Targeted and untargeted metabolomic analysis of Procambarus clarkii exposed to a "chemical cocktail" of heavy metals and diclofenac.

Water pollution poses an important problem, but limited information is available about the joined effects of xenobiotics of different chemical groups to evaluate the real biological response. Procambarus clarkii (P. clarkii) has been demonstrated to be a good bioindicator for assessing the quality of aquatic ecosystems. In this work, we studied the bioaccumulation of cadmium (Cd), arsenic (As) and diclofenac (DCF) in different tissues of P. clarkii during 21 days after the exposure to a "chemical cocktail" of As, Cd and DCF, and until 28 days considering a depuration period. In addition, a combined untargeted and targeted metabolomic analysis was carried out to delve the metabolic impairments caused as well as the metabolization of DCF. Our results indicate that As and Cd were mainly accumulated in the hepatopancreas followed by gills and finally abdominal muscle. As and Cd show a general trend to increase the concentration throughout the exposure experience, while a decrease in the concentration of these elements is observed after 7 days of the depuration process. This is also the case in the abdominal muscle for Cd, but not for As and DCF, which increased the concentration in this tissue in the depuration phase. The hepatopancreas showed the greatest number of metabolic pathways affected. Thus, we observed a crucial bioaccumulation of xenobiotics and impairments of metabolites in different tissues. This is the first study combining the exposure to metals and pharmaceutically active compounds in P. clarkii by untargeted metabolomics including the biotransformation of DCF.

Selective, rapid and simultaneous determination of ergosterol and ergocalciferol in mushrooms by UPLC-Q-TOF-MS.

An ultra-performance liquid chromatography coupled to atmospheric pressure chemical ionization-quadrupole time-of-flight mass spectrometry method has been optimized and validated for the determination of ergosterol and ergocalciferol in mushroom samples, using cholecalciferol as surrogate standard. The separation was carried out with a Synergi Hydro-RP column (100 mm x 3.00 mm i.d, 2.5 µm particle size), (Phenomenex, CA, USA) column, thermostated at 35 °C. The mobile phase was 0.1 % formic acid aqueous solution and methanol in gradient elution mode and it was achieved in 5 min approximately. Detection was achieved by atmospheric pressure chemical ionization in positive mode and quadrupole time-of-flight mass spectrometry. Desolvation and interface temperatures were set at 500 °C and 150 °C, respectively. The recoveries obtained were within 92-105 % for ergosterol, 77-81 % for ergocalciferol and 83-87 % for cholecalciferol. Method limits of detection were 0.4 and 0.5 µg g-1 for ergosterol and ergocalciferol, respectively, and method limits of quantitation were 1.2 and 1.3 µg g-1 for ergosterol and ergocalciferol, respectively. A rapid and simple extraction procedure using small amount of sample (100 mg) with hexane was optimized and the method was applied to the determination of ergosterol and ergocalciferol in white button mushrooms (Agaricus bisporus var. bisporus) exposed to UV irradiation. Results were compared to the corresponding non-irradiated mushrooms.

Hollow fiber liquid-phase microextraction and determination of nonsteroidal anti-inflammatories by capillary electrophoresis and sulfonamides by HPLC in human urine.

In this paper two applications of three-phase HF-LPME for the determination of pharmaceuticals in human urine are proposed: a capillary electrophoresis with a photodiode array detection method for the analysis of seven nonsteroidal anti-inflammatory drugs (NSAIDs) and a high-performance liquid chromatographic with photo diode array and fluorescence detection method for the determination of four sulfonamides and their corresponding N(4)-acetyl-metabolites. Q3/2 Accurel® polypropylene hollow fibers were used for both procedures. Dihexyl ether was used as the supported liquid membrane for the determination of anti-inflammatories and 1-octanol for sulfonamides. An aqueous solution (pH 12) was used in both procedures as the acceptor phase and as the donor phase an aqueous solution (pH 2), and a 2 M Na(2)SO(4) aqueous solution (pH 4) was used for the determination of the anti-inflammatories and sulfonamides. The detection limits obtained were between 0.25 (naproxen) and 0.86 ng/mL (aceclofenac) for the determination of anti-inflammatories and 7 × 10(-4) (sulfamethoxazole) and 0.048 ng/mL (N(4)-acetyl-sulfamethazine) for sulfonamides. The method was successfully applied to the determination of the analytes in human urine.

Application of enzymatic probe sonication extraction for the determination of selected veterinary antibiotics and their main metabolites in fish and mussel samples.

