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PubMed2006

Recent applications of capillary electrophoresis-electrospray ionisation-mass spectrometry in drug analysis.

This review considers applications in 2004-2005 of capillary electrophoresis-electrospray ionisation-mass spectrometry (CE-ESI-MS) to the detection and determination of small molecular mass drug molecules, taken from the Web of Knowledge database. The molecules of small molecular mass less than 1000 Da are chosen according to selected structural classes in which they give ESI signals primarily as [M + H](+) ions. These structural classes are drugs with amine-containing side chains, drugs with N-containing saturated ring structures, 1,4-benzodiazepines, other heterocyclic hypnotics, steroids, bioactive compounds containing phenolic groups, and miscellaneous molecules. Details are given on the fragmentations, where available, that these ionic species exhibit in-source and in ion-trap, triple quadrupole and time-of flight mass spectrometers. The review then gives a critical evaluation of these recent CE-ESI-MS analytical methods for the detection and determination of these small molecular mass drug molecules. Analytical information on, for example, sample concentration techniques, CE separation conditions, recoveries from biological media and limits of detection are provided.

Electrophoresis, Capillary↗

PubMed2001

Kinetics method for the quantitation of anthocyanidins, flavonols, and flavones in foods.

Flavonoids are important dietary constituents owing to their health-promoting properties. As a result, simplified analytic techniques are required for the population of databases with food values so that associations between dietary intake and disease risk/incidence can be established. The current research provides a simplified sample preparation procedure for the accurate estimation of food anthocyanidins, flavones, and flavonols as aglycons. Traditionally, flavonoid aglycons have been formed by acidic hydrolysis. However, some flavonoid aglycons are slowly degraded by acid. A procedure has been developed whereby anthocyanidins and flavonols are deglycosylated with HCl in 50% aqueous methanol and the resulting aglycons subsequently quantified by application of pseudo-first-order kinetics to their degradation. Flavones are also deglycosolated under similar conditions but, at appropriate temperatures, their aglycons are stable in acid, so kinetics were not required for the quantitation of this subclass of flavonoids. Catechins and flavanones were rapidly degraded under the hydrolytic conditions used in these studies.

Anthocyanins↗

PubMed2002

Innovations in the speciation of organolead compounds in water: towards a more rational, rapid, and simple analytical process.

Speciation analysis calls for rapid, simple systems for minimizing errors made in the most troublesome of all steps in the analytical process: sample preparation. In this context, continuous-flow systems are of great help. The evolution in the different methodologies enabled solutions to the main shortcomings occurring from the lack of selectivity of using RP-C18 as sorbent material. One solution was a shift to more sensitive and selective, but only partially automated, systems employing C60 fullerene and Grignard's reagent; another was a shift to completely automated systems employing sodium tetrapropylborate; and a final solution was to employ the simplest possible configuration by removing the reagent stream. The analytical methods developed allowed the identification and quantification of different organolead species at the pg/ml levels in rainwater samples, with precision (RSD) of about 5% and recoveries ranging from 92 to 100%.

Automation↗

PubMed2006

Simultaneous determination of albendazole metabolites, praziquantel and its metabolite in plasma by high-performance liquid chromatography-electrospray mass spectrometry.

The analysis of albendazole sulfoxide, albendazole sulfone, praziquantel and trans-4-hydroxypraziquantel in plasma was carried out by high-performance liquid chromatography-mass spectrometry ((LC-MS-MS). The plasma samples were prepared by liquid-liquid extraction using dichloromethane as extracting solvent. The partial HPLC resolution of drug and metabolites was obtained using a cyanopropyl column and a mobile phase consisting of methanol:water (3:7, v/v) plus 0.5% of acetic acid, at a flow rate of 1.0 mL/min. Multi reaction monitoring detection was performed by electrospray ionization in the positive ion mode, conferring additional selectivity to the method. Method validation showed relative standard deviation (precision) and relative errors (accuracy) lower than 15% for all analytes evaluated. The quantification limit was 5 ng/mL and the linear range was 5-2500 ng/mL for all analytes. The method was used for the determination of drug and metabolites in swine plasma samples and proved to be suitable for pharmacokinetic studies.

Albendazole↗

PubMed2005

Development of a high-throughput method for the determination of itraconazole and its hydroxy metabolite in human plasma, employing automated liquid-liquid extraction based on 96-well format plates and LC/MS/MS.

