Simultaneous HPLC-F analysis of three recent antiepileptic drugs in human plasma

An original high-performance liquid chromatographic method with fluorescence detection is presented for the simultaneous determination of the three antiepileptic drugs gabapentin, vigabatrin and topiramate in human plasma. After pre-column derivatisation with dansyl chloride, the analytes were separated on a Hydro-RP column with a mobile phase composed of phosphate buffer (55%) and acetonitrile (45%) and detected at λ_em = 500 nm, exciting at 300 nm. An original pre-treatment procedure on biological samples, based on solid-phase extraction with MCX cartridges for gabapentin and vigabatrin, and with Plexa^® cartridges for topiramate, gave high extraction yields (>91%), satisfactory precision (RSD < 6.4%) and good selectivity. Linearity was found in the 0.2–50.0 μg mL⁻¹ range for gabapentin, in the 1.0–100.0 μg mL⁻¹ range for vigabatrin and in the 1.0–50.0 μg mL⁻¹ range for topiramate, with limits of detection (LODs) between 0.1 and 0.3 μg mL⁻¹. After validation, the method was successfully applied to some plasma samples from patients undergoing therapy with one or more of these drugs. Accuracy results were satisfactory (recovery >91%). Therefore, the method seems to be suitable for the therapeutic drug monitoring (TDM) of patients treated with gabapentin, vigabatrin and topiramate.     

Rifampicin determination in plasma by stir bar-sorptive extraction and liquid chromatography

A sensitive and reproducible stir bar-sorptive extraction and high performance liquid chromatography-UV detection (SBSE/HPLC-UV) method for therapeutic drug monitoring of rifampicin in plasma samples is described and compared with a liquid:liquid extraction (LLE/HPLC-UV) method. This miniaturized method can result in faster analysis, higher sample throughput, lower solvent consumption and less workload per sample while maintaining or even improving sensitivity. Important factors in the optimization of SBSE efficiency such as pH, temperature, extraction time and desorption conditions (solvents, mode magnetic stir, mode ultrasonic stir, time and number of steps) were optimized recoveries ranging from 75 to 80%. Separation was obtained using a reverse phase C₈ column with UV detection (254 nm). The mobile phase consisted of methanol:0.25 N sodium acetate buffer, pH 5.0 (58:42, v/v). The SBSE/HPLC-UV method was linear over a working range of 0.125–50.0 μg mL⁻¹. The intra-assay and inter-assay precision and accuracy were studied at three concentrations (1.25, 6.25 and 25.0 μg mL⁻¹). The intra-assay coefficients of variation (CVs) for all compounds were less than 10% and all inter-CVs were less than 10%. Limits of quantification were 0.125 μg mL⁻¹. Stability studies showed rifampicin was stable in plasma for 12 h after thawing; the samples were also stable for 24 h after preparation. Based on the figures of merit results, the SBSE/HPLC-UV proved to be adequate to the rifampicin analyses from therapeutic to toxic levels. This method was successfully applied to the analysis of real samples and was as effective as the LLE/HPLC-UV method.     

Quantification of fumaric acid in liver, spleen and urine by high-performance liquid chromatography coupled to photodiode-array detection

Quantification of fumaric acid, an endogenous dicarboxylic acid with interesting biomedical applications either through its own biological activity or as a linker constitutive of the porous iron(III) fumarate metal organic framework (MOF) MIL-88A based drug nanocarrier (MIL stands for Material from Institut Lavoisier), has been developed in different rat biological complex media (liver, spleen and urine). After a liquid-liquid extraction procedure, fumaric acid concentration was determined by a simple and accurate high-performance liquid chromatography (HPLC) method coupled to a photodiode-array detector (PDA) using aminosalicylic acid as internal standard (IS) and a gradient elution. The recovery of fumaric acid reaches 89% and 92% for urine (for concentrations of 0.05 and 1 μg ml⁻¹, respectively) and 90% for liver and spleen tissues, exceeding 89% in all instances in comparison with the IS. Linearity has been kept from 0.05 to 1 μg ml⁻¹ and from 0.5 to 10 μg g⁻¹ of fumaric acid in urine and tissues, respectively. The limit of detection of the method was 0.01 μg per injection. This method has finally allowed the quantification of fumaric acid in rat urine and tissue samples after the intravenous administration of MIL-88A nanoparticles.     

