Relative Retention Times Research Articles

Retention Time of Four Common Poisons （Drugs） by GC-MS Qualitative Analysis.

To investigate the maximum allowable deviation of retention time （RT） or relative retention time （RRT） between the common poisons （drugs） and standard solvent by gas chromatography-mass spectrometry （GC-MS）. After pretreatment with liquid-liquid extraction, four common poisons （drugs）-dichlorvos, phorate, diazepam and estazolam-were detected by full scan mode GC-MS. RT and RRT were analyzed according to combined uncertainty and expanded uncertainty. The expanded uncertainty of RT and RRT were 6.0×10-4-14.1×10-3 and 2.5×10-6-5.9×10-5 （k=3）, respectively. The RT of poisons （drugs） was relatively stable in blood samples with different mass concentrations. Among dichlorvos, phorate, diazepam and estazolam, the absolute deviation and relative deviation of RT were ≤0.03 min and ≤0.4%, respectively, and those of RRT were ≤0.003 min and ≤0.3%, respectively. The maximum allowable deviations of RT and RRT for common poisons （drugs） in blood samples are recommended to be ±0.05 min and ±0.5%.

Fingerprint Analysis and Multi-Component Determination of Ribonucleic Acid for Injection II Recipe by HPLC-DAD and LC-ESI-MS Methods.

Using high-performance liquid chromatography (HPLC), a quantitative method was developed to control the quality of ribonucleic acid for injection II (RA-II), which is used as an anti-cancer drug clinically in China. Using nuclease enzyme and under optimal hydrolysis conditions, the unstable RNA was hydrolyzed into stable nucleosides and nucleotides, which were easily detected by HPLC with diode array detection. Furthermore, by analyzing HPLC chromatograms of 10 batches of samples, six common peaks, namely, cytosine nucleotide, uracil nucleotide, adenine nucleotide, guanine nucleotide, guanosine and adenine, were identified by a HLPC coupled with electrospray ionization mass spectrometry. The similarity, the relative retention time and the relative peak area of each common peak, relative to the reference peak, were calculated to obtain the HPLC fingerprints, and their values were within the scope of the provisions. More importantly, all six components were simultaneously determined. Overall, the developed method in this investigation proved to be a useful tool for monitoring the quality of RA-II in terms of their batch variations and the raw material sources.

Improvement of Tests for the Control of Impurities in Antimicrobial Medicinal Products by Chromatographic Methods: Ciprofloxacin Case Study

The need to reduce analysis time when controlling impurities in medicinal products remains an urgent challenge. The aim of the study was to use the example of ciprofloxacin in order to select chromatographic conditions that will significantly accelerate and simplify determination of impurities in medicinal products. Methods: the study was performed using the Chromolith ® Performance RP-18e 100×4.6 mm and Acquity UPLC BEH C18 1.7 μ m, 100×2.1 mm columns and the Agilent 1290 Infinity chromatography system with a diode array detector. Results: the authors of the study worked out gradient elution modes for determination of ciprofloxacin impurities that involved 15 min run time for fast high-performance liquid chromatography and 12 min run time for ultra-performance liquid chromatography. The identification of impurities was performed based on relative retention times of impurity peaks and their comparison with the reference standard chromatogram. Conclusions: the study demonstrated the possibility of replacing the thin-layer chromatography method used for determination of fluoroquinolonic acid (ciprofloxacin impurity A) with high-performance liquid chromatography. The proposed test conditions allow for identification of ciprofloxacin impurities specified in well-established pharmacopoeias and manufacturers’ product specification files. The obtained data may be used for further development of rapid methods of ciprofloxacin impurities determination in medicinal products.

Doping control analysis of four JWH-250 metabolites in equine urine by liquid chromatography-tandem mass spectrometry.

