Liquid chromatography method for determination of bivalirudin in human plasma and urine using automated ortho-phthalaldehyde derivatization and fluorescence detection

A high-performance liquid chromatographic (HPLC) method was developed using solid-phase extraction, o-phthalaldehyde (OPA) derivatization and fluorescence detection for the determination of the direct thrombin inhibitor bivalirudin in human plasma and urine. The use of this assay will facilitate the study of the pharmacodynamics of bivalirudin in studies of special patient populations. A C(18) bioanalytical column at a flow rate of 1 ml/min with an aqueous trifluoroacetic acid (0.1% TFA in deionized water, pH 2.2, v/v) mobile phase and methanol gradient was used. The assay demonstrated linearity from 3 to 20 microg/ml bivalirudin in plasma, with a detection limit of 1 microg/ml. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of bivalirudin in patients undergoing percutaneous coronary interventions (PCIs).     

Synthesis and bioactivity studies of 1-adamantanamine derivatives of peptides

Small enkephalin-related peptides containing a 1-adamantanamine moiety coupled through an amide linkage at the C-terminus were synthesized. Several of the compounds showed high μ opioid activity and μ receptor selectivity. The new adamantanamine derivatives were also examined for antiviral activity against HIV-1 in a cell culture system. Some of them inhibited syncytia formation even when the antigen assay gave evidence for viral replication.     

Determination of arginine-vasotocin and isotocin in fish plasma with solid-phase extraction and fluorescence derivatization followed by high-performance liquid chromatography

A new HPLC assay for plasma arginine-vasotocin (AVT) and isotocin (IT) determination based on fluorescence detection preceded by combination of solid-phase extraction (SPE) and fluorescence derivatization is presented. Plasma samples retained on solid support were purified and then derivatized by the fluorescent compound 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The peptide derivatives were eluted from cartridges, pre-concentrated and analyzed by HPLC system with fluorescent detection. The separation was carried out on a reversed-phase column with solvent gradient system. The assay was linear in the range 15-220 pmol ml(-1) for AVT r2=0.998 and 10-220 pmol ml(-1) for IT r2=0.996. The detection limits for AVT and IT were 0.8 and 0.5 pmol ml(-1) (3:1, signal-to-noise), respectively. The recoveries of derivatized hormones were in the range 89-93%. Both of the inter- and intra-day assay precision were below 5.5 and 9% for AVT and IT, respectively. The assay should be also applicable to plasma and tissue samples from other animals with only minor modification.     

Stereoselective RP-HPLC determination of esmolol enantiomers in human plasma after pre-column derivatization

A stereoselective reversed-phase HPLC assay to determine S-(-) and R-(+) enantiomers of esmolol in human plasma was developed. The method involved liquid-liquid extraction of esmolol from human plasma, using S-(-)-propranolol as the internal standard, and employed 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate as a pre-column chiral derivatization reagent. The derivatized products were separated on a 5-microm reversed-phase C18 column with a mixture of acetonitrile/0.02 mol/L phosphate buffer (pH 4.5) (55:45, v/v) as mobile phase. The detection of esmolol derivatives was made at lambda=224 nm with UV detector. The assay was linear from 0.035 to 12 microg/ml for each enantiomer. The analytical method afforded average recoveries of 94.8% and 95.5% for S-(-)- and R-(+)-esmolol, respectively. For each enantiomer, the limit of detection was 0.003 microg/ml and the limit of quantification for the method was 0.035 microg/ml (RSD<14%). The reproducibility of the assay was satisfactory.     

Simultaneous determination of the enantiomers of esmolol and its acid metabolite in human plasma by reversed phase liquid chromatography with solid-phase extraction

A stereoselective RP-high performance liquid chromatography (HPLC) assay to determine simultaneously the enantiomers of esmolol and its acid metabolite in human plasma was developed. The method involved a solid-phase extraction and a reversed-phase chromatographic separation with UV detection (lambda = 224 nm) after chiral derivatization. 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate (GITC) was employed as a pre-column chiral derivatization reagent. The assay was linear from 0.09 to 8.0 microg/ml for each enantiomer of esmolol and 0.07-8.0 microg/ml for each enantiomer of the acid metabolite. The absolute recoveries for all enantiomers were >73%. The intra- and inter-day variations were <15%. The validated method was applied to quantify the enantiomers of esmolol and its metabolite in human plasma for hydrolysis studies.     

