Resumen La determinación de aminoácidos (AA) en granos de cacao es de gran interés ya que estos son considerados como unos de los precursores de su sabor y aroma. Por esta razón, el presente trabajo tuvo como objetivo optimizar y validar un método por cromatografía líquida con detección DAD a 254 nm para la determinación selectiva de AA durante la fermentación y tostado en seis variedades de granos de cacao con diferentes genotipos, todos estos cultivados en Venezuela. Los AA se extrajeron del polvo de cacao molido y desgrasado con agua pura a 70 ºC, utilizando la técnica de ultrasonido. Luego, se derivatizaron con fenilotiocianato para separar sus derivados con buena resolución en menos de 29 min en una columna de fase reversa, utilizando gradiente de elución. Se analizaron 110 muestras de cacao. Los resultados mostraron un contenido significativo de AA libres, entre 3,87 y 5,97 g/kg, en ausencia de fermentación con predominio de AA ácidos, y un aumento progresivo en el contenido de AA, mientras ocurre el proceso de fermentación, con un predominio de AA hidrófobos como alanina, valina, isoleucina, leucina, fenilalanina y tirosina. Además, todos los tipos de cacao mostraron una degradación parcial de AA libres durante la etapa de tostado, especialmente los AA hidrófobos.

Abstract Amino acids (AA) composition in cocoa beans can predict the synthesis of compounds which affect cocoa flavor. Thus, their determination is of great interest for the community implied in the commercialization and production of cocoa. In consequence, in this work, the analysis of AA produced during cocoa beans fermentation and roasting was carried out. A high-performance liquid chromatographic method with DAD detection at 254 nm was optimized and validated for their selective determination in six varieties of cocoa beans with different genotypes, all of them grown in Venezuela. AA were extracted by defatted milled cocoa powder ultrasonication using purified water at 70 °C. Then, they were derivatized with phenyl isothiocyanate, and their derivatives were separated, using a reversed-phase column with gradient elution, achieving a satisfactory resolution among the peaks (greater than 1.0) in less than 29 min. 110 cocoa samples were analyzed. Results showed a significant content of free AA, ranging from 3.87 to 5.97 g/kg in absence of fermentation with a predominance of acidic AA. Moreover, there is a progressive increase in the AA content while fermentation process occurs, with a predominance of hydrophobic AA such as alanine, valine, isoleucine, leucine, phenylalanine, and tyrosine. On the other hand, all cocoa types showed a partial degradation of free AA during the roasting step, especially the hydrophobic ones.

Resumo A determinação dos aminoácidos (AA) nos grãos de cacau é importante, pois são considerados um dos precursores de seu sabor e aroma. Neste trabalho, um método foi otimizado e validado por cromatografia líquida com detecção DAD a 254 nm para a determinação seletiva de AA durante a fermentação e torrefação em seis variedades de grãos de cacau com diferentes genótipos, todos cultivados na Venezuela. Os AAs foram extraídos do pó de cacau moído e desengordurados com água pura a 70 ºC usando a técnica de ultrassom. Em seguida, foram derivatizados com feniltiocianato, e os derivados foram separados com boa resolução em menos de 29 minutos em uma coluna de fase invertida usando eluição em gradiente. Foram analisadas 110 amostras de cacau. Os resultados mostraram um conteúdo significativo de AA livre entre 3,87 e 5,97 g/kg na ausência de fermentação com predominância de AA ácidos e um aumento progressivo no conteúdo de AA enquanto o processo de fermentação ocorre com predominância de AA hidrófobos como alanina, valina, isoleucina, leucina, fenilalanina e tirosina. Além disso, todos os tipos de cacau apresentaram uma degradação parcial do AA livre durante a fase de torrefação, principalmente o AA hidrofóbico.

