Determination of oseltamivir and oseltamivir carboxylate in human plasma by liquid chromatography-tandem mass spectrometry

Introduction: Oseltamivir phosphate (OP), the prodrug of oseltamivir carboxylate (OC; active metabolite), is marketed since 10 years for the treatment of seasonal influenza flu. It has recently received renewed attention because of the threat of avian flu H5N1 in 2006-7 and the 2009-10 A/H1N1 pandemic. However, relatively few studies have been published on OP and OC clinical pharmacokinetics. The disposition of OC and the dosage adaptation of OP in specific populations, such as young children or patients undergoing extrarenal epuration, have also received poor attention. An analytical method was thus developed to assess OP and OC plasma concentrations in patients receiving OP and presenting with comorbidities or requiring intensive care. Methods: A high performance liquid chromatography coupled to tandem mass spectrometry method (HPLC-MS/MS) requiring 100-µL aliquot of plasma for quantification within 6 min of OP and OC was developed. A combination of protein precipitation with acetonitrile, followed by dilution of supernant in suitable buffered solvent was used as an extraction procedure. After reverse phase chromatographic separation, quantification was performed by electro-spray ionization-triple quadrupole mass spectrometry. Deuterated isotopic compounds of OP and OC were used as internal standards. Results: The method is sensitive (lower limit of quantification: 5 ng/mL for OP and OC), accurate (intra-/inter-assay bias for OP and OC: 8.5%/5.5% and 3.7/0.7%, respectively) and precise (intra-/inter-assay CV%: 5.2%/6.5% and 6.3%/9.2%, respectively) over the clinically relevant concentration range (upper limits of quantification 5000 ng/mL). Of importance, OP, as in other previous reports, was found not to be stable ex vivo in plasma on standard anticoagulants (i.e. EDTA, heparin or citrate). This poor stability of OP has been prevented by collecting blood samples on commercial fluoride/oxalate tubes. Conclusions: This new simple, rapid and robust HPLC-MS/MS assay for quantification of OP and OC plasma concentrations offers an efficient tool for concentration monitoring of OC. Its exposure can probably be controlled with sufficient accuracy by thorough dosage adjustment according to patient characteristics (e.g. renal clearance). The usefulness of systematic therapeutic drug monitoring in patients appears therefore questionable. However, pharmacokinetic studies are still needed to extend knowledge to particular subgroups of patients or dosage regimens.

Evaluation de l'efficacite antihypertensive de l'isradipine SRO par enregistrement tensionnel en ambulatoire. [Evaluation of the antihypertensive efficacy of isradipine SRO as assessed by ambulatory blood pressure determination]

The calcium channel blocker isradipine has become recently available in a form with delayed release (isradipine SRO). The anti-hypertensive efficacy and tolerance of this preparation at a single daily dose of 5 mg was studied in 40 patients with uncomplicated essential hypertension over a period of 6 weeks. Blood pressure during office visits decreased under Isradipine SRO from 164/105 +/- 16/7 to 144/93 +/- 12/7 mmHg (mean +/- 1 standard deviation p < 0.001). Using ambulatory blood pressure recording we could show that antihypertensive efficacy of the new galenic form persisted over 24 hours. During the day the blood pressure dropped from 150/95 +/- 13/7 to 141/91 +/- 13/7 mmHg (p < 0.001), during the night from 131/85 +/- 13/3 to 121/81 +/- 15/9 mmHg (p < 0.001). Heart-rate was not changed by treatment and the drug was well tolerated. Isradipine SRO at a single dose is thus well suited for antihypertensive treatment.

