Objective To develop a reversed-phase high-performance liquid chromatography(RP-HPLC) method for the determination of residual phenylmethanesulfonyl fluoride(PMSF) content in recombinant virus-like particle(VLP) vaccine stock solution,and to verify the method for the determination of PMSF residues in recombinant VLP vaccine stock solution.Methods A RP-HPLC method for the determination of PMSF residues was developed by screening detection wavelength,flow rate,injection amount and chromatographic column.The specificity,linearity and range,limit of quantitation(LOQ),limit of detection(LOD),accuracy,precision and durability of the method were verified.The developed method was used to detect PMSF residues in three batches of recombinant hepatitis E vaccine stock solution.Results The RP-HPLC method was developed,with ChromCore 300 C18(4.6 mm × 250 mm,5 μm) as the chromatographic column at the column temperature of 25 ℃,the detection wavelength of 210 nm,the injection amount of 100 μL,0.1% trifluoroacetic acid(TFA)/water and0.1% TFA/acetonitrile as mobile phase at a flow rate of 1 mL/min,and the detection time of 18 min.The absorption peaks of PMSF control and vaccine stock solution containing PMSF appeared around 5.0 min,while the vaccine stock solution and stock solution buffer showed no absorption peak.There was a good linear relationship between the concentration of PMSF control and peak area in the range of 0-5.0 μg/mL,with R~2 of 0.999.The LOQ and LOD of this method was 0.250 μg/mL and 0.125 μg/mL,respectively.The recovery rates of PMSF control with high,medium and low concentrations were all between 90% and 110%.The relative standard deviations(RSDs) of retention time,peak height and peak area for the determination of instrument repeatability,sample repeatability and intermediate precision were all less than 5%.The RSDs of retention time,peak height and peak area were all less than 5% under the TFA concentration of 0.09%,0.1% and 0.11% at column temperature of 20,25 and 30 ℃.PMSF was not detected in the three batches of recombinant hepatitis E vaccine stock solution.Conclusion The developed RP-HPLC method has good specificity,linearity and durability,high accuracy and precision,and can be used to detect residual PMSF content in recombinant VLP vaccine stock solution.
Phenylmethanesulfonyl fluoride(PMSF) ; Reversed-phase high-performance liquid chromatography(RP-HPLC) ; Virus-like particle(VLP) vaccine

OBJECTIVE

This study aimed to determine and quantify the presence of the active components in Thai propolis extracts using high performance liquid chromatography (HPLC). Moreover, the anti-caries potential of Thai propolis extract and its active ingredients were tested.

Fifty milligrams of Thai propolis were extracted using either 100%, 90%, 80%, or 70% ethanol and subsequently analyzed using HPLC with a mobile phase gradient system of 10-100% acetonitrile in 0.05% aqueous ortho-phosphoric acid, flow rate of 0.8 mL/min, and detection wavelength of 280 nm. Varying concentrations of Thai propolis extracts as well as four active ingredients were subjected to agar well diffusion test against the growth of Streptococcus mutans (S. mutans) or Lactobacillus caseii (L. caseii).

The concentrations of the four active ingredients: vicenin-2, vitexin, apigenin, and cinnamic acid, were significantly affected by ethanolic concentrations. The chromatographic peaks of all active ingredients from 70% and 80% ethanolic extracts appeared more defined, as compared to those which used higher concentrations of ethanol for extraction. Except for the absolute ethanolic extract, all of the examined propolis extracts, as well as its active ingredients inhibited both S. mutans and L. caseii.

Thai propolis extracts contain vicenin-2, vitexin, apigenin, and cinnamic acid as part of its active ingredients. These were found to be significantly affected by the increase in ethanol during its extraction. The presence of these active ingredients might have contributed to the anti-caries potential of Thai propolis extracts.

