Simultaneous determination of timolol maleate, rosuvastatin calcium and diclofenac sodium in pharmaceuticals and physiological fluids using HPLC-UV

A novel HPLC-UV method was developed for the simultaneous determination of timolol (TM), rosuvastatin (RST), and diclofenac sodium (DS) in pharmaceuticals, human plasma and aqueous humor using naproxen sodium as internal standard (IS). The target compounds were analyzed on Hypersil BDS C(18) column (250 mm × 4.6 mm, 5 μm), applying 0.2% triethylamine (TEA) and acetonitrile (ACN) (40:60, v/v), in isocratic mode as mobile phase, pH 2.75 adjusted with 85% phosphoric acid at a flow rate of 1 ml/min. The column oven temperature was kept at 45°C and the peak response was monitored at 284 nm after injecting a 50 μl sample into HPLC system. The direct liquid-liquid extraction procedure was applied to human plasma and bovine aqueous humor samples using mobile phase as an extraction solvent after deproteination with methanol. The different HPLC experimental parameters were optimized and the method was validated according to standard guidelines. The recoveries of the suggested method in human plasma were 98.72, 96.04, and 95.14%, for TM, RST, and DS, while in aqueous humor were 94.99, and 98.23%, for TM, and DS, respectively. The LOD values were found to be 0.800, 0.500, and 0.250 ng/ml, for TM, RST, and DS, respectively, while their respective LOQ values were 2.00, 1.50, and 1.00 ng/ml. The co-efficient of variation (CV) were in the range of 0.1492-1.1729% and 1.0516-4.0104%, for intra-day and inter-day studies, respectively. The method was found accurate in human plasma and bovine aqueous humor and will be applied for the quantification of these compounds in plasma, and aqueous humor samples using animal models and in pharmaceuticals.     

Improved HPLC method for the simultaneous determination of tramadol and O-desmethyltramadol in human plasma

This paper describes an HPLC method for the determination of tramadol and its major active metabolite, O-desmethyltramadol (ODT), in human plasma. Sample preparation involved liquid-liquid extraction with diethyl ether-dichloromethane-butanol (5:3:2, v/v/v) and back extraction with sulphuric acid. Tramadol, ODT and the internal standard, sotalol, were separated by reversed phase HPLC using 35% acetonitrile and an aqueous solution containing 20 mM sodium phosphate buffer, 30 mM sodium dodecyl sulphate and 15 mM tetraethylammonium bromide pH 3.9. Detection was by fluorescence with excitation and emission wavelengths of 275 and 300 nm, respectively. The method was linear for tramadol (3-768 ng/ml) and ODT (1.5-384 ng/ml) with mean recoveries of 87.2% and 89.8%, respectively. Intra- and inter-day precisions were 10.34% and 8.43% for tramadol and 9.43% and 8.75% for ODT at the respective limits of quantitation (3 and 1.5 ng/ml). Accuracy for tramadol ranged from 96.2% to 105.3%. The method was applied to a pharmacokinetic study of tramadol in human volunteers.     

Determination of quercetin in human plasma using reversed-phase high-performance liquid chromatography

A method is reported for the measurement of quercetin in human plasma using reversed-phase high-performance liquid chromatography (HPLC). Quercetin and kaempferol (as internal standard) were spiked into plasma samples and extracted using C₁₈ Sep-Pak Light cartridges (efficiency > 85%). Flavonoids were eluted with aqueous acetone (50% v/v, pH 3.5), dried down and redissolved in aqueous acetone (45% v/v, pH 3.5). The increased osmolarity promoted a phase separation and the water-saturated acetone layer, containing the flavonoids, was analysed by HPLC with aqueous acetone mobile phase (45% v/v acetone in 250 mM sodium dihydrogen sulphate. The mixture was adjusted to pH 3.5 with phosphoric acid and used at a flow-rate of 1.0 ml/min) and μBondapak C₁₈ column (150 × 3.9 mm I.D., 10 μm particle size). The detection limit (A_{375 nm}) for quercetin in plasma was 0.1 μg/ml (300 nM). The method also detects metabolites of quercetin, although these are not yet identified.     

