New approach on similarity analysis of chromatographic fingerprint of herbal medicine

A new approach to the construction and similarity analysis of chromatographic fingerprint for herbal medicine is presented in this paper. Samples of chuanxiong, a herbal medicine for headache, from three producing areas of China were used to evaluate the utility of this study. The samples were analyzed with high-performance liquid chromatography (HPLC) and the peak areas of the chromatograms were used to construct the fingerprints of the herbal medicines. A vector of differences was defined between the two fingerprints. The scalar mean of the difference vector was taken as a statistic and both the t-test and Bayesian hypothesis testing were implemented to provide a one-to-one comparison of the fingerprints. Compared with principal component analysis (PCA), correlation coefficient and vector cosine, the new method offers a better differentiation of the similarity or difference between the fingerprints from same sample of chuanxiong. When the new method was used in the similarity analysis of the fingerprints of chuanxiong from different production areas, a clear-cut signature was obtained that reveals the significant difference between them.     

Distinction between melanins derived from different precursors using pyrolysis/gas chromatography/mass spectrometry and the NIST mass spectral search algorithm

A method was required to objectively determine whether various melanins were synthesised from different precursors, and whether synthesis from the same precursor was reproducible. Melanins have a complex, heterogeneous, polymeric structure, making them difficult to characterise and compare. Pyrolysis chromatography may be useful for such large molecules that are not amenable to analysis by other methods, but the resulting chromatograms are usually complex and difficult to compare. Techniques used to objectively differentiate between such chromatograms often employ statistical methods that are difficult to use and interpret without specialised knowledge. Melanins were analysed by pyrolysis/gas chromatography/mass spectrometry (PY/GC/MS). Software was developed to automate the conversion of the resulting total ion current (TIC) chromatograms to pseudo-mass spectra (PMS), consisting of a row vector of chromatographic peak areas (analogous to ion abundances) ordered along a retention time axis (analogous to m/z ratio). The National Institute of Standards and Technology (NIST, USA) mass spectral search program, which is used widely as an objective measure of the degree of similarity between mass spectra, was then used to generate match factors for comparisons of the generated PMS. Match factors between melanins synthesised from the same precursors were not significantly different, while match factors between melanins synthesised from different precursors were significantly smaller. The reproducibility of the pyrolysis technique was reasonable, with the majority of relative standard deviation (RSD) values of match factors from melanins synthesised from the same precursor, being below 10% (n=5 for each melanin type). While the method does not allow the unequivocal identification of individual melanin types in isolation, it can be used to compare melanins from different sources and objectively estimate the degree of similarity between them on the basis of significant differences between their pyrograms.     

Chemometric approach in quantification of structural identity/similarity of proteins in biopharmaceuticals

We present a chemometrics study in which we show the identity or degree of similarity of 3D protein structures of various G-CSF (Granulocyte Colony-Stimulating Factor) isolates. The G-CSF isolates share the same amino acid sequence, but the preparation was carried out by somehow diverse technologies. The comparison of 3D structures was made on the basis of 2D NMR NOESY (Nuclear Overhauser Enhancement Spectroscopy) spectra of proteins. In searching for the most appropriate criteria to determine the identity or degree of similarity of selected spectral regions of different isolates, two methods for quantitative evaluation of identity/similarity were used. The first method compares all peaks in the two investigated protein spectral regions; the extent of peaks that overlap is determined. The second method includes spectral invariants originating from graph theory. The criteria of identity/similarity were calculated from graphs, derived from a collection of up to 200 peaks of investigated 2D NMR spectral region. The peaks were linked into a graph according to the sequential nearest neighborhoods. According to the first method all peaks were relevant, considering that spectral noise was previously removed; the largest similarity was found between the protein of a commercially available G-CSF drug and one of the three new isolates produced in the laboratory. The second method indicated that the pairwise similarity of the three new isolates is larger than the similarity of any of the new isolates with the commercially available drug. This is an expected result taking into account that the new isolates are produced by the same technology, while the commercial product has additives for long-term storage that could not be completely compensated. The proposed measure of similarity may help the developers of biosimilar products to optimize the controllable parameters of the production technology and eventually to argue the identity of the new isolate in comparison with the originator commercial product.     

