Sensitive method for the determination of vincristine in human serum by high-performance liquid chromatography after on-line column-extraction.

A column-switching high-performance liquid chromatographic method was developed for the determination of vincristine in serum. Sample preparation was carried out by means of on-line column-extraction, using a C18 reversed-phase preconcentration column. This technique is simple (minimizing manual sampling errors), rapid (reduction of time and costs) and can be easily automated. Both ultraviolet and electrochemical detection are possible, but the latter shows a cleaner chromatogram and is, by the use of a new electrochemical detector, far more sensitive (detection limit 0.3 microgram/l at a signal-to-noise ratio of 3). A matrix study was carried out (using human serum and urine and two kinds of calf's serum). Although it appeared that the system was matrix-dependent, no difference in matrix effects could be found in the serum or plasma of different patients. Controls for human serum analysis should be prepared in human serum. With the method described, pharmacokinetic studies of vincristine in children can be performed.     

Development and validation of a method to determine amoxicillin in physiological fluids using micellar liquid chromatography

A simple and robust method was developed for the routine identification and quantification of amoxicillin by micellar LC. Amoxicillin, a beta-lactamase inhibitor, is one of the most commonly prescribed drugs in the treatment of urine and skin structure infections. In this work, amoxicillin was determined in urine samples without any pretreatment step in a phenyl column using a micellar mobile phase of 0.10 M SDS and 4% butanol at pH 3. A UV detection set at 210 nm was used. Amoxicillin was eluted at 5.1 min with no interference by the protein band or endogenous compounds. Linearities (r >0.9998), intra- and interday precisions were determined (RSD (%) 0.4-2.7% and 0.3-5%, respectively, in micellar media, and 0.14-2.6% and 0.13-6%, respectively, in urine), and robustness was studied in the method validation. LOD and LOQ were 0.04 and 0.1 microg/mL in micellar media and 0.14 and 0.34 microg/mL in urine, respectively. Recoveries in the urine matrix were in the range of 95-110%. The validated method proved to be reliable and sensitive for the determination of amoxicillin in urine samples.     

Analysis of omeprazole and its main metabolites by liquid chromatography using hybrid micellar mobile phases

Omeprazole is a selective inhibitor of gastric acid secretion and is one of the most widely prescribed drugs internationally. A chromatographic procedure that uses micellar mobile phases of sodium dodecyl sulphate and propanol buffered at pH 7 and a C18 column is reported for the determination of omeprazole and its principal metabolites (omeprazole sulphone and hydroxyomeprazole) in urine and serum samples.In this work, direct injection and UV detection set at 305 nm was used. Omeprazole and its metabolites were eluted in less than 11 min with no interference by the protein band or endogenous compounds. Adequate resolution was obtained with a chemometric approach, in which the retention factor and shape of the chromatographic peaks were taken into account. The analytical parameters including linearity (r > 0.9998), intra- and inter-day precision (RSD, %: 0.6-7.9 and 0.14-4.7, respectively) and robustness were studied in the validation of the method for the three compounds. The limits of detection and quantification were less than 6 and 25 ng mL⁻¹, respectively. Recoveries in micellar medium, plasma and urine matrices were in the 98-102% range. Finally, the method was successfully applied to the determination of omeprazole and its metabolites in physiological samples. Omeprazole was also analysed in pharmaceutical formulations.     

Determination of Piribedil in Human Serum, Urine and Pharmaceutical Dosage Form by LC-DAD

A rapid, selective and sensitive reversed-phase liquid chromatographic (LC) method was developed for the determination of piribedil in human serum, urine and pharmaceutical dosage form. LC analysis was carried out using reversed-phase isocratic elution with a C₁₈ column and a mobile phase of 0.01 M phosphate buffer-acetonitrile (50:50, v/v). The chromatograms showed good resolution and sensitivity with no interference of human serum and urine. Piribedil concentrations were determined using diode array detection at 240 nm. Sildenafil citrate was used as internal standard. The limit of quantification (LOQ) and limit of detection (LOD) concentrations were 107.2 and 321.6 pg mL^?1, 96.6 and 290.4 pg mL^?1, 161.7 and 53.9 pg mL^?1 for urine, serum and pharmaceutical dosage forms, respectively. The method was validated for its linearity, precision and accuracy and applied to the tablets, urine and human serum. In addition, the results were compared to those obtained from UV-spectrophotometry.     

