Quantitative determination of the loop diuretic bumetanide in urine and pharmaceuticals by high-performance liquid chromatography with amperometric detection.

A high-performance liquid chromatographic method with amperometric detection has been developed for the determination of the diuretic bumetanide using a microBondapak C18 column. The mobile phase consists of a 50:50 acetonitrile-water mixture containing 5mM KH2PO4-K2HPO4 (pH 4.0). The compound is monitored at +1350 mV with an amperometric detector equipped with a glassy carbon working electrode. A liquid-liquid or solid-liquid extraction is done prior to chromatographic analysis in order to avoid the interferences found in the urine matrix. The percentages of recovery obtained are 71%+/-1% for liquid-liquid extraction and 84.2%+/-0.7% for solid-liquid extraction. The method developed has a linear concentration range from 50 to 499 ng/mL with a reproducibility in terms of relative standard deviation of 1.73% and 3.85% for a concentration level of 70 ng/mL and 237 ng/mL, respectively, and a detection limit of 0.25 ng/mL (3:1 signal-to-noise ratio). The method is applied to the determination of bumetanide in pharmaceutical formulations and urine obtained from hypertensive patients and healthy volunteers after the ingestion of a therapeutic dose of Fordiuran (1 mg bumetanide).     

Development and method validation for testosterone and epitestosterone in human urine samples by liquid chromatography applications

An isocratic high-performance liquid chromatographic method for the determination of testosterone (T) and epitestosterone (ET) in human urine using liquid-liquid or solid-phase extraction (SPE) is developed and validated. The optimum separation is achieved using a Hypersil C(18) column, water-acetonitrile (57:43, v/v) as the mobile phase and UV-absorbance detection at 245 nm. The recoveries obtained for T and ET in liquid-liquid and SPE demonstrate that these procedures are interchangeable. Quantitation limits for T and ET are 8.6 and 5.4 ng/mL using solvent extraction and 7.3 and 5.7 ng/mL using SPE, respectively. The proposed method is used to evaluate the urinary T, ET, and the T/ET ratio for a healthy male population using liquid-liquid extraction, and the T and ET excretion profile for nine healthy men using SPE.     

Solid-phase extraction and high-performance liquid chromatography applied to the determination of quinapril and its metabolite quinaprilat in urine

Quinapril is an antihypertensive drug that belongs to the family of angiotensin-converting enzyme inhibitors. It is metabolized to quinaprilat, which is the compound that is really responsible for the therapeutic action. In this study, a rapid and simple liquid chromatographic method with photometric detection is described and applied to the determination of quinapril and quinaprilat in urine. The cleanup procedure for the urine samples consists of a solid-liquid extraction using C8 cartridges. Under these conditions, both compounds and the internal standard (enalapril maleate) are separated in less than 9 min. Recoveries for quinapril and quinaprilat are greater than 80%. The method is sensitive enough (detection limit of 60 ng/mL for quinapril and 50 ng/mL for quinaprilat) to be applied for the determination of quinapril and quinaprilat in urine samples obtained from four hypertensive patients after the intake of a therapeutic dose.     

Selective liquid chromatographic method for determination of fluoxetine in plasma.

A selective and sensitive liquid chromatographic method was developed for the determination of fluoxetine (FLU) in plasma. FLU was isolated from plasma by liquid-liquid extraction. The chromatographic separation was performed on an analytical 250 x 3.9 mm id Novapak C18 column (4 microm particle size) with an isocratic mobile phase consisting of phosphate buffer-acetonitrile-methanol -triethylamine (58 + 30 + 10 + 2, v/v) adjusted to pH 7. Using UV detection at 226 nm, the detection limit for FLU in plasma was 3 ng/mL. No interferences were found with tricyclic antidepressant drugs, which allows this method to be used in clinical studies. The calibration curve was linear over the concentration range of 10-200 ng/mL. The average recovery was about 80% for plasma. The inter- and intraday assay coefficients of variation were <8%.     

