Quantification by LC–MS/MS of astragaloside IV and isoflavones in Astragali radix can be more accurate by using standard addition

Introduction Astragali radix (AR), the root of Astragalus, is an important medical herb widely used in traditional Chinese medicine. Bioactive components include isoflavones and a unique class of triterpenoid saponins (named astragalosides). Objectives Accurate measurement of bioactive components, especially astragaloside IV, is necessary for confirming AR authenticity, quality control and future medical research. Methodology Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) is a suitable technique but suffers from ion suppression effects due to sample matrix. This can be corrected by using isotopic labelled internal standards, but these are not available for many phytochemicals. We explored the use of standard addition to circumvent this issue. Results LC–MS/MS and liquid chromatography coupled with ultraviolet (LC-UV) detection provided linear calibration curves (R2 > 0.99). LC–MS/MS provided superior selectivity and detection limits below 10 ng/mL, which was 2–3 magnitudes lower than LC-UV detection. Precision and accuracy were overall improved by using LC–MS/MS with diluted sample extracts, resulting in an inter series coefficient of variation (CV) of 12% or less and mean recovery estimates in the 85–115% range. LC–MS/MS quantification by standard addition resulted in significantly higher concentrations of astragaloside IV measured in the samples. Concentrations calculated by standard addition were unaffected by large variation in signal response caused by matrix effects, independent of variation in slope of the standard addition curves. Conclusion Sample dilution was helpful but not sufficient for reducing effects of ion suppression. We have shown that LC–MS/MS quantification by standard addition can be a powerful approach for accurate measurement of phytochemicals in the absence of isotopic labelled internal standards.publishedVersio

Fourier-transform infrared spectroscopy for characterization of liquid protein solutions: A comparison of two sampling techniques

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The role of biospectroscopy and chemometrics as enabling technologies for upcycling of raw materials from the food industry

It is important to utilize the entire animal in meat and fish production to ensure sustainability. Rest raw materials, such as bones, heads, trimmings, and skin, contain essential nutrients that can be transformed into high-value products. Enzymatic protein hydrolysis (EPH) is a bioprocess that can upcycle these materials to create valuable proteins and fats. This paper focuses on the role of spectroscopy and chemometrics in characterizing the quality of the resulting protein product and understanding how raw material quality and processing affect it. The article presents recent developments in chemical characterisation and process modelling, with a focus on rest raw materials from poultry and salmon production. Even if some of the technology is relatively mature and implemented in many laboratories and industries, there are still open challenges and research questions. The main challenges are related to the transition of technology and insights from laboratory to industrial scale, and the link between peptide composition and critical product quality attributes.publishedVersio

Exploring Analytical Methods for Quality Control and Accurate Quantification of Major Biologically Active Components in Astragalus Radix

