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Development and in-house validation of a liquid chromatography-electrospray-tandem mass spectrometry method for the simultaneous determination of Sudan I, Sudan II, Sudan III and Sudan IV in hot chilli products.

An accurate method based on the use of reversed-phase (RP) liquid chromatography-tandem mass spectrometry interfaced with electrospray (LC-ESI-MS/MS) was devised for the determination of Sudan I, Sudan II, Sudan III and Sudan IV in hot chilli food samples. A simple sample treatment procedure entailing the use of an extraction step with acetone without clean-up was developed. A C18 column with an aqueous formic acid/methanol mixture as the mobile phase was used under isocratic conditions. Mass spectral acquisition was done in positive ion mode by applying selected reaction monitoring of three fragmentation transitions per compound to provide a high degree of selectivity. The method was in-house validated in terms of detection limits (LOD), quantitation limits (LOQ), linearity, sensitivity, accuracy, recovery, and selectivity on two kinds of hot chilli sauces. Good results in the low ng/g level were obtained for LOD and LOQ of all analytes in matrices. Under both intra-day repeatability (R.S.D. between 1 and 13%) and intermediate precision (about 5-15% R.S.D. for both chilli sauce matrices) conditions, precision proved to be typical of determinations based on electrospray LC-MS and acceptable for routine monitoring purposes. Extraction recoveries for all four azo-dyes in chilli tomato sauce ranged from 92 to 103% at a spiking level of 5 microg/kg, whereas values between 72 and 97% were calculated in chilli tomato and cheese sauce at the same concentration level. The applicability of the method to the determination of Sudan azo-dyes in hot chilli products was demonstrated.

Azo Compounds↗

Serosurvey of prevalence of human immunodeficiency virus amongst high risk groups in Port Sudan, Sudan.

During October, 1987, 593 sera were collected from risk groups in Sudan's only major deepwater port, Port Sudan. The risk groups included prostitutes, lorry drivers and prisoners. A large proportion of the study participants practised high risk behaviour which included sexual promiscuity, medical treatment by injection, scarification and tattooing. Despite high risk behaviour and evidence of a high prevalence of hepatitis B infection, a virus transmitted in a manner similar to HIV, no study participants were positive for HIV infection. This data suggests that the prevalence of HIV infection amongst high risk groups in Port Sudan is very low. These findings confirm a lack of clinical cases of AIDS in hospitalized patients in Port Sudan and the small number of reported cases in other areas of northern Sudan.

Acquired Immunodeficiency Syndrome↗

Some production characteristics of Sudan Desert sheep under range conditions in north Kordofan, Sudan.

The records on the flocks of sheep at El-Obeid Research Station during 1988-1990 were analysed using least-squares mixed-model procedures. The objective was to study some production characteristics of Sudan Desert sheep in North Kordofan, Sudan, in relation to the seasonal variation and husbandry systems. No significant differences (p > 0.05) were found between sedentary and nomadic flocks in the number of ewes serviced and the conception, lambing or abortion rates. Mortality rates were significantly (p < 0.05) higher in the nomadic flock. The breeding season had a significant effect on all the parameters measured. Higher conception (p < 0.05) and lambing (p < 0.01) rates were recorded for the rainy and late dry seasons. However, the highest ewe mortality (p < 0.01) was in the rainy season. The highest (p < 0.01) lamb weights at birth and at 30 days of age were recorded under the nomadic system. None the less, lambs kept under the sedentary system had somewhat higher weight gains from 60 to 150 days of age. Lambs born during the rainy season had the highest birth weights (p < 0.05) and the highest weight at 30 days of age. However, the live weights from 60 to 150 days of age were significantly higher in lambs born in the late dry season. The highest lamb mortality was recorded in the lambs born in the rainy season. Lambs born under the nomadic system and during the rainy season had the highest (p < 0.05) weights at 30 and 60 days of age, and also higher (p < 0.01) mortality rates compared to the other flock x season combinations. It was concluded that the production characteristics of Sudan Desert sheep reflect the seasonal nutritional status and husbandry system. The effects of supplementary feeding on breeding and pregnant ewes, and on the performance of weaned lambs in the area should be studied. Moreover, the socioeconomic factors governing mobility in nomadic flocks should be thoroughly investigated.

Animal Husbandry↗

To South Sudan with operation lifeline Sudan and UNICEF.

