Robustness of a continuous direct compression line against disturbances in feeding.

The aim of the current study was to characterize the robustness of an integrated continuous direct compression (CDC) line against disturbances from feeding, i.e. impulses of API and short step disturbances. These disturbances mimicked typical variations that can be encountered during long-term manufacture. The study included a primary formulation, with API of standard particle size, which was manufactured at 5 and 10 kg/h production rates, and a modified formulation, with API of large particle size, which was manufactured at 5 kg/h production rate. Overall, the CDC line smoothened all the disturbances, fulfilling the USP uniformity of dosage units (UDU) limit for single tablets. However, runs with the modified formulation failed the pharmacopoeia UDU requirements for the entire run due to high variation between tablets. The primary formulation passed the requirements in all cases. The residence time distribution (RTD) results indicated that the primary formulation allowed better smoothening ability, and an increase in production rate led to poorer smoothening due to shorter RTD. The RTDs revealed that a substantial part of back-mixing took place after the blender. Thus, the tablet press has an important role in smoothening disturbances longer than the mean residence time of the blender, which was very short.

Atomic scale surface engineering of micro- to nano-sized pharmaceutical particles for drug delivery applications.

Atomic layer deposition on pharmaceutical particles for drug delivery applications is demonstrated using assisted fluidized bed dry powder processing. Complete and conformal layering is achieved on particle sizes from the lower micron to upper nanometer range under near ambient conditions. As few as 2-14 atomic alumina layers alter particle properties: dissolution, dispersibility and heat transfer.

Statistical molecular design, parallel synthesis, and biological evaluation of a library of thrombin inhibitors.

A library of thrombin inhibitors has been designed using statistical molecular design. An aromatic scaffold was used, with three varied positions corresponding to three pockets at the active site of thrombin (the S-, P-, and D-pockets). The selection was performed in the building block space, and previously acquired data were included in the design procedure. The design resulted in six, four, and six building blocks for the first (S), second (P), and third (D) pockets, respectively. A second round of selection applied to the combined selected building blocks resulted in a subset of 18 compounds. The selected library was synthesized in parallel and biologically evaluated. The compounds were analyzed with respect to their inhibition (pIC(50)) of thrombin; membrane permeability, estimated by migration behavior in micellar media (CE log k') and pK(a); and specificity with respect to inhibition (K(i)) of trypsin. Multivariate QSAR studies of the responses yielded valuable results and information that could only be found using statistical molecular design in combination with multivariate analysis.

On-line capillary electrophoresis with mass spectrometry detection for the analysis of carbohydrates after derivatization with 8-aminonaphthalene-1,3,6-trisulfonic acid.

Capillary electrophoresis (CE) with mass spectrometry (MS) detection is an ideal tool for analytical use, which combines a nano quantity assay with mass determination. Carbohydrate analysis has always been a challenge because of the inherent structural complexity and the lack of a chromophore, unless derivatization is used. Here we use the derivatization of carbohydrates with a fluorophore, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). This chromophore has two advantages, first, it facilitates UV and fluorescence detection and, second, it introduces negative charge to the analyte, which enhances zone electrophoretic separation. In this study, CE combined with negative ion electrospray MS (ESI-MS) was evaluated for the on-line analysis of ANTS labeled carbohydrates and cellulose fragments. The CE system was connected to the MS by a sheath-liquid electrospray arrangement. The ANTS reagent and Dextrin-15, which contains oligomers of maltose, were used as model samples for ESI-MS optimization in flow-injection-MS and CE-MS modes, respectively. Various sheath-liquid compositions regarding organic modifier (isopropanol, methanol, or acetonitrile) and electrolyte (acetic acid-formic acid, ammonium acetate, or triethylamine) were studied. The response as well as the analyte charge state distribution was found to be dependent on the composition and the orifice voltage. Low-pH conditions with isopropanol as organic modifier were sensitive, stable, and the most favorable for analysis.

Quantitative analysis of film coating in a fluidized bed process by in-line NIR spectrometry and multivariate batch calibration.

A method is described which enables real-time analysis of film coating on pharmaceutical pellets during an industrial manufacturing process. Measurements were conducted on the solid particulate material by near-infrared (NIR) spectrometry utilizing a diffuse reflectance fiber-optic probe positioned inside a fluidized bed process vessel. Time series of NIR spectra from 11 batches generated a three-way data matrix that was unfolded and modeled by partial least squares (PLS) in a multivariate batch calibration. The process conditions were deliberately varied according to an experimental design. This yielded good predictability of the coating thickness with a best model fit, R2 = 0.97, for one PLS-projection, and a root-mean-square error of calibration = 2.2 microm (range tested 0-50 microm). The regression vector was shown to be highly influenced by responses that are both direct (aliphatic C-H stretch overtones) and indirect (aromatic C-H stretch overtones), from film component and core material, respectively. The impact of different data pre-treatment methods on the normalization of the regression vector is reported. Justification of the process calibration approach is emphasized by good correlation between values predicted from NIR data and reference image analysis data on dissected pellets and a theoretical nonlinear coating thickness growth model. General aspects of in-line NIR on solids and multivariate batch calibration are discussed.

