ABSTRACT
Since linear calibration is mostly preferred for analytical determinations, linearity in the calibration range is an important performance characteristic of any instrumental analytical method. Linearity can be proved by applying several graphical and numerical approaches. The principal graphical criteria are visual inspection of the calibration plot, the residuals plot, and the response factors plot, also called sensitivity or linearity plot. All of them must include confidence limits in order to visualize linearity deviations. In this work, the graphical representation of percent relative errors of back-calculated concentrations against the concentration of the calibration standards is proposed as linearity criterion. This graph considers a confidence interval based on the expected recovery related to the concentration level according to AOAC approach. To illustrate it, four calibration examples covering different analytical techniques and calibration situations have been studied. The proposed %RE graph was useful in all examples, helping to highlight problems related to non-linear behavior such as points with high leverage and deviations from linearity at the extremes of the calibration range. By this way, a numerical decision limit which takes into account the concentration of calibration standards can be easily included as linearity criterion in the form of %RETh=2·C-0.11. Accordingly, this %RE parameter is accurate for the decision-making related to linearity assessment according to the fitness-for-purpose approach.
ABSTRACT
Centruroides tecomanus is a medically important scorpion of the state of Colima (Mexico). This communication reports the identification of venom components of this scorpion with biological activity over insects/crickets (Acheta domestica), crustaceans/fresh water shrimps (Cambarellus montezumae), and mammalians/mice (Mus musculus, strain CD1). It also describes the pharmacological effects on cell lines in culture (L5178Y cells, HeLa cells, HuTu cells and Jurkat E6-1 cells), as well as on several types of bacteria (see below). The soluble venom of this scorpion was fractionated by high-performance liquid chromatography (HPLC) and collected separately in twelve independent fractions collected over 60 min run (5 min time apart each other). The HPLC components of fraction VII were lethal to all three species used for assay. The IVth fraction had a toxic effect on freshwater shrimps. In this species, fractions VI, VII and VIII were all lethal. For crickets, fractions V and VI were toxic and fraction VII was lethal. In mouse, the lethal components were found in fraction VII, whereas fraction VIII was toxic, but not lethal, at the doses assayed. The molecular weight of peptides from the various group of fractions were identified by mass spectrometry determination. Components lethal to mice showed molecular weights from 7013 to 7487 Da. Two peptides were obtained in homogeneous form and shown to be lethal to the three species of animal used for assay. The soluble venom tested on L5178Y cell line survival was shown to be cytotoxic, at 10-100 µg/mL concentration, when compared to control murine splenocytes (p = 0.007). The soluble venom applied to Hela, Hutu and Jurkat cell lines did not show cytotoxic effects at these concentrations. On the contrary, it seems to have a proliferative effect. However the HPLC fractions I, III, VI and XII do have a cytotoxic effect on Jurkat E06-1 cells in culture at 200 µg/mL concentration. The antimicrobial activity of the venom fractions on Staphylococcus aureus (gram-positive), Escherichia coli, Pseudomonas aeruginosa y Salmonella spp (gram-negative) was measured, using the liquid inhibition growth system. The four strains of bacteria used were susceptible to fractions III and IV, affecting all four bacterial strains at concentrations below 5 µg/mL.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Antineoplastic Agents/isolation & purification , Apoptosis/drug effects , Drug Discovery , Insecticides/isolation & purification , Scorpion Venoms/chemistry , Amino Acid Sequence , Animals , Anti-Bacterial Agents/adverse effects , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/adverse effects , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Arthropod Proteins/chemistry , Arthropod Proteins/isolation & purification , Arthropod Proteins/pharmacology , Arthropod Proteins/toxicity , Astacoidea/drug effects , Astacoidea/growth & development , Cell Line, Tumor , Cells, Cultured , Gram-Negative Bacteria/drug effects , Gram-Negative Bacteria/growth & development , Gryllidae , Humans , Injections, Intraperitoneal , Insecticides/chemistry , Insecticides/pharmacology , Insecticides/toxicity , Mexico , Mice , Microbial Sensitivity Tests , Scorpion Venoms/administration & dosage , Scorpion Venoms/toxicity , Scorpions/growth & development , Spleen/cytology , Spleen/drug effects , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & developmentABSTRACT
An HPLC-DAD method for determining corticoids in calf feed and in animal feeding water samples using a monolithic column has been developed and validated. The method optimization included the study of binary mobile phases of water and acetonitrile. The optimum separation was achieved at 40 degrees C, with acetonitrile:H(2)O 29:71 v/v used as mobile phase and a 3 ml/min flow-rate, which resulted in their separation in about 5 min. Two reported sample procedures were applied to feed and for animal feeding water samples prior to HPLC. Method validation was carried out according to the EU criteria established for quantitative screening methods. The results indicate that this method is highly specific, reproducible and accurate. The proposed method was found to be robust and unaffected by small variations in the extraction procedure and in HPLC conditions. The developed method for the determination of corticoids in feed and water samples was also found to be suitable for different kinds of feeds and waters.
