Development of a database of gas chromatographic retention properties of organic compounds

A comprehensive database of gas chromatographic retention properties of chemical compounds has been developed using multiple literature sources. The National Institute of Standards and Technology (NIST) database of retention data for non-polar and polar stationary phases currently contains 292,924 data records for 42,888 compounds. The database includes data for Kováts indices, linear indices, Lee indices, retention times and retention volumes. The first release of this database for non-polar stationary phases is available with NIST/US Environmental Protection Agency (EPA)/National Institutes of Health (NIH) Mass Spectral Database (June 2005) and through the internet (NIST Chemistry WebBook). The paper describes the database and the process by which it has been compiled. The format of data presentation and the quality control procedures are described. Data sources of gas chromatographic retention data are also discussed.     

Development of a new reference material for the validation of molecular detection methods for microbiological analysis

The standard methodology used for the detection of bacteria in environmental samples and food is primarily based on bacterial growth on specific culture media and confirmation by biochemical and/or immunological tests of all presumptive colonies. However, this methodology presents a number of drawbacks, such as low sensitivity and specificity, and the long time needed to obtain results. For this reason, the implementation of molecular methods in diagnostic laboratories has increased over the past several years. Nevertheless, most of these newer methods have not been included in current legislation, and, in most of cases, they have not yet been normalized. In this sense, the availability of appropriate reference materials (RMs) can help to overcome these deficiencies. The aim of this study was to develop and validate, following ISO Guide 34, a new RM, in a tablet format, for the quantification of Legionella pneumophila and Salmonella spp. by quantitative PCR (qPCR). This new RM can be used as a work reference sample in internal quality control, in the organization of proficiency testing schemes (PTS), as well as for the validation of molecular methods based on qPCR.     

KF-Silica as a stationary phase for the chromatographic removal of tin residues from organic compounds

Through the simple expedient of using a mixture of KF and silica as the stationary phase in column chromatography, levels of organotin impurities from tributyltin hydride mediated reductions have been reduced from stoichiometric levels to approximately 30 ppm.     

Development and validation of micellar liquid chromatographic methods for the determination of antibiotics in different matrixes

Antibiotics are the most important bioactive and chemotherapeutic compounds to be produced by microbiological synthesis, and they have proved their worth in a variety of fields, such as medicinal chemistry, agriculture, and the food industry. Interest in antibiotics has grown in parallel with an increasingly high degree of productivity in the field of analytical applications. Therefore, it is necessary to develop chromatographic procedures capable of determining various drugs simultaneously in the shortest possible time. Micellar liquid chromatography (MLC) is an RP-HPLC technique that offers advantages over conventional HPLC as far as sample preparation, selectivity, and versatility are concerned. Its main advantage is that samples can be injected directly into the chromatographic system with no previous preparation step. This paper mainly focuses on the results of the authors' own recent research and reports the chromatographic conditions for determination of various antibiotics (penicillins, quinolones, and sulfonamides) in different matrixes (pharmaceuticals, biological fluids, and food). The work of other authors on MLC-based antibiotic determination has been included.     

Development and validation of stability indicating chromatographic methods for simultaneous determination of citicoline and piracetam

Citicoline and piracetam were subjected separately to different stress conditions as recommended by the international conference on harmonization. In addition, new stability indicating thin layer chromatographic and ultra high performance liquid chromatographic methods have been developed and validated for simultaneous determination of citicoline and piracetam in presence of their degradation products. Separation on the proposed thin layer chromatographic method was carried out using a developing system containing methanol:chloroform:ammonium chloride buffer (9:1:2, v/v/v) on silica gel plates at 230 nm. On the other hand, the mobile phase in the ultra high performance liquid chromatographic method was composed of water (containing 0.1% triethylamine):ethanol (92:8, v/v). The flow rate was 1 mL/min and ultraviolet detection was at 230 nm. Moreover, results of the developed methods were statistically compared to those obtained by the reported high‐performance liquid chromatography method and no significant difference between them was found. The greenness profile of ultra high performance liquid chromatographic method was assessed and compared with those of the previously published high‐performance liquid chromatography methods, it was noticed that the proposed ultra high performance liquid chromatographic method more environmentally friendly and greener than other methods.     

