Highly cross-linked monoliths prepared from single cross-linking monomers were found to increase surface area and stability. Therefore, seven cross-linking monomers, i.e., 1,3-butanediol dimethacrylate (1,3-BDDMA), 1,4-butanediol dimethacrylate (1,4-BDDMA), neopentyl glycol dimethacrylate (NPGDMA), 1,5-pentanediol dimethacrylate (1,5-PDDMA), 1,6-hexanediol dimethacrylate (1,6-HDDMA), 1,10-decanediol dimethacrylate (1,10-DDDMA), and 1,12-dodecanediol dimethacrylate (1,12-DoDDMA), were used to synthesize highly cross-linked monolithic columns in 75-µm i.d. capillaries by one-step UV-initiated polymerization using dodecanol and methanol as porogens for reversed-phase liquid chromatography (RPLC) of small molecules. Selection of porogen type and concentration was investigated in detail. Isocratic elution of alkylbenzenes at a flow rate of 300 nL/min was conducted for all of the monoliths. Gradient elution of alkylbenzenes and alkylparabens provided high resolution separations. Several of the monoliths demonstrated column efficiencies in excess of 50,000 plates/m. Monoliths with longer alkyl-bridging chains showed very little shrinking or swelling in solvents of different polarities. In addition, highly cross-linked monolithic capillary columns poly(1,6-HDDMA), poly(cyclohexanediol dimethacrylate) [poly(CHDDMA)] and poly(1,4-phenylene diacrylate) [poly(PHDA)], were synthesized and compared for RPLC of small molecules. Isocratic elution of alkylbenzenes was performed using 1,6-HDDMA and CHDDMA monolithic columns. Gradient elution of alkylbenzenes using all three monolithic columns showed good separations. Monolithic columns formed from 1,6-HDDMA, which had a linear alkyl-bridging chain structure, exhibited the highest column efficiencies (86,000 plates/m). Optimized columns showed high permeability and high run-to-run and column-to-column reproducibilities. Monoliths prepared from controlled/living polymerization was demonstrated exhibiting narrower molecular weight distribution and more homogeneous cross-linked structures due to the reversible character of this polymerization method. Thus, monolithic columns were developed from three cross-linking monomers, i.e., 1, 12-DoDDMA, trimethylolpropane trimethacrylate (TMPTMA) and pentaerythritol tetraacrylate (PETA) using organotellurium-mediated living radical polymerization (TERP) in 150-µm i.d. capillaries for RPLC of small molecules. Selection of the polymerization conditions for the 1,12-DoDDMA monolirh was investigated in detail. Isocratic elution of alkylbenzenes was achieved with good efficiency (47,700 to 64,200 plates/m for uracil) using all monolithic columns prepared using TERP.

Capsaicinoids are a class of compounds responsible for the "heat" of hot peppers. Capsaicin and dihydrocapsaicin have the highest burning effect. The aim of this work is to separate and quantify the two major capsaicinoids in fruits harvested at different stages of development and at different seasons. Simple and rapid HPLC method involves 73:27% methanol water mobile phase with C18 stationary phase and UV-Vis detector set at 210 nm. The method showed good reproducibility with 1.74% - 4.72% relative standard deviations, a linear response within 0.65–45.5 and 0.25-17.5 μg/mL for capsaicin and dihydrocapsaicin, respectively. The method achieved average recovery of 106% for capsaicin and 102% dihydrocapsaicin. Determination of capsaicinoids in four naturally grown chili and commercial source habanero were analyzed. The amount in the sample ranged from 1184-8156 μg/g for capsaicin and 430-3299 μg/g for dihydrocapsaicin.

Cholesterol is a fat-like molecule found among lipids in animal (including human) tissues. It is needed for maintaining good health. However, health issues have been raised because of the strong correlation between high levels of cholesterol in the body and cardiovascular disease. An HPLC method for quantitative determination of cholesterol in foods is presented. This involves a C-18 stationary phase using a 70:30 methanol: 2-propanol mobile phase with an UV detector set at 212 nm. The method showed linearity in the range 5.0 to 100.0 μg/mL and also good reproducibility with relative standard deviation of 4.22%, 2.71%, 4.8%, and 3.7% for the different samples analyzed. The mean recovery of the butter sample was 106.5%. The samples under investigation were common food items such as butter, lard, and two different types of cheese.