JWH-250 is a synthetic cannabinoid. Its use is prohibited in equine sport according to the Association of Racing Commissioners International (ARCI) and the Fédération Équestre Internationale (FEI). A doping control method to confirm the presence of four JWH-250 metabolites (JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, JWH-250 5-OH-indole, and JWH-250N-pentanoic acid) in equine urine was developed and validated. Urine samples were treated with acetonitrile and evaporated to concentrate the analytes prior to the analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The chromatographic separation was carried out using a Phenomenex Lux® 3μm AMP column (150 x 3.0mm). A triple quadrupole mass spectrometer was used for detection of the analytes in positive mode electrospray ionization using multiple reaction monitoring (MRM). The limits of detection, quantification, and confirmation for these metabolites were 25, 50, and 50pg/mL, respectively. The linear dynamic range of quantification was 50-10000pg/mL. Enzymatic hydrolysis indicated that JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, and JWH-250 5-OH indole are highly conjugated whereas JWH-250N-pentanoic acid is not conjugated. Relative retention time and product ion intensity ratios were employed as the criteria to confirm the presence of these metabolites in equine urine. The method was successfully applied to post-race urine samples collected from horses suspected of being exposed to JWH-250. All four JWH-250 metabolites were confirmed in these samples, demonstrating the method applicability for equine doping control analysis.

UPLC characteristic fingerprint of leaves of Moringa oleifera

This study aims to establish the characteristic fingerprint of the leaves of Moringa oleifera by Ultra High Performance Liquid Chromatography (UPLC) for its quality control. The method was developed on a column of Agilent Eclipse XDB-C₁₈ with acetonitrile-0.01% TFA solution as the mobile phase by gradient elution at a flow rate of 0.5 mL·min⁻¹. The detective wavelength was 210 nm, and the column temperature was 35 °C. The 14 batches of the leaves of M. oleifera were compared for the similarity by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2004A). The UPLC characteristic fingerprint was established, and twelve common peaks were identified by comparison with the references and UPLC-MS. The relative retention times were 0.08 (No. 1, adenosine), 0.14 (No. 2, L-phenylalanine), 0.22 (No. 3, 5-caffeoylquinic acid), 0.28 (No. 4, L-tryptophane), 0.42 (No. 5, 4-caffeoylquinic acid), 0.65 (No. 6, vicenin-2), 0.94 (No. 7, vitexin), 0.96 (No. 8, isovitexin), 1.00 (No. 9, isoquercitrin), 1.11 [No. 10, quercetin 3-O-β-D-(6"-malonyl)-glucopyranoside], 1.21 (No. 11, astragalin) and 1.37 [No. 12, kaempferol 3-O-β-D-(6"-malonyl)-glucopyranoside]. It is the first time to establish the UPLC characteristic fingerprint of the leaves of M. oleifera. The method is simple, quick and reproducible with high precision, which can provide a scientific basis for the quality control of the leaves of M. oleifera.

Implementation of normalized retention time (iRT) for bottom-up proteomic analysis of the aminoglycoside phosphotransferase enzyme facilitating method distribution.

Improvements in mass spectrometry technology to include instrument duty cycle, resolution, and sensitivity suggest mass spectrometry as a highly competitive alternative to conventional microbiological proteomic techniques. Targeted mass spectral analysis, sans prior empirical measurements, has begun to solely use the enormous amount of available genomic information for assay development. An in silico tryptic digestion of a suspected antibiotic-resistant enzyme using only its genomic information for assay development was achieved. Both MRM and full-scan MS2 independent data acquisitions were obtained for an antibiotic-resistance microbe not previously measured using mass spectrometry. In addition, computation methods to determine highest responding peptides in positive ion mode liquid chromatography-mass spectrometry (LC-MS) were evaluated. Employment of the relative retention time (iRT) concept using a homemade peptide standard set revealed facile method transfer between two fundamental different mass spectral platforms: an ultra-high-pressure liquid chromatography triple quadrupole-mass spectrometer (UHPLC-MS) and nano-liquid chromatography parallel reaction monitoring (nano-LC-PRM) hybrid quadrupole orbitrap Q-exactive mass spectrometer supporting easy dissemination and rapid method implementation between laboratories. Graphical Abstract.