Determination of orotic acid in aqueous solutions by micellar electrokinetic capillary chromatography using sensitized lanthanide-ion luminescence detection

Summary A sensitive and selective laser-induced luminescence detection scheme for orotic acid in urine, separated by micellar electrokinetic capillary chromatography (MEKC) has been developed. The 325 nm line from a helium cadmium laser is used to excite orotic acid, which transfers its energy to terbium. Resultant luminescence of terbium is linear with orotic acid concentration over more than 2.5 orders of magnitude. This novel and practical system enables the detection of 50 nm orotic acid in urine in less than 1.5 minutes while using only nanoliters of sample. The significant decrease in analysis time over traditional methods (spectrophotometric and chromatographic) comes from the high efficiency of MEKC. A dramatic improvement in sensitivity and selectivity over UV detection in capillary electrophoresis is achieved through the use of laser-induced lanthanide ion energy transfer luminescence detection. Finally, no sample pretreatment is needed and the method is free from any known interferences in urine.     

Quantitative determination of epinephrine and norepinephrine in the picogram range by flame ionization gas-liquid chromatography.

A gas-liquid chromatographic method has been developed using the hydrogen flame detector to determine epinephrine (E) and norepinephrine (NE) in blood plasma, red blood cells, serum, and urine. The chromatographic method presents several advantages over other existing techniques. The derivatives enable separation of E and NE and are stable at room temperature with no signs of decomposition. The detection limit for the catecholamines with the hydrogen detector was approximately 0.1 pg. The catecholamines can be determined simultaneously from the same gas-liquid chromatogram. Purification of the catecholamines using the conventional procedure of chromatographing on alumina has been eliminated. With this gas chromatographic method, no by-products are formed that interfere with E and NE determinations. Dopamine, which constitutes the major source of interference in the commonly used fluorometric methods, does not interfere with the E and NE determinations. Norepinephrine and epinephrine values for several physiological fluids are given with the analysis expanded to include red blood cells, the contents of which have not been previously reported.     

利尿剂的胶束色谱分析

用十二烷基硫酸钠（SDS）胶束溶液作为流动相，建立了11种利尿剂的胶束色谱系统分析方法；研究了SDS浓度、流动相的pH、有机改性剂及温度对色谱保留行为的影响，优化了色谱条件，并且不经化学处理直接进样测定了人尿中的烯睾丙内酯；该法适用于兴奋剂中利尿剂的常规分析。     

Analytical procedure for the determination of rufloxacin, a new pyridobenzothiazine, in human serum and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantification of rufloxacin in human serum and urine has been developed and validated. The compounds, rufloxacin and internal standard, are extracted from buffered serum and urine using dichloromethane. They are then separated on an anion-exchange column using 0.05 M phosphate buffer-acetonitrile (80:20, v/v). The eluate is quantified by measuring the ultraviolet absorbance at 296 nm. The lower limit of detection for the analyte is 0.1 microgram/ml in serum and 0.05 micrograms/ml in urine. The method is linear from 0.3 to 10 micrograms/ml for serum and 0.1 to 10 micrograms/ml for urine. The method has been applied in a pharmacokinetic study in volunteers.     

Determination of cabergoline in plasma and urine by high-performance liquid chromatography with electrochemical detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of cabergoline in plasma and urine has been developed. After buffering plasma and urine samples, cabergoline was extracted with a methylene chloride-isooctane mixture, back-extracted into 0.1 M phosphoric acid, then analysed by reversed-phase high-performance liquid chromatography. Quantitation was achieved by electrochemical detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the biological matrices (human plasma and urine) was observed. The assay was still inadequate in terms of sensitivity for the quantitation of cabergoline plasma concentrations after a single oral dose of 1 mg of the drug to humans, but was successfully used in the determination of the urinary excretion of the drug.     

Detection of flavins by liquid chromatography using an electrochemical detector with two electrodes in series

A liquid chromatographic method for the selective detection of riboflavin (vitamin B₂) and flavin adenine dinucleotide (FAD) utilizing a thin-layer amperometric detector with two electrodes in series is described. The upstream electrode was held at ?0.4 V (vs. SCE) with the downstream electrode at +0.1 V (vs. SCE). The linear ranges are 40 ng?10 μg for riboflavin and 200 ng?8 μg for FAD and the limits of detection are 8 and 40 ng, respectively. The interference of thirteen different vitamins on the spectrophotometric or electrochemical detection of FAD and vitamin B₂ was studied and no interference was found using the two-electrode detector. The effects of light on riboflavin and FAD is discussed. The method is convenient, rapid and economic, and has high selectivity.     

