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PubMed2003

Large-volume injection gas chromatography-mass spectrometry for automated broad-spectrum drug screening in horse urine.

A rapid, sensitive, and rugged method for detecting drugs and drug metabolites in extracts of horse urine is described. The use of large-volume injection (LVI) gas chromatography-mass spectrometry (GC-MS) for analysis of horse urine extracts allowed automation of the derivatization procedure and reduction of the sample volume from 5 mL to 1 mL of urine. An autosampler and temperature-programmable inlet were used to automatically dissolve the sample extract and form trimethylsilyl derivatives of over 200 analytes. The suitability of this procedure for routine GC-MS detection of approximately 80 basic analytes in extracts of racehorse urine was investigated. The formation of derivatives using LVI with in-liner derivatization was compared to a manual procedure involving the dissolution of sample extracts in N,O-bis(trimethylsily)trifluoroacetamide, heating the resulting mixture, and injecting 1 or 2 microL of the mixture through a splitless injector into the GC-MS instrument. In all cases, the in-liner derivatization reactions were found to be as complete as conventional heating block procedures. Ruggedness testing of the method demonstrated that peak resolution, shape, and area were maintained through 40 consecutive injections of sample extracts. No evidence of the accumulation of interfering substances was observed. The limits of detection using LVI GC-MS for routine screening of basic drugs in urine were generally in the range of 5-25 ng/mL. The method is currently being used to detect basic analytes in horse urine extracts with a throughput of approximately 50 urine sample extracts per instrument per day.

Animals↗
PubMed2001

[Determination of bambuterol in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to pharmacokinetic study].

AIM: To develop a sensitive, specific and accurate method for quantifying bambuterol in human plasma and to study pharmacokinetics of bambuterol in male healthy Chinese. METHODS: Plasma samples were prepared based on a simple liquid-liquid extraction. The extracted samples were analyzed on liquid chromatography using a Zorbax SB C18 column interfaced with a triple quadrupole tandem mass spectrometer and detected by use of selected reaction monitoring mode. RESULTS: The linear calibration curves were obtained in the concentration range of 0.05-4.0 ng.mL-1. The limit of quantification was 0.05 ng.mL-1. The intra- and inter-run precision was measured to be below 7%. The inter-run accuracy was less than 8% for the analyte. After an oral administration of 10 mg bambuterol hydrochloride to 18 healthy Chinese volunteers the main pharmacokinetic parameters of bambuterol were as follows: Tmax was (2.3 +/- 1.3) h; Cmax was (3.95 +/- 2.20) ng.mL-1; T1/2 was (11.4 +/- 6.1) h and AUC0-t was (26.85 +/- 11.77) ng.h.mL-1. CONCLUSION: The method is shown to be accurate, robust and convenient, and suitable for pharmacokinetic studies of bambuterol. It was found that there was marked inter-individual difference in the pharmacokinetics of bambuterol in Chinese volunteers after a single oral dose, which may be attributed to the difference of activity of cholinesterase, an enzyme catalyzing bambuterol metabolism.

Adult↗
PubMed1996

Chromatographic separation and behavior of microsomal cytochrome P450 and cytochrome b5.

The methods used for separation of the multiple mammalian cytochrome P450 enzymes by liquid chromatography are reviewed. In addition to the chromatographic techniques, preparation and handling of samples and prefractionation procedures are considered. Conditions that affect stability and chromatographic resolution of cytochromes P450 are also discussed. Special emphasis is put on useful methods which are not routinely used for P450 separation, such as immobilized metal affinity or hydrophobic-interaction chromatography. Applications of low- and high-pressure methods with regard to preparative and analytical separations are compared. It is shown that high- and medium-pressure ion-exchange chromatography are suitable tools for separation of closely related P450 enzymes, especially when specific detection methods are available. In addition to fractionation of cytochromes P450, the isolation and chromatographic behavior of cytochrome b5 is discussed.

Animals↗
PubMed2003

Analysis of multiple pesticide residues in tobacco using pressurized liquid extraction, automated solid-phase extraction clean-up and gas chromatography-tandem mass spectrometry.