A new method based on enzymatic probe sonication extraction prior to high-performance liquid chromatography (HPLC) has been developed for the determination of 11 antibiotics (drugs) and the main metabolites of five of them in fish tissue and mussel samples. The analytes belong to four different classes of antibiotics (sulfonamides, tetracyclines, penicillins and amphenicols). The analysed compounds were sulfadiazine (SDI) and N(4)-acetylsulfadiazine (NDI) metabolite, sulfamethazine (SMZ) and N(4)-acetylsulfamethazine (NMZ), sulfamerazine (SMR) and N(4)-acetylsulfamerazine (NMR), sulfamethoxazole (SMX), trimetroprim (TMP), amoxicillin (AMX) and its main metabolite amoxicilloic acid (AMA), ampicillin (AMP) and its main metabolite ampicilloic acid (APA), chloramphenicol (CLF), thiamphenicol (TIF), oxytetracycline (OXT) and chlortetracycline (CLT). The main factors affecting the extraction efficiency (type of enzyme, type and volume of extractant, ultrasounds power and extraction time) were optimised in tissue of hake (Merluccius merluccius), anchovy (Engraulis encrasicolus), mussel (Mytilus sp.) and wedge sole (Solea solea). The extraction was carried out using an extraction time of 5 min with 5 mL of water and subsequent clean-up with dichloromethane. High-performance liquid chromatography (HPLC) with diode array (DAD) and fluorescence (FLD) detectors was used for the determination of the antibiotics. The separation of the analysed compounds was conducted by means of a Phenomenex Gemini C(18) (150 mm x 4.6 mm I.D., particle size 5 microm) analytical column with LiChroCART LiChrospher C(18) (4 mm x 4 mm, particle size 5 microm) guard-column. Analysed drugs were determined using formic acid 0.1% (v/v) in water and acetonitrile in gradient elution mode as mobile phase. The proposed method was also evaluated by a laboratory assay consisting of the determination of the targeted analytes in samples of Cyprinus carpio which had previously administered the antibiotics.

Urea as new stabilizing agent for imipenem determination: electrochemical study and determination of imipenem and its primary metabolite in human urine.

Imipenem shows a fast chemical conversion to a more stable imin form (identical to that of biochemical dehydropeptidase degradation) in aqueous solutions and stabilizing agents used avoid its electrochemical study and determination. The aim of this work is the proposal of urea as stabilizing agent which allows the electrochemical study of imipenem and the proposal of electrochemical methods for the determination of imipenem and its primary metabolite (M1) in human urine samples. Electrochemical studies were realized in phosphate buffer solutions over pH range 1.5-8.0 using differential-pulse polarography, DC-tast polarography, cyclic voltammetry and adsorptive stripping voltammetry. In acidic media, a non-reversible diffusion-controlled reduction involving a two steps mechanism which involves one electron and one proton in the first step and two electrons and two protons in the second step occurs and the mechanism for the reduction was suggested. A differential-pulse polarographic method for the determination of imipenem in the concentration range 3.2x10(-6) to 2x10(-5)M (0.95-3.4 mg/L) and its primary metabolite in the concentration range 1.4x10(-6) to 10(-4)M (0.43-26.1 mg/L) with detection limits of 9.6x10(-7)M (0.28 microg/L imipenem) and 4.3x10(-7)M (0.14 microg/L M1) was proposed. Also, a method based on controlled adsorptive pre-concentration of imipenem on the hanging mercury drop electrode followed by voltammetric measure, allows imipenem determination in the concentration range 1.8x10(-8) to 1.2x10(-6)M (5.42-347 microg/L) with a detection limit of 5.4x10(-9)M (1.63 microg/L). The proposed methods have been used for the direct determination of the analytes in a pharmaceutical formulation and human urine.

Determination of imipenem and rifampicin in mouse plasma by high performance liquid chromatography-diode array detection.

A high performance liquid chromatographic (HPLC) method for the determination of imipenem and rifampicin was developed and validated. The method involves plasma deproteinisation with methanol, gradient elution on a RP-18 column and diode array detection. Separation was carried out in 8 min using a mobile phase composed of methanol and 0.2M borate buffer (pH 7.2). Imipenem and rifampicin were detected at 300 nm and 255 nm, respectively. A linear response was observed at plasma levels ranged between 0.3 and 30 microgmL(-1) for imipenem and 1.5 and 20 microgmL(-1) for rifampicin. The detection limits were 0.07 microgmL(-1) and 0.47 microgmL(-1) for imipenem and rifampicin, respectively. The method was applied to the determination of both compounds in mouse plasma samples.

Electrochemical behaviour and determination of acrivastine in pharmaceuticals and human urine.

The differential pulse polarography, DC-tast polarography and cyclic voltammetry behaviour of acrivastine was studied in Britton-Robinson buffer solutions (pH 2-11.7). In acidic media, a non-reversible diffusion controlled reduction process involving four electrons takes place. Two reduction waves appear at a E(1/2)=-0.6 and -0.99 V. The reduction mechanism is discussed. The linear relationship between peak current height and acrivastine concentration allowed the differential pulse polarographic determination of acrivastine over a wide concentration range, from 0.35 to 26.1 mg l(-1)at pH 2.5. The procedure was applied to determination of the drug in pharmaceutical formulations and human urine samples.

Electrochemical oxidation of cisatracurium on carbon paste electrode and its analytical applications.

The electrochemical oxidation of cisatracurium was investigated by cyclic voltammetry and differential pulse voltammetry at a carbon paste electrode and the experimental parameters have been optimized in order to obtain the optimum analytical signal. A differential pulse voltammetric method with carbon paste electrode is described for the determination of cisatracurium with detection limit of 0.38 mug/ml and quantitation limit of 1.26 mug/ml. The proposed method was applied to determine the content of cisatracurium in human urine and human serum, obtaining accurate and precise results.