Current Analytical Chemistry - Volume 7, Issue 3, 2011

A Phospholipase A2 termed as BSS PLA2 was purified from the venom of Bungarus sindanus sindanus (Sindhi Krait) through two steps: size exclusion chromatography on sephadex G-75 and reversed-phase chromatography on Vydac C18 HPLC column. The homogeneity and molecular mass of BSS PLA2 were analysed by SDS-PAGE and MALDI-TOF mass spectrometry. The N-terminal sequence of 32 amino acids of the BSS PLA2 revealed close resemblance with other species of bungarus snakes. The BSS PLA2 showed an optimum activity in the alkaline range (pH 8.0-9.5), while the optimum temperature stability was found to be 60 ° C. The effect of calcium ion (Ca2+) concentration revealed that in the absence of Ca2+ the enzyme showed some activity however, the enzymatic activity increases with increase in Ca2+ concentration. The addition of exogenous metal ions, including Mg2+ with Ca2+ increases, while that of Ba2+ with Ca2+ slightly decreases the enzymatic activity of BSS PLA2. Additionally, kappa bungarotoxin with the mass of 7358.5 Da was identified by MALDI TOF MS/MS from another peak of the same HPLC elution. Two of the tryptic peptides matched with kappa-6-bungarotoxin when searched through Mascot search engine.

Duloxetine (DLX) is a selective orally administered serotonin and norepinephrine reuptake inhibitor usually used in the treatment of major depressive disorder. 1-Naphthol (NPH) is a toxic impurity produced when DLX is exposed to acid medium and it also proceeds from the synthesis material. A simple and rapid stability indicating capillary micellar electrokinetic chromatography method was developed and validated for the simultaneous analysis of DLX and NPH, using cimetidine as internal standard. The method was performed with a fused-silica capillary with applied voltage of 25 kV. The background electrolyte consisted of 50 mM Tris and 20 mM SDS, pH 10.6. The linear calibration range was 50-550 μg.mL-1 for DLX and 0.55-5.5 μg.mL-1 for NPH, with r=0.9999 for both molecules. The limits of detection and quantification were 0.50 and 1.45 μg.mL-1 for DLX and 0.20 and 0.55 μg.mL-1 for NPH, respectively. The performed degradations were: acid and basic media with 0.1 M HCl and 1 M NaOH, respectively, oxidation with 6 % H2O2 (v/v), temperature at 60 °C and the exposure to UV light. No interference in the DLX migration was verified. Adequate results for accuracy, precision, selectivity and robustness were obtained. The proposed method was successfully applied for the simultaneous quantitative analysis of DLX and NPH. The DLX data were compared to those obtained by a validated reversephase liquid chromatography method, showing non-significant differences between them. The developed method may be applied to routine quality control of DLX and to assure that NPH does not exceed the allowed amounts.

Interaction between prulifloxacin (PUFX) and pepsin was studied under a simulated physiological condition using fluorescence spectrophotometric techniques when the Eu(III) exists. The fluorescence emission intensity of pepsin was strongly quenched by the addition of prulifloxacin. Spectrophotometric observations are rationalized in terms of a static quenching process. The binding constants and the number of binding sites have been evaluated by the data obtained from the fluorescence quenching experiments. According to the van't Hoff equation, the standard enthalpy change (ΔH°) and standard entropy change (ΔS°) for the reaction were calculated and it indicated that the hydrophobic interactions play a main role in the binding of PUFX and pepsin. In addition, the binding distance R between pepsin (donor) and PUFX (acceptor) was obtained on the basis of the Forster's resonance energy transfer theory. The results obtained herein show that the interaction between the pepsin and PUFX was not influenced by Eu(III).