Determination of acyclovir in human serum by high-performance liquid chromatography using liquid-liquid extraction and its application in pharmacokinetic studies

A fast, simple and sensitive high performance liquid chromatographic (HPLC) method has been described for determination of acyclovir in human serum. Since acyclovir is a polar compound and soluble in aqueous medium and practically insoluble in most of organic solvents, its analysis in biological fluids in currently published HPLC methods, involve pre-treatment of acyclovir plasma sample including deproteinization or solid phase extraction. In present method liquid-liquid extraction of acyclovir and internal standard (vanillin) is achieved using dichloromethane-isopropyl alcohol (1:1, v/v) as an extracting solvent. Analysis was carried out on ODS column using methanol-phosphate buffer (0.05 M) containing sodium dodecyl sulfate (200 mg/L) and triethylamine (2 mL/L, v/v) as mobile phase (pH=2.3; 5:95, v/v) at flow rate of 2 ml/min. The method was shown to be selective and linear into the concentration range of 10-2560 ng/mL. Accuracy and precision of the method were also studied. The limit of quantitation was evaluated to be 10 ng/mL. This method was applied in bioequivalence study of two different acyclovir preparations after administration of 400mg in 12 healthy volunteers.     

High performance liquid chromatographic methods for the determination of aripiprazole with ultraviolet detection in rat plasma and brain: application to the pharmacokinetic study

High performance liquid chromatographic (HPLC) methods were validated for the determination of aripiprazole (OPC-14597, Abilify) in rat plasma and brain. Separation was by Nova-pak phenyl column; flow rate, 1.0 ml/min; mobile phase, acetonitrile-methanol-20 mM sodium sulfate-acetic acid (27:25:48:1, v/v/v/v); UV detection at 254 nm. Reproducibility in plasma and brain showed excellent precision (within 7.8 and 10.6%) and accuracy (96.0-102.4% and 99.0-108.7%) with calibration curve ranges 10.0-2000 ng/ml and 30.0-6000 ng/g, respectively. Validated HPLC methods were successfully applied to pharmacokinetic study of aripiprazole in rats, demonstrating brain concentrations after oral administration five times higher than plasma concentrations.     

High-performance liquid chromatographic determination of quercetin in human plasma and urine utilizing solid-phase extraction and ultraviolet detection

An HPLC method for determining quercetin in human plasma and urine is presented for application to the pharmacokinetic study of rutin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using kaempferol as an internal standard. Solid-phase extraction was performed on an Oasis HLB cartridge (>95% recovery). The HPLC assay was carried out using a Luna ODS-2 column (150 x 2.1 mm I.D., 5 microm particle size). The mobile phase was acetonitrile-10 mM ammonium acetate solution containing 0.3 mM EDTA-glacial acetic acid, 29:70:1 (v/v, pH 3.9) and 26:73:1 (v/v, pH 3.9) for the determination of plasma and urinary quercetin, respectively. The flow-rate was 0.3 ml/min and the detection wavelength was set at 370 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a concentration range of 4-700 ng/ml of quercetin in plasma and 20-1000 ng/ml of quercetin in urine. The lower limit of quantification was approximately 7 ng/ml of quercetin in plasma and approximately 35 ng/ml in urine. The detection limit (defined at a signal-to-noise ratio of about 3) was approximately 0.35 ng/ml in plasma and urine. A preliminary experiment to investigate the plasma concentration and urinary excretion of quercetin after oral administration of 200 mg of rutin to a healthy volunteer demonstrated that the present method was suitable for determining quercetin in human plasma and urine.     

Validated liquid chromatographic-ultraviolet method for the quantitation of tadalafil in human plasma using liquid-liquid extraction

A highly selective, sensitive and rapid HPLC method has been developed and validated to quantify tadalafil in human plasma. The tadalafil and internal standard (loratadine, I.S.) were extracted by liquid-liquid extraction technique followed by an aqueous back-extraction allowing injection of an aqueous solvent in the HPLC system. The chromatographic separation was performed on a reverse phase BDS Hypersil C-18 column (250 mm x 4.6 mm, 5 microm, Thermo Separation Co., USA) with a mobile phase of acetonitrile and aqueous solution containing 0.012 M triethylamine+0.020 M orthophosphoric acid (50/50, v/v). The analytes were detected at 225 nm. The assay exhibited a linear range of 5-600 ng/mL for tadalafil in human plasma. The lower limit of quantitation (LLOQ) was 5 ng/mL. The within- and between batch precision (expressed as coefficient of variation, C.V.) did not exceed 10.3% and the accuracy was within -7.6% deviation of the nominal concentration. The recovery of tadalafil from plasma was greater than 66.1%. Stability of tadalafil in plasma was excellent with no evidence of degradation during sample processing (auto-sampler) and 30 days storage in a freezer. This validated method is applied for the clinical study of the tadalafil in human volunteers.     