Determination of S-nitrosoglutathione in human and rat plasma by high-performance liquid chromatography with fluorescence and ultraviolet absorbance detection after precolumn derivatization with o-phthalaldehyde.

An analytical method is described for the quantification of S-nitrosoglutathione (GSNO), a potent physiological vasodilator and inhibitor of platelet aggregation, in the presence of a high excess of reduced glutathione (GSH). The method is based on the quantitative elimination of GSH by N-ethylmaleimide, the conversion of GSNO by 2-mercaptoethanol to GSH, its reaction with o-phthalaldehyde (OPA) to form a highly fluorescent and UV-absorbing tricyclic isoindole derivative, and subsequent high-performance liquid chromatographic (HPLC) separation with fluorescence and/or UV absorbance detection. The OPA derivatives of GSH and GSNO obtained by this method were found to be identical by mass spectrometry. GSH (up to 50 microM) did not interfere with the analysis of GSNO (up to 1000 nM). The limits of detection of the method for buffered aqueous solutions of GSNO were determined as 3 nM using fluorescence and 70 nM using UV absorbance detection. Isolation of GSNO by HPLC analysis (pH 7.0) of plasma ultrafiltrate samples (200 microl) prior to derivatization allows specific and artifact-free quantification of GSNO in human and rat plasma. Reduced and oxidized glutathione, nitrite, and cysteine did not interfere with the measurement of GSNO in human and rat plasma. The limit of quantitation (LOQ) of the combined method was determined as 100 nM of GSNO in human plasma ultrafiltrate using fluorescence detection. No endogenous GSNO could be detected in ultrafiltrate samples of plasma of 10 healthy humans at concentrations exceeding the LOQ of the method. After iv infusion of GSNO (125 micromol/kg body wt) in a rat for 20 min GSNO and GSH were detected in rat plasma at 60 and 130 microM, respectively. The method should be useful to investigate formation, metabolism, and reactions of GSNO in vitro and in vivo at physiologically relevant concentrations.     

Determination of amlodipine in human plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of amlodipine in human plasma. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl), solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Nortriptyline hydrochloride was used as an internal standard. The assay was linear over the concentration range of 0.25–18.00 ng/ml. Both of the within-day and day-to-day reproducibility and accuracy were less than 11.80% and 12.00%, respectively. The plasma profile following a single administration of 10 mg amlodipine to a healthy volunteer was presented.     

Development and validation of a sensitive assay of valproic acid in human plasma by high-performance liquid chromatography without prior derivatization

Sensitive and selective determination of valproic acid in plasma by high-performance liquid chromatography (HPLC) is usually achieved with pre-column derivatization. In the present work, the derivatization is omitted due to using a simple but highly selective plasma extraction procedure and an optimized chromatographic condition. Valproic acid and the internal standard octanoic acid were extracted from plasma samples with n-hexane under acidic condition followed by back-extraction into diluted triethylamine. Chromatography was performed on a CN column (250 x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-40 mM aqueous sodium dihydrogen phosphate (30:70, v/v), pH 3.5. Detection was made at 210 nm and analyses were run at a flow-rate of 1 ml/min. The method was specific and sensitive with a quantification limit of 1.25 microg/ml and a detection limit of 0.1 microg/ml in plasma. The mean absolute recovery for valproic acid using the present plasma extraction procedure was 75.8%. The intra- and inter-day coefficient of variation and percent error values of the assay method were all in acceptable range. Calibration curves were linear (r>0.999) from 1.25 to 320 microg/ml in plasma.     

