Optimization of morphine extraction method for the assay of its urinary 3-glucuronideconjuguate by gas chromatography-mass spectrometry.

In the field of doping, a great interest is carried for the analysis of morphine, a powerful narcotic analgesic opiate which use is prohibited during competitions. In order to confirm the abnormal analytical result in our anti-doping laboratory, a sensitive and selective gas chromatography-mass spectrometry (GC-MS) method was performed for the quantification of urinary morphine. As sample preparation is a key step for the determination of drugs in biological samples, the aim of this work consists of the optimization of the urinary human sample pretreatment conditions before quantification by GC/MS. Enzymatic hydrolysis associated with liquid-liquid extraction constitute the major pre-treatment steps. Our study has first focused on the optimization of the extraction solvents then to enzymatic hydrolysis which morphine is released from its glucuronide conjugated form. Onboard premiums, a study involving the effect of "amount of enzyme", "incubation temperature" and "duration of hydrolysis" was conducted. This univariate study has enabled us to evaluate the influence of each of these operating variables on the area ratio of morphine to the internal standard (Amorphine/AIS) response and to set the experimental fields for each one of them. Based on these results, an experimental design was established using the Box-Behnken model to determine, by multivariate analysis, the optimal operating conditions maximizing the "Amophine/AIS" response. After validation, the analysis of response surface makes it possible to set the optimum operating conditions, which the ratio "Amorphine/AIS" is maximized. The retained conditions for enzymatic hydrolysis are 160µl of Escherichia coli glucuronidase enzyme during 6hours of incubation at a temperature of 36°C. The solvent mixture Methyl-t-Butyl Ether/isopropanol (4:1, v/v) was selected since it has improved morphine extraction from the urinary matrix allowing a gain of 50% when compared to that used in our routine laboratory. Our developed extraction method can be successfully applied for our forensic anti-doping analysis of morphin in human sample urine.

Study of forced degradation behaviour of florfenicol by LC and LC-MS and development of a validated stability-indicating assay method.

A stability-indicating high performance liquid chromatography (HPLC) method was developed for the analysis of florfenicol in presence of its two available identified degradation products (thiamphenicol and chlorfenicol). The drug was subjected to different International Conference On Harmonisation (ICH) prescribed stress conditions (hydrolysis, oxidation and photolysis). The products formed under different stress conditions were investigated by liquid chromatography (LC) and liquid chromatography-mass spectrometry (LC-MS). The LC method involved a Knauwer Eurospher C18 thermostated column at 25°C; and ammonium acetate buffer 6.49mM (pH adjusted to 4.5)-methanol (70:30 v/v) as mobile phase. The flow rate and detection wavelength were 1ml/min and 225nm respectively. The drug showed instability under acidic, alkaline and photolytic stress conditions mainly in solution state form; however, it remains stable in solid state form and under oxidative stress conditions. The developed method was validated for linearity, precision, accuracy and specificity. The degradation products were characterized by LC-MS. Through the mass/ionization (m/z) values and fragmentation patterns, two principal degradation products listed in bibliography have been shown: the florfenicol amine and thiamphenicol. Based on the results, a more complete degradation pathway of the drug could be proposed.

Stress degradation study on sulfadimethoxine and development of a validated stability-indicating HPLC assay.

Sulfadimethoxine was subjected to different International Conference on Harmonisation prescribed stress conditions (hydrolysis, oxidation and photolysis). A stability-indicating high-performance liquid chromatography method was developed for analysis of the drug in the presence of its major degradation products. It involved a Knauer Eurospher C18 thermostated column at 25°C, and 9.57 mM phosphate buffer (pH adjusted to 2.0 with orthophosphoric acid)-acetonitrile (70:30 v/v) as mobile phase. The mobile phase flow rate and sample volume injected were 1.2 mL/min and 20 µL respectively. The selected wavelength for the determination was 248 nm. The method was validated for its linearity, precision, accuracy, specificity and selectivity, and then applied to the assay of sulfadimethoxine in pharmaceutical formulations. The results of the study show that sulfadimethoxine is highly sensitive to basic hydrolysis and oxidation. The mechanisms and schemas of hydrolytic, oxidative and photolytic degradation are also studied. The method developed, which separates all of the most degradation products, is simple, accurate, precise and specific. Thus, it can be applied to study the stability of veterinary preparations containing sulfadimethoxine.