Effective Sample Size in Diffuse Reflectance Near-IR Spectrometry.

Two independent methods for determination of the effectively sampled mass per unit area are presented and compared. The first method combines directional-hemispherical transmittance and reflectance measurements. A three-flux approximation of the equation of radiative transfer is used, to separately determine the specific absorption and scattering coefficients of the powder material, which subsequently are used to determine the effective sample size. The second method uses a number of diffuse reflectance measurements on layers of controlled powder thickness in an empirical approach. The two methods are shown to agree well and thus confirm each other. From the determination of the effective sample size at each measured wavelength in the visible-NIR region for two different model powder materials, large differences was found, both between the two analyzed powders and between different wavelengths. As an example, the effective sample size ranges between 15 and 70 mg/cm(2) for microcrystalline cellulose and between 70 and 300 mg/cm(2) for film-coated pellets. However, the contribution to the spectral information obtained from a certain layer decreases rapidly with increasing distance from the powder surface. With both methods, the extent of contribution from various depths of a powder sample to the visible-NIR diffuse reflection signal is characterized. This information is valuable for validation of analytical applications of diffuse reflectance visible-NIR spectrometry.

Chiral o-phthaldialdehyde reagents for fluorogenic on-column labeling of D- and L-amino acids in micellar electrokinetic chromatography.

Following a recent communication from this laboratory (A. Tivesten et al., J. High Resol. Chromatogr. 1996, 19, 229-233) where on-column chiral derivatization of D- and L-amino acids in micellar electrokinetic chromatography (MEKC) was demonstrated for the first time, we now present further details of the labeling procedure. The basis of the method is the consecutive injection of a sample and the reagent onto the capillary as two discrete plugs. By utilizing their difference in mobility, the zones are mixed by the electrophoretic process in a controllable way. In this way the amino acids are both derivatized within a few seconds and subsequently separated in a single step. Compared with pre-column derivatization, dilution of the original sample is minimized, which is why the method is highly useful for microchemical analytical work, i.e., labeling of nano- to picoliter samples. Four different chiral thiols were compared in this study, 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranose (TATG), N-acetyl-L-cysteine (AC), N-acetyl-D-penicillamine (AP), and N-isobutyryl-L-cysteine (IBC). Together with o-phthaldialdehyde (OPA) these constitute the chiral reagent. The reaction rate as well as the spectroscopic and chromatographic properties of the formed derivatives were examined. It was found that the fastest reaction is obtained with OPA/TATG, as was the case with L-alanine (L-ala), and that the rate is greatly affected by the presence and concentration of acetonitrile or methanol. Moreover, OPA/TATG yields superior resolution of D- and L-amino acids over the other OPA/thiol combinations in a sodium dodecyl sulfate (SDS) micellar buffer, whereas the OPA/AC and OPA/IBC-amino acid derivatives have a higher fluorescence quantum yield. With laser-induced fluorescence detection (He-Cd, 325 nm) the mass limit of detection is at the low amol level.

Fluorescence, photodestruction, photoionization and thermal degradation of o-phthalaldehyde/beta-mercaptoethanol-labelled aliphatic alpha-oligopeptides.

Photophysical and photochemical properties of o-phthalaldehyde/beta-mercaptoethanol-labelled aliphatic alpha-peptides were investigated. It is found that alpha-peptide derivatives have lower fluorescence quantum yields, higher photodestruction quantum yields and lower yields for formation of solvated electrons as compared to amino acid and simple alkylamine derivatives in aqueous alkaline solution. These properties of the alpha-peptide derivatives sets narrow limits for their utilization in laser-based (high light intensity) detector systems. In contrast, the thermal stability of the peptide derivatives was found to be severalfold higher than for the parent amino acid derivatives. The differential rates of thermal derivative degradation could be utilized in a new approach towards selective determination of peptides. determination of peptides. determination of peptides.

Increased intra- and extracellular concentrations of gamma-glutamylglutamate and related dipeptides in the ischemic rat striatum: involvement of glutamyl transpeptidase.

The present work relates to the possibility that the ATP-independent enzyme gamma-glutamyl transpeptidase (EC 2.3.2.2), which has been postulated to be part of an amino acid uptake system, is active during cerebral ischemia. This was evaluated in the ischemic rat striatum by determination of intra- and extracellular concentrations of gamma-glutamyl dipeptides (the products of the transpeptidation) and glutathione (the physiological gamma-glutamyl donor). An ischemic period (0-30 and 31-60 min) resulted in prominent increases in the respective concentration of extracellular gamma-glutamylglutamate (24- and 67-fold), gamma-glutamyltaurine + gamma-glutamylglycine (5.8- and 19-fold), and gamma-glutamylglutamine (2.6- and 6.8-fold) as revealed using in vivo microdialysis. The changes coincided with increased respective extracellular concentrations of glutamate (83- and 115-fold), taurine (17- and 25-fold), glycine (4.6- and 6.1-fold), and glutamine (1.7- and 2.1-fold). Furthermore, under anoxic conditions in vitro (0-30 and 0-60 min), respective striatal tissue concentrations were increased for gamma-glutamylglutamate (20- and 17-fold), gamma-glutamyltaurine (6.7- and 11-fold), gamma-glutamylglutamine (1.7- and 1.2-fold), and gamma-glutamylglycine (14- and 18-fold), whereas glutathione levels were, on an average, decreased by approximately 350 microM. In summary, gamma-glutamyl transpeptidase is involved in de novo dipeptide synthesis in the mammalian brain during anoxic conditions, indicating transport of amino acids such as glutamate.

