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PubMed2006

Complete quantification of group A and group B soyasaponins in soybeans.

A combination of high-pressure extraction and preparative chromatography was used to purify the group A and group B soyasaponins from soy germ for use as analytical standards and for use in biological assays. A standardized sample preparation and extraction method was developed for the analysis of phytochemicals found in soy and processed soy products, which is reproducible in other laboratories. The extracts can be analyzed with standard liquid chromatography-mass spectrometry and high-performance liquid chromatography methods to identify and quantitate the group A and group B forms of the soy saponins, as well as the soy isoflavones. Complete saponin analysis of the extracts prepared from soy germ (hypocots), hulls, and cotyledons shows that a significant portion of the saponins is concentrated in the germ. The germ contains nearly all of the group A soyasaponins, while the group B soyasaponins are nearly equally distributed between the germ and the cotyledons. The hulls contain little of either isoflavones or saponins. Whole (full fat) soybeans grown on a tract in central Illinois in 2003 contain approximately 4-6% saponins on a weight basis, of which about one-fifth or less of the total saponin content are group A soyasaponins; the balance is group B soyasaponins.

Chromatography, High Pressure Liquid↗
PubMed2001

Influence of the procedure used to prepare the calibration sample set on the performance of near infrared spectroscopy in quantitative pharmaceutical analyses.

Calibrating near infrared diffuse reflectance spectroscopy (NIRS) methods usually involves preparing a set of samples with a view to expanding the analyte concentration range spanned by production samples. In this work, the performances of the two procedures most frequently used for this purpose in near infrared pharmaceutical analysis, viz., synthetic samples obtained by weighing of the pure constituents of the pharmaceutical and doped samples made by under- or overdosing previously powdered production samples, were compared. Both procedures were found to provide similar results in the quantification of the active compound in the pharmaceutical, which was determined with a relative standard error of prediction (RSEP) of < 1.6%. However, the two types of sample preparation provide different spectra, which precludes the accurate quantification of synthetic samples from calibrations obtained with doped samples and vice versa. None of the mathematical pre-treatments tested with a view to reducing this different scattering (viz., second derivative, standard normal variate and orthogonal signal correction) could effectively solve this problem. This hinders accurate validation of the linearity of the procedure and makes it advisable to use doped samples which are markedly less different to production samples.

Calibration↗
PubMed1992

[Development of multicomponent immunoassay and micro-localization analysis by laser-photoacoustic and microscopic image analyzer].

Laser photoacoustic spectroscopy (PAS) has a potential to be developed as a sensitive and solid-phase analytical method and was applied to enzyme immunoassay. The test sample for immunoassay was prepared by adsorbing multi-component immunoglobulins on a nitrocellulose membrane filter. Human lambda- and kappa-chains, which are used as a principal indication of malignant lymphoreticular disease, and immunoglobulin G were used as model proteins, and PAS immunoassay was applied to the individual detection of these three proteins in the urine. Furthermore, in order to develop a sensitive analysis for particular biological components in tissues or cells, laser photoacoustic microscopy (PAM) and video intensified microscopy (VIM) were developed. PAM was shown to be applicable to the detection and quantification of human lambda-chain in a micro-region of the tissue sections of the human fetal spleen and pancreas. VIM was applied to the detection of stimulation-response processes in a cell. By using neutrophils which are stimulated by many substances and produce active oxidants as the results, dynamic changes in the stimulation-response process in a living cell were visualized as fluorescence or chemiluminescence images by the VIM system.

Humans↗
PubMed2000

[Determinations of oxalate in urine and plasma by capillary electrophoresis].