Development and validation of a gas chromatographic—mass spectrometric procedure for the identification and quantification of residues of chloramphenicol

A method for the identification and quantification of residues of the antibiotic chloramphenicol was developed and validated. The method is based on combined gas chromatography-mass spectrometry with negative-ion chemical ionization and the use of [³⁷Cl₂]chloramphenicol as an internal standard. A set of identification criteria, in accordance with guidelines of the European Community, is described. For urine, muscle and eggs limits of detection and quantification of 0.1 μg kg^?1 are obtained. The method shows good repeatability and reproducibility. Results for urine were compared with those obtained with a radioimmunochemical procedure and an enzyme immunoassay (Quik-Card). Screening with an immunochemical procedure followed by confirmation with gas chromatography-mass spectrometry was found to be an effective strategy for monitoring residues of chloramphenicol in biological matrices.     

Development and validation of a solid-phase microextraction method for the analysis of volatile organic compounds in groundwater samples

Summary Solid-phase microextraction is a relatively recent extraction technique for sample preparation. It has been used successfully to analyse environmental pollutants in a variety of matrices such as soils, water and air. In this work, a simple and rapid method for the analysis of volatile organic and polar compounds from polluted groundwater samples by SPME coupled with gas chromatography (GC) is described. Different types of fibres were studied and the extraction process was optimised. The fibre that proved to be the best to analyse this kind of samples was CAR-PDMS. The method was validated by analysis of synthetic samples and comparison with headspace—GC. The optimised method was successfully applied to the analysis of ground-water samples.     

Development of a gas standard reference material containing eighteen volatile organic compounds

Summary A procedure to prepare primary gas cylinder standards for eighteen volatile organic compounds (VOC's) at the 1–15 nmol/mol (ppb) level was developed. The gas standards that were prepared by this procedure were intercompared by using gas chromatography with flame ionization detection (GC-FID) and electron capture detection (GC-ECD). The data, gravimetric concentration of the standards versus the respective GC peak area response, were plotted for each compound and fitted using linear regression. The regression analysis showed excellent agreement among the standards for each compound. These gas standards were evaluated over a period of 2 years and were determined to be stable and accurate. This research resulted in the development of Standard Reference Material (SRM) 1804, which contains these 18 volatile organic compounds in nitrogen at a nominal concentration of 5 nmol/mol for each compound. A batch of cylinders containing the mixture was procured from a commercial supplier. Each cylinder in the batch was analyzed for each of the 18 components. The data showed that the batch was homogeneous and stable for 15 of the 18 organic compounds, resulting in certification and issuance of SRM 1804.     

Two high-performance liquid chromatographic methods for quantitation of theophylline in plasma

Two high-performance liquid chromatographic methods are described for the assay of theophylline in plasma. Both allowed the separation of theophylline from the caffeine metabolites, theobromine and 1,7-dimethylxanthine. Method A, using 8-chlorotheophylline as internal standard, involved back extraction of theophylline from organic extract with 0.1 M sodium hydroxide. Method B used generally accepted solvent extraction followed by evaporation and beta-hydroxyethyltheophylline as internal standard. High-performance liquid chromatographic analyses were performed on reversed-phase phenyl columns (25 X 0.46 and 25 X 0.41 cm) using 20% methanol in 20 mM phosphate buffer at pH 5.6 for Method A and 2% acetonitrile and 8% methanol in 20 mM phosphate buffer for Method B. The column effluent was monitored at UV 273 nm. Standard curves for both Methods A and B were fitted by linear regression (r greater than 0.999) in the concentration range of 0.05-50 micrograms/ml. Either method was selective, accurate and reproducible over the concentration range 0.08-26 micrograms/ml. However, compared with Method B, Method A provided significant advantages in terms of simplicity, speed and efficiency.     