Differentiation of Normal and Neoplastic Cells by Synchronous Fluorescence: Rat Liver Epithelial and Rat Hepatoma Cell Models

ABSTRACT

In the present study, conventional and synchronous luminescence (SL) were utilized to investigate spectral differences in normal and neoplastic cells. The synchronous fluorescence (SF) method involves scanning simultaneously both emission and excitation wavelengths while keeping a constant wavelength interval between them. This SF procedure simplifies the emission spectrum and provides for greater selectivity and is used to detect subtle differences in the fluorescence emission of the biochemical species of cells from rat tissues. A difference between the fluorescent spectra of the normal rat liver epithelial (RLE) and hepatoma cell lines were detected using synchronous fluorescence. The potential use of SF as a screening tool for cancer diagnosis is discussed.     

Resonance Raman and surface-enhanced resonance Raman spectra of LH2 antenna complex from Rhodobacter sphaeroides and Ectothiorhodospira sp. excited in the Qx and Qy transitions

Well-resolved vibrational spectra of LH2 complex isolated from two photosynthetic bacteria, Rhodobacter sphaeroides and Ectothiorhodospira sp., were obtained using surface-enhanced resonance Raman scattering (SERRS) exciting into the Qx and the Qy transitions of bacteriochlorophyll a. High-quality SERRS spectra in the Qy region were accessible because the strong fluorescence background was quenched near the roughened Ag surface. A comparison of the spectra obtained with 590 nm and 752 nm excitation in the mid- and low-frequency regions revealed spectral differences between the two LH2 complexes as well as between the LH2 complexes and isolated bacteriochlorophyll a. Because peripheral modes of pigments contribute mainly to the low-frequency spectral region, frequencies and intensities of many vibrational bands in this region are affected by interactions with the protein. The results demonstrate that the microenvironment surrounding the pigments within the two LH2 complexes is somewhat different, despite the fact that the complexes exhibit similar electronic absorption spectra. These differences are most probably due to specific pigment-pigment and pigment-protein interactions within the LH2 complexes, and the approach might be useful for addressing subtle static and dynamic structural variances between pigment-protein complexes from different sources or in complexes altered chemically or genetically.     

General theory of similarity measures for library search systems

A general theory of similarity measures for library search systems is presented. It shows that once the spectral features to be used in the comparison of two spectra have been selected and their relative importance has been fixed, the characteristics of the similarity measure are fully controlled by two independent parameters. These two parameters control whether a forward or a reverse search and whether an identity, a similarity, or a classification search is conducted.     

Comparing similar spectra: From similarity index to spectral contrast angle

We investigated a spectral-contrast-angle (theta) method to determine whether mass spectra of structural isomers are the same or significantly different. This method represents collisionally activated dissociation (CAD) spectra as vectors in space. Mass spectra of different isomers are represented as different vectors, having characteristic lengths and direction. The derived spectral contrast angle, which is a measure of the angle between two vectors corresponding to two closely related spectra, is a measure of whether the mass spectra are the same or significantly different. We compare this method with the similarity index (SI) method and show that the spectral contrast angle method is superior and can differentiate between very similar spectra in cases where the SI cannot. Both methods can be implemented simply in situations where the analyst is called on to decide, on the basis of mass or product-ion spectra, whether reference and unknown compounds are the same or to evaluate the reproducibility of spectra comprised of many peaks.     

Identification of UV/VIS-spectra based on a fuzzy function

Summary It is shown, how an averaged UV/VIS spectrum may be used as a the nucleus of a fuzzy function. The relative cardinality between the unknown test spectrum and the fuzzy database spectra combined with a weighting vector, which considers the variance information, gives a much more effective similarity measure than the correlation coefficient.     

Similarity of High-Resolution Tandem Mass Spectrometry Spectra of Structurally Related Micropollutants and Transformation Products

High-resolution tandem mass spectrometry (HRMS2) with electrospray ionization is frequently applied to study polar organic molecules such as micropollutants. Fragmentation provides structural information to confirm structures of known compounds or propose structures of unknown compounds. Similarity of HRMS2 spectra between structurally related compounds has been suggested to facilitate identification of unknown compounds. To test this hypothesis, the similarity of reference standard HRMS2 spectra was calculated for 243 pairs of micropollutants and their structurally related transformation products (TPs); for comparison, spectral similarity was also calculated for 219 pairs of unrelated compounds. Spectra were measured on Orbitrap and QTOF mass spectrometers and similarity was calculated with the dot product. The influence of different factors on spectral similarity [e.g., normalized collision energy (NCE), merging fragments from all NCEs, and shifting fragments by the mass difference of the pair] was considered. Spectral similarity increased at higher NCEs and highest similarity scores for related pairs were obtained with merged spectra including measured fragments and shifted fragments. Removal of the monoisotopic peak was critical to reduce false positives. Using a spectral similarity score threshold of 0.52, 40% of related pairs and 0% of unrelated pairs were above this value. Structural similarity was estimated with the Tanimoto coefficient and pairs with higher structural similarity generally had higher spectral similarity. Pairs where one or both compounds contained heteroatoms such as sulfur often resulted in dissimilar spectra. This work demonstrates that HRMS2 spectral similarity may indicate structural similarity and that spectral similarity can be used in the future to screen complex samples for related compounds such as micropollutants and TPs, assisting in the prioritization of non-target compounds.

