chromatographic method incorporating column switching and electrochemical detection was used for the direct analysis of the dopamine (D2) agonist (-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin hydrochloride in plasma. Sample work-up consisted of addition of internal standard, filtration, then direct injection of the plasma sample onto an internal surface reversed-phase (ISRP) guard column where the dopamine agonist and internal standard were separated from plasma proteins. An automated pneumatic valve was then used to switch to a stronger eluent which stripped the retained substances from the ISRP support onto a C18 analytical column where the analytes were separated from endogenous biological interferences. A dual-electrode electrochemical detector was used to minimize interferences and provide the desired sensitivity. The method has a detection limit of 1.5 ng/ml and requires a total assay time of 20 min per plasma sample. The method is linear from 1.5 to 1000 ng/ml and yielded greater than 80% drug recovery for plasma concentrations greater than 10 ng/ml. Precision for the method at 100 ng/ml yielded a relative standard deviation of 4.4%. Reproducibility was within 6.5% on a 20 ng/ml spiked plasma sample assayed on different days by different people. The method has successfully been applied to human plasma samples and for pharmacokinetic studies in rats and monkeys.     

High-performance liquid chromatographic analysis of amphotericin B in plasma, blood, urine and tissues for pharmacokinetic and tissue distribution studies.

A sensitive and reproducible high-performance liquid chromatographic method was developed to assay ampherotericin B in plasma, blood, urine and various tissue samples. Amphotericin B was isolated from each sample matrix by solid-phase extraction (Bond-Elut). The eluate from Bond-Elut containing amphotericin B was injected onto a reversed-phase C18 column (Waters, mu Bondpak, 10 microns, 300 mm x 3.9 mm I.D.) with a mobile phase of 45% acetonitrile in 2.5 mM Na2EDTA at 1 ml/min. Detection of amphotericin B was by ultraviolet absorption at 382 nm. Blood and tissues were homogenized and extracted with methanol prior to Bond-Elut extraction. The extraction efficiencies of amphotericin B from plasma, blood and tissues were approximately 90, 70 and 75%, respectively. The sensitivity of the assay was less than or equal to 5 ng/ml for plasma, less than or equal to 25 ng/ml for blood, 2.5 ng/ml for urine and 50 ng/g for tissues. The linearity of the assay method was up to 2.5 micrograms/ml for plasma, 5 micrograms/ml for blood, 500 ng/ml for urine and 500 micrograms/g for tissues. The assay was reproducible with an intra-day coefficient of variation (C.V., n = 3) of less than 5% in general for plasma, blood and tissues. The inter-day C.V. of the assay was less than 5% for plasma (n = 5), less than 10% for blood (n = 4) and less than 5% for tissues (n = 3). The overall variability in the urine assay was generally less than 10%. This method has demonstrated significant improvement in the sensitivity and reproducibility in assaying amphotericin B in plasma and especially in blood, urine and tissues. We have employed this assay to compare the pharmacokinetic and tissue distribution profiles of amphotericin B in rats and dogs following administration of Fungizone and ABCD (amphotericin B-cholesteryl sulfate colloidal dispersion), a lipid-based dosage form. In addition, the assay method for plasma and urine samples can also be applied to pharmacokinetics studies of amphotericin B in man.     

Simple High-Performance Liquid Chromatographic Method for the Analysis of Quinine in Human Plasma without Extraction

Abstract

A simple, rapid and sensitive assay, capable of quantitating quinine (Q) in human plasma samples is reported. The assay uses a reversed-phase C18 HPLC column packed with 5 μ ODS Hypersil. The chromatographic separation was accomplished with an isocratic mobile phase comprising acetonitrile-aqueous phosphate buffer pH 2 (50:50, v/v) containing 25 mM sodium dodecyl sulfate and 3 mM tetrabutylammonium bromide at a flow rate of 0.5 ml/min. The eluant was monitored by a fluorescence detector (excitation wavelength at 350 nm and emission wavelength at 450 nm). The assay was based on a simple plasma protein precipitation technique. To 200 μ of plasma sample, 400 μ of internal standard (cinchocaine 30 μ/ml in methanol) was added. After brief vortexing and centrifugation, the clear supernatant was injected onto the HPLC column. The inter- and intra-assay coefficients of variation were found to be less than 10%. The lowest limit of detection for Q in plasma was 18 ng/ml.     

Rapid Determination of Ranitidine in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry and Its Application to a Clinical Pharmacokinetic Study

A rapid and sensitive assay based on high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS) was developed for the determination of ranitidine(RAN) in human plasma with codeine as internal standard(IS).After protein precipitation with acetonitrile,the analyte and IS were separated on a Zorbax SB-Aq C18 column(150 mm×4.6 mm i.d.,5 μm) eluted with a mobile phase consisting of methanol/acetonitrile/10 mmol/L ammonium acetate containing 1% formic acid(pH=2.4)(volume ratio 12.5:12.5:75) at a flow rate of 1.0 mL/min.Detection was performed by electrospray ionization in the positive ion mode followed by the multiple reaction monito-ring(MRM) of the transitions of RAN at m/z 315.1→176.3 and of IS at m/z 300.1→165.1.The method was linear over a concentration range of 1―1000 ng/mL(r=0.9991) with a lower limit of quantitation(LLOQ) of 1 ng/mL and a limit of detection(LOD) of 0.3 ng/mL.Accuracy as relative error was from-0.01% to-1.7% and intra-day and inter-day precisions as relative standard deviation were ≤8.9% and ≤5.5%,respectively.The method was successfully applied to a pharmacokinetic study of ranitidine,getting a single oral dose(160 mg) to healthy volunteers.     