Measurement of urinary cystine and cysteinyl-penicillamine in patients with cystinuria

A high-performance liquid chromatographic method with electrochemical detection (LC/EC) was developed to measure cystine and cysteinyl-penicillamine disulfide in the urine of patients screened or treated for cystinuria. Urine was acidified, centrifuged to remove urinary protein, diluted and injected. The disulfides were separated on a reversed-phase column, reduced at the upstream electrode of a dual electrochemical detector with gold-mercury amalgam (Au/Hg) electrodes and the resultant thiols measured at the downstream electrode. The sample preparation is simple, the analysis rapid, specimens can be easily batched and the specificity of the method is better than those of two other separative procedures with which it was compared. The coefficient of variation for cystine in cystinuric urine is 6.7%, 5.5% and 3.2% for levels of 0.09, 0.52 and 1.02 mmol/l respectively, and for cysteinyl-penicillamine disulfide 2.6% and 7.5% for levels of 0.45 and 0.98 mmol/l respectively. Urine for analysis of these disulfides should not be collected within 24 hours of administration of the radiopaque agent diatrizoate but no other interference to the assay has been noted. This method is suitable as a screen for cystinuria in patients with renal tract calculi, for ongoing monitoring of cystinuric patients and to check patient compliance with d-penicillamine therapy.     

Therapeutic monitoring of imipramine and desipramine by micellar liquid chromatography with direct injection and electrochemical detection

A micellar liquid chromatographic (MLC) procedure was developed for the clinical monitoring of imipramine and its active metabolite, desipramine. The determination of these highly hydrophobic substances was carried out after direct injection of the serum samples using a mobile phase composed of 0.15 m SDS--6% (v/v) pentanol buffered at pH 7, pumped at 1.5 mL/min into a C(18) column (250 x 4.6 mm), and electrochemical detection at 650 mV. Using this MLC method, calibration was linear (r > 0.995) and the limits of detection (ng/mL) were 0.34 and 0.24 for imipramine and desipramine, respectively. Repeatabilities and intermediate precision were tested at three different concentrations in the calibration range and a CV (%) below 2.2 was obtained. In this MLC procedure, the serum is determined without treatment, thus allowing repeated serial injections without changes in retention factors, and reducing the time and consumables required to carry out the pretreatment process. The assay method can be applied to the routine determination of serum imipramine and its metabolite in therapeutic drug monitoring.     

Photostability studies for micellar liquid chromatographic determination of nifedipine in serum and urine samples

Nifedipine is a photosensitive compound that is converted into its 4-(2-nitrophenyl) pyridine and 4-(2-nitrosophenyl) pyridine homologue. In order to obtain the most adequate conditions for handling nifedipine solutions in the analytical laboratory, a number of studies on the decomposition of this compound were performed. A simple micellar liquid chromatographic procedure was described to determine nifedipine in different biological matrices such as serum and urine, and to control its decomposition. To perform the analysis, nifedipine was dissolved in 0.1 m SDS at pH 3 and chromatographed using a mobile phase containing 0.125 m SDS-3% pentanol, pH 3 on a C18 column and UV detection at 235 nm. The chromatographic analysis time was 8 min. The response of the drug for both biological matrices was linear in the 1-100 microg/mL range, with r2>0.997 at all times. Repeatability, intermediate precision (CV, %) and limits of quantification and detection (ng/mL) were 0.19, 4.3, 104 and 31 in serum and 0.81, 2.1, 136 and 41 in urine. The method developed here does not show interferences or matrix effects produced by endogenous compounds. Micellar media and mobile phases have the advantage of stabilising the compounds, thus preventing photodegradation and allowing the direct injection of biological samples.     

Micellar electrokinetic chromatographic method for the dabrafenib determination in biological samples

Two different micellar electrokinetic chromatographic methods to determine dabrafenib in urine and serum, both using borate buffer (pH 9.2, 20 mM) and SDS as separation electrolyte, are developed and validated. The analyses were carried out in a fused‐silica capillary of 75 μm of internal diameter and total length of 47 and 37 cm for urine and serum determination, respectively. The detection of the target compound was performed at 227 nm in urine samples and at 251 nm in serum samples. The linearity range was from 1 to 21 mg/L of dabrafenib in urine and from 2 to 40 mg/L in serum. In all cases, inter‐ and intraday RSDs were <4%. Sample preparation of serum samples consists of an only step of 1:1 dilution with water before its injection in the electrophoretic system. These simple, sensitive, accurate, and cost‐effective methods can be used in routine clinical practice to monitor dabrafenib concentrations in urine and serum of metastatic melanoma skin cancer patients.     

