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Due to the side effects of overdosing, the therapeutic dose of warfarin preparations must be very strictly controlled. In order to make it easier for the patient to take the required dose, two different strategies can be followed: The medicine can be commercialized in different dosages and/or tablets can be scored in order to make them easy to split. The splitting of the tablets introduces the question of how to control that the fractions contain the desirable amount of warfarin. The regulations regarding the content uniformity of dosage unit for scored tablets have changed considerably in the last 10 years, and they are still evolving. Warfarin is commercialized under the trademark of Aldocumar in four different preparations, containing 1, 3, 5 and 10 mg sodium warfarin per tablet. All these tablets are also scored, thus suggesting the possibility of splitting. A quantitative Raman method has been developed for the determination of warfarin in tablets and in the potential fragments, taking into account the score lines on the tablet surface. This method is suggested as an auxiliary procedure to verify the uniformity of API distribution in dividable tablets. A combination of a second derivative and standard normal variate (SNV) was used as spectral pre-treatments, and partial least squares (PLS) as the regression algorithm. The relative standard deviation in API content among portions was found to be less than 5%. An HPLC procedure has been used as a reference analytical method.

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The determination of the antioxidant activity of Turnera diffusa using partial least squares regression (PLSR) on chromatographic data is presented. The chromatograms were recorded with a diode array detector and, for each sample, an enhanced fingerprint was constructed by compiling into a single data vector the chromatograms at four wavelengths (216, 238, 254 and 345 nm). The wavelengths were selected from a contour plot, in order to obtain the greater number of peaks at each of the wavelengths. A further pretreatment of the data that included baseline correction, scaling and correlation optimized warping was performed. Optimal values of the parameters used in the warping were found by means of simplex optimization. A PLSR model with four latent variables (LV) explained 52.5% of X variance and 98.4% of Y, with a root mean square error for cross validation of 6.02. To evaluate its reliability, it was applied to an external prediction set, retrieving a relative standard error for prediction of 7.8%. The study of the most important variables for the regression indicated the chromatographic peaks related to antioxidant activity at the used wavelengths.

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A strategy for multi-wavelength chromatographic fingerprinting of herbal materials, using high performance liquid chromatography with a UV-Vis diode array detector is presented. Valeriana officinalis was selected to show the proposed methodology since it is a widely used commercially available herbal drug, and because misfit with other valerian species is a current issue. The enhanced fingerprints were constructed by compiling into a single data vector the chromatograms from four wavelengths (226, 254, 280 and 326 nm), at which characteristic chemical constituents of studied herbs presented maximum absorbance. Chromatographic data pretreatment included baseline correction, normalization and correlation optimized warping. A simplex optimization was performed to retrieve the optimal values of the parameters used in the warping. General success rates of a classification above 90% were achieved by soft independent modeling of class analogy (SIMCA) and partial least squares discriminant analysis (PLS-DA). The sensitivity and specificity of constructed models were above 94%. Tests on laboratory-made mixtures showed that it is possible to detect adulterations or counterfeits with 5% foreign herbal material, even if it is from the Valerianaceae family. The results suggest that the proposed enhanced fingerprinting approach can be used to authenticate herb materials with complex chromatographic profiles.

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The use of a mixed calibration sample set (intact production tablets and powdered doped samples used to enlarge calibration range) is a usual procedure for the NIR reflectance determination of the API content of a pharmaceutical solid preparation. However, the high difference in scattering properties and the intrinsic low sensitivity of NIR make difficult the achievement of a good precision when API is at a low mass proportion (≈1%, w/w). The compression of the calibration powdered samples has been studied as a very simple procedure to enhance the sensitivity of NIR reflectance measurements and, consequently, to improve precision. Different pretreatments (SNV, 1D, 2D and their combinations) have been applied to reduce the spectral difference between powdered and compressed samples. Although none eliminates completely this difference, the combined pretreatment SNV+2D has proved to be the one with a better performance. Results obtained by using both calibration sample sets (powdered and compacted) in the quantification of estradiol valerate (VE, 2mg/tablet, ≈1.6%, w/w) and medroxyprogesterone (MPA, 10mg, ≈8%, w/w) in intact tablets of the hormonal preparation show that a slight but significant improvement in precision is obtained when using compacted samples for calibration. A HPLC procedure was developed to be used as reference method.

