Metabolites In Biological Fluids Research Articles

Column-switching procedures for the fast analysis of drugs in biologic samples.

Liquid chromatography coupled with mass spectrometry in single and dual mode (LC-MS and LC-MS/MS) is the method of choice for the quantification of drugs and their metabolites in biologic fluids. Following the new challenges encountered in the process of drug development, liquid chromatography-mass spectrometry has been found to achieve high-throughput analysis. With this impressive tool, the sample preparation step before analysis is simplified, and the analytic process speeded up. Several generic approaches have recently been developed for the sample extraction coupled on line with a LC-MS system. In this paper, different extraction supports allowing the direct injection of biologic fluids were investigated, namely, restricted-access media, large-size particle, and monolithic phases. In the column-switching configuration, these supports, coupled with microbore analytic columns, were found suitable for the fast analysis (total analysis time of less than 10 minutes) of different drugs and their metabolites in biologic matrices at the nanogram per milliliter level.

Recent advances in the determination of enantiomeric drugs and their metabolites in biological fluids by capillary electrophoresis-mediated microanalysis.

This review summarizes the recent developments in the application of electromigration techniques for the enantioselective analysis of drugs and metabolites in biological fluids. During the period covered by this review, it has been observed an increase in the use of negatively charged chiral selectors, particularly sulfobutyl ether-beta-cyclodextrin and sulfated-beta-cyclodextrin, and the combination of two different chiral selectors in the running buffer to obtain the resolution of drugs and their metabolites. Low detection limits as required for pharmacokinetic studies were obtained by using concentration techniques, including sample stacking procedures, and more sensitive detection systems, such as laser-induced fluorescence and mass spectrometry. Finally, the major points are discussed that can be considered to obtain reliable methods for enantioselective bioanalysis.

Use of liquid chromatography/time-of-flight mass spectrometry and multivariate statistical analysis shows promise for the detection of drug metabolites in biological fluids.

The process of metabolite identification is essential to the drug discovery and development process; this is usually achieved by liquid chromatography/tandem mass spectrometry (LC/MS/MS) or a combination of liquid chromatography/mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR) spectroscopy. Metabolite identification is, however, a time-consuming process requiring an experienced skilled scientist. Multivariate statistical analysis has been used in the field of metabonomics to elucidate differences in endogenous biological profiling due to a toxic effect or a disease state. In this paper we show how a combination of liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) and multivariate statistical analysis can be used to detect drug metabolites in a biological fluid with no prior knowledge of the compound administered.

Biomarkers, validation and pharmacokinetic-pharmacodynamic modelling.

Four elements are crucial to successful pharmacokinetic-pharmacodynamic (PK/PD) modelling and simulation for efficient and effective rational drug development: (i) mechanism-based biomarker selection and correlation to clinical endpoints; (ii) quantification of drug and/or metabolites in biological fluids under good laboratory practices (GLP); (iii) GLP-like biomarker method validation and measurements and; (iv) mechanism-based PK/PD modelling and validation. Biomarkers can provide great predictive value in early drug development if they reflect the mechanism of action for the intervention even if they do not become surrogate endpoints. PK/PD modelling and simulation can play a critical role in this process. Data from genomic and proteomics differentiating healthy versus disease states lead to biomarker discovery and identification. Multiple genes control complex diseases via hosts of gene products in biometabolic pathways and cell/organ signal transduction. Pilot exploratory studies should be conducted to identify pivotal biomarkers to be used for predictive clinical assessment of disease progression and the effect of drug intervention. Most biomarkers are endogenous macromolecules, which could be measured in biological fluids. Many exist in heterogeneous forms with varying activity and immunoreactivity, posting challenges for bioanalysis. Reliable and selective assays could be validated under a GLP-like environment for quantitative methods. While the need for consistent reference standards and quality control monitoring during sample analysis for biomarker assays are similar to that of drug molecules, many biomarkers have special requirements for sample collection that demand a well coordinated team management. Bioanalytical methods should be validated to meet study objectives at various drug development stages, and possess adequate performance to quantify biochemical responses specific to the target disease progression and drug intervention. Protocol design to produce sufficient data for PK/PD modelling would be more complex than that of PK. Knowledge of mechanism from discovery and preclinical studies are helpful for planning clinical study designs in cascade, sequential, crossover or replicate mode. The appropriate combination of biomarker identification and selection, bioanalytical methods development and validation for drugs and biomarkers, and mechanism-based PK/PD models for fitting data and predicting future clinical endpoints/outcomes provide powerful insights and guidance for effective and efficient rational drug development, toward safe and efficacious medicine for individual patients.