A new method was developed for the analysis of pesticide residues in tobacco. The objective was to significantly increase the number of samples that can be processed by the laboratory and to enable the extension of the current coverage to additional pesticides. A new analytical approach was therefore defined based on two main axes, the automation of the sample preparation and the selectivity of the analyte detection using tandem mass spectrometry. This latter aspect reduces the stringency of the requirements placed on the clean-up of the extracts and on the chromatographic resolution when less selective detectors are used. The extraction of the analytes from the matrix is performed using the pressurized liquid extraction technique. Tobacco samples are extracted at elevated temperature and pressure (100 C and 100 atm; 1 atm = 101,325 Pa) using acetone as an extraction solvent. The resulting extract is then concentrated using a Vortex evaporator. Three different solid-phase extraction (SPE) procedures, adjusted to the chemical properties of the different active ingredients to be measured, are applied to the concentrated extract, thus leading to three extract fractions. The first fraction contains such main classes of active ingredients as organohalogenated and 2,6-dinitroaniline compounds while the second one collects the organophosphorus and acylalanines residues; these two fractions are analyzed by capillary gas chromatography coupled to tandem mass spectrometry using negative chemical ionization and electron impact ionization in the positive mode, respectively. The third extract fraction gathers the N-methylcarbamates residues which are analyzed by HPLC with post-column derivatization and fluorescence detection. The different sample preparation stages from extraction to SPE clean-up have been automated through the use of recent analytical technologies. In combination with the analysis by tandem mass spectrometry, this provided a potential for a high sample throughput.

Automation↗
PubMed2004

On-line monitoring of enzymatic conversion of adenosine triphosphate to adenosine diphosphate by micellar electrokinetic chromatography.

Capillary electrophoresis can be a valuable tool for the on-line monitoring of bioprocesses. The enzymatic conversion of nucleotide adenosine triphosphate (ATP) to adenosine diphosphate (ADP) by hexokinase (HK) was monitored in the bioreactor interfaced by a laboratory-built microsampler to a capillary electrophoresis unit. The use of this specially designed sampling device enabled rapid consecutive injections to be performed without high-voltage (HV) interruptions. No additional sample preparation was required. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow (EOF) by cationic surfactant and reversed polarity mode provided a good resolution and short analysis time of less than 5 min. The samples were injected electrokinetically, using -25 kV voltage for 3 s and detected by their UV absorbance at 254 nm. The analytes were detected at a microg/ml level with a reproducibility of about 7%. To demonstrate the potential of CE in understanding the processes of biological interest, such as nucleotide degradation and metabolism, the investigation of the efficiency and the time course of the enzymatic transformation was carried out.

Adenosine Diphosphate↗
PubMed2006

Fast gas chromatographic/mass spectrometric determination of diuretics and masking agents in human urine: Development and validation of a productive screening protocol for antidoping analysis.

An analytical procedure was developed for the fast screening of 16 diuretics (acetazolamide, althiazide, amiloride, bendroflumethiazide, bumetanide, canrenoic acid, chlorthalidone, chlorthiazide, clopamide, ethacrynic acid, furosemide, hydrochlorthiazide, hydroflumethiazide, indapamide, triamterene, trichlormethiazide) and a masking agent (probenecid) in human urine. The whole method involves three analytical steps, including (1) liquid/liquid extraction of the analytes from the matrix, (2) their reaction with methyl iodide at 70 degrees C for 2 h to form methyl derivatives, (3) analysis of the resulting mixture by fast gas chromatography/electron impact mass spectrometry (fast GC/EI-MS). The analytical method was validated by determining selectivity, linearity, accuracy, intra and inter assay precision, extraction efficiencies and signal to noise ratio (S/N) at the lowest calibration level (LCL) for all candidate analytes. The analytical performances of three extraction procedures and five combination of derivatization parameters were compared in order to probe the conditions for speeding up the sample preparation step. Limits of detection (LOD) were evaluated in both EI-MS and ECNI-MS (electron capture negative ionization mass spectrometry) modes, indicating better sensitivity for most of the analytes using the latter ionization technique. The use of short columns and high carrier gas velocity in fast GC/MS produced efficient separation of the analytes in less than 4 min, resulting in a drastic reduction of the analysis time, while a resolution comparable to that obtained from classic GC conditions is maintained. Fast quadrupole MS electronics allows high scan rates and effective data acquisition both in scan and selected ion monitoring modes.

