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.     

Determination and validation of a simple high-performance liquid chromatographic method for simultaneous assay of iprodione and vinclozolin in human urine

A method based on solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) was developed for the simultaneous determination of 3-(3,5-diclorophenyl)-5-ethenyl-5-methyl-2,4-oxazolidinedione (vinclozolin) and 3-(3,5-diclorophenyl)-N-(1-methylethyl)-2,4-dioxo-1-imidazolidinecarboxamide (iprodione) in human urine. Urine samples containing vinclozolin and iprodione were collected by solid phase extraction using C(18) cartridges. The chromatographic separation was achieved on a Spherisorb ODS2 (250 mm x 4.6 mm, 5 microm) column with an isocratic mobile phase of acetonitrile-water (60:40, v/v). Detection was UV absorbance at 220 nm. The calibration graphs were linear from 30 to 1000 ng/mL for the two fungicides. Intra- and inter-day R.S.D. did not exceed 2.9%. The quantitation limit was 50 ng/mL for vinclozolin and 30 ng/mL for iprodione, respectively.     

Determination of linezolid in growth media by high-performance liquid chromatography with on-line extraction

An isocratic high-performance liquid chromatography (HPLC) method with on-line extraction has been developed to determine linezolid in Mueller-Hinton broth. The loading mobile phase consisting of water-acetonitrile 99:1 (v/v) allowed retention of the analyte on a LiChrocart 4-4 pre-column filled with a LiChrospher 100 RP-8, 5 microm. The transfer of the analyte by a backflush mode to a 150 mm x 4.6 mm I.D. Kromasil C8 5 microm column was performed using a mobile phase of water-acetonitrile 80:20 (v/v). UV detection at 254 nm allowed a quantification limit of 0.39 microg/mL with a 50-microL sample size. The method was successfully applied to in vitro pharmacokinetic-pharmacodynamic studies.     

Determination of echinacoside in rat serum by reversed-phase high-performance liquid chromatography with ultraviolet detection and its application to pharmacokinetics and bioavailability

A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of echinacoside (ECH) in rat serum. After protein precipitation of serum sample with trichloroacetic acid, the supernatant was directly injected and analyzed on a C(18) CapcellACR analytical column (150 mm x 4.6mm I.D. 5 microm) with a mobile phase consisting of acetonitrile-0.5% acetic acid (15.5:84.5, v/v). The UV detector was set at 330 nm. The lower limit of detection and quantification were 9 and 29.2 ng/mL, respectively, and the calibration curves were linear over the concentration range of 29.2-18250 ng/mL. The assay method was successfully applied to the study of the pharmacokinetics and bioavailability of ECH in rat.     

Determination of tianeptine in human plasma using high-performance liquid chromatography with fluorescence detection

A new, selective and sensitive high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of tianeptine (TIA) in human plasma using solid phase extraction (SPE) procedures. The method is based on the derivatization of TIA with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer of pH 8.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Phenomenex C(18) column (250 mm x 4.6 mm) using a mobile phase of acetonitrile-10mM orthophosphoric acid (pH 2.5) (77:23, v/v) solvent system at 1 mL/min flow rate. Gabapentin (GA) was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 520 nm (emission). The assay was linear over the concentration range of 5-300 ng/mL. The detection limit (LOD) was found to be 2 ng/mL. The mean recovery was determined to be 88.6%. The proposed method was applied for pharmacokinetic study of 12.5mg TIA in a healthy volunteer.     

