High-performance liquid chromatographic determination of 3 alpha,5 beta-tetrahydroaldosterone in human urine with chemiluminescence detection.

A sensitive method for the determination of 3 alpha,5 beta-tetrahydroaldosterone (THALD) in human urine is described. The method uses high-performance liquid chromatography with chemiluminescence detection. Urinary THALD, released by enzyme hydrolysis, is isolated and concentrated using a Sephadex G-25M column and Bond-Elut C1 cartridges, and then oxidized by copper(II) acetate to form the corresponding glyoxal derivative. The glyoxal derivative is converted into the chemiluminescent quinoxaline by reaction with 4,5-diaminophthalhydrazide. The chemiluminescent quinoxaline is separated within 50 min on a reversed-phase column (TSKgel ODS-120T) with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the quinoxaline with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline solution. The detection limit for THALD is 0.6 pmol (220 pg) ml-1 in urine [1.5 fmol (0.53 pg) per 20 microliters injection] at a signal-to-noise ratio of 3. This method permits the sensitive and precise determination of THALD in human urine (50 microliters) from normal subjects and a patient with primary aldosteronism.     

Determination of 21-hydroxycorticosteroids in human urine by high-performance liquid chromatography with fluorescence detection

A simple and sensitive high-performance liquid chromatographic method with fluorescence detection for the determination of nineteen 21-hydroxycorticosteroids is described. The corticosteroids are oxidized by cupric acetate to form the corresponding glyoxal derivatives. The derivatives are converted into fluorescent quinoxalines by reaction with 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for alpha-dicarbonyl compounds. The quinoxalines are separated within 70 min on a reversed-phase column (TSK gel ODS-120T) by stepwise elution with mixtures of methanol, acetonitrile, and 1.0 M ammonium acetate. The detection limits are 0.14-29.4 pmol at a signal-to-noise ratio of 3 in a 50-microliter injection volume. This sensitivity permits precise determination of hydrocortisone, cortisone, corticosterone, and their tetrahydro derivatives in 500 microliters of normal human urine.     

Determination of free and total catecholamines in human urine by HPLC with fluorescence detection

A simple and highly sensitive method for the determination of free and total (free + conjugated) catecholamines (norepinephrine, epinephrine and dopamine) in human urine is described which employs HPLC with fluorescence detection. Conjugated catecholamines (sulfate form) are hydrolyzed by a sulfatase-mediated reaction to the corresponding free amines. After cation exchange chromatography on a Toyopak IC-SP S cartridge, catecholamines and isoproterenol (internal standard) in urine samples were converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine. These compounds were separated within 8 min on a reversed phase column with isocratic elution using a mixture of water, methanol and acetonitrile containing a Tris-hydrochloric acid buffer (pH 7.0). The detection limit for each catecholamine is ca 2 fmol per 100 microL injection volume.     

Determination of free and total phenylacetic and p- and m-hydroxyphenylacetic acids in human urine by liquid chromatography with fluorimetric detection

A highly sensitive and rapid liquid chromatographic method for the determination of free and total phenylacetic and p- and m-hydroxyphenylacetic acids in human urine is described. After extraction of urine with diethyl ether, these acids and phenylpropionic acid (internal standard) are converted into the corresponding fluorescent derivatives by treatment with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and 18-crown-6 in acetonitrile. The derivatives are separated on a reversed-phase column (Radial-Pak cartridge C18) with aqueous 65% (v/v) methanol and detected fluorimetrically. The detection limits for phenylacetic and p- and m-hydroxyphenylacetic acids are 5, 30 and 100 fmol, respectively, at a signal-to-noise ratio of 5 in a 20-microliter injection volume. This sensitivity permits precise determination of the free and total acids in 20 microliter of normal human urine.     

High-performance liquid chromatographic determination of alpha-keto acids in human serum and urine using 1,2-diamino-4,5-methylenedioxybenzene as a precolumn fluorescence derivatization reagent

A simple and highly sensitive high-performance liquid chromatographic (HPLC) method for the determination of alpha-keto acids in human serum and urine is described. In an acidic solution, twelve species of alpha-keto acids examined were converted by reaction with 1,2-diamino-4,5-methylenedioxy-benzene into highly fluorescent derivatives. The derivatives were separated isocratically by reversed-phase HPLC on a TSK gel ODS-80TM column and detected fluorimetrically. Eight alpha-keto acids in human serum and eleven alpha-keto acids in human urine can be determined simultaneously. The detection limits (signal-to-noise ratio = 5) are 6-44 fmol in an injection volume of 5 microliters. The intra-assay relative standard deviations for both serum and urine sample analyses are usually ca. 5%.     