An online field-amplification sample stacking method for the determination of diuretics in urine by capillary electrophoresis-amperometric detection

A simple and sensitive online field-amplification sample stacking (FASS) pre-enrichment method following by capillary electrophoresis with amperometric detection has been developed for the determination of diuretics, such as indapamide (IDP), hydrochlorothiazide (HCT) and bumetanide (BMTN) in urine. Under the optimum conditions, it was found that the low concentration buffer solution could be used as the diluents for simultaneous field-amplification injection of three diuretics after electrokinetically injecting a short water plug (15 kV, 3 s). Three analytes could be well separated within 10 min in an uncoated fused-silica capillary with H(3)BO(3)-Na(2)B(4)O(7) (BB) buffer solution (pH 8.98). The detection limits (S/N=3) were 9.0 ng/mL for IDP, 20 ng/mL for HCT and 1.5 ng/mL for BMTN, respectively. The detection limits of three diuretics were much lower by FASS than that by conventional sample injection, of which the detection limits were 340, 890 and 330 ng/mL for IDP, HCT and BMTN, respectively. Especially, for bumetanide the detection limit was 220-time lower by FASS. The linear ranges of three diuretics were all over three orders of magnitude. The proposed method has been successfully applied to analyze the diuretics in human urine samples without off-column sample pre-concentration.     

Extraction of testosterone and epitestosterone in human urine using aqueous two-phase systems of ionic liquid and salt

Based on aqueous two-phase systems (ATPS) consisting of 1-butyl-3-methylimidazolium chloride, a hydrophilic ionic liquid (IL), and K2HPO4, a new and simple extraction technique, coupled with a reversed-phase high performance liquid chromatography (RP-HPLC), was developed for the simultaneous concentration and analysis of testosterone (T) and epitestosterone (ET) in human urine. Under the optimal conditions, the extraction efficiencies for both analytes were 80-90% in a one-step extraction. The method required only 3.0 mL of urine and a single hydrolysis/deproteinization/extraction step followed by direct injection of the IL-rich upper phase into HPLC system for analysis. The method has been satisfactorily applied to the analysis of T and ET in human urine with detection limits of 1 ng/mL and linear ranges of 10-500 ng/mL for both compounds. Compared with conventional liquid-liquid extraction or solid phase extraction, this new method is much "greener" due to no use of volatile organic solvent and low consumption of IL. The proposed extraction technique opens up new possibilities in the separation of other drugs.     

Rapid determination of anastrozole in plasma by gas chromatography with electron capture detection and its application to an oral pharmacokinetic study in healthy volunteers

A rapid, sensitive and accurate capillary gas chromatographic assay with (63)Ni electron capture detection was developed for the determination of anastrozole in human plasma. It comprises a one-step liquid-liquid extraction procedure and gas chromatography on a capillary column using constant oven temperature. This method has been applied to the oral pharmacokinetic study of anastrozole in healthy Chinese male volunteers. Pharmacokinetic parameters of two anastrozole preparations were evaluated after single, oral administrations to 18 subjects at a dose of 1 mg in a single-blind cross-over trial. Plasma anastrozole concentration-time profiles were best described by a two-compartment model. After oral administrations of imported and domestic anastrozole tablets, the t(max) and C(max) were 1.52 +/- 1.04 h and 8.75 +/- 3.03 ng/mL for the former, and 1.43 +/- 1.12 h and 9.44 +/- 3.59 ng/mL for the latter; the elimination half-life was 46.0 +/- 25.2 h vs 41.2 +/- 8.8 h, and the area under the curve (AUC) was 423 +/- 114 ng h/mL vs 444 +/- 157 ng h/mL. The result indicates that the two products are bioequivalent.     

Development of a chromatographic method for the determination of saquinavir in plasma samples of HIV patients

A simple, sensitive and reproducible high-performance liquid chromatographic method for detecting and quantifying saquinavir in human plasma is described. Verapamil was used as internal standard. The method employes a single liquid-liquid extraction step with tert-butil methyl ether followed by chromatography on a Lichrospher 60 Select B C8 reversed-phase column. Ultraviolet detection was used to identify the compounds of interest. The quantitation limit of saquinavir was 1 ng/mL and only 0.5 mL of plasma sample was required for the determination. The average saquinavir recoveries over a concentration range of 2.5-500 ng/mL ranged from 86 to 95%. Precision and accuracy did not exceed 5%.     