Quantitation of tamsulosin in human plasma by liquid chromatography-electrospray ionization mass spectrometry

A highly sensitive method for quantitation of tamsulosin in human plasma using 1-(2,6-dimethyl-3-hydroxylphenoxy)-2-(3,4-methoxyphenylethylamino)-propane hydrochloride as the internal standard (I.S.) was established using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). After alkalization with saturated sodium bicarbonate, plasma were extracted by ethyl acetate and separated by HPLC on a C18 reversed-phase column using a mobile phase of methanol-water-acetic acid-triethylamine (620:380:1.5:1.5, v/v). Analytes were quantitated using positive electrospray ionization in a quadrupole spectrometer. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 228 for tamsulosin and m/z 222 for the I.S. Calibration curves, which were linear over the range 0.2-30 ng/ml, were analyzed contemporaneously with each batch of samples, along with low (0.5 ng/ml), medium (3 ng/ml) and high (30 ng/ml) quality control samples. The intra- and inter-assay variability ranged from 2.14 to 8.87% for the low, medium and high quality control samples. The extraction recovery of tamsulosin from plasma was in the range of 84.2-94.5%. The method has been used successfully to study tamsulosin pharmacokinetics in adult humans.     

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.     

顺式藁苯内酯口服给药在大鼠血浆和脑内的药动学特性(英文)

建立大鼠血浆和脑中Z-槀苯内酯(LIG)浓度测定的高效液相色谱法。采用Agilent Hypersil ODS C18色谱柱(150mm×4.6mm,5μm),流动相为甲醇-5%异丙醇水溶液(60:40,v/v),流速为1.0mL/min,检测波长为280nm。血浆与脑中槀苯内酯浓度线性检测范围分别为93.75~3750ng/m(r=0.9999)和93.75~3750ng/g(r=0.9997),日内及日间精密度RSD10%。本法适用于大鼠口服LIG后血浆及脑中药物浓度的研究。     

Sensitive HPLC-APCI-MS method for the determination of cyclovirobuxine D in human plasma

A sensitive high performance liquid chromatography-atmospheric pressure chemical ionisation-mass spectrometry (HPLC-APCI-MS) assay for determination of cyclovirobuxine D (CVB-D) in human plasma using mirtazapine as internal standard (I.S.) was established. After adjustment to a basic pH with sodium hydroxide, plasma was extracted by ethyl acetate and separated by high performance liquid chromatography (HPLC) on a reversed-phase C(18) column with a mobile phase of 30 mM ammonium acetate buffer solution containing 1% formic acid-methanol (48:52, v/v). CVB-D was determined with atmospheric pressure chemical ionisation-mass spectrometry (APCI-MS). HPLC-APCI-MS was performed in the selected-ion monitoring (SIM) mode using target ions at [M+H](+)m/z 403.4 for CVB-D and [M+H](+)m/z 266.2 for I.S. Calibration curves were linear over the range 10.11-4044 pg/ml. The lower limit of quantification was 10.11 pg/ml. The intra- and inter-run variability values were less than 9.5 and 12.4%, respectively. The mean plasma extraction recovery of CVB-D was in the range of 85.3-92.8%. The method was successfully applied to determine the plasma concentrations of CVB-D in Chinese volunteers.     

HPLC determination of pirenzepine dihydrochloride in rabbit aqueous humor

Pirenzepine was considered as a pharmacologic agent of preventing form-deprivation myopia. To assess the ocular bioavailability of pirenzepine, a HPLC method for determination of pirenzepine in rabbit aqueous humor was developed. An HPLC system was used in the reverse phase mode for the determination of pirenzepine. A Luna RP18 5 microm 4.6 mm x 150 mm column was employed at 35 degrees C. The mobile phase was methanol/0.02 M KH2PO4/sodium 1-pentanesulfonate (350/650/1, v/v/m, pH was adjusted to 8.0 by dropping 1M NaOH). The flow rate was 1 ml/min. Pirenzepine was monitored at 280 nm. Sample treatment procedure consists of deproteinisation with methanol. Calibration curves fitted by plotting the peak area versus concentration were linear in the range 20-400 ng/ml. The limit of quantification (LOQ) of present method was 20 ng/ml. Within-day and inter-day coefficient of variation was lower than 10%. Analytical recoveries were determined as 92.4, 95.4 and 101.4% at concentrations of 40, 200 and 400 ng/ml. In conclusion, this HPLC method using a simple sample treatment procedure appears suitable for monitoring ocular concentration of pirenzepine.     

