L. K. Bittner

Near-infrared reflection spectroscopy (NIRS) as a successful tool for simultaneous identification and particle size determination of amoxicillin trihydrate

A successful application of NIR spectroscopy (NIRS) in combination with multivariate data analysis (MVA) for the simultaneous identification and particle size determination of amoxicillin trihydrate particles was developed. Particle size analysis was ascertained by NIRS in diffuse reflection mode on different particle size fractions of amoxicillin trihydrate with D90 particle diameters ranging from 6.9 to 21.7 μm. The present problem of fractionating the powder into good enough size fractions to achieve a stable calibration model was solved. By probing dried suspensions measurement parameters were optimized and further combined with the best suitable chemometric operations. Thereby the quality of established regression models could be improved considerably. A linear coherence between particle size and absorbance signal was found at specific wavenumbers. Satisfactory clustering by particle size was achieved by principal component analysis (PCA) whereas partial least squares regression (PLSR) and principal component regression (PCR) was compared for quantitatively calibrating the NIRS data. PLSR turned out to predict unknown test samples slightly better than PCR.

Comparison of NIR chemical imaging with conventional NIR, Raman and ATR-IR spectroscopy for quantification of furosemide crystal polymorphs in ternary powder mixtures.

The aim of this study was to evaluate the ability of near-infrared chemical imaging (NIR-CI), near-infrared (NIR), Raman and attenuated-total-reflectance infrared (ATR-IR) spectroscopy to quantify three polymorphic forms (I, II, III) of furosemide in ternary powder mixtures. For this purpose, partial least-squares (PLS) regression models were developed, and different data preprocessing algorithms such as normalization, standard normal variate (SNV), multiplicative scatter correction (MSC) and 1st to 3rd derivatives were applied to reduce the influence of systematic disturbances. The performance of the methods was evaluated by comparison of the standard error of cross-validation (SECV), R(2), and the ratio performance deviation (RPD). Limits of detection (LOD) and limits of quantification (LOQ) of all methods were determined. For NIR-CI, a SECVcorr-spec and a SECVsingle-pixel corrected were calculated to assess the loss of accuracy by taking advantage of the spatial information. NIR-CI showed a SECVcorr-spec (SECVsingle-pixel corrected) of 2.82% (3.71%), 3.49% (4.65%), and 4.10% (5.06%) for form I, II, III. NIR had a SECV of 2.98%, 3.62%, and 2.75%, and Raman reached 3.25%, 3.08%, and 3.18%. The SECV of the ATR-IR models were 7.46%, 7.18%, and 12.08%. This study proves that NIR-CI, NIR, and Raman are well suited to quantify forms I-III of furosemide in ternary mixtures. Because of the pressure-dependent conversion of form II to form I, ATR-IR was found to be less appropriate for an accurate quantification of the mixtures. In this study, the capability of NIR-CI for the quantification of polymorphic ternary mixtures was compared with conventional spectroscopic techniques for the first time. For this purpose, a new way of spectra selection was chosen, and two kinds of SECVs were calculated to achieve a better comparability of NIR-CI to NIR, Raman, and ATR-IR.

Characterization of normal and malignant prostate tissue by Fourier transform infrared microspectroscopy

Prostate cancer has become one of the most common malignancies worldwide. Morphological and histomorphological evaluation of this disease is a well established technique for the cancer classification and has remained relatively unchanged since several decades, although it remains a time consuming and subjective technique, with unsatisfactory levels of inter- and intra-observer discrepancy. Novel approaches for histological recognition are necessary to identify and to investigate cancer in detail. Fourier transform infrared (FTIR) spectroscopic imaging has become an essential tool for the detection, identification and characterization of the molecular components of biological processes, such as those responsible for the dynamic properties of cancer progression. Major advantage of this new technique is the acquisition of local molecular expression profiles while maintaining the topographic integrity of the tissue and avoiding time-consuming extraction, purification and separation steps. By using this method it is possible to investigate the spatial distribution of proteins, lipids, carbohydrates, cholesterols, nucleic acids, phospholipids and small molecules within biological systems by in situ analysis of tissue sections. We applied this technique on prostate cancer patients radical prostatectomy specimens in order to develop new tools for histomorphological analysis and the characterization of snap frozen prostate cancer tissues. As a first step, an optimization of sample preparation, tissue section thickness and IR slide material was performed. Special preparation methods for FTIR imaging are the essential requirements to maintain the spatial arrangement of compounds and avoid delocalization and degradation of the analytes. Subsequently, selected cancer samples were characterized with the prior optimized parameters and analyzed by univariate and cluster analysis. For the interpretation and calibration of the system we correlated the FTIR-images with the histopathological information. With this method it is possible to distinguish between cancer and noncancer areas within a prostate cancer tissue with a resolution of 6.25 μm × 6.25 μm on frozen sections.