Two nicotine (NC)-selective electrode systems were developed with different techniques. Precipitation based technique with tungstophosphoric acid as cationic exchanger in poly (vinyl chloride) (PVC) matrix was used for sensor 1 fabrication. 2,9,16,23-tetra-tert-butylphethalocynine (TTBPC) based technique with and without potassium tetrakis[3,5- bis(trifluoromethyl)phenyl]borate (KTFPB), as fixed anionic site in poly (vinyl chloride) (PVC) matrix was used for sensors 2, 3 fabrication. Linear responses of 1× 10-2 to 5× 10× 5 M, 1× 10× 2 to 9× 10× 6 M and 1× 10× 2 to 7× 10× 6 M with slopes of 56.3, 57.1 and 58.6 mV/decade within pH 4-7 were obtained by using electrodes 1, 2 and 3, respectively. The percentage recoveries for determination of NC by the proposed NC-selective electrodes were 100.5±0.7, 100.8±1.1 and 99.0± 0.8% for sensors 1, 2, and 3, respectively. Determination of NC in pure nicotine solutions by the proposed electrodes revealed their applicability for determination of NC human serum and urine. The proposed method was compared with a reported one. Method was validated and no significant difference in both accuracy and precision was observed.

The kinetic study of the thermal oxidative rancidification of extra-virgin olive oils at 98 °C carried out according to the A.O.M. (American Oxygen Method - official method) of the A.O.C.S. (American Oil Chemistry Society) was completed. The orders of magnitude for the values of the kinetic rate constant k (10-4 min-1) for this process, the activation energy Ea (38 kJ mol-1) and the reaction order n (1st order) were definitively established. A best model mechanism for the main processes occurring during oil rancidification was also proposed. The same procedure was successfully extended also to two other olive oil types: olive oil and olive oil residue.

A new high-performance thin-layer chromatographic (HPTLC) method has been established for determination of mycophenolate mofetil in human plasma. Mycophenolate mofetil is used as an important immunosuppressive agent. Chromatographic separation was performed on aluminium plates coated with silica gel 60F254; the mobile phase was a combination of triethylamine buffer (pH 5.3) and acetonitrile in the ratio of 20:80 (v/v) respectively. Densitometric analysis of mycophenolate mofetil was performed at 250 nm. The method was rapid (single-step extraction with acetonitrile), sensitive (limit of quantification 15.4 ng per zone), precise (CV ≤ 3.71 %), accurate (drug recovery 95.08-100.6%), and linear over the range 100-1200 ng per zone. Recovery of mycophenolate mofetil from plasma samples was 95.8 ± 4.5%. The half-life of mycophenolate mofetil in plasma was 20.4 h at 4°C and 17.8 h at 20°C. Mycophenolate mofetil is stable in human plasma for at least two months at -20°C and can tolerate two freeze-thaw cycles with losses <10%. The method was successfully used to determine therapeutic levels of mycophenolate mofetil.

A method has been developed for the direct determination of Cu, Cd, Ni and Pb in aquatic humic substances (AHS) by graphite furnace atomic absorption spectrometry. AHS were isolated from water samples rich in organic matter, collected in the Brazilian Ecological Parks. All analytical curves presented good linear correlation coefficient. The limits of detection and quantification were in the ranges 2.5-16.7 μg g-1 and 8.5-50.0 μg g-1, respectively. The accuracy was determined using recovery tests, and for all analytes recovery percentages ranged from 93 - 98 %, with a relative standard deviation less than 4 %. The results indicated that the proposed method is a suitable alternative for the direct determination of metals in AHS.

Hybrid sorbent based on nano alumina - single walled carbon nanotube was applied to preconcentration of Pb(II), Cu(II) and Co(II) from various samples via solid phase extraction technique. The determination of Pb(II), Co(II) and Cu(II) ions was made by flame atomic absorption spectrometry (FAAS). Experimental parameters including pH of sample solution, sample volume, flow rate of sample solution, volume and concentration of eluent that affect the recovery of the analytes have been optimized. Under optimized experimental conditions, analytical parameters including limit of detection, limit of quantification, linear working range, precision and accuracy have been determined. Interfering effects of matrix constituent on the recovery of the analytes were studied. The reusability and adsorption capacity of the new hybrid sorbent were also investigated. The hybrid sorbent was successfully applied for preconcentration of Pb(II), Cu(II) and Co(II) from various samples with recovery values of 101 %, 95 % and 98 %, respectively. The analytical limit of detection was found to be 2.1 μg L-1, 0.9 μg L-1 and 1.1 μg L-1 for Pb(II), Co(II) and Cu(II), respectively. The hybrid sorbent was stable up to 100 runs. Adsorption capacities of the hybrid sorbent were 101.2 mg g-1 for Pb, 76.3 mg g-1 for Co and 53.2 mg g-1 for Cu. The accuracy of the method was confirmed by analyzing certified materials (BCR 150 Skim milk powder, CMI 7003 Silty clay) and spiked real samples. The method has been applied for the determination of analytes in tap water, dam water, soil and tea leaves.