The protracted civil war in the Sudan has had an immense human and economic cost. The consequent collapse of infrastructure in the south of the country has disrupted the provision of basic health care, and primary health care schemes to eradicate trypanosomiasis and dracunculiasis have been rendered impossible. Operation Lifeline Sudan, an official United Nations umbrella organisation, has been in a unique position to provide long-term medical, agricultural, livestock, fishery and emergency food aid in both government held and rebel held areas over about the past decade.

Developing Countries↗

Unstable malaria in Sudan: the influence of the dry season. Plasmodium falciparum population in the unstable malaria area of eastern Sudan is stable and genetically complex.

This paper reviews surveys carried out, over a period of 6 years between 1989 and 1995, to examine Plasmodium falciparum population structure in Asar village in eastern Sudan, an area of unstable malaria, the incidence of which is confined to a few weeks following the short rainy season (June-October). The first phase of the study involved regular cross sectional surveys, between 1989 and 1993 during the seasons of malaria incidence, while the second involved surveys during the malaria-free months of the dry seasons. The parasites were examined for 20 polymorphic loci, including enzyme electrophoretic variants, proteins detected by 2 dimensional polyacrylamide gel electrophoresis, antigens detected by monoclonal antibodies, and in vitro responses to antimalarial drugs. In addition, alleles of the polymorphic genes for merozoite surface proteins 1 and 2 (MSP-1, MSP-2) were examined using the polymerase chain reaction and oligonucleotide probes. Great genetic complexity was observed among the parasites which appeared during the short transmission seasons. A large proportion of the patients who were infected during the transmission season maintained asymptomatic, subpatent parasitaemias throughout the subsequent dry season, often as genetically complex infections.

Adolescent↗

Evaluation of febrile patients in Port Sudan, Sudan: isolation of dengue virus.

One hundred consecutive patients admitted to the Port Sudan Hospital with a temperature greater than or equal to 100 degrees F were evaluated. Enteric fever was diagnosed in 19 patients and malaria in 13. Virologic studies identified 21 cases of dengue infection. One dengue 1 and 17 dengue 2 infections were diagnosed by viral isolation. Three untyped dengue infections were identified serologically. The clinical presentation and course of patients infected with dengue virus were most consistent with classic dengue fever. There was no evidence of hemorrhagic phenomena or shock in any of the dengue-infected patients. Both dengue 1 and 2 must be considered causes of acute fever in East Africa.

Adolescent↗

Utilization of primary health services in Port Sudan, Sudan.

The utilization of primary health services provided by two health centres and a hospital out-patient department was studied. The epidemiologic pattern was similar in the three health care facilities; minor gastrointestinal tract, respiratory tract and skin conditions formed the majority of cases. Distance was a limiting factor for utilization of health services in the health centres and not in the hospital. The average consultation time per patient was two minutes. Lack of drugs was the universal problem. Suggestions and recommendations to solve some of the problems and improve services are presented.

Adolescent↗

Sudan I is a potential carcinogen for humans: evidence for its metabolic activation and detoxication by human recombinant cytochrome P450 1A1 and liver microsomes.

1-Phenylazo-2-hydroxynaphthol (Sudan I, C.I. Solvent Yellow 14) is a liver and urinary bladder carcinogen in mammals. We compared the ability of hepatic microsomal samples from different species including human to metabolize Sudan I. Comparison between experimental animals and human cytochromes P450 (CYP) is essential for the extrapolation of animal carcinogenicity data to assess human health risk. Human microsomes generated the pattern of Sudan I metabolites reproducing that formed by hepatic microsomes of rats. Using hepatic microsomes of rats pretreated with specific CYP inducers, microsomes from Baculovirus-transfected insect cells expressing recombinant human CYP enzymes, purified CYP enzymes, and selective CYP inhibitors, we found that rat CYP1A1 and recombinant human CYP1A1 are the most efficient enzymes metabolizing Sudan I. Microsomes from livers (the target of Sudan I carcinogenicity) of different human donors were used to estimate whether authentic human CYPs oxidize Sudan I. Using Western blot analysis and NH(2)-terminal sequencing, we were able to detect and quantify CYP1A1 in human hepatic microsomes. The sequence of nine amino acids of the protein band cross-reacting with antirat CYP1A1 in human microsomes, LFPISMSAT, matched the sequence of human CYP1A1 perfectly (residues 2-10). CYP1A1 expression levels varied significantly among the different human microsomes (0.04-2.4 pmol/mg protein), and constituted <0.6% of the total hepatic CYP complement. All of the human hepatic microsomal samples oxidized Sudan I to C-hydroxymetabolites. Moreover, using the nuclease P1-enhanced version of the (32)P-postlabeling assay, we found that human microsomes were competent in activating Sudan I to form adducts with DNA. The role of specific CYP enzymes in the human hepatic microsomal metabolism was investigated by correlating the CYP-catalytic activities (or CYP contents) in each microsomal sample with the levels of individual metabolites and/or Sudan I-DNA adducts formed by the same microsomes, and by examining the effects of agents that can inhibit specific CYP in Sudan I metabolism. On the basis of these studies, we attribute most of Sudan I metabolism in human microsomes to CYP1A1, but participation of CYP3A4 cannot be ruled out. These results, the first report on the metabolism of Sudan I by human CYP enzymes, strongly suggest a carcinogenic potency of this rodent carcinogen for humans.