Simultaneous quantification of verbenalin and verbascoside in Verbena officinalis by ATR-IR and NIR spectroscopy

Attenuated-total-reflectance infrared spectroscopy (ATR-IR) and near-infrared diffuse reflectance spectroscopy (NIR) in hyphenation with multivariate analysis was utilized to quantify verbenalin and verbascoside in Verbena officinalis. A new high performance liquid chromatography (HPLC) method as a reference was established and validated. For both vibrational spectroscopic methods test-set and cross validation were performed. Different data-pre-treatments like SNV, 1st and 2nd derivative were applied to remove systematic errors and were evaluated. Quality parameters obtained for the test-set validation revealed that ATR-IR (verbenalin: R(2)=0.94, RPD=4.23; verbascoside: R(2)=0.93, RPD=3.63) has advantages over NIR (verbenalin: R(2)=0.91, RPD=3.75; verbascoside: R(2)=0.80, RPD=2.35) in the given application.

Comparison of NIR and ATR-IR spectroscopy for the determination of the antioxidant capacity of Primulae flos cum calycibus

In this study, near-infrared (NIR) and attenuated-total-reflectance infrared (ATR-IR) spectroscopy techniques in hyphenation with partial least squares (PLS) regression were utilized to determine the antioxidant capacity of Primulae flos cum calycibus samples. Folin–Ciocalteu (FC), ferric ion reducing antioxidant power (FRAP), 2,2-diphenyl-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and cupric reducing antioxidant capacity (CUPRAC) assays were performed as reference methods. Different spectral pretreatments such as standard normal variate (SNV), 1st or 2nd derivative, were applied to remove scattering effects. For all assays, cross and test-set validations were performed. The ability of the two spectroscopic techniques to replace the five assays was evaluated and compared. The standard error of prediction (SEP) and the ratio performance deviation (RPD) were determined and corrected for the imprecision of the reference data to obtain the respective SEPcorr and RPDcorr values. In general, NIR demonstrated advantages over ATR-IR spectroscopy and resulted best for the ABTS assay (R2: 0.94, RPDcorr: 4.66; test-set validation). Also with ATR-IR spectroscopy, the best prediction power was obtained for the ABTS assay (R2: 0.94, RPDcorr: 4.10; test-set validation). The feasibility of vibrational spectroscopy as a fast and simple tool to replace wet chemistry assays for the measurement of the antioxidant capacity of Primulae flos cum calycibus samples was demonstrated.

Fourier transform infrared imaging analysis in discrimination studies of St. John's wort (Hypericum perforatum)

In the present study, Fourier transform infrared (FTIR) imaging and data analysis methods were combined to study morphological and molecular patterns of St. Johns wort (Hypericum perforatum) in detail. For interpretation, FTIR imaging results were correlated with histological information gained from light microscopy (LM). Additionally, we tested several evaluation processes and optimized the methodology for use of complex FTIR microscopic images to monitor molecular patterns. It is demonstrated that the combination of the used spectroscopic method with LM enables a more distinct picture, concerning morphology and distribution of active ingredients, to be gained. We were able to obtain high-quality FTIR microscopic imaging results and to distinguish different tissue types with their chemical ingredients.

