Search

Echinacea purpurea is a traditional medicinal plant widely used as adjuvant for the treatment of respiratory and urinary infections. Caffeic acid derivatives are considered the main active markers, such as chicoric acid, caftaric acid and chlorogenic acid. An analytical method using ultra performance liquid chromatography (UPLC) and diode array detector was developed and validated, to quantify caffeic acid derivatives in commercial dried extracts of EP. UPLC method was developed using a C18 column (50 × 2.1 mm, 1.8 μm), at 30°C. Mobile phase was composed of acetonitrile and 0.05% (v/v) formic acid aqueous solution (10:90), flow rate 0.2 mL/min. Injection volume was 10 μL and detection was performed at 300 and 330 nm. The developed method complied with all required validation parameters, and showed to be linear, precise, accurate, selective and robust for all caffeic acid derivatives. Using the validated method, the levels of caftaric acid (0.110–0.507%w/w), chicoric acid (0.040–0.179%w/w) and chlorogenic acid (0.013–0.084%w/w) were determined in five commercial dried extracts of E. purpurea, with significant variation in the contents between different samples, indicating the need of standardization and control of individual caffeic acid derivatives in commercial extracts.

The therapeutic regimen for the treatment of AIDS using efavirenz, lamivudine and tenofovir disoproxil fumarate has demonstrated efficacy and safety. In this work, an analytical method was developed and validated for simultaneous quantitation of efavirenz (EFV), lamivudine (3TC) and tenofovir disoproxil fumarate (TDF) in fixed-dose combination tablet. Analysis by high performance liquid chromatography was performed using a reversed phase column Sun Fire® C18 (250 mm x 4,6 mm; 5 μm) at 30 °C, 20 μL injection volume and detection at 260 nm. The mobile phase, composed of acetate buffer pH 5.4 and methanol, was employed at 1.0 mL/min flow rate and gradient elution. Adequate linearity was obtained in the range from 66 to 198 μg/mL for EFV, 66 to 198 μg/mL from 3TC and 33 to 99 μg/mL for TDF. Relative standard deviation values were lower than 5% and mean recoveries were within 98% to 102%, confirming the precision and accuracy of the method, respectively. In addition, the developed method showed to be selective and robust and can be successfully applied for routine quality control analysis.

Piper methysticum G. Forst, popularly known as kava, is a traditional medicinal plant widely used for the treatment of anxiety and insomnia. The aim of this study was to investigate new therapeutic applications of this plant. Nociceptive response induced by heat (hot-plate) was used as pain model. Susceptibility of different strains to kava ethanolic dried extracts was evaluated by broth microdilution method. Acute oral toxicity was performed according to Organisation for Economic Cooperation and Development (OECD) guideline. Administration of kava dried extracts and kavain inhibited the nociceptive response in the hot-plate model and did not affect the time mice spent in the rota-rod apparatus. The samples showed no significant antibacterial activity, however slight antifungal activity was verified. The extracts may be considered of low oral acute toxicity. Kava extracts exhibited promising antinociceptive activity in model of nociceptive pain, which should be deeper explored as a new therapeutic application of kava.

Introduction Dried extracts of Piper methysticum G. Forst, also known as kava, has been widely used due to its anxiolytic and sedative properties. In order to assure the quality of these extracts, it is essential to accurately quantify kavalactones, known as the active principle. Objectives To develop and validate an analytical method for the simultaneous quantification of six major kavalactones (kavain, dihydrokavain, methysticin, dihydromethysticin, yangonin and demethoxyyangonin) in kava extracts, comparing multi-standards and single standard validation approaches. Material and methods Separation was performed using a C18 column, water/methanol/acetonitrile/2-propanol (66:07:09:18 v/v/v/v) and detection at 245 and 350 nm. A full method validation was performed, employing analytical standards for each compound. Commercial kava dried extracts were assayed and the results obtained using the method validated for six kavalactone standards were compared with those obtained when only kavain was used as standard. Results Baseline resolution for all kavalactones was obtained in short run time (15 min). Although the total kavalactone content varied between samples, a similar distribution profile was observed. When the method validated with all six analytical standards was compared to the calibration using only kavain standard, kavalactone contents were considerably different (from 7.57 to 36.53%). Conclusion The obtained results demonstrate the importance of a validated method using individual kavalactone standards for the effective quality control of kava extracts. In a next step, the method needs to be adapted to also include flavokavin B (FKB), as an important authentication marker to distinguish between the accepted variety "noble Kava" and the toxic "two-day Kava".

Introduction Arbutin is a phenol glucoside found in high concentrations in bearberry leaves and associated with the antimicrobial activity of the plant. Hydroquinone can also be found in leaves or be formed by degradation of arbutin. Lengthy exposure to free hydroquinone is associated with induction of toxicity in different organs. Objective To develop and validate a stability-indicating method by high-performance liquid chromatography diode array detector (HPLC-DAD) for simultaneous quantification of arbutin and hydroquinone in bearberry leaves and perform a comprehensive forced degradation study comparing synthetic arbutin and the arbutin in bearberry leaves. Methods Separation was performed using a C18 column, mobile phase with water-methanol (95:5), flow rate 1.0 mL/min and detection at 280 nm. Bearberry leaves were assayed and a forced degradation study of arbutin was performed in different conditions. Results The method complied with all required validation parameters. Contents varied from 1.19 to 4.15% (w/w) of arbutin and from 0.022 to 0.604% (w/w) of hydroquinone. Synthetic arbutin was susceptible to acid hydrolysis and oxidative degradation, forming hydroquinone as the main degradation product. The same study using bearberry leaves showed that constituents of the plant matrix may act as antioxidants, reducing the oxidative degradation of arbutin, however acid hydrolysis of arbutin occurred in higher intensity. Conclusion Analysis of bearberry leaves evidenced high variation in arbutin and hydroquinone levels, demonstrating the need for standardisation and control. The stability profiles of synthetic arbutin and the arbutin in bearberry leaves were considerably different and the results may be useful for determining the most appropriate conditions for extraction and production of bearberry-based formulations.

