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。     

Separation of phenolic compounds and corresponding glucuronides by coupled-column micellar reversed-phase liquid chromatography

Summary An isocratic HPLC technique for separation of phenolic compounds and corresponding glucuronides in urine is developed. Sample pre-treatment, often a tedious and rate-limiting factor, was eliminated by use of a coupled column system. Spiked urine samples were injected directly into a C₄ precolumn and a selected fraction was transferred on-line from the precolumn to a silanized C₁₈ analytical column in the backflush mode. Analyte peak enrichment was attained by employing mobile phases of different elution strengths. The weaker mobile phase (7% v/v acetonitrile) was used to strongly retain the analytes on the precolumn while most of the polar endogenous compounds were washed to waste. Elution and transfer of the trapped analytes from the precolumn to the analytical column was achieved by introducing a stronger mobile phase (20% v/v acetonitrile) with the aid of a switching valve. The use of cetyltrimethylammonium bromide as counter ion and micellar agent in the mobile phase involved a high selectivity for the analytes relative to the urine matrix components and allowed simultaneous analysis of the glucuronides and parent compounds without the need of gradient elution. The system demonstrated a good repeatability on spiked urine samples. Who passed away July 21, 1996     

Simultaneous micellar electrokinetic chromatography and liquid chromatography of adriblastina and tarabine PFS Their application to some biological fluids

The current work presents analytical procedures for simultaneous determination of tarabine PFS and adriblastina by micellar electrokinetic chromatography (MEKC) and liquid chromatography (LC). For MEKC analysis, separations and identifications were accomplished using uncoated fused-silica capillary with hydrodynamic injections in the presence of 50mM borate/phophate pH 8.7 and 100mM SDS. The migration times of tarabine PFS and adriblastina were found to be 2.70 and 6.40min, respectively. Calibration curves were established for 10-300ng/mL (r=0.998) tarabine PFS and for 8-120microg/mL (r=0.999) adriblastina. For LC analysis, separations were performed on teicoplanin stationary phase with reversed mobile phase containing methanol:buffer pH 4.05 (20:80%, v/v) at 285nm. The retention times of tarabine PFS and adriblastina were 5.18 and 7.20min, respectively. Calibration curves were established for 3-90microg/mL (r=0.998) tarabine PFS and for 10-120microg/mL (r=0.999) adriblastina. Both MEKC and LC methods were applied for the simultaneous determination of analytes in urine samples.     

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.     

Simultaneous determination of three opiates in serum by micellar liquid chromatography using direct injection

A simple and reliable micellar liquid chromatographic method was developed for the simultaneous determination of 3 opiates (codeine, morphine, and thebaine) in serum, using direct injection and ultraviolet detection. The separation of the drugs was optimized on a C18 column, thermostatically controlled at 25 degrees C, by evaluating mobile phases containing sodium dodecyl sulfate (SDS) and various modifiers (propanol, butanol, or pentanol). Adequate resolution of the opiates was obtained with a chemometrics approach, in which retention was modeled as a first step by using the retention factors for several mobile phases. Next, an optimization criterion that takes into account the position and shape of the chromatographic peaks was applied. The 3 opiates were totally resolved and determined in 12 min with the mobile phase 0.15M SDS-7% (v/v) butanol buffered at pH 7. The limits of detection for codeine and morphine were greatly improved by using fluorimetric detection. Repeatability and intermediate precision were tested for 3 different concentrations of the drugs, and the relative standard deviations were <0.8% for most of the assays. Finally, the method was successfully applied to the determination of morphine and codeine in serum samples.     

Separation optimization of quercetin,hesperetin and chrysin in honey by micellar liquid chromatography and experimental design

The chemometrics approach was applied for the separation optimization of flavonoid markers (quercetin, hesperetin and chrysin) in honey using micellar liquid chromatography (MLC). The investigated method combines SPE of flavonoids from honey using C₁₈ cartridge and their separation and quantification by micellar liquid chromatography. A two level full factorial design was carried out to evaluate the effect of four experimental factors including concentration of SDS, alkyl chain length of the alcohol used as the organic modifier (N), volume percentage of the organic modifier (V_m) and volume percentage of acetic acid (AcOH) in mobile phase on analytes retention times. Experiments for analytes retention times modeling and optimization of separation were performed according to central composite design. Multiple linear regression method was used for the construction of the best model based on experimental retention times. Pareto optimal method was used to find suitable compatibility between resolution and analysis time of analytes in honey. The optimum mobile phase composition for separation and determination of analytes in honey were [SDS]=0.124 mol/L; 7.8% v/v ethanol and 5.0% v/v AcOH. Limits of detection and linear range of flavonoid markers were 0.0079–0.0126, 0.05–50.0 mg/L, respectively.     

