Reference Chromatogram Research Articles

The Formation of Small Amounts of Cyclopropane during Pulsed Pyrolysis of C4–C5 Acyclic Alkanes in the Adiabatic Compression Reactor

During high-temperature pulse pyrolysis of acyclic butanes and pentanes under adiabatic compression conditions, cyclopropane, a stressed cyclic hydrocarbon, was found among the reaction products in small quantities for the first time. The analysis of the reaction products was performed by gas chromatography using three capillary columns of different polarity, selectivity and sufficient efficiency. The identification of reaction products, including cyclopropane, was performed using retention times of individual substances and model mixtures, as well as comparing chromatograms with reference chromatograms from the literature and the ScanView Application Database. It was shown that the chromatographic peak attributed to cyclopropane could not be a ghost peak. Additional confirmation of this conclusion was obtained in a series of experiments on the pyrolysis of n-butane at a reduced initial temperature of the adiabatic compression reactor (from 120 °C to 50 °C) and a modified mode of GC analysis. Cyclopropane yields as a function of maximum temperature have a bell-shaped asymmetric dependence. The maximum value of the yield of cyclopropane increases with the transition from normal alkanes to isoalkanes, and from pentanes to butanes; for n-pentane, 0.009 wt. %, and for isobutene, ≈0.017 wt. %. During the pulse pyrolysis of isobutane, n-butane, isopentane and n-pentane, cyclopropane is not a primary product. Further theoretical and experimental studies are needed to establish the mechanism of cyclopropane formation during pyrolysis of C4–C5 acyclic alkanes.

System optimization and validation to improve thin-layer chromatography of roselle calyces (Hibiscus sabdariffa L.) required by the Indonesian Herbal Pharmacopoeia Edition II

Context: Roselle (Hibiscus sabdariffa L.) is one of the traditional crude drugs monographed in the Indonesian Herbal Pharmacopoeia Edition II (IHP II). As per IHP II requirements, a TLC pattern should be used in its authentication. However, the TLC system described for this purpose does not produce a clear reference chromatogram, often leading to inconclusive results. Aims: To develop and validate TLC systems of H. sabdariffa calyces for a better-quality chromatogram than those listed in the IHP II, thereby facilitating crude drugs authentication. Methods: To optimize TLC for H. sabdariffa, sixteen systems, differing in stationary phase, mobile phase composition, and/or visualization reagent were tested. The TLC system was then validated using several parameters such as analyte stability during chromatography, analyte stability in the extract solution and on the TLC plate, stability of the derivatization result, and precision on a plate as well as intermediate precision. Results: Two systems (I and II) were successfully designed and applied to evaluate H. sabdariffa quality. System I used Si gel 60 F254 for the stationary phase, ethyl acetate-formic acid-glacial acetic acid-water (100:11:11:2) for the mobile phase, H. sabdariffa’s ethanol extract (5%, 20 µL) for the test solution, cyanidin 3-O-glucoside (100 ppm, 4 µL) as a reference, no derivatization, and detection with visible light. System II combined Si gel 60 F254 for the stationary phase, ethyl acetate-formic acid-glacial acetic acid-toluene-water (80:11:11:20:19), H. sabdariffa’s ethanol extract (5%, 20 µL) for the test solution, chlorogenic acid (1000 ppm, 2 µL) and caffeic acid (50 ppm, 2 µL) as references, the visualizing reagent NP/PEG, and investigation under 366 nm UV light. Conclusions: Both systems are simple but have good stability and precision, thus facilitating H. sabdariffa calyx authentication and paving the way for developing content analysis methods for H. sabdariffa markers.

Identification of the Authenticity and Geographical Origin of Bear Bile Powder by Using High Performance Liquid Chromatography - Charged Aerosol Detector Fingerprints Combined with Chemometrics.

