Stability-indicating method for the novel antifungal compound RI76: Characterization and in vitro antifungal activity of its active degradation product.

RI76 is a novel 2-thiazolylhydrazone compound with reported antifungal activity. In preclinical drug development, it is fundamental to know the impurity profile and to understand degradation mechanisms of the molecule. In our study, RI76 was subjected to forced degradation conditions, and a stability-indicating HPLC-DAD method was developed and validated. Separation was carried out on a C18 column (150 × 4.6 mm i.d., 5 µm) maintained at 40°C using a 1 mL/min flow rate of 2 mM ammonium acetate with 0.1% formic acid (pH 3.0) and acetonitrile in gradient mode. The method was linear in the range of 0.7-91 µg/mL for RI76 and 0.7-25 µg/mL for its degradation product PD76. The formation of a major degradation product was quickly observed when RI76 was in aqueous solution. The chemical structure of this product, named PD76, was proposed based on LC-UV-MS experiments, synthesized in-house, and confirmed by NMR spectroscopy and chromatographic analysis. In vitro antifungal activity assays demonstrated that this resultant product shows a promising activity against clinically important Candida and Cryptococcus strains, matching or surpassing the activity of its precursor and of well-established antifungal drugs.

Simultaneous quantitation of kavalactones in kava dry extracts: comparison of multi-standards and single standard validation approaches.

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".

Purity determination of a new antifungal drug candidate using quantitative 1 H NMR spectroscopy: Method validation and comparison of calibration approaches.

Quantitative nuclear magnetic resonance (qNMR) is an analytical technique that offers numerous advantages in pharmaceutical applications including minimum sample preparation and rapid data collection times with no need for response factor corrections, being a powerful tool for assaying drug content in both drug discovery and early drug development. In the present work, we have applied qNMR, using both the internal standard and the electronic reference to access in vivo concentrations 2 calibration methods, to assess the purity of RI76, a novel antifungal drug candidate. NMR acquisition and processing parameters were optimized in order to obtain spectra with intense, well-resolved signals of completely relaxed nuclei. The analytical method was validated following current guidelines, demonstrating selectivity, linearity, accuracy, precision, and robustness. The calibration approaches were statistically compared, and no significant difference was observed when comparing the obtained results and their dispersion in terms of relative standard deviation. The proposed qNMR method may, therefore, be used for both qualitative and quantitative assessments of RI76 in early drug development and for characterization of this compound.

Comparative stability of arbutin in Arctostaphylos uva-ursi by a new comprehensive stability-indicating HPLC method.

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.

Development and validation of an LC-ESI-MS/MS method for the simultaneous quantification of naproxen and sumatriptan in human plasma: application to a pharmacokinetic study.

A sensitive and fast liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the simultaneous quantification of naproxen and sumatriptan in human plasma. A simple liquid-liquid extraction procedure, with a mixture of ethyl acetate, methyl tert-butyl ether, and dichloromethane (4:3:3, v/v), was used for the cleanup of plasma. Naratriptan and aceclofenac were employed as internal standards. The analyses were carried out using an ACE C18 column (50 × 4.6 mm i.d.; particle size 5 µm) and a mobile phase consisting of 2 mM aqueous ammonium acetate with 0.025 % formic acid and methanol (38:62, v/v). A triple-quadrupole mass spectrometer equipped with an electrospray source in the positive mode was set up in the selective reaction monitoring mode to detect the ion transitions m/z 231.67 → m/z 185.07, m/z 296.70 → m/z 157.30, m/z 354.80 → m/z 215.00, and m/z 336.80 → m/z 97.94 for naproxen, sumatriptan, aceclofenac, and naratriptan, respectively. The method was validated and proved to be linear, accurate, precise, and selective over the ranges of 2.5-130 µg mL(-1) for naproxen and 1-50 ng mL(-1) for sumatriptan. The validated method was successfully applied to a pharmacokinetic study with simultaneous administration of naproxen sodium and sumatriptan succinate tablet formulations in healthy volunteers.

An improved LC-MS/MS method for quantitation of indapamide in whole blood: application for a bioequivalence study.

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 Laboratórios S.A.) were bioequivalent to Natrilix®, according to the rate and extent of absorption.