Ultrasonic-assisted precolumn derivatization-HPLC determination of acrylamide formed in Radix Asparagi during heating process

An ultrasonic-assisted precolumn derivatization-HPLC method was established and validated for the determination of acrylamide formed in traditional Chinese herb Radix Asparagi during heating process. This method entails extraction with water, ultrasonic-assisted derivatization with 2-mercaptobenzoic acid. The final extracted acrylamide derivative was separated on C₁₈ column by using a mixture of acetonitrile and acetic acid (1 g L⁻¹ water solution) (20:80, v/v) as mobile phase. The flow rate was 0.7 mL min⁻¹ and the detection wavelength was set at 238 nm. Factors influencing the derivative reaction were evaluated and the optimum derivatization conditions were as follows: molar ratio of derivative reagent to acrylamide was 35:1, pH 8–12, ultrasonic-assisted reaction time was 100 min. The calibration curve of acrylamide showed good linearity in the range of 0.015–4.5 μg mL⁻¹ with correlation coefficient of 0.9999. The limit of detection was estimated to be 25 μg kg⁻¹ based on the signal-to-noise ratio of 3 recorded at 238 nm. Recovery of acrylamide from the sample was 106.6 ± 6.6%. Relative standard deviation of five duplicate determinations for the same sample solution was 1.59%.     

Determination of topiramate in human plasma by capillary electrophoresis with indirect UV detection

A rapid capillary zone electrophoresis method with indirect UV detection for the determination of topiramate in human plasma was developed and validated. The analyses were carried out with a background electrolyte composed of 10 mM sulfamethoxazole as chromophore in phosphate buffer (25 mM, pH 12.0); gabapentin was selected as the internal standard. Application of a voltage of +15 kV led to an analysis time shorter than 5 min; indirect UV detection was operated at 256 nm. Isolation of topiramate from plasma was accomplished by a carefully implemented solid-phase extraction procedure on C18 cartridges. The method provided a linear response over the concentration range of 2–60 μg of topiramate per mL of plasma. The limit of detection (LOD) was 0.8 μg mL⁻¹ and the limit of quantitation (LOQ) was 2.0 μg mL⁻¹. Precision, expressed as relative standard deviation, was always lower than 7.3%, extraction yields were always greater than 92%. The results obtained analysing plasma samples from epileptic patients undergoing therapy with topiramate were satisfactory in terms of precision and selectivity.     

Development and validation of an ultra performance liquid chromatography-tandem mass spectrometry method for the quantification of daptomycin in human plasma

A rapid, simple and accurate analytical method based on ultra performance liquid chromatography (UPLC) combined with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a hybrid q TOF instrument has been developed and fully validated for the quantification of daptomycin (DPT) in human plasma. The samples were analyzed after simple pretreatment involving protein precipitation, while chromatographic separation of DPT and the internal standard (reserpine) was achieved on an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using gradient elution with 0.1% aqueous formic acid (FA) and acetonitrile with 0.1% FA (with DPT eluting at 2.60 min). The method presented good fit (r > 0.999) over the quantification range of 0.01-10 μg mL⁻¹ with the lower limit of quantitation (LLOQ) being 0.01 μg mL⁻¹ of human plasma for DPT. The intra- and inter-day precision, measured as % relative standard deviation, was less than 11% for DPT. The validation results showed that the developed method demonstrated adequate selectivity, sensitivity, precision and accuracy and therefore was successfully applied to the analysis of clinical samples following intravenous (iv) administration of 5.4 mg kg⁻¹ DPT to patients suffering from post-traumatic osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA). The developed methodology is the first report of an accurate mass tandem MS method for the analysis of this potent antibiotic in human plasma and can be used to further study pharmacokinetic, bioequivalence and even metabolic aspects related to this drug.     