Bear bile powder (BBP) is a rare animal-derived traditional Chinese medicine, and it has been widely used to treat visual disorders and hepatobiliary diseases in East Asia. However, there is still a lack of reliable quality control methods for BBP. This study was designed to establish a comprehensive quality map of BBP based on bile acids. High-performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD) was used for fingerprint establishment and quantitative analysis of BBP. The similarities of HPLC-CAD chromatograms for 50 batches of BBP were more than 0.95, while the similarities of reference chromatograms between 6 other animal bile and BBP were low than 0.7. Additionally, five bile acids in BBP, including tauroursodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, ursodesoxycholic acid, and chenodeoxycholic acid, were simultaneously quantified. This method has been validated with good regression as well as satisfactory precision, sensitivity, stability, repeatability, and accuracy. Using this method, the contents of five bile acids in BBP samples from five producing areas were determined and compared. Furthermore, Fisher linear discriminant analysis was performed to discriminate the geographic origins of BBP. The result demonstrated that HPLC-CAD fingerprint combined with multi-components quantification is an effective and reliable method for quality control of BBP, it could be a meaningful reference for the quality evaluation of medicinal bile.

Stability research of Xiaoer Ganmaoning Oral Liquid based on chromatographic peak matching and image sensory evaluation

The high performance liquid chromatography(HPLC) characteristic chromatogram of Xiaoer Ganmaoning Oral Liquid(oral liquid for short) was established. The medicinal materials corresponding to characteristic peaks, their index components and ranges of similarity with the reference chromatograms were clarified. The similarity between the characteristic chromatograms of 10 batches of the oral liquid and the reference chromatogram was higher than 0.994. Eighteen characteristic peaks were identified, which were derived from different medicinal materials including Scutellariae Radix, Arctii Fructus, Lonicerae Japonicae Flos, Gardeniae Fructus and Forsythiae Fructus. Further, 11 characteristic peaks were assigned by the comparison with reference substances as chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, baicalin, baicalein, wogonin, scutellarin, forsythiaside A and arctiin. Also, the characteristic chromatogram of precipitate in the oral liquid was established, and the similarity between characteristic chromatograms of 10 batches of the precipitate and the reference chromatogram was higher than 0.940. The 14 characteristic peaks originating from the precipitate and those from the oral liquid were consistent in retention time, and the content of all index components in the precipitate was lower than 5% of that in the oral liquid. Moreover, the stability of precipitate during the accelerated stability test was explored with filtration and Matlab-based image sensory evaluation. The precipitate mass and precipitation degree both increased over the stability test duration significantly. The stability of the oral liquid was used as a model system in this study to establish the integrated quality control system which related to medicinal materials, preparations and precipitate with HPLC characteristic chromatograms and image sensory evaluation, which lays a foundation for the exploration of the quantity value transfer of the oral liquid.

Quality assessment of fried licorice based on fingerprints and chemometrics

Fried licorice is obtained by frying licorice without using any auxiliary materials, and it is widely used both as food and medicine in China. To explore the influence of licorice origin on the quality of fried licorice, a method based on fingerprinting and chemometrics was developed. Twenty batches of licorice were selected from four locations. The reference chromatograms of each location were established via similarity analysis. Chemometric methods, such as cluster, principal component, and orthogonal partial least squares discriminant analyses were used to evaluate the changes in the composition of fried licorice, predict its origin, and reflect its quality. Mass spectrometry was used to identify the chemical components. Finally, an origin prediction function was established via discriminant analysis to trace the origin of licorice. The model was demonstrated to be stable, reliable, and accurate in predicting licorice origin and to provide a reference for origin traceability.

Application of fingerprint combined with quantitative analysis and multivariate chemometric methods in quality evaluation of dandelion (Taraxacum mongolicum).