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Graphical Abstract ?

     

Raman microscopy for the chemometric analysis of tumor cells

Raman spectroscopy is recognized as a tool for chemometric analysis of biological materials due to the high information content relating to specific physical and chemical qualities of the sample. Thirty cells belonging to two different prostatic cell lines, PNT1A (immortalized normal prostate cell line) and LNCaP (malignant cell line derived from prostate metastases), were mapped using Raman microscopy. A range of spectral preprocessing methods (partial least-squares discriminant analyses (PLSDAs), principal component analyses (PCAs), and adjacent band ratios (ABRs)) were compared for input into linear discriminant analysis to model and classify the two cell lines. PLSDA and ABR were able to correctly classify 100% of cells into benign and malignant groups, while PLSDA correctly classified a greater proportion of individual spectra. PCA was used to image the distribution of various biochemicals inside each cell and confirm differences in composition/distribution between benign and malignant cell lines. This study has demonstrated that PLSDAs and ABRs of Raman data can identify subtle differences between benign and malignant prostatic cells in vitro.     

Multi-component kinetic-spectrophotometric analysis. Selection of wavelength and time ranges

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.     

Structural diversity of small molecule libraries

A novel method for assessing structural diversity is presented. Maximum common subgraph identity is used as the measure of similarity between two chemical structures. A conditional probability treatment of similarity distributions for libraries of chemical structures is used to define diversity. This evaluation method together with the evaluation of traditional physicochemical properties is used to assess a large number of chemical libraries and to understand structural differences between these.     

Analysis of neoplastic changes in mouse lung using Fourier-transform infrared microspectroscopy

Methods based on infrared microspectroscopy were explored as a means to distinguish normal from neoplastic lung tissue. Mice were exposed to urethane, a known environmental carcinogen. After 3–8 months, lungs were removed, snap-frozen, sectioned and analyzed by standard histological methods and by infrared microspectroscopy. Neoplasms were readily observed in mice treated with urethane. Ultra-structurally, the neoplasms were composed entirely of type II alveolar cells displaying intracellular lamellar bodies. A fusion of two spectral pre-processing techniques, optimal region selection and linear discriminant analysis (LDA), was used to search for infrared spectral signatures distinguishing normal from neoplastic tissue. These techniques showed clear and reproducible differences between the complex spectra of these tissue types, suggesting that infrared microspectroscopy in conjunction with spectral processing technology may be useful to reveal subtle spectral differences occurring following induction of neoplastic changes and to interpret their biochemical origins.     

HPLC指纹图谱相似度研究

将HPLC指纹图谱看作多维空间内的向量,利用向量夹角余弦的基本公式计算两个指纹图谱间的相似度。用7个不同品种的金银花提取物的HPLC指纹图谱对计算方法进行了检验,结果表明相似度能较好地定量评价指纹图谱间的相似性,可应用于中药质量控制。     

A novel method for human gender classification using Raman spectroscopy of fingernail clippings

This paper illustrates a novel method for human gender classification by measuring the Raman spectrum of fingernail clippings. As Raman spectroscopy reveals the characteristics of vibrational frequencies of the fingernails, it provides unique chemical fingerprints that can be used to describe the molecular structure differences of fingernail between males and females. As the differences of Raman spectra of human fingernails are very subtle, they are enhanced by using a pattern recognition method. In the present study, a combination algorithm of principal component analysis (PCA) and support vector machines (SVM) was implemented to perform the data classification. This combined algorithm provides a classification accuracy of up to 90%. The success of this present method may be used as an alternative rapid tool to identify human gender in forensic applications.     

