Latest ArticlesObjective: To establish an HPLC method for simultaneous determination of loganic acid, gentiopicroside, hesperidin, paeonol, aloe-emodin, rhein, emodin, chrysophanol and physcion in Tongshu Koushuang tablets (Rhei Radix et Rhizoma, Aurantii Fructus Immaturus and Gentianae Macrophyllae Radix, etc.). Methods: The samples were extracted by refluxing with methanol and the separation was performed on a Thermo Acclaim-C18(250 mm×4.6 mm, 5 μm) column at 30 ℃, with mobile phase consisting of 0.15% phosphoric acid solution and acetonitrile with a gradient elution at a flow rate of 1.0 mL·min-1. The injection volume was 10 μL, and the detection wavelength was set at 236 nm (detecting loganic acid), 280 nm (detecting gentiopicroside, hesperidin and paeonol) and 254 nm (detecting aloe-emodin, rhein, emodin, chrysophanol and physcion). Results: Nine constituents (loganic acid, gentiopicroside, hesperidin, paeonol, aloe-emodin, rhein, emodin, chrysophanol and physcion) were in good linearity within their own ranges (r were 0.999 1-0.999 9), and the average recoveries were 99.6% (RSD=1.9%), 101.1% (RSD=2.0%), 103.0% (RSD=1.5%), 101.6% (RSD=0.51%), 99.8% (RSD=1.4%), 100.9% (RSD=1.4%), 100.8% (RSD=1.8%), 102.7% (RSD=1.2%) and 102.9% (RSD=0.86%), respectively. The content ranges of nine constituents were 1.871-3.517 mg·g-1, 6.274-12.55 mg·g-1, 2.140-3.946 mg·g-1, 1.393-2.018 mg·g-1, 0.394 8-0.807 2 mg·g-1, 0.657 5-1.246 mg·g-1, 0.484 7-0.915 8 mg·g-1, 0.839 8-1.429 mg·g-1 and 0.327 3-0.547 9 mg·g-1, respectively. Conclusion: This simple and accurate method can be used for the quality control and evaluation of Tongshu Koushuang tablets.
Objective: To establish a dual derivatization method combined with tandem mass spectrometry for the simultaneous determination of 18 different steroid hormones in human serum. Methods: Serum samples were treated with hydroxylamine and 1, 2-dimethylimidazole-5-sulfonyl chloride for derivatization, and the resulting compounds were analyzed by liquid chromatography-tandem mass spectrometry in positive ion selected reaction monitoring (SRM) mode. The Kinetex®C8 column (100 mm×2.1 mm, 2.6 μm) was used for the separation. Mobile phase A (0.1% acetic acid in water) and mobile phase B (0.1% acetic acid in methanol) with gradient elution（0-0.5 min, 35% B; 0.5-5 min, 35% B→100% B; 5.0-5.1 min, 100% B→35% B; 5.1-7 min, 35% B）at the flow rate of 0.4 mL·min-1 were applied. Column temperature was set at 40 ℃. Injection volume was 20 μL and collection time was 6 min. Results: The linearity correlation coefficients for all 18 steroid hormones were greater than 0.99, the recovery rates ranged from 85% to 115%, and the precision RSD was less than 15%. This method was successfully applied to the analysis of serum samples from 156 healthy subjects (75 males and 61 females), and reference intervals were established. Conclusion: This method can be used to simultaneously determine 18 types of steroid hormones, such as pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, corticosterone, cortisol, 11-deoxycorticosterone, 21-deoxycorticosterone, aldosterone, testosterone, androstanedione, dehydroepiandrosterone, sulfated dehydroepiandrosterone, adrenocorticotropin, 18-hydroxicorticotropin, estrone, estradiol, and estriol using non-solid-phase extraction.