Determination of 3-α-hydroxytibolone in human plasma by LC-MS/MS: application for a pharmacokinetic study after administration of a tibolone formulation.

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.

Dynamic interaction between fluconazole and amphotericin B against Cryptococcus gattii.

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.

A rapid HPLC-ESI-MS/MS method for determination of dihydrouracil/uracil ratio in plasma: evaluation of toxicity to 5-flurouracil in patients with gastrointestinal cancer.

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.

Quantitation of glucosamine sulfate in plasma by HPLC-MS/MS after administration of powder for oral solution formulation.

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-[1-¹³C] glucosamine hydrochloride as an internal standard. Chromatographic separation was performed on an ACE Ciano column using isocratic elution with acetonitrile and aqueous 2 mM 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 50-5000 ng/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.

Development of a stability-indicating assay method by HPLC-DAD and MS characterization of forced degradation products of ravuconazole.

Ravuconazole (RAV) is a triazole antifungal with broad spectrum and a novel alternative in the treatment of systemic fungal infections. A stability-indicating method by high-performance liquid chromatography-diode array detection was developed and fully validated to assay ravuconazole in the presence of its degradation products. Separation was achieved with a Sunfire C18 column (250 mm × 4.6 mm id, 5 µm), mobile phase composed of acetonitrile and water (80:20), at 1 mL/min. The volume of injection was 5 µL and DAD detection was performed at 287 nm. RAV was well resolved from its degradation products and the method proved to be linear, selective, accurate, precise and robust. A forced degradation study was conducted on the pure drug under oxidative conditions in presence of H2O2 and metallic ions and under acid, alkaline and neutral hydrolysis. RAV was degraded mainly under alkaline hydrolysis, forming two main degradation products. The chemical structures were proposed according to the data obtained by liquid chromatography coupled to mass spectrometry (LC-MS) analysis. This study provided a new and selective stability-indicating method to evaluate the intrinsic stability of ravuconazole in active pharmaceutical ingredients. The developed method was found to be suitable for quality control routine analysis and to stability studies of ravuconazole.

Biopharmaceutical evaluation of kavain in Piper methysticum G. Forst dried extract: Equilibrium solubility and intestinal permeability in Caco-2 cell model.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper methysticum G. Forst, popularly known as kava, is a traditional medicinal plant native from South Pacific islands and widely used to treat anxiety, depression and stress. The psychoactive properties are related to the kavalactones, mainly kavain. AIM OF THE STUDY: To evaluate the biopharmaceutical properties of synthetic kavain and when present in kava dried extracts by means of equilibrium solubility and intestinal permeability studies in the Caco-2 cell model. MATERIALS AND METHODS: The equilibrium solubility of kavain was performed using a shake flask incubator at 37 °C in different media at physiological pH range (1.2-6.8). The intestinal permeability of kavain evaluated in Caco-2 cells in the presence and absence of the P-glycoprotein inhibitor verapamil. Kavain concentrations were determined by reversed phase high performance liquid chromatography (HPLC). RESULTS: HPLC methods were developed and fully validated for kavain quantitation. Kavain demonstrated low solubility and the pH of the aqueous media did not affect its solubility. Kavain was found to be highly permeable and efflux of kavain mediated by P-glycoprotein was not significant during intestinal permeation. CONCLUSION: The results of biopharmaceutical studies provided useful information for predicting availability of kavain from the gastrointestinal tract and this compound was ranked as BCS Class II, exhibiting dissolution rate-limited absorption.

Acute oral toxicity, antinociceptive and antimicrobial activities of kava dried extracts and synthetic kavain.

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.

A Rapid UPLC Method for the Simultaneous Quantitation of Caffeic Acid Derivatives in Dried Extracts of Echinacea Purpurea.

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.

Microextraction by packed sorbent and high performance liquid chromatography for simultaneous determination of lumefantrine and desbutyl-lumefantrine in plasma samples.