On-line molecular imprinted solid-phase extraction flow-injection fluorescence sensor for determination of florfenicol in animal tissues

A simple and convenient on-line molecular imprinted solid-phase extraction flow-injection fluorescence sensor was developed in this study. Florfenicol (FF)-imprinted polymer was prepared by self-assembly with acrylamide (AM) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker. The binding characteristics of the imprinted polymer to florfenicol were evaluated by equilibrium binding experiments, and the morphology of the imprinted polymer was studied by scanning electron microscope (SEM). Fluorescence intensity of 3-p-nitrylphenyl-5-(2′-sulfonophenylazo) rhodanine (4NRASP) and bovine serum albumin (BSA) was inhibited by FF. Under optimum conditions the intensity of fluorescence shows a linear relationship with the concentration of FF over the range of 1.2 × 10⁻⁶ to 2.6 × 10⁻⁵ g mL⁻¹ with a regression equation of ΔI = 23.54 + 17.86c (c 10⁻⁶ g mL⁻¹) (r = 0.9965, n = 6). The low detection limit of FF was found to be 3.4 × 10⁻⁷ g mL⁻¹ according to 11 parallel determinations for the blank solution. The relative standard deviation for the determination of 2.0 × 10⁻⁶ g mL⁻¹ FF solution was 3.5% (n = 11). This proposed sensor could be satisfactorily applied to the determination of FF in liver and meat samples.     

Solid phase extraction–spectrophotometric determination of salicylic acid using magnetic iron oxide nanoparticles as extractor

This method shows a novel, fast and simple solid phase extraction–spectrophotometric procedure for preconcentration and determination of salicylic acid (SA) in blood serum using magnetic iron oxide nanoparticles (MIONs) as extractor. It is shown that the novel magnetic nano-adsorbent is quite efficient for fast adsorption of SA at 25 °C. Various parameters affecting the adsorption of SA on MIONs, such as pH of solution, type, volume and concentration of desorbing reagent and amount of adsorbent and matrix effects, have been investigated. The calibration graph for the determination of SA was linear in the range of 0.025–1.250 μg mL⁻¹. The limit of detection (LOD) based on three times the standard deviation of the blank (3S_b) was 5.5 × 10⁻³ μg mL⁻¹ (n = 10) for SA. The intra-day precision (R.S.D.) was below 10.1% and inter-day R.S.D. was less than 17.5%, while accuracy (relative error R.E.) was within ±3.6 determined from quality control samples for salicylic acid (SA) which corresponded to requirement of the guidance of Food and Drug Administration (FDA). The preconcentration factor of 100 was achieved in this method. The proposed procedure has been successfully applied to the determination of SA in blood serum.     

Extraction and determination of some psychotropic drugs in urine samples using dispersive liquid–liquid microextraction followed by high-performance liquid chromatography

A simple, rapid and sensitive method termed as dispersive liquid–liquid microextraction (DLLME) combined with high-performance liquid chromatography-ultraviolet detector (HPLC-UV) has been proposed for the determination of three psychotropic drugs (amitryptiline, clomipramine and thioridazine) in urine samples. The determination was performed on a C₈ column under the optimal chromatographic conditions (mobile phase: ammonium acetate (0.03 mol L⁻¹, pH 5.5)–acetonitrile (60:40, v/v); flow rate: 1.0 mL min⁻¹; detection wavelength: 238 nm). Several factors influencing the extraction efficiency of the target drugs, such as pH, extraction and disperser solvent type and their volume, extraction time and ion strength were studied and optimized. Under the optimal DLLME conditions, the absolute recoveries of amitryptiline, clomipramine and thioridazine from the urine samples were 96, 97 and 101%, respectively. The detection limits (LODs) and quantification (LOQs) of the proposed approach were 3 and 10 ng mL⁻¹ for amitryptiline, 7 and 21 ng mL⁻¹ for clomipramine, and 8 and 25 ng mL⁻¹ for thioridazine, respectively. The relative standard deviations (RSDs) for nine replicate determinations at 0.100 μg mL⁻¹ level of target drugs were less than 4.8%. Good linear behaviors over the investigated concentration ranges were obtained with the values of R² > 0.998 for the target drugs. The proposed method was successfully applied to the real urine samples from two female patients under amitryptiline and clomipramine treatment, respectively.     

