Determination of remoxipride in plasma and urine by reversed-phase column liquid chromatography

A reversed-phase column liquid chromatographic method for the determination of remoxipride, a novel antipsychotic drug, in biological fluids is described. A simple one-step extraction is used followed by liquid chromatography on a 3-microns octadecylsilica column and ultraviolet absorbance detection. The method is accurate and precise for clinical remoxipride levels in both plasma and urine. For situations where a higher sensitivity is necessary a two-step extraction and a modified mobile phase are used. With this modification plasma concentrations down to 2 nM can be determined with acceptable precision.     

High-performance liquid chromatographic analysis of amiloride in plasma and urine

A high-performance liquid chromatographic method has been developed for amiloride in rabbit plasma and urine which uses a reversed-phase C18 column, a mobile phase (flow-rate 2 ml/min) consisting of 32% acetonitrile in 0.15 M perchloric acid, pH 2.2, and spectrofluorometric detection via excitation at 286 nm. A simple extraction step with ethyl acetate eliminates interfering peaks. Short retention times of about 2.3 and 3.8 min are observed for amiloride and the internal standard, triamterene, respectively. The method can measure 4 ng/ml amiloride in plasma. This assay has been used to explore the pharmacokinetics of amiloride in rabbits. The plasma disposition profile is biexponential after a 50-mg intravenous bolus dose and there is no evidence for saturable elimination at zero-order infusion rates of 1.8, 3.6 and 7.2 mg/h.     

Simultaneous determination of cefamandole and cefamandole nafate in human plasma and urine by high-performance liquid chromatography with column switching

A high-performance liquid chromatographic method with column switching has been developed for the simultaneous determination of cefamandole and cefamandole nafate in plasma and urine. The plasma and urine samples were injected onto a precolumn packed with Corasil RP C18 (37-50 microns) after simple dilution with an internal standard solution in 0.05 M phosphoric acid. Polar plasma and urine components were washed out using 0.05 M phosphoric acid. After valve switching, the concentrated drugs were desorbed in back-flush mode and separated by a reversed-phase C8 column with methanol-5 mM tetrabutylammonium bromide (45:55, v/v) as the mobile phase. The method showed excellent precision with good sensitivity and speed, and a detection limit of 0.5 microgram/ml. The total analysis time per sample was less than 30 min, and the mean coefficients of variation for intra- and inter-assay were both less than 4.9%. The method has been successfully applied to plasma and urine samples for human volunteers after intravenous injection of cefamandole nafate.     

Determination of gambogic acid in dog plasma by high-performance liquid chromatography for a pharmacokinetic study

A rapid, simple and sensitive high-performance liquid chromatography method for the quantification of gambogic acid in dog plasma was developed and validated. After acidification with hydrochloric acid, dog plasma was extracted with ethyl acetate and determined by HPLC. The analysis was carried out on a reversed-phase C(18) analytical column. The mobile phase consisted of a mixture of methanol-0.05% phosphoric acid (94:6, v/v), and the column temperature was maintained at 35 degrees C. A constant mobile phase flow rate of 1.0 mL/min was employed throughout the analyses. The ultraviolet detector was set at 360 nm. Chromatographic separation was achieved in less than 10 min and the calibration curve was linear over a concentration range of 0.156-20 microg/mL. The intra-assay and inter-assay variability values were less than 10.0%. The accuracy ranged from 93.0 to 104.2%. The established method has been successfully applied to a pharmacokinetic study of gambogic acid in dogs.     