Near-Infrared Imaging Spectroscopy as a Tool to Discriminate Two Cryptic Tetramorium Ant Species

Correct species identification is a precondition for many ecological studies. Morphologically highly similar, i.e., cryptic, species are an important component of biodiversity but particularly difficult to discriminate and therefore understudied ecologically. To find new methods for their rapid identification, thus, is important. The cuticle’s chemical signature of insects often is unique for species. Near-infrared spectroscopy (NIRS) can capture such signatures. Imaging NIRS facilitates precise positioning of the measurement area on biological objects and high-resolution spatial capturing. Here, we tested the applicability of imaging NIRS to the discrimination of cryptic species by using the ants Tetramorium caespitum and T. impurum. The classification success of Partial Least Squares Regression was 98.8%. Principal Component Analysis grouped spectra of some T. impurum individuals with T. caespitum. Combined with molecular-genetic and morphological evidence, this result enabled us to pose testable hypotheses about the biology of these species. We conclude that discrimination of T. caespitum and T. impurum with imaging NIRS is possible, promising that imaging NIRS could become a time- and cost-efficient tool for the reliable discrimination of cryptic species. This and the direct facilitation of potential biological insight beyond species identification underscore the value of imaging NIRS to ecology.

Near Infrared Spectroscopy (NIRS) as a Tool to Analyze Phenolic Compounds in Plants

Phenolics are related to a number of pharmacological effects and form an important category among secondary plant metabolites. For many years well established and highly efficient methods such as UV spectroscopy, high perform- ance liquid chromatography, gas chromatography, liquid chromatography, mass spectrometry and capillary electrophore- sis have been used for determination. In the last years, near infrared spectroscopy with its benefits such as being non- invasive, rapid, almost no necessary sample preparation, on-/inline measurements, being able to determine physical and chemical parameters simultaneously, became a widely used analytical technique in phyto-analytics and is included in pharmacopeias to an increasing extent (1). This manuscript reviews recent applications of NIRS in the analysis of pheno- lic compounds in plant material.

A chromatographic and spectroscopic analytical platform for the characterization of St John's wort extract adulterations

In the present study, a novel analytical platform is introduced, which enables both analysis and quality control of St Johns wort extracts and tissue. The synergistic combination of separation techniques (including thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC)) with mass spectrometry (MS) and vibrational spectroscopy is demonstrated to get deeper insights into the ingredients composition. TLC was successfully employed to identify some unknown ingredients being present in samples with Chinese provenience. On the one hand, the novel HPLC method described here allowed us to clearly differentiate between European and Chinese samples; on the other hand, this method could successfully be employed for the semi-preparative isolation of an unknown ingredient. Matrix-free laser desorption ionization time of flight mass spectrometry (mf-LDI-TOF/MS) using a specially designed titanium oxide layer was employed to identify the structure of the substance. The analytical knowledge generated so far was used to establish an infrared spectroscopic model allowing both quantitative analysis of ingredients as well as differentiating between European and Chinese provenience. Finally, infrared imaging spectroscopy was conducted to obtain knowledge about the highly resolved distribution of ingredients. The analytical platform established so far can be used for fast and non-destructive quantitation and quality control to identify adulteration being of interest according to the Deutsche Arzneimittel Codex (DAC).

Application of Near-Infrared Spectroscopy (NIRS) as a Tool for Quality Control in Traditional Chinese Medicine (TCM)

Traditional Chinese Medicine (TCM) is becoming more and more popular all over the world. Novel analytical tools for quality control are highly demanded enabling analysis starting at breeding and ending at biological fluids including urine or serum. Compared to analytical separation methods (chromatography, electrophoresis) near-infrared spectroscopy (NIRS) allows analyzing matter of interest non-invasively, fast and physical/chemical parameters simultaneously. It can be used for the quantitative control of certain (active) ingredients. In many cases identification can only be achieved by pattern recognition. Therefore, NIRS combined with cluster analysis offers huge potential to identify e.g. species, geographic origin, special medicinal formula etc. In the present contribution the fundamentals, possibilities of NIR applied in quality control of TCM are pointed out and its adand disadvantages are discussed in detail by several practical examples.