Feed contaminated with Fusarium toxins alter lymphocyte proliferation and apoptosis in primiparous sows during the perinatal period

Two groups of pregnant primiparous sows (day 89 ± 2 of gestation) were 54 days (±1 day) fed either with an experimental diet (5.08 mg kg⁻¹ deoxynivalenol – DON, 0.09 mg kg⁻¹ zearalenone and 21.61 mg kg⁻¹ fusaric acid) or control diet (0.25 mg kg⁻¹ DON). The consummation of feed was significantly higher in the control group. Lymphocyte stimulation in culture from peripheral blood lymphocytes (PBL) was measured by BrdU incorporation test using two different concentrations of mitogen PHA 10 and 20 μg ml⁻¹, two different concentrations of DON (5 and 1 μg ml⁻¹) and a combination of both, PHA and DON (PHA 10 + DON 5, PHA 10 + DON 1 and PHA 10 + DON 0.1 μg ml⁻¹). The lymphocyte proliferation, except for PHA 10 μg ml⁻¹, was significantly higher in the experimental group. Further on, using the photometric enzyme immunoassay, the apoptosis was studied in non-stimulated 72 h lymphocyte culture or stimulated with 1 μg ml⁻¹ of DON. The significantly higher apoptosis was in non-stimulated lymphocyte cultures from the experimental group (P = 0.036). The results suggest that such feed may affect the peripheral lymphocyte population in the direction of their proliferation response and programmed cell death.     

Daily intake of inorganic arsenic and some organic arsenic species of Japanese subjects

The daily dietary intake of inorganic arsenic (InAs) and some of organic arsenic (OrAs) species of Japanese subjects were estimated by determining the concentrations of As species in two different sets of total diet sample: duplicated diet samples collected from 25 subjects in Japan and a certified reference material with total diet matrix (NIES CRM No. 27 Typical Japanese Diet, TJD). The concentration of InAs and OrAs in diet samples were determined by LC-ICP-MS using a photo-oxidation and hydride generation system. The median intake of InAs for the 25 subjects was 3.8 μg day⁻¹ (2.0–57 μg day⁻¹) and intake of 27 μg day⁻¹ was estimated from TJD. The median intake of MMA, DMA and TMAsO were <0.18, 1.1 and <0.053 μg day⁻¹ for the 25 subjects and that of MMA, DMA, AB and TMAsO was estimated to be 3.9, 11, 140 and 5.9 μg day⁻¹, respectively, based on TJD analysis. On the basis of InAs intakes estimated and the oral slope factor of the US EPA and Health Canada, excess cancer risk was estimated to exceed acceptable level. Cancer risk posed by the dietary InAs of the general Japanese may not be negligible.     

Simultaneous quantification of daptomycin and rifampicin in plasma by ultra performance liquid chromatography: Application to a pharmacokinetic study

A rapid and simple method based on ultra performance liquid chromatography (UPLC) with ultra violet detection has been developed for the determination of daptomycin (DPT) and rifampicin (RFM) in rabbit plasma using 4-nitrophenol as internal standard (IS). Sample preparation involved protein precipitation with an acetonitrile:methanol mixture and centrifugation. Chromatographic separation was achieved on an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using gradient elution with methanol and 0.1% aqueous TFA. The total analysis time was 4.5 min with DPT and RFM eluting at 1.9 and 2.1 min, respectively. The method was fully validated with a lower limit of quantitation (LLOQ) of 2 μg mL⁻¹ for both DPT and RFM. The intra- and inter-day precision, measured as % relative standard deviation, were less than 12.1 for DPT and 10.7 for RFM, respectively. This validated method was successfully applied to a pharmacokinetic study involving intravenous administration of 14 mg kg⁻¹ DPT and 30 mg kg⁻¹ RFM to rabbits.     