Conventional and thermal lens spectrophotometric determination of p-aminobenzoic acid and arylamine diuretics previous azodye formation in a micellar medium

The determination of the diuretics hydrochlorothiazide, bendroflumethiazide and furosemide by both conventional and thermal lens spectrophotometry (TLS, 100 mW of pump power at 514.5 nm) following previous hydrolysis, diazotization and coupling with N-(naphthyl)ethylenedine (NED) in a sodium dodecyl sulphate (SDS) micellar medium of pH approximately 1 was studied. p-Aminobenzoic acid (PABA) was used as a model compound to optimize the derivatization procedures. 3-Substituted indoles, such as 5-hydroxyindole-3-acetic acid and tryptophan, gave N-nitroso derivatives which interfered with the determination of the diuretics in urine. The derivatized diuretics in urine were separated using HPLC with a Spherisorb ODS-2 C(18) column, and a 0.1M SDS mobile phase containing 5% n-propanol and 0.001M sodium dihydrogen phosphate (pH 3). The diuretics gave limits of detection (LODs) of ca. 5 x 10(-9)M for the TLS procedure. The LODs were 20-50-fold higher for the corresponding spectrophotometric procedure.     

Quinolones control in milk and eggs samples by liquid chromatography using a surfactant-mediated mobile phase

Four quinolones (danofloxacin, difloxacin, flumequine and marbofloxacin) were determined in milk and egg samples by a simplified high-performance liquid chromatographic procedure using a micellar mobile phase. No extraction was needed to precipitate the proteins from the matrices since they were solubilised in micelles. The only pretreatment steps required were homogenisation, dilution and filtration before injecting the sample into the chromatographic system. An adequate resolution of the quinolones was achieved by a chemometrics approach where retention was modelled as a first step using the retention factors in only five mobile phases. Afterwards, an optimisation criterion was applied to consider the position and shape of the chromatographic peaks. Analytical separation involved a C18 reversed-phase column, a hybrid micellar mobile phase of 0.05 M sodium dodecyl sulphate, 10% (v/v) butanol and 0.5% (v/v) triethylamine buffered at pH 3 and fluorimetric detection. Quinolones were eluted in less than 15 min without the protein band or other endogenous compounds from the food matrices interfering. The calculated relevant validation parameters, e.g., decision limit (CC(α)), detection capability (CC(β)), repeatability, within-laboratory reproducibility, recoveries and robustness, were acceptable and complied with European Commission Decision 2002/657/EC. Finally, the proposed method was successfully employed in quantifying the four quinolones in spiked egg and milk samples.     

UV partial least-squares calibration and liquid chromatographic methods for direct quantitation of levofloxacin in urine

Levofloxacin was determined in human urine samples by application of a spectrophotometric multivariate calibration partial least-squares (PLS-1) method. A calibration set consisting of standards was prepared by using a multilevel multifactor experimental design. In order to ensure accurate results, the calibration matrix included a urine sample free of levofloxacin (i.e., urine blank). The components of the calibration matrix were levofloxacin and urine. The concentration of levofloxacin ranged from 0.5 to 16.5 microg/mL. Different urine concentrations were used as the second component of the calibration matrix in order to include the information inherent in the changes in the UV spectrum for urine upon dilution. In addition, a high-performance liquid chromatographic method was proposed. In this method, a Shim-pack amino column was used at ambient temperature with a mobile phase of 25 mM potassium dihydrogen phosphate (pH adjusted to 3.1 with phosphoric acid)-acetonitrile (70 + 30, v/v), and the flow rate was 1 mL/min. UV detection at 293 nm was used for quantitation. The proposed methods were applied to the determination of the dissolution rate for tablets containing levofloxacin. The urinary excretion pattern for the cumulative amount of levoflacin excreted was also calculated.     