A method for routine quantitation of urinary 8-hydroxy-2'-deoxyguanosine based on solid-phase extraction and micro-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

8-Hydroxy-2'-deoxyguanosine (8OHdG), one of the major oxidative DNA lesions induced by radical agents, is commonly used as a biomarker for oxidative stress, nowadays preferably in urine. In the absence of a commercially available internal standard a micro-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (micro-HPLC/ESI-MS/MS) method, suitable for routine analysis of 8OHdG in human urine using external calibration, was developed. Evaluation of the matrix effect showed that the method allows highly sensitive and accurate quantitation despite the absence of an internal standard. HPLC analysis was performed using gradient elution at a flow rate of 10 microL min(-1) using a capillary reversed-phase column and an injection volume of 0.5 microL, with detection of 8OHdG in positive multiple reaction monitoring (MRM) mode. The absolute limit of detection was 0.35 fmol using m/z 168 as a quantifier (fragment) ion. A linear (R2> 0.999) calibration curve in urine was obtained over a range 0.2-10 ng mL(-1). This method is about 20 times more sensitive than previously described procedures, and is characterized by high accuracy (mean 90%) and good reproducibility (RSD <10%). The optimized method was applied to determination of 8OHdG in 18 urinary samples derived from three healthy volunteers. 8OHdG urinary excretion ranged from 3.0-7.9 microg/day, and a large intra-individual variation was found. This method, which effectively circumvents the need for isotopically labeled 8OHdG (internal standard), is suitable for routine monitoring of exposure to DNA-damaging factors in a large number of subjects.     

Validation and application of a method for the determination of nicotine and five major metabolites in smokers' urine by solid-phase extraction and liquid chromatography-tandem mass spectrometry

An SPE-LC-MS/MS method was developed, validated and applied to the determination of nicotine and five major metabolites in human urine: cotinine, trans-3'-hydroxycotinine, nicotine-N-glucuronide, cotinine-N-glucuronide and trans-3'-hydroxycotinine-O-glucuronide. A 500 microL urine sample was pH-adjusted with phosphate buffer (1.5 mL) containing nicotine-methyl-d3, cotinine-methyl-d3 and trans-3'-hydroxycotinine-methyl-d3 internal standards. For the unconjugated metabolites, an aliquot (800 microL) of the buffered solution was applied to a 30 mg Oasis HLB-SPE column, rinsed with 2% NH4OH/H2O (3.0 mL) and H2O (3.0 mL) and eluted with methanol (500 microL). The eluate was analyzed isocratically (100% methanol) by LC-MS/MS on a diol column (50 x 2.1 mm). For the total metabolites, a beta-glucuronidase/buffer preparation (100 microL) was added to the remaining buffered solution and incubated at 37 degrees C (20 h). An aliquot (800 microL) of the enzymatically treated buffered solution was extracted and analyzed in the same manner. The conjugated metabolites were determined indirectly by subtraction. The quantitation range of the method (ng/mL) was 14-10,320 for nicotine, 15-9800 for cotinine and 32-19,220 for trans-3'-hydroxycotinine. The validated method was used to observe diurnal variations from a smoker's spot urine samples, elimination half-lives from a smoker's 24 h urine samples and metabolite distribution profiles in the spot and 24 h urine samples.     

Determination of M+4 stable isotope labeled cortisone and cortisol in human plasma by microElution solid-phase extraction and liquid chromatography/tandem mass spectrometry

A sensitive microElution solid-phase extraction (SPE) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of M+4 stable isotope labeled cortisone and cortisol in human plasma. In this method, M+4 cortisone and M+4 cortisol were extracted from 0.3 mL of human plasma samples using a Waters Oasis HLB 96-well microElution SPE plate using 70 microL methanol as the elution solvent, and chromatographed on a Waters Symmetry C18 column (4.6 x 50 mm, 3.5 microm). M+9 cortisone and M+9 cortisol were used as the internal standards. A PE Sciex API 4000 tandem mass spectrometer interfaced with the liquid chromatograph via a turboionspray source was used for mass analysis and detection. The selected reaction monitoring (SRM) of precursor --> product ion transitions were monitored at m/z 365.2 [M+H](+) --> 167.0 and at m/z 367.3 [M+H](+) --> 125.1 for M+4 cortisone and M+4 cortisol, respectively. The lower limit of quantitation was 0.1 ng mL(-1) and the linear calibration range was from 0.1 to 100 ng mL(-1) for both analytes. This method demonstrated to be very reproducible and reliable.     

Simultaneous determination of serotonin and 5-hydroxyindole-3-acetic acid in human urine by automated precolumn derivatization and semi-microbore column liquid chromatography with fluorescence detection.