Validation of a liquid chromatography method for the simultaneous determination of sulfadimethoxine and trimethoprim and application to a stability study.

A liquid chromatography method is described for the simultaneous determination of sulfadimetoxine and trimethoprim from a veterinary formulation at the proportion of 187 mg and 40 mg respectively in presence of some excipient. The solution was subjected to different International Conference On Harmonisation prescribed stress conditions (hydrolysis, oxidation and photolysis). A stability-indicating high-performance liquid chromatography method was developed for the analysis of active substances in presence of their major degradation products. It involved a Knauer Eurospher C18 thermostated column at 25 degrees C, and 9.57 mM phosphate buffer (pH adjusted to 2.0 with orthophosphoric acid)-acetonitrile (70:30 v/v) as mobile phase. The mobile phase flow rate and sample volume injected were 1.2 mL/min and 20 microL, respectively. The selected wavelength for the determination was 248 nm. The method was validated for linearity, precision, accuracy and specificity, and then applied to a stability study of sulfadimetoxine and trimethoprim in the veterinary solution packaged in high density polyethylene plastic bottles of 1 L and 100 mL thermosealed and no thermosealed and corked by a white cap, at both accelerated and long-term conditions required by the International Conference On Harmonisation. The method developed, which separates all of the most degradation products formed under variety of conditions, proved to be simple, accurate, precise and specific. The results of the stress degradation show that the solution is more sensitive to hydrolysis. The stability studies carried out on three batches of each presentation show that the finished product remains stable for six months.

Atypical hemolytic uremic syndrome: A monocentric adult Tunisian study and review of literature.

Atypical hemolytic uremic syndrome (aHUS) is characterized by microvascular thrombosis resulting in thrombocytopenia, hemolytic anemia, and multiorgan dysfunction. It is associated with genetic or acquired disorders of regulatory components of the complement system. For our study, we collected data from 16 patients diagnosed with aHUS between January 2010 and January 2014. The mean age was 33.6 years. The female-to-male ratio was 3. The median follow-up duration was 27 ± 3.5 months. The most common clinical presentation was hypertension. Renal involvement was noted in all cases. Ten patients had extrarenal manifestations. Semi-quantitative dysfunction of the alternative pathway of complement was found in all cases. A genetic study was not available for our patients. During the acute stage, all patients received plasma therapy, and among them, seven required dialysis and five were still on dialysis at the time of discharge. One patient underwent renal transplantation. None of our patients received eculizumab perfusion. The renal survival was inversely correlated to young age (<30 years) (P = 0.001), presence of anti-factor H antibodies (P = 0.003) and serum creatinine at diagnosis >5 mg/dL (P = 0.02). Mortality rate was significantly correlated to young age (<30 years old) (P = 0.01). Collecting multicentric data on adult patients with aHUS will enable better characterization of the spectrum of adult aHUS in our country and the evaluation of current treatments and different outcomes.

[Cerebral venous thrombosis during pregnancy: about 7 cases]. / Thrombophlebite cerebrale survenant lors de la grossesse: a propos de 7 cas.

BACKGROUND: cerebral venous thrombophlebitis (CVT) is a rare but serious pathology. The pregnancy and especially the postpartum constitute supporting circumstances occured of the CVT. AIM: the aim of our study is to remember the symptomatology of CVT, the importance of the IRM and the angio IRM in the diagnosis of CVT, and its treatment. OBSERVATIONS: We present a retrospective study relating to 7 cases of CVT during gravidopuerperality over 7 years (1996 to 2002). The average age of our patients is 32.1 years. The CVT occured during the first trimester of the pregnancy in one case and in the postpartum in 6 cases. The symptomatology was dominated by cephalgia and the signs of intracranian hypertension. Convulsions occured in 3 cases. The diagnosis of CVT was confirmed by IRM coupled with the Angio-IRM in all cases. The treatment by heparinotherapy was instaured immediately and relayed by the Anti-Vit K as the evolution was favorable in all cases. CONCLUSION: cerebral venous thrombophebitis (CVT) is a serious pathology (especially in pregnancy and post partum). The diagnosis is performed by the magnetic resonance imaging (IRM) and the early introduction of the heparinotherapy.

[Prevalence of diabetes-related autoantibodies in celiac disease]. / Prévalence des marqueurs sérologiques du diabète de type 1 chez les enfants atteints de maladie coeliaque.

