Analysis of chemosensory markers in cigarette smoke from different tobacco varieties by GC×GC-TOFMS and chemometrics.

Commercial cigarettes are made from a blend of different tobacco varieties, which in turn are the results of different agronomic practices and post-harvest curing processes. The highly complex mixture of smoke compounds reflects each tobacco variety and the levels of sensory-relevant markers. Therefore, the aim of this work was to identify potential relevant chemosensory markers in the mainstream smoke of four main types of commercial tobaccos and establish any possible relationship between them and the tobacco growing/curing practices. The tobacco samples were segregated into four segments: (1) three curing stages of flue-cured Virginia, (2) three curing stages of air-cured Burley, (3) three geo-regions of sun-cured Oriental and (4) three different process applied to tobacco. One hundred and twenty cigarettes (10 batches per flavour category) were produced and smoked under standard machine-smoking protocols. The mainstream smoke samples collected were extracted and analysed by GC × GC TOFMS. The processed data was analysed by partial least square discriminant analysis (PLS-DA) and the selectivity ratio was used to identify key chemosensory markers responsible for the four segments. All models had sensitivity and specificity equal to unity. Flue-cured Virginia (193 markers) and air-cured Burley (184 markers) showed a similar trend for O-heterocycles markers in the lighter leaf colours and N-heterocycles in the darker leaf colours post-processing, but they had compounds of different flavour descriptions, e. g. sweet and nutty. The three geo-regions of sun-cured Oriental (290 markers) also presented O-heterocycles markers in correlation with leaf sugar contents in addition of sucrose esters markers. The three unusually processed tobacco generated many chemical markers (436 markers), some derived from the so-called Cavendish fermentation process with sweet, spicy and peppery notes, whereas the dark fermented air-cured tobacco presented similar descriptors as air-cured Burley. In addition, some polycyclic aromatic hydrocarbons (PAH) were detected as markers from the fire-curing process. The PLS-DA with selectivity ratio evidenced total of 1098 chemosensory markers in cigarette smoke, in which 173 were tentatively identified.

High-throughput simultaneous quantitation of multi-analytes in tobacco by flow injection coupled to high-resolution mass spectrometry.

The high-throughput screening by flow injection coupled to high-resolution mass spectrometry (HTS-FIA-HRMS) is a powerful technique that enables the identification of several types of samples in a short period of time, either with qualitative or quantitative purposes. Sensory attributes of tobacco are affected by its chemical composition, and it is very important to quantify multi-analytes in a high-throughput methodology. HTS-FIA-HRMS coupled to multivariate analysis was used to create calibration models for 27 analytes, or group of compounds, of tobacco sensory interest. The models were validated by different approaches, including permutation test to avoid overfitting, evaluation of the equipment repeatability by control samples, reproducibility comparison of results from two different equipment and analysts, and with a blind test analysis. All tests demonstrated a good response to the proposed method. No statistical difference between the errors of both equipment was observed, with less than 7% error from the control samples, and a blind test error between 5.96% and 20.10%. The partial least squares (O-PLS) regression models were applied to 815 samples, and a principal component analysis (PCA) was performed from the predicted concentration values, aiming at the non-supervised classification based on tobacco type. We expect that this proposed methodology shows not only the applicability in tobacco samples, but also demonstrates a guideline to an efficient performance of multi-analytes target analysis using the flow injection mass spectrometry with reliable and robust validation steps.

Innovative Approaches for Estimating the Levels of Tobacco-Specific Nitrosamines in Cured Tobacco Samples.

Tobacco-specific nitrosamines (TSNAs), mainly the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are known carcinogens. Part of the NNK found in smoke is provided from matrix-bound NNK, and its determination is extremely relevant. However, the reference extraction procedure of matrix-bound NNK is time-consuming and labor-intensive and has a limited analytical capacity. Three different methodologies were proposed to predict matrix-bound NNK: simple linear regression (LR) with soluble NNK; multiple linear regression (MLR) considering soluble NNK and characteristic parameters of the samples; and orthogonal partial least-squares (O-PLS) regression using high-throughput screening by flow injection analysis coupled to high-resolution mass spectrometry (HTS-FIA-HRMS) data. Simple linear regression showed a high influence of matrix and leaf origin. Although an existing linearity trend has been observed ( R2 = 0.62) for the global model, higher correlation values were achieved for matrix and country segregation models. Multiple linear regression predicted matrix-bound NNK with more satisfactory efficiency than simple linear regression models. The coefficients of determination were 0.87 and 0.94 for flue-cured Virginia and air-cured Burley, respectively. However, this method has a limited application, since previous information about the sample is required. The proposed method based on HTS-FIA-HRMS and O-PLS has shown the most suitable performance in the prediction of matrix-bound NNK, with errors comparable to the reference method, and a higher throughput. In addition, this approach allows to determine other soluble nitrosamines, namely N'-nitrosoanatabine, N'-nitrosoanabasine, and N-nitrosonornicotine, with relative percentage errors between 5.25 and 11.98%. Therefore, the third approach is the best method for a large number of cured tobacco for accuracy in determination of TSNAs.

Characterization of sildenafil citrate tablets of different sources by near infrared chemical imaging and chemometric tools.