Resumo O desenvolvimento do sabor e do aroma do cacau é um fenômeno complexo que depende de como os processos de fermentação e torrefação são realizados. Durante a fermentação, ocorre a formação de alguns compostos chamados “precursores de aroma e sabor”, que são finalmente expressos durante a fase de torrefação. Portanto, a avaliação de precursores de aroma, como os aminoácidos formados durante a fermentação, é crucial para determinar neste contexto como o método foi desenvolvido e validado para o estudo desses compostos em amostras de cacau, sendo os aminoácidos convertidos quantitativamente em seus derivados de trimetilsilil antes de serem determinados por cromatografia gasosa com detecção por espectrometria de massa. Os resultados foram verificados por meio de estudos de precisão e acurácia. Os coeficientes de variação inter e intraensaio (CV; n = 5) foram inferiores a 4,7% e 4%, respectivamente. As recuperações analíticas (95% to 108% com CV < 4,2; n = 5) demonstraram alto desempenho do procedimento de extração. O método foi aplicado com sucesso na análise dos aminoácidos em 110 amostras de cacau venezuelano Criollo durante os três dias de fermentação e torrefação (110 °C por 25 min). Todas as amostras estudadas apresentaram um conteúdo apreciável de aminoácidos livres variando entre 3,87 e 5,97 g/kg na ausência de fermentação. A degradação dos aminoácidos ácidos foi observada durante o primeiro dia de fermentação, enquanto os demais aminoácidos aumentaram progressivamente durante o processo de fermentação com predomínio dos hidrofóbicos, principalmente leucina, fenilalanina, valina, alanina e isoleucina. Além disso, durante a fase de torrefação, uma fração dos aminoácidos, especialmente os hidrofóbicos, foi parcialmente degradada pela reação de Maillard para formar os compostos associados ao aroma e ao sabor do cacau.

Abstract The development of cocoa flavor and aroma is a complex phenomenon that depends on how the fermentation and roasting processes are carried out. During fermentation, the formation of some compounds so-called “aroma and flavor precursors” takes place, which are finally expressed during the roasting stage. Therefore, the evaluation of aroma precursors such as the amino acids formed during fermentation is crucial in order to determine the quality of the cocoa. In this context, we developed and validated a method for the study of these compounds in cocoa samples. The amino acids were quantitatively converted into their trimethylsilyl derivatives before their determination by gas chromatography with mass spectrometry detection. The results were verified performing precision and accuracy studies. The inter and intra assay coefficients of variation (C.V, n = 5) were lower than 4.7% and 4%, respectively. The analytical recoveries (95% to 108% with C.V < 4.2, n = 5) demonstrated the high performance of the extraction procedure. The method was successfully applied to the analysis of the amino acids in 110 samples of Venezuelan Criollo cocoa during the three days of fermentation and roasting (110 °C for 25 min). All samples had an appreciable content of free amino acids ranging between 3.87 and 5.97 g/kg in the absence of fermentation. We observed degradation of the acidic amino acids during the first day of fermentation, while the rest of amino acids increased progressively during the fermentation process with a predominance of the hydrophobic ones, mainly leucine, phenylalanine, valine, alanine and isoleucine. Additionally, during the roasting stage a fraction of the amino acids, especially the hydrophobic ones, was partially degraded through Maillard reaction to form the compounds associated with the cocoa aroma and flavor.

A selective and sensitive gas chromatography method with mass spectrometric detection (GC/MS) was optimized and validated for the determination of fructose , glucose and sucrose in cocoa beans. Analytes were extracted by ultrasonication from defatted milled cocoa powder using purified water (18 Ω) at 70 ºC. Then, these were derivatized with N-methyl-trimethylsilyl trifluoacetamida (MSTFA) assisted by microwave irradiation at a power of 630 W for 60 s, in order to achieve its volatility. Under optimal conditions , the method provided quantitative recoveries of analytes (98-108 %, CV < 3,68 % n = 5) and a precision with a CV < 3,31 % (n = 5). Ninety samples of four varieties, with different genotypes, of cocoa beans grown in Venezuela , taken before, during and after the fermentation and roasting stages, were analyzed.

Se optimizó y validó un método sensible y selectivo por cromatografía de gases con detección por espectrometría de masas (CG-EM) para la determinación de fructosa, glucosa y sacarosa en muestras de cacao. Los analitos se extrajeron desde el polvo de cacao desgrasado y molido con agua (70 ºC) y sonicación durante 15 minutos. Seguidamente se derivatizaron con con N-metil-trimetilsilil-trifluoacetamida (MSTFA) utilizando irradiación con microondas a una potencia de 630 W durante 60 s para lograr su volatilidad. Bajo las condiciones óptimas , el método proporcionó recuperaciones cuantitativas de los analitos (98-108 %, CV < 3,68 % n = 5) y una precisión con un CV < 3,31 % (n = 5). Se analizaron 90 muestras de diferentes variedades de cacao criollo venezolano tomadas antes, durante y después de los procedimientos de fermentación y tostado.