Diuretics↗
PubMed2005

Evaluation of LC-MS for the analysis of cleaning verification samples.

The cleaning verification of pharmaceutical manufacturing equipment prior to further use is a cGMP requirement. Typically, relevant data are generated by HPLC with UV detection using methods individually developed and validated for each product. This work describes the use of HPLC with mass spectrometry to analyse cleaning verification samples, a novel means of utilising this analytical technology. The initial aim was to produce a single, generic method capable of quantifying a broad range of pharmaceuticals. Ultimately, however, a more effective strategy, in terms of efficiency and reliability, proved to be application of a well-defined approach to the rapid generation of compound specific methods. Results of studies to optimise the sample preparation for a basic compound in drug development (compound 1), together with experimental results for two further compounds are presented. These demonstrated that the combination of a well defined approach to chromatographic method development and mass spectrometric detection provided methodology with advantages in terms of sensitivity. Additionally, and by virtue of its potential for general applicability, the approach proposed has the potential to improve the overall efficiency with which methods for cleaning verification samples can be developed and applied.

Chromatography, High Pressure Liquid↗
PubMed2000

Quantitative analysis of mutagenic heterocyclic aromatic amines in cooked meat using liquid chromatography-atmospheric pressure chemical ionisation tandem mass spectrometry.

Five mutagenic heterocyclic aromatic amines (HAAs) were quantified from meat extracts, and grilled and pan fried bacon samples using stable isotopically labeled internal standards. These compounds were isolated from the matrices by a tandem solid-phase extraction procedure, followed by separation on reversed-phase liquid chromatography (HPLC) and quantified by atmospheric pressure chemical ionization tandem mass spectrometry (APCIMS-MS). Tandem mass spectrometry (MS-MS) acquisition was done in selected reaction monitoring (SRM) mode to provide a high degree of sensitivity and selectivity for accurate quantification of HAAs. The detection and quantification limits of these HAAs approached 0.015 and 0.045 microg/kg (part-per-billion), respectively, with only 4 g of meat. The HAA levels ranged widely from 0.045 to 45.500 microg/kg, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was the predominant HAA found in these samples. The amount of HAAs formed was highly dependent upon the type of meat and method of preparation. An intralaboratory comparison of the extraction procedure showed that estimates of these HAAs obtained by three different individuals at HAA levels below 2 microg/kg were within 5% with coefficients of variation below 19%, indicating the robustness of the analytical method. Moreover, because all of these HAAs from this class of molecules undergo facile cleavage at the N-methylimidazole moiety under collision-induced dissociation (CID) conditions, MS-MS analysis in the constant neutral loss mode of [M+H]+-15 enabled the identification of two other HAAs, 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx) and 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx), which have rarely been reported in cooked meats.

Amines↗
PubMed1998

Capillary electrophoretic determination of acetic acid and trifluoroacetic acid in synthetic peptide samples.

Synthetic peptide samples may contain counter-ions such as acetate or trifluoroacetate as a result of their method of preparation. Furthermore, because acetic acid (HOAc) and trifluoroacetic acid (TFA) are frequently used reagents in peptide synthesis, these acids may be found in synthetic peptide samples as impurities. This paper describes a method validation to determine HOAc and TFA in synthetic peptide samples by capillary electrophoresis (CE) using an internal standard (I.S.) with indirect UV detection. Typical analytical parameters such as precision, linearity, accuracy, specificity, limit of detection and ruggedness were evaluated during the validation. In addition, the contents of HOAc and TFA in two synthetic opioid peptide samples, TIPP[psi] and Orphanin FQ, were determined using the validated method. A unique feature of the method is that it offers determination of both acids in a single assay using a common I.S. The method is very efficient because of relatively short electrophoretic migration times (typically 2 to 8 min) for the acids investigated. This paper also discusses the factors that affect precision in a CE assay.