Reversed-phase liquid chromatography (RPLC) is a commonly used separation technique in chemistry. Nevertheless, the mechanistic interactions at the molecular level among the eluent, analyte, and the stationary phase are not fully understood. Because of this limited understanding, optimization of the separation must be done experimentally. Learning more about molecular interactions should aid in improving separations. We are currently using second-harmonic generation (SHG) spectroscopy to investigate how analytes adsorb to the surface. SHG is a spectroscopic technique that produces signal only at places of non-isotropic symmetry; this typically occurs at surfaces. SHG can be used to produce surface isotherms of test analytes adsorbed to a model C18 stationary phase surface. Fitting these isotherms with a Langmuir model produces an adsorption equilibrium constant. However, the equilibrium constant can only be accurately determined if the true bulk concentration is known; this thesis describes an approach to ensure this. The equilibrium constant relates to Gibbs free energy and is the start to obtaining other thermodynamic information. The long equilibration times of analytes with the stationary phase observed in this study emphasize the importance of both thermodynamic information and kinetic values for understanding retention. Once equilibrium constants and other parameters are accurately obtained, this information can be used to improve predictions and calculations from numerical models.

A combinação entre o delapril (DEL), um inibidor da enzima conversora de angiotensina e o manidipino (MAN), um antagonista dos canais de cálcio, produz um efeito anti-hipertensivo sinérgico, podendo ser considerado um ótimo tratamento para pacientes com hipertensão essencial leve e moderada. No presente trabalho foram desenvolvidos e validados métodos cromatográfico e eletroforético para avaliação simultânea de DEL e MAN em produto farmacêutico. As análises por cromatografia líquida em fase reversa (CL-FR) foram executadas utilizando coluna C8 (250 mm x 4,6 mm), mantida a 35 oC. A fase móvel foi constituída por acetonitrila e solução de trietilamina 0,3%, pH 3,0 (55:45; v/v), eluída na vazão de 1,2 mL/min com detecção a 220 nm. Paralelamente, desenvolveu-se método por eletroforese capilar, utilizando modo de separação por cromatografia eletrocinética micelar (MEKC) e ácido salicílico como padrão interno. Foi utilizado capilar de sílica fundida (comprimento efetivo de 72 cm) mantido a 35 °C, com solução eletrolítica composta de tampão borato 50 mM e dodecil sulfato de sódio (SDS) 5 mM, pH 9,0. Voltagem de 25 kV foi aplicada e a injeção foi de 50 mbar durante 5 s, com detecção a 208 nm. A especificidade e a capacidade dos métodos serem indicativos de estabilidade foram demonstradas através de estudos de degradação forçada dos fármacos e pela não interferência dos excipientes nas análises. Além disso, o desenho experimental Plackett-Burman foi utilizado para a avaliação da robustez, observando-se resultados adequados para ambos métodos. Os procedimentos foram validados de acordo com guias aceitos internacionalmente, observando-se resultados em uma faixa aceitável. Os métodos propostos foram aplicados com sucesso na determinação quantitativa simultânea de DEL e MAN em comprimidos, não havendo diferença significativa dos resultados (P>0,05), contribuindo, portanto, para aprimorar o controle da qualidade, assegurando a eficácia terapêutica. / The combination of delapril (DEL), an angiotensin converting enzyme inhibitor and manidipine (MAN), an antagonist of calcium channels, produces a synergic antihypertensive effect and may be regarded as an optimal antihypertensive drug treatment in mild to moderate essential hypertensive patients. The chromatographic and eletrophoretic methods for the simultaneous evaluation of DEL and MAN in pharmaceutical product were developed and validated in the present work. The reversed-phase liquid chromatography (RP-LC) method was carried out on a C8 column (250 mm x 4.6 mm i.d., 5 μm), maintained at 35 ºC. The mobile-phase consisted of acetonitrile and a solution of triethylamine 0.3% pH 3.0 (55:45; v/v), running at a flow rate of 1.2 mL/min, with detection at 220 nm. The capillary electrophoresis method was developed using the micellar electrokinetic chromatography (MEKC) as the separation mode, and salicylic acid as internal standard. The analysis were performed on a fused-silica capillary (effective length of 72 cm) maintained at 35 °C, with 50 mM of borate buffer and 5 mM of sodium dodecyl sulfate (SDS) at pH 9.0 as background electrolyte. The separation was achieved at 25 kV applied voltage and the injection was performed at 50 mbar for 5 s, with detection at 208 nm. The specificity and stability-indicating capability of the methods were demonstrated through forced degradation studies, which also show that there is no interference of the excipients in the analysis. Moreover, the Plackett- Burman experimental design was used for robustness evaluation, giving acceptable results for both methods. The procedures were validated according to Internationals guidelines, giving results within the acceptable range. Therefore, the proposed methods were successfully applied for the simultaneous quantitative analysis of DEL and MAN in the tablet dosage form, showing non-significant difference (P>0.05), contributing to improve the quality control and to assure the therapeutic efficacy.