A validated HPLC method with electrochemical detection for simultaneous assay of 5-aminosalicylic acid and its metabolite in human plasma

A high-performance liquid chromatographic method was developed, validated and applied to the simultaneous determination of 5-aminosalicylic acid (5-ASA) and its acetylated metabolite (acetyl-5-ASA) in human plasma. The method involves liquid-liquid extraction with methanol followed by isocratic reversed-phase chromatography on a Kromasil KR100 C(18) column with electrochemical detection. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma samples. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of 5-ASA, acetyl 5-ASA and internal standard were investigated. Limits' of detection were 5 ng/mL for 5-ASA and 10 ng/mL for acetyl-5-ASA, respectively. The method can be used for supporting therapeutical drug monitoring and pharmacokinetic studies.     

Study of the solid phase extraction of pentoxifylline and its major metabolite as a basis of their rapid low concentration gas chromatographic determination in serum.

A gas-liquid chromatographic method for the determination of pentoxifylline and its secondary alcohol metabolite in serum has been developed. The method is based on the combination of solid phase extraction, capillary column separation and nitrogen-phosphorus detection of the analytes. Optimization of the solid phase extraction conditions permitted a low concentration determination, with limits of determination of 2 ng/mL and 10 ng/mL for pentoxifylline and its metabolite, respectively. The simplicity and rapidity of the extraction step was preserved.     

Analysis of a new H2 receptor antagonist, 3-amino-5-[3-[4-(1-piperidinoindanyloxy)]propylamino]-1-methyl-1 H-1,2,4-triazole, in human plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the determination of a new H2 receptor antagonist, 3-amino-5-[3-[4-(piperidinoindanyloxy)]propylamino] -1-methyl-1H-1,2,4-triazole (I), in human plasma and urine was developed. The method employs liquid-liquid extraction of the analyte and an internal standard and chromatographic separation using an alkylphenyl-bonded HPLC column. The total time of chromatography was less than 10 min. Sensitivity was 10 ng/ml for the plasma analysis and 1 microgram/ml for the analysis of I from urine. The coefficients of variation, based on interpolated concentrations, were less than 10%. The method was used for more than 5000 samples during clinical pharmacokinetic studies.     

Determination of atenolol in human urine by gas chromatography-mass spectrometry method

A sensitive and efficient method was developed for the determination of atenolol in human urine by gas chromatography-mass spectrometry (GC-MS). Atenolol and metoprolol (internal standard, IS) were extracted from human urine with a mixture of chloroform and butanol at basic pH with liquid-liquid extraction. The extracts were derivatized with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analyzed by GC-MS using a capillary column. The standard curve was linear (r = 0.99) over the concentration range of 50-750 ng/mL. Intra- and inter-day precision, expressed as the relative standard deviation were less than 5.0%, and accuracy (relative error) was better than 7.0%. The analytical recovery of atenolol from human urine has averaged 91%. The limit of quantification was 50 ng/mL. Also, the method was successfully applied to a patient with hypertension who had been given an oral tablet of 50 mg atenolol.     

Atmospheric pressure photoionization applied to quantitation of cyproterone acetate in human plasma

Cyproterone acetate [6-chloro-1beta,2beta-dihydro-17alpha-hydroxy- 3'H-cyclopropa(1,2)-pregna-1,4,6-triene-3,20-dione acetate] is a powerful antiandrogen used in the treatment of women suffering from disorders associated with androgenization such as hirsutism and acne. A fast, sensitive, and robustness method is developed for the determination and quantitation of cyproterone acetate in human blood plasma by liquid chromatography coupled with tandem mass spectrometry. Cyproterone acetate is extracted from 0.2 mL human plasma by liquid-liquid extraction. The method has a chromatographic run of 4.5 min, using a C18 analytical column (100- yen 2.1-mm i.d.), and the linear calibration curve over the range is linear from 1 to 500 ng/mL (r2 > 0.994). The between-run precision, based on the relative standard deviation replicate quality controls, is 96.2% (3 ng/mL), 97.5% (120 ng/mL), and 99.1% (400 ng/mL). The between-run accuracy was +/- 2.7%, 3.1%, and 4.8% for the previously mentioned concentrations, respectively. The method is employed in a bioequivalence study of two tablet formulations of cyproterone acetate (100 mg).     