Objectives: The aim of this study is to establish a Reversed Phase – High Performance Liquid Chromatographic (RP-HPLC) method for the quantification of Rhein from Cassia fistula L. leaves. Methods: A Shimadzu system equipped with a C18 Column (150 x 4.6 mm, 5 μm) with an isocratic elution of Acetonitrile (solvent A) and 0.1% trifluoroacetic acid aqueous solution (solvent B) (Merck, 1.08178.0050) with a 55:45 ratio, respectively and a flow rate of 1.0 mL/min and sample injection of 10 μL detection was done at 230 nm. Standard solution of Rhein (Chengdu Biopurify) was prepared for method development. This study was validated using the guidelines set under “ICH Topic Q2 R2 or the Validation of Analytical Procedures”. Procedures for linearity, precision, accuracy, limit of detection, and limit of quantitation were performed. Results: The retention time of Rhein standard was determined at 5.10 minutes. LOD and LOQ were determined to be 1.278 mcg/mL and 3.872 mcg/mL, respectively with good linearity (R2 ≥0.996) with a linear range of 2.5-20 ug/mL of the Rhein standard. The accuracy of the method was determined based on % recovery method and ranged from 94.75%-100.32% (intraday, n=3) with %RSD of 0.71. The intraday precision %RSD was 2.92 (n=6) while interday precision %RSD was 3.75 (n=3). The method was able to check the Rhein quantity among 10 samples of Cassia fistula L. leaves from different locations in the Philippines. Conclusion The method was found to be sensitive and accurate for the quantification of Rhein. The method was found to be useful for the quantification of the amount of Rhein and can be used as a Quality Control tool for the assessment of Cassia fistula.
Cassia ; Chromatography, High Pressure Liquid

OBJECTIVES

The aim of this study is to establish a Reversed Phase – High Performance Liquid Chromatographic (RP-HPLC) method for the quantification of Rhein from Cassia fistula L. leaves.

A Shimadzu system equipped with a C18 Column (150 x 4.6 mm, 5 μm) with an isocratic elution of Acetonitrile (solvent A) and 0.1% trifluoroacetic acid aqueous solution (solvent B) (Merck, 1.08178.0050) with a 55:45 ratio, respectively and a flow rate of 1.0 mL/min and sample injection of 10 μL detection was done at 230 nm. Standard solution of Rhein (Chengdu Biopurify) was prepared for method development. This study was validated using the guidelines set under “ICH Topic Q2 R2 or the Validation of Analytical Procedures”. Procedures for linearity, precision, accuracy, limit of detection, and limit of quantitation were performed.

The retention time of Rhein standard was determined at 5.10 minutes. LOD and LOQ were determined to be 1.278 mcg/mL and 3.872 mcg/mL, respectively with good linearity (R2 ≥0.996) with a linear range of 2.5-20 ug/mL of the Rhein standard. The accuracy of the method was determined based on % recovery method and ranged from 94.75%-100.32% (intraday, n=3) with %RSD of 0.71. The intraday precision %RSD was 2.92 (n=6) while interday precision %RSD was 3.75 (n=3). The method was able to check the Rhein quantity among 10 samples of Cassia fistula L. leaves from different locations in the Philippines.

The method was found to be sensitive and accurate for the quantification of Rhein. The method was found to be useful for the quantification of the amount of Rhein and can be used as a Quality Control tool for the assessment of Cassia fistula.

Mice ; Animals ; Tandem Mass Spectrometry ; Alpha-Amanitin ; Chromatography, Liquid ; Metabolomics ; Chromatography, High Pressure Liquid/methods*