A combinação entre o delapril (DEL), um inibidor da enzima conversora de angiotensina e o manidipino (MAN), um antagonista dos canais de cálcio, produz um efeito anti-hipertensivo sinérgico, podendo ser considerado um ótimo tratamento para pacientes com hipertensão essencial leve e moderada. No presente trabalho foram desenvolvidos e validados métodos cromatográfico e eletroforético para avaliação simultânea de DEL e MAN em produto farmacêutico. As análises por cromatografia líquida em fase reversa (CL-FR) foram executadas utilizando coluna C8 (250 mm x 4,6 mm), mantida a 35 oC. A fase móvel foi constituída por acetonitrila e solução de trietilamina 0,3%, pH 3,0 (55:45; v/v), eluída na vazão de 1,2 mL/min com detecção a 220 nm. Paralelamente, desenvolveu-se método por eletroforese capilar, utilizando modo de separação por cromatografia eletrocinética micelar (MEKC) e ácido salicílico como padrão interno. Foi utilizado capilar de sílica fundida (comprimento efetivo de 72 cm) mantido a 35 °C, com solução eletrolítica composta de tampão borato 50 mM e dodecil sulfato de sódio (SDS) 5 mM, pH 9,0. Voltagem de 25 kV foi aplicada e a injeção foi de 50 mbar durante 5 s, com detecção a 208 nm. A especificidade e a capacidade dos métodos serem indicativos de estabilidade foram demonstradas através de estudos de degradação forçada dos fármacos e pela não interferência dos excipientes nas análises. Além disso, o desenho experimental Plackett-Burman foi utilizado para a avaliação da robustez, observando-se resultados adequados para ambos métodos. Os procedimentos foram validados de acordo com guias aceitos internacionalmente, observando-se resultados em uma faixa aceitável. Os métodos propostos foram aplicados com sucesso na determinação quantitativa simultânea de DEL e MAN em comprimidos, não havendo diferença significativa dos resultados (P>0,05), contribuindo, portanto, para aprimorar o controle da qualidade, assegurando a eficácia terapêutica. / The combination of delapril (DEL), an angiotensin converting enzyme inhibitor and manidipine (MAN), an antagonist of calcium channels, produces a synergic antihypertensive effect and may be regarded as an optimal antihypertensive drug treatment in mild to moderate essential hypertensive patients. The chromatographic and eletrophoretic methods for the simultaneous evaluation of DEL and MAN in pharmaceutical product were developed and validated in the present work. The reversed-phase liquid chromatography (RP-LC) method was carried out on a C8 column (250 mm x 4.6 mm i.d., 5 μm), maintained at 35 ºC. The mobile-phase consisted of acetonitrile and a solution of triethylamine 0.3% pH 3.0 (55:45; v/v), running at a flow rate of 1.2 mL/min, with detection at 220 nm. The capillary electrophoresis method was developed using the micellar electrokinetic chromatography (MEKC) as the separation mode, and salicylic acid as internal standard. The analysis were performed on a fused-silica capillary (effective length of 72 cm) maintained at 35 °C, with 50 mM of borate buffer and 5 mM of sodium dodecyl sulfate (SDS) at pH 9.0 as background electrolyte. The separation was achieved at 25 kV applied voltage and the injection was performed at 50 mbar for 5 s, with detection at 208 nm. The specificity and stability-indicating capability of the methods were demonstrated through forced degradation studies, which also show that there is no interference of the excipients in the analysis. Moreover, the Plackett- Burman experimental design was used for robustness evaluation, giving acceptable results for both methods. The procedures were validated according to Internationals guidelines, giving results within the acceptable range. Therefore, the proposed methods were successfully applied for the simultaneous quantitative analysis of DEL and MAN in the tablet dosage form, showing non-significant difference (P>0.05), contributing to improve the quality control and to assure the therapeutic efficacy.