Normal phase high performance liquid chromatography for determination of paclitaxel incorporated in a lipophilic polymer matrix

A normal phase (NP) high performance liquid chromatography (HPLC) method was developed for analysis of paclitaxel incorporated in poly(sebacic-co-ricinoleic acid), a lipophilic polymer matrix utilized for preparation of an injectable formulation for the localized delivery of paclitaxel. Thin layer chromatography experiments revealed that separation of paclitaxel from the polymer is dependent on the eluting strength (solvent strength) of the mobile phase. The HPLC system consists of a Purospher STRAR Si analytical HPLC column (5 microm, 250mm x 4mm, Merck), and 1-2.5% (v/v) methanol in dichloromethane as the mobile phase. Detection was by UV absorbance at 240 and 254 nm. The effect of the mobile phase composition on paclitaxel retention, peak shape and column efficiency, and the influence of the sample loading on the shape of the paclitaxel peak were studied. The mobile phases used for the chromatography consisted of 1.5% (v/v) methanol in dichloromethane. Paclitaxel was determined in the formulation and in the samples from degradation studies using UV detection at a wavelength of 254 nm. UV detection at 240 nm has advantages for following polymer matrix degradation products due to higher detector response at this wavelength. The utility of the proposed NP HPLC approach was demonstrated by assessment of intra- and inter-batch content uniformity, and by the determination of paclitaxel content after 7 and 60 days exposure of the paclitaxel-loaded polymer matrix to in vitro and in vivo degradation.     

Determination of furosemide in urine samples by direct injection in a micellar liquid chromatographic system

A sensitive, selective and efficient micellar liquid chromatographic (MLC) procedure was developed for the determination of furosemide (4-chloro-N-furfuryl-5-sulfamoylanthranilic acid) in urine samples by direct injection and UV detection. The procedure makes use of a C18 reversed-phase column and a micellar mobile phase of 0.05 mol l(-1) sodium dodecyl sulfate-6% v/v propanol and phosphate buffer at pH 3 to resolve furosemide from its photochemical degradation products. The importance of protecting the standards and urine samples to be analysed from light in the assay of furosemide, avoiding its degradation, was verified. The limit of quantification was 0.15 microg ml(-1) and the relative standard deviation of the inter-day assay was 0.8-0.04% in the 6-82 microg ml(-1) range. Detection of urinary excretion of furosemide was followed up to 12 h after ingestion of the drug by a healthy volunteer. No potential interference from the major metabolite (furosemide acylglucuronide) and its hydrolytic product (4-chloro-5-sulfamoylanthranilic acid) was observed. Commonly administered drugs also did not interfere. The proposed MLC procedure permits the rapid and reproducible measurement of low levels of furosemide in a small amount of urine.     

Quantitation of antihistamines in pharmaceutical preparations by liquid chromatography with a micellar mobile phase of sodium dodecyl sulfate and pentanol.

A reversed-phase liquid chromatographic procedure with a micellar mobile phase of sodium dodecyl sulfate (SDS), containing a small amount of pentanol, was developed for the control of 7 antihistamines of diverse action in pharmaceutical preparations (tablets, capsules, powders, solutions, and syrups): azatadine, carbinoxamine, cyclizine, cyproheptadine, diphenhydramine, doxylamine, and tripelennamine. The retention times of the drugs were <9 min with a mobile phase of 0.15M SDS-6% (v/v) pentanol. The recoveries with respect to the declared compositions were in the range of 93-110%, and the intra- and interday repeatabilities and interday reproducibility were <1.2%. The results were similar to those obtained with a conventional 60 + 40 (v/v) methanol-water mixture, with the advantage of reduced toxicity, flammability, environmental impact, and cost of the micellar-pentanol solutions. The lower risk of evaporation of the organic solvent dissolved in the micellar solutions also increased the stability of the mobile phase.     

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.     

Determination of aromatic amines in environmental water samples using solid-phase extraction modified with sodium dodecyl sulphate and micellar liquid chromatography

Extraction and determination of seven aromatic amines in environmental water samples were performed with solid-phase extraction (SPE) and micellar liquid chromatography (MLC) using experimental design. Extraction of aromatic amines was carried out with a C₁₈ cartridge modified with sodium dodecyl sulphate (SDS). The washing solution and elution solvent for extraction of aromatic amines were aqueous solution containing 5% (v/v) acetonitrile and 5% (v/v) acetone and 3 mL methanol, respectively. The chemometrics approach was applied for the separation optimisation of these compounds using MLC. Different mobile phase compositions were used for modelling based on retention times to obtain the best separation using central composite design. The optimum mobile phase composition for separation and determination of analytes in water samples was 69 mM SDS, 9% v/v 1-propanol and pH = 6.4. Recoveries were between 84.8–93.5% with relative standard deviation (RSD) less than 5.8% (n = 5). Limits of detection and linear range were 1–4.5 and 3.1–125.0 µg/L, respectively. The proposed method was applied to determine the aromatic amines in real samples (river and well waters). Amount of 4-nitroaniline and 3-nitroaniline in river water sample were 2.15 and 1.91 µg/L, respectively.     