An improved high-performance liquid-chromatographic (HPLC) assay for the simultaneous determination of serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) in human urine is reported. Following the automated precolumn derivatization of urinary 5-HT and 5-HIAA with benzylamine, the derivatives are separated by isocratic elution on a reversed-phase C18 semi-microbore column, and are fluorometrically detected at an excitation wavelength of 345 nm and an emission wavelength of 480 nm. The detection limits of 5-HT and 5-HIAA are 7 and 5 nmol/l in urine (0.7 and 0.5 fmol/20-microl injection). The proposed automatic method permits a highly selective and sensitive determination of 5-HT and 5-HIAA in human urine without any sample purification.     

Validation of a novel, fully automated high throughput high-performance liquid chromatographic/tandem mass Spectrometric method for quantification of pantoprazole in human plasma

An automated high-throughput HPLC/MS/MS method was developed for the quantitative determination of pantoprazole in human plasma. Only 100 microL plasma was placed in 2.2 mL 96 deep-well plates, and both pantoprazole and omeprazole (IS) were extracted from human plasma by liquid-liquid extraction, using diethyl ether-dichloromethane (70:30, v/v) as the organic solvent. Robotic liquid-handling workstations were used for all liquid transfer and solution preparation steps and resulted in a short sample preparation time. After vortexing, centrifugation, and freezing, the supernatant organic solvent was evaporated and reconstituted in a small volume of reconstitution solution. Sample analysis was performed by utilizing the combination of RP-HPLC/MS/MS, with positive-ion electrospray ionization and multiple reaction monitoring detection. The chromatographic run time was set at 1.8 min with a flow rate of 0.6 mL/min on a Nucleosil octylsilyl (C8) analytical column. The method was proven to be sensitive, specific, accurate, and precise for the determination of pantoprazole in human plasma. The method was applied to a bioequivalence study after per os administration of a 40 mg pantoprazole gastric retentive tablet.     

Automated sample treatment with the injection of large sample volumes for the determination of contaminants and metabolites in urine

This work reports the development of a simple and automated method for the quantitative determination of several contaminants (triazine, phenylurea, and phenoxyacid herbicides; carbamate insecticides and industrial chemicals) and their metabolites in human urine with a simplified sample treatment. The method is based on the online coupling of an extraction column with RP LC separation–UV detection; this coupling enabled fast online cleanup of the urine samples, efficiently eliminating matrix components and providing appropriate selectivity for the determination of such compounds. The variables affecting the automated method were optimized: sorbent type, washing solvent and time, and the sample volume injected. The optimized sample treatment reported here allowed the direct injection of large volumes of urine (1500 μL) into the online system as a way to improve the sensitivity of the method; limits of detection in the 1–10 ng/mL range were achieved for an injected volume of 1500 μL of urine, precision being 10% or better at a concentration level of 20 ng/mL. The online configuration proposed has advantages such as automation (all the steps involved in the analysis – injection of the urine, sample cleanup, analyte enrichment, separation and detection – are carried out automatically) with high precision and sensitivity, reducing manual sample manipulation to freezing and sample filtration.     

Simple and highly sensitive determination of free fatty acids in human serum by high performance liquid chromatography with fluorescence detection.

A highly sensitive and simple reversed phase high performance liquid chromatographic (HPLC) method for the quantitative determination of free fatty acids in human serum is presented. The method is based on the direct derivatization of serum fatty acids with 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 37 degrees C. The resulting derivatives are separated within 75 min on a reversed phase column (YMC Pack C8) with a gradient elution of aqueous acetonitrile and detected fluorimetrically. The detection limits are 2.5-5 fmol in a 10 microL injection volume. The sensitivity permits precise determination of free fatty acids in 5 microL serum. The method is simple and is without the conventional liquid-liquid extraction steps of serum fatty acids.     

Measurement of N-acetylneuraminic acid in human serum and urine by high performance liquid chromatography with chemiluminescence detection.

A highly sensitive method for the determination of N-acetylneuraminic acid in human serum and urine is investigated. This method employs high performance liquid chromatography with chemiluminescence detection. N-Acetylneuraminic acid, released by hydrochloric acid hydrolysis of serum and urine, and N-glycolylneuraminic acid (internal standard) are converted into chemiluminescent derivatives with 4,5-diaminophthalhydrazide dihydrochloride, a chemiluminescence derivatization reagent for alpha-keto acids. The derivatives are separated within 35 min on a reversed phase column, TSKgel ODS-120T, with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the derivatives with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline solution. The detection limit for N-acetylneuraminic acid is 9 fmol (signal-to-noise ratio = 3). This sensitivity permits precise determination of N-acetylneuraminic acid in 10 nL of serum or 50 nL of urine. The method is applied to the determination of the N-acetylneuraminic acid in human sera from normal subjects and cancer patients and in normal urine.     

