A new quantitative thermospray LC-MS method for nicotine and its metabolites in biological fluids

A rapid thermospray liquid chromatography-mass spectrometry (TSP LC-MS) method is described for the simultaneous determination of nicotine and 17 of its metabolites. Chemical ionization of nicotine and its metabolites separated by reversed-phase HPLC is achieved by postcolumn addition of ammonium acetate buffer with the filament of the ion source turned off. Quantification is accomplished by selectively monitoring the unique protonated molecular ion of each metabolite. Trideuterated cotinine serves as an internal standard. Linear responses for cotinine, demethylcotinine, and trans-3'-hydroxycotinine were observed over a concentration range of 20-8000 ng/mL, and 80-8000 ng/ml for nicotine and nicotine-1'-N-oxide. Of the 17 metabolites examined, only nicotine, cotinine, demethylcotinine, and trans-3'-hydroxycotinine were detected in smokers' urine.     

Development of an ultrasensitive stacking technique for 5‐nitroimidazole determination in untreated biological fluids by micellar electrokinetic chromatography

Cation‐selective exhaustive injection and sweeping followed by a MEKC separation is evaluated for the sensitive analysis of 5‐nitroimidazoles in untreated human serum and urine. Deproteinized serum and urine samples were diluted 76 and 143 times, respectively, in a low‐conductivity solvent (5.00 mM orthophosphoric acid containing 5.0% v/v methanol). Samples were electrokinetically injected at 9.8 kV for 632 s in a previously conditioned fused‐silica capillary (65.0 cm × 50 μm id). Separation was performed at –30 kV and 20°C using 44 mM phosphate buffer (pH 2.5), 123 mM SDS, and 8% v/v tetrahydrofurane as BGE. Signals were monitored at 276 nm and peak area was selected as analytical response. Good linearity (R² ≥ 0.988) and LODs lower than 1.5 and 1.8 μg/mL were achieved in serum and urine, respectively.     

Development of an electrochemical membrane-based nanobiosensor for ultrasensitive detection of dengue virus

A sensitive membrane-based electrochemical nanobiosensor is developed for the detection of dengue type 2 virus (DENV-2) using nanoporous alumina-modified platinum electrode. Its sensing mechanism relies on the monitoring of electrode's Faradaic current response toward redox probe, ferrocenemethanol, which is sensitive toward the formation of immune complexes within the alumina nanochannels. Anti-DENV-2 monoclonal antibody (clone 3H5, isotype IgG) is used as the biorecognition element in this work. The stepwise additions of antibody, bovine serum albumin (BSA) and DENV-2 are characterized by differential pulse voltammetry (DPV). A low detection limit of 1 pfu mL⁻¹ with linear range from 1 to 10³ pfu mL⁻¹ (R² = 0.98) can be achieved by the nanobiosensor. The nanobiosensor is selective toward DENV-2 with insignificant cross reaction with non-specific viruses, Chikungunya virus, West Nile virus and dengue type 3 virus (DENV-3). Relative standard deviation (RSD) for triplicate analysis of 5.9% indicates an acceptable level of reproducibility. The first direct quantitation of DENV-2 concentration in whole mosquito vector is demonstrated using this electrochemical nanobiosensor.     

Development of an ultrasensitive electrochemiluminescence inhibition method for the determination of tetracyclines

An electrochemiluminescence (ECL) inhibition method is developed for quantitative determination of four tetracyclines (TCs) in honey samples, including tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC) and doxycycline (DC). It was found that the four TCs strongly inhibited the ECL signal of the Ru(bpy)₃²⁺/DBAE system. Based on the ECL signal changes, a simple and ultrasensitive detection method for TCs was thus established. The optimum experimental conditions including the scan mode and scan rate of the applied potential, the type of the buffer solution and its pH, and the concentration of Ru(bpy)₃²⁺ and DBAE for the ECL inhibition method, were investigated in detail. Under the optimized conditions, the quenched ECL intensity versus the logarithm of the concentration of TCs is in good linear relationship over a concentration range from 4.0 × 10⁻¹¹ to 4.0 × 10⁻⁹ g mL⁻¹. The detection limits were found to be 2.0 × 10⁻¹² g mL⁻¹. The results obtained by the proposed ECL system, in terms of sensitivity, were much better than those of previously reported methods. In addition, the method was applied successfully to determine the total residuals of the four TCs in honey samples. The relative standard deviations were found in a range of 4.9–14.3%, and the recoveries were obtained from 87.5% to 115.0%. A possible mechanism for the quenching effects of Ru(bpy)₃²⁺/DBAE system was also proposed.     