Validation protocol of analytical procedures for quantification of drugs in polymeric systems for parenteral administration: Dexamethasone phosphate disodium microparticles

In this work a protocol to validate analytical procedures for the quantification of drug substances formulated in polymeric systems that comprise both drug entrapped into the polymeric matrix (assay:content test) and drug released from the systems (assay:dissolution test) is developed. This protocol is applied to the validation two isocratic HPLC analytical procedures for the analysis of dexamethasone phosphate disodium microparticles for parenteral administration. Preparation of authentic samples and artificially “spiked” and “unspiked” samples is described. Specificity (ability to quantify dexamethasone phosphate disodium in presence of constituents of the dissolution medium and other microparticle constituents), linearity, accuracy and precision are evaluated, in the range from 10 to 50 μg mL⁻¹ in the assay:content test procedure and from 0.25 to 10 μg mL⁻¹ in the assay:dissolution test procedure. The robustness of the analytical method to extract drug from microparticles is also assessed. The validation protocol developed allows us to conclude that both analytical methods are suitable for their intended purpose, but the lack of proportionality of the assay:dissolution analytical method should be taken into account.     

Speciation of Sb(III) and Sb(V) in meglumine antimoniate pharmaceutical formulations by PSA using carbon nanotube electrode

A new and simple electroanalytical method for speciation of Sb(III) and Sb(V) in pharmaceutical formulation by potentiometric stripping analysis (PSA) using a multiwall carbon nanotube paste electrode was developed. All instrumental and chemical parameters influencing the performance of the method were carefully assessed and optimized. Trivalent antimony was determined in acid medium (pH 3.6) under the optimized condition (deposition potential of −0.7 V, deposition time of 180 s, ionic strength of 0.3 M and oxidant mercury concentration of 10 mg l⁻¹). Total antimony was determined after quantitative reduction of Sb(V) with l-cysteine (1.5%, w/v) and its concentration was calculated from difference between the total antimony and Sb(III). The developed method provided two distinct linear calibration one ranging from 10 up to 50 μg l⁻¹ and other from 100 up to 800 μg l⁻¹ with respective correlation coefficient of 0.9978 and 0.9993, presenting a detection limit of 6.2 μg l⁻¹. Repeatability for the six independent samples expressed in terms of relative standard deviation was found to be 3.01 and 1.39% for 40.0 and 300.0 μg l⁻¹ antimony concentration, respectively. Results on the effect of foreign substances [Al(III), Mg(II), Fe(III), Cd(II), Zn(II) and meglumine] on analytical signal of antimony showed no interference even using high content of foreign ions in the analyte:interferent ratio up to 1:100. The proposed method was successfully applied for the speciation of Sb(III) and Sb(V) in pharmaceutical formulation and the accuracy was assessed from addition and recovery tests as well as comparing with graphite furnace atomic absorption spectrometry (GF AAS) technique used as reference analytical method.     

Can we predict community-wide effects of herbicides from toxicity tests on macrophyte species?