The role of LEKTI in fate determination of keratinocyte stem cells

SPINK5 (serine protease inhibitor Kazal-type 5) encodes the putative proteinase inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor). In skin, LEKTI expression is restricted to the stratum granulosum of the epidermis and the inner root sheath of hair follicles. Mutations that create premature termination codons in SPINK5 have been reported as the cause of Netherton syndrome (NS), a human autosomal recessive disorder characterized by congenital ichthyosis with defective cornification, a specific hair shaft defect known as trichorrexis invaginata or 'bamboo hair', and severe atopic manifestations, including atopic dermatitis and hayfever. Althought recombinant human LEKTI inhibits a battery of serine proteases including plasmin, trypsin, subtilisin A, cathepsin G, and elastase, the precise role of LEKTI in the physiopathology of NS remains unclear. Spink5−/− mice display a NS-like phenotype. Surprisingly, a psoriasis-like hyperplasia, basement membrane breakdown followed by evagination of spindle-shaped epidermal cells into the dermal compartment, and the presence of numerous sweat gland-like structures were also observed when the skin of Spink5−/− newborn mice, which die at birth, was transplanted onto the back of nude mice. Collectively, these observations suggest that LEKTI may play a role on cell proliferation and stem cell fate. Our current work aims at elucidating the mechanisms by which LEKTI impact these biological processes. Using keratinocyte stem cells obtained from NS patients, we have identified LEKTI as a regulator node in several signaling pathways involved in stem cell behavior.

Simultaneous determination of antidementia drugs in human plasma: Procedure transfer from HPLC-MS to UPLC-MS/MS.

A previously developed high performance liquid chromatography mass spectrometry (HPLC-MS) procedure for the simultaneous determination of antidementia drugs, including donepezil, galantamine, memantine, rivastigmine and its metabolite NAP 226-90, was transferred to an ultra performance liquid chromatography system coupled to a tandem mass spectrometer (UPLC-MS/MS). The drugs and their internal standards ([(2)H(7)]-donepezil, [(13)C,(2)H(3)]-galantamine, [(13)C(2),(2)H(6)]-memantine, [(2)H(6)]-rivastigmine) were extracted from 250μL human plasma by protein precipitation with acetonitrile. Chromatographic separation was achieved on a reverse phase column (BEH C18 2.1mm×50mm; 1.7μm) with a gradient elution of an ammonium acetate buffer at pH 9.3 and acetonitrile at a flow rate of 0.4mL/min and an overall run time of 4.5min. The analytes were detected on a tandem quadrupole mass spectrometer operated in positive electrospray ionization mode, and quantification was performed using multiple reaction monitoring. The method was validated according to the recommendations of international guidelines over a calibration range of 1-300ng/mL for donepezil, galantamine and memantine, and 0.2-50ng/mL for rivastimgine and NAP 226-90. The trueness (86-108%), repeatability (0.8-8.3%), intermediate precision (2.3-10.9%) and selectivity of the method were found to be satisfactory. Matrix effects variability was inferior to 15% for the analytes and inferior to 5% after correction by internal standards. A method comparison was performed with patients' samples showing similar results between the HPLC-MS and UPLC-MS/MS procedures. Thus, this validated UPLC-MS/MS method allows to reduce the required amount of plasma, to use a simplified sample preparation, and to obtain a higher sensitivity and specificity with a much shortened run-time.

Simultaneous determination of antidementia drugs by HPLC-MS: validation data of the method and plasma level determinations in 300 patients

Aims: A rapid and simple HPLC-MS method was developed for the simultaneousdetermination of antidementia drugs, including donepezil, galantamine, rivastigmineand its major metabolite NAP 226 - 90, and memantine, for TherapeuticDrug Monitoring (TDM). In the elderly population treated with antidementiadrugs, the presence of several comorbidities, drug interactions resulting frompolypharmacy, and variations in drug metabolism and elimination, are possiblefactors leading to the observed high interindividual variability in plasma levels.Although evidence for the benefit of TDM for antidementia drugs still remains tobe demonstrated, an individually adapted dosage through TDM might contributeto minimize the risk of adverse reactions and to increase the probability of efficienttherapeutic response. Methods: A solid-phase extraction procedure with amixed-mode cation exchange sorbent was used to isolate the drugs from 0.5 mL ofplasma. The compounds were analyzed on a reverse-phase column with a gradientelution consisting of an ammonium acetate buffer at pH 9.3 and acetonitrile anddetected by mass spectrometry in the single ion monitoring mode. Isotope-labeledinternal standards were used for quantification where possible. The validatedmethod was used to measure the plasma levels of antidementia drugs in 300patients treated with these drugs. Results: The method was validated accordingto international standards of validation, including the assessment of the trueness(-8 - 11 %), the imprecision (repeatability: 1-5%, intermediate imprecision:2 - 9 %), selectivity and matrix effects variability (less than 6 %). Furthermore,short and long-term stability of the analytes in plasma was ascertained. Themethod proved to be robust in the calibrated ranges of 1 - 300 ng/mL for rivastigmineand memantine and 2 - 300 mg/mL for donepezil, galantamine and NAP226 - 90. We recently published a full description of the method (1). We found ahigh interindividual variability in plasma levels of these drugs in a study populationof 300 patients. The plasma level measurements, with some preliminaryclinical and pharmacogenetic results, will be presented. Conclusion: A simpleLC-MS method was developed for plasma level determination of antidementiadrugs which was successfully used in a clinical study with 300 patients.