One of the highest incidences of illegal drug products is related to phosphodiesterase-5 inhibitors, used in treatment of erectile dysfunction, including those containing sildenafil citrate and tadalafil. In this context, comprehensive evaluation of the quality of genuine and illegal medicines was performed. A simple and rapid ultra-high performance liquid chromatography (UHPLC-UV) method to quantify sildenafil and tadalafil in the presence of six degradation products was developed and validated. Sildenafil and tadalafil were submitted to forced degradation. The separation was carried out on a Kinetex C18 (50 × 2.1 mm; 1.7 μm) column with mobile phase composed of acetonitrile and aqueous triethylamine solution. The calibration curves were linear in the range of 14–126 μg mL−1 for sildenafil citrate and 4–36 μg mL−1 for tadalafil and the method proved to be selective, precise, accurate and robust. Sildenafil degraded in oxidative media, whereas tadalafil degraded in acidic, alkaline and oxidative environment. The chemical structures and the mechanisms for the formation of the main degradation products were proposed by UHPLC coupled to tandem mass spectrometry. The UHPLC-UV method was applied in the pharmaceutical analysis of genuine and seized medicines. Some of them did not meet quality standards, mainly due to contents below specifications and the large variation on contents between units within a batch.

Lumefantrine is the first-choice treatment of Falciparum uncomplicated malaria. Recent findings of resistance to lumefantrine has brought attention for the importance of therapeutic monitoring, since exposure to subtherapeutic doses of antimalarials after administration is a major cause of selection of resistant parasites. Therefore, this study focused on the development of innovative, selective, less expensive and stable molecularly imprinted polymers (MIPs) for solid-phase extraction (SPE) of lumefantrine from human plasma to be used in drug monitoring. Polymers were synthesized by precipitation polymerization and chemometric tools (Box-Behnken design and surface response methodology) were employed for rational optimization of synthetic parameters. Optimum conditions were achieved with 2-vinylpyridine as monomer, ethylene glycol dimethacrylate as crosslinker and toluene as porogen, at molar ratio of 1:6:30 of template/monomer/crosslinker and azo-bisisobutyronitrile as initiator at 65 °C. The MIP obtained was characterized and exhibited high thermal stability, adequate surface morphology and porosity characteristics and high binding properties, with high affinity (adsorption capacity of 977.83 μg g−1) and selectivity (imprinting factor of 2.44; and selectivity factor of 1.48 and selectivity constant of 1.44 compared with halofantrine). Doehlert matrix and fractional designs were satisfactorily used for development and optimization of a MISPE-HPLC-UV method for determination of lumefantrine. The method fulfilled all validation parameters, with recoveries ranging from 83.68% to 85.42%, and was applied for quantitation of the drug in plasma from two healthy volunteers, with results of 1407.89 and 1271.35 ng mL−1, respectively. Therefore, the MISPE-HPLC-UV method optimized through chemometrics provided a rapid, highly selective, less expensive and reproducible approach for lumefantrine drug monitoring.

Malaria is the most incident parasite infection worldwide. Artemisinin based combination therapy (ACT) has been proposed as a promising treatment for malaria, and artemether + lumefantrine (20 + 120 mg) is the recommended association in endemic areas. Despite its widespread use, there is still scarce information about dissolution of artemether and lumefantrine, reflecting in the absence of a specific method in pharmacopoeias and international compendia. Because the of their low solubility, both artemether and lumefantrine are candidates for in vitro-in vivo correlation (IVIVC) studies. Previous equilibrium solubility studies have been carried out for both drugs using the shake-flask method and dissolution profiles. Experiments were conducted with a range of parameters such as medium composition, pH and surfactants. In vivo data obtained in a previous pharmacokinetic study was used to select the optimum conditions for dissolution test, based on IVIVC. For drug quantitation, a selective method by high performance liquid chromatography was optimized and validated. For this dosage form, the best dissolution conditions found for artemether were: paddles, 900 mL of dissolution medium containing phosphate buffer pH 6.8 with 1.0% sodium lauryl sulfate and rotation speed of 100 rpm. The same was obtained for lumefantrine, except the dissolution medium, which was pH 1.2 with 1.0% polysorbate 80. After obtaining the curve of in vitro dissolved fraction versus in vivo absorbed fraction, the calculated coefficient of determination (R squared) was close to 1.00 for both drugs, indicating a level A correlation. Therefore, a novel method for assessing dissolution of arthemeter and lumefantrine tablets was established and validated.

Etoposide-loaded poly(lactic-co-glycolic acid) implants were developed for intravitreal application. Implants were prepared by a solvent-casting method and characterized in terms of content uniformity, morphology, drug-polymer interaction, stability, and sterility. In vitro drug release was investigated and the implant degradation was monitored by the percent of mass loss. Implants were inserted into the vitreous cavity of rabbits' eye and the in vivo etoposide release profile was determined. Clinical examination and the Hen Egg Test-Chorioallantoic Membrane (HET-CAM) method were performed to evaluate the implant tolerance. The original chemical structure of the etoposide was preserved after incorporation in the polymeric matrix, which the drug was dispersed uniformly. In vitro, implants promoted sustained release of the drug and approximately 57% of the etoposide was released in 50 days. In vivo, devices released approximately 63% of the loaded drug in 42 days. Ophthalmic examination and HET-CAM assay revealed no evidence of toxic effects of implants. These results tend to show that etoposide-loaded implants could be potentially useful as an intraocular etoposide delivery system in the future.

A simple and fast bioanalytical method for the quantification of kavain in mice plasma was developed using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). A full method validation was performed, according to regulatory guidelines, employing isotopically labeled kavain as the internal standard (racemic-kavain-d3). For the quantification, [M+H]+ was formed using an electrospray ionization (ESI) source in the positive ion mode and multiple reaction monitoring (MRM) was employed using a quadrupole-linear ion trap (4000 QTRAP®) instrument. The monitored MRM transitions were 231.0 → 115.1 and 231.0 → 152.8 for kavain; and 234.2 → 199.2 for the internal standard. A linear response was obtained at the concentration range of 10 to 200 ng/mL with intra- and inter-day variations within the acceptable criteria for all quality control samples. After validation, the method was successfully applied for the quantification of kavain in mice plasma after oral administration of the kavain standard and Kava-kava extract. The plasma concentration over time results were applied for a pharmacokinetics study. The obtained pharmacokinetic parameters indicated a considerably higher bioavailability for kavain when Kava-kava extract was administered due to a pharmacokinetic synergism between the analyte and the other compounds present in the extract.

