Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids.

Potato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicum) seeds for seven pospiviroid species known to occur naturally in these crops. The test consists of two multiplex reactions running in parallel, PospiSense 1 and PospiSense 2, that target Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd) and tomato planta macho viroid (TPMVd, including the former Mexican papita viroid). Dahlia latent viroid (DLVd) is used as an internal isolation control. Validation of the test showed that for both pepper and tomato seeds the current requirements of a routine screening test are fulfilled, i.e. the ability to detect one infested seed in a sample of c.1000 seeds for each of these seven pospiviroids. Additionally, the PospiSense test performed well in an inter-laboratory comparison, which included two routine seed-testing laboratories, and as such provides a relatively easy alternative to the currently used tests.

Molecular State of Active Pharmaceutical Ingredients in Ketoprofen Dermal Patches Characterized by Pharmaceutical Evaluation.

The molecular states of ketoprofen and the interaction between ketoprofen and other pharmaceutical excipients in the matrix layer were examined to determine their effect on the pharmaceutical properties of original and generic ketoprofen dermal patches (generic patches A and B). Molecular states of ketoprofen were evaluated using polarized light microscopy, Raman spectroscopy and powder X-ray diffraction. For the original ketoprofen patch, crystalline components were not observed in the matrix layer. However, crystalline ketoprofen was observed in the two generic ketoprofen patches. Moreover, the ketoprofen exhibited hydrogen bonding with the pharmaceutical excipients or patch materials in the generic products. Skin permeation of ketoprofen from the patches was evaluated using hairless mouse skin. Twelve hours after application, the original patch demonstrated the highest level of cumulative skin permeation of ketoprofen. This was followed by generic patch B while generic patch A showed the lowest level of permeation. Fluxes were calculated from the skin permeation profiles. The original patch was approx. 2.4-times faster compared with generic patch A and approximately 1.9-times faster compared with generic patch B. This investigation suggested that pharmaceutical properties such as skin permeability for these types of products are affected by the precipitation of crystalline ketoprofen in the matrix layer and the interaction of ketoprofen with the pharmaceutical excipients or patch materials.

Assessment of the functionality of a pilot-scale reactor and its potential for electrochemical degradation of calmagite, a sulfonated azo-dye.

Electrochemical degradation (ECD) is a promising technology for in situ remediation of diversely contaminated environmental matrices by application of a low level electric potential gradient. This investigation, prompted by successful bench-scale ECD of trichloroethylene, involved development, parametric characterization and evaluation of a pilot-scale electrochemical reactor for degradation of calmagite, a sulfonated azo-dye used as a model contaminant. The reactor has two chambers filled with granulated graphite for electrodes. The system has electrical potential, current, conductivity, pH, temperature, water-level and flow sensors for automated monitoring. The reactor supports outdoor and fail-safe venting, argon purging, temperature regulation and auto-shutdown for safety. Treatment involves recirculating the contaminated solution through the electrode beds at small flow velocities mimicking low fluid-flux in groundwater and submarine sediments. The first phase of the investigation involved testing of the reactor components, its parametric probes and the automated data acquisition system for performance as designed. The results showed hydraulic stability, consistent pH behavior, marginal temperature rise (<5 degrees C) and overall safe and predictable performance under diverse conditions. Near complete removal of calmagite was seen at 3-10V of applied voltage in 8-10h. The effects of voltage and strength of electrolyte on degradation kinetics have been presented. Further, it was observed from the absorption spectra that as calmagite degrades over time, new peaks appear. These peaks were associated with degradation products identified using electrospray ionization mass spectrometry. A reaction mechanism for ECD of calmagite has also been proposed.

Robust Raman measurement of hydrogen peroxide directly through plastic containers under the change of bottle position and its long-term prediction reproducibility.

Hydrogen peroxide concentration in antiseptic solutions has been non-destructively measured with the use of wide area illumination (WAI) scheme by applying a laser beam directly through a plastic container. The prediction accuracy was comparable to that from the use of quartz cell instead of a plastic bottle as well as near-infrared (NIR) prediction result in the previous study. Although the position of a plastic bottle was artificially changed in three-dimensional directions up to +/-10 mm to simulate real situation of practical measurement, the prediction accuracy was not degraded within +/-5 mm positional variation. This robust prediction performance was powered by the merits of WAI scheme that could provide improved reproducibility by illuminating large sample areas as well as less sensitivity of sample placement by the design of long focal length. Moreover, the prediction accuracy was successfully maintained over a long period of time. Finally, the prediction was also reproducible under the situation of laser power variation. The overall results demonstrate that WAI Raman scheme can provide robust and non-destructive quantitative analysis for not only hydrogen peroxide in antiseptic solution, but also other active pharmaceutical ingredients in diverse containers.