Nature versus nurture in social insect caste determination

Recent evidence for genetic effects on royal and worker caste differentiation from diverse social insect taxa has put an end to the view that these phenotypes stem solely from a developmental switch controlled by environmental factors. Instead, the relative influences of genotypic and environmental effects on caste vary among species, ranging from largely environmentally controlled phenotypes to almost purely genetic systems. Disentangling the selective forces that generate variation for caste predisposition will require characterizing the genetic mechanisms underlying this variation, and identifying particular life-history strategies and kin structures associated with strong genetic effects on caste.

Determination of extreme floods using a distributed hydrological model

Hydrological models developed for extreme precipitation of PMP type are difficult to calibrate because of the scarcity of available data for these events. This article presents the process and results of calibration for a distributed hydrological model at fine scale developed for the estimation of probable maximal floods in the case of a PMP. This calibration is done on two Swiss catchments for two events of summer storms. The calculation done is concentrated on the estimation of the parameters of the model, divided in two parts. The first is necessary for the computation of flow speeds while the second is required for the determination of the initial and final infiltration capacities for each terrain type. The results, validated with the Nash equation show a good correlation between the simulated and observed flows. We also apply this model on two Romanian catchments, showing the river network and estimated flow.

Determination of plasma levels of citalopram and its demethylated and deaminated metabolites by gas chromatography and gas chromatography-mass spectrometry.

Sensitive and specific methods based on gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) for the determination of levels of citalopram, desmethylcitalopram and didesmethylcitalopram in the plasma of patients treated with citalopram are presented, as well as a GC-MS procedure for the assay of the citalopram propionic acid derivative. After addition of a separate internal standard for each drug, liquid-solvent extraction is used to separate the basic compounds from the acid compounds. The demethylated amines are derivatized with trifluoroacetic anhydride, and the acid metabolite with methyl iodide. GC-MS is performed in the electron impact mode, as mass spectrometry by the (positive-ion) chemical ionization mode (methane and ammonia) appeared to be unsuitable. The limits of quantification were 1 ng/ml for citalopram and desmethylcitalopram and 2 ng/ml for the other metabolites. The correlation coefficients for the calibration curves (range 10-500 ng/ml) were &gt; or = 0.999 for all compounds, whether determined by GC or GC-MS.

Determination of testosterone misuse in sport, an international comparative study

