Tetradenia riparia (Lamiaceae) essential oil: an alternative to Rhipicephalus sanguineus

In Brazil, Rhipicephalus sanguineus resistance to some pyrethroids have been detected, motivating research on new phyto-insecticides such as essential oil from Tetradenia riparia leaves (EOL) and flower buds (EOFB). The essential oils were obtained by hydrodistillation (3h) and identified by GC/MS. In addition, a multivariate exploratory analysis was done to determine the analysis of the major compounds (PCA). The bioassays on R. sanguineus larvae were done by immersion test at different EO concentrations which ranged from 50,000 to 0.47 mg/mL (v/v). The action mechanism of EOs were determined by bioautographic method evaluating the inhibitory potential on the acetylcholinesterase enzyme. The EO yield in leaves was 0.29±0.22 (%) and in flower buds 0.38±0.17 (%). The class projections showed oxygenated sesquiterpenes (43.62%) and diterpenes (15.60%) in EOFB, and hydrocarbon sesquiterpenes (26.44%) and oxygenated monoterpenes (16.44%) in EOL. Four components presented a greater distancing of mass flow: fenchone (11.57 and 6.01 %), α-cadinol (12.21 and 13.69 %), 14-hidroxy-9-epi-caryophyllene (8.56 and 15.38 %), and caryophyllene oxide (1.32 and 4.50 %) in EOL and EOFB, respectively. The lethal concentrations (LCs) to kill R. sanguineus larvae were (LC50: 2.18±0.24 and LC99.9: 9.98±0.10 mg/mL) for EOL, and (LC50: 5.36±2.50 and LC99.9: 20.12±0.54 mg/mL) for EOFB. The action mechanism of EOs by bioautographic methods indicated an inhibition of 0.70 mg/mL (EOL) and 1.40 mg/mL (EOFB) on the acetylcholinesterase enzyme (AChE). Therefore, this species can be considered promising to be part of the chemical larvicides to control this ectoparasite

Validation of Thin-Layer Chromatography-Bioautographic Method for Determination of Streptomycin

Background: A simple bio-assay for determination of streptomycin hyphenated with planar chromatography techniques was developed. Objective: This study aims to validate the method for identification and determination of streptomycin in injection preparations with TLC-bioautography. Methods: Thin Layer Chromatography (TLC) was performed on the silica Gel GF-254 using KH2PO4 solution as mobile solvent. The visualization was performed by spraying 2% resorcinol. Direct bi autography was developed using Escherichia coli ATCC 25922 as a bacterial test, grown on the nutrient agar medium at 37oC for 24 hours. The method was validated corresponding to linearity, limit of detection (LOD), intra day precision, and accuracy parameters. The accuracy was measured using streptomycin injection as a sample. Results: The Results showed that the KH2PO4 solution at 7.5% concentration was found to be the optimized solvent with Rf value of 0.5. The linear equation was y = 10.176x + 4.046 at 150 - 350 µg/mL concentration range with the linearity coefficient, Limit of Detection, accuracy, and variation coefficient were 0.9907; 40 ppm; 96.37 + 2.22% (with an RSD value of 2.31%); and 1.63 respectively. Conclusion: The prospective TLC-bioautographic method was applied for the identification and determination of streptomycin in a preparation using a single eluent KH2PO4. The eluent system optimization remains necessary for the identification and determination of the mixture of streptomycin with other antibiotics, such as aminoglycoside groups.

Determination of Monensin in Bovine Tissues: A Bridging Study Comparing the Bioautographic Method (FSIS CLG-MON) with a Liquid Chromatography-Tandem Mass Spectrometry Method (OMA 2011.24)

The U.S. Department of Agriculture, Food Safety Inspection Service regulatory method for monensin, Chemistry Laboratory Guidebook CLG-MON, is a semiquantitative bioautographic method adopted in 1991. Official Method of AnalysisSM (OMA) 2011.24, a modern quantitative and confirmatory LC-tandem MS method, uses no chlorinated solvents and has several advantages, including ease of use, ready availability of reagents and materials, shorter run-time, and higher throughput than CLG-MON. Therefore, a bridging study was conducted to support the replacement of method CLG-MON with OMA 2011.24 for regulatory use. Using fortified bovine tissue samples, CLG-MON yielded accuracies of 80–120% in 44 of the 56 samples tested (one sample had no result, six samples had accuracies of >120%, and five samples had accuracies of 40–160%), but the semiquantitative nature of CLG-MON prevented assessment of precision, whereas OMA 2011.24 had accuracies of 88–110% and RSDr of 0.00–15.6%. Incurred residue results corroborated these results, demonstrating improved accuracy (83.3–114%) and good precision (RSDr of 2.6–20.5%) for OMA 2011.24 compared with CLG-MON (accuracy generally within 80–150%, with exceptions). Furthermore, χ2 analysis revealed no statistically significant difference between the two methods. Thus, the microbiological activity of monensin correlated with the determination of monensin A in bovine tissues, and OMA 2011.24 provided improved accuracy and precision over CLG-MON.

Determination of Narasin in Chicken Fat: A Bridging Study Comparing the Bioautographic Method (FSIS CLG Method R22) to a Liquid Chromatography with Tandem Mass Spectrometry Method (AOAC Method 2011.24)

Lilly Method AM-AA-CA-R108-AB-755, which is substantially the same as U.S. Department of Agriculture, Food Safety and Inspection Service (FSIS) Chemistry Laboratory Guidebook (CLG) method R22, is the current regulatory method for determining narasin in cattle and chicken tissues and is based on bioautography, creating a zone of inhibition of bacterial growth, with the size of the zone correlating to the amount of narasin extracted from the tissue. AOAC Method 2011.24 is an LC-tandem mass spectrometry (MS/MS) method for determining narasin content from bovine, swine, or chicken tissues. It has many advantages over the regulatory method, including higher throughput, less solvent use, no use of carbon tetrachloride, a wider method range, inclusion of swine tissues, and it is less labor intensive. In this study, AOAC Method 2011.24 was compared to FSIS CLG method R22 for the determination of narasin in chicken abdominal fat. Fortified chicken-fat samples ranging from 20 to 960 ng/g and incurred chicken-fat samples ranging from 40 to 480 ng/g were assayed by both methods in triplicate. Mean accuracies for the two methods were similar, 77–110% for CLG R22 and 84–96% for AOAC Method 2011.24, and the method results showed a linear correlation. The methods differed in precision, however, with the CLG R22 method yielding 2.6–34% RSD and AOAC Method 2011.24 yielding 0.15–6.4% RSD. It is recommended that AOAC Method 2011.24–granted AOAC Official MethodSM Final Action status—be adopted as the official U.S. regulatory method.