Liquid chromatographic determination of acidic beta-aspartyl and gamma-glutamyl peptides in extracts of rat brain.

This work describes further development of our previously presented method for determination of acidic sulfur/phosphor-containing amino acids, gamma-glutamyl di/tripeptides, and beta-aspartyl dipeptides. Automated precolumn fluorogenic derivatization was performed with o-phthaldialdehyde/beta-mercaptoethanol and the derivatives were separated by reversed-phase liquid chromatography. The method was optimized for the analysis of brain tissue extracts. Due to the complex sample matrix, three separation schemes with complementary selectivities were developed. Different extraction protocols were evaluated and sonication of frozen tissue powder in methanol-H2O (9:1, v/v) yielded the highest recoveries and precision. beta-Mercaphtoethanol and EDTA were added to the extraction media to inhibit spontaneous oxidation of thiol-containing amino compounds. Analyte identification was based on retention times and recovery of standards added to extracts. The following compounds were identified in rat cerebral cortex (mean tissue concentration +/- SD, n = 6): gamma-glutamylglutamine (38.5 +/- 12.6 microM), gamma-glutamylglutamate (14.4 +/- 6.0 microM), gamma-glutamyltaurine (4.9 +/- 2.2 microM), beta-aspartylglycine (4.0 +/- 0.4 microM), beta-aspartyltaurine (3.7 +/- 0.6 microM), O-phosphoserine (3.2 +/- 0.8 microM), gamma-glutamylcysteine (1.9 +/- 0.3 microM), gamma-glutamylglycine (1.1 +/- 0.1 microM), and gamma-glutamylcysteateglycine (0.8 +/- 0.1 microM). In addition over 15 unidentified components were found. Cysteate, cysteine sulfinate, homocysteate, homocysteine sulfinate, O-Sulfoserine, gamma-glutamylaspartate, gamma-glutamylcysteate, gamma-glutamylhistidine, and beta-aspartylalanine were not present at concentrations above 1 microM.

Age determination based on amino acid racemization: A new possibility.

A method has been developed to determine the age of fossil bone samples based on amino acid racemization (AAR). Approximately one hundred fossil bone samples of known age from Hungary were collected and analysed for D- and L-amino acids. As the racemization of amino acids is affected by temperature, pH, metal content of the soil, and time passed since death, these factors were eliminated by comparing the estimated age to age determined by the radiocarbon method. Determining the D- and L-amino acid contents in samples of known age, determining the half life of racemization and plotting the D/L ratio as a function of time, calibration curves were obtained. These curves can be used for the age estimation of samples after determining their D- and L-amino acid content. The D/L ratio for 2 to 3 amino acids was determined for each sample and the mean value of estimated ages based on calibration curves was considered to estimate age of the fossil samples.

Semiconductor laser-induced fluorescence detection in picoliter volume flow cells.

A visible semiconductor laser-induced fluorescence detection system for ultratrace chemical analysis of nanoliter-to-picoliter samples is optimized and characterized in detail. With low-power semiconductor lasers emitting at 675 and 639 nm for excitation, the best limit of detection is 8000 molecules for a model fluorescent compound (1,1',3,3,3',3'-hexamethylindotricarbocyanine iodide) in an ethanol solution. Fused silica capillaries with inner diameters between 250 and 11 microm were employed as picoliter volume flow cells, which permits the use of the system in miniaturized separation techniques such as packed and open tubular liquid chromatography and capillary electrophoresis.

Automated determination of neuroactive acidic sulphur-containing amino acids and gamma-glutamyl peptides using liquid chromatography with fluorescence and electrochemical detection.

A column liquid chromatographic method is presented for the determination of trace levels of acidic sulphur-containing amino acids and gamma-glutamyl di- and tripeptides in microdialysates sampled from rat brain in vivo. Automated precolumn derivatization was performed with o-phthaldialdehyde-beta-mercaptoethanol. The derivatives were separated by reversed-phase liquid chromatography with electrochemical and fluorescence detection. The mean relative standard deviation (n = 10) was 1.03 and 4.59% for retention times and peak heights, respectively. The mean correlation coefficient of linearity (r) was 0.9982 in the range 4.5-450 pmol (n = 15), and the lowest detectable amount was 200 fmol for the homocysteinesulphinic acid derivative, (k' = 5.4, at a signal-to-noise ratio of 3). A microcolumn electrochemical detection method, developed for volume-limited samples, produced a fifteen-fold increase in mass sensitivity. Neurochemical applications using microdialysis in vivo are presented.