Quantitative analysis of ES-285, an investigational marine anticancer drug,in human,mouse, rat,and dog plasma using coupled liquid chromatography and tandem mass spectrometry

A method was developed for the quantitative analysis of the novel anticancer agent ES-285 (spisulosine; free base) in human, mouse, rat, and dog plasma using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry in order to support pre-clinical and clinical studies with the drug. Sample preparation was carried out by protein precipitation with acetonitrile, containing isotopically labeled (d(3)) ES-285 as internal standard. Aliquots of 10 micro l of the supernatant were injected directly on to an Inertsil ODS-3 column (50 x 2.0 mm i.d., 5 micro m). Elution was carried out using methanol-10 mM ammonium formate (pH 4) in water (80 : 20, v/v) pumped at a flow-rate of 0.2 ml min(-1) with a run time of 8 min. Multiple reaction monitoring chromatograms obtained on an API365 triple-quadrupole mass spectrometer were used for quantification. The lower limit of quantitation (LLOQ) was 10 ng ml(-1) in human, mouse, rat, and dog plasma and the linear dynamic range extended to 500 ng ml(-1). A full validation of the method was performed in human plasma, and partial validations were performed in mouse, rat and dog plasma. Accuracies and precisions were <20% at the LLOQ concentration and <15% for all other concentrations in all matrices. ES-285 was stable during all steps of the assay. Thus far this method has been used successfully to analyze over 500 samples in pre-clinical trials, and will be implemented in the planned clinical phase I studies.     

High-Performance Liquid Chromatographic Determination of Acetazolamide in Human Plasma

Abstract

A simple, rapid and specific HPLC method has been developed to determine acetazolamide concentrations in human plasma. The assay procedure requires only 250 μl of sample with direct injection of the organic supernatant after protein precipitation with acetonitrile. Chlorothiazide was used as an internal standard. A reversed-phase C₁₈ μBondapak column was employed for the chromatographic separation. The eluent was monitored at 265 nm using a UV variable wavelength detector. The retention times for acetazolamide (ACZ) and chlorothiazide (CTZ) were 6 and 8 min respectively. A linear relationship (r).995) was obtained over the 1-20 μg/ml concentration range. The limit of sensitivity for ACZ was 0.5 μg/ml, with greater than 85% recovery of ACZ and internal standard. The method was applied to human plasma samples obtained after administration of a 250 mg acetazolamide tablet.     

Liquid chromatographic-tandem mass spectrometric determination of amprenavir (agenerase) in serum/plasma of human immunodeficiency virus type-1 infected patients receiving combination antiretroviral therapy

A selective assay method for quantitation of amprenavir (agenerase) in human immunodeficiency virus type-1 infected patient serum or plasma using liquid chromatography-tandem mass spectrometry (LC-MS-MS) is described. Amprenavir and an internal standard (reserpine) are extracted by liquid-liquid extraction and chromatographically separated by a reversed-phase C18-analytical column. The triple quadrupole LC-MS-MS system is operated in the positive-ion mode and multiple reaction monitoring is used for drug quantitation. The method has been validated over the range of 0.05-10.0 microg/ml. The RSDs for the intra-day and inter-day determinations ranged from 5.3 to 6.1% and from 4.7 to 6.2%, respectively. The average assay accuracy at two different concentrations ranged from 96.0 to 103.0% and the extraction recovery of amprenavir was 90.8%. The lower limit of quantitation was 0.05 microg/ml. Using a short microbore column, the analysis was completed in less than 5 min.     

Liquid chromatographic analysis of propafenone enantiomers in human plasma

A convenient and sensitive high-performance liquid chromatographic method for analysis of the enantiomers of propafenone (PPF) in human plasma was developed. Racemic propafenone and (-)-ephedrine (internal standard) were first extracted from plasma samples into a mixture of hexane-2-propanol-heptafluorobutanol (95:5:1.25, v/v). After evaporation of the organic layer, the samples were derivatized with R(-)-naphthylethyl isocyanate. The derivatization reached its maximum within 30 s at room temperature with an efficiency of 93.9 +/- 2.8% (mean +/- S.D.). The formed diastereomers were subsequently separated on a silica column with a mobile phase of hexane-2-propanol-isobutanol (96:2:2, v/v) at a flow-rate of 1.5 ml/min. The ultraviolet detection wavelength was set at 220 nm. Using 1 ml plasma, the detection limit was 6.25 ng/ml for the propafenone enantiomers. The assay was successfully employed to measure propafenone enantiomers in plasma samples of a healthy subject after oral administration of a single 150-mg dose of the racemate.