LC-MS/MS determination in rabbit plasma of the main photoproduct of RLP068/Cl, a cationic sensitizer proposed for photodynamic therapy (PDT) of microbial infections

The clinical development of a sensitizer for photodynamic therapy (PDT) requires the structural identification of the photoproducts and their quantification in biological fluids and tissues. We describe the LC-MS identification of the most important photoproducts of a cationic phthalocyanine sensitizer (RLP068/Cl) and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of the main photoproduct (the cationic phthalimide derivative 3-[(1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl)oxy]-N,N,N-trimethylbenzenaminium chloride) in rabbit plasma. The tri-deuterated product was used as co-eluting internal standard. The cationic photoproduct was isolated from plasma samples by protein precipitation with perchloric acid in methanol (7%, v/v). HPLC step was performed on a Phenomenex Synergi Hydro-RP column (20 mm x 2.0 mm, 2 microm particles) with a mobile phase of 0.5% (v/v) aqueous TFA/methanol (85:15, v/v). Flow rate was 0.2 mL/min and 40 microL injection were performed. Run time was 10 min. Detection was achieved by means of a Bruker Esquire 3000+ ion trap mass spectrometer equipped with an ESI source working in positive mode. A multiple reaction monitoring method following the transitions 297.1 --> 282.1 for the analyte and 300.1 --> 282.1+285.1 for the internal standard was used. The analytical method was validated over the concentration range 0.46-91.2 ng/mL and lower limits of detection (LLOD) and quantification (LLOQ) respectively of 0.2 and 0.5 ng/mL were found.     

Simple and sensitive method for determination of metronidazole in human serum by high-performance liquid chromatography

A simple and sensitive HPLC method for determination of metronidazole in human plasma has been developed. A step of freezing the protein precipitate allowed an efficient separation of aqueous and organic phases minimizing the noise level and improved therefore the limit of quantitation (10 ng ml⁻¹ using 1 ml of plasma sample). The separation of compounds was performed on a RP 18 column with acetonitrile–aqueous 0.01 M phosphate solution (15:85, v/v) as mobile phase. Detection was performed by UV absorbance at 318 nm. Metronidazole was well resolved from the plasma constituents and internal standard. An excellent linearity was observed between peak-height ratios plasma concentrations over a concentration range of 0.01 to 10 μg ml⁻¹. Within-day and between-day precision (expressed by relative standard deviation) and accuracy (mean error in per cent) did not exceed 4% between 1 and 10 μg ml⁻¹ and 8.3 and 7.2% respectively for the limit of quantitation. The method is suitable for bioavailability and pharmacokinetic studies in humans.     

Simultaneous quantification of voriconazole and posaconazole in human plasma by high-performance liquid chromatography with ultra-violet detection

A sensitive and selective high-performance liquid chromatographic (HPLC) method with ultra-violet detection has been developed and validated for the simultaneous determination of posaconazole and voriconazole, two systemic anti-fungal agents. An internal standard diazepam was added to 100 microL of human plasma followed by 3 mL of hexane-methylene chloride (70:30, v/v). The organic layer was evaporated to dryness and the residue was reconstituted with 100 microL of mobile phase before being injected in the chromatographic system. The compounds were separated on a C8 column using sodium potassium phosphate buffer (0.04 M, pH 6.0): acetonitrile:ultrapure water (45:52.5:2.5, v/v/v) as mobile phase. All compounds were detected at a wavelength of 255 nm. The assay was linear and validated over the range 0.2-10.0 mg/L for voriconazole and 0.05-10.0 mg/L for posaconazole. The biases were comprised between -3 and 5% for voriconazole and -2 and 8% for posaconazole. The intra- and inter-day precisions of the method were lower than 8% for the routine quality control (QC). The mean recovery was 98% for voriconazole and 108% for posaconazole. This method provides a useful tool for therapeutic drug monitoring.     

Determination of nalmefene in plasma by high-performance liquid chromatography with electrochemical detection and its application in pharmacokinetic studies

A method for measuring human plasma levels of nalmefene after oral and intravenous administration is presented. The method consists of a solid-phase extraction procedure followed by HPLC analysis. A cyanopropyl column is used for the solid-phase extraction and 60% (v/v) acetonitrile in dilute sodium pentanesulfonic acid solution for elution. The concentrated and filtered eluate is injected into the HPLC system, which is equipped with an electrochemical dual-electrode detector. A phenyl column is used in this HPLC system with a mobile phase containing 30% (v/v) acetonitrile in dilute sodium pentanesulfonic acid solution. A signal-to-noise ratio of 4.5 is obtained when a 1 ng/ml spiked plasma sample is analyzed. To determine the applicability of this method for human pharmacokinetic studies, nalmefene levels in plasma were measured at time points up to 24 h following oral and intravenous administration of 30 mg of nalmefene hydrochloride to two subjects. These studies demonstrated that the proposed method is sufficiently sensitive to study the pharmacokinetic profile of nalmefene in man.     

