Validation of a quantitative and confirmatory method for residue analysis of aminoglycoside antibiotics in poultry, bovine, equine and swine kidney through liquid chromatography-tandem mass spectrometry.

The use of aminoglycoside antibiotics in food animals is approved in Brazil. Accordingly, Brazilian food safety legislation sets maximum levels for these drugs in tissues from these animals in an effort to guarantee that food safety is not compromised. Aiming to monitor the levels of these drugs in tissues from food animals, the validation of a quantitative, confirmatory method for the detection of residues of 10 aminoglycosides antibiotics in poultry, swine, equine and bovine kidney, with extraction using a solid phase and detection and quantification by LC-MS/MS was performed. The procedure is an adaptation of the US Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) qualitative method, with the inclusion of additional clean-up and quantification at lower levels, which proved more efficient. Extraction was performed using a phosphate buffer containing trifluoroacetic acid followed by neutralization, purification on a cationic exchange SPE cartridge, with elution with methanol/acetic acid, evaporation, and dilution in ion-pair solvent. The method was validated according to the criteria and requirements of the European Commission Decision 2002/657/EC, showing selectivity with no matrix interference. Linearity was established for all analytes using the method of weighted minimum squares. CCα and CCß varied between 1036 and 12,293 µg kg(-1), and between 1073 and 14,588 µg kg(-1), respectively. The limits of quantification varied between 27 and 688 µg kg(-1). The values of recovery for all analytes in poultry kidney, fortified in the range of 500-1500 µg kg(-1), were higher than 90%, and the relative standard deviations were lower than 15%, except spectinomycin (21.8%). Uncertainty was estimated using a simplified methodology of 'bottom-up' and 'top-down' strategies. The results showed that this method is effective for the quantification and confirmation of aminoglycoside residues and could be used by the Brazilian programme of residue control.

Optimisation and validation of a quantitative and confirmatory LC-MS method for multi-residue analyses of ß-lactam and tetracycline antibiotics in bovine muscle.

A multi-residue method for the determination of the ß-lactam antibiotics ampicillin, cefazolin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V and the tetracyclines chlotetracycline, tetracycline and oxytetracycline was optimised and validated in bovine muscle. The method is based on the extraction of the residues from muscle using water/acetonitrile (2/8, v/v) with subsequent use of dispersive solid-phase C18 and hexane for purification. Extracts were analysed using ultra-performance liquid chromatography (UPLC-MS/MS) coupled with the mass spectrometer in positive electrospray ionisation mode (ESI+) for all analytes. The method was validated according to the requirements of European Commission Decision 2002/657/EC. The validation results were obtained within the MRL range of 0-1.5 of the MRL, with recoveries varying from 90% to 110% and CV < 20% (n = 54), except for cloxacillin, dicloxacillin and nafcillin. However, matrix interference was observed. The decision limit (CCα) ranged from 10% to 15% of the MRL. The uncertainty measurement was estimated based on both bottom-up and top-down strategies and the uncertainty values were found to be lower than 20% of the MRL. The method has a simple extraction procedure whereby analytes are separated with reasonable resolutions in a single 11-min chromatographic run. According to the validation results, this method is suitable for monitoring ß-lactams and tetracyclines according to National Program for Residue and Contaminant Control - Brazil (NPRC-Brazil) in bovine muscle.

Analysis of the MMP-9 (C-1562 T) and TIMP-2 (G-418C) gene promoter polymorphisms in patients with chronic periodontitis.

BACKGROUND: Matrix metalloproteinases (MMP)-9 is an important member of the matrix metalloproteinase family. A functional polymorphism has been described in the promoter region of the human MMP-9 gene. A C-to-T base exchange at -1562 creates two different alleles, and the C/T and T/T genotypes promote high activity of the MMP-9 gene promoter, increasing the risk for inflammatory diseases. The metalloproteinase-2 tissue inhibitor (TIMP-2) regulates the activity of MMPs in the extracellular matrix, and a polymorphism at the -418 position of the TIMP-2 gene promoter has been found in a Sp-1 binding site. In this study we have investigated the association between the above-mentioned polymorphisms and chronic periodontitis severity. METHODS: Genomic DNA from oral mucosa of 100 subjects was amplified by polymerase chain reaction and analysed by restriction endonuclease digestion. The significance of the differences in observed frequencies of polymorphisms in moderate and severe disease and healthy groups was assessed by chi(2) test (p<0.05). RESULTS: No association was observed between the polymorphism in the promoter region of MMP-9 (p=0.6693) and chronic periodontitis. The analysis of TIMP-2 showed that the G/G genotype was found at a frequency of 99%. CONCLUSION: The results show that the polymorphism in the promoter region of MMP-9 gene is not associated with chronic periodontitis. The high frequency of GG genotype in the TIMP-2 gene promoter in the population studied did not allow any conclusion regarding its effect on chronic periodontitis.

Analysis of the TGF-beta1 promoter polymorphism (C-509T) in patients with chronic periodontitis.