Screening and quantitation of forty-six drugs of abuse and toxic compounds in human whole blood by capillary electrophoresis: Application to forensic cases

A novel capillary electrophoresis method, coupled with photo-diode array detection, was established for the analysis of forty-six drugs of abuse, including opiates, cocaine, amphetamines, benzodiazepines, and alkaloids, in human whole blood. Doxapram was used as the internal standard, and an Oasis HLB column was used for solid-phase extraction of drugs from human whole blood. This method was applied to analyze real blood samples in forensic cases. The separation was performed on a fused-silica capillary (75 μm I.D. × 50.2 cm, with 40 cm of effect length). In this study, the running buffer was 150 mM phosphate buffer (pH 2.4) containing 20% methanol. Samples were injected at a pressure of 0.5 psi for 10 s and separated at a running voltage of 16 kV. The precision, accuracy, sensitivity, linear range, limit of detection, and recovery of the method were evaluated. The time taken for the entire separation process was 18.5 min. All compounds were identified by relative retention times, and the peak height ratios (the ratio between the target analyte peak height and the internal standard peak height) were monitored at 200 nm and 210 nm in corresponding ultraviolet spectra. 210 nm was used as quantitative detection wavelength. Calibration curves were established for all compounds of interest. For human blood samples, the detection limits were 8–80 ng/mL, and the linear range was 0.03–10 μg/mL. The intraday and interday precisions were both less than 13%. This method offers simple and effective screening and quantitation of drugs of abuse and toxic compounds in human blood at toxicologically relevant concentrations, and was applied to analyze blood samples from forensic cases.

Simultaneous qualitative and quantitative analysis of 11 active compounds in rhubarb using two reference substances by UHPLC.

Multi-component analysis is one of the key techniques for the overall quality control of traditional Chinese medicines. However, the shortage and high cost of reference substances are the greatest obstacles. The substitute method is an alternative solution. In the present study, 11 compounds of rhubarb were simultaneously determined by a method named "two reference substances for determination of multiple components", which includes a qualitative method with linear calibration using two reference substances and a quantitative method with a relative correction factor combined with ultra high performance liquid chromatography. Using aloe-emodin-8-O-β-D-glucopyranoside and chrysophanol as reference compounds, chromatographic peak identification was performed. The results demonstrated that linear calibration using two reference substances method showed higher accuracy, less deviation, and better column adaptability compared to the relative retention time method. Using chrysophanol as a reference compound, the relative correction factors were determined and showed good reproducibility and stability in different laboratories with different instruments, columns, and wavelength fluctuations. The results had no significant difference compared with the external standard method. The strategy of two reference substances for determination of multiple components coupled with ultra high performance liquid chromatography is economical, efficient, accurate, reliable, and environmentally friendly and is suitable for the quality control of traditional Chinese medicines.

UPLC-MS/MS Based Identification of Dietary Steryl Glucosides by Investigation of Corresponding Free Sterols

Dietary plant foods are characterized by a vast molecular diversity of glycosylated sterols (SG) that differ in the structure of the steryl backbone. The identification of these polar steryl conjugates represents a major challenge as they are structurally highly similar, and commercial standards are limited to a few naturally abundant species. Spectral databases do not yet contain MS/MS spectra of these sterol conjugates obtained by electrospray ionization (ESI), which would facilitate their reliable identification. Thus, this study aimed at providing novel information on ESI-MS/MS spectra of both abundant and minor SG found in foods. As a first step, however, free sterols (FS) were investigated for their fragmentation behavior as they share the same intermediate ion as SG. Pure SG were obtained from commercially available standard mixtures and minor SG were extracted from different food sources (oat bran, wheat bran, pumpkin seeds, melon, rapeseeds, and potato peel). ESI-MS/MS spectra of 15 FS were assessed and fragment ions reflective of structural features were identified and rationalized. Subsequently, 14 SG were identified at four different levels, while relative retention times from chromatographic separation and spectral features of FS served to identify five SG. Spectral data from FS were directly transferable to SG when analyzed as aglycone ions as shown by similarity scores while SG were characterized by shorter retention times in reverse phase chromatography and the additional analysis as sodiated adduct confirmed their glycosidic nature. Moreover, we report for the first time the occurrence of 24-methylenecholesterol and a 4-monomethyl sterol as glycosidic conjugates in higher plants. The presented data will serve as a valuable tool for SG profiling of foods by facilitating their identification.