Determination of oestrogens in pregnancy urine by high-performance liquid chromatography with fluorescence detection

A simple and sensitive high-performance liquid chromatographic method is described for the determination of three oestrogens (oestriol, oestrone and oestradiol) in pregnancy urine. Free oestrogens are extracted with chloroform from the urine sample. The phenolic group of each oestrogen in chloroform is formylated in the presence of an alkaline aqueous solution, and the resulting aldehyde is converted into a fluorescent derivative by reaction with 1,2-diamino-4,5-dimethoxybenzene. In order to determine free and conjugated oestrogens, conjugated oestrogens are hydrolysed by heating in hydrochloric acid before the extraction and then treated in the same way as free oestrogens. The fluorescent derivatives are separated on a reversed-phase column, TSKgel ODS-120T, with stepwise gradient elution using an aqueous methanol-containing phosphate buffer (pH 2.2). The lower limit of detection for each oestrogen is ca. 200 fmol per 100-microliters injection volume.     

High-throughput liquid-chromatography method with fluorescence detection for reciprocal determination of furosemide or norfloxacin in human plasma

A simple, high-throughput, highly selective and sensitive HPLC-FLD method for isolation and determination of furosemide and/or norfloxacin in human plasma samples following a simple organic solvent deproteinization step with acetonitrile as sample 'clean-up' procedure is reported. One of the two drug substances plays the internal standard role for the determination of the other. Separation of analyte and internal standard was achieved in less than 5.3 min (injection to injection) on a Chromolith Performance RP-18e column, using an aqueous component containing 0.015 mol/L sodium heptane-sulfonate and 0.2% triethylamine brought to pH = 2.5 with H(3)PO(4). The composition of the mobile phase was: acetonitrile-methanol-aqueous component = 70:15:15 (v/v/v) and the flow-rate was set up to 3 mL/min. The chromatographic method applied to the determination of furosemide relies on fluorescent detection parameters of 235 nm for the excitation wavelength, and 402 nm for the emission wavelength. In case of norfloxacin, the excitation wavelength is set up to 268 nm and the emission wavelength is set up to 445 nm. The overall method leads to quantitation limits of about 27 ng/mL for furosemide, and 19.5 ng/mL for norfloxacin, using an injection volume of 250 microL. The method was applied to the bioequivalence study of two furosemide-containing formulations.     

High-performance liquid chromatography/tandem mass spectrometry method for the determination of arbidol in human plasma

An HPLC/MS/MS method for the determination of arbidol in human plasma was developed. Arbidol and internal standard (loratadine) were extracted from alkaline plasma with tert-butyl methyl ether and analyzed on a Zorbax SB C18 column (30 x 2.1 mm id, 3.5 microm particle size). The detection was by monitoring arbidol at m/z 479.1 --> 434.1 and the internal standard at m/z 383.2 --> 337.2. The method was validated according to U.S. Food and Drug Administration guidelines. The calibration curve was linear over the range of 0.5-500 ng/mL using a 100 microL sample volume. The intraday and interday precisions were less than 6.5%, and acceptable values were obtained for accuracy, recovery, and sensitivity. The developed method was selective, simple, sensitive, and easily applicable.     

Development and validation of HPLC method for the determination of α-tocopherol in human erythrocytes for clinical applications

In this work, a simple isocratic reversed-phase HPLC method for determination of alpha-tocopherol in human erythrocytes has been developed and validated. After separation of plasma the erythrocytes were washed three times with 0.9% sodium chloride containing 0.01% butylated hydroxytoluene (BHT) as antioxidant and then were diluted 1:1 (v/v) with the same solution. In the liquid-liquid extraction (LLE) procedure, 2500 microL of n-hexane was added to 500 microL of erythrocytes. After 2 min this mixture was deproteinized by addition of cool ethanol (500 microL, 5 min) denatured with 5% methanol containing alpha-tocopherol acetate (20 micromol L(-1)), as internal standard, and then extracted for 5 min by vortex mixing. After centrifugation (10 min, 1600xg) an aliquot (2000 microL) of the clean extract was separated and evaporated under nitrogen. The residue was dissolved in 400 microL methanol and analysed by reversed-phase HPLC on a 4.6 mmx150 mm, 5 microm Pecosphere C18 column; the mobile phase was 100% methanol, flow rate 1.2 mL min(-1). The volume injected was 100 microL and detection was by diode-array detector at a wavelength of 295 nm. The extraction recovery of alpha-tocopherol from human erythrocytes was 100.0+/-2.0%. The detection limit was 0.1 micromol L(-1) and a linear calibration plot was obtained in the concentration range 0.5-20.0 micromol L(-1). Within determination precision was 5.2% RSD (n=10), between determination precision was 6.1% RSD (n=10). The method was applied successfully in a clinical study of patients with acute pancreatitis and for determination of the reference values in the healthy Czech population.     