A quality assessment method based on quantitative analysis of multi-components by single marker (QAMS) and fingerprint was constructed from 15 batches of dandelion (Taraxacum mongolicum), using multivariate chemometric methods (MCM). MCM were established by hierarchical cluster analysis (HCA) and factor analysis (FA). HCA was especially performed using the R language and SPSS 22.0 software. The relative correction factors of chlorogenic acid, caffeic acid, p-coumaric acid, luteolin and apigenin were calculated with cichoric acid as a reference, and their contents were determined. The differences between external standard method (ESM) and QAMS were compared. There was no significant difference (t-test, p > 0.05) in quantitative determination, proving the consistency of the two methods (QAMS and ESM). Dandelion material from Yuncheng, Shandong was used as a reference chromatogram. The fingerprints in 15 batches of dandelion were established by HPLC analysis. The similarity of the fingerprints in different batches of dandelion material was greater than or equal to 0.82. A total of 10 common peaks were identified. This strategy is simple, rapid and efficient in multiple component detection of dandelion. It is beneficial in simplifying dandelion's quality control processes and providing references to enhance quality control for other herbal medicines.

Quality evaluation of Acanthopanax senticosus via quantitative analysis of multiple components by single marker and multivariate data analysis

A comprehensive method for the evaluation of Acanthopanax senticosus (AS) was established by the quantitative analysis of multiple components by single marker (QAMS), fingerprint, similarity analysis (SA), hierarchical cluster analysis (HCA), and factor analysis (FA) based on high performance liquid chromatography (HPLC). A total of 27 common peaks were identified in the standard fingerprint of 20 batches of AS from different regions in China, of which 8 peaks were identified as protocatechuic acid, syringin, chlorogenic acid, caffeic acid, eleutheroside E, hyperoside, isofrqxidin, and acacetin, and the concentrations of these eight components were determined simultaneously by QAMS. The results showed that the QAMS method was effective and feasible compared with the external standard method (ESM) (RD < 3.3 %, P ≤ 0.01). Sample 1 (S1) was used as the reference chromatogram, the similarity of other samples was between 0.765 and 0.968. Through HCA, AS could be mainly divided into two production areas, the north Liaoning (including Liaoning) and the south Liaoning areas. Furthermore, FA showed that the quality of AS in the north Liaoning area was better than that in the south Liaoning area. In summary, the method established in this study can comprehensively and systematically evaluate quality differences in AS samples, and may be used to help to improve the quality control of AS.

Study on quality evaluation method of classical prescription Mahuang Decoction primary standard substances

This study aimed to establish the HPLC characteristic chromatogram and content determination method for index components with the primary standard substances of the classical prescription Mahuang Decoction, and to provide data basis for the establishment of its quality standard and the development and utilization of compound preparations. First, HPLC was used to establish the material reference chromatograms of Mahuang Decoction, and 15 batches of standard samples of Mahuang Decoction were determined. Their similarity was calculated by the median method. Secondly, the content of the standard substances was determined and a simplecontent determination method was established by HPLC. Relevant methodology was investigated, and the extraction ratio, index component transfer rate and moisture content of 15 batches of primary standard samples were calculated. The results showed that the two sets of HPLC methods had their own characteristics. The six chromatographic peaks identified from the 10 common peaks in the former characteristic chromatogram covered all the herbal medicines in the standard substances, which can better indicate the quality characteristics of the standard substances of Mahuang Decoction. The latter method(content determination method) was simple and practical, so it was suitable for establishing the quality standard of its compound preparation. Two sets of methods were jointly used to evaluate the quality of 15 batches of Mahuang Decoction. The results were as follows: the similarity of 15 batches of samples was greater than 0.90; the average extraction ratio was 18.1%; the average moisture content was 9.7%; the average content and transfer rate of the standard ingredients ephedrine hydrochloride and total pseudoephedrine hydrochloride were 2.3% and 26.7% respectively, and the average content and transfer rate of amygdalin were 2.2% and 48.3% respectively. None of the data showed dispersion(beyond 70%-130% of the mean value), which met the application data requirements for the substance standards of ancient classical Chinese herbal compound preparations(draft for comments). Based on the above research, the primary substance quality standard of Mahuang Decoction was established in order to provide reference for the development and research of the compound preparation of Mahuang Decoction.