PURPOSE: The determinations of oxalate in urine and plasma are important in the evaluation and treatment of patients with calcium oxalate nephrolithiasis. Although many analytical methods for determining oxalate have been developed, most of them need complicated sample preparation, and are expensive for routine examination. Especially for estimation of plasma oxalate, much more sensitive measurement is required because of the extremely low concentration. A simple and rapid assay for oxalate in urine and plasma by capillary electrophoresis has been described here, and utilized for assessment of renal oxalate clearance. In addition, simultaneous determination of urinary oxalate and citrate was developed. METHODS: A Waters Quanta 4000E system was used with a detection at 185 nm. Separation was obtained on a fused silica capillary, 60 cm long x 75 microns and 100 microns (i.d.) for urine and plasma samples respectively. Urine samples were diluted with 60 mM hydrochloric acid, and ultrafiltrates of plasma were acidified and diluted with 300 mM boric acid and 50 mM phosphoric acid. RESULTS: The intraassay coefficient variation was 2.7-4.0% for urinary oxalate, and 1.3-3.9% for citrate. The mean recovery ratio of 0.2 mM oxalate and 1.0 mM citrate added to 10 samples were 99.0% (92.6-107.4%) and 98.4% (91.2-103.9%), respectively. In the determination of plasma oxalate, the minimum detectable limit was 0.9 microM, the coefficient variation was 5.8-16.0%, and the recovery rate was 101.5% (87.8-125.6%). The plasma oxalate levels in 8 adult males were 2.39 +/- 1.46 microM (Mean +/- SD). Renal oxalate clearances with one hour method were 72.9 +/- 20.0 ml/min in 6 healthy controls and 83.2 +/- 27.8 ml/min in 8 stone formers. Oxalate/creatinine clearance ratios in each groups were 0.70 +/- 0.16 and 1.11 +/- 0.34 respectively. CONCLUSION: The simultaneous determination of urinary oxalate and citrate was satisfactory. Capillary electrophoresis is suited for routine examination of urinary oxalate and citrate with the advantage on simplicity and economy. The assay of plasma oxalate by this method was also acceptably sensitive, specific under a low temperature and an acidification.

Adult↗
PubMedDate not supplied

Quantitative determination of fluoride as a major pollutant in the emissions from the thermal treatment of clayey raw materials.

Three clayey raw materials for production of traditional ceramics, all with medium to high fluorine content, have been investigated for determining their attitude to fluoride emission during firing, at varying parameters of the firing cycle, maximum firing temperature included, and in the presence or in the absence of calcium carbonate either added to the sample or naturally present in it. Different analytical methods for the determination of fluoride have been tested, together with different sample preparation procedures aimed to obtain minimum loss of fluoride in the pre-treatment step.

Air Pollutants↗
PubMed2006

Analysis of mycophenolic acid in saliva using liquid chromatography tandem mass spectrometry.

Salivary levels of the immunosuppressive agent, mycophenolic acid (MPA), may provide a convenient and noninvasive method for drug monitoring. An analytical method was developed and validated for quantification of salivary MPA using liquid chromatography tandem mass spectrometry. Sample preparation included addition of 50 microL internal standard solution [500 microg/L indomethacin in methanol] to 100 microL saliva sample, followed by protein precipitation with 200 microL acetonitrile. Supernatants were dried and reconstituted in 100 microL of 85:15% (vol/vol) mixture of methanol and water containing 0.05% formic acid and 20 microL was injected onto the analytical column. The mobile phase comprised a gradient mixture of methanol and 0.05% formic acid, giving a total run time of 7.5 minutes. Chromatograms were obtained using mass transitions of m/z 319.0-->190.8 for MPA and m/z 355.9-->312.2 for indomethacin. The calibration curve was linear over a concentration range of 2.5 to 800 microg/L (r=0.9999) and the recovery of MPA from saliva was >90%. The inaccuracy was <10% and intra- and interday coefficient of variation ranged from 2.8% to 5.2%. Mean+/-SD of MPA concentrations in saliva (n=100) obtained from 11 kidney transplant recipients was 31.4+/-32.3 microg/L (range: 2.6 to 220.4 microg/L) and correlated well with total (r=0.909) and unbound (r=0.910) MPA concentrations in plasma. In conclusion, a simple, sensitive, and specific method was developed and validated for quantification of MPA in saliva. Additional clinical studies are required to establish the usefulness of this specimen in the clinical management of organ transplant recipients.

Chromatography, High Pressure Liquid↗
PubMed1990

Development of a laboratory robotic system for automated bioanalytical methods--I. The determination of theophylline in human plasma: a comparison between the robotized and manual method.