En el presente trabajo se evaluó algunos parámetros asociados con los compuestos de aroma y sabor del cacao , Theobroma cacao L., Criollo Merideño (CM) cultivado bajo dos condiciones edafoclimáticas. Un primer lote establecido en el Campo Experimental San Juan de Lagunillas del Instituto Nacional de Investigaciones Agrícolas (INIA-Mérida), Venezuela , a una altura de 1.050 m.s.n.m con temperatura media anual de 26 ºC y otro lote ubicado en la población Zea del mismo estado, a 850 m.s.n.m y 22 ºC, respectivamente. Los análisis se efectuaron en muestras de cotiledones frescos durante el proceso de fermentación. En ambos casos se evaluó el porcentaje de: cascarilla, humedad , grasa y punto de fusión , contenido de ácidos volátiles, azúcares totales, polifenoles, purinas totales, teobromina, cafeína y pirazinas. Los resultados muestran que el cacao CM en San Juan de Lagunillas (SJL), presentó menor acidez titulable al inicio del secado y volátil al final de la fermentación, en comparación con el CM en Zea. Así mismo, se observó ligeras diferencias en el contenido de grasas, azúcares totales , pirazinas, cafeína y teobromina, lo que indica que las condiciones edafoclimáticas generaron cambios en el comportamiento químico de los precursores de aroma y sabor . Todos los parámetros químicos fueron corregidos en función de la humedad de la muestra durante el proceso de fermentación y relacionados con una muestra de cacao fermentado Forastero de Ghana

The present study evaluated some parameters associated with the aroma and fl avor compounds in cocoa, Theobroma cacao L., Merideño Creole (MC) grown under soil and climatic conditions. A lot set in the Experimental Station of the San Juan de Lagunillas (INIA-Mérida), Venezuela at an altitude of 1,050 m.a.s.l. and average annual tempe rature of 26 ºC and another in the town of Zea, Zea municipality of Mérida and 850 m.a.s.l. 22 ºC average tempe rature. Tests were conducted on samples of fresh cotyledons during the fermentation process. Both cases evaluated the percentage of fi ber, moisture, fat and its melting point, content of volatile acidity, total sugars, polyphenols, total purines, theobro mine, caffeine and pyrazine. The results show that the MC cacao in San Juan of Lagunillas, a low acidity at the start of drying compared to MC in Zea as well as a less volatile acidity at the end of fermentation. It was also noted slight differences in fat content, total sugars, pyrazine and content of caffeine and theobromine, indicating that soil and climatic conditions have led to changes in the chemical behavior of the precursors of aroma and fl avor. All chemical parameters were corrected according to the moisture of the sample during the fermentation process and were associated with a sample of fermented cocoa of Ghana

En este trabajo se desarrolla una propuesta metodológica alternativa, simple y rápida, para la determinación de atenolol en productos farmacéuticos. El método se basa en la disolución, o extracción, del principio activo con cloroformo y análisis directo de los estándares y muestras por espectrometría infrarroja con transformada de Fourier. La cuantificación del analito se llevó a cabo, en paralelo, en las bandas del analito centradas en 1512 (banda principal) y 1300 cm-1, utilizando como señal analítica la absorbancia a 1512 y 1300 cm-1, respectivamente, corregida mediante líneas de base apropiadas. Las figuras de mérito del método en la banda principal (intervalo de trabajo: 0,1-1,0% (p/v), límite de detección (3σ): 0,004% (p/v); límite de cuantificación (10 σ): 0,0125% (p/v), precisión: 0,6% y una frecuencia de análisis de 30 muestras h-1) resultan adecuadas para realizar el análisis propuesto. La exactitud del método se certificó mediante análisis comparativos de muestras reales con el método oficial descrito en la Farmacopea de los Estados Unidos de América (USP). Se analizaron, de forma satisfactoria, diversos productos farmacéuticos que se comercializan en el país. En todos los casos, la concentración encontrada está en el rango de tolerancia aceptado por la USP.

In this work, a simple and rapid alternative method for determining atenolol in solid pharmaceutical dosages has been developed. The method is based on the dissolution, or extraction, of the active principle with chloroform and direct analysis of standard and sample solutions by FTIR spectrometry. Quantification of the analyte was carried out at the 1512 (principal band) and 1300 cm-1 bands, using as analytical signal the absorbance at 1512 and 1300 cm-1, respectively, corrected by mean appropriate baselines. The main figures of merit at the principal band (working range 0.1-1.0% (w/v), limit of detection (3 σ): 0.004% (w/v), limit of quantification (10 σ): 0.0125% (w/v), precision: 0.6% and sample throughput of 30 samples h-1) are attractive and really adequate for the proposed analysis. The accuracy of the method was certified by means comparative analysis of real samples using the official method described by the United Stated of America Pharmacopeia (USP). The method was satisfactorily applied to the determination of atenolol in a series of commercially available solid pharmaceutical samples. All the values obtained for the commercial samples were within the limit prescribed by the official pharmacopoeia.