Peak alignment of gas chromatography–mass spectrometry data with deep learning

We present ChromAlignNet, a deep learning model for alignment of peaks in Gas Chromatography-Mass Spectrometry (GC–MS) data. In GC–MS data, a compound’s retention time (RT) may not stay fixed across multiple chromatograms. To use GC–MS data for biomarker discovery requires alignment of identical analyte’s RT from different samples. Current methods of alignment are all based on a set of formal, mathematical rules. We present a solution to GC–MS alignment using deep learning neural networks, which are more adept at complex, fuzzy data sets. We tested our model on several GC–MS data sets of various complexities and analysed the alignment results quantitatively. We show the model has very good performance (AUC ∼ 1 for simple data sets and AUC ∼ 0.85 for very complex data sets). Further, our model easily outperforms existing algorithms on complex data sets. Compared with existing methods, ChromAlignNet is very easy to use as it requires no user input of reference chromatograms and parameters. This method can easily be adapted to other similar data such as those from liquid chromatography. The source code is written in Python and available online.

Quality evaluation of Styrax based on quantitative determination of 7 chemical components using HPLC wavelength switching method and chemometrics

In order to evaluate the quality of Styrax more comprehensively,this study attempted to establish an HPLC wavelength switching method to simultaneously determine the content of seven compounds in Styrax,and chemometrics were used to analyze the quality difference between different sources of Styrax,and finally establish a characteristic chromatogram of Styrax. The column was Agilent ZORBAX Extend C18( 4. 6 mm×250 mm,5 μm) with phase a mixture of acetonitrile-0. 1% phosphoric acid aqueous solution as the mobile phase in a gradient elution procedure; the detection wavelength was set as follows: 0-13. 5 min,194 nm( benzoic acid);13. 5-20. 5 min,278 nm( cinnamic acid); 20. 5-32 min,194 nm( benzyl benzoate,benzyl cinnamate,cinnamyl cinnamate,dehydroabietic acid); 32-55 min,241 nm( abietic acid). The methodological verification results showed that when the injection masses of benzoic acid,cinnamic acid,benzyl benzoate,benzyl cinnamate,cinnamyl cinnamate,dehydroabietic acid and abietic acid were0. 006 948-0. 694 8,0. 001 426-0. 142 6,0. 013 16-0. 658 0,0. 006 148-0. 614 8,0. 008 035-0. 803 5,0. 002 121-0. 212 1,and0. 010 172-1. 017 2 μg,respectively,there were good linear relationship between injection mass and peak area. The average recovery rates of seven compounds were in the range from 94. 34% to 105. 8%,and all RSD were less than 3. 0%( n = 6). The methodological verification results showed that the developed HPLC wavelength switching method has good accuracy and repeatability. The results of the sample analysis showed that the quality of Styrax from different sources was quite different. The chromatogram of Styrax reference material( S1) was used as the reference chromatogram to calculate the fingerprint similarity of each batch of samples. The results showed that the similarities of samples S2-S10 were 0. 952,0. 949,0. 981,0. 351,0. 751,0. 969,0. 979,0. 992 and 0. 971,respectively.The similarity values of other batches samples were satisfactory,except for sample S5 and S6,indicating that the quality difference among these samples is small. The similarity values of S11-S20 were 0. 060,0. 055,0. 054,0. 285,0. 092,0. 002,0. 044,0. 044,0. 044,and 0. 040,respectively. The results showed that compared with the sample S1,there was a large quality difference among S11-S20. Based on the chromatograms of S1-S10,the HPLC characteristic chromatograms of Styrax was established and the purpose is to give reference to other pharmaceutical researchers. The newly developed HPLC wavelength switching method have the advantages of simplicity,reproducibility and specificity,and the developed HPLC characteristic chromatograms provided a reference method for the overall quality evaluation of Styrax.

