Validation of an ELISA method for quantification of the major Timothy grass pollen allergen Phl p 5a (BSP090).

Progress towards standardisation of allergen products has been made in recent years. Nevertheless, no standardised test method to quantify the allergen content of grass pollen allergen products is available at present. One aim of the BSP090 project was to validate a quantitative assay for a major Timothy grass (Phleum pratense) pollen allergen, Phl p 5. Qualification of a candidate ELISA system was performed with regard to range, robustness and cross-reactivity in preliminary studies. The assay specifically detected Phl p 5 with a quantification range from 3.9 ng/mL to 62.5 ng/mL. Suitability to quantify recombinant and natural Phl p 5 was further assessed in a collaborative study including 14 laboratories in Europe and the USA. Precision and accuracy of the assay was satisfactory with 93% of calculated Phl p 5 concentrations and 100% of total recoveries being within the ± 30% acceptance range. Similar results were obtained for spike recoveries, with exclusion of the lowest concentration spike, showing spike recoveries exceeding the acceptance range for six laboratories. Inter-assay (repeatability) and inter-laboratory (reproducibility) variability were satisfactory, in the format used in the present study. Robustness towards different statistical methods for data analysis was demonstrated. In conclusion, the assay can easily be established in routine testing and results of the preliminary testing and collaborative study support the proposal of the assessed Phl p 5-specific ELISA as a European Pharmacopoeia general method.

Quantitative 31P NMR Method for Individual and Concomitant Determination of Phospholipid Classes in Polar Lipid Samples.

Herein, to achieve individual and concomitant quantifications of phospholipid classes, an absolute quantification 31P NMR method using an internal standard was examined. Phospholipid standards and dietary foods were dispersed to prepare test solutions in an anionic surfactant (sodium cholate) solution containing EDTA, as a modification based on a reported method. Each phospholipid class showed a reproducible chemical shift at a near-neutral test solution pH of 6.90±0.04 and temperature of 30.0±0.1°C. The quantity of synthetic phosphatidylcholine measured using 31P NMR was consistent with that measured by 1H NMR using an internal standard. As the principal phospholipid class of soybean and egg yolk lecithin is phosphatidylcholine, the measurement conditions of 31P NMR (pulse interval time and number of scans) were optimized such that minor phospholipids, including lysophospholipids, also present in lecithin could be quantified simultaneously. Phospholipid classes in commercial polar lipid samples derived from porcine brain, yeast, and soybean were individually quantified using the above conditions. Using phosphoserine as the internal standard material allowed the absolute molar quantity of the phospholipid class to be precisely determined with traceability to the SI. The determined molar amounts of phospholipid classes were then translated to the weight amount by assuming that each phospholipid class contained two stearic acid molecules as the constituent fatty acid. The calculated total contents of each phospholipid class by 31P NMR were in good agreement with those obtained by molybdenum blue colorimetry. Furthermore, the quantitative values of the principal phospholipid classes in the polar lipid samples obtained by 31P NMR corresponded in a broad view, however, was more informative for the separation of individual phospholipid species rather than the quantitative 2D thin-layer chromatography.

Multiwavelength detection and multivariate analysis for tongzhi surunjiang preparation with substitute reference substance method by high performance liquid chromatography / 中国药学杂志

OBJECTIVE: To propose a holistic strategy for quality control of Chinese patent medicines, and establish an HPLC analytical method for Tongzhi Surunjiang preparation according to the strategy. METHODS: The strategy contained three steps.The first step was multi-wavelength chromatographic detection.The second step was multivariate analysis for identification and assay. The third step was to establish substitute reference substance method.The preparations were extracted by ultrasound with methanol, chromatography was performed on ODS column with gradient elution with acetonitrile and 0.1%phosphoric acid aqueous solution.The detection wavelengths were set at 270, 350, 410 and 440 nm.The radar chart, HCA heatmap, principal component analysis and cosine similarity were used for data analysis.At last, linear calibration using two reference substances, relative retention time method and PDA spectrum method were used for peak identification, and relative correction factor method was used for quantitative analysis. RESULTS: The multi-components determination method and fingerprint analysis met the method validation requirements. Data analysis showed that there were some differences among the samples of different manufacturers. Strong characteristic peaks for classification were gallic acid, chebulinic acid, chebulea fructus, sennae folium, sennae folium and crocin.CONCLUSION: The method is specific, with low cost, and could be used to accurately control the quality of Tongzhi Surunjiang preparation.

[Guideline principle and technical requirement for preparing traditional Chinese medicine reference drug].