Objective: To establish a quantitative analysis of multi-components by single marker (QAMS) method to simultaneously determine the contents of scopolamine, anisodamine, scopolin, hyoscyamine and scopoletin in Physochlaina infundibularis Kuang. Methods: Agilent 5 TC-C18 (250 mm×4.6 mm, 5 μm) column was used with methanol-0.1% phosphoric acid aqueous solution as mobile phase for gradient elution at a flow rate of 1.0 mL·min-1. The detection wavelengths were 210 nm and 344 nm, the column temperature was 30 °C, and the injection volume was 10 μL. The relative correction factors for scopolamine, scopolin, hyoscyamine and scopoletin and anisodamine were calculated by using anisodamine as the internal reference substance. The contents were determined and compared with the results of external standard method (ESM). Results: The five components had good linear relationship in their respective ranges(r=1.000). The relative correction factors of scopolamine, anisodamine, scopolin, hyoscyamine and scopoletin were 0.951 8, 0.562 6, 0.830 4 and 0.433 9, respectively, with good repeatability. The contents of scopolamine, anisodamine, scopolin, hyoscyamine and scopoletin in P. infundibularis from 10 habitats calculated by QAMS method were not significantly different from those determined by ESM. Conclusion: The method is accurate, stable and reproducible, and can be used for the quality control of P. infundibularis.
Objective: To determine the concentration of vanillic acid by polyoxometal-based host-guest frame material modified glass carbon electrode. Methods: A stable polyacid-based host-guest metal-organic framework material PMoV@MIL-100(Fe) was synthesized by combining vanadium-substituted phosphomolybdic acid (PMoV) with MIL-100(Fe) according to the synthesis method of polyacid-based host-guest framework materials. Functional composite material PMoV@MIL-100(Fe)@Pt was prepared by composite metal nanoparticles with PMoV@MIL-100(Fe) by solution method, and was used to modify glassy carbon electrode, detect vanillic acid, and prepare vanillic acid electrochemical sensor. Results: Under optimal conditions, the vanillic acid electrochemical sensor showed wide linear range and high sensitivity. Rapid and sensitive detection of vanillic acid could be performed under mild conditions with excellent stability. Conclusion: The electrochemical sensor constructed in this experiment provides a new idea for the detection of vanillic acid.
Objective: To explore the fragmentation patterns of synthetic cannabinoids by electron impact (EI) ionization mass spectrometry. Methods: Forty synthetic cannabinoids were systematically investigated by gas chromatography coupled to mass spectrometry (GC-MS). Ionization mode was EI (70 eV) and the acquisition range was m/z 50-600. Results: According to the different structures of the “head group” and “linked group”, forty synthetic cannabinoids were divided into six categories, namely cumyl-carboxamide type, adamantyl-carboxamide type, carbamoyl/methyl butyrate-carboxamide type, naphthylformyl type, benzoyl/phenylacetyl type and tetramethylcyclopropane-acyl type. Through the analysis of the mass spectrum of synthetic cannabinoids, the fragmentation pathways and characteristic ions of different types of synthetic cannabinoids were given. The main EI-MS fragmentation patterns of synthetic cannabinoids were that both sides of the carbonyl group in the “linking group” undergo α-cleavage, and the N atom on the indole/indazole parent nucleus was prone to γ-H rearrangement, and loss of a R1. In addition, fragment ions m/z 116, 130, 144 and fragment ions m/z 117, 131, 145 were the characteristic fragments of indazole and indole parent nucleus, which could be used to identify the parent nucleus of synthetic cannabinoids. Conclusion: These kind of compounds have strong fragmentation regularity. When standard substances are lacking or commercial mass spectral libraries are difficult to obtain, the proposed synthetic cannabinoids EI-MS fragmentation pathways can help to rapidly identify the structures of unknown synthetic cannabinoids.
To establish a low-field and high-field nuclear magnetic resonance (NMR) technique for the determination of pyridoxine hydrochloride. To compare the differences between the results of method validation and sample content.