THE AIM: of this study is to investigate the prevalence of diabetes-related autoantibodies in a group of children with celiac disease and to compare it with a control group. MATERIAL AND METHODS: We recruited 31 celiac children at diagnosis (on gluten containing diet) and 31 age and sex matched healthy children. Anti-islet cell antibodies (ICA) were detected by indirect immunofluorescence on monkey pancreas. Anti-glutamate decarboxylase (anti-GAD) and anti-tyrosine phosphatase (anti-IA2) antibodies were assessed by a radio-immuno- precipitation method. RESULTS: Three out of 31 celiac patients (9.7%) had one or more diabetes-related autoantibodies. ICA, anti-GAD and anti-IA2 were found in respectively 3.2%; 3.2% and 9.7% of patients. Only one control (3.2%) had anti-GAD. There was no statistically significant difference between the 2 groups. CONCLUSION: The risk to develop diabetes seems to be the same in celiac patients and in healthy subjects thus screening of diabetes-related autoantibodies is not justified in celiac patients.

[Intra-uterine growth retardation: etiologic factors and management]. / Retard de croissance intra-utérin: etiologie et prise en charge obstetricale.

OBJECTIVE: The aim of this study is to define the etiologic factors, the diagnostic proceduresand obstetrical rules to be observed during a pregnancy in relation with low birth weight. PATIENTS AND METHODS: This is a retrospective study of 124 cases, carried out at the military hospital of Tunis, between January 1st 1999 and December 31 st 2001. RESULTS: The frequency of intra-uterine growth retardation (IVGR) was 2.1%. 46.8% of the patients were primiparous. The mean age of the patients was 30 years. 25.8% of the patients developed toxemia. The etiology was predominantly renovascular-syndromes, urinary infections, and idiopathic hypotrophy. The positive diagnosis was suggesred by the measure of the uterine height in 96% and was confirmed by sonogaphy in all cases. The ombilical Doppler was pathological in 17.76% of the cases. A corticosteroid administration for pulmonary maturation was prescribed in 28% of the cases. Obstetrical common rule was fetal extraction: 42.7% of the patients were delivered by cesarean. CONCLUSION: There is a real need to insist on the importance of early detection of fetal hypotrophy and of better management of the women at risk.

[Not Available]. / Determination polarographique de facteurs vitaminiques P (rutine et ses derives).

Several flavonols of therapeutic interest on account of their vascular properties have been studied. They were all derived from the o-diphenol quercetol. Their electrochemical oxidation at a rotating solid electrode has been examined, and the choice of solvent and pH is discussed. Results obtained by d.c. and pulse polarography are presented. A relation between their structure and electrochemical behaviour has been established. Application to drug analysis is proposed, giving identification, determination and purity assay simultaneously. There is a linear response over the range 5 x 10(-6)-10(-4)M, a sufficient range for analysis of pharmaceuticals.

Zero-crossing derivative spectrophotometry for the determination of haloperidol in presence of parabens.

First derivative spectrophotometry with a zero-crossing technique of measurement is used for the quantitative determination of haloperidol in the presence of methylparaben and propylparaben, which is added as antimicrobial preservatives in pharmaceuticals. This technique permits the quantification of haloperidol in the presence of parabens, with closely overlapping spectral bands, and without any separation step. Linear calibration graphs of first derivative values (at 255.2 nm for haloperidol) versus concentration (in the range 4.0-20.0 micrograms ml-1) were obtained with negligible intercepts. Relative standard deviation of 0.83% was obtained for intra-day precision and 1.86% for inter-day precision. The recovery of haloperidol in synthetic mixtures with parabens and pharmaceutical dosage forms is also reported.

Hydrocarbon contamination of coastal sediments from the Sfax area (Tunisia), Mediterranean Sea.

The coastal area off the city of Sfax (730,000 inhabitants), well-known for fisheries and industrial activities, receives high inputs of organic matter mostly anthropogenic. Eighteen stations were selected in the vicinity of the direct discharge of industrial sewage effluents in the sea in order to study the spatial distribution of the organic contamination. Surface sediments sampled in the shallow shelf were analysed for hydrocarbons by Fourier transform infrared spectroscopy, gas chromatography and gas chromatography/mass spectrometry. Total hydrocarbon distributions revealed high contamination as compared to other coastal Mediterranean sites, with an average concentration of 1865 ppm/dry weight sediment. Gas chromatographic distribution patterns, values of unresolved mixture/n-alkane ratio and distributions of steranes and hopanes confirmed a petroleum contamination of the Arabian light crude oil type. Biogenic compounds were also identified with a series of short-chain carbon-numbered n-alkenes in the carbon range 16-24.