BACKGROUND: A polymorphism in the promoter region of the transforming growth factor beta-1 (TGF-beta1) gene was described at position -509. This polymorphism represents a C-to-T base exchange, which creates a YY1 consensus sequence in an area involved with down transcription regulation. This polymorphism has been associated with risk for asthma and allergies. In this study we investigated the association between this polymorphism and chronic periodontitis severity. METHODS: Genomic DNA from oral mucosa of 87 Caucasian subjects was amplified by PCR, and digested with Eco81I restriction endonuclease. The alleles were separated by polyacrylamide gel electrophoresis. The differences in genotype distribution from those expected by Hardy-Weinberg equilibrium, and the significance of the differences in observed frequencies of the polymorphism in moderate and severe disease and healthy groups were assessed by the chi2 test. RESULTS: There was a difference in the presence of the different alleles and genotypes among the healthy, moderate and severe periodontitis groups. The allele T was seen at 57.7% in the group with severe periodontitis and 37.8% and 35.4% in the healthy group and moderate periodontitis group, respectively (p=0.0387). The genotype T/T was found at 38.5% in the group with severe periodontitis, and at a frequency of 8% in the healthy group (p=0.0258). CONCLUSION: These results demonstrate that the polymorphism at bp -509 in the TGF-beta1 promoter may have a small effect on the modulation of the inflammatory process during periodontitis.

Polymorphism at position -174 of IL-6 gene is associated with susceptibility to chronic periodontitis in a Caucasian Brazilian population.

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that mediates inflammatory tissue destruction. A G to C substitution at position -174 in the promoter of IL-6 gene reduces in vitro transcription of IL-6. This polymorphism has been associated with inflammatory diseases like chronic arthritis. OBJECTIVE: The aim of this study was to investigate the association between the IL-6-174 polymorphism and susceptibility to chronic periodontitis in Brazilians. MATERIAL AND METHODS: Eighty-four nonsmoking subjects over 25 years (mean age 42.4) were divided according to the severity level of periodontal disease: 36 healthy individuals (control group), 24 subjects with moderate and 24 with severe periodontitis. Genomic DNA was obtained from epithelial cells through a mouthwash with 3% glucose and scraping of oral mucosa. The samples were analyzed for IL-6-174 polymorphism using PCR-RFLP. The significance of the differences in the frequencies of the polymorphism in the control and groups with periodontitis was assessed by chi2 test (p<0.05). RESULTS: Differences were found between control and groups with periodontitis in the genotype (p=0.0036, OR=3.0) and in the allele (p=0.0838, OR=1.9) frequencies. CONCLUSION: We concluded that the IL-6-174 polymorphism is associated with susceptibility to chronic periodontitis in the population studied.

MMP-1 promoter polymorphism: association with chronic periodontitis severity in a Brazilian population.

BACKGROUND: A single nucleotide polymorphism was described in the promoter region of the human MMP-1 gene, and this polymorphism has been associated with risk of cancer metastasis and inflammatory diseases. In this paper, we studied the possible relationship between the MMP-1 promoter polymorphism and the severity of chronic periodontitis. METHODS: Genomic DNA from oral mucosa was amplified by PCR and analyzed by restriction endonuclease. The alleles were separated by polyacrylamide gel electrophoresis. The significance of the differences in observed frequencies of polymorphism in moderate and severe disease and healthy groups was assessed by Chi-squared test. RESULTS: In the healthy group, the 2G allele was observed with a frequency of 48.7%, while in severely diseased patients the 2G allele was seen in 69.2% (P = 0.0344). The genotype 2G/2G was found in 46.15% of the group with severe periodontitis, and 24.3% and 25.0%, respectively, of the healthy and moderate groups (P = 0.0647). CONCLUSION: These results show that a polymorphism in the promoter region of MMP-1 gene is associated with the severe chronic periodontitis phenotype in non-smokers.

Frequencies of the -330 (T --> G) IL-2 and -590 (T --> C) IL-4 gene polymorphisms in a population from south-eastern Brazil.

Polymorphisms in the promoter regions of cytokine genes may affect their transcription. A T/G substitution at position -330 of the interleukin-2 (IL-2) gene and a T/C substitution at position -590 of the interleukin-4 (IL-4) gene have been described previously. The -590 (T --> C) IL-4 gene polymorphism was associated with asthma and atopy in US and Japanese populations. Population genetics is a useful tool for determination of the biological significance of genetic polymorphisms. The aim of this study was to investigate the frequencies of polymorphisms in the promoter regions of the IL-2 and IL-4 genes in a population from south-eastern Brazil and to compare them with those published for other populations. Allele frequencies were estimated in 114 unrelated individuals from São Paulo State. These subjects had an average age of 41.2 years (+/- 12.4 years) and the ethnic composition of the sample was: 78.07% Caucasian, 11.4% Black and 10.53% Mulatto. DNA from subjects was extracted from epithelial buccal cells, and the PCR-RFLP technique was employed to investigate the -330 (T --> G) IL-2 and -590 (T --> C) IL-4 gene polymorphisms. The allele frequency of the IL-2 gene polymorphism obtained in our study was similar to that found in UK Caucasoid groups. The T allele frequency of the IL-4 gene polymorphism observed in the Caucasian Brazilian group was similar to that found in UK and Australian populations, while the frequency observed for the Black Brazilian group was similar to that found in Japanese and Kuwaiti Arab populations. The results for the -330 (T --> G) IL-2 and -590 (T --> C) IL-4 polymorphisms are consistent with the high contribution of European lineages to the population in south-eastern Brazil.