Master's thesis in Biological ChemistryAbstract Introduction Over the past decade, the worldwide consumption and sales of Traditional Chinese Medicine (TCM) herbs have grown enormously. Astragalus Radix (Chinese: Huang qi) (AR), the dried root of Astragalus membranaceous is a typical example. It is widely used in TCM to boost the body’s immune system, to reinforce the vital energy (“Qi” in Chinese) and for the treatment of bronchitis, pneumonia, and fatigue. The bioactive compounds of AR are flavonoids, saponins, polysaccharides, amino acids, and trace elements. In many countries (China, Japan, the USA, and Europe), Pharmacopeia’s and monographs have been published, which describes the morphological characteristics and procedure for assays to test the quality and standardization of medicinal plants. The purpose of the study is to routinely investigate the quality of herbs, analysing the bioactive compounds in a highly accurate, reproducible, qualitative, and quantitative manner. Objectives 1. Identification and quantification of isoflavonoids (formononetin, ononin, and calycosin-7-O-β-D glucoside) and saponins (astragaloside IV and cycloastragenol) in Astragalus Radix samples. 2. Technical comparison and use of improved methods for the quantitative determination of chemical components in AR samples and, 3. Comparison of commercial samples from different vendors. Methods Chemical standards were used for comparison and confirmation. Ultrasonication extraction was performed for a higher yield and sample preparation was optimized. Thin-layer chromatography (TLC) was used for detecting the presence of compounds. These were confirmed afterward when TLC plates were used in mass spectrometric (MS) detection. Fourier Transform Infrared Spectroscopy (FTIR) analysis was used to identify the functional groups of bioactive chemical compounds. The samples were further analysed using rapid and sensitive high-performance liquid chromatography–ultraviolet detector (HPLC-UV) and tandem mass spectrometric (LC-MS/MS) methods. The chromatographic conditions were optimized using the gradient elution of 0.2% formic acid in water and acetonitrile as mobile phases for HPLC-UV and methanol instead of acetonitrile in LC-MS/MS. The isoflavonoids were measured with a detection wavelength of 254 nm using a diode-array detector. The triple quadrupole tandem mass spectrometer was operated using positive electron-ionization modes and monitored using multiple reaction monitoring (MRM). The method was validated for linearity, selectivity, accuracy, and precision. External calibration and standard addition were performed during LC-MS/MS analysis. Results TLC-MS analysis of Astragalus Radix samples showed four of the five compounds detected. Compared with the other bioactive compounds, cycloastragenol gave very low-intensity peaks. The FTIR spectra of the sample extracts were not good enough to make a baseline fingerprint, but some functional groups could still be detected. The three isoflavonoids could successfully be quantified by HPLC-UV in different extracts of AR samples, but not formononetin in hydrophilic concentration. The presence of the latter was confirmed by LC-MS/MS. Astragaloside IV and cycloastragenol could not be detected by the UV method, while all five standards of compounds were detected using tandem mass spectrometry. LC-MS/MS method is a more selective, sensitive, reliable, and accurate method than HPLC-UV for the analysis of bioactive compounds of AR. Compared to external calibrations in LC-MS/MS, the quantitative results were significantly improved by using standard addition performed by adding known concentrations of standards to the sample solutions. These improved results can be due to a reduction in matrix effects by dilutions and ion suppression. Cycloastragenol was not detectable in any of the Astragalus Radix samples. The concentration in sample extracts might be below the detection limit or be naturally absent. The peaks in both MRM channels in exact retention times were not seen. Samples from different vendors contain widely different concentrations of the bioactive compounds, indicating much lower quality of Astragalus Radix samples from certain vendors. The highest concentration of astragaloside IV (203 ± 6 µg/g) was present in granulate samples from one vendor, whereas capsules from another vendor contained more ononin and calycosin 7-O-β-D glucoside. Formononetin in the capsule sample was comparable with samples from different vendors. The lowest concentrations of all compounds were observed in tablets from one vendor. This is partly but not completely understandable, as the tablets contain raw herbs rather than granulates (granulates are on average 3-5 times more concentrated than raw powder). Conclusions In the absence of isotopic labelled internal standards, the accuracy of quantification of bioactive components in Astragalus Radix samples of LC-MS/MS can be improved by using standard addition. The standard addition method was applied in diluted samples and quantification was independent of variations in signal response caused by matrix effects. LC-MS/MS was found to be significantly more sensitive and accurate than HPLC-UV for the measurement of essential bioactive compounds of AR. LC-MS/MS therefore should be used for Quality control of AR samples. This is probably also true for many other traditional Chinese medicine herbs, in the absence of isotope labelled internal standards. However, this should be further investigated. Another incredibly important research project would be to assess the pharmacokinetics of AR compounds in different persons, under various conditions, in blood, tissues, cells, and excretions (feces, urine, saliva). The results will assist the personalized dosing of AR herb in individual patients with different diseases. Keywords: Astragalus Radix, Isoflavonoids, Saponins, HPLC-UV, LC-MS/M

Major bioactive chemical compounds in Astragali Radix samples from different vendors vary greatly

The worldwide traditional Chinese medicine (TCM) herbs sales figures have increased considerably to 50 billion US$ (2018). Astragali Radix (AR) is amongst the most often sold TCM herbs; sales in the European Union (EU) need European Medicines Agency (EMA) approval. However, comparisons of characteristic bioactive molecules concentrations in AR from different EU vendors are lacking. This study uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) with standard addition to evaluate the influence of different sample and preparation types and ammonia treatment on bioactive molecules concentrations in AR. We also compare AR samples from different EU-vendors. Astragaloside IV (AG-IV), ononin and calycosin 7-O-β-D-glucoside concentrations were higher in root powder samples when extracted with boiled water than with ultrasonication using 70% methanol. AG-IV content was by far the highest in granulates from vendor 1 (202 ± 35 μg/g) but very low in hydrophilic concentrates from vendor 1 (32 ± 7 μg/g) and granulates from vendor 4 (36 ± 3 μg/g). Ammonia-treatment significantly increased AG-IV concentrations in all samples (e.g., to 536 ± 178 μg/g in vendor 1 granulates). Comparable effects were found for most other bioactive molecules. AG-IV and other bioactive molecules concentrations differed strongly depending on sample types, extraction processes, ammonia treatment-or-not and especially between different vendors samples. Ammonia-treatment is debatable, as it is supposed to convert other astragalosides, to AG-IV. The results indicate that routine quantitative analysis of major bioactive compounds present in AR, helps in quality control of AR and to guarantee the quality of commercial products.publishedVersio