Development and validation of a liquid chromatography-tandem mass spectrometry assay for determination of raloxifene and its metabolites in human plasma

This paper describes the development and validation of a method for the detection of raloxifene (Ral) and its two glucuronide metabolites, raloxifene-6-glucuronide (M1) and raloxifene-4'-glucuronide (M2), in human plasma samples. Both glucuronides were synthesized enzymatically, purified and used as authentic standards. The assay involves a simple solid phase extraction (SPE) procedure of 0.5 mL of human plasma and subsequent analysis by LC-MS-MS. The recoveries were higher than 71% and chromatographic separation of all the analytes was accomplished in less than 7 min. Linear ranges (r(2)>0.99) were found from 0.200 to 340 microg/L, from 1.600 to 2720 microg/L and from 0.088 to 60.00 microg/L, for M1, M2 and Ral, respectively. The limits of detection achieved were 8, 11 and 6 ng/L for M1, M2 and Ral, respectively. The method presented was successfully applied to a genetic polymorphism study of 47 plasma samples from women taking Evista (raloxifene hydrochloride).     

Simultaneous determination of 3'-azido-2',3'-dideoxyuridine and novel prodrugs in rat plasma by liquid chromatography

3'-Azido-2',3'-dideoxyuridine (AZDU) is a nucleoside analog structurally similar to zidovudine (AZT) with proven activity against human immunodeficiency virus (HIV). The purpose of this study was to develop and validate a high-performance liquid chromatographic (HPLC) method to quantitatively determine AZDU and its novel prodrugs in rat plasma simultaneously. A reversed-phase gradient elution HPLC method was developed to quantitate AZDU and its prodrugs, N3-pivaloyloxymethyl-3'-azido-2',3'-dideoxyuridine (I), 5'-pivaloyloxymethyl-3'-azido-2',3'-dideoxyuridine (II), 5'-O-valinyl-3'-azido-2',3'-dideoxyuridine hydrochloride (III) and 5'-O-phenylalanyl-3'-azido-2',3'-dideoxyuridine hydrochloride (IV), in rat plasma. AZDU and its prodrugs were analyzed using an octadecyl silane column with a mobile phase consisting of 0.04 microM sodium acetate buffer, pH 5.0, and acetonitrile, running in a segmented gradient manner at a flow rate of 2 ml/min. Acetonitrile was increased from 10 to 50% during the first 8 min by 5% per min, followed by 10% per min until it reached 90% acetonitrile. 3'-Azido-2',3'-dideoxy-5-ethyluridine (CS-85) was used as an internal standard (25 microg/ml). Compounds were detected by UV absorption at 261 nm. Extraction recoveries for all compounds were greater than 80%. Retention times of AZDU, CS-85, prodrugs I, II, III and IV were 3.3, 5.2, 9.1, 8.8, 6.3 and 7.3 min, respectively. Calibration plots were linear over the range of 0.25-100 microg/ml for AZDU and prodrugs II, III, and IV and 0.5-100 microg/ml for prodrug I. The limit of quantitation was 0.25 microg/ml for prodrugs II, III and IV and 0.5 microg/ml for prodrug I. The intra- and inter-day variations were less than 10% and accuracies were greater than 90%. This method is rapid, sensitive and reproducible for the determination of AZDU and prodrugs in rat plasma.     

Phenylisothiocyanate and dansyl chloride precolumn derivatizations for the high-performance liquid chromatography analysis of the antitumoral agent ES-285 in dog plasma