Establishment and application of a new HPLC qualitative and quantitative assay for Gentiana Macrophyllae Radix based on characteristic constituents of anofinic acid and its derivatives

Gentiana Macrophylla Radix (GMR) is officially used as traditional Chinese medicine, but easily confused with Gentianae Radix et Rhizoma (GRR) and adulterants. This study aimed to establish an HPLC method for qualitative and quantitative analysis of GMR based on characteristic components, anofinic acid and its derivatives. HPLC analysis was performed on a C18 column with gradient elution using acetonitrile and 0.1% phosphoric acid as mobile phase, and detected at 240 nm by conventional methodology validation. For fingerprint analysis, RSDs of relative retention times and relative peak areas of the characteristic peaks were within 0-1.10 and 0-4.08%, respectively. For determination of 2-methoxyanofinic acid, the calibration curve showed good linearity (R2 > 0.9999) within the test range. The RSDs of precision, repeatability and stability test did not exceed 2.46, 0.83 and 1.11%, respectively. The average recoveries were between 95.08 and 103.05% with RSDs ≤2.29%. The results showed that there was no significant difference among the four species of GMR, but there were significant differences among GMR, GRR and spurious breeds by principal component analysis and hierarchical cluster analysis. Anofinic acid and its derivatives, as the characteristic markers, could be used for the identification and quality control of GMR.

A Simple Method for Peak Alignment Using Relative Retention Time Related to an Inherent Peak in Liquid Chromatography-Mass Spectrometry-Based Metabolomics.

Retention time (RT) shifts are inevitable in liquid chromatography-mass spectrometry (LC-MS)-based metabolomics studies. RT alignment, aligning peaks originating from the same feature to an identical RT, is a necessary step prior to multivariate data analysis. Many RT alignment software packages and various algorithms have been developed. In the present study, a simple method for peak alignment was proposed using relative RT related to an inherent peak in the chromatogram, and its performance on RT alignment in untargeted metabolomics was evaluated using an LC-MS dataset acquired from plasma samples from osteoporotic rats, and compared with the common MarkerLynx XS method using different RT window. RT alignment accuracy and relative standard deviation (RSD) value of the intensity of 79 selected ions in a chromatogram were employed to assess the alignment effectiveness of Method I (0.2 RT window), Method II (0.5 RT window) and Method III (relative RT). The overall RT alignment accuracy of these three methods was 67.7%, 77.2% and 90.9%, and 44.3%, 72.2% and 82.3% of the ions had intensity RSD values <30%, respectively. The results demonstrated that Method III using relative RT significantly reduced the influence caused by RT shifts in LC-MS-based metabolomics. Furthermore, Method I and Method III were applied to discover plasma biomarkers for osteoporosis in rats. Thirteen and nineteen ions were selected as potential biomarkers, respectively. Among them, only eight common ions were found, which demonstrated that different sets of biomarkers were obtained using those two RT alignment methods. The relative RT method exhibited fewer missing values, a lower RSD of peak intensity and better performance in pattern recognition, thereby showing the great potential in future metabolomics studies.

Abstract 738: Development of an HPLC method for the assessment of hydrophobicity of ADC payloads