Determination of diazinon, chlorpyrifos, and their metabolites in rat plasma and urine by high-performance liquid chromatography

This study reports a simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of the insecticide diazinon (O,O-diethyl-O[2-isopropyl-6-methylpyridimidinyl] phosphorothioate), its metabolites diazoxon (O,O-diethyl-O-2-isopropyl-6-methylpyridimidinyl phosphate) and 2-isopropyl-6-methyl-4-pyrimidinol, the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate) and its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate), and TCP (3,5,6-trichloro-2-pyridinol) in rat plasma and urine samples. The method is based on using C18 Sep-Pak cartridges for solid-phase extraction and HPLC with a reversed-phase C18 column and programmed UV detection ranging between 254 and 280 nm. The compounds are separated using a gradient of 1% to 80% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.5 mL/min in a period of 16 min. The limits of detection ranged between 50 and 150 ng/mL, and the limits of quantitation were 100 to 200 ng/mL. The average percentage recovery of five spiked plasma samples were 86.3 +/- 8.6, 77.4 +/- 7.0, 82.1 +/- 8.2, 81.8 +/- 8.7, 73.1 +/- 7.4, and 80.3 +/- 8.0 and from urine were 81.8 +/- 7.6, 76.6 +/- 7.1, 81.5 +/- 7.9, 81.8 +/- 7.1, 73.7 +/- 8.6, and 80.7 +/- 7.7 for diazinon, diazoxon, 2-isopropyl-6-methyl-4-pyrimidinol, chlorpyrifos, chlorpyrifos-oxon, and TCP, respectively. The relationship between the peak area and concentration was linear over a range of 200 to 2,000 ng/mL. This method was applied in order to analyze these chemicals and metabolites following dermal administration in rats.     

Preconcentration of indapamide from human urine using molecularly imprinted solid‐phase extraction

A simple, sensitive, and selective molecularly imprinted solid‐phase extraction and spectrophotometric method has been developed for the clean‐up and preconcentration of indapamide from human urine. Molecularly imprinted polymers were prepared by a non‐covalent imprinting approach using indapamide as a template molecule, 2‐(trifluoromethyl) acrylic acid as a functional monomer, ethylene glycol dimethacrylate as a crosslinker, N,N‐azobisisobutyronitrile as a thermal initiator and acetonitrile as a porogenic solvent. A non‐imprinted polymer was also prepared in the same way, but in the absence of template. Molecularly imprinted polymer and non‐imprinted polymer sorbents were dry‐packed into solid‐phase extraction cartridges. Eluates from cartridges were analyzed using a spectrophotometer for the determination of indapamide by referring to the calibration curve in the range 0.14–1.50 μg/mL. Preconcentration factor, limit of detection, and limit of quantification were 16.30, 0.025 μg/mL, and 0.075 μg/mL, respectively. A relatively high imprinting factor (9.3) was also achieved and recovery values for the indapamide spiked into human urine were in the range of 80.1–81.2%. In addition, relatively low within‐day (0.17–0.42%) and between‐day (1.1–1.4%) precision values were obtained as well. The proposed molecularly imprinted solid‐phase extraction and spectrophotometric method was successfully applied to selective extraction, preconcentration, and determination of indapamide from human urine samples.     