A semi-automated liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous quantification of the antifungal drug itraconazole (ITZ) and its coactive metabolite hydroxyitraconazole (OH-ITZ) in human plasma. The plasma samples underwent liquid-liquid extraction (LLE) in 2.2 mL 96 deepwell plates. ITZ, OH-ITZ and the internal standard (IS) R51012 were extracted from plasma, using a mixture of acetonitrile (ACN) and methyl t-butyl ether (MTBE) as the organic solvent. This specific mixture, due to its composition, had a significant impact on the performance of the assay. All liquid transfer steps, including preparation of calibration standards and quality control samples as well as the addition of the IS, were performed automatically using robotic liquid handling workstations for parallel sample processing. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated. The analytes and IS were dissolved in a small volume of a reconstitution solution, an aliquot of which was analyzed by combined reversed phase LC/MS/MS, with positive ion electrospray ionization and a TurboIonSpray interface, using multiple reactions monitoring (MRM). The method was shown to be sensitive and specific to both ITZ and OH-ITZ, it revealed excellent linearity for the range of concentrations 2-500 ng mL(-1) for ITZ and 4-1000 ng mL(-1) for OH-ITZ, it was very accurate and it gave very good inter- and intra-day precisions. The proposed high-throughput method was employed in a bioequivalence study after per os administration of two 100 mg tablets of ITZ, and it allowed this study to be completed in under four days.

Chromatography, Liquid↗

PubMed2001

Determination of recombinant human epidermal growth factor (rhEGF) in a pharmaceutical preparation by capillary electrophoresis.

A simple assay method of recombinant human epidermal growth factor (rhEGF) in a pharmaceutical preparation was studied and validated by capillary electrophoresis (CE) using micellar electrokinetic chromatography (MEKC) techniques. Factors affecting the migration behavior and separation performances of the peptide; type of buffer, pH, buffer concentration, and concentration of sodium dodecyl sulfates (SDS) were investigated to optimize the analytical performance. CE was performed using running buffer, 50.0 mM borate (pH 8.5) containing 12.5 mM SDS at 20 kV of the applied voltage. Calibration curves for the rhEGF showed good linearity (r>0.999) over the wide dynamic range from 1.25 to 100 microg/ml. Sample analysis was performed by using standard addition method to eliminate the matrix effects of dosage vehicle. This method is assumed to be useful for quality control (QC) of various forms of pharmaceutical products of the peptide.

Electrophoresis, Capillary↗

PubMed2005

Simultaneous determination of metal ions, amino acids, and other small biogenic molecules in human serum by capillary zone electrophoresis with transient isotachophoretic preconcentration.

CE with indirect UV detection was used for the simultaneous determination of lithium, magnesium, calcium, creatinine, carnitine, and a number of amino acids in human serum. The target analytes, positively charged under acidic electrolyte conditions, were separated with positive separation voltage polarity using 10 mM 4-methylbenzylamine, 4.5 mM citric acid, 25% (v/v) methanol at pH 4.05 as background electrolyte providing optimal separation. When analyzing real samples, however, some peaks were broadened due to essentially destacking conditions. In order to maintain the separation efficiency and also enhance the detection sensitivity, transient isotachophoresis (tITP) sample stacking was applied and yielded theoretical plate numbers in the range from 160,000 (arginine) to 350,000 (creatinine). The limit of detection values with tITP preconcentration were 0.11-0.26 mg L(-1) for metal cations, 1.0 mg L(-1) for creatinine, and 1.3-3.9 mg L(-1) for histidine, lysine, arginine, and ornithine. The method precision for peak areas was from 0.4 to 5.0% relative standard deviation using the matrix sodium as internal standard. The accuracy of the developed tITP-CZE system was verified by consistent results for Li+, Mg2+, Ca2+, and creatinine obtained on analyzing two serum certified reference materials. The only sample preparation required was ultrafiltration and acidification (to release protein-bound alkaline earths), and working ranges for individual analytes corresponded well to clinical concentration ranges.

Amino Acids↗

PubMed2003

Evaluation of in vitro secondary caries using confocal laser scanning microscope and X-ray analytical microscope.

PURPOSE: To evaluate in vitro secondary caries formation and inhibition by using a confocal laser scanning microscope (CLSM), and further map the calcium distribution with an X-ray analytical microscope (XAM). MATERIALS AND METHODS: 36 box-shaped cavities were prepared on bovine root dentin and restored with Fuji II, Fuji II LC (improved), or Clearfil Liner Bond 2V system with a non-fluoride releasing resin-based composite; Clearfil AP-X was used as a negative control. After immersion in a buffered demineralizing solution of 50 mmol/L of acetic acid adjusted to pH 4.5 for 3.5 days, longitudinal half sections were cut through each restoration and polished for analysis under CLSM. In order to confirm the CLSM results, the same samples were subsequently examined by means of the X-ray analytical microscope (XAM). The values of the outer lesions and the inhibition zones obtained by the two different CLSM and XAM measurements were then analyzed by one-way ANOVA, Fisher's PLSD Test at the 95% level of confidence and correlation coefficient. RESULTS: Inhibition zones and outer lesions were clearly detected under CLSM around the glass-ionomer and resin-modified glass-ionomer restorations. As expected, an inhibition zone was not detected adjacent to the resin-based composite restoration. Additionally, using the transmission x-ray images of XAM, the inhibition zones were observed as high density areas, and the outer lesions as low density areas. The width of the inhibition zones and the depth of the outer lesions measured under the CLSM were similar to those obtained from the XAM.