Determination of clomipramine and its hydroxylated and demethylated metabolites in plasma and urine by liquid chromatography with electrochemical detection

A procedure for the determination of clomipramine and its 8-hydroxy, demethyl, 8-hydroxydemethyl and didemethyl metabolites in plasma and urine by high-performance liquid chromatography with electrochemical detection is described. A 1-ml plasma or urine sample is made alkaline with a carbonate buffer (pH 9.8) and extracted with 20% ethyl acetate in n-heptane. After back-extraction into an acid phosphate buffer (pH 2.4), an aliquot is injected into a 5-microns ion-paired reversed-phase column and eluted with a mobile phase containing a phosphate buffer with tetramethylammonium chloride-acetonitrile (57:43). The detection is coulometric with a first cell at +0.40 V, a second at +0.73 V and a guard cell set at 0.75 V for oxidation of the mobile phase. The method provides recoveries in the general range of 80-110% and a day-to-day precision of 3.7-8.8%, depending on the compound. The minimum quantifiable level for all compounds was 0.2 ng/ml with a 20-microliters injection. Steady-state plasma concentration data and urinary levels are reported for 24 depressed patients receiving daily either 75-150 mg orally or 50-75 mg by infusion.     

Ion-paired high-performance liquid chromatographic determination of mitoxantrone in physiological fluids

Mitoxantrone is one of the newer anthracenedione derivatives which has already been studied in phase I and II trials, where it has shown significant antitumor activity against a variety of human tumours. To determine the prolonged terminal half-life of mitoxantrone, we developed a sensitive high-performance liquid chromatographic method, providing a detection limit of 1 ng/ml of extracted serum. This system uses a C18 reversed-phase column. The mobile phase consists of a mixture of acetonitrile (30%, v/v) and an ammonium formate buffer (70%, v/v) with a pH of 2.7. Hexane sulphonic acid is added as an ion-pair former. Detection at a wavelength of 658 nm provides a highly selective system, showing no interfering peaks. Ametantrone, another anthracenedione derivative, is used as an internal standard. The extraction procedure for serum also uses hexane sulphonic acid in an ion-paired system. Because of the highly selective detection wavelength, urine samples can be injected without a sample clean-up procedure. This very sensitive method, combined with high selectivity and a fast and inexpensive serum clean-up procedure, has allowed us to document the prolonged terminal plasma half-life of mitoxantrone (levels of 2-5 ng/ml of plasma can still be detected six days after an intravenous infusion of 15 mg/m2 over 30 min). In urine an as yet unidentified metabolite was discovered.     

Validated and optimized high-performance liquid chromatographic determination of tizoxanide, the main active metabolite of nitazoxanide in human urine, plasma and breast milk

A high-performance liquid chromatographic method was optimized and validated for the determination of desacetyl nitazoxanide (tizoxanide), the main active metabolite of nitazoxanide in human plasma, urine and breast milk. The proposed method used a CN column with mobile phase consisting of acetonitrile-12mM ammonium acetate-diethylamine in the ratio of 30:70:0.1 (v/v/v) and buffered at pH 4.0 with acetic acid, with a flow rate of 1.5 mL/min. Quantitation was achieved with UV detection at 260 nm using nifuroxazide as internal standard. A simplified direct injection of urine samples without extraction in addition to the urinary excretion pattern were calculated using the proposed method. Also, the effectiveness of protein precipitation and a clean-up procedure were investigated for biological plasma and human breast milk samples. The validation study of the proposed method was successfully carried out in an assay range between 0.2 and 20 μg/mL.     

Rapid method for the measurement of methylprednisolone and its hemisuccinate in plasma and urine following "pulse therapy" by high-performance liquid chromatography

A rapid method for the measurement of methylprednisolone and its 21-hemisuccinate ester in plasma and urine following high dose pulse therapy is described. The drugs were extracted using Extrelut columns, eluted with ethyl acetate which was evaporated to dryness and the residue was reconstituted in chromatographic mobile phase. High-performance liquid chromatography was performed on a reversed-phase column using a mobile phase of acetonitrile-acetate buffer with detection at 251 nm. No interference from any drugs or endogenous compounds has been observed. The method has been used to analyse over 200 plasma and 150 urine samples from patients with rheumatoid disease or renal failure who have received high dose methylprednisolone hemisuccinate infusions.     