Using deuterated water as the solvent and maleic acid as the internal standard，method validation of quantitative nuclear magnetic resonance (qNMR) was accomplished with both 80 MHz low-field NMR and 500 MHz high-field NMR instruments. The content of pyridoxine hydrochloride was also determined.
The linearity of pyridoxine hydrochloride was good in the range of 10-30 mg·mL-1，and the linear correlation coefficients were 0.999 8 and 0.999 6 for the low-field and high-field NMR，respectively. RSDs of the low-field and high-field NMR measurements were 1.4% and 0.040%，respectively. The contents were 100.4% and 100.8%，which were in agreement with the results from the mass balance method (100.0%). And the repeatability RSD was 0.20%. For the same concentration of the sample，the signal-to-noise ratio (S/N) of high-field NMR was about 100 times that of low-field NMR，and the half-peak width was 1/15 of that of low-field NMR.
Both low-field and high-field NMR instruments gave accurate results in the determination of pyridoxine hydrochloride content. However，low-field NMR was only suitable for compounds with simple structures. In the actual content determination，it is necessary to choose the appropriate instrument according to the structure and solubility of the sample to be tested.
To establish an inductively coupled plasma mass spectrometry (ICP-MS) method for the determination of migration Co，As，Br，Cd，Sb，Ba and Pb in recombinant human coagulation factor Ⅷ-Fc fusion protein for injection，and to quantitatively analyze the compatibility of long-term stored samples.
After adding 0.75 mL of water to the sample for 4 h，the purified water was filled to 5 mL，and the contents of 7 elements in the solution were determined by standard addition method. The working parameters of ICP-MS：radio frequency (RF) power of 1 550 W，sampling depth of 10.0 mm，carrier gas flow rate of 0.50 L·min-1，atomizer pump speed of 0.30 r·s-1，atomization chamber temperature of 2 ℃，dilution gas flow rate of 0.74 L·min-1，repeated sampling times of 3 times，analysis mode of full quantitative.
The linear relationship of each element was good in the concentration range of the detected mass，and the recoveries of the element added were 92.0%-101.8%. When applied to the determination of the samples at each accelerated placement point，the increased migration amounts were 43.2，68.7 and 66.8 ng·mL-1，respectively，except the element content of Br increased slightly. The contents of the remaining 6 elements were far below the prescribed limit.
The method is reliable and can be used for the determination of migration of 7 elements in recombinant human coagulation factor Ⅷ-Fc fusion protein for injection.
To establish a method for the quantitative detection of bacterial endotoxins in human peripheral blood mononuclear cell (PBMC) suspensions and to validate the feasibility of the detection method.
According to the standards for photometric determination of bacterial endotoxin in General Notice 1143 of Chinese Pharmacopoeia (ChP) part Ⅲ (2020 edition) and 1085 of USP. A preliminary detection method of bacterial endotoxin content in PBMC suspension was established from standard curve reliability verification and primary interferential screening test. Bacterial endotoxins in three batches of PBMC suspensions were quantitatively detected by this method.
In the establishment of standard curve and reliability verification test，the linear regression equation was lgt= 2.910 9-0.300 1lgC，r=-0.999 8，negative control t>3 600 s，repeated reaction tube RSDs were less than 3%，the standard curve was established successfully. The results of the primary interferential screening test showed that the PBMC suspension had interference on limulus test at the dilution ratio of 10 and the recovery rate was close to 100% at the dilution ratio of 80，indicating that 80-fold was the best interference dilution ratio without interference. The results of the three batches of samples showed that the endotoxin contents were all less than 1.0 EU·mL-1 at the dilution ratio of 80，and the RSDs were less than 10%. The positive recoveries were 80.8%-100.3%，which complied with ChP 2020.
This method can quickly and quantitatively detect the content of endotoxin in PBMC suspension.
To establish methods of thin-layer chromatography(TLC) qualitative identification and quantitative analysis of multi-components by single-marker (QAMS) according to the characterization results of the main components of Iris tectorum Maxim..
