Endocrine Control of Exaggerated Trait Growth in Rhinoceros Beetles.

Juvenile hormone (JH) is a key insect growth regulator frequently involved in modulating phenotypically plastic traits such as caste determination in eusocial species, wing polymorphisms in aphids, and mandible size in stag beetles. The jaw morphology of stag beetles is sexually-dimorphic and condition-dependent; males have larger jaws than females and those developing under optimum conditions are larger in overall body size and have disproportionately larger jaws than males raised under poor conditions. We have previously shown that large males have higher JH titers than small males during development, and ectopic application of fenoxycarb (JH analog) to small males can induce mandibular growth similar to that of larger males. What remains unknown is whether JH regulates condition-dependent trait growth in other insects with extreme sexually selected structures. In this study, we tested the hypothesis that JH mediates the condition-dependent expression of the elaborate horns of the Asian rhinoceros beetle, Trypoxylus dichotomus. The sexually dimorphic head horn of this beetle is sensitive to nutritional state during larval development. Like stag beetles, male rhinoceros beetles receiving copious food produce disproportionately large horns for their body size compared with males under restricted diets. We show that JH titers are correlated with body size during the late feeding and early prepupal periods, but this correlation disappears by the late prepupal period, the period of maximum horn growth. While ectopic application of fenoxycarb during the third larval instar significantly delayed pupation, it had no effect on adult horn size relative to body size. Fenoxycarb application to late prepupae also had at most a marginal effect on relative horn size. We discuss our results in context of other endocrine signals of condition-dependent trait exaggeration and suggest that different beetle lineages may have co-opted different physiological signaling mechanisms to achieve heightened nutrient-sensitive weapon growth.

Impact of electrospray ion source platforms on matrix effect due to plasma phospholipids in the determination of rivastigmine by LC-MS/MS.

BACKGROUND: This study evaluates the performance of three electrospray ionization source designs to monitor the interference of plasma phospholipids for reliable estimation of rivastigmine by LC-MS/MS for method ruggedness. The variation in the area response due to matrix effects was assessed by post-column infusion, post-extraction spiking and standard-line slope methods. RESULTS: The observed interference due to coeluting phospholipids (m/z: 524.0/184.0) at the retention time of rivastigmine was 39.5, 12.9 and 0.4% using angular spray, orthogonal spray and dual orthogonal ion source spray design, respectively. Similarly, %CV for standard line slopes was 6.9, 4.6 and 2.0, respectively. CONCLUSION: Z-spray source design provided better and efficient transfer of gas phase ions into the mass analyzer compared with angular and orthogonal spray. The study showed that Z-spray ion source provided minimum interference from phospholipids compared with other ion source designs.

Trace analysis of acetylcholinesterase inhibitors with antipsychotic drugs for Alzheimer's disease by capillary electrophoresis with on column field-amplified sample injection.

A simple and sensitive capillary zone electrophoresis (CZE) with UV detection (214 nm) was developed and validated for the simultaneous determination of the acetylcholinesterase inhibitors (AChEI), donepezil, and rivastigmine, with antipsychotic drugs in plasma. A sample pretreatment by liquid-liquid extraction and subsequent quantification by CZE with field-amplified sample injection (FASI) was used. The optimum separation for these analytes was achieved in <20 min at 25 °C with a fused-silica capillary column of 60.2 cm × 50 µm I.D. (effective length 50 cm) and a run buffer containing 120 mM phosphate (pH 4.0) with 0.1 % γ-cyclodextrin, 40 % methanol (MeOH), and 0.02 % polyvinyl alcohol as a dynamic coating to reduce analytes' interaction with the capillary wall. Using phenformin as an internal standard (40.0 ng/mL), the linear ranges of the proposed method for the simultaneous determination of donepezil, rivastigmine, aripiprazole, quetiapine, risperidone, clozapine, ziprasidone, and trazodone were over the range 4.0-80.0 ng/mL, and olanzapine was over the range 1.0-20.0 ng/mL. The method was applied for concentrations monitoring of AChEIs and antipsychotic drugs in ten Alzheimer's disease patients with behavioral and psychological symptoms of dementia after oral administration of the commercial products.

Development of a capillary electrophoresis-mass spectrometry method for the determination of rivastigmine in human plasma--optimization of the limits of detection and quantitation.

A capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) method was developed for the analysis of the acetylcholinesterase inhibitor rivastigmine. Several electrophoretic and ESI-MS parameters were investigated in order to improve sensitivity. These parameters were categorized in three areas: (i) background electrolyte (BGE) parameters, (ii) sheath liquid parameters, and (iii) spray chamber parameters. The optimized results were obtained by using 40-mM ammonium acetate at pH 9 as BGE, a sheath liquid of 1% acetic acid in water:MeOH (50:50 v/v) at a flow rate of 10 µL/min, and a drying gas flow rate that was set at 6 L/min and at a temperature of 200°C. These parameters provided limit of detection and limit of quantitation of 2.8 ng/mL and 8.4 ng/mL, respectively. The optimal CZE-ESI-MS conditions were applied to a plasma sample obtained from an Alzheimer's disease patient following rivastigmine patch administration, and the mean (±standard deviation) plasma concentration was estimated to be 14.6 (±1.7) ng/mL. Several sample preparation procedures were examined, and solid-phase extraction using a C18 cartridge proved to be the most effective procedure, since higher sensitivity and recovery were obtained. In addition, precision was evaluated based on migration time and peak area in plasma, and the relative standard deviations were in the range of 0.10-0.16% and 0.62-9.0%, respectively.

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 250 µL human plasma by protein precipitation with acetonitrile. Chromatographic separation was achieved on a reverse phase column (BEH C18 2.1 mm × 50 mm; 1.7 µm) with a gradient elution of an ammonium acetate buffer at pH 9.3 and acetonitrile at a flow rate of 0.4 mL/min and an overall run time of 4.5 min. 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 1-300 ng/mL for donepezil, galantamine and memantine, and 0.2-50 ng/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.

Tissue distribution and pharmacodynamics of rivastigmine after intranasal and intravenous administration in rats.

The aim of the study was mainly to investigate the relationship between concentration of rivastigmine and its inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) following intranasal (IN) and intravenous (IV) administration in rats, and to provide a novel nasal delivery route for the brain disease therapy. Rivastigmine was administered to male rats at 2 mg/kg by IN and IV route. Drug concentration, AChE and BuChE activity were measured in the plasma, central nervous system (CNS) regions i.e. olfactory region, hippocampus, cerebrum and cerebellum, and peripheral tissues. It was determined that rivastigmine was characterized by extremely rapid and complete absorption into the systemic circulation followed by a rapid decline in the plasma concentrations, and can also quickly distribute into CNS and peripheral tissues by the two routes. IN administration showed higher concentration in CNS regions and longer action on inhibiting the activity of AChE and BuChE than IV administration. More significant decrease of the two enzymes was observed in CNS regions than in peripheral tissues for both administrations. A close relationship was found between the concentration of rivastigmine and enzyme inhibition in plasma and CNS tissues in rats. Based on these findings, it was concluded that rivastigmine could cause relatively strong inhibition of AChE and BuChE in plasma and brain tissues, especially in hippocampus, cortex and cerebrum. The pharmacodynamics was closely related to its concentration in vivo. The intranasal route can be strategy for delivering the drug into brain.

[Preparation of rivastigmine liposome and its pharmacokinetics in rats after intranasal administration].

To prepare rivastigmine liposome, rivastigmine was loaded into liposome via ammonium sulfate gradient method. Its pharmacokinetic profile in rats was evaluated after intranasal administration. The size, zeta potential, entrapped efficiency and release of rivastigmine from the liposome in vitro were determined. Plasma concentration of rivastigmine was determined by high performance liquid chromatography-tandem mass spectrometry (HPLC/MS) using antipyrine as internal standard. The pharmacokinetic parameters were calculated by DAS 2.0. The entrapped efficiency of rivastigmine liposome was (33.41 +/- 6.58) %, with the mean diameter of 154-236 nm and zeta potential of (-10.47 +/- 2.41) mV. The release behavior of rivastigmine was fitting the first order equation in vitro. The pharmacokinetic studies indicated that the C(max), T(max) and AUC(0-infinity), of rivastigmine liposome were (1.50 +/- 0.15) mg x L(-1), 15 min and (89.06 +/- 8.30) mg x L(-') x min, respectively. Rivastimine liposome was absorbed rapidly, and could reach a certain concentration in rat plasma after intranasal delivery.

Pesticide exposure among pregnant women in Jerusalem, Israel: results of a pilot study.