Simple high-performance liquid chromatographic method for the determination of tolbutamide and its metabolites in human plasma and urine using photodiode-array detection

A high-performance liquid chromatographic method has been developed for the determination of tolbutamide and its metabolites in human plasma and urine. The compounds examined were extracted with diethyl ether from the acidified biological fluid. Chlorpropamide was used as internal standard, and 235 nm was chosen as the wavelength for diode-array detection. A study of the relationship between the capacity factor and the mobile phase composition and pH showed that acetonitrile-2-propanol-0.1% orthophosphoric acid (17: 17: 66, v/v) was the best eluent on a C8 reversed-phase column. The method is precise, sensitive and suitable for pharmacological investigations.     

A Simple Isocratic HPLC Method for the Determination of Metoclopramide in Plasma and Urine

Abstract

Metoclopramide concentrations in plasma and urine were determined by high performance liquid chromatography using a cyanopropylsilane column and UV detection. The mobile phase consisted of 0.03M sodium acetate (pH 7.4) and acetonitrile. The plasma samples were extracted with dichloromethane after pH adjustment. Urine proteins were precipitated with acetonitrile. The reproducibility and precision of the methods were demonstrated by the analysis of samples containing 5 – 200 ng/ml plasma and 0.25 – 200 ug/ml urine.

The glucuronide and sulfate conjugates of metoclopramide were also quantitated after differential acid hydrolysis of urine samples. The conditions for acid hydrolysis were studied. The methods have been applied to the analysis of plasma and urine samples obtained from human volunteers.     

HPLC‐MS/MS analysis of anthocyanins in human plasma and urine using protein precipitation and dilute‐and‐shoot sample preparation methods,respectively

A high‐performance liquid chromatography tandem–mass spectrometry (HPLC‐MS/MS) method has been developed to analyze anthocyanins in urine and plasma to further understand their absorption, distribution, metabolism and excretion. The method employed a Synergi RP‐Max column (250 × 4.6 mm, 4 μm) and an API 4000 mass spectrometer. A gradient elution system consisted of mobile phase A (water–1% formic acid) and mobile phase B (acetonitrile) with a flow rate of 0.60 mL/min. The gradient was initiated at 5% B, increased to 21% B at 20 min, and then increased to 40% B at 35 min. The analysis of anthocyanins presents a challenge because of the poor stability of anthocyanins during sample preparation, especially during solvent evaporation. In this method, the degradation of anthocyanins was minimized using protein precipitation and dilute‐and‐shoot and sample preparation methods for plasma and urine, respectively. No interferences were observed from endogenous compounds. The method has been used to analyze anthocyanin concentrations in urine and plasma samples from volunteers administered saskatoon berries. Cyanidin‐3‐galactoside, cyanidin‐3‐glucoside, cyanidin‐3‐arabinoside, cyanidin‐3‐xyloside and quercetin‐3‐galactoside, the five major flavonoid components in saskatoon berries, were identified in plasma and urine samples.     

Selective quantification of doxycycline in human plasma and urine with optimised chromatography

Summary A method for the selective quantification of doxycycline in human plasma and urine has been developed using HPLC with UV-detection at 350 nm. Analyte and internal standard were extracted from plasma by extraction columns filled with octadecylsilica. Urines were only mixed with diluted acid prio to injection. The influence of pH on peak shapes is discussed as well as comparative investigations on selectivity and peak symmetry on commerical octadecylsilica. The method was successfully applied to the samples of a clinical study with an oral single dose of 100 mg. Precision and accuracydata of the assay and calculated pharmacokinetic parameters are presented.     

High-performance liquid chromatographic determination of vinca-alkaloids in plasma and urine

A liquid chromatographic method is described for separating and determining vinblastine, vincristine and vindesine in plasma and urine. The drugs are extracted from the biological material using an ion-pair extraction, with sodium octylsulphate as counter-ion at pH 3. The extracts are injected on a reversed-phase system with a cyano column as stationary phase and a mobile phase composed of acetonitrile-phosphate buffer, pH 3 (65:35, vol. %). Stability studies are carried out for stock solutions of the drugs in water at different temperatures and pH values. The stability of these compounds in plasma is also investigated in the presence of an antioxidant. The method is applied to determine drug levels of vindesine and vinblastine in preliminary pharmacokinetic studies, using vincristine as the internal standard.     

