Microanalytical high-performance liquid chromatographic assay for cefpirome in human milk and urine.

To permit the characterization of cefpirome disposition in lactating females, a previously published high-performance liquid chromatographic (HPLC) method for determining the drug in serum was adapted for use with milk and urine. This automated, microanalytical technique requires 50 microliters of biological matrix, which is subjected to an isopropanol extraction. Chromatography was accomplished using a microbore HPLC system, a reversed-phase C18 column and a mobile phase of 0.3% triethylamine in water (pH 5.1). Cefpirome and the internal standard (beta-hydroxypropyltheophylline) were monitored using UV detection at 240 nm and had retention times of 2.84 and 5.05 min, respectively. The method was linear up to 500 mg/l for both matrices and had a limit of detection of 0.6 mg/l. The interday variation (relative standard deviation) at concentrations of 5.0, 50.0 and 500.0 mg/l was consistently less than 5% in both urine and breast milk. The method was found to be free from interference by other commonly administered medications and readily adaptable for use in clinical investigations. The ease of sample preparation, small sample volume requirement, short chromatographic time, apparent lack of interferences, analytical sensitivity and high precision and accuracy make this method ideal for use in pharmacokinetic investigations involving the determination of cefpirome in human milk and urine.     

New simplified microassay for the quantitation of theophylline and its major metabolites in serum by high-performance liquid chromatography

A high-performance liquid chromatographic technique is presented for simultaneous determination of theophylline, 3-methylxanthine, 1-methyluric acid, 1,3-dimethyluric acid and caffeine in serum using beta-hydroxyethyltheophylline (BHET) as an internal standard. An aliquot of a serum sample (100 microliters) was added to 40 microliters of an internal standard solution (BHET; 100 micrograms/ml) and vortexed. A 20% trichloroacetic acid solution (60 microliters) was then added; the mixture was vortexed, centrifuged and 100 microliters were injected onto a C8 column maintained at 45 degrees C. Inter- and intra-day variability of the assay for all compounds was less than 4.3%. Sensitivity ranged from 10 ng/ml for 1-methyluric acid to 25 ng/ml for theophylline. Drugs commonly coadministered with theophylline did not interfere with the assay.     

Mix-mode TiO-C18 monolith spin column extraction and GC-MS for the simultaneous assay of organophosphorus compounds and glufosinate, and glyphosate in human serum and urine

A rapid, specific, and sensitive method for the simultaneous quantitation of organophosphates (fenitrothion (MEP), malathion, and phenthoate (PAP)), glufosinate (GLUF), and glyphosate (GLYP) in human serum and urine by gas chromatography-mass spectrometry (GC-MS) has been validated. All of the targeted compounds together with the internal standard were extracted from the serum and urine using a mix-mode TiO-C(18) monolithic spin column. The recovery of organophosphates from serum and urine ranged from 12.7 to 49.5%. The recovery of GLUF and GLYP from serum and urine ranged from 1.9 to 7.9%. The intra- and inter-accuracy and precision (expressed as relative standard deviation, %RSD) were within 96.7-107.7% and 4.0-13.8%, respectively. The detection and quantitation limits for serum and urine were 0.1 and 0.1 μg/ml, respectively, for organophosphates, 0.1 and 0.5 μg/ml, respectively for GLUF and GLYP. The method had linear calibration curves ranging from 0.1 to 25.0 μg/ml for organophosphates and 0.5-100.0 μg/ml for GLUF, and GLYP. The validated method was successfully applied to a clinical GLYP poisoning case.     

Rapid Turbidimetric Determination of Lipase Activity In Biological Fluids

Abstract

An improved turbidimetric technique for the determination of lipase activity is presented. A stable and reproducible triolein emulsion obtained with the use of isopropanol is suitable substrate for the lipase activities present in human serum and pancreatic juice.

The within-run precision CV is 5.2 % whereas the day-to-day precision CV is 7.3 % for enzyme activities near 400 U/l. Linear response is obtained for lipase activities ranging from 60 to 780 U/l. the detection lower limit is 18 U/l. the use of optimal colipase, sodium taurodeoxycholate and calcium chloride concentrations offer the system increased specificity and analytical sensitivity.     