Chiral assay of omeprazole and metabolites and its application to a pharmacokinetics related to CYP2C19 genotypes

Studies investigating the relationship between CYP2C19 genotype and the stereoselective metabolism of omeprazole have not been reported. In the present study, we developed a simple and sensitive analytical method based on column switching reversed phase high-performance liquid chromatography (HPLC) with UV detection to determine the concentrations of (R)- and (S)-omeprazole and of its principal metabolites, (R)- and (S)-5-hydroxyomeprazole, and the non-chiral, omeprazole sulfone, in human plasma. Sample preparation involved liquid-liquid extraction with diethyl ether:dichloromethane (60:40, v/v) followed by clean-up on a TSK BSA-ODS/S column (5 μm, 10 mm × 4.6mm i.d.) using phosphate buffer:acetonitrile (97:3, v/v, pH 6.4). After column switching, separation was performed on a Shiseido CD-ph chiral column (5 μm, 150 mm × 4.6mm i.d.) using phosphate buffer:methanol (45:55, v/v, pH 5.0) as mobile phase. The limit of quantitation (LOQ) was 5 ng/mL for all analytes with intra- and inter-day precisions (as coefficient of variation) of <9.5% and <9.6%, respectively for all analytes. The present method was successfully applied to a chiral pharmacokinetic study of omeprazole in human volunteers with different CYP2C19 genotypes. The results show that the formation of (R)-5-hydroxyomeprazole gives the best correlation with CYP2C19 genotype.     

An HPLC method for determination of inosine and hypoxanthine in human plasma from healthy volunteers and patients presenting with potential acute cardiac ischemia

A simple and sensitive high-performance liquid chromatography (HPLC) method utilizing ultraviolet (UV) detection was developed for the determination of inosine and hypoxanthine in human plasma. For component separation, a monolithic C(18) column at a flow rate of 1.0 mL/min with an aqueous mobile phase of trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and methanol gradient was used. The method employed a one-step sample preparation utilizing centrifugal filtration with high component recoveries (approximately 98%) from plasma, which eliminated the need of an internal standard. The method demonstrated excellent linearity (0.25-5 microg/mL, R>0.9990) for both inosine and hypoxanthine with detection limits of 100 ng/mL. This simple and cost effective method was utilized to evaluate potential endogenous plasma biomarker(s), which may aid hospital emergency personnel in the early detection of acute cardiac ischemia in patients presenting with non-traumatic chest pain.     

Liquid chromatography method for determination of mefenamic acid in human serum

A simple, rapid and specific method for analysis of mefenamic acid (I) in serum by a sensitive high-performance liquid chromatography is described. Only 70 microl of serum and a little sample work-up is required. A simple procedure of extraction by dichloromethane followed by evaporation to dryness under gentle stream of nitrogen and dissolving the dried residue in mobile phase was used. The mefenamic acid peak was separated from endogenous peaks on a C(8) column by a mobile phase of acetonitrile-water (50:50, v/v, pH 3). Mefenamic acid and internal standard (IS) (diclofenac) were eluted at 7.4 and 5.4 min, respectively. The limit of quantitation of mefenamic acid in serum was 25 ng/ml at 280 nm. The method was linear over the range of 25-2000 ng/ml with r(2) of 0.998. Mean recovery for mefenamic acid was 110%.     

High throughput LC-MS/MS method for simultaneous quantification of lamivudine, stavudine and nevirapine in human plasma

A selective and high throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and validated to separate, detect and simultaneously quantify lamivudine (3TC), stavudine (d4T) and nevirapine (NVP) in human plasma using metaxalone as internal standard (IS). After solid phase extraction (SPE), the analytes and the IS were chromatographed on a Symmetry C18 (150 mmx3.9 mm i.d., 5 microm particle size) column using 5 microL injection volume with a run time of 4.5 min. An isocratic mobile phase consisting of 0.5% glacial acetic acid in water:acetonitrile (20:80, v/v) was used to separate all these drugs. The precursor and product ions of these drugs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring mode (MRM) without polarity switch. The method was validated over the range of 25-3000 ng/mL for 3TC, 20-2000 ng/mL for d4T and 50-5000 ng/mL for NVP. The absolute recoveries for analytes (>or=86%) and IS (98.12%) achieved from spiked plasma samples were consistent and reproducible. Inter-batch and intra-batch precision (%CV) across four validation runs (LLOQ, LQC, MQC and HQC) was less than 10. The accuracy determined at these levels was within +/-8% in terms of relative error. The method was successfully applied to a pivotal bioequivalence study of [60 (3TC)+12 (d4T)+100 (NVP)] mg dispersible tablets in 60 healthy human subjects under fasting condition.     