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Interleukin 11 (IL-11) is a multifunctional cytokine in the IL-6 type family of long-chain helical cytokines, which modulates the proliferation, differentiation and maturation of various types of hematopoietic cells. Recombinant human interleukin-11 (rhIL-11) produced by DNA technology in Escherichia coli is currently being used worldwide for the prevention of thrombocytopenia and to reduce the need for platelet transfusions after myelosuppressive chemotherapy in patients with nonmyeloid malignancies. A stability-indicating reversed-phase liquid chromatography (RP-LC) method was validated for the assessment of recombinant human interleukin-11 (rhIL-11) in biopharmaceutical formulations. The RP-LC method was carried out on a Jupiter C4 column (250 mm x 4.6 mm i.d.), maintained at 25ºC. The mobile phase A consisted of 0.1% TFA and the mobile phase B was acetonitrile with 0.1% TFA, run as follows: time 0 to 0.1 min 40% of B; from 0.1 to 30 min linear up to 65% of B; from 30.01 to 31 min linear down to 40% of B, maintained up to 40 min. The flow rate was 1 mL/min, and using photodiode array (PDA) detection at 214 nm. Chromatographic separation was obtained with a retention time of 27.6 min, and was linear over the concentration range of 1 200 μg/mL (r2 = 0.9995). The limits of detection and quantitation were 0.34 and 1.12 μg/mL, respectively. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.22% with bias lower than 1.25%. Moreover, the in vitro cytotoxicity test of the degraded products showed non-significant differences (p>0.05). The proposed method was applied to the assessment of rhIL-11 and related proteins in biopharmaceutical dosage forms, and the results were correlated to those of a bioassay, showing a higher mean difference of the estimated content/potencies of 2.60% for the RP-LC method, aiming to establish new alternatives to monitor stability, improve quality control and thereby assure therapeutic efficacy of the biological medicine. / A interleucina 11 (IL-11) é uma citocina multifuncional que pertence a família da interleucina- 6 e estimula a proliferação, diferenciação e maturação de células hematopoiéticas. A interleucina-11 humana recombinante (rhIL-11) é produzida pela tecnologia do DNA em Escherichia coli e é usada clinicamente para a prevenção de trombocitopenia grave e redução da necessidade de transfusão de plaquetas após quimioterapia mielossupressiva em pacientes com neoplasias malignas não mielóides. No presente trabalho foi desenvolvido e validado método por cromatografia líquida em fase reversa (CL-FR) para a avaliação de rhIL-11 em formulações de produtos farmacêuticos. Utilizou-se coluna Júpiter C4 (250 mm x 4,6 mm d.i.), mantida a 25ºC. A fase móvel A foi constituída de TFA 0,1% e a fase móvel B de acetonitrila com 0,1% TFA, eluídas no seguinte gradiente: 0 0,1 min, 40% de B; 0,1 30 min, 40 65% de B; 30,01 a 31 min, 65 40% de B, mantendo-se nesta proporção até 40 min. Utilizou-se vazão de 1 mL/min e detector de arranjo de diodos (DAD) a 214 nm. A eluição cromatográfica foi obtida no tempo de 27,6 min, sendo linear na faixa de concentração de 1 200 μg/mL (r2 = 0,9995). Os limites de detecção e quantificação foram 0,34 e 1,12 μg/mL, respectivamente. A especificidade foi avaliada em estudos de degradação, que também demonstraram que não houve interferência dos excipientes. A exatidão foi 100,20%, com bias inferior a 1,25%. Além disso, realizou-se o teste de citotoxicidade in vitro das formas degradadas, as quais não apresentaram diferença significativa em relação a forma intacta (p>0,05). O método proposto foi aplicado para a avaliação da potência de rhIL-11 e de proteínas relacionadas em formulações farmacêuticas, e os resultados foram comparados com o bioensaio, observando-se diferenças das médias de conteúdo/potência 2,60% superiores para o método por CL-FR. Contribuíu-se assim para estabelecer procedimentos que aprimoram o controle da qualidade, garantindo a segurança e eficácia terapêutica do produto biotecnológico.