Multi‐scale Gaussian/Haar wavelet strategies coupled with sub‐window factor analysis for an accurate alignment in nontargeted metabolic profiling to enhance herbal origin discrimination capability

Metabolic dataset can provide an overview of different herbal origin, which is conducted by some statistical procedures. Such results often deviate to a certain degree, due to peaks shifts in chromatographic signals. In order to solve this problem, an improved algorithm of combining sub-window factor analysis with the mass spectrum information is proposed. The algorithm uses a peak detection approach derived either from multi-scale Gaussian function or Haar wavelet to locate the peaks with different application scope; the candidate drift points at each peak are estimated by Fast Fourier transform cross correlation; Specifically, the best drift points at each candidate peaks are confirmed by sub-window factor analysis and mass spectrum information in nontargeted metabolic profiling. Finally, the peak regions were aligned against a reference chromatogram, and the non-peak regions were used linear interpolation. The chromatographic signals of 30 Bupleurum samples were aligned as an illustration of this algorithm, and they could be well distinguished using some statistical procedures. The result demonstrates that the presented method is stronger than other mass-spectra based algorithms, when facing the alignment of some co-eluted peaks.

Analysis of lipophilic components of Salvia miltiorrhiza roots and S. yunnanensis roots by UPLC and LC-MS/MS

Fingerprints of lipophilic components in the roots of Salvia miltiorrhiza and S.yunnanensis were analyzed by UPLC-DADand UPLC coupled with mass spectroscopy to evaluate the differences and similarities of the lipophilic components in the two kinds of herbs.The UPLC analysis of 18 batches of S.miltiorrhiza and 16 batches of S.yunnanensis was performed on a 25℃Thermo Accucore C_(18)column(2.1 mm×100 mm,2.6μm)by Shimadzu LC-20AD;mobile phase was 0.026%phosphoric acid(A)-acetonitrile(B)with gradient elution;flow rate was 0.4 m L·min~(-1);detection wavelength was set at 270 nm;injection volume was 2μL.The molecular structures of the lipophilic components were analyzed on a 25℃Thermo Accucore C_(18)column(2.1 mm×100 mm,2.6μm)by Thermo U3000 UPLC Q Exactive Orbitrap LC-MS/MS with a mobile phaseconsisting of 0.1%formic acid water(A)and 0.1%formic acidacetonitrile(B).The mass spectrometry was acquired in positive modes using ESI.There are 10 common peaks in the lipophilic components of S.miltiorrhiza.The similarity between the 16 batches of S.miltiorrhiza and their own reference spectra was greater than 0.942,and the average similarity was 0.973.There are 12 common peaks in the lipophilic components of S.yunnanensis.The similarity between the 18 batches of S.yunnanensis and their own reference spectra was greater than 0.937,and the average similarity was 0.976.The similarity between the reference chromatograms of S.miltiorrhiza and S.yunnanensis was only 0.900.There are three lipophilic components in S.yunnanensis,which are not found in S.miltiorrhiza,and one of which isα-lapachone.There is a lipophilic component in S.miltiorrhiza not found in S.yunnanensis,which may be miltirone.The two herbs contain 8 common lipophilic components including dihydrotanshinoneⅠ,cryptotanshinone,tanshinoneⅠ,tanshinoneⅡ_A,nortanshinone in which the content of tanshinoneⅡ_A,dihydrotanshinoneⅠand cryptotanshinone of S.yunnanensisis significantly lower than that of S.miltiorrhiza(P<0.01),and the contents of tanshinoneⅠand nortanshinone are significantly lower than that of S.miltiorrhiza too(P<0.05).There are significant differences in the types and contents of lipophilic components between the roots of S.miltiorrhiza and S.yunnanensis,and the similarity between the fingerprints of interspecies is much lower than that between the same species.Therefore,the roots of S.miltiorrhiza and S.yunnanensis are two kinds of herbs which are quite different in chemical compounds and compositions.