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A kinetic-spectrophotometric method for the determination of theophylline, dyphylline and proxyphylline, based on their azo coupling reaction with the diazonium ion of sulfanilic acid after a treatment with alkali, is proposed. The absorbance is recorded from 340 to 600 nm every second during reaction for 90 s, and calibration is performed by partial least-squares regression, using first derivative spectra values. Mixtures containing 2.5-13 micro g mL(-1) dyphylline and proxyphylline, and 2-9 micro g mL(-1) theophylline were successfully resolved with root mean squared errors of prediction (RMSEP) of 0.4, 0.3, and 0.2 for dyphylline, proxyphylline, and theophylline, respectively. The proposed method was satisfactorily applied to the determination of the three compounds in a commercially available pharmaceutical preparation and provided results similar to those obtained by HPLC.

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Calibrating near infrared diffuse reflectance spectroscopy (NIRS) methods usually involves preparing a set of samples with a view to expanding the analyte concentration range spanned by production samples. In this work, the performances of the two procedures most frequently used for this purpose in near infrared pharmaceutical analysis, viz., synthetic samples obtained by weighing of the pure constituents of the pharmaceutical and doped samples made by under- or overdosing previously powdered production samples, were compared. Both procedures were found to provide similar results in the quantification of the active compound in the pharmaceutical, which was determined with a relative standard error of prediction (RSEP) of < 1.6%. However, the two types of sample preparation provide different spectra, which precludes the accurate quantification of synthetic samples from calibrations obtained with doped samples and vice versa. None of the mathematical pre-treatments tested with a view to reducing this different scattering (viz., second derivative, standard normal variate and orthogonal signal correction) could effectively solve this problem. This hinders accurate validation of the linearity of the procedure and makes it advisable to use doped samples which are markedly less different to production samples.

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An empirical method for the selection of the best wavelength and time ranges which can be used in the quantification of binary mixtures, in a kinetic-spectrophotometric system, is proposed. It is based on finding those ranges which provide the least correlation between the kinetic profiles and the spectra of the products of reaction. The method was applied to the analysis of binary mixtures using simulated data with different rate constant ratios and in the presence of an interference that shows spectral overlap with the analytes. Subsequently, the proposed method was applied to the resolution of dyphylline and proxyphylline mixtures. The system studied was characterized by an elevated similarity in the kinetic behavior of the analytes under pseudo-first-order conditions and an elevated degree of spectral overlap of the products of reaction. In spite of this, satisfactory results were obtained in the quantification of the two analytes. The standard error of prediction (SEP) and the standard deviation between replicates (SDBR) did not show significant differences, being of the order of 4 and of 3% for dyphylline and proxyphylline, respectively.

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The fact that bitumens behave as non-Newtonian fluids results in non-linear relationships between their near-infrared (NIR) spectra and the physico-chemical properties that define their consistency (viz. penetration and viscosity). Determining such properties using linear calibration techniques [e.g. partial least-squares regression (PLSR)] entails the previous transformation of the original variables by use of non-linear functions and employing the transformed variables to construct the models. Other properties of bitumens such as density and composition exhibit linear relationships with their NIR spectra. Artificial neural networks (ANNs) enable modelling of systems with a non-linear property-spectrum relationship; also, they allow one to determine several properties of a sample with a single model, so they are effective alternatives to linear calibration methods. In this work, the ability of ANNs simultaneously to determine both linear and non-linear parameters for bitumens without the need previously to transform the original variables was assessed. Based on the results, ANNs allow the simultaneous determination of several linear and non-linear physical properties typical of bitumens.