Determination of ritodrine in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and sensitive HPLC/MS/MS method was developed and evaluated to determine the concentration of ritodrine (RTD) in human plasma. Liquid-liquid extraction with ethyl acetate was employed as the sample preparation method. The structural analogue salbutamol was selected as the internal standard (IS). The liquid chromatography was performed on a Hanbon Sci. & Tech. Lichrospher CN (150 mm x 4.6 mm, i.d., 5 microm) column (Hanbon, China) at 20 degrees C. A mixture of 0.03% acetic acid and methanol (50:50, v/v) was used as isocratic mobile phase to give the retention time 3.60 min for ritodrine and 2.94 min for salbutamol. Selected reaction monitoring (SRM) in positive ionization mode was employed for mass detection. The calibration functions were linear over the concentration range 0.39-100 ng mL(-1). The intra- and inter-day precision of the method were less than 15%. The lower limit of quantification was 0.39 ng mL(-1). The method had been found to be suitable for application to a pharmacokinetic study after oral administration of 20mg ritodrine hydrochloride tablet to 18 healthy female volunteers. The half-life is 2.54+/-0.67 h.     

A simple and sensitive HPLC method for determination of gliclazide in human serum

A simple, rapid and specific method for analysis of gliclazide in serum by a sensitive high-performance liquid chromatographic method is described. Only 100 microl of serum and a little sample work-up is required. A simple procedure of extraction by toluene followed by evaporation to dryness under a gentle stream of air and dissolving the dried residue in mobile was used. The gliclazide peak was separated from endogenous peaks on a C(8) column by a mobile phase of acetonitrile-water (45:55, v/v), pH 3. Gliclazide and internal standard (phenytoin) were eluted at 6.8 and 3.8 min, respectively. The limit of quantitation (LOQ) for gliclazide in serum was 75 ng/ml at 230 nm. The method was linear over the range of 75-10,000 ng/ml with r(2) of 0.999. Mean recovery for gliclazide and internal standard was 84.5 and 87%, respectively.     

Quantitative determination of erythromycylamine in human plasma by liquid chromatography-mass spectrometry and its application in a bioequivalence study of dirithromycin

A sensitive, rapid liquid chromatographic-electrospray ionization mass spectrometric method for determination of erythromycylamine in human plasma was developed and validated. Erythromycylamine in plasma (0.2 mL) was extracted with ethyl acetate, the organic phase was transferred to another clear 1.5 mL Eppendorf tube and evaporated to dryness under gentle nitrogen stream at 45 degrees C, and the residue was dissolved in 100 microL of mobile phase. The samples were separated using a Thermo Hypersil HyPURITY C18 reversed-phase column (150 mm x 2.1 mm I.D., 5 microm). A mobile phase containing 10 mM of ammonium acetate (pH = 6.4)-acetonitrile-methanol (50:10:40, v/v/v) was used isocratically eluting at a flow rate of 0.2 mL/min. Erythromycylamine and its internal standard (IS), midecamycin, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 4.5 to 720 ng/mL with r = 0.9997. The limit of quantification for erythromycylamine in plasma was 4.5 ng/mL with good accuracy and precision. The mean extraction recovery of the method was higher than 75.1% and 72.7% for erythromycylamine and IS, respectively. The intra-day and inter-day precision ranged from 5.2% to 6.4% and 5.6-9.3% (relative standard deviation, RSD), respectively. The established method has been successfully applied to a bioequivalence study of two dirithromycin formulations for 18 healthy volunteers.     