Qualitative analysis of the roots of Salvia miltiorrhiza and Salvia yunnanensis based on NIR, UHPLC and LC–MS-MS

Near infrared spectroscopy (NIR) was applied to discriminate the roots of salvia miltiorhiza Bunge (Danshen for short) and Salvia yunnanensis C. H. Wright (Zidanshen for short) by means of principal component analysis (PCA), improved and simplified K nearest neighbors (IS-KNN). Furthermore, an ultra-high performance liquid chromatographic (UHPLC) coupled with photodiode-array detector was developed for building fingerprints of lipophilic components of Danshen and Zidanshen, respectively. Basing on NIR information, both PCA and IS-KNN method classified the two kinds of Chinese medical herbs with 100% accuracy. The chromatographic fingerprints of the lipophilic components of Danshen and Zidanshen have 10 and 12 common peaks, respectively. Liquid chromatography coupled with mass spectroscopy (LC–MS-MS) was applied to identify these peaks. Among these, three small peaks in the fingerprints of Zidanshen are not found in Danshen, one of which was identified as α-lapachone, and the other two compounds were not yet identified; a small peak after tanshinone IIA in the fingerprints of Danshen was not found in Zidanshen, which was identified as miltirone. The two herbs have 10 common lipophilic components. The similarity between the two reference chromatograms of Zidanshen and Danshen is 0.902, but the mean similaritie between Zidanshen (or Danshen) fingerprints and its own reference chromatogram is 0.973 (or 0.976). The contents of main lipophilic components are significantly lower in Zidanshen than in Danshen (P < 0.01 or P < 0.05). The results indicate that the two Chinese medical materials are not only different in NIR spectra, but also different in species and quantities of lipophilic components. NIR spectra analysis can identify Danshen and Zidanshen rapidly and accurately. UHPLC coupled with MS analysis demonstrates the detail differences between the two herbs both in species and contents of their lipophilic components.

Application of Subwindow Factor Analysis and Mass Spectral information for accurate alignment of non-targeted metabolic profiling

The peak shifts may lead to an incorrect statistical result for nontargeted metabolomics profiling, such as classification and discrimination in pattern recognition. In the paper, a more accurate alignment algorithm is developed based on Subwindow Factor Analysis and Mass Spectral information (SFA-MS). Compared with other methods, this new algorithm aligns the peaks more accurately without changing their shapes, especially for the overlapping peak clusters. To begin, the Continuous Wavelet Transform with Haar wavelet as the mother wavelet (Haar CWT) is used to determine the position and width of peaks. On this basis, the candidate drift points are confirmed by Fast Fourier Transform (FFT) cross correlation. Furthermore, the MS fitting degree of the common components between the reference chromatogram and the raw chromatogram is determined by the Subwindow Factor Analysis (SFA). When the MS information between reference and raw peaks is identical, the corresponding moving points are the optimum shifts. It is remarkable that all the peaks are moved through linear interpolation in the non-peak parts, so that the aligned chromatograms remain unchanged. The SFA-MS algorithm was implemented in the Matlab language and is available as an open source package.

QUANTIFICATION OF XYLOPIC ACID AND CHROMATOGRAPHIC FINGERPRINT EVALUATIONS OF THE DRIED FRUITS OF XYLOPIA AETHIOPICA FROM FOUR AFRICAN COUNTRIES