Acetic Acid↗
PubMed1996

Effects of multiple sterilization on surface characteristics and in vitro biologic responses to titanium.

PURPOSE: This study evaluates the surface changes and effects on in vitro cell attachment and spreading brought about on prepared commercially pure titanium by multiple exposures to common sterilization methods. MATERIALS AND METHODS: Discs of commercially pure titanium were prepared to approximate the surface roughness of commercially available bone miniplates. Samples underwent sterilization by exposure to ultraviolet light; ethylene oxide sterilization (1, 5, or 10 cycles); or by steam autoclaving (1, 5, or 10 cycles). Representative surfaces from these sterilization groups were examined using a series of surface analytical techniques including scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), auger electron spectroscopy (AES), and contact angle measurements. Cell attachment assays using murine fibroblasts were then performed on titanium surfaces from each sterilization group and on tissue culture plastic controls. Sterilized surfaces contained O, C, and N contaminants, which affected surface energetics. Mean percent cell attachment values for each group were obtained for periods of up to 1 hour. Representative samples from each group were examined using SEM to ascertain cell spreading and morphology for each sterilization group. RESULTS: Ultraviolet (UV) sterilized surfaces showed no changes from the unsterilized state macroscopically or under SEM. UV surfaces showed cell attachment levels similar to control surfaces at all intervals, and a chronologic progression of cell spreading. Ethylene oxide-sterilized surfaces showed occasional bluish discoloration and a microscopic particulate contaminant, resulting in modest decreases in cell attachment levels without strong correlation to numbers of sterilization cycles. Autoclaved surfaces generally showed the greatest discoloration and heaviest particulate contamination. Cell attachment levels were lower, and cell spreading was diminished compared with the ethylene-oxide-treated group. CONCLUSIONS: Both ethylene oxide and steam autoclave sterilization contaminated and altered the titanium surface, resulting in decreased levels of cell attachment and spreading in vitro. Although corroborative in vivo experiments should be conducted, the results of this study indicate that some multiple sterilization regimens for metallic materials may pose serious biologic concerns.

3T3 Cells↗
PubMed2001

Assessment of colloid formation and physical state distribution of trace polycyclic aromatic hydrocarbons in aqueous samples.

A tandem-cartridge system was established for studying colloid formation and physical state distribution of trace polycycic aromatic hydrocarbons (PAHs) in water. The effectiveness of the method for measuring the trace PAH colloids was demonstrated. With aqueous samples prepared by adding the PAH solutes with a small amount of organic solvent carrier, the potential of colloid formation increased with the hydrophobicity and concentration of the solute, but the incipient concentration for the colloid formation may be far lower than the aqueous solubility of the solute. After formation in water, the colloids showed remarkable stability at room temperature, and the stability was greatly reduced by elevated temperature and the presence of a small amount of inorganic electrolytes. The possible mechanism of destabilization of the colloids was discussed, and the mechanism might be of utility in providing insights into the physical state distribution of the solutes in various water samples. However, further effort to investigate the mechanism through fluorescence spectrophotometry was unsuccessful. The significance of the colloid formation in methods of preparation of aqueous samples and for measuring aqueous solubilities of PAHs and other hydrophobic compounds was illustrated. The results of this work demonstrate that it is undesirable to prepare aqueous solutions of PAHs or similarly hydrophobic compounds by vigorous mixing or the use of cosolvents, methods that are widely used in many studies for which aqueous solutions of such analytes are required.

Chromatography, Gas↗
PubMed2001

Direct simultaneous analysis of plasma samples for a drug discovery compound and its hydroxyl metabolite using mixed-function column liquid chromatography-tandem mass spectrometry.

