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This paper reports the validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows the quantification of 10 antiretroviral (ARV) drugs in peripheral blood mononuclear cells (PBMCs) using 6 different isotopic internal standards (IS) and its clinical application. PBMCs are isolated from blood by density gradient centrifugation and drugs are extracted with a 60% methanol (MeOH) solution containing the 6 IS. The cell extract is then injected in the HPLC system and analytes are separated on a Symmetry Shield RP18 2.1 mm x 50 mm column. The different molecules are then detected by MS/MS in electrospray positive or negative ionisation modes and data are recorded using the multiple reaction monitoring (MRM) mode. Calibration curves are constructed in the range of 0.25-125 ng/ml of cell extract by a 1/x(2) weighted quadratic regression. The regression coefficients obtained are always greater than 0.99 and back calculated values always comprised in the range of +/-15% from their nominal concentration. Mean extraction recoveries are greater than 80% for all analytes and the method is accurate and precise with CV and bias lower than 9.4%. The lower limits of quantification (LLOQ) of the different drugs range from 0.0125 to 0.2 ng/ml of cell extract. This method was successfully applied to a cohort of 98 HIV-infected patients treated with Kaletra (400/100 mg of lopinavir/ritonavir (LPV/RTV) twice a day, n=48) or with Stocrin (600 mg once a day, n=50) and has been tested for cellular quantification of tipranavir (TPV) in 2 patients treated with Aptivus (500 mg twice a day). The patients treated by Kaletra showed mean cell-associated concentrations (CC) of 1819.0 and 917.2 ng/ml, for LPV and RTV, respectively. Patients treated with Stocrin showed mean CC of 2388.11 ng/ml while both patients under Aptivus showed TPV CC of 4322.7 and 1078.0 ng/ml, respectively. This method can be used to analyze ARV drug concentrations within the target tissue.

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Numerous difficulties arise during in vivo measurements of transepithelial nasal potential difference (PD) in mice, such as inadequate duration and depth of anaesthesia, bronchoaspiration of solutions perfused in the nose, and respiratory and/or cardiovascular depression. Anaesthesia was induced in adult C57 mice with intraperitoneal injection of a combination of fentanyl, droperidol and medetomidine, each of these at either a small dose (0.20, 10 and 0.33 mg/kg, respectively) or at a large dose (0.40, 20 and 0.40 mg/kg, respectively), combined with a fixed dose of 0.375 microg clonidine. In order to establish a pharmacokinetic-pharmacodynamic relationship, blood concentrations of the first three drugs were measured in 24 animals by liquid-chromatography tandem mass spectrometry. At the end of the experiment, naloxone, a competitive morphinic antagonist, and atipamezole, an alpha-2 adrenergic antagonist, were administered. Bronchoaspiration was prevented by tilting the animal head downwards and by absorbing the excess fluid from the opposite nostril and from the oral cavity. Optimal assessment of anaesthesia associated with regular respiration, loss of blink, pupillary and pedal withdrawal reflexes was obtained with doses of fentanyl, droperidol and medetomidine corresponding to 0.20, 20 and 0.40 mg/kg, respectively. Blood concentrations of fentanyl around 17 ng/mL induced loss of respiratory efforts and were followed by death during the experiment. Integrity of ion transport was demonstrated under continuous perfusion by successive depolarization after amiloride and repolarization after chloride-free solution. The combination investigated in this study lead to adequate surgical anaesthesia (stage III, plane 2) for prolonged nasal PD measurements in spontaneously breathing mice.

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A new immunoassay of sirolimus based on the microparticle enzyme immunoassay (MEIA) principle has been developed with collaboration of Abbott Diagnostics. Laboratories and Axis-Shield. Our laboratory evaluated this new assay on 153 whole blood samples (EDTA) drawn from a population of renal (n = 141) and hepatic (n = 12) transplant patients. Each blood sample was analyzed simultaneously by MEIA (Y) and by a reference method (X) used routinely in our laboratory, namely, liquid chromatography tandem mass spectrometry (LC-MS/MS). The statistical analysis of Passing-Bablok produced the following results: Spearman r value = 0.95, slope = 1.15 and intercept with the Y axis = +0.2 ng/mL. The observed global overestimation of 15% compared to the reference method could be explained by the cross-reactivity of sirolimus metabolites with the antibody. A complementary analysis taking into account the transplanted organ (kidney versus hepatic) did not show any significant difference between the populations, most likely owing to the low number of hepatic transplantation samples. The analytical performance of the MEIA method showed CV < or =10% and a limit of quantification of 1.5 ng/mL, which were acceptable for routine clinical monitoring. In conclusion, the MEIA method has shown robust, stable, reproducible, features with an excellent correlation with the reference LC-MS/MS method.

