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In this paper, a method was described to determine cocaine (COC) and benzoylecgonine (BZE) in human urine samples by GC-MS detection. The extraction of analytes from urine samples was achieved in an Oasis hydrophilic-lipophilic balance column (20 mmx3.9 mm id, dp=25 microm; Waters, USA), incorporated in a multisyringe flow injection system, used for the sample treatment. Finally, to improve the volatility of the BZE, an in-line derivatization reaction with N,O-bis (trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane was made microwave-assisted in order to reduce the reaction time. The results showed that the proposed method is a good alternative for the analysis of COC and BZE in urine samples because it offers advantages compared with those described in the literature, which include simplicity in the sample treatment, the sensitivity and selectivity necessary to determine the analytes of interest at low levels in the urine and high sample throughput.

A column-switching high-performance liquid chromatographic method with UV detection for the determination of cocaine (COC) and benzoylecgonine (BZE) in human blood plasma samples is described. The method uses an alkyl-diol-silica ADS-C18 extraction precolumn. A 50- micro L plasma sample was introduced to the ADS precolumn in order to separate the analytes from proteins and endogenous compounds. The fraction containing COC and BZE was back-flushed and transferred to an Alltech mixed-mode C(18)/cation-exchange analytical column for final separation. The validation of the method revealed quantitative recoveries from 95.0 to 99.0% for COC at three different concentrations (0.5, 1.0 and 2.0 micro g mL(-1)), and from 96.0 to 99.0% for BZE at the same concentration levels with coefficients of variation <4.00% (n=5). The detection limit (signal to noise ratio (S/N)>3) was 0.03 micro g mL(-1) for all the compounds with an injection volume of 50 micro L. However, it was possible to enhance the sensitivity further by injecting larger plasma volumes, up to 200 micro L, at the same optimal conditions. The overlap of sample preparation, analysis and reconditioning of the extraction column, increase the overall sample throughput to 5 samples h(-1). The developed method has been applied to human blood plasma samples from subjects suspected of cocaine abuse.

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.

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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 ºC. 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.

This study describes a simple and sensitive column-switching high-performance liquid chromatographic method with UV detection for the determination of Lamotrigine in 50 microL of serum. After solid-phase extraction of Lamotrigine on an Oasis HLB extraction precolumn (20 x 3.9 mm; dp: 25 microm), chromatographic separation was achieved at 30 degrees C on a Chromolith RP-18e column (50 mm x 4.6 mm i.d.) using a solution of 20% acetonitrile in 15 mM phosphate buffer (pH 7.0) as the mobile phase, at a flow-rate of 2.0 mL/min. The eluant was detected at 215 nm. The retention time for Lamotrigine was 1.28 min. The total analysis time was ca. 5 min. However, the overlap of sample preparation, analysis, and reconditioning of the precolumn increased the overall sample throughput to one injection every 3 min. The method was validated for system suitability, linearity, precision, accuracy, robustness, and limit of quantitation. The linearity of the calibration lines, expressed by the linear correlation coefficient, was better than 0.9996. Recovery studies achieved from Lamotrigine spiked plasma samples showed values greater than 93%, demonstrating the excellent extraction efficiency of the precolumn. Intra- and inter-day precision were generally acceptable; the coefficient of variation was < 2.3% in all cases. The detection limits for Lamotrigine at a signal-to-noise ratio of 3 was 0.002 microg/mL when a sample volume of 50 microL was injected. However, it was possible to enhance the sensitivity further by injecting larger volumes, up to 200 microL. The method was shown to be robust and the results were within the acceptable range. The method was successfully applied to the determination of Lamotrigine in human serum samples of patients submitted to Lamotrigine therapy.