Acetates↗

PubMed1980

Profiling of uremic ultrafiltrate using high resolution gas chromatography-mass spectrometry - identification of 6 polyphenols.

An analytical method for separation and identification of compounds in uremic ultrafiltrate has been developed using a glass capillary column gas chromatography-mass spectrometry-computer system. The ultrafiltrate samples of blood were obtained during hemodialysis treatment of chronic uremic patients and non-uremic patients using the extracorporeal ultrafiltration method. Sample preparation consisted of acidification, extraction, evaporation, and trimethylsilylation. Many compounds were newly identified in the uremic ultrafiltrate by electron impact ionization, chemical ionization, and high resolution mass spectrometry. Six toxic polyphenols, namely, catechol, resorcinol, hydroquinone, 2-methoxyresorcinol, 3-methoxycatechol, and methoxyhydroquinone were first detected in the uremic ultrafiltrate.

Acute Kidney Injury↗

PubMed1996

Determination of ibogaine in plasma by gas chromatography--chemical ionization mass spectrometry.

Ibogaine is naturally occurring indole alkaloid that is currently being considered as a treatment medication for drug dependence. Although there have been a variety of investigations regarding the mechanisms of action and pharmacology of ibogaine, relatively little has been reported regarding quantitative methods. Because of the paucity of analytical methodologies, studies involving the pharmacokinetics and metabolism of ibogaine have also been limited. A method is described for the determination of ibogaine levels in plasma by gas chromatography -- methane chemical ionization mass spectrometry. [13C2H3]Ibogaine was synthesized and used as an internal standard to control for recovery during sample preparation. The assay requires one ml of plasma and is shown to be a selective and sensitive means of ibogaine quantitation.

Carbon Isotopes↗

PubMed2006

Sample clean-up with sol-gel enzyme and immunoaffinity columns for the determination of bisphenol A in human urine.

The paper describes the development of a simple and highly selective analytical method for the determination of free and total bisphenol A in urine samples. Free bisphenol A levels can be determined after sample clean-up using sol-gel immunoaffinity columns containing anti-bisphenol A antibodies. In determining total bisphenol A levels, the sample pre-treatment procedure consists of sample preparation using an on-line combination of two sol-gel columns, an enzyme column containing glucuronidase and arylsulfatase, and an immunoaffinity column. Bisphenol A can then be quantified by high-performance liquid chromatography and fluorescence detection. The mean recovery was found to be 78% with a standard deviation of 3.4%, the LOD (S/N=3) was 0.2 ng/ml. The method was applied to determine free and total urinary BPA levels of healthy adults and dialysis patients.

Adult↗

PubMed2003

Comprehensive two-dimensional gas chromatography in the analysis of urban aerosols.

Comprehensive two-dimensional gas chromatography utilising a semi-rotating cryogenic modulator was applied to the analysis of urban aerosols. Samples were collected onto glass fibre filters using a high-volume sampler in Helsinki, Finland. Sample preparation included extraction into n-hexane-acetone mixture and clean-up on silver-impregnated silica column. Analyses were performed with GC x GC-FID and GC x GC-QMS equipment. Linearity of the method was good with R2 values greater than 0.994 for all analytes. Polycyclic aromatic hydrocarbons (PAHs) and oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) were identified and quantified in urban particulates. PAH and oxy-PAH concentrations ranged from 0.5 to 5.5 ng/m3.

Aerosols↗

PubMed2003

Predicting petroleum phototoxicity.

Phototoxicity to Daphnia magna was studied on 14 polycyclic aromatic hydrocarbons (PAHs) and 22 petroleum products (ranging from diesel to crude oil). The phototoxicity ranking of pure PAHs was about the same as found in another study on fish gill cells in vitro (Toxicology 127 (1998) 143), suggesting that the relative acute phototoxicity does not differ significantly between different species. Most petroleum products were found to be phototoxic, although the results differ somewhat between test methods (preparation of water-accommodated fractions or petroleum ether dissolved oil slurries). The degree of phototoxicity of a sample is related to both the source and the refining process of the crude oil. The best chemical predictors found were the concentrations of eight phototoxic parent PAHs. An even simpler analytical technique suggested for initial screening would be direct measurements of "total PAH," where samples having more than 3% PAH should be studied further.