The quality of bioanalytical methods is often determined by the quality of sample preparation. Using a robot for sample treatment may give better results than manual sample preparation, since the robot lacks human behaviour and incidental errors that are part of it. The use of a laboratory robot has the additional advantage of giving each sample the same analytical history, resulting in better reproducibility. An automated method has been developed for the analysis of drugs in plasma using a laboratory robot. Theophylline was used as a probe drug. Sample preparation was automated with a Zymate II robot, followed by separation and quantitation on an HPLC-system. The robotic method showed a good correlation with the manual method, while sample throughput was doubled.

Chemistry, Clinical↗
PubMed1992

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.

Cephalosporins↗
PubMed1996

Validation of liquid chromatographic and gas chromatographic methods. Applications to pharmacokinetics.

Validations of analytical methods are important for the generation of data for bioavailability, bioequivalence and pharmacokinetic studies. It is essential to use well defined and fully validated analytical methods to obtain reliable results that can be satisfactorily interpreted. This manuscript is intended to provide guiding principles for the evaluation of a method's overall performance. For this purpose, all of the variables of the method are considered, including sampling procedure, sample preparation, chromatographic separation, detection and data evaluation. The criteria considered are as follows: stability, selectivity, limits of quantification and of detection, accuracy, precision, linearity, recovery and ruggedness. Models used for analytical calibration curves are explained in term of validity and limitations, along with a presentation of the most common statistical considerations used to validate the model. Appropriate means of testing precision and accuracy, the most important factors in assessing method quality, are presented. Other issues, such as re-validation, cross-validation, partial sample volume, endogenous drugs and biological matrix of limited availability, are also discussed.

Calibration↗
PubMed2002

Quantification of monohydroxy-PAH metabolites in urine by solid-phase extraction with isotope dilution-GC-MS.

For measurement of biomarkers from polycyclic aromatic hydrocarbon (PAH) exposure, an analytical method is described quantifying hydroxylated PAH (OH-PAH) in urine samples. This method determined monohydroxy metabolites of naphthalene, fluorene, phenanthrene, fluoranthene, pyrene, chrysene, benzo[c]phenanthrene, and benz[a]anthracene. The sample preparation consisted of enzymatic hydrolysis, solid-phase extraction and derivatization with a silylating reagent. Five carbon-13 labeled standards were used for isotope dilution. Analytes were separated by gas chromatography (GC) and quantified with high-resolution mass spectrometry (HRMS). This method produced good recoveries (41-70%), linearity, and specificity. Data were corrected for blank levels from the naphthalene, fluorene, and phenanthrene metabolites. Method detection limits ranged from 2 ng L(-1) for 1-hydroxypyrene to 43.5 ng L(-1) for 1-hydroxynaphthalene. Using quality control charts from two urine pools, the method can be readily applied to biomonitoring PAH exposure.

Chromatography, High Pressure Liquid↗
PubMed2004

Automated sample preparation based on the sequential injection principle. Solid-phase extraction on a molecularly imprinted polymer coupled on-line to high-performance liquid chromatography.

A molecularly imprinted polymer (MIP) prepared using caffeine, as a template, was validated as a selective sorbent for solid-phase extraction (SPE), within an automated on-line sample preparation method. The polymer produced was packed in a polypropylene cartridge, which was incorporated in a flow system prior to the HPLC analytical instrumentation. The principle of sequential injection was utilised for a rapid automated and efficient SPE procedure on the MIP. Samples, buffers, washing and elution solvents were introduced to the extraction cartridge via a peristaltic pump and a multi-position valve, both controlled by appropriate software developed in-house. The method was optimised in terms of flow rates, extraction time and volume. After extraction, the final eluent from the extraction cartridge was directed to the injection loop and was subsequently analysed on HPLC. The overall set-up facilitated unattended operation, operation and improved both mixing fluidics and method development flexibility. This system may be readily built in the laboratory and can be further used as an automated platform for on-line sample preparation.

Automation↗
PubMed1995

Determination of pesticide metabolites in human urine using an isotope dilution technique and tandem mass spectrometry.