Development and validation of a liquid chromatographic/tandem mass spectrometric assay for the quantitation of nucleoside HIV reverse transcriptase inhibitors in biological matrices

Besides liquid chromatographic (LC)/UV methods adapted to therapeutic drug monitoring, there is still a need for more powerful techniques that can be used for pharmacological research and clinical purposes. We developed an LC method coupled with tandem mass spectrometry (MS/MS) to separate, detect and quantify with high sensitivity the nucleoside analogues used in multitherapies (zidovudine, stavudine, zalcitabine, didanosine, lamivudine and abacavir) in plasma and in the intracellular medium. We worked on two essential issues: (i) the need to use two ionization modes in order to achieve the best sensitivity, which leads to the optimization of the chromatographic separation of drugs detected in the positive ionization mode and drugs detected in the negative ionization mode, and (ii) the need to optimize the extraction step in order to enhance sample recovery. The peripheral blood mononuclear cells were lysed in Tris buffer-MeOH. A clean-up procedure was performed by solid-phase extraction only for plasma samples. The LC separation was carried out on a Zorbax Stable Bond C(18) column followed by MS/MS analysis after electrospray ionization in either the negative or positive mode. The positive ionization mode was applied at the beginning of the run to detect zalcitabine and lamivudine, then the ionization mode was changed to negative for the detection of didanosine, stavudine, internal standard and zidovudine. The calibration range for all the analytes was 0.5-200 ng ml(-1). The recoveries were between 64 and 90%, with coefficients of variation (CVs) lower than 15%. The inaccuracy (bias) was +/-15% with CVs always lower than 12%. The analytes were stable at room temperature and in the extraction solvent for at least 24 h, after storage at -80 degrees C for 3 months, after three freeze-thaw cycles and in the injection solvent after 48 h at 4 degrees C. Together with the measurement of intracellular triphosphorylated metabolites thanks to the powerful plasma and intracellular assay method for intact drugs, it is possible to describe the behaviour of nucleoside analogues against HIV through plasma pharmacokinetics, cell membrane diffusion including drug transport involvement, and also the intracellular metabolism.     

Identification of an organic compound leached from a membrane filter

An organic compound leached from a Millipore HA filter has been identified as the non-ionic surfactant polyoxyethylene nonylphenyl ether with a degree of polymerization of 4-10 for the ethylene oxide unit. It is suggested that the Millipore HA filter should be used only after several rinses with sample or doubly-distilled water.     

Characterization and evaluation of material used for the recovery of organic compounds from water

Summary A general procedure that should be applied when a sorbent material is examined in order to recover organic pollutants from water is shown. This method recommends determination of specific retention volumes, the break-through of test compounds and the influence on these of the interfering substances. An expanded hydrophobic perlite was characterized as a suitable adsorbent for the recover of polycyclic aromatic hydrocarbons (PAHs).     

Development and validation of column high-performance liquid chromatographic and ultraviolet spectrophotometric methods for citalopram in tablets

Column high-performance liquid chromatographic (LC) and UV spectrophotometric methods for the quantitative determination of citalopram, a potent and selective serotonin reuptake inhibitor, in tablets were developed. The parameters linearity, precision, accuracy, specificity, robustness, limit of detection, and limit of quantitation were studied according to International Conference on Harmonization guidelines. Chromatography was carried out by the reversed-phase technique on an ACE C18 column with a mobile phase composed of 0.30% triethylamine solution-acetonitrile (55 + 45, v/v) adjusted to pH 6.6 with 10% ortho-phosphoric acid at a flow rate of 1.0 mL/min and 25 degrees C. The UV spectrophotometric method was performed at 239 nm. The linearity of the LC method was in the range of 10.00-70.00 microg/mL, and 2.50-17.50 microg/mL for the UV spectrophotometric method. The interday and intraday assay precision was < 1.5% (relative standard deviation) for the LC and UV spectrophotometric methods. The recoveries were in the range 100.70-101.35% for the LC method and 98.48-98.65% for the UV spectrophotometric method. Statistical analysis by Student's t-test showed no significant difference between the results obtained by the 2 methods. The proposed methods are highly sensitive, precise, and accurate and can be used for the reliable quantitation of citalopram in tablets.     