Dunal) A. Rich, (herein called XYA), family Annonaceae, commonly known as “Guinea pepper”, “Ethiopian pepper” or “Negro pepper”, are widely used in traditional African medicines to treat a wide array of diseases including malaria, fungal infections, rheumatism, arthritis, etc. Scientific investigations have ascribed the following activities to the fruits of XYA; anti-diabetic, anti-inflammatory, antimicrobial, antiplasmodial, analgesic, anti-nociceptive, anti-proliferative, spermatogenic and neuropharmacological effects. The main active principle reported is xylopic acid (XA), a kaurene diterpene. This study aimed to develop and validate a simple HPLC/UV (high performance liquid chromatography – ultraviolet detection) analytical method for the quantification of XA that can be reproduced in poor-resource settings where advanced analytical detection techniques such as HPLC-MS are unavailable.&#x0D; Materials and Methods: Thus in this study, a simple C18 solid-phase extraction (SPE) column-pretreatment ─ HPLC/UV analytical procedure was developed for the quantification of XA in the dried fruits of XYA from four African countries, Benin, Cameroon, Ghana and Nigeria. The samples of XYA from the four countries were assessed for similarities using chromatographic fingerprinting.&#x0D; Results: The HPLC method was validated for linearity, limits of detection and quantification, precision and accuracy. The samples of XYA from Cameroon were found to have the highest average content of XA while those from Benin had the lowest average content of XA.&#x0D; Conclusion: Using the chromatographic fingerprint evaluation, the similarities of the samples from the four countries to the reference chromatogram was in the order: Benin &gt; Cameroon &gt; Nigeria &gt; Ghana.&#x0D; Key words: Xylopia aethiopica, xylopic acid, C18

Application of HPLC fingerprint based on acid amide components in Chinese prickly ash (Zanthoxylum)

In order to establish a high performance liquid chromatography (HPLC) fingerprint of Chinese prickly ash (Zanthoxylum) based on acid amide components, 38 specimen were collected from Western China. Similarity analysis, hierarchical clustering analysis (HCA) and principal component analysis (PCA) were carried out to analyze the obtained fingerprints. Furthermore, uncertainty and reliability values of the established HPLC fingerprints were evaluated. Similarity values ranged between 0.587 and 0.999 for specimen of Zanthoxylum bungeanum Maxim. and between 0.991 and 0.999 for specimen of Zanthoxylum armatum DC. 17 samples from different habitats were classified by HCA into three groups, i.e. Group I (8 samples), Group II (6 samples) and Group III (3 samples) and were divided into three clusters by PCA. The compound of peak 6 was identified as hydroxyl-β-sanshool. The characteristic compounds of peaks 5, 7, 8, 10, 12 and 13 were tentatively identified by LC–MS as other acid amide compounds. As for credibility analysis, the results showed that the values of macroscopic qualitative reliability and macroscopic quantitative reliability of the reference chromatogram fingerprint were more than 0.9772. Statistical analysis indicated that the HPLC fingerprint technology may be applied for quality assessment and classification of Zanthoxylum.

Development of high-performance thin-layer chromatography method for herbals identification in extemporaneous oral solution

The preparation of extemporal medicines in store includes carrying out intrapharmacy quality control and stability studies. Since there is no single analytical method that ideally suited for all drugs, for each formulation depend on its composition and analytical goal, appropriate quality control methods should be developed. The object of analysis was pediatric oral solution with sedative action that contains Valerian tincture, Motherwort tincture, and sodium bromide.The purpose of research was the development of simple, specific and reproducible methods of active herbal ingredients identification formulated into oral solution.Materials and methods. The high-performance thin-layer chromatography (HPTLC) was used as the method for analysis. It was performed on 200 × 100 mm silica gel 60 F 254 HPTLC glass plates (Merck, Germany). Standard solutions and samples were applied onto the plate as bands 8.0 mm wide using spray-on technique with Automatic TLC sampler ATS 4; the development was made in saturated Automatic developing chamber ADC 2, temperature and the relative humidity were 23 °C and 33 % respectively; the derivatization was performed using Chromatogram Immersion Device; the documentation was conducted using Visualizer with visionCATS software (CAMAG, Switzerland).As a result two HPTLC identification methods have been developed for valerenic acids that are specific for Valerian tincture and flavonoids that are specific for Motherwort tincture. Thus, the sample preparation has been developed, the optimal chromatographic conditions have been chosen, the acceptance criteria have been proposed, and the reference chromatograms have been shown. Valerenic acids of oral solution were determined in mobile phase: cyclohexane, ethyl acetate, acetic acid (60:38:2); the derivatization of plates was performed through dipping with Anisaldehyde-sulfuric acid reagent and heated to 100 °C for 3 min; documentation was performed after derivatization under white light and UV 366 nm. Flavonoids of oral solution were determined in mobile phase: ethylacetate, methyl ethyl ketone, formic acid, water (5:3:1:1); the derivatization of plates was performed through dipping with Natural Products/PEG reagents, heated to 100 °C for 3 min before dipping; documentation was performed after derivatization under UV 366 nm.Conclusions. The developed methods can be used for identification of oral solution, and for stability study with the purpose of determination of its storage conditions and shelf-life.