This research established a high-performance liquid chromatography(HPLC) method for simultaneous determination of isoorientin, orientin, vitexin, and isovitexin in Commelina communis to conduct content difference analysis and quality evaluation of 62 batches of C. communis from different origins. The HPLC content determination was performed on a Dikma Platisil ODS chromatographic column(4.6 mm×250 mm, 5 μm), with acetonitrile-0.1% formic acid(14∶86) as the mobile phase. The detection wavelength was set at 348 nm, the flow rate was 1.0 mL·min~(-1), and the column temperature was 35 ℃. The differences in origins and quality of 62 batches of C. communis were studied by chemometrics. The results showed that the determination of four components mani-fested a good linear relationship in the range of mass concentration(r>0.999 9), and the average recovery rate was 96.17%-103.0%. The relative standard deviations(RSDs) of precision, stability, and repeatability were all less than 2.0%. The content of four components from high to low was isoorientin>isovitexin>orientin>vitexin. Forty-seven batches of C. communis with clear origins were classified into six categories by chemometrics. C. communis from different origins had different qualities. Generally, C. communis from Western China, Central China, and South of China had superior qualities. The HPLC method established in this study is specific, simple, and efficient, which provides references for the comprehensive evaluation of the quality of C. communis. The chemometrics shows that the qualities of C. communis from different origins are largely different. Isoorientin can be used as an index to determine the content of C. communis, and its content limit should be set no less than 0.023%.
Commelina ; Chemometrics ; Drugs, Chinese Herbal/chemistry* ; China ; Chromatography, High Pressure Liquid/methods*

An analytical method for 10 mycotoxins in Hippophae Fructus medicinal and edible products was established in this study, and the contamination of their mycotoxins was analyzed. First of all, the mixed reference solution of ten mycotoxins such as aflatoxin, ochratoxin, zearalenone, and dexoynivalenol was selected as the control, and the Hippophae Fructus medicinal and edible products were prepared. Secondly, based on the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) technology, 10 mycotoxins in Hippophae Fructus medicinal and edible products were quantitatively investigated and their content was determined. Finally, the contamination of mycotoxins was analyzed and evaluated. The optimal analysis conditions were determined, and the methodological inspection results showed that the 10 mycotoxins established a good linear relationship(r>0.99). The method had good repeatability, test sample specificity, stability, and instrument precision. The average recovery rates of 10 mycotoxins in Hippophae Fructus medicinal products, edible solids, and edible liquids were 90.31%-109.4%, 87.86%-107.8%, and 85.61%-109.1%, respectively. Relative standard deviation(RSD) values were 0.22%-10%, 0.75%-13%, and 0.84%-8.5%, repsectively. Based on UPLC-MS/MS technology, the simultaneous determination method for the limits of 10 mycotoxins established in this study has fast detection speed, less matrix interference, high sensitivity, and accurate results, which is suitable for the limit examination of 10 mycoto-xins in Hippophae Fructus medicinal and edible products.
Mycotoxins/analysis* ; Chromatography, Liquid/methods* ; Tandem Mass Spectrometry/methods* ; Hippophae ; Limit of Detection ; Chromatography, High Pressure Liquid/methods*

We prepared 15 batches of Kaixin Powder benchmark samples with the decoction pieces of different batches. Further, we established the specific chromatograms and index component content determination method of Kaixin Powder benchmark samples and analyzed the peaks and similarity of the chromatograms. With sibiricose A5, sibiricose A6, polygalaxanthone Ⅲ, 3,6'-disinapoyl sucrose, ginsenoside Rb_1, β-asarone, α-asarone, and dehydropachymic acid as index components, the index component content determination method was established and 70%-130% of the mean content of each component was set as the range. The chromatograms of 15 batches of Kaixin Powder benchmark samples had a total of 22 characteristic peaks, among which 8 peaks were identified, which represented sibiricose A5, sibiricose A6, polygalaxanthone Ⅲ, 3,6'-disinapoyl sucrose, ginsenoside Rb_1, β-asarone, α-asarone, and dehydropachymic acid, respectively. The chromatograms shared the similarity of 0.992-0.999. The 15 batches of benchmark samples had sibiricose A5 of 0.34-0.55 mg·g~(-1), sibiricose A6 of 0.43-0.57 mg·g~(-1), polygalaxanthone Ⅲ of 0.12-0.19 mg·g~(-1), 3,6'-disinapoyl sucrose of 1.08-1.78 mg·g~(-1), ginsenoside Rb_1 of 0.33-0.62 mg·g~(-1), β-asarone of 2.34-3.72 mg·g~(-1), α-asarone of 0.11-0.22 mg·g~(-1), and dehydropachymic acid of 0.053-0.079 mg·g~(-1). This study established the specific chromatograms and index component content determination method of Kaixin Powder benchmark samples, and the method was simple, feasible, reproducible, and stable. This study provides a scientific basis for further research on the key chemical properties of the benchmark samples and preparations of Kaixin Powder.
Powders ; Ginsenosides ; Benchmarking ; Drugs, Chinese Herbal/chemistry* ; Sucrose ; Chromatography, High Pressure Liquid/methods*