The application of mass spectrometry in pharmacokinetics studies

Pharmacokinetics is the study of the absorption, distribution, metabolism, and excretion (ADME) of drugs; the respective rates of which are of fundamental importance in determining their effect on the organism to which they are administered. Pharmacokinetic studies with mass spectrometry can provide quantitative information about a compound’s half-life in the body and how quickly it is metabolized or excreted. These studies are also used to determine drug distribution, partitioning within an organism, and drug dosing regime. The latter is important in determining a drug’s therapeutic range, which is a balance between the concentration required for positive therapeutic effect versus higher doses which may have toxicological effects. Finally, pharmacokinetics is used to investigate drug dosing with regards to administration factors of age, gender, ethnicity, concomitant drugs, or diseases. Pharmacokinetic analysis involves the determination of the concentration of drugs and/or their metabolites in biological fluids, which may be present at very low concentrations. Quantitative analysis is important since a major consideration in these types of studies is to determine how much drug gets into the systemic circulation. The integration of drug concentration over time (the so-called Area-Under-TheCurve or AUC) is a measure of both how well the drug is cleared by an individual and how well the analytical system is performing. Therefore, reliable, sensitive and specific methods are required for these types of analyses. With the increased pressure on researchers and pharmaceutical companies to discover and develop novel therapeutic compounds in a rapid manner, mass spectrometry now plays a central role in pharmacokinetics studies. The initial mass spectrometric technique used in pharmacokinetics, gas chromatography/mass spectrometry (GC/MS) was limited to molecules that were either volatile or could be made thermally stable by pre-column derivatization. Derivatization is often used to improve a molecule’s ability to be vaporized and ionized with a GC/MS, yet it has significant disadvantages because many molecules are still not amenable to GC/MS analysis after derivatization and the sample handling associated with derivatization results in sample loss. More recently, liquid chromatography coupled with soft ionization techniques such as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) with triple quadrupole mass analysis (Fig. 1), have become the most common approaches in pharmacokinetic studies. This is because

Development of a capillary gas chromatographic method with flame ionisation detection for the simultaneous determination of sildenafil and itsN-demethylated metabolite in biological fluids

A simple, rapid, and sensitive method has been developed for the analysis of sildenafil (S) and its N-Demethylated metabolite UK-103,320 (UK) using capillary gas chromatography with a flame ionisation detector. Optimal conditions were investigated, achieving analysis times shorter than 12 minutes. Aspects such as stability of the solutions, linearity, accuracy (<91%), precision, specificity, and limits of detection (0.02 mg/L and 0.12 mg/L for S and UK respectively) were tested in order to validate the method. A solid-phase extraction (SPE) prior to the analysis allows quantification of S in serum and urine, and UK in serum at clinically relevant concentrations. Furthermore the specificity has been assessed by means of mass spectrometry in SCAN mode.

Metabonomic assessment of physiological disruptions using 1H-13C HMBC-NMR spectroscopy combined with pattern recognition procedures performed on filtered variables.

Metabonomic characterization of long-lasting although weak physiological events such as anabolic disruptions remains poorly investigated. We have validated 1H-13C HMBC-NMR as a suitable generator of instrumental variables that are strongly linked to the concentration of endogenous metabolites in biological fluids. This method is interfaced to multivariate pattern recognition procedures. Fingerprints established from urine sample collected on cattle treated with anabolic steroids were used to validate this method. Four main results arise from this study. (i) 2D NMR is as informative as 1D NMR. (ii) 2D NMR variable clustering highlights successfully a contingent redundancy of variables, although a relevant hierarchical model of statistical correlations covering from structural relationships to physiologic links can also be evidenced. (iii) To enhance pattern recognition performances, we have validated a variable selection algorithm for accurate prediction of unknown individuals belonging to predetermined groups achieved by linear discriminant analysis (LDA). This algorithm synthesizes the whole information contained in the data set by selecting preferentially nonredundant variables. Parameters generating variable subsets are validated by predicted variance efficiency obtained when minimizing error rates calculated by cross-validation methods. (iv) Provided variables are correctly filtered, LDA fairly competes with partial least-squares methods for both classification of individuals and statistical interpretation of metabolic responses obtained in such a physiological disruption context.

Characterization of isoflavones and their conjugates in female rat urine using LC/MS/MS.