Analysis of selected designer benzodiazepines by ultra high performance liquid chromatography with high‐resolution time‐of‐flight mass spectrometry and the estimation of their partition coefficients by micellar electrokinetic chromatography

A new ultra high performance liquid chromatography with electrospray ionization time of flight mass spectrometry method for the selective and sensitive separation, identification, and determination of selected designer benzodiazepines (namely, pyrazolam, phenazepam, etizolam, flubromazepam, diclazepam, deschloroetizolam, bentazepam, nimetazepam, and flubromazolam) in human serum was developed. The separation of the studied designer benzodiazepines was achieved on C₁₈ chromatographic column using gradient elution within 6 min without any significant matrix interferences. Liquid–liquid extraction with butyl acetate was applied for serum samples cleanup and preconcentration of studied designer benzodiazepines. The method was validated in terms of linearity, limit of detection, limit of quantification, matrix effects, specificity, precision, accuracy, recovery, and sample stability. The limit of detection values were 0.10–0.15 ng/mL. The method was applied to a spiked serum sample to demonstrate its applicability for systematic toxicology analysis. Furthermore, a capillary chromatographic method with micellar electrokinetic chromatography was used for the estimation of partition coefficients of studied designer benzodiazepines as important parameters to evaluate their pharmacological and toxicological properties.     

Sensitive analysis of blood for amodiaquine and three metabolites by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method using oxidative electrochemical detection has been developed for selective and sensitive quantification of the antimalarial drug amodiaquine and three of its metabolites in the blood of dosed individuals. The method requires only one extraction step and has detection limits of 1 ng/ml for amodiaquine and its metabolites desethylamodiaquine and bisdesethylamodiaquine and 3 ng/ml for 2-hydroxydesethylamodiaquine. Minor modification of the mobile phase preserves the chromatographic separation and allows ultraviolet spectroscopic detection, which, although appreciably less sensitive, permits monitoring of levels of amodiaquine and the three metabolites in blood and urine samples if an electrochemical detector is unavailable. Levels of amodiaquine and the three metabolites were determined for two volunteers undergoing a nine-week chemoprophylactic regimen in connection with travel to a malarious area. Data are included to compare the in vitro antimalarial activities against three strains of Plasmodium falciparum of amodiaquine and the three metabolites considered.     

Direct determination of 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid in urine by high-performance liquid chromatography with amperometric detection

The improvement of high-performance liquid chromatographic analysis with electrochemical detection for urinary homovanillic acid is described. The method permits the chromatographic resolution of authentic homovanillic acid from coeluting interfering compounds in human and nonhuman primate, and rat urine. The electrochemically derived results are compared with post-column derivatized fluorescence results, and quality-control checks necessary to maintain assay precision in automated analysis are described.     

Determination of furosemide in urine by HPLC with preconcentration by micellar-extraction

The micellar extraction of furosemide by the nonionic surfactant (NS) Triton X-100 at the cloud point was studied; it was performed in a conventional heating mode and upon induction with phenol. It was shown that the quantitative recovery of the preparation was attained using hydrophobic micellar phases of NS modified with phenol. A procedure was developed for determining furosemide in urine by reversed-phase HPLC with preconcentration by micellar extraction in the presence of phenol. The preconcentration ratio was 80 and the detection limit for furosemide, 3 mg/L. The procedure was used to analyze urine samples by the standard addition method.     

A rapid, sensitive high performance liquid chromatographic method for the determination of meropenem in pharmaceutical dosage form, human serum and urine.

A new, simple, precise and rapid high performance liquid chromatographic method was developed for the determination of meropenem in human serum, urine and pharmaceutical dosage forms. Chromatography was carried out on an LC(18) column using a mixture of 15 mM KH(2)PO(4):acetonitrile:methanol (84:12:4; v/v/v), adjusted to pH 2.8 with H(3)PO(4). The proposed method was conducted using a reversed-phase technique, UV monitoring at 307.6 nm and cefepime as an internal standard. The retention times were 5.98 and 7.47 min for cefepime and meropenem, respectively. The detector response was linear over the concentration range of 50-10,000 ng/mL. The detection limit of the procedure was found to be 22 ng/mL. The detection limit for meropenem in human plasma was 108.4 ng/mL and the corresponding value in human urine was 179.3 ng/mL. No interference from endogenous substances in human serum, urine and pharmaceutical preparation was observed. The proposed method is sufficiently sensitive for determination of the concentrations of meropenem and may have clinical application for its monitoring in patients receiving the drug.