This study aims to explore the chemical composition of Rehmanniae Radix braised with mild fire and compare the effect of processing method on the chemical composition of Rehmanniae Radix. To be specific, ultra-high performance liquid chromatography with linear ion trap-orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) was used to screen the chemical constituents of Rehmanniae Radix. The chemical constituents were identified based on the relative molecular weight and fragment ions, literature information, and Human Metabolome Database(HMDB). The ion peak area ratio of each component before and after processing was used as the index for the variation. SIMCA was employed to establish principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) models of different processed products. According to the PCA plot, OPLS-DA plot, and VIP value, the differential components before and after the processing were screened out. The changes of the content of differential components with the processing method were analyzed. A total of 66 chemical components were identified: 57 of raw Rehmanniae Radix, 55 of steamed Rehmanniae Radix, 55 of wine-stewed Rehmanniae Radix, 51 of repeatedly steamed and sundried Rehmanniae Radix Praeparata, 62 of traditional bran-braised Rehmanniae Radix, and 63 of electric pot-braised Rehmanniae Radix. Among them, the 9 flavonoids of braised Rehmanniae Radix were from Citri Reticulatae Pericarpium. PCA suggested significant differences in the chemical composition of Rehmanniae Radix Praeparata prepared with different processing methods. OPLS-DA screened out 32 chemical components with VIP value >1 as the main differential components. Among the differential components, 9 were unique to braised Rehmanniae Radix(traditional bran-braised, electric pot-braised) and the degradation rate of the rest in braised(traditional bran-braised, electric pot-braised) or repeatedly steamed and sundried Rehmanniae Radix was higher than that in the steamed or wine-stewed products. The results indicated the chemical species and component content of Rehmanniae Radix changed significantly after the processing. The 32 components, such as rehmapicrogenin, martynoside, jionoside D, aeginetic acid, hesperidin, and naringin, were the most important compounds to distinguish different processed products of Rehmanniae Radix. The flavonoids introduced by Citri Reticulatae Pericarpium as excipient may be the important material basis for the effectiveness of braised Rehmanniae Radix compared with other processed products.
Humans ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal/chemistry* ; Plant Extracts/chemistry* ; Rehmannia/chemistry* ; Flavonoids/analysis*

The chemical constituents in stem leaf, root, and flower of Ixeris sonchifolia were identified by the ultra performance li-quid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry(UPLC-LTQ-Orbitrap-MS~n). The separation was performed on an Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) with a mobile phase of water(containing 0.1% formic acid, A)-acetonitrile(B) with gradient elution. With electrospray ionization source, the data of 70% methanol extract from stem leaf, root and flower of I. sonchifolia were collected by high-resolution full-scan Fourier transform spectroscopy, data dependent acquisition, precursor ion scan, and selected ion monitoring in the negative and positive ion modes. The compounds were identified based on accurate molecular weight, retention time, fragment ions, comparison with reference standard, Clog P and references. A total of 131 compounds were identified from the 70% methanol extract of I. sonchifolia, including nucleosides, flavonoids, organic acids, terpenoids, and phenylpropanoids, and 119, 110, and 126 compounds were identified from the stem leaf, root and flower of I. sonchifolia, respectively. In addition, isorhamnetin, isorhamnetin-7-O-sambubioside and caffeylshikimic acid were discovered from I. sonchifolia for the first time. This study comprehensively analyzed and compared the chemical constituents in different parts of I. sonchifolia, which facilitated the discovery of effective substances and the development and application of medicinal material resources of I. sonchifolia.
Drugs, Chinese Herbal/chemistry* ; Methanol ; Chromatography, High Pressure Liquid/methods* ; Mass Spectrometry ; Asteraceae