[Antiphosphoplipid antibodies and retinal occlusive vasculopathy]. / Anticorps antiphospholipides et occlusion vasculaire rétinienne.

PURPOSE: to determine the prevalence of antiphospholipid antibodies in patients with occlusive retinal vascular events, exempt from conventional risk factors of retinal thrombosis. METHODS: eleven patients with retinal vascular occlusion, free of main accepted risk factors for retinal thrombosis, were retrospectively screened for antiphospholipid antibodies (anticardiolipin and anti-beta2 glycoprotein 1 antibodies) by an Elisa method. Prevalence of antiphospholipid antibodies were compared with those in a homogenous control group of 100 patients. RESULTS: the prevalence of antiphospholipid antibodies in the study group was 27% (three of 11). Comparison with control group prevalence (3%) showed a statistically significant difference (p < 0,001). One patient in the study disclosed positivity for IgG anticardiolipine antibodies, one for IgM anticardiolipine antibodies and one for anti-beta2 glycoprotein 1 antibodies. CONCLUSION: our results lead us to recommend a systematic search for specific antiphospholipid antibodies in such young patients which could have an importance for the diagnosis of primary antiphospholipid syndrome.

Treatment of saline wastewaters from marine-products processing factories by activated sludge reactor.

An activated sludge reactor, operated at room temperature (20-30 degrees C) was used to treat saline wastewaters generated by marine-products industries. The system was operated continuously and the influence of the organic loading rates (OLRs), varying from 250 to 1000 mg COD l(-1) day(-1), on chemical oxygen demand (COD) removal was investigated. The system, inoculated with NaCl-acclimated culture, removed up to 98% and 88% of the influent COD concentrations at OLRs of 250 and 1000 mg COD L(-1) day(-1), respectively. Since the organic pollution is essentially composed of proteins, microorganisms, which produced proteolytic enzymes, were isolated from the activated sludge culture. One bacterium with the highest protease activity, identified as Bacillus cereus, was chosen for protease production in fishery wastewaters of different concentrations containing combined heads and viscera powder. Protease synthesis was strongly enhanced when cells were cultivated in two times diluted fishery wastewaters. The enhancement of protease synthesis could have been due to the presence in effluent of organic matters or salts, which stimulated the growth of the strain and protease production.

[Multifocal bony hemangioendothelioma: a case report]. / Hémangioendothéliome osseux multicentrique.

Hemangioendothelioma is a rare tumor (0.5% of primary malignant bone tumors) generally found in long bones and tarsal bones. We report a case in a 67-year-old man who underwent surgery in 1987 for a tumor of the left ankle. He again consulted in 1998 for pain in the left knee. Standard x-rays of the left lower limb evidenced multiple partitioned bone defects with cortical lysis in the femoral condyle, the upper and lower portions of the tibia and the talus. Computed tomography showed a lytic image of the lower femoral and superior tibial epiphysis without cortical or soft tissue involvement. Laboratory findings were normal. Pathology examination of a biopsy specimen led to the diagnosis of grade I multifocal bony hemangioendothelioma of the medial femoral condyle and the medial upper tibial facet of the left knee. Search for extension was negative. Curettage followed by cement filling of all tumor sites and postoperative radiotherapy were successful. No recurrence has been observed 3 years later. The histological polymorphism of hemangioendotheliomas leads to varying therapeutic management schemes.

[A long survival after the treatment of a squamous cell carcinoma of the gallbladder]. / Une longue survie après traitement d'un carcinome épidermoïde de la vésicule biliaire.

We report the case of a long survival after the treatment of a squamous cell carcinoma of the gallbladder. The patient is a 58-year-old man, who was treated by cholecystectomy, followed by postoperative radiotherapy of the tumour bed at a dose of 45Gy, combined with 5-fluoro-uracil chemotherapy. After 18 years, the patient is alive in complete remission. The end point of this work was to study the clinical and therapeutic characteristics of squamous cell carcinoma of the gallbladder and its prognosis through this case and a review of literature.

Behçet syndrome with ankylosing spondylitis.

Two HLA B27-positive patients with concomitant Behçet syndrome and ankylosing spondylitis are reported. The relevant literature is reviewed and the meaning of this combination discussed.