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Pharmaceutical preparations usually contain preservatives and sweeteners. When parabens are used as preservatives and a polyol as a sweetener, a transesterification reaction may happen, yielding the transester polyol-paraben. The products formed in the transesterification reaction of methylparaben and sorbitol were analyzed by micellar electrokinetic chromatography and by HPLC. Up to six positional isomers of sorbitolparaben (SPB) can be produced. However, only three peaks were found by HPLC. The higher efficiency and resolution power of MEKC allowed one to resolve five peaks. Results were compared with those obtained by capillary zone electrophoresis in borate buffer, where the separation of isomers occurred in a different way, because of a complexation between SPB and borate.

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A new procedure for the analytical control of a pharmaceutical formulation by capillary zone electrophoresis (CZE) is proposed. It allows the simultaneous determination of the major compounds in the formulation: active compound (sodium picosulfate) and preservative (methylparaben), and the degradation products of the preservative, which slowly degrades by hydrolysis or by transesterification with sorbitol (sweetener in excess in the formulation) yielding p-hydroxybenzoic acid and sorbitolparaben, respectively. UV-Vis detection in the absorption maxima of the analytes and 20 mM borate solution at pH 10 as background electrolyte are used. Results are compared with those provided by the HPLC procedure. The method has also been validated using the HPLC procedure as the reference method, evaluating selectivity, accuracy, linearity and precision. The CZE procedure developed is sufficiently accurate and the precision achieved is about 1% for major and 3% for minor compounds.

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A procedure for the simultaneous kinetic-spectrophotometric determination of levodopa (I) and benserazide (II), from their oxidation reaction with KIO(4) in an acidic medium, is described. Both species instantly oxidize, giving rise to compounds which present maximum values of absorbance close to 400 nm. In the presence of an excess of the oxidizing agent, the levodopa derivative evolves to form the corresponding aminochrome (lambda(m)=480 nm), while the benserazide derivative decomposes to yield colorless compounds. The appearance of new compounds, with absorption bands in the region of 500-700 nm, is additionally seen upon adding the oxidizing agent to a mixture of I and II. These compounds also evolve decomposing and forming colorless products. In spite of the complexity of the system studied, the calibration by bi-linear partial least squares (PLS) as well as by three-way partial least squares (nPLS) permit the quantification of both analytes with a precision on the order of 0.7% for levodopa and of 1.5% for benserazide. nPLS also allows for the qualitative interpretation of the phenomena which occur. The proposed method is applied to the quantification of I and II in the commercial, pharmaceutical preparation Madopar, using high performance liquid chromatography (HPLC) as the analytical reference technique.

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A near infrared diffuse reflectance spectroscopy (NIRS) procedure for the quantitative control analysis of the active compound (otilonium bromide) in a pharmaceutical preparation in three steps of the production process (blended product, cores and coated tablets) and a methodology for its validation are proposed. The analytical procedure is composed by two consecutive steps. First, the sample is identified by comparing its spectrum with a second derivative spectral library. If the sample is positively identified, the active compound is quantified by using a previously established partial least squares (PLS) calibration model. The procedure was validated by studying repeatability, intermediate precision, accuracy and linearity. To this end, an adaptation of ICH (International Conference on Harmonisation) validation methodology to an NIR multivariate calibration procedure is proposed. The relative standard error of prediction (RSEP) was < or = 1% and the suitability of the procedure for control analysis was confirmed by the results obtained analysing new production samples produced over a three-month period.

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A near-infrared (NIR) spectroscopic method based on the use of a fiber optical probe for the analysis of a commercially available pharmaceutical preparation is proposed. The analyte is identified by comparison with a second-derivative spectral library, using the correlation coefficient as the discriminating parameter. Once a sample has been positively identified, the active principle is quantified with partial least-squares (PLS) calibration. The proposed method was validated for use as a control method; to this end, the selectivity of the identification process, and the repeatability, intermediate precision, accuracy, linearity, and robustness of the active principle quantitation, were assessed.

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New methods for the determination of the nominal content of miokamycin in three commercial pharmaceutical preparations available in many different forms are proposed. Solid samples, grinding of which is the sole pretreatment required, are analysed by near infrared (NIR) spectroscopy, using a fibre-optic probe. The active principle is quantified by partial least-squares regression (PLSR). The three proposed methods were validated with a view to their use as control methods; the selectivity of the method, and the repeatability, intermediate precision, accuracy, linearity and robustness of each PLSR calibration model used were determined. The relative standard error of prediction (RSEP) was < 1.5% and the validation results testify to the suitability of the proposed methods.