Stereoselective determination of R-(+)- and S-(−)-remoxipride, a dopamine D2-receptor antagonist, in human plasma by chiral high-performance liquid chromatography

A stereoselective high-performance liquid chromatographic (HPLC) method is described for the selective and sensitive quantitation in human plasma of R-(+)- and S-(−)-enantiomers of remoxipride. Remoxipride was extracted from basified plasma into hexane-methyl-tert.-butyl ether (20:80, v/v), washed with sodium hydroxide (1.0 M), then back-extracted into phosphoric acid (0.1 M). A structural analog of remoxipride was used as an internal standard. The sample extracts were chromatographed using a silica-based derivatized cellulose chiral column, Chiralcel OD-R, and a reversed-phase eluent containing 30–32% acetonitrile in 0.1 M potassium hexafluorophosphate. Ultraviolet (UV) absorbance detection was performed at 214 nm. Using 0.5-ml plasma aliquots, the method was validated in the concentration range 0.02-2.0 μg/ml and was applied in the investigation of systemic inversion of remoxipride enantiomers in man.     

Determination of acyclovir in maternal plasma,amniotic fluid,fetal and placental tissues by high-performance liquid chromatography

Acyclovir [9-[(2-hydroxyethoxy)-methyl]-guanosine, Zovirax, ACV] is a synthetic purine nucleoside analog active against herpes simplex virus types 1 (HSV-1), 2 (HSV-2), and varicella zoster virus. Acyclovir has frequently been used in HSV-2 seropositive mothers to prevent prenatal transmission of herpes virus to their unborn children. A fast and reproducible HPLC method for the determination of the highly polar acyclvoir in maternal rat plasma, amniotic fluid, placental tissue, and fetal tissue has been developed and validated. Plasma and amniotic fluid samples were prepared by protein precipitation using 2 M perchloric acid and syringe filtering. Tissue samples were homogenized in distilled water, centrifuged, and extracted using a C(18) solid-phase extraction method prior to analysis. Baseline resolution was achieved for acyclovir and the internal standard gancyclovir, an anti-viral of similar structure to acyclovir, using an Agilent Eclipse XDB C(8) column (150 x 2.1 mm, 5 microm). The mobile phase used for the plasma and amniotic fluid was 10 mM acetate/citrate buffer-3.7 mM aqueous octanesulfonic acid (87.5:12.5, v/v) at a flow-rate of 0.2 ml/min. The mobile phase used for the tissue samples was 30 mM acetate/citrate buffer with 5 mM octanesulfonic acid-acetonitrile (99:1, v/v). Both aqueous mobile phase portions were pH adjusted to 3.08. All separations were done using an Agilent 1100 Series HPLC system with UV detection of 254 nm. The assay was validated for each matrix over a range of 0.25-100 microg/ml over 3 days using five replicates of three spiked concentrations. The relative standard deviation and percent error for each validation data set was <15% for middle and high quality control (QC) points and <20% for all low QC points. All calibration curves showed good linearity with an R(2)>0.99. The extraction efficiency for recovery of acyclovir from all matrices was >80%.     

Sensitive determination of clarithromycin in human plasma by high-performance liquid chromatography with spectrophotometric detection

A rapid, selective and sensitive high-performance liquid chromatographic method with spectrophotometric detection was developed for the determination of clarithromycin in human plasma. Liquid-liquid extraction of clarithromycin and norverapamil (as internal standard) from plasma samples was performed with n-hexane/1-butanol (98:2, v/v) in alkaline condition followed by back-extraction into diluted acetic acid. Chromatography was carried out using a CN column (250 mm x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-50 mM aqueous sodium dihydrogen phosphate (32:68, v/v), pH 4.5. Detection was made at 205 nm and analyses were run at a flow-rate of 1.0 ml/min at 40 degrees C. The analysis time was less than 11 min. The method was specific and sensitive with a quantification limit of 31.25 ng/ml and a detection limit of 10 ng/ml in plasma. The mean absolute recovery of clarithromycin from plasma was 95.9%, while the intra- and inter-day coefficient of variation and percent error values of the assay method were all less than 9.5%. Linearity was assessed in the range of 31.25-2000 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method was used to analyze several hundred human plasma samples for bioavailability studies.