Simultaneous determination of haloperidol and its metabolite, reduced haloperidol, in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the simultaneous determination of haloperidol and reduced haloperidol in human plasma, urine and rat tissue homogenates using bromperidol as an internal standard. The method involved extraction followed by injection of 50-80 microliters of the aqueous layer onto a C18 reversed-phase column. The mobile phase was 0.5 M phosphate buffer-acetonitrile-methanol (58:31:11, v/v/v) and the flow-rate was 0.6 ml/min. The column effluent was monitored by ultraviolet detection at 214 nm. The retention times for reduced haloperidol, haloperidol and bromperidol were 5.4, 7.2 and 8.4 min, respectively. The detection limits for haloperidol and reduced haloperidol in human plasma were both 0.5 ng/ml, and the corresponding values in human urine were both 5 ng/ml. The coefficients of variation of the assay were generally low (below 10.7%) for plasma, urine, blood and tissue homogenates. No interferences from endogenous substances or any drug tested were found.     

纤维素衍生物手性固定相高效液相色谱法分离盐酸川丁特罗对映体

以纤维素三-(3,5-二甲基苯基氨基甲酸酯)为手性固定相(Lux Cellulose-1),建立了在正相色谱条件下直接分离盐酸川丁特罗对映体的高效液相色谱法。考察了乙醇、异丙醇等有机改性剂,三氟乙酸、二乙胺等流动相添加剂和柱温对对映体分离的影响。结果显示,酸性和碱性添加剂对对映体分离的影响最为显著: 添加二乙胺时两对映体无分离趋势;添加三氟乙酸时对映体保留强,且分离趋势明显;而同时添加三氟乙酸和二乙胺则两对映体分离显著改善,分离度可达4.0。优化后的色谱条件: 色谱柱为Lux Cellulose-1手性柱(250 mm×4.6 mm, 5 μm),流动相为正庚烷-乙醇-三氟乙酸-二乙胺(88:12:0.3:0.05, v/v/v/v),流速为1.0 mL/min,紫外检测波长为246 nm,柱温为25 ℃。该方法简便,快速,可用于左旋盐酸川丁特罗原料中右旋异构体杂质的检查。     

Mobile phase versus stationary phase approaches to the direct injection of biological fluids in liquid chromatography

The liquid chromatographic analysis of drugs in urine through direct injection without any sample pretreatment was extended to micellar chromatography with nonionic surfactants, the Pinkerton ISRP column and the shielded hydrophobic phase (Hisep) column. The feasibility of using each was demonstrated through the determination of the diuretic, hydrochlorothiazide, in urine. Good separation, recovery, precision and linearity, and adequate limits of detection were obtained for this analysis with all three techniques. The advantages and limitations of the mobile phase approach of micellar chromatography and the two stationary phase approaches are discussed for the direct injection of urine as well as other biological fluids.     

Determination of trazodone in urine and pharmaceuticals using micellar liquid chromatography with fluorescence detection

A simple and reliable liquid chromatographic procedure is described for the determination of trazodone in pharmaceutical formulations and urine samples. The optimized procedure uses fluorimetric detection, a C18 column and a micellar mobile phase of sodium dodecyl sulfate (SDS) and 1-butanol. The mobile phase selected for use was 0.2M SDS and 8% 1-butanol fixed at pH 3 with phosphate buffer. The total analysis time was 10 min. For the analysis of urine samples, one great advantage of the method is that no extraction step is required. The quantification limit was 9.5 ng mL(-1), ensuring the analysis of the drug in biological fluids. The procedure shows good accuracy, repeatability and selectivity. Repeatability and intermediate precision were tested for several concentrations of the drug. Good claim percentages were obtained in the analysis of pharmaceutical formulations. Calibration repeatability in urine matrix was also studied in the 0.06-22.4 microg mL(-1) range. Good recoveries were obtained from spiked urine samples. No interferences from common additives frequently administered with trazodone or from endogenous compounds in urine samples were found. The results show that the procedure is suitable for routine analysis of the drug.