A polymer-coated mixed-function (PCMF) column was evaluated for direct plasma injection for the simultaneous determination of a drug candidate and its hydroxyl metabolite by high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS-MS) in support of pharmacokinetic studies. Each diluted monkey plasma sample containing internal standard was directly injected on to the PCMF column for sample clean-up, enrichment and chromatographic separation. The proteins and macromolecules were first eluted from the column while the drug molecules were retained on the bonded hydrophobic phase. The analytes retained on the column were then eluted with a strong mobile phase using a gradient separation technique at a constant flow rate of 1.0 ml min(-1). When not diverted, the column effluent was connected either to the atmospheric pressure chemical ionization (APCI) source or the electrospray ionization (ESI) source as part of the mass spectrometer system used for quantification. The calibration curve was linear over the range 5-2500 ng ml(-1) for both analytes. The retention times for the analytes and the internal standard were both consistent and no column deterioration was observed for at least 500 injections. The recovery through the column and reproducibility of the dosed compound and its hydroxyl metabolite in monkey plasma samples were > 90% (RSD < 6%). The total analysis time was < 8 min per sample. The analytical results obtained by the proposed direct plasma injection method were in good agreement with those obtained by the conventional LC-MS-MS method.

Chromatography, Liquid↗
PubMed1988

Sample loss during measurement of airborne antineoplastic agents.

Handling, preparation and administration of antineoplastic drugs are suspected health hazards for hospital and clinic personnel. Measurement of personal and area exposures to such materials in air is difficult because the average concentration is often close to the analytical detection limit. For this reason, and because of the random nature of drug spills and releases, the sampling method of choice typically extends over a 40-hour workweek. In this study a method for monitoring two airborne antineoplastic drugs, fluorouracil and methotrexate, was tested for possible loss of the drug from filters during air sampling. Glass-fiber filters (37 mm) were spiked with known amounts of drug and subjected to the same airflow conditions as are used in workplace sampling. Filter extracts were analyzed using high performance liquid chromatography (HPLC). Losses during storage up to a month were less than 5% for both drugs. Losses during airflow were directly proportional to cumulative air volume, 2% of the amount on the spiked filter/m3 air for fluorouracil and 6%/m3 for methotrexate. Losses during actual collection will be less and ordinarily are not expected to constitute a major hindrance to extended sampling in occupational settings.

Air Movements↗
PubMed2003

Liquid chromatography-tandem mass spectrometry--application in the clinical laboratory.

This review provides a concise survey of liquid chromatography-tandem mass spectrometry (LC-TMS) as an emerging technology in clinical chemistry. The combination of two mass spectrometers with an interposed collision cell characterizes LC-TMS as an analytical technology on its own and not just as a more specific detector for HPLC compared with conventional techniques. In LC-TMS, liquid chromatography is rather used for sample preparation but not for complete resolution of compounds of interest. The instrument technology of LC-TMS is complex and comparatively expensive; however, in routine use, methods are far more rugged compared to conventional chromatographic techniques and enable high-throughput analyses with very limited manual handling steps. Moreover, compared to both gas chromatography-mass spectrometry (GC-MS) and conventional HPLC techniques, LC-TMS is substantially more versatile with respect to the spectrum of analyzable compounds. For these reasons it is likely that LC-TMS will gain far more widespread use in the clinical laboratory than HPLC and GC-MS ever did. In this article, the key features of LC-TMS are described, method development is explained, typical fields of application are discussed, and personal experiences are related.

Chemistry, Clinical↗
PubMed1995

An improved HPLC assay for the assessment of liver slice metabolic viability using 7-ethoxycoumarin.