Application of synchronous fluorescence to parchment characterization

A nondestructive method for quantitative parchment characterization and sensitive indication of its deterioration stage was developed. Synchronous fluorescence (SF) measurements were applied for the first time to parchment samples. The method provides detailed spectral features, which are useful for parchment characterization. The discrimination of parchment samples into groups (modern, historical, and artificially aged) was successfully performed. The SF spectra could be resolved into specific fluorophores, which were related to the parchment condition. The spectral data indicate a continuous change in the collagen-to-gelatin ratio during the aging process. Depth-resolved synchronous fluorescence spectra were also measured. The data indicate that parchments possess a layered structure, and the dominant fluorophore in the upper layer is different from those in the lower layers. Layer-resolved profiling allows for quantifying the contribution of each fluorophore in each given layer. This way, significant differences between modern, artificially aged, and historical samples can be observed.     

A computer-based storage and retrieval system for electronic absorption spectra

A computer-based system for storage and retrieval of spectra in the ultra-violet region, with absorbance as the observed variable, is described. Differences in concentration between solutions relating to observed and stored spectra are automatically adjusted. The entire spectral curve is stored, not only selected points such as maxima etc.; this makes it possible to investigate the spectral range continuously during the retrieval procedure. The increment between two observations (absorbance values) is 2 nm on the wavelength axis.The input medium is punched cards containing values taken directly from observed (catalogued) spectra. The output is a printed list, giving information about observed and retrieved spectra, system parameters used during the retrieval procedure, etc. An important feature of the system is that displacements between observed and stored spectra are allowed for, in two dimensions during comparison, thus taking into account 'chromic' effects. Information about partial identity between observed and stored (retrieved) spectra is given, even if this partial identity exists in dislocated wavelength regions. To test the program, constructed (test) curves as well as real spectra were used. The system is capable of collecting in groups substances with common spectral characteristics, and thus substances belonging to the same class of chemical compounds. This makes the system a valuable tool not only for identification work but also for the study of spectral behaviour in general.     

Investigation of chemometric calibration performance based on different chemical matrix and signal-to-noise ratio.

The chemometric calibration performance was systematically investigated by two parameters (changing the chemical matrix as well as the signal-to-noise ratio) of the NIR (near-infrared) spectrum. Three different analytes (hexane, cyclohexane, toluene) were selected and heptane was used as a solvent. The degree of spectral difference significantly affected the calibration performance. The largest structural difference between the analyte and the solvent provided the best calibration result for a given signal-to-noise ratio. Additionally, the signal-to-noise ratio of the spectra also directly influenced the calibration performance. Overall, the spectral difference and signal-to-noise ratio were the major factors for governing the chemometric calibration performance, especially in the low-concentration range.     

Hydration of simple amides. FTIR spectra of HDO and theoretical studies

The hydration of formamide (F), N-methylformamide (NMF), N,N-dimethylformamide (DMF), acetamide (A), N-methylacetamide (NMA), and N,N-dimethylacetamide (DMA) has been studied in aqueous solutions by means of FTIR spectra of HDO isotopically diluted in H2O. The difference spectra procedure has been applied to remove the contribution of bulk water and thus to separate the spectra of solute-affected HDO. To facilitate the interpretation of obtained spectral results, DFT calculations of aqueous amide clusters were performed. Molecular dynamics (MD) simulation for the cis and trans forms of NMA was also carried out for the SPC model of water. Infrared spectra reveal that only two to three water molecules from the surrounding of the amides are statistically affected, from among ca. 30 molecules present in the first hydration sphere. The structural-energetic characteristic of these solute-affected water molecules differs only slightly from that in the bulk and corresponds to the clathrate-like hydrogen-bonded cage typical for hydrophobic hydration, with the possible exception of F. MD simulations confirm such organization of water molecules in the first hydration sphere of NMA and indicate a practical lack of orientation and energetic effects beyond this sphere. The geometry of hydrogen-bonded water molecules in the first hydration sphere is very similar to that in the bulk phase, but MD simulations have affirmed subtle differences recognized by the spectral method and enabled their understanding. The spectral data and simulations results are highly compatible. In the case of F, NMF, and A, there is a visible spectral effect of water interactions with N-H groups, which have destabilizing influence on the amides hydration shell. There is no spectral sign of such interaction for NMA as the solute. The energetic stability of water H-bonds in the amide hydration sphere and in the bulk fulfills the order: NMA > DMA > A > NMF > bulk > DMF > F. Microscopic parameters of water organization around the amides obtained from the spectra, which have been used in the hydration model based on volumetric data, confirm the more hydrophobic character of the first three amides in this sequence. The increased stability of the hydration sphere of NMA relative to DMA and of NMF relative to DMF seems to have its origin in different geometries, and so the stability, of water cages containing the amides.