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A kinetic spectrophotometric method for the determination of hydrocortisone acetate based on its condensation with isonicotinic acid hydrazide is proposed. The method is applied to the determination of hydrocortisone acetate in a commercially available pharmaceutical preparation, presented as a pomade, that also contains another corticosteroid and additional active compounds. The operating procedure involves dissolving the pomade in chloroform and the addition of the reagent solution directly to the cuvette, in this way avoiding the previous extraction of analytes from the insoluble pomade matrix required by the alternative HPLC procedure. Calibration is performed by partial least-squares regression, using absorbance or first derivative spectra values recorded each minute during the first 30 min of reaction. Use of first derivative spectra overcomes possible scattered light problems produced by excipients precipitating, and produced slightly better results than absorbance data. The relative standard deviation obtained for 11 replicates analysed on different days was approx. 1.5%. The proposed method improves both accuracy and precision of the classical initial rate method and the precision of the HPLC procedure.

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The new method for the enantiomeric resolution of various 2-arylpropionic acids by capillary zone electrophoresis (CZE) using heptakis-tri-O-methyl-beta-cyclodextrin as chiral selector was applied to the determination of ketoprofen in different commercially-available pharmaceutical preparations. The analyte was determined under chiral and nonchiral conditions (viz. in the presence and absence of 50 mM heptakis-tri-O-methyl-beta-cyclodextrin in the background electrolyte), with significantly similar results and relative standard deviations from 1.2 to 6.5% in both cases. The limits of detection and determination for the inactive enantiomer, R-(-)-ketoprofen, were calculated to be 7.0 x 10(-7) and 1.6 x 10(-6) M, respectively. The proposed method was successfully used to determine enantiomeric purity in the drugs studied, with results comparable to those provided by the chiral HPLC method.

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A method for resolving the enantiomers of various 2-arylpropionic acids (viz. ketoprofen, ibuprofen and fenoprofen) by capillary zone electrophoresis (CZE) using a background electrolyte (BGE) containing a cyclodextrin as chiral selector is proposed. The effects of the type of cyclodextrin used and its concentration on resolution were studied and heptakis-2,3,6-tri- O-methyl-beta-cyclodextrin was found to be the sole effective choice for the quantitative enantiomeric resolution of all the compounds tested. The influence of pH, BGE concentration, capillary temperature and addition of methanol to the BGE on resolution and other separation-related parameters was also studied. The three compounds studied can be enantiomerically resolved with a high efficiency in a short time (less than 20 min) with no capillary treatment. This makes the proposed method suitable for assessing the enantiomeric purity of commercially available pharmaceuticals.

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A new method for analysis of the active compound in a commercial pharmaceutical formulation in different steps of the production cycle, based on near-infrared diffuse reflectance spectroscopy, is proposed. The analysis includes three different steps of the production cycle: granules ready for compression (mixed phase), cores (intermediate) and coated tablets (final product). Satisfactory predictive results for production samples, independently of its origin in the production cycle, require calibration with laboratory-made samples covering the concentration range involved in the manufacturing process, and also production samples from various production batches and different steps, which introduce the variation sources inherent in such a process. A global and sole model was found to determine the active compound during the production cycle, with errors of prediction less than 1.8% in all cases. Tablets can be individually analysed with high accuracy and precision, so a content uniformity analysis may be performed.

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Near infrared diffuse reflectance spectroscopy with a fibre-optic probe was used to determine the water content in ferrous lactate dihydrate. Spectra were recorded by immersing the probe in a beaker containing the ferrous lactate sample. Spectral data were processed by using two different multivariate calibration procedures, viz. stepwise multiple linear regression (SMLR) and partial least-squares regression (PLSR). The results provided by the two calibration procedures were similar and departed by less than 1.5% from the values obtained by Karl Fischer titration.

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