Determination of the antiplatelet agent. KB-3022, and its metabolite by high-performance liquid chromatography

A rapid, accurate and reproducible high-performance liquid chromatographic method for the simultaneous determination of a new antiplatelet agent, ethyl 2-[4,5-bis(4-methoxyphenyl)thiazol-2-yl]pyrrol-1-ylacetate (KB-3022), and its main metabolite, 2-[4,5-bis(4-methoxyphenyl)thiazol-2-yl]pyrrol-1-ylacetic acid (desethyl KB-3022), in biological fluids has been developed. KB-3022 and desethyl KB-3022 in plasma or urine were extracted with a mixture of n-hexane and dichloromethane (1:1), separated on a reversed-phase C18 column with a mixture of 0.01 M (NH4)2HPO4 (pH 3.0), acetonitrile and isopropyl alcohol as a mobile phase, and quantitated by ultraviolet absorbance measurement at 340 nm. 4-[2-(4,5-bis(4-methoxyphenyl)thiazol-2-yl)pyrrol-1-yl]butyric acid was used as an internal standard. The detection limit of KB-3022 in plasma was 3 ng/ml, and that of KB-3022 in urine and desethyl KB-3022 in plasma or urine was 1 ng/ml. The coefficients of variation for the determination of KB-3022 or desethyl KB-3022 in plasma or urine were below 5.6%. This method was applied to the determination of the plasma concentration of KB-3022 and desethyl KB-3022 after intravenous administration to rats.     

Extractionless HPLC Method for the Determination of Naproxen in Human Plasma and Urine

Abstract

A simple and rapid (extractionless) high-performance liquid chromatographic method with ultraviolet detection, at 278 nm, is described for the determination of naproxen in human plasma and urine. Niflumic acid is used as internal standard. The chromatographic system consists of a reversed-phase C18-Spherisorb column with acetonitrile/0.1 M sodium acetate (35:65 v/v, pH 6.14) as the mobile phase. The retention time is 3.0 min for naproxen and 3.8 min for niflumic acid. The total run time is 5 min and the typical assay time is 10 min. The method is sufficiently sensitive for biopharmaceutical studies, after the oral administration of a single sustained release dose.     

Rapid and specific approach for direct measurement of glimepiride in human plasma by LC-ESI-MS-MS employing automated 96 well format: application to a bioequivalence study

A rapid liquid chromatographic method with electrospray ionization tandem mass spectrometric (LC-MS-MS) detection is developed and validated for quantification of glimepiride in heparinized human plasma. Plasma samples, without a drying and reconstitution step, are extracted by solid-phase extraction (SPE) and eluted with 0.9 mL of acetonitrile-methanol (1:1, v/v) containing 0.05% formic acid. The analyte and glimepiride d8 (internal standard, IS) are chromatographed on a C(18) column; the mobile phase is acetonitrile-2 mm ammonium formate (88:12, v/v), with the pH adjusted to 3.5 with formic acid, at a flow rate of 0.5 mL/min. The retention times of glimepiride and the IS are 0.93 min, and the runtime is 1.6 min per sample. Selected reaction monitoring of MH(+) at m/z 491.20 and 499.26 result in stable fragment ions with m/z 351.80 and 359.96 for glimepiride and the IS, respectively. The response was a linear function of the concentration in the range of 2.0-650.0 ng/mL, with r ≥ 0.9994. The recovery of glimepiride and the IS ranged from 81.91 to 83.36%. The assay has excellent characteristics and has been successfully used for the analysis of glimepiride in healthy human subjects in a bioequivalence study. It was well suited to clinical studies of the drug involving large numbers of samples.     