Febuxostat is a novel non purine drug indicated for the treatment of hyperuricemia in gout. A reversed-phase liquid chromatography (RP-LC) method was validated for the determination of febuxostat in pharmaceutical dosage forms. The LC method was carried out on a XTerra C18 column (150 mm x 3.9 mm I.D.), maintained at 25 ºC. The mobile phase consisted of water (pH 3.5) acetonitrile (40:60, v/v), run at a flow rate of 0.8 mL/min and using photodiode array (PDA) detection at 316 nm. The chromatographic separation was obtained with retention time of 3.9 min, and was linear over the range of 0.25 - 30 μg/mL (r2=0.9995). The specificity and stability-indicating capability of the method was proven through degradation studies were carried out by LC and MS and showing also, that there was no interference of the excipients and degradation products in the quantification of the drug. Moreover, the in vitro cytotoxicity test of the degraded products showed significant differences (p<0.05). The accuracy was 100.54% with bias lower than 0.65%. The limits of detection and quantitation were 0.08 and 0.28 μg/mL, respectively. The procedure was validated evaluating parameters such as the specificity, linearity, precision, accuracy, limits of detection and quantitation, robustness, and system suitability test, giving results within the acceptable range. The proposed method was applied for dissolution studies and the analysis of tablet dosage forms, contributing to assure the safety and therapeutic efficacy. / Febuxostate é um novo fármaco, não purínico, indicado para o tratamento da hiperuricemia em pacientes com gota. No presente trabalho foi desenvolvido e validado método por cromatografia líquida em fase reversa (CL-FR) para determinação de febuxostate em produtos farmacêuticos. No método por CL-FR foi utilizada coluna XTerra C18 (150 mm x 3,9 mm d.i), mantida a 25 oC. A fase móvel foi composta de água ultra-pura (pH 3,5): acetonitrila (40:60, v/v), eluída na vazão de 0,8 mL/ min com detecção no ultravioleta a 316 nm. A separação cromatográfica foi obtida no tempo de 3,9 min, sendo linear na faixa de concentração de 0,25-30 μg/mL (r2=0,9995). A especificidade do método foi comprovada através de estudos de degradação realizados por cromatografia líquida e espectrometria de massas, demonstrando que não houve interferência dos excipientes e dos produtos de degradação na quantificação do fármaco. Além disso, o teste de citotoxicidade in vitro das amostras degradadas, apresentou diferenças significativas (p <0,05) em relação à forma intacta. A precisão foi de 100,54%, com bias menor do que 0,65%. Os limites de detecção e de quantificação foram de 0,08 e 0,28 μg/mL, respectivamente. O procedimento foi validado, avaliando-se os parâmetros de especificidade, linearidade, precisão, exatidão, limite de detecção e quantificação, robustez e teste de adequabilidade do sistema, cujos resultados estão de acordo com os requisitos preconizados. O método proposto foi aplicado no estudo de dissolução e análise de formas farmacêuticas de comprimidos, contribuindo, assim, para aprimorar o controle da qualidade de medicamentos, bem como garantir a segurança e eficácia no uso terapêutico.