A method that measures 12 analytes in urine and reflects possible exposure to pesticides was developed. The sample preparation involves enzyme hydrolysis and solvent extraction through the use of laboratory robotics, followed by phase-transfer catalysis derivatization and silica cleanup. Samples are analyzed by capillary gas chromatography and tandem mass spectrometry using an isotope dilution technique with 13C-labeled internal standards. The limit of detection is 1 microgram/L (1 part per billion) for most analytes, and most analytes have a linear response up to 100 micrograms/L. The precision of the method is reflected in the variation observed in quality control materials over 33 months; the variation averaged 17% for these analytes. On the basis of the detectable analyte levels of unspiked urine samples collected from unexposed volunteers, this method can be used to measure the low levels necessary for establishing reference range values of the selected pesticides or metabolites.

Calibration↗
PubMed2006

Quantification of [3H]docetaxel in feces and urine: development and validation of a combustion method.

Most radiolabeled biological samples require extensive sample preparation to reduce quenching interference before quantification of radioactivity is possible. Clearly, a more rapid and simple method ensuring a constant count rate and optimal counting efficiency has important advantages. We report on the development and analytical method validation of a rapid and simple combustion method to quantify [3H]docetaxel excreted in human feces and urine. A 3-day validation procedure was performed; quality control (QC) samples, prepared in blank feces and urine, were combusted 5 times and aliquots of the produced tritiated combustion water were counted in a liquid scintillation counter. The validation runs demonstrated adequate precision (below 7.6%) across all QC levels. Sensitivity at the lowest QC level was excellent and recovery of radioactivity constant (ranging from 85 to 91.8%). Clinical applicability of the method was tested in a cancer patient receiving docetaxel and a tracer amount of [3H]docetaxel; during the first 72 h after [3H]docetaxel infusion, 60% of total radioactivity was excreted in the collected feces and urine, which is within the expected range. Combustion of tritiated feces and urine samples is a simple, rapid, sensitive, precise and reproducible method with high recovery. It can be applied to quantify [3H]docetaxel excretion after i.v. administration.

Antineoplastic Agents, Phytogenic↗
PubMed2002

Determination of lacidipine from urine by HPTLC using off-line SPE.

A simple, rapid and sensitive high performance thin layer chromatographic method was developed and validated for the estimation of lacidipine. The sample preparation involved protein precipitation followed by an efficient solid phase extraction on C18 cartridge. The analytes were isolated from 1 ml of urine and recovered by pure ethyl acetate solution. The method employed TLC aluminium plate precoated with silica gel 60F254 as the stationary phase. The solvent system employed consists of toulene-ethyl acetate [6.5:3.5v/v]. This system gave a dense and compact spot of the drug at R(f) value of 0.45. The linear regression data for the calibration plots showed good linear relationship (r=0.999) over the concentration range 10-80 ng. Recovery studies were performed at two different levels. The recovery data reveals that the R.S.D. for intra-day and inter-day analysis at 10 ng was found to be 0.84 and 0.22%, respectively. The proposed method was found to be useful for the routine analysis of pharmaceuticals and pharmacokinetic studies in human urine samples.

Calcium Channel Blockers↗
PubMed2003

High-performance liquid chromatographic method for mycophenolic acid and its glucuronide in serum and urine.

OBJECTIVE: To develop a simple analytical method for monitoring serum and urine concentrations of mycophenolic acid (MPA), an active metabolic constituent of the immunosuppressive pro-drug mycophenolate mofetil, and its glucuronide. METHODS: Serum samples were prepared by solid-phase extraction (SPE), while urine samples were simply diluted with water. Serum was added to an SPE cartridge, then washed twice with 5% methanol solution. The analytes were eluted with methanol containing benzoic acid as internal standard for mycophenolic acid glucuronide (MPAG). The resultant eluate was directly injected into a high-performance liquid chromatograph (HPLC) to determine MPAG. For the assay of MPA, the remaining eluate was dried under nitrogen and resolved in a mixture of acetonitrile and 20 mM phosphate buffer (pH 3.0). RESULTS: The present methods were reproducible and accurate based on the intra- and inter-assay, and had detection limits of 0.225 microg/mL for MPA and 9.0 microg/mL for MPAG. The present methods enabled us to monitor the time course of changes in the concentrations of MPA and MPAG in serum and urine in a patient with a renal transplant during 12 h after ingestion of mycophenolate mofetil. CONCLUSION: The HPLC method described should be useful for the routine monitoring of serum and urine concentrations of MPA and MPAG during immunosuppressive medication for renal transplantation.