Chromophor and fluorophor addition reactions involving phenylisothiocyanate (PITC) and dansyl chloride (DC) were optimized to adapt two high-performance liquid chromatography (HPLC) procedures designed for the accurate determination of the novel antitumoral agent ES-285 in Beagle dog plasma. ES-285 was reacted with PITC at 60 degrees C for 15 min in the presence of triethylamine. The dansyl derivative was obtained by reaction of ES-285 with dansyl chloride in a basic medium at 80 degrees C for 20 min. Both reactions also worked for ES-299, a compound structurally related to ES-285 which was used as internal standard. The treatment of ES-285 and ES-299 spiked plasma samples with a basic phosphate buffer and MeOH permitted the extraction of the drug from the matrix. The purification of the analytes was carried out by solid-phase extraction followed by precolumn derivatization with PITC and DC. The phenylisothiocyanate adducts were analyzed by isocratic HPLC with UV detection at 254 nm. The ES-285 and ES-299 derivatives were eluted from a C(18) column at approximately 6.9 and approximately 8.1 min, respectively. The eluent ACN-water (95:5, v/v) was delivered to the column at a flow-rate of 1 ml/min and the analysis was completed in 15 min. The dansyl derivatives were analysed by a two-HPLC column system with fluorescence detection and gradient elution. The column temperature was maintained at 40 degrees C. The analysis lasted 55 min with the elution of ES-285 and ES-299 derivatives at approximately 35.2 and approximately 37.9 min, respectively. The PITC- and DC-based procedures were characterized by limits of quantification of 20 and 15 ng/ml, respectively. Both procedures were validated according to the ICH and FDA guidelines. They were selective for ES-285 and provided accurate, precise and reproducible results. ES-299 was shown to be a suitable internal standard. The HPLC procedure involving derivatization with DC was more sensitive and permitted to process plasma sample volumes as low as 100 microl. On the other hand, the PITC-based procedure characterised by a quite similar LOQ permitted a higher throughput but implied the processing of a 500-microl plasma volume.     

Simultaneous enantiomer determination of 20 (R)- and 20 (S)-ginsenoside-Rg2 in rat plasma after intravenous administration using HPLC method

20 (R,S)-Ginsenoside-Rg2, an anti-shock agent, is prescribed as a racemate. To analyze simultaneously the enantiomers of 20 (R)-ginsenoside-Rg2 and 20 (S)-ginsenoside-Rg2 in plasma, a simple and reproducible high-performance liquid chromatographic (HPLC) method has been developed. The enantiomeric separation and determination were successfully achieved using a Diamonsil ODS C18 reversed-phase column (5 microm, 250 mm x 4.6 mm) with an RP18 (5 microm) guard column and a mobile phase of MeOH-aq. 4% H3PO4 (65:35, v/v, pH 5.1) with UV detection at 203 nm. Both enantiomers, 20 (R)-ginsenoside-Rg2 and 20 (S)-ginsenoside-Rg2, were well separated at 14.5 min and 13.6 min, respectively. The linear ranges of the standard curves were 2.0-250 microg/ml. The intra- and inter-day precision (R.S.D.) were 相似文献   

Stereoselective high-performance liquid chromatography determination of propranolol and 4-hydroxypropranolol in human plasma after pre-column derivatization

A stereoselective reversed-phase HPLC assay to quantify S-(−) and R-(+) enantiomers of propranolol and 4-hydroxypropranolol in human plasma was developed. The method involved liquid–liquid extraction for sample clean-up and employed 2,3,4,6-tetra-O-acetyl-β-glucopyranosyl isothiocyanate as a pre-column chiral derivatization reagent. The internal standard used was 4-methylpropranolol. The derivatized products were separated on an Altex C₁₈ column using a mixture of acetonitrile–water–phosphoric acid–triethylamine (58:42:0.1:0.06 and 50:50:0.15:0.06, v/v, for propranolol and 4-hydroxypropranolol, respectively) as mobile phase. The detection of propranolol derivatives was made at λ_ex=280 nm and λ_em=325 nm, and the corresponding 325 and 400 nm were used for 4-hydroxypropranolol derivatives. The assay was linear from 1 to 100 ng/ml and from 2 to 50 ng/ml using 0.5 ml of human plasma for propranolol and 4-hydroxypropranolol enantiomers, respectively. The present assay is used to quantify the enantiomers of propranolol and 4-hydroxypropranolol, respectively, in human plasma for pharmacokinetic studies.     

Validation of a simple liquid chromatography-tandem mass spectrometric method for the determination of propiverine hydrochloride and its N-oxide metabolite in human plasma

A simple high-performance liquid chromatography (HPLC)-tandem mass spectrometric method has been developed for determination of propiverine hydrochloride and its metabolite, propiverine N-oxide (M-1) in human plasma using stable isotopes, propiverine hydrochloride-d10 and M-1-d10, as internal standards. The analytes were extracted with dichloromethane from 0.2 ml of plasma in neutral condition (pH 7.0) and separated by HPLC on a C18 reversed-phase column using methanol-1% acetic acid (50:50) as a mobile phase, and detected using positive electrospray ionization in selected reaction monitoring (SRM) mode. The method was validated over a concentration range of 2-500 ng/ml for propiverine hydrochloride and 4-1000 ng/ml for M-1 using 0.2 ml of human plasma per assay. The method developed was successfully applied to analysis of propiverine hydrochloride and M-1 in clinical studies.     