A combinação entre o delapril (DEL), um inibidor da enzima conversora de angiotensina e o manidipino (MAN), um antagonista dos canais de cálcio, produz um efeito anti-hipertensivo sinérgico, podendo ser considerado um ótimo tratamento para pacientes com hipertensão essencial leve e moderada. No presente trabalho foram desenvolvidos e validados métodos cromatográfico e eletroforético para avaliação simultânea de DEL e MAN em produto farmacêutico. As análises por cromatografia líquida em fase reversa (CL-FR) foram executadas utilizando coluna C8 (250 mm x 4,6 mm), mantida a 35 oC. A fase móvel foi constituída por acetonitrila e solução de trietilamina 0,3%, pH 3,0 (55:45; v/v), eluída na vazão de 1,2 mL/min com detecção a 220 nm. Paralelamente, desenvolveu-se método por eletroforese capilar, utilizando modo de separação por cromatografia eletrocinética micelar (MEKC) e ácido salicílico como padrão interno. Foi utilizado capilar de sílica fundida (comprimento efetivo de 72 cm) mantido a 35 °C, com solução eletrolítica composta de tampão borato 50 mM e dodecil sulfato de sódio (SDS) 5 mM, pH 9,0. Voltagem de 25 kV foi aplicada e a injeção foi de 50 mbar durante 5 s, com detecção a 208 nm. A especificidade e a capacidade dos métodos serem indicativos de estabilidade foram demonstradas através de estudos de degradação forçada dos fármacos e pela não interferência dos excipientes nas análises. Além disso, o desenho experimental Plackett-Burman foi utilizado para a avaliação da robustez, observando-se resultados adequados para ambos métodos. Os procedimentos foram validados de acordo com guias aceitos internacionalmente, observando-se resultados em uma faixa aceitável. Os métodos propostos foram aplicados com sucesso na determinação quantitativa simultânea de DEL e MAN em comprimidos, não havendo diferença significativa dos resultados (P>0,05), contribuindo, portanto, para aprimorar o controle da qualidade, assegurando a eficácia terapêutica. / The combination of delapril (DEL), an angiotensin converting enzyme inhibitor and manidipine (MAN), an antagonist of calcium channels, produces a synergic antihypertensive effect and may be regarded as an optimal antihypertensive drug treatment in mild to moderate essential hypertensive patients. The chromatographic and eletrophoretic methods for the simultaneous evaluation of DEL and MAN in pharmaceutical product were developed and validated in the present work. The reversed-phase liquid chromatography (RP-LC) method was carried out on a C8 column (250 mm x 4.6 mm i.d., 5 μm), maintained at 35 ºC. The mobile-phase consisted of acetonitrile and a solution of triethylamine 0.3% pH 3.0 (55:45; v/v), running at a flow rate of 1.2 mL/min, with detection at 220 nm. The capillary electrophoresis method was developed using the micellar electrokinetic chromatography (MEKC) as the separation mode, and salicylic acid as internal standard. The analysis were performed on a fused-silica capillary (effective length of 72 cm) maintained at 35 °C, with 50 mM of borate buffer and 5 mM of sodium dodecyl sulfate (SDS) at pH 9.0 as background electrolyte. The separation was achieved at 25 kV applied voltage and the injection was performed at 50 mbar for 5 s, with detection at 208 nm. The specificity and stability-indicating capability of the methods were demonstrated through forced degradation studies, which also show that there is no interference of the excipients in the analysis. Moreover, the Plackett- Burman experimental design was used for robustness evaluation, giving acceptable results for both methods. The procedures were validated according to Internationals guidelines, giving results within the acceptable range. Therefore, the proposed methods were successfully applied for the simultaneous quantitative analysis of DEL and MAN in the tablet dosage form, showing non-significant difference (P>0.05), contributing to improve the quality control and to assure the therapeutic efficacy.

Reversed-phase liquid chromatography (RPLC) is a widely used technique for analytical separations but routinely requires empirical optimization. Gaining a better understanding of the molecular reasons for retention may mean more efficient separations with fewer trial and error runs to obtain optimized separations. Vibrationally resonant sum frequency generation (VR-SFG) is a surface specific technique that has allowed for in situ examination of model RPLC stationary phases under various solvent and pressure conditions. In order to improve on past work with model RPLC stationary phases two challenges had to be overcome. First, improved vibrational mode assignments of the C18 stationary phase were needed for proper understanding of this model system. Second, the synthesis of back-surface reference mirrors used in these VR-SFG experiments allowed us to better correct the relative intensities of the various spectral peaks present in typical spectra. After examination of model RPLC systems under various conditions, we have found that these model substrates have a significant amount of interference from nonresonant signal. This interference of resonant and nonresonant signals on fused silica surfaces has not been previously examined and further studies of the model RPLC stationary phase must properly deal with the non-negligible nonresonant interference that is present. We have seen changes in the VR-SFG spectra of these model systems under a variety of conditions including elevated pressure, however the changes are mostly due to nonresonant interference. These spectral changes, although apparently not solely from structural changes, need to be investigated further to better understand the molecular basis of retention in model RPLC systems.