A stability indicating HPLC method to determine diltiazem hydrochloride (DTZ) in tablets and compounded capsules was developed and validated according to Brazilian and the International Conference on Harmonization (ICH) guidelines. The separation was carried out on a Purospher Star® C18 (150 x 4.6 mm i.d., 5 µm particle size, Merck Millipore) analytical column. The mobile phase consisted of a 0.05% (v/v) trifluoroacetic acid aqueous solution and a 0.05% trifluoroacetic acid methanolic solution (44:56, v/v). The flow rate was 1.0 mL.min-1 with a run time of 14 minutes. The detection of DTZ and degradation products (DP) was performed at 240 nm, using a diode array detector. The method proved to be linear, precise, accurate, selective, and robust, and was adequate for stability studies and routine quality control analyses of DTZ in tablets and compounded capsules. Um método de HPLC indicador de estabilidade para determinação de cloridrato de diltiazem (DTZ) em comprimidos e cápsulas manipuladas foi desenvolvido e validado de acordo com as diretrizes brasileiras e da Conferência Internacional de Harmonização (ICH). A separação foi realizada em uma coluna analítica Purospher Star® C18 (150 x 4,6 mm i.d., tamanho de partícula de 5 µm, Merck Millipore). A fase móvel consistiu de uma solução aquosa de ácido trifluoroacético a 0,05% (v/v) e uma solução metanólica de ácido trifluoroacético a 0,05% (44:56, v/v). A vazão foi de 1,0 mL.min-1 com tempo de corrida de 14 minutos. A detecção de DTZ e produtos de degradação (DP) foi realizada em 240 nm, utilizando um detector de arranjo de diodos. O método mostrou-se linear, preciso, exato, seletivo e robusto, sendo adequado para estudos de estabilidade e análises de rotina de controle de qualidade de DTZ em comprimidos e cápsulas manipuladas.

Nicorandil (N-(2-hydroxyethyl)nicotinamide nitrate) is an antianginal drug, which activates guanylyl cyclase and opens the ATP-dependent K(+) channels, actions that have been suggested to mediate its vasodilator activity. We synthesized nicorandil and its two isomers, which vary in the positions of the side chain containing the nitric oxide (NO) donor, and also their corresponding denitrated metabolites. The activities of these compounds were evaluated in an experimental model of pain in mice. Pharmacokinetic parameters of nicorandil and its isomers, as well as the plasma concentrations of the corresponding denitrated metabolites and also nicotinamide and nitrite were determined. Nicorandil exhibited the highest antinociceptive activity, while the ortho-isomer was the least active. Nicorandil and para-nicorandil, which induced higher plasma concentrations of nitrite, exhibited higher antinociceptive activity, which suggests that the release of NO may mediate this activity.

An improved LC-MS/MS method for the quantitation of indapamide in human whole blood was developed and validated. Indapamide-d3 was used as internal standard (IS) and liquid–liquid extraction was employed for sample preparation. LC separation was performed on Synergi Polar RP-column (50 × 4.6 mm i.d.; 4 µm) and mobile phase composed of methanol and 5 mm aqueous ammonium acetate containing 1 mm formic acid (60:40), at flow rate of 1 mL/min. The run time was 3.0 min and the injection volume was 20 μL. Mass spectrometric detection was performed using electrospray ion source in negative ionization mode, using the transitions m/z 364.0 m/z 188.9 and m/z 367.0 m/z 188.9 for indapamide and IS, respectively. Calibration curve was constructed over the range 0.25–50 ng/mL. The method was precise and accurate, and provided recovery rates >80% for indapamide and IS. The method was applied to determine blood concentrations of indapamide in a bioequivalence study with two sustained release tablet formulations. The 90% confidence interval for the geometric mean ratios for maximum concentration was 95.78% and for the area under the concentration–time curve it was 97.91%. The tested indapamide tablets (Eurofarma Laboratorios S.A.) were bioequivalent to Natrilix®, according to the rate and extent of absorption. Copyright © 2014 John Wiley & Sons, Ltd.

The present study developed and validated an HPLC method for the simultaneous determination of artesunate (AS) and mefloquine hydrochloride (MQ) in fixed-dose combination tablets, according to ICH guidelines. The chromatographic separation was carried out on an XBridge C18 (250 x 4.6 mm i.d., 5 µm particle size, Waters) analytical column. The mobile phase included a 0.05 M monobasic potassium phosphate buffer (pH adjusted to 3.0 with phosphoric acid) and acetonitrile (50 + 50, v/v). The flow rate was 1.0 mL/min, and the run time was 13 minutes. A dual-wavelength approach was employed: AS detection was performed at 210 nm and MQ was detected at 283 nm, using a diode array detector. Stability of sample solutions was evaluated for 8 hours after preparation, during which time the solutions remained stable. Youden's test was employed to evaluate robustness. The method proved to be linear (r²>0.99), precise (RSD0,99), preciso (DPR

A new method was developed for the quantitation of 3-α-hydroxy tibolone, in human plasma, after oral administration of a tablet formulation containing tibolone (2.5 mg). 3-α-Hydroxy tibolone was extracted by a liquid–liquid procedure, using cyproterone acetate as internal standard and chlorobutane as extraction solvent. After extraction, samples were submitted to a derivatization step with p-toluenesulfonyl isocyanate. A mobile phase consisting of acetonitrile and water (72:28 v/v) was used and chromatographic separation was achieved using Agilent XDB C18 column (100 × 4.6 mm i.d.; 5 µm particle size), at 40°C. Mass spectrometric detection was performed using atmospheric pressure chemical ionization in negative mode for 3-α-hydroxy tibolone and in positive mode for cyproterone acetate. The fragmentation transitions were m/z 510.2 m/z 170.1 and m/z 417.0 m/z 357.1 for 3-α-hydroxy tibolone and cyproterone acetate, respectively. Calibration curves were constructed over the range 100–30,000 pg/mL and the method was shown to be specific, precise and accurate, with a mean recovery rate of 94.2% for 3-α-hydroxy tibolone. No matrix effect or carry-over was detected in the samples. The validated method was applied in a pharmacokinetic study with a tibolone formulation in healthy female volunteers. Copyright © 2013 John Wiley & Sons, Ltd.