The chemical imaging technique by near infrared spectroscopy was applied for characterization of formulations in tablets of sildenafil citrate of six different sources. Five formulations were provided by Brazilian Federal Police and correspond to several trademarks of prohibited marketing and one was an authentic sample of Viagra. In a first step of the study, multivariate curve resolution was properly chosen for the estimation of the distribution map of concentration of the active ingredient in tablets of different sources, where the chemical composition of all excipients constituents was not truly known. In such cases, it is very difficult to establish an appropriate calibration technique, so that only the information of sildenafil is considered independently of the excipients. This determination was possible only by reaching the second-order advantage, where the analyte quantification can be performed in the presence of unknown interferences. In a second step, the normalized histograms of images from active ingredient were grouped according to their similarities by hierarchical cluster analysis. Finally it was possible to recognize the patterns of distribution maps of concentration of sildenafil citrate, distinguishing the true formulation of Viagra. This concept can be used to improve the knowledge of industrial products and processes, as well as, for characterization of counterfeit drugs.

Characterization of semi-solid Self-Emulsifying Drug Delivery Systems (SEDDS) of atorvastatin calcium by Raman image spectroscopy and chemometrics.

A methodology based on Raman image spectroscopy and chemometrics for homogeneity evaluation of formulations containing atorvastatin calcium in Gelucire(®) 44/14 is presented. In the first part of the work, formulations with high amounts of Gelucire(®) 44/14 (80%) and solvents of different polarities (diethylene glycol monoethyl ether, propyleneglycol, propylene glycol monocaprylate and glyceryl mono/dicaprylate/caprate) were prepared for miscibility screening evaluation by classical least squares (CLS). It was observed that Gelucire(®) 44/14 presented higher affinity for the lipophilic solvents glyceryl mono/dicaprylate/caprate and propylene glycol monocaprylate, whose samples were observed to be homogeneous, and lower affinity for the hydrophilic solvents diethylene glycol monoethyl ether and propyleneglycol, whose samples were heterogeneous. In the second part of the work, the ratio of glyceryl mono/dicaprylate/caprate and Gelucire(®) 44/14 was determined based on studies in water and allowed the selection of the proportions of these two excipients in the preconcentrate that provided supersaturation of atorvastatin upon dilution. The preconcentrate was then evaluated for homogeneity by partial least squares (PLS) and an excellent miscibility was observed in this proportion as well. Therefore, it was possible to select a formulation that presented simultaneously homogeneous preconcentrate and solubility enhancement in water by Raman image spectroscopy and chemometrics.

Fingerprinting of sildenafil citrate and tadalafil tablets in pharmaceutical formulations via X-ray fluorescence (XRF) spectrometry.

The production of counterfeited drugs is a criminal problem that carries serious risks to public health in the worldwide. In Brazil, Viagra and Cialis are the most counterfeit medicines, being used to inhibit the phosphodiesterase type 5 (PDE-5), treating thus, problems related to erectile dysfunction. X-ray fluorescence (XRF) is a suitable technique to control the quality of new pharmaceutical formulations and distinguish between authentic and counterfeit tablets. XRF has advantageous features like multielemental capability, good detectivity, high precision, short analysis times, and is nondestructive, which makes it suitable to be extended to a great variety of samples. In this work, the inorganic fingerprinting chemical of forty-one commercial samples (Viagra, Cialis, Lazar, Libiden, Maxfil, Plenovit, Potent 75, Rigix, V-50, Vimax and Pramil) and fifty-six counterfeit samples (Viagra and Cialis) were obtained from XRF data. XRF presented an excellent analytical methodology for semi-quantitative determination of active ingredient (in case of sildenafil citrate that presents S in its structure) and excipients such as calcium phosphate, titanium oxide and iron oxide (P, Ca, Ti and Fe). The matrix data were allied to chemometric methods (Principal Component Analysis and Hierarchical Cluster Analysis) to classify the tablets investigated between authentic and counterfeit, grouping the samples into of seven groups: A, B, C, D and E (counterfeit group) and F and G (authentic group).

Analysis of pharmaceutical pellets: an approach using near-infrared chemical imaging.

Pharmaceutical pellets are spherical or nearly spherical multi-unit dosage forms designed to optimize pharmacokinetics and pharmacodynamics features of drug release. The distribution of the pharmaceutical ingredients in the layers and core is a very important parameter for appropriate drug release, especially for pellets manufactured by the process of layer gain. Physical aspects of the sample are normally evaluated by Scanning Electron Microscopy (SEM), but it is in many cases unsuitable to provide conclusive chemical information about the distribution of the pharmaceutical ingredients in both layers and core. On the other hand, methods based on spectroscopic imaging can be very promising for this purpose. In this work, a Near-Infrared Chemical Imaging (NIR-CI) method was developed and applied to the analysis of diclophenac sodium pellets. Since all the compounds present in the sample were known in advance, Classical Least Squares (CLS) was used for calculations. The results have shown that the method was capable of providing chemical information about the distribution of the active ingredient and excipients in the core and coating layers and therefore can be complementary to SEM for the pharmaceutical development of pellets.