Introduction: Urinary steroid profiling is used in doping controls to detect testosterone abuse. A testosterone over epitestosterone (T/E) ratio exceeding 4.0 is considered as suspicious of testosterone administration, irrespectively of individual heterogeneous factors such as the athlete's ethnicity. A deletion polymorphism in the UGT2B17 gene was demonstrated to account for a significant part of the inter-individual variability in the T/E between Caucasians and Asians. However, the anti-doping strategy includes the determination of carbon isotope ratio on androgen metabolites which has been demonstrated to be reliable for the direct detection of testosterone misuse. Herein, we examined the profiles and the variability in the 13C/12Cratios of urinary steroids in a widely heterogeneous cohort of professional soccer players residing in different world countries (Argentina, Italy, Japan, South-Africa, Switzerland and Uganda). Aim: The determination of threshold values based on genotype information and diet specific of the ethnicity is expected to enhance significantly the detection of testosterone misuse. Methods: The steroid profile of 57 Africans, 32 Asians, 50 Caucasians and 32 Hispanics was determined by gas chromatography-mass spectrometry. The carbon isotope ratio of selected androgens in urine specimens were determined by means of gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS). Results: Significant differences have been observed between all ethnic groups. After estimation of the prevalence of the UGT2B17 deletion/deletion genotype (African:22%; Asian:81%; Caucasian:10%; Hispanic:7%), ethnicspecific thresholds were developed for a specificity of 99% for the T/E (African:5.6; Asian:3.8; Caucasian:5.7; Hispanic:5.8). Italian and Swiss populations recorded an enrichment in 13C of the urinary steroids with respect to the other groups, thereby supporting consumption of a relatively larger proportion of C3 plants in their diet. Noteworthy, detection criteria based on the difference in the carbon isotope ratio of androsterone and pregnanediol for each population were well below the established threshold value for positive cases. Conclusion: These profiling results demonstrate that a unique and nonspecific threshold to evidence testosterone misuse is not fit for purpose. In addition, the carbon isotopic ratio from these different diet groups highlight the importance to adapt the criteria for increasing the sensitivity in the detection of exogenous testosterone. In conclusion, it may be emphasized that combining the use of isotope ratio mass spectrometry including refined interpretation criteria for positivity and the subject-based profiling of steroids will most probably improve the efficiency of the confirmatory test.

A program for the computation of power and determination of sample size in hierarchical experimental designs.

We have devised a program that allows computation of the power of F-test, and hence determination of appropriate sample and subsample sizes, in the context of the one-way hierarchical analysis of variance with fixed effects. The power at a fixed alternative is an increasing function of the sample size and of the subsample size. The program makes it easy to obtain the power of F-test for a range of values of sample and subsample sizes, and therefore the appropriate sizes based on a desired power. The program can be used for the 'ordinary' case of the one-way analysis of variance, as well as for hierarchical analysis of variance with two stages of sampling. Examples are given of the practical use of the program.

Faktorenanalytische Untersuchung und Rehabilitätsbestimmung der statischen und dynamischen Pupillometrie bei Normalpersonen und psychopathologischen Gruppen [Factor analytic study and determination of rehabilitation of static and dynamic pupillometry in normal persons and psychopathologic groups]

Variables measured during static and dynamic pupillometry were factor-analyzed. Following factors were obtained regardless whether investigations were carried out in normals or in psychiatric patients: A static factor, a dynamic factor, a stimulus-specific factor and a restitution-dependent factor. Evaluation of reliability in normals demonstrated a high reliability for the static variables of pupillometry.

Characterization and distribution of Pogonomyrmex harvester ant lineages with genetic caste determination.

Genetic caste determination has been described in two populations of Pogonomyrmex harvester ants, each comprising a pair of interbreeding lineages. Queens mate with males of their own and of the alternate lineage and produce two types of diploid offspring, those fertilized by males of the queens' lineage which develop into queens and those fertilized by males of the other lineage which develop into workers. Each of the lineages has been shown to be itself of hybrid origin between the species Pogonomyrmex barbatus and Pogonomyrmex rugosus, which both have typical, environmentally determined caste differentiation. In a large scale genetic survey across 35 sites in Arizona, New Mexico and Texas, we found that genetic caste determination associated with pairs of interbreeding lineages occurred frequently (in 26 out of the 35 sites). Overall, we identified eight lineages with genetic caste determination that always co-occurred in the same complementary lineage pairs. Three of the four lineage pairs appear to have a common origin while their relationship with the fourth remains unclear. The level of genetic differentiation among these eight lineages was significantly higher than the differentiation between P. rugosus and P. barbatus, which questions the appropriate taxonomic status of these genetic lineages. In addition to being genetically isolated from one another, all lineages with genetic caste determination were genetically distinct from P. rugosus and P. barbatus, even when colonies of interbreeding lineages co-occurred with colonies of either putative parent at the same site. Such nearly complete reproductive isolation between the lineages and the species with environmental caste determination might prevent the genetic caste determination system to be swept away by gene flow.