In the present work, the development of a method based on the coupling of flow analysis (FA), hydride generation (HG), and derivative molecular absorption spectrophotometry (D-EAM) in gas phase (GP), is described in order to determine total antimony in antileishmanial products. Second derivative order (D²224nm) of the absorption spectrum (190 - 300 nm) is utilized as measurement criterion. Each one of the parameters involved in the development of the proposed method was examined and optimized. The utilization of the EAM in GP as detection system in a continuous mode instead of atomic absorption spectrometry represents the great potential of the analytic proposal.

En el presente trabajo se desarrolló un método para determinar cafeína en café, basado en un sistema de análisis en flujo con detección por espectrometría infrarroja de transformada de Fourier (AF-IRTF). El método involucra la extracción de la cafeína, fuera de línea, a partir de la muestra de café sólido con agua a 80°C, la retro-extracción de la cafeína -en línea- en cloroformo y el análisis directo de la fase orgánica por IRTF. El criterio de medida seleccionado fue el valor absoluto de la medida pico-valle (1668-1649 cm-1) de la derivada de primer orden del espectro de absorción en la banda de 1659 cm-1. El límite de detección fue de 0,001% (p/v), la desviación estándar relativa del procedimiento completo, desde la preparación de la muestra, de 1,2% ([Cafeína] » 0,05% (p/v), n = 5) y la frecuencia de análisis real (triplicado), de aproximadamente 12 muestras h-1. El método se validó mediante estudios de adición de estándar, recuperación y precisión entre días; así como por análisis comparativos con el método oficial para el análisis de cafeína en café. Se analizaron 20 muestras comerciales de café tostado, encontrando concentraciones de cafeína en el intervalo de 0,60 a 1,16% (p/p).

In this work a flow analysis-Fourier transform infrared spectrometric (FA-FTIR) method for the determination of caffeine in coffee was developed. The method involves the extraction, in batch, of caffeine from solid samples of roasted coffee with hot water (80°C), on-line retro-extraction of caffeine in chloroform and direct analysis of the organic phase by FTIR spectrometry. The measurement criterion selected for the quantification of caffeine was the absolute peak-valley value (1668-1649 cm-1) of the first order derivate of the absorption spectrum, corresponding to the 1659 cm-1 band. The method provides a limit of detection (3s) of 0.001% (w/v) caffeine, a relative standard deviation corresponding to the whole procedure, until the sample preparation, of 1.2% ([Caffeine] » 0.05% (w/v), n = 5) and a sample throughput (triplicate) of approximately 12 samples h-1. The method was validated by means of standard addition, recovery and inter-day precision studies and by comparative analysis of real samples with the official chromatographic-spectrophotometric procedure. The method was applied to the analysis of commercial samples, obtaining a caffeine content ranging from 0.60 to 1.16% (w/w).

En el presente trabajo se hace una revisión crítica de los trabajos que han aparecido en la literatura especializada sobre la generación de fases gaseosas en línea, y su determinación vía espectrometría infrarroja de transformada de Fourier (IR-TF). Se examinan diversos procedimientos analíticos desarrollados para la determinación de alcoholes y carbonatos en varias matrices, mezclas de tolueno, benceno y metil, tert-butil éter en gasolinas, y acetato de butilo, tolueno y metil etil cetona en solventes comerciales para pintura; cuyas Figuras de mérito son adecuadas para las aplicaciones propuestas. En todos los casos, los resultados reportados en los trabajos revisados muestran una buena coincidencia con aquellos obtenidos mediante métodos de referencia. También se presenta el desarrollo reciente del acoplamiento del análisis en flujo con generación de hidruros y detección por IR-TF para la determinación individual y simultánea de antimonio, arsénico y estaño, así como algunas aplicaciones propuestas para esta técnica. Todo esto con la finalidad de mostrar la real potencialidad analítica que presenta la técnica de generación de fases gaseosas con detección por espectrometría IR-TF en el análisis de muestras reales.

In this work; a critical review about on-line gaseous phases generation with Fourier transform infrared (FTIR) spectrometry detection is presented. In this sense, different experimental approaches for the determination of ethanol and carbonates in different samples, and for the simultaneous determination of benzene, toluene and methyl tert-butyl ether in gasoline; and butyl acetate, toluene and methyl ethyl ketone in commercial paint solvents has been examined. In all these methods, the figures of merit are adequate for the proposed applications, and the results are in good agreement with those obtained by diverse reference procedures. Also, it is presented the recent development of the flow analysis-hydride generation-FTIR coupling for the individual and simultaneous determination of antimony, arsenic and tin, as well as some applications proposed for this technique. All this procedures are focused to demonstrate the real analytic potentiality depicted by the on line gaseous phase generation-FTIR spectrometry technique for the analysis of real samples.