Animals↗

PubMed1999

Matrix interferences in the analysis of benzene in urine.

The analysis of benzene in urine of the general population or of exposed workers can be performed with different methods using the 'purge and trap' or 'solid-phase microextraction' techniques in combination with gas chromatographic analysis and photoionisation or mass spectrometric detection. The published results, however, are deeply conflicting. Differences in sample preparation by different research groups and our own preliminary observations prompted us to investigate pre-analytical and analytical factors potentially capable of modifying the urinary benzene quantification results. Benzene concentrations were measured in 20 urine samples in relation to different conditioning conditions (at 24, 40 and 80 degrees C) and at basic or acid pH. Urinary protein concentrations were measured in the same samples. Urine heating at 80 degrees C yields benzene concentrations on average five times higher than at 24 degrees C. On acidification of urine, the benzene released increases up to 28-fold in comparison to that obtained at uncorrected 'physiological' pH. Despite a widely scattered data distribution, a statistically significant linear correlation was found between 'heat-released' and 'acid-labile' benzene values. There was no correlation between total urinary proteins present in 'physiological' concentrations (between 12 and 110 mg/l) and the different kinds of benzene in urine. Our results could perhaps be explained if it is supposed that part of the benzene in urine is absorbed onto sediment, or bound to specific proteins, or derived from parent molecules and is released with pH modification or heat administration. Our observations may also help to explain why the urinary benzene concentrations reported by different investigators vary considerably even when environmental levels are comparable.

Artifacts↗

PubMed1989

Biological monitoring of toxic metals in urine by simultaneous inductively coupled plasma-atomic emission spectrometry.

A simple and rapid method for the simultaneous determination of 11 metals (As, Be, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, and Zn) in urine by inductively coupled argon plasma-atomic emission spectrometry is presented. Acidification of the urine was the only sample preparation required. Background correction was applied to ensure accuracy. Analytical calibration was based on matrix matching using a "simulated urine" solution. Detection limits in the low mg/L range and linearity over three orders of magnitude were obtained for all 11 metals. In the Occupational Health Laboratory, this procedure has been applied for routine screening of workers for occupational exposure to toxic metals.

Calibration↗

PubMed2005

Separation of proteins with a molecular mass difference of 2 kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: application to the isolation of 24 kDa human growth hormone.

A method for separating proteins with a molecular mass difference of 2 kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24 kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2 kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24 kDa hGH. The 22 and 24 kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13-18% linear gradient gel, and a 15-10% linear inverted gradient gel. Fractions containing purified 24 kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2 kDa.

Chromatography↗

PubMed2000

The use of nuclear magnetic resonance spectroscopy in the detection of drug intoxication.

The use of nuclear magnetic resonance (NMR) spectroscopy as a method for drug analysis has the advantages of reduced pre-analytical preparation time and the potential to detect and quantitate drug conjugates and metabolites simultaneously. NMR was investigated as a method to screen for organic substances (and metabolites) in 25 patients who presented to the Emergency Department with clinical indications of a drug overdose. Urine specimens were examined by 1H NMR spectroscopy at 300 MHz and the results compared with gas chromatography-mass spectrometry (GC-MS) results. There was a 56% concordance (14 of 25 samples) between NMR and GC-MS. NMR identified acetaminophen, ibuprofen, aspirin, valproate, carbamazepine, and pseudoephedrine as parent compounds or metabolites. For a patient for whom GC-MS results were negative, NMR strongly suggested the presence of erythromycin. NMR was most successful in identifying analgesics and antiepileptic drugs (sensitivity 83-100%). In 10 patients, signals from 1,2-propanediol, a common vehicle for some pediatric medications, were observed by NMR spectroscopy. NMR had 0% sensitivity in identifying tricyclic antidepressants and antipsychotic drugs. In these samples, GC-MS detected a variety of compounds, including tricyclic antidepressants and their metabolites and chlorpromazine. In addition, other substances that had not been disclosed as having been ingested, such as caffeine, diphenhydramine, and nicotine, were detected by GC-MS. NMR spectroscopy represents an emerging supplementary analytical technique that is applicable to a wide range of possible intoxicants and to the evaluation of the intoxicated patient, particularly when larger amounts of the intoxicant (> 200 mg) are ingested.

Adolescent↗

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↗