BACKGROUND: Pesticides have been shown to disrupt neurodevelopment in laboratory animals and in human populations. To date, there have been no studies on exposure to pesticides in pregnant women in Israel, despite reports of widespread exposure in other populations of pregnant women and the importance of evaluating exposure in this susceptible sub-population. METHODS: We measured urinary concentrations of organophosphorus (OP) insecticide metabolites and plasma concentrations of OP and other pesticides in 20 pregnant women, recruited in Jerusalem, Israel in 2006, and collected questionnaire data on demographic factors and consumer habits from these women. We compared geometric mean concentrations in subgroups using the Mann-Whitney U-test for independent samples. We compared creatinine-adjusted OP pesticide metabolite concentrations, as well as plasma pesticide concentrations, with other populations of pregnant women. RESULTS: Creatinine-adjusted total dimethyl (DM) metabolite concentrations were between 4 and 6 times higher in this population compared to other populations of pregnant women in the United States while total diethyl (DE) metabolite concentrations were lower. Dimethylphosphate (DMP) was detected in 74% of the urine samples whereas dimethylthiophosphate (DMTP) was detected in 90% of the urine samples. The carbamate bendiocarb was detected in 89% of the plasma samples, while the OP insecticide chlorpyrifos was detected in 42% of the samples. Mean plasma concentrations of bendiocarb and chlorpyrifos in our sample were 4.4 and 3.9 times higher, respectively, than that of an urban minority cohort from New York City. Twelve women (63%) reported using some form of household pest control during their pregnancy and five (26%) reported using household pest control during the past month. Women with a graduate degree had significantly higher geometric mean concentrations of total urinary DM metabolite concentrations compared to other women (P=0.006). Finally, one woman in the study had exceptionally high concentrations of DMP, DMTP, DMDTP compared to the other women in the study, despite reporting no current occupational exposure to OP pesticides and no other significant exposure sources. CONCLUSIONS: Pregnant women in the Jerusalem area are exposed to OP pesticides and to the carbamate pesticide bendiocarb. It is unclear why total DM metabolites concentrations were much higher in this population compared to other populations of pregnant women in the United States and Netherlands. Finally, the finding of very high DM metabolite concentrations in one woman who reported being moved from her regular laboratory work to administrative work upon becoming pregnant, raises questions about the adequacy of measures to protect pregnant women from pesticide exposures during pregnancy.

Rapid and sensitive LC-MS/MS method for determination of felbamate in mouse plasma and tissues and human plasma.

Felbamate (2-phenyl-1,3-propanediol dicarbamate) is a second generation antiepileptic drug used to treat seizures refractory to other antiepileptic drugs. With approximately 3500 new patients exposed annually, several important pharmacologic interaction questions remain unanswered necessitating the need for rapid and accurate methods of felbamate analysis in biological matrices. To this end, a rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the measurement of felbamate in mouse plasma and tissues and human plasma. Plasma (100 µL) and tissues homogenates (100 µL of 100 mg/mL) were spiked with internal standard (carisoprodol) prior to protein precipitation with acetonitrile. Samples were chromatographed on a XBridge Phenyl, 2.5 µm, 4.6 mm×50 mm column with quantitation by internal standard reference monitoring of the ion transitions m/z 239→117 for felbamate and m/z 261→176 for carisoprodol. Calibration curves were linear from 2.5 to 500 ng/mL in mouse or human plasma and 25-5000 pg/mg in tissue homogenates. Recoveries were greater than 97% for plasma and homogenates with accuracies >92% in any of the mouse matrices and >88% in human plasma. Comparable accuracies and precision were found with and without the use of the internal standard in preparation of the calibration curves and suggest that the internal standard may not be required.

Simultaneous HPLC-UV analysis of rufinamide, zonisamide, lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in deproteinized plasma of patients with epilepsy.

We present an implementation of a method we previously reported allowing the newer antiepileptic drugs (AEDs) rufinamide (RFN) and zonisamide (ZNS) to be simultaneously determined with lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine (MHD) and felbamate (FBM) in plasma of patients with epilepsy using high performance liquid chromatography (HPLC) with UV detection. Plasma samples (250 microL) were deproteinized by 1 mL acetonitrile spiked with citalopram as internal standard (I.S.). HPLC analysis was carried out on a Synergi 4 microm Hydro-RP, 250 mm x 4.6 mm I.D. column. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5), acetonitrile and methanol (65:26.2:8.8, v/v/v) at an isocratic flow rate of 0.8 mL/min. The UV detector was set at 210 nm. The chromatographic run lasted 19 min. Commonly coprescribed AEDs did not interfere with the assay. Calibration curves were linear for both AEDs over a range of 2-40 microg/mL for RFN and 2-80 microg/mL for ZNS. The limit of quantitation was 2 microg/mL for both analytes and the absolute recovery ranged from 97% to 103% for RFN, ZNS and the I.S. Intra- and interassay precision and accuracy were lower than 10% at all tested concentrations. The present study describes the first simple and validated method for RFN determination in plasma of patients with epilepsy. By grouping different new AEDs in the same assay the method can be advantageous for therapeutic drug monitoring (TDM).

Gender differences in the pharmacokinetics of rivastigmine in rats.