Firstly，the main components of Iris tectorum were characterized by ultra performance liquid chromatography tandem time-of-flight mass spectrometry(UPLC-Q TOF MS/MS) technology，using an ACQUITY UPLC® BEH C18 column (100 mm×2.1 mm，1.7 μm) with gradient elution using 0.1% formic acid-water (A)-acetonitrile (B) as mobile phase，and the data were collected in positive ion mode. In addition，the qualitative identification method was established by the TLC. Finally，quantitative analysis was performed on an Agilent 5 TC-C18 column (250 mm×4.6 mm，5 μm). The mobile phase was 0.05% phosphoric acid water(A)-acetonitrile(B) with gradient elution. The QAMS method was established by using tectorigenin as an internal standard to establish its relative correction factor with tectoridin and irigenin，and the contents of three isoflavones were calculated by the correction factor. At the same time，the accuracy and feasibility of the QAMS method was verified by the external standard method(ESM).
Five isoflavones were characterized (tectoridin，iristectorin B，tectorigenin，irigenin and iristectorigenin A) by UPLC-Q TOF MS/MS. A TLC identification method for four components，tectoridin，iristectorin B，tectorigenin and irigenin was established. In this study，the linearity of the three components of tectoridin，tectorigenin and irigenin were good in a certain concentration range (r>0.999)，with the average recoveries of 97.7%-100.8% and the RSDs of 1.1%-2.9%，and the relative correction factors of tectoridin and irigenin were 1.114 5 and 0.827 0，respectively. And the repeatability of the correction factors was good under different experimental conditions (RSD<5%). There was no significant difference between the contents of the three analytes in ten different batches of Iris tectorum determined by QAMS and ESM. The contents of tectorigenin，tectoridin and irigenin in 10 batches of Iris tectorum from different habitats determined by QAMS were not significantly different from those determined by ESM.
Based on the characterized components of Iris tectorum medicinal materials，the TLC and QAMS methods with high-content and stable ingredients are established. The methods are simple and stable，which can be used for qualitative and quantitative analysis and quality evaluation of Iris tectorum.
To establish a microbial limit test method and quality standard conforms to the USP for compound Kushen extract and its related components.
Comparative analysis was conducted on the standards for traditional Chinese medicine preparations and decoction pieces in the USP and the Chinese Pharmacopoeia 2020 Edition to determine the microbial limit standards for compound Kushen extract and its related components. Then，guided by the General Principles of the USP<61> and <62>，a microbial limit test method suitable for compound Kushen extract and its related components was developed. The plate pouring method was used to perform a methodological applicability test on the total aerobic bacterial count，mold and yeast count of compound Kushen extract and its two components Sophorae Flavescentis Radix and Heterosmilacis Rhizoma. The method applicability test was conducted on the control bacteria using the direct inoculation method. Finally，the MALDI/TOF MS microbial identification method and bacterial 16S rRNA gene sequence alignment analysis method were comprehensively used to identify the bacterial species of the contaminated microorganisms in the sample，and further analysis was conducted on the distribution and harm of the contaminated microbial population.
As a result，microbial limit standards for compound Kushen extract and its related components were established in accordance with the requirements of the USP. The recoveries of the counting methods for compound Kushen extract and its two components，Sophorae Flavescentis Radix and Heterosmilacis Rhizoma，were between 0.5 and 2.0. The positive control strains in the control bacterial method suitability test were able to grow，meeting the requirements of the USP. According to the identification results，40 contaminated microorganisms involved 13 species and 5 genera，mainly covering different types of Bacillus genus (87.5%)，Enterobacterium genus (10%)，and Pseudomonas genus (2.5%). Polluting microorganisms were all less harmful conditional pathogenic bacteria.
Established a microbial limit test method and quality standard conforms to the USP for compound Kushen extract and its related components. Defined the distribution and harm analysis of the pollution microorganism poulation of the above products. Improved the microbial quality control of the Chinese medicine preparation and its components.
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