Nicorandil (2-nicotinamide ethyl nitrate), an antianginal drug characterized by the coupling of nicotinamide with a nitric oxide (NO) donor, activates guanylyl cyclase and opens ATP-dependent K(+) channels. In the present study, we investigated the effects induced by per os (p.o.) administration of nicorandil (12.5, 25 or 50 mg/kg) or equimolar doses (corresponding to the highest dose of nicorandil) of N-(2-hydroxyethyl) nicotinamide (NHN), its main metabolite, or nicotinamide in the model of nociceptive response induced by formaldehyde in mice. Nicorandil, but not NHN or nicotinamide, inhibited the second phase of the nociceptive response. This activity was observed when nicorandil was administered between 30 and 120 min before the injection of formaldehyde. Ipsilateral intraplantar injection of nicorandil (125, 250 or 500 μg/paw) did not inhibit the nociceptive response. After p.o. administration of nicorandil (50 mg/kg), peak plasma concentrations of this compound and NHN were observed 0.63 and 4 h later, respectively. Nicotinamide concentrations were not increased after administration of nicorandil. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 or 2 mg/kg), a guanylyl cyclase inhibitor, partially attenuated the antinociceptive activity of nicorandil. However, this activity was not changed by glibenclamide (30 or 60 mg/kg), an inhibitor of ATP-dependent K(+) channels. In conclusion, we demonstrated the antinociceptive activity of nicorandil in a model of pain that exhibits both a nociceptive and an inflammatory profile. This activity is not mediated by nicotinamide or NHN. The coupling of an NO-donor to nicotinamide results in a compound with an increased potency. The NO-cGMP pathway, but not ATP-dependent K(+) channels, partially mediates the antinociceptive activity of nicorandil.

Cryptococcus gattii is the main pathogen of cryptococcosis in healthy patients and is treated mainly with fluconazole and amphotericin B. The combination of these drugs has been questioned because the mechanisms of action could lead to a theoretical antagonistic interaction. We evaluated distinct parameters involved in the in vitro combination of fluconazole and amphotericin B against Cryptococcus gattii . Fourteen strains of C. gattii were used for the determination of MIC, fractional inhibitory concentration, time-kill curve, and postantifungal effect (PAFE). Ergosterol quantification was performed to evaluate the influence of ergosterol content on the interaction between these antifungals. Interaction between the drugs varied from synergistic to antagonistic depending on the strain and concentration tested. Increasing fluconazole levels were correlated with an antagonistic interaction. A total of 48 h was necessary for reducing the fungal viability in the presence of fluconazole, while 12 h were required for amphotericin B. When these antifungals were tested in combination, fluconazole impaired the amphotericin B activity. The ergosterol content decreased with the increase of fluconazole levels and it was correlated with the lower activity of amphotericin B. The PAFE found varied from 1 to 4 h for fluconazole and from 1 to 3 h for amphotericin B. The interaction of fluconazole and amphotericin B was concentration-dependent and special attention should be directed when these drugs are used in combination against C. gattii .

BACKGROUND A liquid chromatography-tandem mass spectrometry method for the simultaneous quantitation of endogenous uracil (U) and dihydrouracil (UH2) was developed and tested in a Brazilian population of patients with gastrointestinal cancer previously exposed to 5-fluorouracil (5FU). METHODS The analytes were extracted by a liquid-liquid method using 5-clorouracil as internal standard. The separation was performed on a reversed-phase XTerra C18 column with a mobile phase composed of methanol and aqueous 0.1% ammonium hydroxide (15:85). Mass spectrometry detection was carried out using negative electrospray ionization and selected reaction monitoring. Bovine serum albumin was employed as an alternative matrix to prepare the calibration standards, aiming to avoid the measurement of physiologic U and UH2. Calibration curves were constructed over the range of 5-200 ng/mL for U and 10-500 ng/mL for UH2. RESULTS The mean RSD values in the intrarun precision were 6.5% and 10.0% and in the interrun precision were 7.8% and 9.0% for U and UH2, respectively. The mean accuracy values were within the range of 90%-110% for both analytes. The analytes were stable in plasma under different conditions of temperature and time. The validated method was successfully applied to determine the plasma concentrations of U and UH2 in patients with gastrointestinal cancer (n = 32) previously treated with 5FU and for whom clinical toxicity was well documented. U concentrations varied from 21.8 to 56.6 ng/mL, whereas UH2 concentrations varied from 57.7 to 271.5 ng/mL. UH2/U ratio ranged from 1.56 to 6.18. CONCLUSIONS The method has proved to provide a quick, reliable, and reproducible quantitation of the plasma concentrations of U and its metabolite UH2. The UH2/U ratios did not discriminate patients previously exposed to 5FU with and without severe toxicities, possibly due to the small sample. Further studies in a larger population are desirable.

Association of amlodipine besylate and olmesartan medoxomil in fixed-dose combination tablets is effective, safe and well tolerated for the treatment of hypertension. The aim of this study was to optimize and validate a novel and fast UHPLC-DAD method for simultaneous quantification of these antihypertensive drugs in tablets, using a transfer procedure from a conventional HPLC-DAD method. The HPLC separation was carried out using a C18 column (150 × 4.6 mm2; 5 μm) and a mobile phase composed of acetonitrile, methanol and 0.3% trimethylamine pH 2.75 (30:30:40), at 1.0 mL/min. UV detection was performed at 238 nm and injection volume was 10 μL. Then, the analytical method was transferred to UHPLC, using a BEH C18 column (50 × 2.1 mm2; 1.7 μm). Mathematical equations were applied to calculate the UHPLC mobile phase flow rate and injection volume, which were 0.613 mL/min and 0.7 μL, respectively. UHPLC method was fully validated and showed to be selective, linear (r2 > 0.99), precise (RSD < 2.0%), accurate and robust. UHPLC method was statistically equivalent to the HPLC method after analysis of three batches of BenicarAnlo® tablets. However, UHPLC method promoted faster analyses, better chromatographic performance and lower solvent consumption.