Determination of drugs and metabolites in biological fluids

The determination of drugs and metabolites in biological fluids (biopharmaceutical analysis) is becoming increasingly important in the pharmaceutical and biomedical sciences. Successful analyses require sensitivities at ppb-level or less, high selectivity, and minimal interferences from artifacts. Modern approaches to biopharmaceutical analysis rely principally on chromatographic and immunochemical methods.     

Determination of chlorproguanil and metabolites in biological fluids

Summary The retention behaviour of chlorproguanil and its putative metabolites chlorcycloguanil and 3,4-dichlorophenylbiguanide has been studied on a reversed phase chromatographic system incorporating sodium lauryl sulphate as hydrophobic pairing ion. On the basis of data obtained and comparison of standard compounds with components present in urine following the administration of chlorproguanil, the identity of the metabolites have been confirmed chromatographically. A third unidentified metabolite is also observed. The retention study allows selection of a solvent system which, when used with a small volume, 100 × 2 mm, 3μm ODS column, enables chlorproguanil and its two major metabolites to be determined in plasma, whole blood and urine. The analytical characteristics of the method are reported and the usefullness of the method in obtaining pharmacokinetic data on the drug and its metabolites is discussed.     

Application of liquid separation techniques to the determination of the main urinary nicotine metabolites

A rapid procedure for the analysis of the main nicotine metabolites (cotinine, trans-3′-hydroxycotinine) in urine has been worked out. The procedure includes isolation of nicotine and its metabolites from urine by means solid–liquid extraction technique using resin Amberlite XAD-2 and then quantitation by the use of thin-layer chromatography with densitometry (in reflection mode). GC–MS was applied to confirm the results obtained by TLC. The procedure was applied to the analysis of cotinine concentrations in urine samples taken from children living in Upper Silesia region (Poland). Among 444 investigated children we did not find cotinine almost in 60% but in 15% of this population, there were children who could have been exposed to cigarette smoke.     

Profiles of volatile metabolites in biological fluids using capillary columns

Nickel capillary columns coated with moderately polarstationary phases such as Witconol can be used for the separation of the organic volatile fraction from biological fluids. A “transevaporator” sampling technique for the collection of the organic volatiles on glass beads from as little as 5 to 500 μl of biological fluids (e.g. urine, serum, amniotic fluid, breast milk, saliva, etc.) is described. The organic volatiles are thermally desorbed from the glass beads into a short precolumn cooled in liquid nitrogen, which overcomes the problems associated with sample introduction onto narrow-bore capillary columns. The application of the full analytical technique to problems associated with the early detection of disease is illustrated.     

Using nanoparticle aggregation to give an ultrasensitive amperometric metal ion sensor

Herein we show an electrochemical sensor for the detection of copper ions that has a limit of detection below 1 pM. This extraordinarily low limit of detection is achieved in two ways. The first is to use SAM modified electrodes to give low background current where electron transfer to the underlying polycrystalline electrode is achieved through gold nanoparticles attached to the terminus of the SAM. The gold nanoparticles are modified with cysteine to allow copper accumulation. The second way the low detection limits are achieved is by the addition of cysteine-modified nanoparticles to the sample solution, as well as the SAM modified electrode. The complexation of copper with the nanoparticles in solution causes them to aggregate with these aggregates accumulating on the electrode. Thus a higher percentage of copper in the solution is collected at the electrode giving the enhanced detection limits.     

Utilization of 2,6-bis(2-benzimidazolyl)pyridine to detect toxic benzene metabolites

The tridentate ligand 2,6-bis(2-benzimidazolyl)pyridine has the ability to detect toxic benzene metabolites such as phenol, hydroquinone, resorcinol, catechol and p-benzoquinone by simple techniques like UV/vis and fluorescence spectroscopy. The formation of a stable supramolecular complex between 2,6-bis(2-benzimidazolyl)pyridine and hydroquinone was confirmed by X-ray analysis.     