液相色谱-串联质谱法测定尿液中的内源性类固醇激素

建立了液相色谱-串联质谱(LC-MS/MS)测定尿液中的内源性类固醇激素的方法。尿样经葡萄糖醛酸甙酶酶解后进行液-液提取,以甲醇-0.1%甲酸缓冲液(含0.02 mol/L乙酸铵)(体积比为68:32)为流动相,采用Cosmosil C18色谱柱分离,并以三重四极杆串联质谱多反应监测扫描方式对尿样中的脱氢表雄酮(DHEA)、睾酮、表睾酮、雄酮和苯胆烷醇酮等5种激素进行检测。方法的最低检出限为0.01～10 ng/mL,平均回收率为96.7%～106.5%,日内和日间相对标准偏差(RSD)分别小于7%和11%。应用所建立的方法测定了健康志愿者口服DHEA后尿液中内源性类固醇激素的变化情况,结果表明该方法样品处理简便,色谱分离完全,结果准确可靠,可替代气相色谱-质谱法用于体液中内源性类固醇激素兴奋剂的常规分析。     

Development and validation of automated SPE‐LC‐MS/MS method for determination of indapamide in human whole blood and its application to real study samples

A fast and simple liquid chromatography–electrospray ionization tandem mass spectrometry method for determination of indapamide in human whole blood was developed and validated. The sample extraction of indapamide from human whole blood was achieved using automated solid‐phase extraction. Chromatographic separation was performed on Kinetex C₁₈ column (100 × 2.1 mm, 1.7 µm particle size) using acetonitrile and 2 mm ammonium formate in ratio 90:10 (v/v) as a mobile phase. The mass spectrometer was operated in the multiple reaction monitoring mode using positive electrospray ionization for indapamide and the internal standard (zolpidem tartarate). The total run time was 2.5 min. The present method was found to be linear in the concentration range of 1–50 ng/mL with the coefficient of determination 0.9987. The absolute recoveries of indapamide were 90.51–93.90%. The method was validated according the recommendations for validation of bioanalytical methods of European Medicines Agency guideline and was successfully used to analyze human whole blood samples for application in a pharmacokinetic study. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid HPLC measurement of buflomedil in plasma in poisoning cases

A rapid high-performance liquid chromatographic method for the determination of buflomedil in human plasma is described. It requires a single liquid-liquid extraction step from 1 mL of plasma with diethyl ether followed by chromatography on a Nova Pak C(18) reversed-phase column and detection by ultaviolet light. Metoclopramide was used as internal standard. The method is sensitive with a quantification limit at 500 ng/mL. It was used for the determination of buflomedil in biological fluids in poisoning cases.     

Determination of nitroimidazole residues in porcine urine by liquid chromatography/tandem mass spectrometry

A reliable and sensitive method was developed for determination of nitroimidazoles in porcine urine. Sample preparation involves an extraction with ethyl acetate, followed by a solid-phase extraction cleanup step. The final extract is injected into the liquid chromatography/tandem mass spectrometry system in the positive-ion electrospray ionization mode. A C8 column with water and acetonitrile as the mobile phase is used for chromatographic separation under gradient conditions. Overall average recoveries ranged from 83 to 107%. The limits of detection ranged from 0.03 to 0.05 ng/mL, and the limits of quantitation, from 0.1 to 0.2 ng/mL.     

Determination of selected stimulants in urine for sports drug analysis by solid phase extraction via cation exchange and means of liquid chromatography-tandem mass spectrometry

Stimulatory substances applied during competition possess a reasonable potential as performance enhancing agents and their misuse in elite sport has been frequently reported during the last few decades. An analytical method for the qualitative determination of selected stimulants containing a primary or secondary amine moiety in human urine for doping control purposes was developed. A rapid and highly specific procedure based on a sample preparation using weak cation exchange solid phase extraction (SPE-XCW) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a C6-Phenyl analytical column allowed the unambiguous identification of the target analytes down to low ng mL(-1) concentration levels. Validation provided recovery rates of better than 75%, precisions of less than 20% and a linear approximation in the required working range (10-750 ng mL(-1)) were obtained for 19 different target compounds. This method provides a rugged and highly specific alternative to the established method utilising gas or liquid chromatography after liquid-liquid extraction.