Abstract Antibody-Drug Conjugates (ADCs) are antibodies attached to cytotoxic agents (payloads) through chemically labile linkers. The development of ADCs can fail at various stages, a common cause being a lack of reproducibility in the methods used to attach the payload to the antibody (i.e., the conjugation process) to provide a good yield and uniform drug-antibody ratio (i.e., the DAR). One of the major problems associated with conjugation is the hydrophobicity of a payload which can cause aggregation. The aim of this study was to create a hydrophobicity model to study the physicochemical properties of payloads at an early stage. This should allow their hydrophobicity to be evaluated and optimised at the design stage. A predictive model has been developed based on High Performance Liquid Chromatography (HPLC). Initial work involved development of a gradient reversed-phase HPLC method, and its use in the analysis of multiple reference compounds to establish their relative retention times. This involved the evaluation of a range of reversed-phase columns and solvent systems in order to obtain reproducibility. These initial studies, which utilized a C18 monolithic column for optimum results, demonstrated a clear relationship between the retention time (RT) of the reference compounds and their experimental LogP values obtained from the literature (r = 0.983). Next, a number of commercially available ADC payloads were analysed using the same HPLC method to determine their RT values, and their LogP values were calculated using the ChemDraw software. Payloads studied included Monomethyl Auristatin E (MMAE), Talirine and Tesirine (PBD dimers), Mertansine (DM1) and Calicheamicin. For this group of compounds, the results confirmed that their RT values correlated well with their calculated LogP values (r = 0.94). For example, the payload Talirine, which has a calculated LogP of 3.77, had an RT value of 17 min on the HPLC system equating to a LogP of 3.13 based on the reference compounds. Finally, some novel DNA-interactive payloads (e.g., the pyridinobenzodiazepine FGX-2-62) were evaluated using the same methodology. For FGX-2-62, the calculated LogP varied significantly between different software packages (i.e., 1.47, 3.38 or 4.46 for ChemSketch, MarvinSketch or ChemDraw, respectively). However, its HPLC retention time using the same method was 1.88 min, equating to a LogP of 1.63. This suggests that FGX-2-62 is significantly less hydrophobic than Talirine (e.g., RTs of 1.88 min versus 17 min, respectively), an observation consistent with the ease of conjugation of FGX-2-62 to antibodies without significant aggregation. Overall, these results suggest that HPLC retention time derived from a suitable HPLC method is more reliable than the currently available software-based LogP prediction methods in providing a realistic assessment of the hydrophobicity of novel payloads and their propensity to cause aggregation during conjugation. Citation Format: Ilona Pysz, Paul J. Jackson, Khondaker M. Rahman, David E. Thurston. Development of an HPLC method for the assessment of hydrophobicity of ADC payloads [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 738.

DEVELOPMENT OF A QUALIFICATION PROCEDURE FOR METHIONINE FORM OF INTERFERON ALFA-2b STANDARD TO CONFIRM ITS AUTHENTICITY BY MEANS OF A PEPTIDE MAPPING METHOD

Authenticity evaluation of proteins obtained with recombinant DNA technology is an important step in confirming efficacy and safety of the drugs based on them. One of the main ways to assess the authenticity is to compare molecular structure of the test and standard samples using the peptide mapping method with chromatographic separation of the products obtained by enzymatic degradation. Proper selection of a standard reference sample is essential in order to achieve reliable results. A standard sample of Interferon (CRS, Chemical Reference Substances) recommended by the European Agency for the Quality of Medicines for interferon alpha-2b substances containing N-terminal methionine is inappropriate, since the Interferon CRS sample doesn’t contain methionine. We present the results of development of qualification procedure for methionine form of Interferon alfa-2b industrial standard sample (ISS). The range of use for this ISS is authenticity confirmation for the methionine form of interferon alpha-2b substance using peptide mapping method with reverse-phase high-performance liquid chromatography (reverse-phase HPLC). The quality assessment was performed for all the parameters specified by the manufacturer of this candidate substance at the initial stage of qualification procedure, due to changed application area, and changed package size. Further, 30 peptide cards of the ISS candidate substance were obtained after pre-trypsinolysis of the protein followed by validated HPLC method with proven repeatability. It was shown that the hydrolysis conditions, i.e., the choice of trypsin preparations, may significantly affect the peptide map profile. Therefore, a reference to specific manufacturer and the catalog number of the product should be provided in description of application conditions for the ISS proposed. A set of eight reference peaks (peaks of comparison) has been justified, as based on evaluation of peptide maps and results of high-resolution mass spectrometry. The peak with maximally stable yield and intensity was selected as the main peak with an established absolute retention time. Two peaks with relative retention times were chosen as essential peaks for evaluation, i.e., the 1st peak containing N-terminal methionine, and the 2nd peak of highest molecular weight with an established amino acid sequence covering 11% of the studied interferon molecule. We have also qualified ISS parameters expressed as absolute (minimum for one reference peak), and relative (for the remaining reference peaks) retention time periods. Authenticity of the ISS candidate was further confirmed by the peptide mapping method, as compared with interferon CRS reference standard. Their peak patterns proved to be near-similar, except of a peak with eluted peptide containing N-terminal methionine as revealed by high-resolution mass spectrometry