The use of precision-cut liver slices constitutes a new in vitro metabolism technique for the study of coupled phase I and phase II biotransformations. This technique has the advantage of being easily amenable to studies with human tissue. As a means of characterizing the metabolic viability of diverse liver samples, a standard substrate capable of undergoing oxidative and conjugative pathways of metabolism would be desirable. An assay based on 7-ethoxycoumarin was developed whereby the metabolites--7-hydroxycoumarin, 7-hydroxycoumarin sulfate, and 7-hydroxycoumarin glucuronide--could be quantitated using a single HPLC analytical method. This required the synthesis of metabolite standards. 7-Hydroxycoumarin glucuronide was prepared by coupling 7-hydroxycoumarin with methyl 2,3,4-tri-O-acetyl-1-bromo-1-alpha-D- glucopyranuronate, under phase transfer conditions, to give the protected conjugate that was then hydrolyzed to give the glucuronide as the sodium salt. Assignment of configuration at the anomeric center was based on analysis of the simulated 1H and gated 13C NMR spectra, in addition to enzymatic hydrolysis. The sulfate conjugate was prepared by treatment of 7-hydroxycoumarin with Bu4N+ HSO4-/dicyclohexylcarbodiimide/pyridine. 7-Ethoxycoumarin, 7-hydroxycoumarin, and the glucuronide and sulfate conjugates were resolved by HPLC on a C8 Hypersil BDS column using ion-pairing conditions. Incubation of 7-ethoxycoumarin with rat or human liver slices in Krebs-Henseleit buffer resulted in the formation of these metabolites that were readily quantitated in the media with UV detection at 320 nm, using external standard curves. Although the sulfate was seen as the major metabolite in rats, the glucuronide predominated in human tissue. Two different human livers were examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMedDate not supplied

Immunoassay as an analytical tool in agricultural biotechnology.

Immunoassays for biotechnology engineered proteins are used by AgBiotech companies at numerous points in product development and by feed and food suppliers for compliance and contractual purposes. Although AgBiotech companies use the technology during product development and seed production, other stakeholders from the food and feed supply chains, such as commodity, food, and feed companies, as well as third-party diagnostic testing companies, also rely on immunoassays for a number of purposes. The primary use of immunoassays is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of GM analysis using immunoassays and especially its application to the testing of grains. The 2 most commonly used formats are lateral flow devices (LFD) and plate-based enzyme-linked immunosorbent assays (ELISA). The main applications of both formats are discussed in general, and the benefits and drawbacks are discussed in detail. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effects they may have on the accuracy of the immunoassays.

Agriculture↗
PubMed1999

Chromatographic separation of carotenoids.

The carotenoids are extremely reactive and consequently unstable due to their long system of conjugated double bonds. Several precautions, such as protection against light and oxygen, use of low temperature and antioxidants, analysis in the shortest possible time, should be taken during isolation and chromatography. The food samples, preferably fresh, are homogenized and immediately extracted with a suitable organic solvent. Saponification has been employed in order to hydrolyze the carotenoid esters, remove fatty material and destroy chlorophyll. This optional step facilitates subsequent carotenoid separation, identification and quantification. The separation of carotenoids is usually carried out by column chromatography, thin layer chromatography and high performance liquid chromatography, in analytical or preparative scale, on many stationary phases such as silica-gel, alumina, MgO, Ca(OH)2 and reversed-phase material (C18 and C30). The choice of the most suitable chromatographic method depends on the amount of sample, carotenoid composition, resolution, speed and purity required. Examples of carotenoid separation in different stationary phases will be shown and discussed.

Carotenoids↗
PubMed2005

Fluorimetric liquid chromatographic analysis of amantadine in urine and pharmaceutical formulation.

A simple and sensitive liquid chromatographic method is described for the analysis of amantadine and memantine. The method is based on the derivatization of amantadine and memantine extracted from alkalified samples with (2-naphthoxy)acetyl chloride at mild conditions. The resulting derivatives were analyzed by isocratic HPLC with a fluorimetric detector (lambdaex, 227 nm; lambdaem, 348 nm). The linear range for the determination of amantadine or memantine spiked in urine (1.0 ml) was 1.0-10.0 nmol with a detection limit of about 0.2 nmol (S/N = 3; injected sample 20 microl). Only amantadine preparations are available on our local market, and application of the method to the analysis of amantadine in formulation and in the urine of a dosed subject was demonstrated and proved feasible. Quantitation of AT in tablets or capsules is capable in the linear range of 2.0-50.0 microM. Toluene was used as the solvent for extracting amantadine or memantine in samples and the resulting toluene extract was directly subjected to subsequent derivatization without solvent replacement leading to a simpler analytical procedure.

Amantadine↗