Isoflavone phytoestrogens found in soybeans are the most widely studied phytochemicals in human diets and soy infant formulas. The health benefits of the isoflavones daidzein and genistein have been reported, and concerns about potential adverse effects have also been raised. However, the results of direct analysis of isoflavones and their metabolites in biological fluids after consumption of soy-containing diets are scarce. This study describes an LC/MS/MS method for the analysis of isoflavones and their metabolites in the urine of female rats fed diets made with soy protein isolate. Five isoflavones (daidzein, genistein, glycitein, dihydrodaidzein, and O-desmethylangolensin) were identified by comparison with authentic standards. Seventeen conjugates of isoflavones were characterized in the urine, the most unusual being genistein 5-glucuronide and four glucuronide conjugates of reductive metabolites of daidzein. The application of LC/MS/MS to analyze isoflavone metabolites is simple and sensitive, and appears to be an excellent method for determining the bioavailability and metabolism of food phytochemistry.

Evolving bioanalytical methods for the cardiovascular drug bosentan

The history of the development of analytical methods for the determination of the cardiovascular agent bosentan, and its three major metabolites in biological fluids is described. Before the advent of LC-MS-MS, conventional HPLC-UV methodology was adequate for the determination of the drug alone in samples generated from toxicology studies. The progressive changes in the methods described reflect improvements in MS instrumentation and software, the need for measuring metabolites, as well as the availability of deuterated internal standards. Finally, three LC-MS-MS methods were chosen as being suitable for routine use. The most sophisticated of these (E), involving protein precipitation, liquid-liquid extraction, and on-line solid phase extraction by automated column switching, allowed the simultaneous and sensitive determination of the drug and three metabolites in a variety of biological matrices from several species to support pharmacokinetic studies. Two further, simpler, MS methods were developed (F and G) which, compared with the sophisticated method, focussed on reduced manual effort and reduced instrumental run cycle times. The limits of quantitation were between 1 and 2 ng mL−1 for bosentan and the three metabolites.

Identification of [(GS)2AsSe]− in rabbit bile by size‐exclusion chromatography and simultaneous multielement‐specific detection by inductively coupled plasma atomic emission spectroscopy

AbstractAn arsenic–selenium metabolite that exhibited the same arsenic and selenium X‐ray absorption near‐edge spectra as the synthetic seleno‐bis(S‐glutathionyl) arsinium ion [(GS)2AsSe]− was recently detected in rabbit bile within 25 min after intravenous injection of rabbits with sodium selenite and sodium arsenite. X‐ray absorption spectroscopy did not (and cannot) conclusively identify the sulfur‐donor in the in vivo sample. After similar treatment of rabbits, we analyzed the collected bile samples by size‐exclusion chromatography (SEC) using inductively coupled plasma atomic emission spectroscopy (ICP‐AES) to monitor arsenic, selenium and sulfur simultaneously. The bulk of arsenic and selenium eluted in a single peak, the intensity of which was greatly increased upon spiking of the bile samples with synthethic [(GS)2AsSe]−. Hence, we identify [(GS)2AsSe]− as the major metabolite in bile after exposure of rabbits to selenite and arsenite. The reported SEC–ICP‐AES method is the first chromatographic procedure to identify this biochemically important metabolite in biological fluids and is thus a true alternative to X‐ray absorption spectroscopy, which is not available to many chemists. Copyright © 2001 John Wiley &amp; Sons, Ltd.

Reduction of signal suppression effects in ESI-MS using a nanosplitting device.

Electrospray ionization mass spectrometry is a valuable tool in the identification and quantification of drug metabolites in biological fluids. However, there are many instances where matrix components present in these fluids interfere with analyte detection and prevent the acquisition of accurate or complete results. In some instances, the matrix can suppress ionization to such an extent that analytes are completely undetectable by MS. In this work, we investigate how ionization and ion-transfer efficiencies are affected by drastically reducing the flow into the MS. A postcolumn concentric flow-splitting device was constructed to allow the measurement of analyte signal and ionization suppression across a range of flow rates (0.1-200 microL/min). Using this device, the effects of flow rate on signal intensity and ionization suppression were measured in analytical experiments that included flow injection analysis MS, postcolumn addition LC-MS, and on-line LC-MS analysis of metabolites generated from rat liver microsomes. The device used to deliver 0.1 microL/min flows is referred to as a nanosplitter because it achieved high split ratios (2000:1), producing flow rates comparable to those observed in nanoelectrospray. The nanosplitter maintained chromatographic integrity with high fidelity and allowed the direct comparison of analyte signal across a range of flow rates (0.1-200 microL/min). A significant improvement in concentration and mass sensitivity as well as a reduction in signal suppression is observed when the performance at 200 versus 0.1 microL/min flow rate is compared. Using this specially designed concentric splitting device, the advantages of ultralow flow ESI were easily exploited for applications employing large bore chromatography.