Macrophyte communities play an essential role in the way freshwater ecosystems function. It is thus of great concern to understand how environmental factors, especially anthropogenic ones, influence their composition and diversity. The aim of this study was to examine whether the effects of a herbicide mixture (50% atrazine, 35% isoproturon, 15% alachlor) on single macrophyte species can be used to predict its impact at a community level. In a first experiment we tested the sensitivity of six species (Azolla filiculoides, Ceratophyllum demersum, Elodea canadensis, Lemna minor, Myriophyllum spicatum and Vallisneria spiralis) grown separately and exposed to 0.6-600 μg L⁻¹ of the herbicide mixture. In a second experiment, conducted in microcosms, we tested the effects of herbicides on macrophyte assemblages composed of the same six species exposed to 0, 6 or 60 μg L⁻¹ of the herbicide mixture. Species grown separately exhibited growth inhibition at 60 and 600 μg L⁻¹. At 600 μg L⁻¹ the sensitivity differed significantly between species. V. spiralis was the most resistant species, C. demersum, M. spicatum and E. canadensis exhibited intermediate sensitivities, and A. filiculoides and L. minor were the most sensitive species. In microcosms, community biomass and Shannon evenness index were reduced after 8 weeks at 60 μg L⁻¹. Communities also exhibited changes in their composition: the relative and absolute abundance of C. demersum increased at 6 μg L⁻¹, while the relative abundance of V. spiralis increased at 60 μg L⁻¹. These results are in agreement with the individual responses of these species to the herbicides. It is therefore concluded that short-term effects of herbicides on simple macrophyte communities can be predicted from the sensitivity of individual species. However, further investigations are required to examine whether longer term effects can be predicted as well, especially in more complex communities.     

Determination of alprostadil in rat plasma by ultra performance liquid chromatography–electrospray ionization–tandem mass spectrometry after intravenous administration

A rapid, highly selective ultra performance liquid chromatography–electrospray ionisation–tandem mass spectrometry method (UPLC–ESI–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of alprostadil in rat plasma. After a simple sample preparation procedure involving a one-step liquid–liquid extraction, alprostadil and the internal standard, diphenhydramine, were chromatographed on an ACQUITY UPLC™ BEH C₁₈ column with gradient elution using a mobile phase consisting of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.25 mL min⁻¹. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear (r² = 0.99) over the concentration range 0.4–250.0 ng mL⁻¹, with a lower limit of quantification of 0.4 ng mL⁻¹ for alprostadil. The inter- and intra-day precision (%R.S.D.) was less than 8.5% and 2.4%, respectively, and the accuracy (RE%) was between 9.3% and 1.0% (n = 6). Alprostadil in rat plasma was stable when stored at room temperature for 0.5 h and at −20 °C for two weeks. The method was very rapid, simple and reliable, and was employed for the first time for the pharmacokinetic studies of alprostadil in rats after a single intravenous administration of 50 μg kg⁻¹.     

Development and validation of an ion-pair RP-HPLC method for the determination of oligopeptide-20 in cosmeceuticals

Oligopeptide-20 is a growth-factor mimicking peptide used in cosmeceuticals. This article describes the development and validation of an ion-pair reversed-phase liquid chromatography method that allows, after liquid-liquid extraction, the quantification of oligopeptide-20 in cosmetic creams. Chromatographic separation was achieved on a cyanopropyl Hypersil analytical column (100 mm × 2.1 mm i.d., 5 μm particle size), using a mobile phase of acetonitrile-heptafluorobutyric acid (pH = 2.5, 9.0 mM) (70:30, v/v) containing 0.045% diethylamine at a flow rate of 0.50 mL min⁻¹. Ultraviolet (UV) spectrophotometric detection at 225 nm was used. The method had linear calibration curve over the range 1.35-4.95 μg mL⁻¹ for oligopeptide-20. The intra- and inter-day RSD values were less than 3.3%, while the relative percentage error, %E_r, was less than 1.9. The developed method was applied successfully to the quality control of a cosmetic cream containing 0.003% (w/w) oligopeptide-20.     

Development and validation of a stability-indicating HPLC-UV method for the determination of alizapride and its degradation products