Measurement of amphotericin B in serum or plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the analysis of amphotericin B in 25 microliters of serum or plasma is described. The procedure involves the addition of the internal standard, p-nitrobenzyloxyamine, to the sample followed by a precipitation of protein with acetonitrile. The supernatant is directly injected into a chromatograph attached to a reversed-phase mu Bondapak (Waters) column containing C18 packing. The mobile phase is a 60 : 40 mixture of a sodium acetate buffer (10 mM, pH 7.0)--acetonitrile, and we employ a flow-rate of 1.5 ml/min and a detection wavelength of 405 nm. Total analysis time per sample is 10 min. Coefficients of variation were found to be less than 4% for concentrations less than 2 mg/l. Analytical recoveries were between 75 and 80%. No drug or drug metabolite interference was found. The method will be used to study pharmacokinetic and pharmacodynamic data in a pediatric population.     

Determination of proquazone and its m-hydroxy metabolite by high-performance liquid chromatography. Clinical application: pharmacokinetics of proquazone in children with juvenile rheumatoid arthritis

A method for the determination of proquazone and its m-hydroxy metabolite in serum and urine by reversed-phase high-performance liquid chromatography is described. The technique is based on a single extraction of the unchanged drug, its metabolite and an internal standard from serum or urine with chloroform. The column was packed with mu Bondapak C18 and the mobile phase was acetonitrile--water (50:50) (pH 3). The detection limits for proquazone and its metabolite were 0.02 mumol/l using 500 microliters of sample. For the determination of the total m-hydroxy metabolite only 100 microliters of sample are needed. The method described is suitable for routine clinical and pharmacokinetic studies. The clinical application of this method suggests that the pharmacokinetics of proquazone in adults and children are similar.     

Separation and determination of denatured alpha(s1)-, alpha(s2)-, beta- and kappa-caseins by hydrophobic interaction chromatography in cows', ewes' and goats' milk,milk mixtures and cheeses

Caseins alpha(s1)-, alpha(s2)-, beta- and kappa- from raw cows', ewes' and goats' milk were separated and determined by hydrophobic interaction chromatography (HIC) by using a Propyl column (Eichrom) in the presence of 8.0 M urea in the mobile phase. The method is based on fast and easy solubilization of real raw samples by 4.0 M guanidine thiocyanate followed by the HIC analysis, without any preliminary precipitation or separation of the casein fraction. Elution conditions have been optimized by analyzing commercial single bovine standard caseins and their mixture. In the optimized chromatographic conditions the four casein fractions were separated in less than 45 min. A linear relationship between the concentration of casein and peak area (UV absorbance detector at 280 nm) has been obtained over the concentration range of 0.5 to 40 microM. The detection limit for alpha-, beta- and kappa-caseins ranged between 0.35 and 0.70 microM. The precision of the method was evaluated, the coefficient of variation for alpha-, beta- and kappa-casein determination ranging between 3.0 and 6.0%. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certificated for total nitrogen content. The method was applied to commercial casein mixture and to the qualitative and quantitative analysis of casein fractions in unprocessed, raw cows', goats' and ewes' milk (10 samples analyzed for each species), in one sample of unprocessed buffalos' milk and in commercial cheeses (mozzarella, robiola, ricotta and stracchino). Binary mixtures of milk (cow/goat and cow/ewe) were also analyzed and the ratio between casein peak areas (alpha(s1)/kappa, alpha(s2)/beta, beta/kappa and alpha(s2)/alpha(s1)) of the HIC chromatograms was proposed and discussed in order to evaluate a possible application of this method to detect milk adulteration.     

Determination of beryllium and selenium in human urine and of selenium in human serum by graphite-furnace atomic absorption spectrophotometry.