Bioanalysis and pharmacokinetics of chitosan ester in rabbit serum by HPLC with postcolumn fluorescence derivatization

Interest in antiatherosclerotic activity of chitosan ester (PS916) with a new form of sulfate amino polysaccharide derived from marine chitin has necessitated the development of a sensitive and specific method to study its pharmacokinetics. A sensitive and reproducible high-performance liquid chromatography (HPLC) with postcolumn fluorescence derivatization method was developed and validated for the determination of PS916 in rabbit serum. Chromatography was carried out using a C8 reversed-phase column with an isocratic mobile phase consisting of methanol-water (20:80, v/v) at a flow rate of 0.2 ml/min. The derivatization procedure involved postcolumn reaction with guanidine hydrochloride in an alkaline medium at 110 degrees C. The fluorometric detector was operated at 250 nm (excitation) and 435 nm (emission). The assay was linear over the concentration range of 5-100 microg/ml. The lower limit of detection (LLOD) was found to be 1.0 microg/ml. The proposed method was successfully applied for a pharmacokinetic study of PS916 in rabbits.     

Measurement of the anti-cancer agent gemcitabine in human plasma by high-performance liquid chromatography

A reversed-phase HPLC assay has been developed to determine the concentration of the anti-metabolite 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) in human plasma over the concentration range of 0.5-150 microM (0.13-39.44 microg/ml), and 2',2'-difluorodeoxyuridine (dFdU), the deaminated, inactive metabolite, over the range of 1.0-227 microM (0.26-60 microg/ml). After the addition of 20 nmol 2'-fluorodeoxycytidine (FdC) as an internal standard, 0.5-ml samples of plasma were subjected to acetonitrile precipitation, followed by analysis using a gradient reversed-phase HPLC assay with UV detection. A Phenomenex Columbus C(18) column, 5 microm, 150 x 4.6 mm, and a Waters C(18), 4 microm, Nova-Pak Sentry guard column were used to achieve separation. FdC, dFdC and dFdU were monitored at 282, 269 and 258 nm, respectively, on a Waters 996 photodiode array detector. The mobile phase, run at a total flow-rate of 1.5 ml/min, was composed of two solvents: 50 mM ammonium acetate pH 5.0 in either 2% (solvent A) or 10% methanol (solvent B, v/v); 100% solvent A was run for 17 min, followed by a linear gradient to 100% solvent B over 14 min. FdC, dFdC and dFdU were resolved from endogenous compounds and had retention times of 13.6+/-0.5, 18.1+/-1.1 and 29.0+/-0.6 min, respectively. The assay was useful in measuring the plasma levels of both analytes in samples obtained from adult cancer patients participating in a Phase I trial of gemcitabine given as either a 1- or 2-h infusion weekly for 3 of 4 weeks.     

On-line coupling of sequential injection with liquid chromatography for the automated derivatization and determination of gamma-aminobutyric acid in human biological fluids

The principle of sequential injection analysis (SIA) was exploited to develop a rapid fully automated and efficient pre-column derivatization procedure coupled on-line to liquid chromatography (HPLC). Using the SIA-HPLC derivatization protocol gamma-aminobutyric acid (GABA) was determined fluorimetrically in human biological fluids with o-phthaldialdehyde (OPA) as derivatization reagent and minimum sample pretreatment. A lab-built SIA system was used to handle samples, standard solutions and OPA reagent. Appropriate volumes of the reagents were introduced in the holding coil of the SIA system and were mixed on propulsion to the HPLC loop through a suitable reaction coil. The chemical (pH, c(OPA), c(mercaptoethanol)) and instrumental variables (volumes of sample and reagent, reaction time) of the reaction were studied and optimized in terms of maximum sensitivity. The chromatographic variables (gradient composition of the eluent and flow rate) were studied for optimum selectivity and peak characteristics. The developed experimental configuration facilitated fully-automated operation thus minimizing errors in handling. Additionally the method as a whole provided very satisfactory sensitivity, precision and accuracy. Direct determination of GABA in human urine and cerebrospinal fluid (CSF) at microg L(-1) (ppb) levels was accomplished, with minimum sample pretreatment.     