Off-line and on-line measurements of drug-loaded hot-melt extruded films using Raman spectroscopy.

The objective of this study was to investigate the use of Raman spectroscopy for the quantitative and qualitative analysis of an active ingredient in hot-melt extruded film formulations. Clotrimazole and ketoprofen were used as the active pharmaceutical ingredients (APIs) in the subject formulations. Films were prepared with contents varying from 1 to 20% of the respective API. Raman spectroscopy was used to quantify these APIs, both off-line and on-line. The spectral data were also used to ascertain the physical status of these APIs in the formulations. For off-line analysis, the films were cut into small rectangles, and the amount of the API was measured using a fiber optic probe equipped with a non-contact optic (NCO). For on-line analysis, real-time measurements were accomplished by fixing the probe over the extruded film for continuous data collection. Raman spectroscopy can be a convenient alternative to HPLC and other techniques currently employed for the quantification of the API in these formulations. Because Raman is also sensitive to changes in crystallinity, employment of the technique provided additional information to deduce the crystalline status of the API. The results reported in this paper suggest the suitability of Raman for PAT applications because of the on-line capability.

Direct, non-destructive quantitative measurement of an active pharmaceutical ingredient in an intact capsule formulation using Raman spectroscopy.

The active pharmaceutical ingredient (ambroxol) in an intact capsule formulation has been non-destructively quantified using Raman spectroscopy. To improve the problem of insufficient representative sampling inherent in Raman measurements, we have employed a wide area illumination (WAI) scheme that enables much improved sample coverage through a circular excitation laser spot with a 6 mm diameter. One of the anticipated sources of variation for this measurement was variation in the capsule shells. However, the WAI scheme significantly decreased the spectral variation among empty capsules compared to a measurement with a traditional small-spot excitation. Therefore, measurement variations emanating from the capsule shell did not significantly influence the accuracy of the determination of ambroxol concentrations. The resulting standard error of prediction (SEP) using the WAI scheme was comparable to that from previous Raman measurements which used a conventional small-spot excitation and employed a sampling scheme that involved rotation of an ambroxol pellet. It is further noteworthy that the SEP was also similar to that obtained from the use of transmission NIR spectroscopy, which was achieved by collection of spectra of the powdered capsule contents removed from the shell. The proposed Raman measurement using the WAI scheme in this case was sufficient to achieve the quantitative measurement of the active pharmaceutical ingredient (API) content of capsules non-destructively.

Reliable and fast quantitative analysis of active ingredient in pharmaceutical suspension using Raman spectroscopy.

The concentration of acetaminophen in a turbid pharmaceutical suspension has been measured successfully using Raman spectroscopy. The spectrometer was equipped with a large spot probe which enabled the coverage of a representative area during sampling. This wide area illumination (WAI) scheme (coverage area 28.3 mm2) for Raman data collection proved to be more reliable for the compositional determination of these pharmaceutical suspensions, especially when the samples were turbid. The reproducibility of measurement using the WAI scheme was compared to that of using a conventional small-spot scheme which employed a much smaller illumination area (about 100 microm spot size). A layer of isobutyric anhydride was placed in front of the sample vials to correct the variation in the Raman intensity due to the fluctuation of laser power. Corrections were accomplished using the isolated carbonyl band of isobutyric anhydride. The acetaminophen concentrations of prediction samples were accurately estimated using a partial least squares (PLS) calibration model. The prediction accuracy was maintained even with changes in laser power. It was noted that the prediction performance was somewhat degraded for turbid suspensions with high acetaminophen contents. When comparing the results of reproducibility obtained with the WAI scheme and those obtained using the conventional scheme, it was concluded that the quantitative determination of the active pharmaceutical ingredient (API) in turbid suspensions is much improved when employing a larger laser coverage area. This is presumably due to the improvement in representative sampling.

A new non-invasive, quantitative Raman technique for the determination of an active ingredient in pharmaceutical liquids by direct measurement through a plastic bottle.