For human urine beryllium (Be), each sample (500 microl) was diluted (1+1) with Nash reagent (containing 0.2% (v/v) acetylacetone and 2.0 M ammonium acetate buffer at pH 6.0) and then a 20-microl volume of Triton X-100 (0.4%, v/v) aqueous solution was added. An aliquot (10 microl) of the diluted urine mixture was introduced into a graphite cuvette and was atomized according to a temperature program. The method detection limit (MDL, 3sigma) for Be was 0.37 microg/l in the undiluted urine sample and the calibration graph was linear up to 65.0 microg/l. Calibration graphs were prepared by the standard addition method. Accuracies of 98.6-102% were obtained when testing standard reference material (SRM 2670) freeze dried human urine samples. Precision (relative standard deviation, RSD) for urine Be was < or = 2.3% (withinrun, n = 5) and was < or = 3.0% (between-run, n = 3). For human urine and serum selenium (Se), samples (100 microl) were diluted with HNO3 (0.2%, v/v) to make a (1+1) dilution for urine analysis or a (1+4) dilution for serum analysis. An additional aliquot (10 microl) of Triton X-100 (0.1%, v/v) was added to each 200 microl of (1+1) diluted urine (or 20 microl of the Triton X-100 was added to each 500 microl of (1+4) diluted serum) sample. After the diluted sample mixture (10 microl) was introduced into a graphite cuvette, the corresponding chemical modifier (10 microl, containing Ni2+ + Pd + NH4NO3 in HNO3 (0.2%, v/v)) was added to it and the mixture was atomized. The MDL (3sigma) for Se in urine and in serum was 4.4 and 21.4 microg/l in undiluted sample, respectively, and the calibration graphs were linear up to 150 and 400 microg/l. Accuracies of urine Se were 98.9 - 99.4% by testing SRM 2670 (NIST) urine standards with RSD (between-run, n = 3) within 2.9%; and that of serum Se was 97.2% when testing a certified second-generation human serum (No. 29, #664) with RSD (between-run, n = 3) of 1.4%. The proposed method can be applied easily, directly, and accurately to the measurement of Be and Se in real samples (including six urine Se and four serum Se from patients of Blackfoot Disease in Taiwan).     

A micro liquid column chromatography procedure for twelve anticonvulsants and some of their metabolites

Solvent extracts of 50 microliter volumes of serum were sufficient for the determination of twelve anticonvulsant drugs. The liquid chromatography procedure utilized a C-18 reversed-phase column and isocratic elution with 15% acetonitrile in water. No derivatization was required. Eluted anticonvulsants were detected by UV absorption at 195 nm and quantitated by drug--internal standard peak area ratios. The procedure provided linear working curves over the concentration range from 1 to 100 mg/l of drug in the serum. The procedure for serum provided recoveries of the drugs from 92 to 101%. Within-day precision was about 4% and day-to-day precision was about 6.5%. The procedures has been applied to urine samples to facilitate bioavailability studies. Data are also given for several metabolites. There is a discussion of many practical aspects of the procedure to improve the reliability of the results.     

Quantitative determination of tolazoline in serum and urine

A high-performance liquid chromatographic procedure is described for the analysis of tolazoline in serum and urine. This assay procedure is suitable for the analysis of micro-samples (50 or 100 microliters serum and 100 microliters urine). Samples are extracted in a single step and injected into a reversed-phase high-performance liquid chromatography system for detection at 210 nm. The clinical applicability of this assay is demonstrated by the determination of tolazoline serum and urine concentrations in neonates. In addition, the presence of urine conjugates and the extent of serum protein binding were investigated. This assay procedure has the required sensitivity (0.1 microgram/ml), accuracy and precision for both routine monitoring and pharmacokinetic characterization of tolazoline in neonates and adults.     

Mass fragmentographic determination of prostaglandin F2 alpha in human and rabbit urine

Analysis of prostaglandin F2 alpha (PGF2 alpha) in urine is a useful indicator of renal prostaglandin synthesis. A mass fragmentographic method for PGF2 alpha analysis in human urine was developed using [3,3,4,4-2H4]PGF2 alpha as an internal standard and carrier. PGF2 alpha was extracted from urine (20 ml) with chloroform, purified by preparative thin-layer chromatography and converted to the methyl ester trimethylsilyl ether before analysis by gas chromatography--mass spectrometry. The specificity of the urine analysis was demonstrated by retention time and the use of two pairs of fragments m/e 494/498 and 513/517 with the same results. The coefficient of variation for duplicate analysis averaged 12.6%, n = 17. Urine from recumbent women contained 4.9 +/- 2.6 (S.D.) ng/ml or 4.1 +/- 1.0 ng PGF2 alpha per mg creatinine (n = 10) with little diurnal variation. Male urine contained 5.0 +/- 2.7 (S.D.) ng/ml or 3.7 +/- 2.1 ng/mg creatinine (n = 10). Similar concentrations were found in boys and in girls. These observations indicate that urinary PGF2 alpha originates from the kidneys with little contribution from the male accessory sexual glands. This method can also be applied to analysis of PGF2 alpha in rabbit urine.     