Tuberculosis treatment consists of a fixed dose combination of rifampicin (RIF), isoniazid (INH), pyrazinamide (PYZ), and ethambutol hydrochloride (EMB). The combined treatment using various drugs is necessary for patient curing, without recrudescence, and for prevention of drug-resistant mutants, which may occur during treatment. An HPLC-diode array detector (DAD) method for the simultaneous determination of RIF, INH, PYZ, and EMB in fixed dose combination tablets was developed and validated. Chromatographic experiments were performed on an Agilent 1200 HPLC system, and the separation was carried out on a Purospher STAR RP18e (250 × 4.6 mm id, 5 μm, Merck) analytical column. Gradient elution was carried out with a mobile phase of 20 mM monobasic sodium phosphate buffer with 0.2% triethylamine (pH 7.0) and acetonitrile at a flow rate of 1.5 mL/min. The total run time was 12 min, and the re-equilibration time was 5 min. EMB detection was performed at 210 nm, and RIF, INH, and PYZ were detected at 238 nm, using a DAD. The method proved to be specific, linear (r2 > 0.99), precise (RSD

A liquid chromatography-electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous quantitation of nicorandil and its denitrated metabolite, N-(2-hydroxyethyl)-nicotinamide, in rat plasma. After a liquid-liquid extraction step, chromatographic separation was performed on a ShinPack C(18) column with an isocratic mobile phase composed of methanol and 2 mM aqueous ammonium acetate containing 0.03% (v/v) formic acid (33:67 v/v). Procainamide was used as an internal standard (IS). Selected reaction monitoring was performed using the transitions m/z 212 → m/z 135, m/z 166 → m/z 106 and m/z 236 → m/z 163 to quantify nicorandil, its denitrated metabolite and IS, respectively. Calibration curves were constructed over the range of 5-15,000 ng.ml(-1) for both nicorandil and its metabolite. The mean relative standard deviation (RSD%) values for the intra-run precision were 5.4% and 7.3% and for the inter-run precision were 8.5% and 7.3% for nicorandil and its metabolite, respectively. The mean accuracy values were 100% and 95% for nicorandil and its metabolite, respectively. No matrix effect was detected in the samples. The validated method was successfully applied to a pharmacokinetic study after per os administration of nicorandil in rats.

A rapid method for the quantification of glucosamine in human plasma using high-performance liquid chromatography coupled to tandem mass spectrometry was developed and validated. The sample preparation includes a simple deproteinization step, using D-(1- 13 C) glucosamine hydrochloride as an internal standard. Chromatographic separation was performed on an ACE Ciano column using isocratic elution with acetonitrile and aqueous 2mM ammonium acetate containing 0.025% formic acid (80:20). Selected reaction monitoring was performed using the transitions m/z 180.1 !m/z 72.1 and m/z 181.0 !m/z 74.6 to quantify glucosamine and internal standard, respectively. The method was validated and proved to be linear, accurate and precise over the range 50-5000ng/mL of glucosamine. Recovery rates higher than 90% were obtained for both glucosamine and internal standard. No matrix effect was detected in the samples. The validated method was successfully applied to a pharmacokinetic study after oral administration of a powder for oral solution formulation containing glucosamine sulfate. Copyright © 2011 John Wiley & Sons, Ltd.

A sensitive and fast high-performance liquid chromatography-electrospray ionization-MS/MS method for the simultaneous quantitation of levodopa and carbidopa in human plasma was developed and validated. A simple protein precipitation step with perchloric acid was used for the cleanup of plasma, and methyldopa was added as an internal standard. The analyses were carried out using an ACE C(18) column (50 × 4.6 mm i.d.; 5 µm particle size) and a mobile phase consisting of 0.2% formic acid and acetonitrile (90:10). The triple-quadrupole mass spectrometer equipped with an electrospray source in positive mode was set up in the selective reaction monitoring mode to detect the ion transitions m/z 198.1 → m/z 107.0, m/z 227.2 → m/z 181.0, and m/z 212.1 → m/z 139.2 for levodopa, carbidopa, and methyldopa, respectively. The method was validated and proved to be linear, accurate, and precise over the range 50-5000 ng/mL for levodopa and 3-600 ng/mL for carbidopa. The proposed method was successfully applied in a pharmacokinetic study with a levodopa/carbidopa tablet formulation in healthy volunteers.

Youden's test is a reliable method to evaluate the robustness of analytical methods, by means of an experiment design which involves seven analytical parameters combined in eight tests. In the present study, we assessed the robustness of a chromatographic method to quantify lumefantrine in raw material samples, using Youden's test. Hence, it was possible to determine the effect of each analytical parameter in the final analysis results. Youden's test showed to be a simple and feasible procedure to evaluate the robustness of chromatographic methods.O teste de Youden constitui um método confiável para avaliação da robustez de métodos analíticos, por meio de um delineamento que envolve sete parâmetros analíticos combinados em oito experimentos. No presente estudo, a robustez de um método cromatográfico para quantificação de lumefantrina em matéria-prima farmacêutica foi avaliada utilizando-se o teste de Youden. Assim, foi possível determinar o efeito de cada parâmetro analítico nos resultados finais das análises. O teste de Youden se mostrou um procedimento simples e confiável para a avaliação da robustez de métodos cromatográficos.

This paper describes the development and validation of a HPLC-UV method (210 nm) for the simultaneous quantitation of artemether and lumefantrine in fixed dose combination tablets. The method showed to be linear (r(2)>0.99), precise (R.S.D.

This paper describes the development and validation of an UV-Visible spectrophotometric method for quantitation of genistein and genistin in soy dry extracts, after reaction with aluminum chloride. The method showed to be linear (r²= 0.9999), precise (R.S.D. < 2%), accurate (recovery of 101.56%) and robust. Seven samples of soy dry extracts were analyzed by the spectrophotometric validated method and by RP-HPLC. Genistein concentrations determined by spectrophotometry (0.63% - 16.05%) were slightly higher than values obtained by HPLC analysis (0.40% - 12.79%); however, the results of both methods showed a strong correlation.

Isoflavones are widely used as an alternative treatment to hormone replacement therapy and also for prevention of several chronic diseases, including cancers. Genistein, daidzein and glycitein are the most abundant isoflavone aglycones found in soy extracts, where they also occur as glycosides. This paper describes the development and validation of an isocratic reversed-phase HPLC (RP-HPLC) method for the analysis of isoflavone aglycones, released after acid hydrolysis of soy dry extracts, used as pharmaceutical raw material. The quantification was carried out in a C(18) endcapped column, using a mobile phase composed of 0.1% acetic acid and methanol (52:48), at a flow rate of 1.0 ml/min and diode array detection (DAD) at 254 nm. The method showed to be linear (r(2)>0.99), precise (R.S.D.