Sub-micro methods for the analysis of organic compounds

Summary A new technique is described which enables organic compounds to be analysed for elements and functional groups using sample weights of 30–50 micrograms (0.03 to 0.05 mg). These amounts are only just visible to the naked eye. The accuracy is similar to that of the microscale, and the technique is fairly simple.It is expected that these methods will be of value in biochemistry and associated fields where amounts of sample are often limited. The new technique is not intended to replace micro-methods. Investigations are still proceeding to develop further methods for the determination of functional groups and to refine earlier procedures.     

Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the quantitation of prazepam and its main metabolites in human plasma

A method was developed and fully validated for the quantitation of prazepam and its major metabolites, oxazepam and nordiazepam, in human plasma. Sample pretreatment was achieved by solid-phase extraction using Oasis HLB cartridges. The extracts were analysed by high-performance liquid chromatography (HPLC) coupled with single-quadrupole mass spectrometry (MS) with an electrospray ionization interface. The MS system was operated in the selected ion monitoring mode. HPLC was performed isocratically on a reversed-phase XTerra MS C18 analytical column (150 x 3.0 mm i.d., particle size 5 microm). Diazepam was used as the internal standard for quantitation. The assay was linear over a concentration range of 5.0-1000 ng ml(-1) for all compounds analyzed. The limit of quantitation was 5 ng ml(-1) for all compounds. Quality control samples (5, 10, 300 and 1000 ng ml(-1)) in five replicates from three different runs of analysis demonstrated an intra-assay precision (CV) of < or = 9.1%, an inter-assay precision of < or = 6.0% and an overall accuracy (relative error) of < 4.6%. The method can be used to quantify prazepam and its metabolites in human plasma covering a variety of pharmacokinetic or bioequivalence studies.     

Gas chromatographic/mass spectrometric identification of some organic compounds and odours in poultry wastes

Air and waste samples coming from poultry breeding farms have been analysed by means of thermal desorption coupled to the gas chromatographic/mass spectrometric technique. The solid samples not only showed the presence of organic compounds such as acids, sulphides, benzopyrroles and phenols originated by anaerobic biotransfor-mation of the natural products, but also the occurrence of anti-oxidant substances: 2,6-di-tert-butylcresol, 2,6-di-tert-butylphenol and two of their oxidation derivatives. In the air, four artificial benzoquinones were detected, two of which had already been identified in the solid waste. In this work, the mechanism by which oxidized compounds are formed, is suggested.     

Isolation, identification and quantitation of urinary organic acids

An application of the HISLIB program for the comparison of gas chromatographic-mass spectrometric profiles of urinary organic acids isolated by extraction and ion-exchange methods is described. Ion-exchange methods are clearly superior to solvent extraction in terms of the variety of compounds isolated. However, the former method has practical difficulties which make solvent extraction more attractive for rapid analyses. For the compounds isolated by both methods, the precision of analysis is similar, with standard deviations of relative concentration in the range 10--30% for most compounds.     

Development and validation of a liquid chromatographic method for monitoring of process-related synthetic organic impurities of profenofos in technical products

A simple and rapid reversed-phase high-performance liquid chromatographic method for the monitoring of process-related synthetic organic impurities of profenofos (PFS) is developed. Impurities are separated and determined on a reversed-phase Hypersil C(18) column using gradient elution of 50 mM ammonium formate buffer-acetonitrile as a mobile phase and detection at 230 nm at ambient temperature. The method is validated with respect to accuracy, precision, linearity, and limits of detection and quantitation. The method is found to be suitable not only for monitoring the reactions involved in the process development of PFS, but also quality assurance, as it can detect impurities at the level of 1.5 x 10(-8) g.