Occurrence and Distribution of Unusual Tri- and Tetracyclic Terpanes and Their Geochemical Significance in Some Paleogene Oils from China

Two unusual tricyclic terpanes (compounds X and Y) and four tetracyclic terpanes (compounds X1, Y1, Z, and Z1) have been detected in crude oils from the Pearl River Mouth, Beibuwan, and Liaohe basins in China. On the basis of their elution order, relative retention times in m/z 191 mass chromatograms, and their diagnostic ion fragments, we have identified two tricyclic terpanes (marked by peaks X and Y in previous literature) as C21 and C25 tricyclic terpanes and four tetracyclic terpanes (marked by peaks X1, Y1, Z, and Z1) as C24-des-A-lupane, C24-des-A-oleanane, C24-des-A-ursane, and C27 tetracyclic terpane, respectively. These six compounds have similar characteristics to oleanane, ursane, and lupane in their chemical structure and are considered likely to originate from alcohols or ketone precursors present in higher plants. The high abundance of these tri- and tetracyclic terpanes is probably related to the distinctive contribution of higher plant material in the organic matter content of source rocks. In addition, the redox conditions and water depth in the depositional environment significantly impact the distribution patterns of the compounds, and it is possible that they are readily formed under oxidation conditions. Because of their unique biological origin and prominent geochemical significance, they may have potential application in oil–source correlation and the classification of crude oil families.

HPLC fingerprints of pig, cattle and sheep biles

To establish the fingerprints of biles of pig, cattle and sheep, HPLC was used with Acclaim™ RSLC 120 C₁₈ column (3.0 mm×100 mm, 2.2 μm, 120 Å), the column temperature 35 °C, acetonitrile-1% perchloric acid as mobile phase, gradient elution, 0.5 mL·min⁻¹ flow rate, and detection wavelength at 200 nm. The fingerprint was generated by using Similarity Evaluation Software of Chromatographic Fingerprint of Chinese Medicine (2004A Edition). The fingerprint peaks were identified by reference substances and verified by ELSD and LC-MS/MS. Then, the biles of pig, cattle and sheep were detected to contain 14, 9 and 8 common fingerprint peaks respectively, and the similarity was greater than 0.92. To analyze each technical parameter, GHDCA in pig bile and TCA in cattle and sheep bile were selected as reference peak. The precision, repeatability and stability all meet the requirements of fingerprint establishment. The RSD of the relative retention time of the fingerprint peaks was less than 1.5%, and the RSD of the relative peak area was less than 5%. The fingerprint peaks in pig bile were THDCA, TCDCA, GHDCA and GCDCA, and TCA, TCDCA, GCA, GCDCA and GDCA in cattle and sheep bile. The main components of pig, cattle and sheep bile were conjugated bile acids, but there were significant differences in bile acids between pig bile and cattle, sheep biles. The HPLC method established in this paper is simple, rapid and reproducible, and could be applied to the identification and quality control of biles.

Amine over-alkylation side products in the synthesis of BMS-955176

Over-alkylation side products are common in the alkylation of amines by substitution. In the synthesis of the novel HIV Maturation inhibitor BMS-955176, two over-alkylation byproducts were routinely observed at the penultimate synthetic step, in which a thiomorpholine dioxide side chain was added to the core molecule by alkylation of a primary amine. These two byproducts had drastically different HPLC relative retention times, despite both containing only one additional side chain. Adding complexity to the challenge of solving their structures was the proclivity of the two byproducts to interconvert. Positive- and negative-ion HRMS, as well as isolation and 1D and 2D NMR were utilized to determine their structures. These byproducts were additionally problematic in that they led to daughter impurities at the API step.

Development and Method Validation of Analysis of Urushiol in Sumac and Food Ingredients in Korea.