Two high performance liquid chromatography (HPLC) methods for the quantitative determination of indinavir sulfate were tested, validated and statistically compared. Assays were carried out using as mobile phases mixtures of dibutylammonium phosphate buffer pH 6.5 and acetonitrile (55:45) at 1 mL/min or citrate buffer pH 5 and acetonitrile (60:40) at 1 mL/min, an octylsilane column (RP-8) and a UV spectrophotometric detector at 260 nm. Both methods showed good sensitivity, linearity, precision and accuracy. The statistical analysis using the t-student test for the determination of indinavir sulfate raw material and capsules indicated no statistically significant difference between the two methods.

Poly(e-caprolactone) implants containing etoposide, an important chemotherapeutic agent and topoisomerase II inhibitor, were fabricated by a melt method and characterized in terms of content uniformity, morphology, drug physical state, and sterility. In vitro and in vivo drug release from the implants was also evaluated. The cytotoxic activity of implants against HeLa cells was studied. The short-term tolerance of the implants was investigated after subcutaneous implantation in mice. The original chemical structure of etoposide was preserved after incorporation into the polymeric matrix, in which the drug was dispersed uniformly. Etoposide was present in crystalline form in the polymeric implant. In vitro release study showed prolonged and controlled release of etoposide, which showed cytotoxicity activity against HeLa cells. After implantation, good correlation between in vitro and in vivo drug release was found. The implants demonstrated good short-term tolerance in mice. These results tend to show that etoposide-loaded implants could be potentially applied as a local etoposide delivery system.

Ofloxacin, second-generation fluoroquinolone derivative, is one of the most commonly used to treat and prevent superficial ocular infection in animals and human beings. However, poor bioavailability, rapid elimination, and non compliance by patients are several problems associated with ocular route. Ophthalmic controlled drug delivery offers the potential to enhance the efficacy of treatment for pathological conditions, while reducing the side effects and the toxicity associated with frequent applications. Specific analytical methods to determine drugs in eye are needed to analyze and compare the new controlled release ocular devices with those conventional eye drops. The topical eye administration of ophthalmic drugs induces lachrymation, and the tear promotes a drug wash out. Quantify drugs in tear is a good tool to study their kinetic comportment in the eye. A liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantitation of ofloxacin in rabbits' tears was developed and validated. The tear was collected with tear strips, extracted by a liquid extraction procedure and then separated on an ACE C(18) column with a mobile phase composed of 0.15% aqueous formic acid and methanol (60:40, v/v). Calibration curve was constructed over the range of 10-5000 ng/mL for ofloxacin. The mean R.S.D. values for the intra-run and inter-run precision were 5.15% and 4.35%, respectively. The mean accuracy value was 100.16%. The validated method was successfully applied to determine the ofloxacin concentration in tears of rabbits treated with a mucoadhesive chitosan films and a conventional eye drop formulation.

Electrospray ionization in the positive ion mode and high resolution and high accuracy tandem mass spectrometry performed in a hybrid quadrupole time-of-flight mass spectrometer were used to investigate the dissociation chemistry of the intact molecules of two most widely used antimalarial drugs: artemether and lumefantrine. The dissociation pathways of their cationized and protonated forms were rationalized based on high accuracy mass measurements and isotopic distributions. The obtained results should benefit LC-MS/MS monitoring and quantitation by mass spectrometry of the artemether and lumefantrine molecules, as well as of new derivatives or other structurally related antimalarial drugs. Ionização por electrospray no modo de íons positivos, e espectrometria de massas de alta resolução e exatidão, e em tandem, realizadas em espectrômetro de massas híbrido quadrupolo e tempo de vôo, foram utilizadas para investigar a dissociação química das moléculas intactas dos dois antimaláricos mais amplamente utilizados: artemeter e lumefantrina. As rotas de dissociação das formas cationizadas e protonadas foram claramente estabelecidas via determinações de massas de alta resolução e distribuição isotópica. Os resultados obtidos podem auxiliar a monitorização e a quantificação de artemeter e lumefantrina por LC-MS/MS, assim como de novos derivados ou outros fármacos antimaláricos estruturalmente relacionados.

A sensitive method for the determination of fluticasone in plasma was developed using high performance liquid chromatography with tandem mass spectrometric detection, whereas beclomethasone was used as internal standard. The analytes were extracted with a simple liquid-liquid extraction from the plasma samples and separated on an ACE C(18) 50 × 4.6 mm i.d.; 5 μm particle size column with a mobile phase consisting of acetonitrile - 0.01% formic acid (48:52, v/v) at a flow rate of 1 ml/min. Detection was achieved by an Applied Biosystems API 5000 mass spectrometer (LC-MS/MS) set at unit resolution in the multiple reaction monitoring mode. Atmospheric pressure chemical ionization (APCI) was used for ion production. The mean recovery for fluticasone propionate was 85%, with a lower limit of quantification set at 2 pg/mL. The validated analytical method was applied to a bioequivalence study of fluticasone propionate administered by nasal spray formulations in human volunteers.

A sensitive and simple method was developed for the quantitation of levodopa and its metabolite 3-O-methyldopa, in human plasma, after oral administration of tablet formulations containing levodopa (200 mg) and benserazide (50 mg). The analytes were extracted by a protein precipitation procedure, using carbidopa as an internal standard. A mobile phase consisting of 0.2% formic acid and acetonitrile (94:6, v/v) was used and chromatographic separation was achieved using ACE C(18) column (50 mm×4.6 mm i.d.; 5 μm particle size). Selected reaction monitoring was performed using the fragmentation transitions m/z 198→m/z 107, m/z 212→m/z 166 and m/z 227→m/z 181 for levodopa, 3-O-methyldopa and carbidopa, respectively. Calibration curves were constructed over the range 50.0-6000.0 ng/mL for levodopa and 25.0-4000.0 ng/mL for 3-O-methyldopa. The method shown to be specific, precise, accurate and provided recovery rates higher than 85% for all analytes. No matrix effect was detected in the samples. The validated method was applied in a pharmacokinetic study with a levodopa/benserazide tablet formulation in healthy volunteers.