The effect of gender on the pharmacokinetics of rivastigmine (CAS 123441-03-2) was studied in male and female Wistar rats following intravenous bolus administration. The area under the plasma concentration-time curve (AUC), apparent volume of distribution (Vd), systemic clearance (CL), and terminal plasma halflife (t1/2) of rivastigmine were compared between male and female rats. Compared to male rats, female rats exhibited higher plasma rivastigmine levels showing significantly (p < 0.05) larger AUC (226.77 vs. 149.68 ng h/ml), Vd (6.70 vs. 4.13 L), t1/2 (0.84 vs. 0.34 h) and a lower CL (5.51 vs. 8.35 L/h). The male rats had a 2.5 fold greater elimination rate constant than female rats (2.02 vs. 0.82 h(-1)). Gender had a significant effect on the pharmacokinetics of rivastigmine. Gender differences were reported due to gonadal hormones, and the observed difference in pharmacokinetics of rivastigmine might be attributed to testosterone in male rats.

Simultaneous determination of galantamine, rivastigmine and NAP 226-90 in plasma by MEKC and its application in Alzheimer's disease.

A simple and sensitive MEKC with UV detection was developed and validated for the simultaneous determination of acetylcholinesterase inhibitors including galantamine, rivastigmine and major metabolite NAP 226-90 in plasma. A sample pretreatment by liquid-liquid extraction with diethylether and subsequent quantification by MEKC was used. The optimum separation for these analytes was achieved in <10 min at 25 degrees C with a fused-silica capillary column of 30.2 cm x 50 microm id (effective length 20 cm) and a run buffer containing 25 mM Tris buffer (pH 5.0) with 160 mM sodium octanesulfonate, 20% ACN and 0.01% PVP as a dynamic coating to reduce analytes' interaction with the capillary wall. For sensitivity consideration regarding the determination of linearity, LOD, quantitation and monitoring drugs concentration in patients, the detection wavelengths for galantamine or rivastigmine and NAP 226-90 were set at 214 or 200 nm, respectively. One male volunteer (26-year-old) was orally administered a single dose of 4.5 mg rivastigmine (Exelon, Novartis) in capsule, and blood samples were drawn over a 12 h period for concentration-time profile study. The method was also successfully applied for monitoring galantamine or rivastigmine and its metabolite NAP 226-90 in 11 Alzheimer's disease patients' plasma after oral administration of the commercial products Reminyl (8 mg galantamine/capsule) or Exelon (3 mg rivastigmine/capsule), respectively.

A study of rivastigmine liposomes for delivery into the brain through intranasal route.

The present study is mainly aimed at delivering a drug into the brain via the intranasal route using a liposomal formulation. For this purpose, rivastigmine, which is used in the management of Alzheimer's disease, was selected as a model drug. Conventional liposomes were formulated by the lipid layer hydration method using cholesterol and soya lecithin as lipid components. The concentration of rivastigmine in brain and plasma after intranasal liposomes, free drug and per oral administration was studied in rat models. A significantly higher level of drug was found in the brain with intranasal liposomes of rivastigmine compared to the intranasal free drug and the oral route. Intranasal liposomes had a longer half-life in the brain than intranasally or orally administered free drug. Delivering rivastigmine liposomes through the intranasal route for the treatment of Alzheimer's disease might be a new approach to the management of this condition.

Fluorimetric determination of rivastigmine in rat plasma by a reverse phase--high performance liquid chromatographic method. Application to a pharmacokinetic study.

A sensitive and selective high performance liquid chromatographic (HPLC) method was developed and validated for the rapid quantification of rivastigmine (CAS 123441-03-2) in micro quantity in rat plasma samples. The chromatographic separation was achieved with a reverse phase monomeric column C18 (4.6 x 250 mm, 5 microm) and the mobile phase consisted of acetonitrile and 20 mmol/L phosphate buffer pH 3.0 (25:75) with a flow rate of 1 mL/min. The effluents were measured by fluorimetric detection with excitation and emission wavelengths at 220 nm and 293 nm, respectively. The calibration curve was linear (r2 > 0.99) ranging from 25-3000 ng/mL and the lower limit of quantification was 25 ng/mL. The method was validated with excellent sensitivity, selectivity, accuracy, precision, recovery and stability. The method has been successfully applied in a pharmacokinetic study of rivastigmine in rats.

Drug monitoring and toxicology: a simple procedure for the monitoring of felbamate by HPLC-UV detection.