This study developed an analytical method to determine the urushiol content in sap and several foods. The full process for urushiol analysis consists of extraction, trimethylsilyl silylation, analysis, and identification via GC-MS, with each step optimized to attain the required accuracy and precision. Urushiol was separated from sap via liquid-liquid extraction and was derivatized via silylation. The components were analyzed using a polar capillary column and identified using GC-MS. The deviations of relative retention times and retention time windows were within 0.001 and 0.02 min, which satisfied the criteria of 0.06 and 0.03 min, respectively. The response of the urushiol standards tested was found to be linear in the investigated concentration range, with a correlation coefficient of 0.998. The LODs were between 1.74 and 2.67 μg/mL.

Fingerprint analysis and pharmacological evaluation of Ailanthus altissima.

Ailanthus altissima (AA) has been used in various anticancer prescriptions and showed excellent therapeutic effect. However, there is no report on the method of quality control and the anti-glioblastoma activity. In this study, we used a combinative method approach consisting of chromatographic fingerprinting and quantitative methods to analyze quality of different samples. The anti-glioblastoma activity and the possible mechanisms were studied by pharmacological methods. The samples were separated on a Kromasil 100-5C18 column and the flow rate was 0.8ml/min at 25˚C. The mobile phase was composed of 1%formic acid and 1%methanol-water at a flow rate of 0.8ml/min. The method was validated and applied to the quantification of different samples. Ten batches of AA from different provinces in China were detected, and we found that the contents of Ailanthone(AT) ranged from 0.21-1.78mg/g and the relative retention times were similar in different origins. Ten batches of AA were analyzed by the high-performance liquid chromatography (HPLC) fingerprinting method and 19 common peaks were detected. The similarity of 10batches is <1.5%. The peak areas in different samples were significantly different (0.682-0.954). We also found that AA induced oxidative stress first in U87 cells, then induced ER stress, finally activated the caspases which caused cell apoptosis. In conclusion, a method combining chromatographic fingerprinting and quantitative analysis can be used to control the quality of AA. AA could be used as a medicine or a constituent part of herb prescription to treat glioblastoma.

Evaluation of phytochemicals in Agastache rugosa (Fisch. & C.A.Mey.) Kuntze at different growth stages by UPLC-QTof-MS

To improve the utility of A. rugosa as a seasonal edible vegetable, it is necessary to determine the components and contents of reference substances during its different growth stages. Standard markers were characterized as six compounds including a new compound 5 (acacetin 2″-O-acetyl-6″-O-malonyl-β-d-glucopyranoside) by NMR and UPLC-QTof-MS analyses. A method for qualitative/quantitative analysis of the extract was evaluated including compounds (3–5), which are easily converted to tilianin (2) during extraction & isolation process by using UPLC-QTof-MS. The contents of each compound were remarkably changed at different growth stages, especially tilianin group (2–4, and 5), which were the predominant substances (>95%) in all stages. Additionally, more comprehensive chemical information, including the relative retention time, peak area and contents, were obtained simultaneously from the raw material using marker compounds. Therefore, application of this analysis method may relieve the increasing demand for pure standards.

Establishment of a reference standard database for use in the qualitative and semi-quantitative analysis of pharmaceutical contact materials within an extractables survey by GC–MS

The analysis of reference standards may be performed to enhance the qualitative and quantitative data generated by non-specific screening methods utilized in extractables studies performed on pharmaceutical contact materials. However, the establishment of a database containing relative response factor and retention index values obtained from these standards has not been published. In this study, the establishment of such a database for GC–MS, a methodology commonly included in extractables studies, on an intra-lab basis was investigated. A set of 154 organic compounds representing a diverse range of chemical functionalities and properties was analyzed at eight time points on four GC–MS instruments that represent the diversity of age and model at our laboratory. The results of this study have shown that any variance in relative response factor between instruments was not significant from a practical perspective as supported by the coefficient of variation values (n = 32), which were ≤15% and ≤10% for 75% and 45% of the compounds tested, respectively. Furthermore, the retention index of the compounds, as expressed by retention time and relative retention time, did not have more than a 2% coefficient of variation between instruments or columns in most cases. It was concluded that a database of these values could be established for future use in extractables studies on an intra-laboratory basis.