Biodegradable polyurethane was synthesized by preparing aqueous polyurethane dispersion having poly(caprolactone) and poly(ethylene glycol) as soft segments. Montmorillonite particles were delaminated within the waterborne polyurethane to produce a nanocomposite. The triamcinolone acetonide (TA), an important corticoid drug, was dispersed into the nanocomposite followed by a drying step to produce an implantable drug delivery system. Infrared (FTIR) results demonstrated that the original chemical structure of the TA was preserved after incorporation into the nanocomposite. Wide angle (WAXS) and small angle X-ray scattering (SAXS) results suggested that TA and clay do not dramatically change the morphology phase of the polymer although they can interact with each other. The presence of montmorillonite particles in the nanocomposite reduced the rate of TA release as compared to the pure polyurethane and enhanced the mechanical properties of the polymer. The overall results indicate that montmorillonite clay-based polyurethane nanocomposite could be potentially applied as local TA delivery system.

Indirect spectrophotometric detection was investigated for application in the anion-exchange chromatography of alkylphosphonic acids and fosfomycin. Four chromophoric eluent anions were studied: phthalate, bezoate, phenylphosphonate and p -toluenesulphonate. Phthalate showed the best displacing power and the larger dynamic reserve whereas benzoate exhibited the highest absorptivity and transfer ratio. The optimal experimental conditions were found to be 0.4 mM phthalate (pH 8.5) as mobile phase, a flow-rate of 1 ml min −1 and a detection wavelength of 272 nm. Selectivity (between 1.02 and 1.85, depending on the pair of vicinal compounds), linearity over a 5–100 μg ml −1 concentration range ( r = 0.999), precision (R.S.D. 0.6–5.3%) and sensitivity (0.4–1.0 μg, ml −1 according to the phosphonic acid) were determined. The minimum detectable concentrations were improved by an average factor of 50 in comparison with direct non-suppressed conductimetric detection. The application of the technique to the determination of fosfomycin in plasma samples is described.

Capillary zone electrophoresis with indirect UV detection was used for the determination of a series of alkylphosphonic acids. For this purpose, a few UV-absorbing background electrolytes were tested and phenylphosphonic acid, which has a mobility close to that of the analysed compounds, was shown to be the most suitable. The influence of several parameters such as concentration of the UV-absorbing background electrolyte and concentration of borate on both sensitivity and efficiency was investigated. An increase in the borate concentration produced an improvement of the signal-to-noise ratio. Conversely, the sensitivity decreased with increasing concentration of the phenylphosphonic acid. The reproducibility of the method was very satisfactory and limits of detection were less than 0.21 pmol injected.

A liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) method for the simultaneous quantitation of artemether and lumefantrine in human plasma was developed and validated. Artesunate was used as an internal standard (IS). The analytes were extracted by a protein precipitation procedure and separated on a reversed-phase Zorbax SB-Ciano column with a mobile phase composed of methanol and 10 mM aqueous ammonium acetate containing 0.2% (v/v) acetic acid and 0.1% (v/v) formic acid. Multiple reaction monitoring was performed using the transitions m/z 316 → m/z 267, m/z 530 → m/z 348 and m/z 402 → m/z 267 to quantify artemether, lumefantrine and artesunate, respectively. Calibration curves were constructed over the range of 10–1000 ng/mL for artemether and 10–18,000 ng/mL for lumefantrine. The lower limit of quantitation was 10 ng/mL for both drugs. The mean R.S.D. values for the intra-run precision were 2.6% and 3.0% and for the inter-run precision were 3.6% and 4.6% for artemether and lumefantrine, respectively. The mean accuracy values were 102.0% and 101.2% for artemether and lumefantrine, respectively. No matrix effect was detected in the samples. The validated method was successfully applied to determine the plasma concentrations of artemether and lumefantrine in healthy volunteers, in a one-dose pharmacokinetic study, over the course of 11 days.

The quantitation of artemether in both pharmaceutical raw material and injections was carried out by high performance liquid chromatography (HPLC) with ultraviolet detection. A Zorbax C18 column (150 x 4.6 mm; 5 μm), at 30 °C, and a mobile phase composed of acetonitrile and water (70:30), at a flow rate of 1ml/min, were used. The detection wavelength was 216 nm and the injection volume was 20 μL. The method proved to be linear (r²=0.9999), precise (RSD < 20% for intra-day and inter-day precision), accurate and selective regarding possible impurities and excipients of the samples. The detection and quantitation limits were 8 μg/mL and 25 μg/mL, respectively. The artemether content obtained in the raw material analysis was 99.26% and in the injections, 102.08%. The optimized and validated method may be successfully employed to perform routine quality control analyses.A quantificação de artemeter em matéria-prima farmacêutica e solução injetável foi realizada por cromatografia líquida de alta eficiência (CLAE) com detecção na região do ultravioleta. Empregou-se coluna Zorbax C18 (150 x 4.6 mm; 5 μm), mantida a 30 °C, e fase móvel composta por acetonitrila e água (70:30), com fluxo de 1 ml/min. A detecção foi realizada a 216 nm, e o volume de injeção foi 20 μl. O método se mostrou linear (r²=0,9999), preciso (DPR < 2,0% para precisão intra-dia e inter-dias) e seletivo em relação a possíveis impurezas e excipientes das amostras. Os limites de detecção e quantificação obtidos foram 8 μg/mL e 25 μg/mL, respectivamente. O teor médio de artemeter obtido na análise da matéria-prima farmacêutica foi 99,26% e na solução injetável, 102,08%. O método otimizado e validado pode ser utilizado com sucesso para análises rotineiras em controle de qualidade.

This paper describes the development and evaluation of a HPLC, UV spectrophotometry and potentiometric titration methods to quantify lumefantrine in raw materials and tablets. HPLC analyses were carried out using a Symmetry C 18 column and a mobile phase composed of methanol and 0.05% trifluoroacetic acid (80:20), with a flow rate of 1.0 ml/min and UV detection at 335 nm. For the spectrophotometric analyses, methanol was used as solvent and the wavelength of 335 nm was selected for the detection. Non-aqueous titration of lumefantrine was carried out using perchloric acid as titrant and glacial acetic acid/acetic anhydride as solvent. The end point was potentiometrically determined. The three evaluated methods showed to be adequate to quantify lumefantrine in raw materials, while HPLC and UV methods presented the most reliable results for the analyses of tablets.

