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.

[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.

[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.