Temperature-promoted large-volume solute enrichment in column-switching miniaturized liquid chromatography: determination of an antioxidant

A two-valve sub-ambient temperature-promoted reversed-phase packed-capillary liquid-chromatography column-switching system has been tailored for sensitive determination of hydrophobic compounds. Such compounds are not easily dissolved in solvent mixtures of non-eluting properties that traditionally are used for solute enrichment in reversed-phase liquid chromatography. Enrichment-column solute focusing of large sample volumes was promoted by use of sub-ambient temperatures only, allowing the use of sample solvents that were stronger or equal to the mobile phase solvent strength. Subsequent column switching and enrichment-column temperature increment provided efficient low-dispersion back-flushed enrichment-column solute desorption onto the analytical column, where the solute was subjected to temperature-programmed gradient action. The antioxidant, Irganox 1076 (octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate) extracted from low density polyethylene with 100% acetonitrile served as a hydrophobic model compound. The mobile phase consisted of acetonitrile containing 10 mM triethylamine and formic acid, and the 0.25 mm id enrichment-column and analytical column in lengths of 27 and 250 mm, respectively, were packed with 3.5 microm Kromasil C18 particles. Sample volumes of up to 500 microL were successfully focused on the enrichment column at 5 degrees C using loading flow rates of up to 40 microL min(-1) prior to temperature programming to 90 degrees C. The concentration limit of detection of Irganox 1076 was 6 ng mL(-1) when using an injection volume of 500 microL. The within-assay precision was in the range 3.5-6.8% (n = 6) while the between-day precision was 7.5% (n = 3) relative standard deviation. The method was linear within the investigated mass range 3-100 ng (R2 = 0.9993).     

A simple and sensitive assay for the quantitative analysis of rivastigmine and its metabolite NAP 226-90 in human EDTA plasma using coupled liquid chromatography and tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the determination of rivastigmine and its major metabolite NAP 226-90 is presented. A 100 microL plasma aliquot was spiked with a structural analogue of rivastigmine as internal standard (PKF214-976-AE-1) and proteins were precipitated by adding 200 microL of methanol. After centrifugation a volume of 100 microL of the clear supernatant was mixed with 100 microL of methanol/water (30:70, v/v) and volumes of 25 microL were injected onto the HPLC system. Separation was acquired on a 150 x 2.0 mm i.d. Gemini C18 column using a gradient system with 10 mM ammonium hydroxide and methanol. Detection was performed by using a turboionspray interface and positive ion multiple reaction monitoring by tandem mass spectrometry. The assay quantifies rivastigmine from 0.25 to 50 ng/mL and its metabolite NAP 226-90 from 0.50 to 25 ng/mL, using human plasma samples of 100 microL. Validation results demonstrate that rivastigmine and metabolite concentrations can be accurately and precisely quantified in human EDTA plasma. This assay is now used to support clinical pharmacologic studies with rivastigmine.     

Sensitive and simultaneous determination of HIV protease inhibitors in rat biological samples by liquid chromatography-mass spectrometry

A sensitive and simultaneous liquid chromatographic-mass spectrometric (LC/MS) method for the determination of current four HIV protease inhibitors (PIs), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV) and amprenavir (APV) in rat plasma and liver dialysate by a microdialysis method was described. An isocratic LC/MS method in combination with atmospheric pressure chemical ionization was developed for the determination of these four PIs in biological samples in the same run. The analytes including an internal standard were extracted from 100 microL of plasma or 150 microL of liver dialysate samples by salting-out with 100 microL of ice-cold 2 M K(3)PO(4) followed by ether extraction. The separation of analytes was carried out on a reversed-phase semi-micro column using 50% of acetonitrile containing 1% acetic acid as mobile phase at a flow rate of 0.2mL/min(-1). The separation was completed within 5 min. Precision, recovery and limits of detection indicated that the method was suitable for the quantitative determination of these PIs in rat plasma or liver dialysate. This simple, sensitive and highly specific LC/MS method is suitable for pharmacokinetic studies and therapeutic drug monitoring in AIDS patients who receive double protease therapy.