A HPLC method was developed to quantify thymine and thymidine impurities in stavudine bulk drug. The separation was carried out in isocratic mode using methanol/water (20:80) as mobile phase, a C18 column and UV detection at 266 nm. The method provided selectivity based on peak purities and resolution among peaks. It was linear over the range of 0.5-5.0 µg/mL. The quantitation limits were 0.021 µg/mL for thymine and 0.134 µg/mL for thymidine. The average accuracies of three concentrations ranged from 97.06 to 102.61% and precision was close to 1%. The method showed robustness, remaining unaffected by deliberate variations in relevant parameters.

A simple procedure for obtaining and purifying two degradation products of efavirenz (amino alcohol and quinoline derivatives) from drug substance hydrolysis is described. These impurities are known to exhibit very different UV absorbance properties from those of the parent compound, making determination using a quantitation factor (QF) inaccurate. The obtained hydrolysis products were characterized by physicochemical methods to assure identity, purity and strength. Quinoline derivative was of high purity degree (100%) and amino alcohol was 98.74% pure. Both were set as reference standards in chromatographic related compounds test for efavirenz drug substance and tablets analyses.

A simple and rapid precipitation titration method was developed and validated to determine sulfate ion content in indinavir sulfate raw material. 0.1 mol L-1 lead nitrate volumetric solution was used as titrant employing potentiometric endpoint determination using a lead-specific electrode. The United States Pharmacopoeia Forum indicates a potentiometric method for sulfate ion quantitation using 0.1 mol L-1 lead perchlorate as titrant. Both methods were validated concerning linearity, precision and accuracy, yielding good results. The sulfate ion content found by the two validated methods was compared by the statistical t-student test, indicating that there was no statistically significant difference between the methods.

An optimization, statistically based on t-student test, to set up dissolution test conditions for indinavir sulfate capsules is presented. Three dissolution media, including that reported in United States Pharmacopeial Forum, and two apparatus, paddle and basket, were applied. Two different indinavir sulfate capsules, products A and B, were evaluated. For a reliable statistical analysis eighteen capsules were assayed in each condition based on the combination of dissolution medium and apparatus. All tested media were statistically equivalent (P0.05) for both drug products when paddle apparatus was employed at the stirring speed of 50 rpm. The use of basket apparatus at the stirring speed of 50 rpm caused significant decrease in the drug release percent for the product B (P0.05). The best dissolution conditions tested, for products A and B, were applied to evaluate capsules dissolution profiles. Twelve dosage units were assayed and dissolution efficiency concept was used, for each condition, to obtain results with statistical significance (P0.05). Optimal conditions to carry out the dissolution test were 900 ml of 0.1 M hydrochloric acid as dissolution medium, basket at 100 rpm stirring speed and 260 nm ultraviolet detection.

e13008 Background: Up to 30% of patients on 5-fluorouracil (5FU) experience severe toxicity. Dihydropyrimidine-dehydrogenase (DPD) deficiency explains 36-61% of cases. Predicting toxicity is an unmet challenge. Uracil breath test (UraBT) consists of measuring 13CO2 in exhaled breath after ingestion of 2-13C-uracil to evaluate pyrimidine (and 5FU) catabolism. Methods: We studied 33 gastrointestinal cancer patients previously exposed to 5FU: thirteen had grade 3-4 and 20, grade 0-1 toxicity. Groups were well-balanced regarding: age (median, 57 years); gender (males, 35%); primary (colorectal, 90%); ethnicity (Caucasians, 55%); chemotherapy (Mayo clinic regimen, 75%). Main toxicities were febrile neutropenia, diarrhea and stomatitis. Tests used to evaluate pyrimidine catabolism: (1) sequencing of three exons of DPYD; (2) plasma dihydrouracil/uracil ratio (UH2/U); (3) UraBT. We tested the performance of UraBT to discriminate patients who had grade 0-1 toxicity versus grade 3-4 toxicity and patients with and without proven DPD-deficiency. DPD-deficients were defined as having had grade 3-4 toxicity plus either UH2/U < 1.8 or deleterious mutation. Results: 4/13 grade 3-4 toxicity patients proved to be DPD-deficient: three had deleterious mutations (IVS14+1G>A in one; SNP 2846A>T in two), and one had low UH2/U ratio. Mean delta over baseline in 50 minutes (DOB50) significantly differed between groups. DOB50 ≤ 161.4 discriminated individuals with grade 3-4 versus grade 0-1 toxicity (sensitivity= 61.5%; specificity= 85%) and DPD-deficient versus non DPD-deficient (sensitivity= 75%; specificity= 85%). Conclusions: UraBT is a non-invasive and easy to perform method with promising accuracy in discriminating individuals with severe toxicity to 5FU, comparing favorably to most tests available to predict 5FU toxicity. [Table: see text]

Antimicrobials are among the most commonly used drugs and have become a class of contaminants with great environmental importance. Metronidazole is an antimicrobial used for the therapeutic management of several human diseases. The toxicity of antimicrobials on aquatic species may affect sensitive microorganisms and reduce metabolic processes. Cyanobacteria is a group of organisms that are of great ecological importance in aquatic environments. Studies indicate that cyanobacteria are very sensitive to some antimicrobials. Therefore, it is necessary to evaluate the effects of metronidazole contamination on phytoplankton. The aim of this study was to investigate the effects of metronidazole on the growth of the cyanobacterium Microcystis protocystis and to evaluate the stability of this antimicrobial agent in the culture medium over a period of 96 hours. M. protocystis was resistant to growth inhibition by metronidazole. The EC50 of this antimicrobial for M. protocystis was 117.3 mg L–1. Under the growth inhibition test conditions, neither a significant change in the MNZ concentration nor the presence of drug metabolites or degradation products was observed. These results indicate low cellular uptake of the antimicrobial agent and its persistence in the culture medium.

A method using HPLC-UV was developed and validated for the determination of etoposide incorporated into polycaprolactone implants. The method was carried out in isocratic mode using a C18 column (250 x 4.6 mm; 5 µm), at 25 ºC, with acetonitrile and acetic acid 4% (70:30) as mobile phase, a flow rate of 2 mL/min, and UV detection at 285 nm. The method was linear (r² > 0.99) over the range of 5 to 65 µg/mL, precise (RSD < 5%), accurate (recovery of 98.7%), robust, selective regarding excipient of the sample, and had a quantitation limit equal to 1.76 µg/mL. The validated method can be successfully employed for routine quality control analyses.