The concentration of an active pharmaceutical ingredient (povidone) in a commercial eyewash solution has been measured directly through a plastic (low-density polyethylene: LDPE) container using a wide area illumination (WAI) Raman scheme. The WAI scheme allows excitation using a 6mm laser spot (focal length: 248 mm) that is designed to cover a wide sample area. As a result, it has the potential to improve the reliability Raman measurements by significantly enhancing representative sample interrogation, thus improving the reproducibility of sampling. It also decreases the sensitivity of sample placement with regard to the excitation focal plane. Simultaneously, isobutyric anhydride was placed in front of the bottles to use for a synchronous external standard configuration. This helps to correct the problematic variation of Raman intensity from the inherent fluctuation in laser power. Using the WAI Raman scheme combined with the synchronous standard method, the povidone concentration was successfully measured with spectral collection that was performed through a plastic barrier. The conventional Raman scheme was difficult to employ for the same purpose because of the degraded spectral reproducibility resulting from the smaller laser illumination area and the sensitivity of such an approach to the position of the sample bottle. The result from this study suggests that the WAI scheme exhibits a strong potential for the non-destructive quantitative analysis of pharmaceuticals measured directly in plastic containers. Preliminary work also shows that similar measurements can also be made in glass bottles. If implemented, this technique could be utilized as a simple and rugged method for quality assurance of final products in a manner consistent with Process analytical technology (PAT) requirements.

New reliable Raman collection system using the wide area illumination (WAI) scheme combined with the synchronous intensity correction standard for the analysis of pharmaceutical tablets.

A novel and reliable Raman collection system for the non-destructive analysis of pharmaceutical tablets has been proposed. The main idea was to develop and utilize the wide area illumination (WAI) scheme for Raman collection to cover a large surface area (coverage area: 28.3mm2) of solid tablet to dramatically improve the reliability in sample representation and the reproducibility of sampling due to less sensitivity of sample placement with regard to the focal plane. Simultaneously, the effective and synchronous standard configuration using isobutyric anhydride was harmonized with the WAI scheme to correct the problematic variation of Raman intensity from the change of laser power. To verify the quantitative performance of the proposed scheme, the compositional analysis of active ingredient in naproxen tablet has been performed. The average sample composition was successfully represented by using the WAI scheme compared to the conventional scheme with a much smaller illumination area. After the intensity correction using the non-overlapping peak of isobutyric anhydride standard and area normalization, a partial least squares (PLS) model was developed using an optimized spectral range and the concentrations of naproxen in tablets were accurately predicted. The prediction accuracy was not sensitive to changes in laser power or tablet position within +/-2mm. Additionally, the prediction accuracy was repeatable without systematic drift or need for re-calibration.

High-flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes LC and C1.

Flavonoids are a group of polyphenolic plant secondary metabolites important for plant biology and human nutrition. In particular flavonols are potent antioxidants, and their dietary intake is correlated with a reduced risk of cardiovascular diseases. Tomato fruit contain only in their peel small amounts of flavonoids, mainly naringenin chalcone and the flavonol rutin, a quercetin glycoside. To increase flavonoid levels in tomato, we expressed the maize transcription factor genes LC and C1 in the fruit of genetically modified tomato plants. Expression of both genes was required and sufficient to upregulate the flavonoid pathway in tomato fruit flesh, a tissue that normally does not produce any flavonoids. These fruit accumulated high levels of the flavonol kaempferol and, to a lesser extent, the flavanone naringenin in their flesh. All flavonoids detected were present as glycosides. Anthocyanins, previously reported to accumulate upon LC expression in several plant species, were present in LC/C1 tomato leaves but could not be detected in ripe LC/C1 fruit. RNA expression analysis of ripening fruit revealed that, with the exception of chalcone isomerase, all of the structural genes required for the production of kaempferol-type flavonols and pelargonidin-type anthocyanins were induced strongly by the LC/C1 transcription factors. Expression of the genes encoding flavanone-3'-hydroxylase and flavanone-3'5'-hydroxylase, which are required for the modification of B-ring hydroxylation patterns, was not affected by LC/C1. Comparison of flavonoid profiles and gene expression data between tomato leaves and fruit indicates that the absence of anthocyanins in LC/C1 fruit is attributable primarily to an insufficient expression of the gene encoding flavanone-3'5'-hydroxylase, in combination with a strong preference of the tomato dihydroflavonol reductase enzyme to use the flavanone-3'5'-hydroxylase reaction product dihydromyricetin as a substrate.