Administration, Oral↗
PubMed2001

Determination of the metabolites of nitrofuran antibiotics in animal tissue by high-performance liquid chromatography-tandem mass spectrometry.

A LC-MS-MS method is presented to analyse simultaneously the metabolites of four nitrofuran antibacterial agents, furazolidone, furaltadone, nitrofurazone and nitrofurantoin in animal muscle tissue. Sample clean-up and analyte enrichment was performed by solid-phase extraction (SPE) with a polystyrene sorbent following combined hydrolysis of the protein-bound drug metabolites and derivatisation of the homogenised tissue with 2-nitrobenzaldehyde. Limits of detection of 0.5-5 ng g(-1) tissue and limits of determination of 2.5-10 ng g(-1) tissue were achieved using electrospray ionisation in positive mode. Analyte identification and quantification was performed according to EU guidelines, using multiple reaction monitoring (MRM) with one precursor ion and two product ions as identifiers. The use of an internal standard in combination with the simplified sample preparation led to a sensitive and reliable analysis method. The yield of the derivatisation reaction was between 66 and 74% and the recovery of SPE reached 92-105% for all values between 10 and 500 ng g(-1). The developed analytical protocol has been applied to contaminated tissue samples of furazolidone- and furaltadone-treated pigs and allowed unequivocal identification and quantification of the metabolites.

Animals↗
PubMed2000

Liquid chromatographic-electrospray tandem mass spectrometric method for the simultaneous quantitation of the prodrug fosinopril and the active drug fosinoprilat in human serum.

A sensitive, specific, accurate and reproducible LC-MS-MS method was developed and validated for the simultaneous quantitation of the prodrug fosinopril and its active drug fosinoprilat in human serum. The method employed acidification of the serum samples to minimize the hydrolysis of fosinopril to fosinoprilat prior to purification by solid-phase extraction to isolate the two analytes and the two internal standards from human serum. The extracted samples were analyzed by turbo ionspray LC-MS-MS in the positive ion mode. Chromatography was performed on a polymer-based C18 column (Asahipak ODP PVA-C18, 2x50 mm) using gradient elution with methanol and 10 mM ammonium acetate, pH 5.5. The calibration curve, 1.17 to 300 ng/ml, was fitted to a weighted (1/x) linear regression model. Serum quality control (QC) samples used to gauge the accuracy and precision of the method were prepared at concentrations of 5.00, 100, 250 and 500 ng/ml of each analyte. The inter-assay accuracies were within 6% (DEV) for both analytes. The intra- and inter-assay precisions were within 7% and 11% (RSD), respectively, for both analytes. The hydrolysis of fosinopril to fosinoprilat during sample processing was < or = 6%. This degree of conversion would cause little error in the analysis of post-dose serum samples since such samples are known to contain low levels of the prodrug compared to the drug.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2004

Application of solvent microextraction in a single drop for the determination of new antifouling agents in waters.

A new, rapid microextraction technique termed solvent microextraction (SME) has been developed for the simultaneous determination of new generation antifouling agents, in water samples. Chlorothalonil, dichlofluanid and Sea nine 211 were employed as model compounds to asses the extraction procedure and were determined by gas chromatography with electron capture detection. Experimental parameters which control the performance of SME, such as selection of solvent, exposure time, agitation, organic drop volume, and salt concentration were optimized. The new method provided good average enrichment factors of >10.7 for all analytes, good precision (RSD < 8.5%) and good linearity (r2 > 0.9880). The limits of detection (LODs) were in the range of 0.00025-0.003 microg/L (S/N = 3). The SME was performed in different type of natural water samples and acceptable recoveries were obtained for the tested analytes. The results demonstrated that SME is a rapid, accurate and effective preparation method and could be successfully performed for the determination of antifouling agents in water samples.

Aniline Compounds↗