Column-switching technique for the sensitive determination of ertapenem in human cerebrospinal fluid using liquid chromatography and ultraviolet absorbance detection

A sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) assay with on-line extraction was developed for quantifying ertapenem in human cerebrospinal fluid (CSF). This assay is at least five times more sensitive than previously published ertapenem methods with a lower limit of quantitation at 0.025 microg/ml. In this assay, a CSF sample is extracted on-line using a RP extraction column and an aqueous acidic mobile phase (0.1% formic acid) to wash away polar endogenous materials, while ertapenem is retained on the column. Ertapenem is then back-flushed off the extraction column and directed to a RP analytical column using an acidic mobile phase with an organic modifier (acetonitrile/0.1% formic acid, 15:85 (v/v)) and detected using UV absorbance. The acidic mobile phase provided a sharper chromatographic peak and on-line extraction allowed large injection volumes (> or = 150 microl) of buffered CSF to be injected without compromising column integrity. These assay conditions were necessary to quantify ertapenem at levels expected to be found in human CSF (< 0.05 microg/ml). The method was successfully validated and implemented for a clinical study: intraday precision and accuracy of the CSF assay for calibration standards (0.025-10 microg/ml) and quality control samples (0.1, 0.5, and 2.5 microg/ml) were < 6.2% coefficient of variation and 96.8-104.0% of nominal concentration, respectively.     

High-performance liquid chromatographic assay for xanomeline, a specific M-1 agonist, and its metabolite in human plasma

A reversed-phase high-performance liquid chromatographic assay (HPLC) was utilized for monitoring xanomeline (LY246708/NNC 11–0232) and a metabolite, desmethylxanomeline, in human plasma. Xanomeline, desmethylxanomeline and internal standard were extracted from plasma with hexane at basic pH. The organic solvent extract was evaporated to dryness with nitrogen and the dried residue was reconstituted with 0.2 M HCl-methanol (50:50, v/v). A Zorbax CN 150 × 4.6 mm I.D., 5-μm column and mobile phase consisting of 0.5% (5 ml/l) triethylamine (TEA) adjusted to pH 3.0 with concentrated orthophosphoric acid-tetrahydrofuran (THF) (70:30, v/v) produced consistent resolution of analytes from endogenous co-extracted plasma components. Column effluent was monitored at 296 nm/0.008 a.u.f.s. and the assay limit of quantification was 1.5 ng/ml. A linear response of 1.5 to 20 ng/ml was sufficient to monitor plasma drug/metabolite concentrations during clinical trials. HPLC assay validation as well as routine assay quality control (QC) samples indicated assay precision/accuracy was better than ±15%.     

Simple and validated HPLC-UV analysis of levetiracetam in deproteinized plasma of patients with epilepsy

We present a simple, fast and validated method for the determination of the new generation antiepileptic drug (AED) levetiracetam (LEV) in plasma of patients with epilepsy using high performance liquid chromatography (HPLC) with UV detection. Plasma sample (500 microL) pretreatment was based on simple deproteinization by methanol spiked with the internal standard (I.S.). HPLC analysis was carried out on a Synergi 4-microm Hydro-RP, 150 mm x 4 mm I.D. column. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5) and acetonitrile (94:6, v/v) at an isocratic flow rate of 1.5 mL/min. The UV detector was set at 205 nm. Calibration curves were linear (mean correlation coefficient=0.999) over a range of 4-80 microg/mL. The quantitation limit was 2 microg/mL and the absolute recovery was >90% for LEV and the I.S. Both intra and interassay precision and accuracy were lower than 7.5%. The chromatographic run lasted 13 min. The present procedure omitting expensive solid phase or time-consuming liquid-liquid extraction and drying steps is cheaper, faster and simpler than mostly published analytical methods for levetiracetam. Applied to a large population of patients with epilepsy this assay proved very practical in our therapeutic drug monitoring setting (TDM).     