Recent developments in chiral capillary electrophoresis and applications of this technique to pharmaceutical and biomedical analysis.

This paper provides an overview of the current status of chiral capillary electrophoresis (CE). The emphasis is placed on the application of CE in chiral separation of various racemic compounds. During the last two years about 280 papers, several review articles, and two entire issues, edited by S. Fanali (Electrophoresis 1999, 20, 2577-2798, and H. Nishi and S. Terabe (J. Chromatogr. A 2000, 879, 1-471.) have been devoted to chiral CE. Enantiomeric separations of various compounds, e.g., pharmaceuticals, drug candidates, drugs and related metabolites in biological fluids, amino acids, di- and tri peptides, pesticides and fungicides, have been performed using different chiral selectors. Native and derivatized cyclodextrins continue to be the most widely used chiral selectors. Other chiral selectors such as natural and synthetic chiral micelles, crown ethers, chiral ligands, proteins, oligo- and polysaccharides, and macrocyclic antibiotics have also been applied to chiral CE separations.

The effect of stationary phase type and mobile phase pH on the separation of some catecholamines

The catecholamines are very important in the control and regulation of a variety of functions acting as neurotransmitters in both central and peripheral nervous systems. Determination of the levels of catecholamines and their metabolites in biological fluids and brain tissue is very important for diagnosis of neurological and psychiatric disorders and in studies on the physiological and pathological role of the autonomic nervous system. High-performance ion-pair chromatography with electrochemical detection and very complicated mobile phases is most frequently used for estimation of these substances. In our studies we tried to apply reversed-phase planar chromatography for the purpose. Silica gel and silanized silica gel were used as stationary phases and solutions of buffer and organic solvents as mobile phases.

Ion interaction reagent reversed-phase high-performance liquid chromatography determination of anti-tuberculosis drugs and metabolites in biological fluids

New methods of ion interaction reagent (IIR) RP-HPLC are presented for the determination of anti-tuberculosis drugs and their metabolites, singly or in multi-component mixtures, in biological fluids. The following analytes are considered: isoniazid, ethionamide, pyrazinamide, morphazinamide, p-aminosalicylic acid, nicotinic and isonicotinic acids. Aqueous solutions of three different ion interaction reagents are alternatively or comparatively used as the mobile phases, namely: (A) 5.00 m M octylamine at pH 3.00 for o-phosphoric acid, (B) 5.00 m M octylamine at pH 8.00 for o-phosphoric acid, and (C) 5.00 m M 1,6 diaminohexane at pH 6.00 for o-phosphoric acid. The response linearity between peak area and analyte concentration is verified for all the analytes in the concentration range within the determination limits and 2.00 mg/l. Detection limits are always lower than 82 μg/l for standard solutions; in the analysis of samples of rat serum, rat plasma and human serum, the matrix effect is negligible, the detection limits are always lower than 94 μg/l and the average recovery yield is always greater than 96%.

Biological indicators of genotoxic risk and metabolic polymorphisms.