Liquid-solid extraction and HPLC determination of amiodarone metabolites in biological fluids

Summary An improved liquid-solid extraction procedure for the purification and determination of amiodarone and desethylamiodarone in biological fluids is proposed. The sample is passed through a silica-C₁₈ column, and after washing, the analytes are eluted with methanol. The determination is then accomplished by RP HPLC using an octadecyl silica column and a mobile phase of methanol containing 0.0015% of ammonium hydroxide. The effect of ammonia concentration on the capacity factors of the analytes has been used for estimating the acid dissociation constants of the investigated secondary and tertiary amines in methanol.     

Detection and determination of antimalarial drugs and their metabolites in body fluids

This review of methods for determining antimalarial drugs in biological fluids has focused on the various analytical techniques for the assay of chloroquine, quinine, amodiaquine, mefloquine, proguanil, pyrimethamine, sulphadoxine, primaquine and some of their metabolites. The methods for determining antimalarials and their metabolites in biological samples have changed rapidly during the last eight to ten years with the increased use of chromatographic techniques. Chloroquine is still the most used antimalarial drug, and various methods of different complexity exist for the determination of chloroquine and its metabolites in biological fluids. The pharmacokinetics of chloroquine and other antimalarials have been updated using these new methods. The various analytical techniques have been discussed, from simple colorimetric methods of intermediate selectivity and sensitivity to highly sophisticated, selective and sensitive chromatographic methods applied in a modern analytical laboratory. Knowledge concerning the method for a particular study is determined by the type of application and the facilities, equipment and personnel available. Often is it useful to apply various methods when conducting a clinical study in malaria-endemic areas. Field-adapted methods for the analysis of urine samples can be applied at the study site for screening, and corresponding blood samples can be preserved for subsequent analysis in the laboratory. Selecting samples for laboratory analysis is based on clinical, parasitological and field-assay data. The wide array of methods available for chloroquine permit carefully tailored approaches to acquire the necessary analytical information in clinical field studies concerning the use of this drug. The development of additional field-adapted and field-interfaced methods for other commonly used antimalarials will provide similar flexibility in field studies of these drugs.     

Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy

Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1-20 ms), and demonstrate its generation via both one- and multistep excitation processes. Apart from reflecting a major pathway for photobleaching, our results also suggest the exploitation of the photoinduced ionization and the subsequent reduction by antioxidants for biomolecular monitoring purposes and as a possible switching mechanism with applications in high-resolution microscopy.     

Improved gas chromatographic method for the determination of nicotine and cotinine in biologic fluids

Improved methods have been developed for the determination of nicotine and its major metabolite, cotinine, in blood, plasma, and urine samples. These methods utilize gas chromatography with alkali flame ionization (nitrogen--phosphorus) detection and structural analogs of nicotine and cotinine as internal standards.     

Development of an ultrasensitive rGO/AuNPs/ssDNA-based electrochemical aptasensor for detection of Pb2+

Journal of Solid State Electrochemistry - The present work is performed on Pb2+ detection via electrochemical sensor. The novel aptasensor was developed&nbsp;by modification of glassy carbon...     

Potentiometric determination of mephedrone in oral fluids with ion-selective membranes

This work presents a potentiometric approach for the detection of two cathinone derivatives, namely mephedrone (4-MMC) and clephedrone (4-CMC). Contrary to neutral-carriers (dibenzo-18-crown-6-ether and calix[4]arene), cation-exchanger (tetraphenylborate sodium) as active component of ion-selective membrane showed near-Nernstian response (+47 mV/decade from 1.7 ⋅ 10⁻⁶ to 5.6 ⋅ 10⁻⁴ mol l⁻¹) and selectivity for 4-MMC in oral fluids (OF). The agreement between the quantitative results obtained with the potentiometric approach and the HPLC/MS-MS method determination of 4-MMC in OF shows the potential of the proposed potentiometric approach for forensic analysis.     

Carbon-13 nuclear magnetic resonance spectra of nicotine metabolites and related compounds

The potential of ¹³C NMR techniques for studies of nicotine metabolism has been investigated by acquisition and assignment of ¹³C NMR spectra of nicotine, its metabolites and some related compounds. It is shown that C-2′ of nicotine would be the best site for ¹³C labelling when using the method to gain further insight into the metabolic pathways of nicotine.