This article describes a simple isocratic high-performance liquid chromatographic (HPLC) method with UV detection for the determination of felbamate in the serum of patients with epilepsy. Sample preparation requires only protein precipitation with a single-step methanol extraction. After centrifugation, the resulting supernatant was injected directly onto the HPLC system. Separation was achieved by reversed-phase HPLC, using a 5-microm Microsorb-MV C(18) column (250 x 4.6 mm) connected to a Silica C(18) guard column (20 x 4.6 mm) and a mobile phase consisting of a mixture of phosphate buffer (pH = 6.9, 0.05 M), methanol, and acetonitrile (64:18:18, v/v/v). The flow rate was at 1.0 mL/min and column temperature was set at 35 degrees C. Quantitation was performed by measurement of the UV absorbance at a wavelength of 210 nm. Calibration curves were linear over a range of 2-200 mg/L, which covered the proposed range of 50-150 mg/L for reference. Both within-run and between-run precision were lower than 5%. Recoveries ranged between 97% and 105% for spiked and pooled samples. No interferences with other common antiepileptic drugs (except zonisamide) were observed. The method requires only 100 microL of serum or less. It is simple and fast (sample preparation and analysis time) and suitable for routine clinical use.

Rivastigmine exposure provided by a transdermal patch versus capsules.

OBJECTIVES: The rivastigmine transdermal patch is the first transdermal treatment for Alzheimer's disease (AD) and dementia associated with Parkinson's disease. The objective of this study was to evaluate the pharmacokinetics of rivastigmine following transdermal delivery by a patch versus oral delivery with conventional capsules in a population of AD patients. METHODS: Both non-compartmental and compartmental analyses were performed on the same database showing relatively large inter-patient variations in pharmacokinetic parameters (up to 73% for the capsule group). The compartmental analysis provided model-based predictions of pharmacokinetic parameters, with the aim of comparing the two modes of administration when adjusting for confounding factors such as patient body weight and gender. RESULTS: According to both non-compartmental and compartmental analyses, the patch provided significantly lower peak rivastigmine plasma concentrations (C(max)) and slower times to C(max) (t(max)), compared with capsules. However, drug exposure (area under the curve; AUC) was not significantly different between the 4.6 mg/24 hour (5 cm(2)) patch and 3 mg BID (6 mg/day) capsule doses, or between the 9.5 mg/24 hour (10 cm(2)) patch and 6 mg BID (12 mg/day) capsule doses, according to both analyses. This suggests comparable exposure from these two rivastigmine delivery systems. CONCLUSION: The analyses were consistent with previous reports of a markedly less fluctuating, more continuous drug delivery with the rivastigmine patch. This characteristic delivery profile is associated with similar efficacy yet improved tolerability, compared with capsules.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of rivastigmine in human plasma.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of rivastigmine in human plasma. Rivastigmine was extracted from human plasma by using solid-phase extraction technique. Zolpidem was used as the internal standard. A Betabasic-8 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 251.20-->206.10, 86.20 for rivastigmine and m/z 308.10-->235.10 for zolpidem. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatographic conditions and mass spectrometric detection that enables detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.2-20.0 ng/ml with a correlation coefficient > or =0.9988. The intra-run and inter-run precision and accuracy were within 10.0%. The overall recoveries for rivastigmine and zolpidem were 86.28% and 87.57%, respectively. The total run time was 2.0 min. The developed method was applied for the determination of the pharmacokinetic parameters of rivastigmine following a single oral administration of a 3 mg rivastigmine capsule in 20 healthy male volunteers.

A simple and sensitive assay for the quantitative analysis of rivastigmine and its metabolite NAP 226-90 in human EDTA plasma using coupled liquid chromatography and tandem mass spectrometry.

A sensitive and specific high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the determination of rivastigmine and its major metabolite NAP 226-90 is presented. A 100 microL plasma aliquot was spiked with a structural analogue of rivastigmine as internal standard (PKF214-976-AE-1) and proteins were precipitated by adding 200 microL of methanol. After centrifugation a volume of 100 microL of the clear supernatant was mixed with 100 microL of methanol/water (30:70, v/v) and volumes of 25 microL were injected onto the HPLC system. Separation was acquired on a 150 x 2.0 mm i.d. Gemini C18 column using a gradient system with 10 mM ammonium hydroxide and methanol. Detection was performed by using a turboionspray interface and positive ion multiple reaction monitoring by tandem mass spectrometry. The assay quantifies rivastigmine from 0.25 to 50 ng/mL and its metabolite NAP 226-90 from 0.50 to 25 ng/mL, using human plasma samples of 100 microL. Validation results demonstrate that rivastigmine and metabolite concentrations can be accurately and precisely quantified in human EDTA plasma. This assay is now used to support clinical pharmacologic studies with rivastigmine.