International scientific publications on the influence of metabolic genotypes on biological indicators of genotoxic risk in environmental or occupational exposure are reviewed. Biomarkers of exposure (substance or its metabolites in biological fluids, urinary mutagenicity, protein and DNA adducts) and of effects (chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (Mn), COMET assay, HPRT mutants) have been evaluated according to different genotypes (or phenotypes) of several activating/detoxifying metabolic activities. In less than half the studies (43 out of 95), the influence of genotype on the examined biological indicator was found, of which four report poorly reliable results (i.e., with scarce biological plausibility, because of the inconsistency of modulated effect with the type of enzymatic activity expressed). As regards urinary metabolites, the excretion of mercapturic acids (MA) is greater in subjects with high GST activity, that of 1-pyrenol and other PAH metabolites turns out to be significantly influenced by genotypes CYP1A1 or GSTM1 null, and that of exposure indicators to aromatic amines (AA) (acetylated and non-acetylated metabolites) is modulated by NAT2. In benzene exposure, preliminary results suggest an increase in urinary t, t-muconic acid (t,t-MA) in subjects with some genotypes. On urinary mutagenicity of PAH-exposed subjects, the effects of genotype GSTM1 null, alone or combined with NAT2 slow are reported. When DNA adduct levels are clearly increased in PAH-exposed group (18 out of 22), 7 out of 18 studies report the influence of GSTM1 null on this biomarker, and of the five studies which also examined genotype CYP1A1, four report the influence of genotype CYP1A1, alone or in combination with GSTM1 null. A total of 25 out of 41 publications (61%) evaluating the influence of metabolic polymorphisms on biomarkers of effect (cytogenetic markers, COMET assay, HPRT mutants) do not record any increase in the indicator due to exposure to the genotoxic agents studied, confirming the scarce sensitivity of these indicators (mainly HPRT mutants, Mn, COMET assay) for assessing environmental or occupational exposure to genotoxic substances. Concluding, in determining urinary metabolites for monitoring exposure to genotoxic substances, there is sufficient evidence that genetically-based metabolic polymorphisms must be taken into account in the future. The unfavourable association for the activating/detoxifying metabolism of PAH is also confirmed as a risk factor due to the formation of PAH-DNA adducts. The clearly protective role played by GSTT1 on DEB (and/or related compound)-induced sister chromatid exchanges (SCEs) should be noted. The modulating effects of genotypes on protein adduct levels in environmental and occupational exposure have not yet been documented, and most studies on the influence of genotype on biological indicators of early genotoxic effects report negative results.

Rapid and highly sensitive method for the determination of verapamil, [2H7]verapamil and metabolites in biological fluids by liquid chromatography–mass spectrometry

A rapid and highly sensitive method for the determination of verapamil [2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] and [2H7]verapamil and their primary metabolites D-617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile], D-703 [2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dimethoxy-phenyl)-6-methyl-2-isopropyl-6-azaoctanitrile], D-702 [2-(3,4-dimethoxy-phenyl)-8-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile], norverapamil [2,8-bis-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile] and secondary metabolites D-620 [2-(3,4-dimethoxyphenyl)-5-amino-2-isopropylvaleronitrile], D-717 [2-(4-hydroxy-3-methoxyphenyl)-5-amino-2-isopropylvaleronitrile], and D-715 [2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dimethoxy-phenyl)-2-isopropyl-6-azaoctanitrile] has been developed using high-performance liquid chromatography–electrospray mass spectrometry. D-832, the gallopamil analogue of D-617 and [2H3]norverapamil were used as internal standards. The analytes were extracted automatically from plasma and intestinal perfusate using end-capped CN- and C2 solid-phase extraction cartridges. Separation of the eight analytes was achieved on a LUNA C8 analytical column (150×2 mm I.D., 5 μm particle size) with 5 mM ammonium acetate–acetonitrile as the mobile phase run with a gradient from 70:30 to 40:60 and run times of 15 min. With the mass spectrometer operated in the selected-ion monitoring mode, the limits of quantification in plasma and intestinal perfusate were 1 pmol/ml for D-620, D-617, D-702, D-703, norverapamil, verapamil, and [2H7]verapamil and 2.5 pmol/ml for D-717 and D-715 using a sample size of 1 ml plasma and intestinal perfusate. The method described was successfully applied to the determination of verapamil, [2H7]verapamil and their metabolites in human plasma and intestinal fluid in pharmacokinetic studies.

The synthesis of deuterium-labelled cocaine, cocaethylene and metabolites

We describe the syntheses of benzoylecgonine, (1,1,1-2H3)methyl ester [(2H3)cocaine], (2H5)benzoylecgonine, (2H5)benzoylecgonine methyl ester [(2H5)cocaine], benzoylecgonine (2,2,2-2H3)ethyl ester [(2H3)cocaethylene], (2H5)benzoylecgonine ethyl ester [(2H5)cocaethylene], (2H5)benzoylecgonine (2,2,2-2H3)ethyl ester [(2H8)cocaethylene], ecgonine (1,1,1-2H3)methyl ester, ecgonine (2,2,2-2H3)ethyl ester, ecgonine (1,1,2,2,2-2H5)ethyl ester and anhydroecgonine (1,1,1-2H3)methyl ester. (2H5)Cocaine and (2H3)cocaethylene have been administered to human subjects to study the interactions of cocaine and ethanol. The other eight compounds were utilized as analytical standards or internal standards for GC-MS quantitation of cocaine and its metabolites in biological fluids. Copyright © 1999 John Wiley & Sons, Ltd.