Animals↗

Modulation of CYP1A1-mediated oxidation of carcinogenic azo dye Sudan I and its binding to DNA by cytochrome b5.

OBJECTIVES: Modulation of the cytochrome P450 (CYP) 1A1-mediated oxidative activation and detoxication of carcinogenic Sudan I by the heme-protein cytochrome b(5) (b(5)) was investigated. Another aim of the study was to examine the formation of Sudan I-DNA adducts in vivo. METHODS: High performance liquid chromatography (HPLC) with ultraviolet (UV) detection was employed for the separation of Sudan I metabolites formed by human recombinant CYPs and rat CYP1A1. The (32)P-postlabeling technique was utilized to determine Sudan I-DNA adducts. RESULTS: The capabilities of the most efficient CYP enzymes oxidizing Sudan I, human and rat recombinant CYP1A1, as well as of human recombinant CYP1A2, 2A6 and 3A4 were significantly increased by b(5), while reactions catalyzed by human CYP1B1, 2C8, 2C9 and 2E1 were insensitive to this heme protein. Sudan I oxidation catalyzed by CYP2B6, 2C19 and 2D6 was even decreased by b(5). The stimulation of the CYP1A1-mediated Sudan I oxidation was dependent on concentration of b(5). Likewise, the increase in CYP1A1-mediated formation of Sudan I-DNA adducts by b(5) was concentration dependent. Other proteins containing heme such as cytochrome c or myoglobin were without this effect. The major Sudan I-DNA adducts formed in vitro are also generated in vivo, in livers of rats treated with Sudan I. CONCLUSIONS: The data are the first report on the stimulation of CYP1A1-mediated oxidative reactions by b(5). In addition, the results demonstrating covalent binding of Sudan I to rat liver DNA in vivo indicate a genotoxic mechanism of Sudan I carcinogenicity in rats.

Animals↗

Prostaglandin H synthase-medicated oxidation and binding to DNA of a detoxication metabolite of carcinogenic Sudan I, 1-(phenylazo)-2,6-dihydroxynaphthalene.

The metabolite of the carcinogenic azo dye Sudan I, 1-(phenylazo)-2,6-dihydroxynaphthalene (6-OH-Sudan I), which is considered to be the detoxification product of this dye is metabolized by prostaglandin H synthase (PHS) in the presence of arachidonic acid or H2O2 in vitro. The apparent Michaelis constant value for 6-OH-Sudan I as a substrate is 98.9 microM. 1-(Phenylazo)-2,6-naphthoquinone is a principal product of the 6-OH-Sudan I oxidation. This oxidation is inhibited by radical scavengers nitrosobenzene, ascorbate, glutathione and NADH. This indicates that PHS metabolizes 6-OH-Sudan I through a one-electron oxidation mechanism, giving rise to free radicals. During the PHS-mediated reaction, 6-OH-Sudan I is activated to metabolites binding to protein and DNA. The 32P-postlabeling analysis of DNA modified by activated 6-OH-Sudan I provides evidence that covalent binding to DNA is the principal type of DNA modification. The PHS-mediated binding of 6-OH-Sudan I to DNA presumably proceeds through formation of 1-(phenylazo)-2,6-naphthoquinone. The results suggest strongly that the C-hydroxylated derivative of Sudan I (6-OH-Sudan I) should be evaluated as a proximate carcinogenic metabolite, which may participate in the initiation of Sudan I-carcinogenesis in the urinary bladder.