A stability-indicating high-performance liquid chromatography procedure has been developed for the determination of alizapride (AL) and its main degradation products alizapride carboxylic acid (AL-CA) and alizapride N-oxide (AL-NO2) in drug substance and product. The method was developed based on forced degradation data obtained by HPLC–MS analysis. Indeed AL underwent chemical degradation by acid/base catalyzed hydrolysis and oxidation the main degradation products being AL-CA and AL-NO2 respectively. The separation and quantisation were achieved on a 150-mm reverse phase column with a hydrophilic linkage between silica particles and hydrophobic alkyl chains. The mobile phase was constituted (flow rate 1.5 mL min⁻¹) of eluant A: aqueous acetate buffer (pH 4.0; 20 mM) and eluant B: CH₃OH using a gradient elution and detection of analytes at 225 nm. The method showed good linearity for the AL, AL-CA, AL-NO2 mixture in the 25–75, 1–15 and 1–15 μg mL⁻¹ ranges respectively, being all the square of the correlation coefficients greater than 0.999. The precision, determined in terms of intra-day and inter-day precisions and expressed as RSDs were 0.8, 1.3 and 2.1% and 1.0, 1.7, 4.8% for AL, AL-CA and AL-NO2 respectively. The method demonstrated also to be accurate; indeed the average recoveries, at 100% and 0.2% of the target assay concentration, were 100.5, 98.6, and 96.8% for AL, AL-CA and AL-NO2 respectively. The robustness was also evaluated by variations of mobile phase composition and pH. Finally, the applicability of the method was evaluated in commercial dosage form analysis as well as in stability studies.     

Determination of losartan,telmisartan, and valsartan by direct injection of human urine into a column-switching liquid chromatographic system with fluorescence detection

Column-switching high-performance liquid chromatographic (HPLC) method has been developed and validated for quantification of losartan, telmisartan, and valsartan in human urine. Urine samples were diluted on the extraction mobile phase (1:4, v/v) and a volume of 20 μL of this mixture were directly injected onto the HPLC system. The analytes were extracted from the matrix using an on-line solid-phase extraction procedure involving a precolumn packed with 25 μm C₁₈ alkyl-diol support (ADS), and a solution 2% methanol in 5 mM phosphate buffer (pH 3.8) at a flow-rate of 0.8 mL/min for isolation and preconcentration of losartan, telmisartan, and valsartan. The enriched analytes were back-flushed after, onto the analytical column with a mixture of 5 mM phosphate buffer (pH 3.8)–acetonitrile–methanol (65:20:15, v/v/v) at a flow-rate of 3.0 mL/min and detected by fluorescence at 259 and 399 nm as excitation and emission wavelength respectively. The separation of losartan, telmisartan, and valsartan was achieved on a Chromolith RP-18e monolithic column. The method provides extraction recoveries from spiked urine samples greater than 93%. Intra-day and inter-day precision were generally acceptable; the intra-day-assay C.V. was <3.5 for all compounds and the inter-day-assay C.V. was <3.7%. The estimated calibration range was 0.001–2.5 μg mL⁻¹ with excellent coefficient of determination (>0.9981). The detection limits for losartan, telmisartan, and valsartan at a signal-to-noise ratio of 5:1 were 0.002, 0.0002 and 0.001 μg mL⁻¹ when a sample volume of 20 μL was injected. The proposed method permitted the simultaneous determination of losartan, telmisartan, and valsartan in 8 min, with an adequate precision and sensitivity. However, the overlap of the sample cleanup step with the analysis increases the sampling frequency to 12 samples/h. The developed column-switching method was successfully applied for the determination of these analytes in human urine samples of patients submitted at ARA-IIs therapy.     

Tocol modified glycol chitosan for the oral delivery of poorly soluble drugs

The aim of this study was to develop tocol derivatives of chitosan able (i) to self-assemble in the gastrointestinal tract and (ii) to enhance the solubility of poorly soluble drugs. Among the derivatives synthesized, tocopherol succinate glycol chitosan (GC-TOS) conjugates spontaneously formed micelles in aqueous solution with a critical micelle concentration of 2 μg mL⁻¹. AFM and TEM analysis showed that spherical micelles were formed. The GC-TOS increased water solubility of 2 model class II drugs. GC-TOS loading efficiency was 2.4% (w/w) for ketoconazole and 0.14% (w/w) for itraconazole, respectively. GC-TOS was non-cytotoxic at concentrations up to 10 mg mL⁻¹. A 3.4-fold increase of the apparent permeation coefficient of ketoconazole across a Caco-2 cell monolayer was demonstrated. Tocol polymer conjugates may be promising vehicles for the oral delivery of poorly soluble drugs.