Totalp-Nitrophenol Determination in Urine Samples of Subjects Exposed to Parathion and Methyl-Parathion by SPME and GC/MS

Journal of High Resolution ChromatographyVolume 22, Issue 11 p. 628-630 Short Communication Total p-Nitrophenol Determination in Urine Samples of Subjects Exposed to Parathion and Methyl-Parathion by SPME and GC/MS Maurizio Guidotti, Corresponding Author Maurizio Guidotti Presidio Multizonale di Prevenzione di Rieti, Via Salaria per l'Aquila n°8, 02100 Rieti, ItalyPresidio Multizonale di Prevenzione di Rieti, Via Salaria per l'Aquila n°8, 02100 Rieti, ItalySearch for more papers by this authorGuido Ravaioli, Guido Ravaioli Presidio Multizonale di Prevenzione di Rieti, Via Salaria per l'Aquila n°8, 02100 Rieti, ItalySearch for more papers by this authorMatteo Vitali, Matteo Vitali Cattedra di Igiene Ambientale, Istituto d'Igiene, Università “La Sapienza”, P.le A. Moro n°1, 00100 Roma, ItalySearch for more papers by this author Maurizio Guidotti, Corresponding Author Maurizio Guidotti Presidio Multizonale di Prevenzione di Rieti, Via Salaria per l'Aquila n°8, 02100 Rieti, ItalyPresidio Multizonale di Prevenzione di Rieti, Via Salaria per l'Aquila n°8, 02100 Rieti, ItalySearch for more papers by this authorGuido Ravaioli, Guido Ravaioli Presidio Multizonale di Prevenzione di Rieti, Via Salaria per l'Aquila n°8, 02100 Rieti, ItalySearch for more papers by this authorMatteo Vitali, Matteo Vitali Cattedra di Igiene Ambientale, Istituto d'Igiene, Università “La Sapienza”, P.le A. Moro n°1, 00100 Roma, ItalySearch for more papers by this author First published: 09 December 1999 https://doi.org/10.1002/(SICI)1521-4168(19991101)22:11<628::AID-JHRC628>3.0.CO;2-0Citations: 20AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume22, Issue111 November 1999Pages 628-630 RelatedInformation

Determination of Opiates and Cocaine and Its Metabolites in Biological Fluids by High-Performance Liquid Chromatography with Electrospray Tandem Mass Spectrometry

A rapid, sensitive, and specific method for the determination of opiates and cocaine and metabolites in urine, plasma, and blood was established. A one-step extraction followed by liquid chromatography-electrospray ionization tandem mass spectrometry operating in multiple reaction monitoring mode was used. Two chromatographic runs were performed, each in less than 6 min. The lower limit for accurate quantitative determination was 5 microg/L for cocaine and metabolites and 10 microg/L for opiates. Linearity was obtained from 10 to 1000 microg/L. Intraday (n = 6) and interday (n = 6) precisions and recoveries (n = 6) were determined at 10 or 25, 100, and 1000 microg/L concentrations. Precisions with a coefficient of variation less than 15% were obtained. Recoveries between 85 and 115% were determined.

Determination of Glucose in Dried Serum Samples by Fourier-Transform Infrared Spectroscopy

Practical improvements are needed to allow measurement of glucose concentrations by Fourier- transform infrared (FT-IR) spectroscopy. We developed a new method that allows determination of the glucose concentration in dried sera. We studied 32 serum samples after fourfold dilution and desiccation before FT-IR analyses on a spectrometer operated at a resolution of 2.0 cm(-1). We integrated all spectral windows at the surface of the spectrum in the C--O region. For comparison, glucose was measured in the sera by a glucose oxidase method. One peak within the spectrum was most specific for glucose (997-1062 cm(-1)). Its surface integration showed a strong relationship with reference data (r = 0.998; P <0.001). FT-IR analyses of five glucose solutions were performed to determine its specific absorption at the same peak. In this way, glucose concentrations in serum spectra could be measured. For the first time while using FT-IR spectroscopy, no manipulation of spectra nor use of internal standard was necessary to obtain results in high accordance with glucose concentration measured by a conventional (glucose-oxidase) method (S(y|x) = 0.25 mmol/L; r = 0. 998). FT-IR spectroscopy appears to be an easy and accurate method to determine glucose concentration and could be widely used to simultaneously identify and quantify several metabolites in biological fluids or tissues.