High-performance liquid chromatography method with ultraviolet detection for the quantification of the BCR-ABL inhibitor nilotinib (AMN107) in plasma, urine, culture medium and cell preparations

An isocratic and sensitive HPLC assay was developed allowing the determination of the new anticancer drug nilotinib (AMN107) in human plasma, urine, culture medium and cell samples. After protein precipitation with perchloric acid, AMN107 underwent an online enrichment using a Zirchrom-PBD precolumn, was separated on a Macherey-Nagel C18-HD column and finally quantified by UV-detection at 258 nm. The total run time is 25 min. The assay demonstrates linearity within a concentration range of 0.005-5.0 microg/ml in plasma (r(2)=0.9998) and 0.1-10.0 microg/ml in urine (r(2)=0.9913). The intra-day precision expressed as coefficients of variation ranged depending on the spiked concentration between 1.27-9.23% in plasma and 1.77-3.29% in urine, respectively. The coefficients of variation of inter-day precision was lower than 10%. Limit of detection was 0.002 microg/ml in plasma and 0.01 microg/ml in urine. The described method is stable, simple, economic and is routinely used for in vivo and in vitro pharmacokinetic studies of AMN107.     

A stability-indicating high performance liquid chromatographic (HPLC) assay for the simultaneous determination of atorvastatin and amlodipine in commercial tablets

A simple, rapid, precise and accurate isocratic reversed-phase stability-indicating HPLC method was developed and validated for the simultaneous determination of atorvastatin (AT) and amlodipine (AM) in commercial tablets. The method has shown adequate separation for AM, AT from their associated main impurities and their degradation products. Separation was achieved on a Perfectsil Target ODS-3, 5 microm, 250 mm x 4.6 mm i.d. column using a mobile phase consisting of acetonitrile-0.025 M NaH(2)PO(4) buffer (pH 4.5) (55:45, v/v) at a flow rate of 1 ml/min and UV detection at 237 nm. The drugs were subjected to oxidation, hydrolysis, photolysis and heat to apply stress conditions. The linearity of the proposed method was investigated in the range of 2-30 microg/ml (r=0.9994) for AT and 1-20 microg/ml (r=0.9993) for AM. The limits of detection were 0.65 microg/ml and 0.35 microg/ml for AT and AM, respectively. The limits of quantitation were 2 microg/ml and 1 microg/ml for AT and AM, respectively. Degradation products produced as a result of stress studies did not interfere with the detection of AT and AM and the assay can thus be considered stability-indicating.     

Liquid chromatographic method for simultaneous determination of mycophenolic acid and its phenol- and acylglucuronide metabolites in plasma

A simple, sensitive and reproducible HPLC method is presented for the simultaneous determination of mycophenolic acid (MPA) and its metabolites phenolic MPA-glucuronide (MPAG) and acyl glucuronide (AcMPAG) in human plasma. Sample purification requires protein precipitation with 0.1 M phosphoric acid/acetonitrile in the presence of Epilan D as an internal standard (IS). Separation was performed by reversed-phase HPLC, using a Zorbax SB-C18 column, 32% acetonitrile and a 40 mM phosphoric acid buffer at pH 3.0 as mobile phase; column temperature was 50 degrees C, flow rate 1.4 ml/min, and measurement by UV detection was at 215 nm (run time 12 min). The method requires only 50 microl plasma. Detection limits were 0.1 microg/ml for MPA and AcMPAG, and 2.0 microg/ml for MPAG, respectively. Mean absolute recovery of all three analytes was >95%. This analytical method for the determination of MPA and its metabolites is a reliable and convenient procedure that meets the criteria for application in routine clinical drug monitoring and pharmacokinetic studies.