反相高效液相法测定四环素类抗生素

 利用高效液相法 ,在C18柱上以甲醇 乙腈 0 0 1mol/L草酸溶液 (pH 2 0 ) (体积比为 11∶2 2∶6 7)为流动相 ,采用 2 6 7nm紫外光进行检测 ,在 2 2min内将 7种四环素类抗生素全部洗脱并达到基线分离。探讨了流动相的pH值、草酸的浓度、流动相中有机相的比例以及检测波长等因素对分离度和灵敏度的影响。采用标准加入法定量 ,对两种实际样品进行了分析。结果表明 :该方法操作简单、灵敏度高、定量准确。     

Determination of salbutamol in human plasma and urine by high-performance liquid chromatography with a coulometric electrode array system

A method is developed to determine salbutamol in human plasma and urine using high-performance liquid chromatography (HPLC) with a coulometric electrode array system, based on the electrochemical behavior of salbutamol at graphite electrode. The mobile phase component A is 30 mM sodium dihydroxy phosphate-30 mM triethylamine and is adjusted to pH 6.0 with 20% phosphate acid. The mobile phase component B is methanol. The optimized mobile phase composition was A and B in the proportion of 90:10 (v/v). Paracetamol is selected as the external standard. The human plasma and urine samples are pretreated using solid-phase extraction cartridges (Sep-Pak Silica), and the eluting solution is monitored by the coulometric electrode array system. The electrode potentials are set at 300, 400, 550, and 650 mV, respectively. Calibration curves show good linearity, and the recovery of salbutamol proves to be constant and unaffected by the concentration of the drug. This method, developed using HPLC-electrochemical detection, is reproducible and sensitive enough for the determination of salbutamol in human plasma and urine.     

High-performance liquid chromatographic determination of glibenclamide in human plasma and urine

A selective and sensitive high-performance liquid chromatographic method for determination of intact glibenclamide in human plasma or urine has been developed. With glibornuride as internal standard, acid-buffered plasma or urine was extracted with benzene. The organic layer was evaporated and the residue was dissolved in equilibrated mobile phase (acetonitrile-phosphate buffer 0.01 M pH 3.5, 50:50). An aliquot of 20 microliters was chromatographed on a Spherisorb ODS reversed-phase column, and quantitation was achieved by monitoring the ultraviolet absorbance at 225 nm. The response was linear (0-1000 ng/ml) and the detection limit was 5-10 ng/ml in plasma or urine. The within-assay variation was less than or equal to 10%. No interferences from metabolites or endogenous constituents could be noted. The utility of the method was demonstrated by analysing glibenclamide in samples from diabetic subjects on therapeutic doses of the drug.     

High-performance liquid chromatographic analysis of a novel carbapenem antibiotic in human plasma and urine

High-performance liquid chromatographic methods for quantification of a novel carbapenem anti-infective agent, I, in plasma and urine have been developed, validated, and applied to clinical samples. The carbapenem is stabilized in the matrix by the addition of a non-nucleophilic buffer, rapid freezing, and storage at -70 degrees C. After addition of another carbapenem, II, as internal standard, plasma proteins are precipitated with acetonitrile, which is subsequently extracted from the sample with methylene chloride. A portion of the aqueous phase is injected onto a reversed-phase phenyl column that is eluted with 4% (v/v) acetonitrile in 15 mM ammonium phosphate (pH 7.4). The urine assay entails addition of the internal standard II to buffered urine, which is subsequently extracted with methylene chloride prior to injection of the aqueous phase onto a cation-exchange column. The urine assay mobile phase is 5% v/v tetrahydrofuran in 100 mM sodium acetate (pH 5.4). The detector response at 313 nm is a linear (r greater than 0.99) function of concentration over the ranges 0.50-100 micrograms/ml and 2.0-200 micrograms/ml for the plasma and urine assays, respectively. Thermal degradation products do not interfere with either assay. These assays have proven to be accurate, precise, reproducible, and rugged during clinical sample analyses.