Animals↗

Visual detection of Sudan dyes based on the plasmon resonance light scattering signals of silver nanoparticles.

A visual light scattering detection method of Sudan dyes is reported in food products based on the formation of silver nanoparticles (NPs) . Sudan dyes including I, II, III and IV have reducibility due to the nitrogen-nitrogen double bond and phenol group in their molecular structure, and a redox reaction could occur with AgNO3. Owing to the formation of silver NPs as a result of the redox reaction, color changes could be observed by eye from the red of Sudan to the brown of silver NPs, resulting in strong plasmon resonance light scattering (PRLS) signals characterized at 452 nm, which could be measured using a common spectrofluorometer. It was found that the PRLS intensities were proportional to the dye concentrations over the range of 0.2-2.4 microM Sudan I, 0.1-2.4 microM Sudan II, 0.1-2.4 microM Sudan III, and 0.2-3.0 microM Sudan IV, with the corresponding limits of determination (3 sigma) of 3.2, 3.0, 3.2, and 2.9 nM, respectively. Using hot chili as a model sample, detection could be made with the recovery of 90.8-103.3% and RSD of 4.0-4.9%, and the results are identical with that of a liquid chromatographic method approved by the European Commission. To make the PRLS method much more practical, we could visually detect the quantity of Sudan dyes based on the PRLS signals using simple devices such as a portable laser pointer (653 nm) and a light emitting diode (458 nm). Mechanism investigations show that the functional group of Sudan oxidized by AgNO3 is the phenol group, not the nitrogen-nitrogen double bond.

Azo Compounds↗

Mechanism of formation and 32P-postlabeling of DNA adducts derived from peroxidative activation of carcinogenic non-aminoazo dye 1-phenylazo-2-hydroxynaphthalene (Sudan I).

Horseradish peroxidase in the presence of hydrogen peroxide mediates the activation of carcinogenic 1-phenylazo-2-hydroxynaphthalene (Sudan I) to DNA-bound products in vitro. The peroxidase activating system is greater than 10 times more effective with respect to DNA modification by Sudan I than the microsomal enzymes containing cytochrome P450. The DNA-binding reaction of the Sudan I metabolite(s) formed by the peroxidase system is dependent on Sudan I and H2O2 concentration and pH. Reactive intermediate(s) or product(s) of the Sudan I oxidation by peroxidase with a short half-life are responsible for the DNA modification. DNA modified by peroxidase-activated Sudan I becomes colored and has an absorption maximum at approximately 480 nm. The modification of DNA by Sudan I metabolites(s) formed by the peroxidase system is inhibited by some compounds of physiological importance (ascorbate, glutathione, Mg2+ ions) and by radical trapping agents (nitrosobenzene, methyl viologen). 32P-Postlabeling assay of the DNA modified by Sudan I activated by the peroxidase system indicates that the covalent DNA adduct formation is the principal type of the DNA modification. Four major and several minor adducts of deoxyribonucleotide 3',5'-bisphosphate from DNA with Sudan I metabolite(s) were detected by the classical Randerath 32P-postlabelling assay as well as by the nuclease P1 version of the same method.

Animals↗

Identification of contact allergens in C.I. Solvent Red 23 (commercial Sudan III) by chemical analysis and animal testing.

C.I. Solvent Red 23, commercial Sudan III, is widely used in cosmetic products. Chemical analyses and guinea pig sensitization tests were carried out to identify its contact allergens. In the Magnusson & Kligman guinea pig maximization test, C.I. Solvent Red 23 showed 20% positive reactions. By conducting chemical analyses with HPLC and GLC, 2-naphthol (82 ppm), azobenzene (48 ppm), Sudan I (570 ppm) and many unknown impurities, as well as the main constituent pigment Sudan III (87%), were found. The chemical structure of one unknown impurity was identified as an isomer of Sudan III. We found that purified Sudan III showed no positive reaction, while the isomer elicited 30% positive reactions, in the same guinea pig test. Furthermore, cross-sensitization with p-phenylenediamine was investigated using the guinea pig test. Animals sensitized with p-phenylenediamine also showed positive elicitation reactions with purified Sudan III. From these results, the contact allergenicity of C.I. Solvent Red 23 is considered to be due to impurities, including the isomer of Sudan III, 1-(o-phenylazophenylazo)-2-naphthol. Positive reactions to Sudan III previously demonstrated in hairdressers are due to cross-sensitivity with p-phenylenediamine.

Allergens↗