Optimizing the process of reference selection for correlation optimised warping (COW) and interval correlation shifting (icoshift) analysis: automating the chromatographic alignment procedure

The present work introduces an automated procedure for generating the reference chromatogram for subsequent correlation optimised warping (COW) and interval correlation shifting (icoshift) analysis.

BARCHAN: Blob Alignment for Robust CHromatographic ANalysis

Two-dimensional gas chromatography (GC×GC) plays a central role into the elucidation of complex samples. The automation of the identification of peak areas is of prime interest to obtain a fast and repeatable analysis of chromatograms. To determine the concentration of compounds or pseudo-compounds, templates of blobs are defined and superimposed on a reference chromatogram. The templates then need to be modified when different chromatograms are recorded. In this study, we present a chromatogram and template alignment method based on peak registration called BARCHAN. Peaks are identified using a robust mathematical morphology tool. The alignment is performed by a probabilistic estimation of a rigid transformation along the first dimension, and a non-rigid transformation in the second dimension, taking into account noise, outliers and missing peaks in a fully automated way. Resulting aligned chromatograms and masks are presented on two datasets. The proposed algorithm proves to be fast and reliable. It significantly reduces the time to results for GC×GC analysis.

Origin discrimination and quality evaluation of &lt;i&gt;Gastrodiae rhizoma&lt;/i&gt; (Orchidaceae) by high-performance liquid chromatographic fingerprint

Purpose : To develop a high-performance liquid chromatography (HPLC) fingerprint method for the quality control and origin discrimination of Gastrodiae rhizoma. Methods : Twelve batches of G. rhizoma collected from Sichuan, Guizhou and Shanxi provinces in china were used to establish the fingerprint. The chromatographic peak (gastrodin) was taken as the reference peak, and all sample separation was performed on a Agilent C18 (250 mm×4.6 mmx5 μm) column with a column temperature of 25 °C. The mobile phase was acetonitrile/0.8 % phosphate water solution (in a gradient elution mode) and the flow rate of 1 mL/min. The detection wavelength was 270 nm. The method was validated as per the guidelines of Chinese Pharmacopoeia. Results : The chromatograms of the samples showed 11 common peaks, of which no. 4 was identified as that of Gastrodin. Data for the samples were analyzed statistically using similarity analysis and hierarchical cluster analysis (HCA). The similarity index between reference chromatogram and samples’ chromatograms were all > 0.80. The similarity index of G. rhizoma from Guizhou, Shanxi and Sichuan is evident as follows: 0.854 - 0.885, 0.915 - 0.930 and 0.820 - 0.848, respectively. The samples could be divided into three clusters at a rescaled distance of 7.5: S1 - S4 as cluster 1; S5 - S8 cluster 2, and others grouped into cluster 3. Conclusion : The findings indicate that HPLC fingerprinting technology is appropriate for quality control and origin discrimination of G. rhizoma . Keywords : Gastrodiae rhizoma, Origin discrimination, Quality control; HPLC fingerprint