A simple chromatographic method for determining norfloxacin and enoxacin in pharmacokinetic study assessing CYP1A2 inhibition

We developed a simple assay method for the determination of serum and urine norfloxacin and enoxacin using reversed‐phase high‐performance liquid chromatography and perchloric acid precipitation for sample pre‐treatment. Optimized conditions can permit detection of norfloxacin and enoxacin in the same chromatogram, so either compound can be used as an internal standard for another determinant. Supernatants of the precipitated samples were analyzed by the octadecylsilyl silica‐gel column under ambient temperature and an ultraviolet wavelength of 272 nm. A mobile phase solvent consisting of 20 mm sodium dihydrogenphosphate (pH 3.0) and acetonitrile (85:15, v/v) was pumped at a flow rate of 1.0 mL/min. The calibration curves for norfloxacin and enoxacin at a concentration of 62.5–1000 ng/mL for serum and 250–4000 ng/mL for urine were linear (r > 0.9997). The recoveries of norfloxacin and enoxacin from serum and urine were >94% with the coefficient of variations (CV) <5%. The CVs for intra‐ and inter‐day assay of norfloxacin and enoxacin were <4.2 and <5.5%, respectively. This method can be applied to the pharmacokinetic study of norfloxacin and enoxacin after repeated administration to assess changes in CYP1A2 activity in healthy subjects. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of thiamine in blood serum and urine by high-performance liquid chromatography with direct injection and post-column derivatization

Thiamine (vitamin B₁) was determined in human serum and urine by HPLC with fluorimetric detection of its oxidation product, thiochrome. The samples were injected directly into the chromatographic system without previous treatment or dilution. A column filled with an ultra-high molecular weight surface-modified polyethylene (PE) was able to separate matrix components from analyte and also to allow a good chromatographic resolution of thiamine. The interaction of thiamine, thiocrome and both matrices (serum and urine) with PE was studied off- and on-line to determine the optimal procedure for vitamin B₁ determination. When carried off-line, matrix adsorption yield was 49 mg serum proteins/g polymer and components of 1000 μl urine/g polymer. In an on-line arrangement, the yield dropped to 10 mg/g and 150 μl/g, respectively. The matrix/analyte separation was carried out in an on-line procedure on a 50×4.6-mm, 25-μm PE column, using a water-sodium phosphate-methanol gradient elution. Part of the matrix was eluted within the first 2 min and thiamine after 3.8 min. The rest of the matrix retained on the column was eluted after thiamine at the last step of the gradient elution. Analysis time was 12 min. The within-day and day-to-day precision gave C.V. varying from 3.6% to 14.5% and recoveries from spiked samples were in the range of 84.8-98.8%.     

Determination of methamphetamine in urine samples with sodium 1,2-naphthoquinone-4-sulphonate

Optimal conditions have been studied for the determination of methamphetamine in urine samples by an extractive-spectrophotometric method with sodium 1,2-naphthoquinone-4-sulphonate (NQS) as reagent. These conditions are: NaHCO₃ pH 10, NQS 6.3 × 10^–3 mol/l and heating for 5 min at 45°C. The accuracy and precision of the method were tested. The detection limits were 0.2 mg/l in the standard and 0.9 mg/l when 5 ml of urine sample were taken. The standard deviation of blank urine was evaluated from 12 different samples. The relative errors found in the determination of methamphetamine in urine were lower than 10% if the methamphetamine-amphetamine ratio was higher than 4.     

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.     

Simultaneous stereoselective analysis by capillary electrophoresis of tramadol enantiomers and their main phase I metabolites in urine.

Capillary zone electrophoresis was successfully applied to the enantiomeric resolution of racemic tramadol and its six phase I metabolites using carboxymethylated beta-cyclodextrin (CMB) added to the background electrolyte (BGE). Baseline resolution of tramadol and its metabolites was obtained in less than 30 min using a 50 mM phosphate buffer (pH 2.5) containing 5 mM of CMB. Chiral determinations of tramadol and its main three metabolites, O-demethyltramadol (M1), N-demethyltramadol (M2) and O-demethyl-N-demethyltramadol (M5), were performed in urine after a simple double liquid-liquid extraction of 200 microliters of biological material. In the tested concentration range (0.5-20 micrograms/ml, except for M2: 0.5-10 micrograms/ml) coefficients of correlation superior than 0.994 were obtained. Within-day variation determined on three different concentrations for each enantiomers showed accuracies ranging from 95.4% to 103.2%. The relative standard deviation (RSD) of these assays was determined to be less than 10.0%. Day-to-day variation presented accuracies ranging from 96.3% to 106.5% with a RSD less than 9.0%. After oral administration of 100 mg of tramadol hydrochloride to an healthy volunteer, the urinary excretion was monitored during 30 h. About 15% of the dose was excreted as unchanged tramadol. The enantiomeric ratios of all the excreted analytes, T, M1, M2 and M5, were found to be very different to 1.0, showing that a stereoselective metabolism of tramadol clearly occurred.     