This work described the electrochemical response characteristic of poly (vinyl chloride) (PVC) membrane sensor and coated wire ion selective electrode (CWISE) based on the use of copper (II) salicylidene-N- anilinoacetohydrazone ionophore. o-nitrophenyloctylether (o-NPOE) is used as a plasticizer in the PVC matrix. The membrane sensors were prepared and the performance characteristics were evaluated according to IUPAC recommendations. The prepared sensors exhibited fast, stable and linear response for copper over a concentration range 1× 10× 6-1× 10× 2 M and 7× 10-5- 1× 10-2 M with cationic slopes of 29.6 and 27.1 mV per concentration decade at pH 5 for membranes doped with and without tetraphenylborate as an additive, respectively. The potentiometric selectivity data revealed minimal or no interference from common cations. Direct potentiometric determination of copper using the prepared membrane sensors gave average recoveries of 99.89 ± 0.9% and 100.04 ± 1.1% for membrane formulated with additive (detection limit; 1× 10-6-1× 10-2 M) and without additive (detection limit; 7× 10-5-1× 10-2 M), respectively. The sensors were used for measurement of copper content in industrial waste water; metal alloys containing from copper factories and pharmaceutical formulations. The developed method was found to be simple, accurate and precise when compared with conventional analytical methods for copper determinations (e.g; atomic absorption spectroscopy and voltammetry).

Metallothioneins (MTs) are a group of low molecular mass, cysteine-rich proteins with a variety of functions including involvement in metal homeostasis, free radical scavenging, protection against heavy metal damage, and metabolic regulation via Zn donation. The overexpression of MTs in proliferating cells is turning the attention to the study of MTs as novel promising marker of tumor diseases. Besides, levels of MTs in invertebrates and aquatic vertebrates well correlate with heavy metal pollution of an environment and, thus, serve as bio-environmental marker. It is not surprising that these proteins are of great interest not only for biochemists, molecular biologists clinical chemists, but also for environmental chemists and ecologists. Detection and quantification of MTs, which is needed in all above mentioned areas, is challenging due to the high cysteine content and relatively low molecular mass. This review is giving an overview of the analytical methods used for determination of MTs comprising a broad range of immunochemical, electrochemical as well as spectrometric methods, which have been optimized and even hyphenated with different separation methods to detect MTs. More attention is paid to the structural analysis of these proteins as well as the employment of analytical instruments for environmental, biochemical and clinical purposes.

A novel ionic liquid-based microextraction methodology was developed for preconcentration of ultra-traces of arsenic prior to its determination by electrothermal atomic absorption spectrometry (ETAAS). As(III) was extracted as ammonium pyrroldinedithiocarbamate (APDC) complex from 10 mL sample into a 50 μL-volume of ionic liquid, 1- hexyl-3-methylimidazolium hexafluorophosphate, with the aid of sonication in an ultrasonic bath. Several variables affecting microextraction efficiency and ETAAS signal were optimized. Under the optimum experimental conditions, the detection limit (3s) was 0.01 μg L-1 and the enhancement factor was 208. The relative standard deviation for six replicate determinations of 0.2 μg L-1 As(III) was 4.6 %. The method was validated by analysis of a certified reference material and applied to the determination of arsenic in water samples. The proposed procedure is effective, very simple and fast; it does not require cooling, centrifugation and other time-consuming steps.