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Bile is, in certain cases, collected together with blood from different sites (heart, brain, femoral), urine and other organs or matrices. This study reports comparative results obtained from the analysis of blood and bile for different drugs found: acetaminophen, amphetamine and related compounds, several antidepressants, several benzodiazepines, cocaine and its metabolites, dextropropoxyphene and its metabolite, hydroxyzine, methadone and metabolite, morphine and codeine, levomepromazine, thioridazine, propranolol, tramadol and its metabolite. Several findings are presented: (1) There were no significant differences in the levels of the compounds among the samples of blood obtained from different sites. (2) Levels in bile are generally several fold higher than those in blood. The mean bile to blood ratios vary from about 1 (for acetaminophen, amphetamine) to about 2000 (for desmethylclobazam). (3) In certain cases (16 over 44), although the drug or its metabolite was not detected in blood from different sites, it was detected in bile. As other authors had advocated, it is very useful to ask the pathologist to take the gall bladder with its contents together with the other samples, in order that the sample of bile can be used in the comprehensive toxicological analysis and therefore be complementary to the other fluids or matrices. An additional advantage for using bile is that the concentrations of drugs or their metabolites are generally several fold higher than their blood concentrations.

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OBJECTIVE: Since the use of 4-methylpyrazole (4-MP) in the treatment of humans with methanol poisoning is poorly documented, we report two cases of acute methanol intoxication partially treated by this potent alcohol dehydrogenase (ADH) inhibitor. SETTING: Intensive Care Unit in a university hospital. PATIENTS: A 56-year-old man and an 18-year-old woman were observed, respectively, 41 and 16 h after the voluntary ingestion of an unknown amount of methanol. INTERVENTION: In both cases, ethanol was used as the first antidote. In the first patient, hemodialysis was also performed on admission because a high methanol level (0.72 g/l) and visual impairment were noted. In the second patient, ethanol therapy was withdrawn after 12 h when clinical and biological signs of acute pancreatitis became evident. Both patients received multiple oral doses of 4-MP. No recurrence of metabolic acidosis occurred and the 4-MP therapy was well tolerated. CONCLUSION: While the use of 4-MP is better documented in cases of ethylene glycol poisoning, it could also become an accepted option for the management of methanol poisoning since 4-MP offers advantages over ethanol therapy.

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The toxicological screening, using the combination of high performance liquid chromatography with diode array ultraviolet detector and ion-pairing technique in liquid-liquid extraction, is an effective tool in the identification and quantification of the acidic and basic substances in a single run. The use of an ion-pairing technique in the conventional extraction shows the co-extraction of the uncharged and charged form of the analytes present in a serum sample. The stationary phase used is C-18-bonded phase. The mobile phase is acetonitrile--phosphate buffer (pH 3; 25 mM) containing 25 mM triethylammonium as ion-pairing agent. The analytical validation shows reproducible recoveries, good day-to-day repeatability and sensible results compatible with clinical and forensic use.

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Serious exposure to the herbicide paraquat usually results in death, either due to gastrointestinal caustic lesions, shock, and acute respiratory distress syndrome or related to the progressive development of pulmonary fibrosis associated with refractory hypoxemia. We report a case of suicidal paraquat ingestion in a 59-year-old man. Most of the indicators of poor prognosis were encountered in this patient. Treatment consisted of early digestive decontamination and hemodialysis, followed by antioxidant therapy, including the administration of deferoxamine (100 mg/kg in 24 h) and a continuous infusion of acetylcysteine (300 mg/kg/d during 3 weeks). The patient only developed a nonoliguric acute renal failure, a mild alteration of liver tests, and an impairment of CO transfer factor without any respiratory complaint. Renal and hepatic disturbances completely resolved within 1 month, whereas CO transfer factor remained altered 14 months later. This observation suggests that early administration of an antioxidant therapy, including deferoxamine and acetylcysteine could be usefully associated with measures that prevent digestive absorption or enhance elimination to limit systemic toxicity in potentially fatal paraquat poisoning.

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Aminoglycosides are still used extensively in the treatment of nosocomial infections with Gram-negative bacteria. However, the treatment is associated with several adverse effects. Aminoglycosides monitoring is therefore essential to prevent toxic accumulations and to reach therapeutic concentrations. A computer program, PHARMONITOR, has been developed to optimize aminoglycosides monitoring, responding to the demands of most clinical daily situations. This program, based on a one-compartment open pharmacokinetics model, is developed for IBM PC-compatible computers, using D-Base III+. It can calculate t1/2, Vd, Cldrug, Cpmax, and the theoretical optimal dose and interval and also evaluates the creatinine clearance. The program has been conceived to allow maximal speed, flexibility, and reliability by the use of (e.g.) a linear least-squares analysis, the possible reference to previous protocols, the extensive use of keywords to classify and recall patients according to their pathologies, the development of messages recommending maximal dose or minimal dosing interval, and increasing the safety of the analysis. We consider the program a valuable tool for adjusting aminoglycoside dosage in individuals.

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