Development of a liquid chromatography/tandem mass spectrometry assay for the determination of bestatin in rat plasma and its application to a pharmacokinetic study

Bestatin is a low molecular weight aminopeptidase inhibitor originally isolated from culture filtrates of Streptomyces olivoreticuli. We have developed a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitative determination of bestatin in rat plasma using granisetron as the internal standard. The analyte and internal standard were isolated from 50 microL plasma samples by solid phase extraction (SPE). Reverse-phase HPLC separation was accomplished on a Lichrospher C18 column (4.6 mm x 50 mm, 5 microm) with a mobile phase composed of methanol-water-formic acid (70:30:0.5, v/v/v) at a flow rate of 0.8 mL/min. The method had a chromatographic total run time of 3 min. A Varian 1200L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 309.2-->120.0 (bestatin) and 313.4-->138.0 (granisetron) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 5 ng/mL, with good linearity (r2 >or= 0.999) over the linear range of 5-2000 ng/mL. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of bestatin in rats.     

Rapid determination of gefitinib and its main metabolite, O-desmethyl gefitinib in human plasma using liquid chromatography-tandem mass spectrometry

A novel, rapid and specific liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous quantification of gefitinib and its predominant metabolite, O-desmethyl gefitinib in human plasma. Chromatographic separation of analytes was achieved on an Alltima C18 analytical HPLC column (150 mm × 2.1 mm, 5 μm) using an isocratic elution mode with a mobile phase comprised acetonitrile and 0.1% formic acid in water (30:70, v/v). The flow rate was 300 μL/min. The chromatographic run time was 3 min. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionization (ESI) in positive mode. Linearity was demonstrated in the range of 5-1000 ng/mL for gefitinib and 5-500 ng/mL for O-desmethyl gefitinib. The intra- and inter-day precisions for gefitinib and O-desmethyl gefitinib were ≤10.8% and the accuracies ranged from 89.7 to 104.7% for gefitinib and 100.4 to 106.0% for O-desmethyl gefitinib. This method was used as a bioanalytical tool in a phase I clinical trial to investigate the possible effect of hydroxychloroquine on the pharmacokinetics of gefitinib. The results of this study enabled clinicians to ascertain the safety of the combination therapy of hydroxychloroquine and gefitinib in patients with advanced (Stage IIIB-IV) non-small cell lung cancer (NSCLC).     

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。     

Determination of rifabutin in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of rifabutin in human plasma. Rifabutin and sulindac (internal standard) are extracted from human plasma using a C₈ Bond Elut extraction column. Methanol (1 ml) is used to elute the compounds. The methanol is dried down under nitrogen and reconstituted in 250 μl of mobile phase. Separation is achieved by HPLC on a Zorbax Rx C₈ column with a mobile phase composed of 0.05 M potassium dihydrogen phosphate and 0.05 M sodium acetate at pH 4.0-acetonitrile (53:47, v/v). Detection is by ultraviolet absorbance at 275 nm. The retention times of rifabutin and internal standard were approximately 10.8 and 6.9 min, respectively. The assay is linear over the concentration range of 5–600 ng/ml. The quantitation limit was 5 ng/ml. Both intra-day and inter-day accuracy and precision data showed good reproducibility.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of D-amphetamine and diphenhydramine in beagle dog plasma and its application to a pharmacokinetic study

A new drug, quick-acting anti-motion capsule (QAAMC) composed of d-amphetamine sulfate, dimenhydrinate and ginger extraction has been studied for anti-motion-sickness use. We have developed a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitative determination of d-amphetamine and diphenhydramine, the main effective components of the QAAMC, using pseudoephedrine as the internal standard. The analytes and internal standard were isolated from 200 microL plasma samples by a simple liquid-liquid extraction (LLE). Reverse-phase HPLC separation was accomplished on a Zorbax SB-C18 column (100 mm x 3.0 mm, 3.5 microm) with a mobile phase composed of methanol-water-formic acid (65:35:0.5, v/v/v) at a flow rate of 0.2 mL/min. The method had a chromatographic total run time of 5 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 136.0-->91.0 (D-amphetamine), 256.0-->167.0 (diphenhydramine) and 166.1-->148.0 (IS) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.5 ng/mL for d-amphetamine and 1 ng/mL for diphenhydramine, with good linearity in the range 0.5-200 ng/mL for D-amphetamine and 1-500 ng/mL for diphenhydramine (r(2)> or =0.9990). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of the QAAMC in beagle dogs.