A bioanalytical method for the determination of lumefantrine and its metabolite desbutyl-lumefantrine in plasma samples using microextraction by packed sorbent (MEPS) and high-performance liquid chromatography was developed and validated. A complete factorial planning and surface response approach were employed to optimize the extraction parameters sample volume, dilution, aspirated sample volume and extraction cycles. The method employed C18 MEPS sorbent and diazepam as internal standard (IS). Separation was performed on a Luna C18 column (250 mm × 4.6 mm, 5 µm) at 35 °C, with mobile phase composed of acetonitrile and 0.05 % trifluoroacetic acid (68:32, v/v), detection at 305 nm and injection volume of 25 µL. The developed method showed to be selective, precise, accurate and linear in the range of 50-5000 ng/mL for lumefantrine and desbutyl-lumefantrine. Using the optimized MEPS procedure, high recovery rates were obtained for both analytes and IS (92.2 %-99.0 %). The method was successfully applied for the determination of lumefantrine and its metabolite in human plasma samples after oral administration of lumefantrine tablets in healthy volunteers.

Liquid chromatography-tandem mass spectrometry bioanalytical method for the determination of kavain in mice plasma: Application to a pharmacokinetic study.

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 â†’ 115.1 and 231.0 â†’ 152.8 for kavain; and 234.2 â†’ 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.

Preclinical pharmacokinetic study of a new thiazolyl hydrazone derivative with antifungal activity in mice plasma by LC-MS/MS.

RN104, named 2-[2-(cyclohexylmethylene)hydrazinyl)]-4-phenylthiazole, is a thiazolyl hydrazone derivative with promising antifungal activity. Pharmacokinetic profile of the RN104 was evaluated in mice plasma using a developed and validated bioanalytical method by LC-MS/MS. Clotrimazole was used as internal standard. The analytes were extracted by a protein precipitation procedure and separated on a C18 end-capped column and mobile phase composed of acetonitrile - 0.1% formic acid (85:15, v/v), in isocratic mode. Electrospray ionization in positive ionization mode (ESI + ) and multiple reaction monitoring (MRM) were employed using the transitions m/z 286.1 â†’ m/z 176.1 (quantifier) and m/z 286.1 â†’ m/z 112.2 (qualifier) for RN104 and m/z 345.2 â†’ m/z 277.1 (quantifier) and m/z 345.2 â†’ m/z 165.2 (qualifier) for internal standard. The method was validated and proved to be linear, accurate, precise, and selective over the range 0.625 to 40.0 ng/mL. The pharmacokinetic model that best fit the data was the bicompartmental model. The maximum plasmatic concentration was reached 20 min after administration (per os and intraperitoneal) and the highest plasma concentration of RN104 was found after per os administration at a dosage of 50 mg/kg compared to i.p. administration at 10 mg/kg.

Chirobiotic V Versus Chiralpak ID for the Enantioselective Chromatographic Separation of Chloroquine: Stability and Validation Study.

Chloroquine is a chiral antimalarial drug and demonstrates enantioselective pharmacodynamic and pharmacokinetic properties. However, this drug is administered as racemate. The knowledge of stereoselective aspects of these agents may be useful to better understand their mechanisms of action and to optimize their safety and/or clinical efficacy. In this study, an enantioselective analytical method for the quantification of chloroquine enantiomers was developed using HPLC-UV. The chromatographic conditions were: Chirobiotic V column (100 × 2.1 mm, 5 µm) at 25°C, mobile phase containing methanol:acetic acid:triethylamine (100:0.12:0.12), flow rate 1 mL/min, injection volume 10 µL and detection at 258 nm. The validation parameters evaluated were selectivity, linearity, precision, accuracy, and robustness. In addition, a stability study after forced degradation of chloroquine enantiomers was performed. The enantioseparation of chloroquine using a polysaccharide-based chiral stationary phase (Chiralpak ID) at different mobile phase composition was evaluated and the chromatographic performance of both columns was compared. Thus, a stability-indicating chiral analytical method was developed and fully validated, allowing the separation of chloroquine enantiomers and its degradation products in tablets available in Brazil.

Simultaneous Quantitation of Amlodipine Besylate and Olmesartan Medoxomil in Fixed-Dose Combination Tablets: HPLC-DAD Versus UHPLC-DAD.

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.

Level A in vitro-in vivo correlation: Application to establish a dissolution test for artemether and lumefantrine tablets.

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.