Direct analysis of reference biofluids by coupled in situ electrodeposition-electrothermal atomic absorption spectrometry

The application of coupled in situ electrodeposition-electrothermal atomic absorption spectrometry (ED-ETAAS) to the determination of Pb in biological standard reference materials is described. In situ electrodeposition at a cell voltage of 3.0 V from 25-μl samples onto electrodeposited Pd is used to quantitatively separate the analyte from blood and urine matrices. With subsequent withdrawal of spent electrolyte, this overcomes the atomisation problems inherent with high salt and organic contents. ED-ETAAS is applied with minimal sample pre-treatment (acidification). The electrolysis process aids decomposition of the organic matrix, and the release of trace elements. Evolution of H₂ at the cathode counters fouling of the Pd modifier surface. The palladium deposit is renewed in situ for each determination. For AMI certified lyophilised blood, diluted 1+3 with 0.1 M HCl (18.1 μg/l Pb), the R.S.D. was 3.0% (peak height; n=5) and the detection limit (3 σ_blank; n=5) was 1.5 μg/l. Results for certified blood samples were AMI 72.3±4.3 μg/l (certified 76.2±7.6 μg/l) and Seronorm 34.2±2.0 μg/l (36±4 μg/l). The result for NIST SRM 2670 normal urine acidified to 1% HNO₃ was 8.1±0.6 μg/l (recommended value 10 μg/l).     

Sensitive analysis of 1alpha,25-dihydroxyvitamin D3 in biological fluids by liquid chromatography-tandem mass spectrometry.

A liquid chromatographic-tandem mass spectrometric assay using 5% bovine serum albumin as the calibration matrix has been developed for the quantitative analysis of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] in biological fluids. The analyte was extracted from the matrix after protein precipitation using an automated solid-phase extraction procedure involving both a reversed-phase and normal-phase procedure on a single C18 cartridge. The analytical chromatography was performed using a Symmetry C8 50 x 2.1 mm, 3.5 microm column. The mobile phase was a linear gradient from 75 to 99% methanol with a constant concentration of 2 mM ammonium acetate. 1alpha,25(OH)2D3 and the internal standard [2H6]1alpha,25(OH)2D3 were detected by using MS-MS. The ion source was operated in the positive electrospray ionisation mode. The assay is specific, sensitive, and has a capacity of more than 100 samples per day, with a limit of quantitation of 20 pg ml(-1) for a 1.0-ml sample aliquot. The assay has been used for the analysis of 1alpha,25(OH)2D3 in serum from rats and pigs simultaneously with the analysis of the vitamin D analog seocalcitol.     

Determination of methimazole in urine by liquid chromatography

A liquid chromatography methodology is developed and validated for detection and quantification of methimazole in urine. The approach is based on derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate, reversed-phase high-performance liquid chromatography (HPLC) separation of so formed methimazole 2-S-quinolinium derivative from other urine matrix components, followed by detection and quantification with the use of ultraviolet–visible detector. Neither extraction, nor preconcentration of the sample are necessary. The methimazole standards added to normal urine before derivatization step show that the response of the detector, set at 345 nm, is linear within the concentration range studied, that is, from 0.25 to 50 mg/l urine. The relative standard deviation values for precision and recovery within the calibration range were from 1.8 to 5.0% and from 95.7 to 103.3%, respectively. Lower limits of detection and quantitation were 0.15 and 0.25 mg/l urine, respectively.     

Determination of trichloroacetate in human serum and urine by ion chromatography

A simple and rapid ion chromatographic method has been developed for the determination of trichloroacetate in human serum and urine. Two short (50 x 5 mm i.d.) low-capacity anion-exchange resin columns in series were used as the analytical column and a mixture of 35 mM sodium hydroxide, 20 mM p-hydroxybenzonitrile and 2% V/V acetonitrile (pH 12.3) as the eluent. The calibration graph was rectilinear from 0.3 to 33 mg l-1 of trichloroacetate at 10 microS with a detection limit (3 sigma) of 0.08 mg l-1. The recovery of trichloroacetate, obtained by analysing spiked serum and urine samples, was in the range 98.2-101% and the coefficient of variation was always less than 3.0%.