Traditional Chinese medicine (TCM) reference drug is a new form of TCM standard reference substance. The purpose of this guideline is to guide the establishment of the reference drug and standardize its investigation and application in national drug standards. Definition of TCM reference drug was specified and relating guideline and technical requirement were introduced in this paper. Its application in quality control of TCM was analyzed and the developing train was proposed. There is a wide prospect for the application of reference drug in quality control of TCM. Thus it has practical significance to explore and conduct the quality evaluation system by using TCM reference drug as the reference substance.

Guideline principle and technical requirement for preparing traditional Chinese medicine reference drug / 中国中药杂志

Traditional Chinese medicine (TCM) reference drug is a new form of TCM standard reference substance. The purpose of this guideline is to guide the establishment of the reference drug and standardize its investigation and application in national drug standards. Definition of TCM reference drug was specified and relating guideline and technical requirement were introduced in this paper. Its application in quality control of TCM was analyzed and the developing train was proposed. There is a wide prospect for the application of reference drug in quality control of TCM. Thus it has practical significance to explore and conduct the quality evaluation system by using TCM reference drug as the reference substance.

A reference substance free diagnostic fragment ion-based approach for rapid identification of non-target components in Pudilan Xiaoyan oral liquid by high resolution mass spectrometry.

Rapid and reliable identification of non-target components in herbal preparations remains a primary challenge, especially when corresponding reference substances are inaccessible. In this work, an efficient post-experiment data processing methodology, named reference substance free diagnostic fragment ion (RSFDFI), was developed based on ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC/LTQ-Orbitrap). The first step of this approach was to cluster the components that share common fragment ions into several groups. After querying the database using a predicted chemical formula, the component with the fewest primary hits was preferentially deduced based on its MS/MS spectrum. Once the structure was characterized, its common fragment ions could be used as the prior structural information to select the possible candidates that would facilitate the subsequent identification for each group. Taking Pudilan Xiaoyan oral liquid (PDL) as a model herbal preparation, which has been extensively used for the treatment of epidemic parotitis and children with hand-foot-mouth diseases, this strategy enables a nearly eight-fold narrowing of the database hits, with fifty-two components, including lignans, flavonoids, alkaloids and steroids, being rapidly identified. In conclusion, our work clearly demonstrates that integrating RSFDFI with high-resolution mass spectrometry is a powerful methodology for rapid identification of non-target components from herbal prescriptions and may open new avenues for chemical analysis in other complex mixtures.

[Research on quality standards of Aleuritopteris Herba].

This study aims to establish quality standards of Aleuritopteris Herba (AH), which could supply scientific evidence for the quality control of AH. The morphological and microscopic identification characters were reformulated. The tests of water content, total ash, acid-insoluble ash and ethanol-soluble extractives of AH were carried out according to the methods recorded in appendix of Chinese Pharmacopeia (2010 edition, volume 1). The TLC method was established by using aleuritopesis A [2,19-diol（2ß,4α）-16-enekaureniod] and reference herb as references. With preparation of aleuritopesis A[2,19-diol（2ß,4α）-16-enekaureniod] reference substance, the content of aleuritopesis A in AH was determined by HPLC. As a result, the macroscopic identification, microscopic features and TLC methods were specific and simple. The water content, total ash, acid-insoluble ash and ethanol-soluble extractive and the content of aleuritopesis A of all samples varied in the ranges of 8.8%-10.9%, 7.6%-11.4%, 2.5%-4.2%, 9.3%-10.2% and 0.56%-0.71%, respectively. The improved quality standard can be used to evaluate and guarantee the quality of AH comprehensively.

[Quantitative analysis of nine ginsenosides in red ginseng using QAMS method based on high-resolution and reference chromatography of red ginseng].

Using reversed-phase high performance liquid chromatography, nine ginsenosides were simultaneously separated on an UltimateC18 column with high-resolution and high purity of each chromatographic peak. Adopting the QAMS quality evaluation model for traditional Chinese medicines, ginsenoside Rb1 was used as the internal reference substance, and the relative correction factors (RCFs) and the relative retention values (RTRs) of ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, Rb3, Rd and 20 (S)-ginsenoside Rg3 to ginsenoside Rb1 were calculated individually. Through a series of methodology evaluations, and positioned by the red ginseng reference chromatograph and RTVs, nine ginsenosides in red ginseng were simultaneously assayed only by quantitative determined ginsenoside Rb1.

Guideline of substitute reference substance method for evaluation of traditional chinese medicines / 中国药学杂志

Substitute reference substance method is an effective approach for quality control of multiple components in accordance with the characteristics of traditional Chinese medicines. The purpose of the guideline is to guide the establishment of substitute reference substance method, prove the conformance of the method to the requirements for testing, and standardize the study method and its application in national drug standards. The topics of the guideline include the definition and classification of substitute reference substance method, the principles and approaches of quantitative analysis, the identification and confirmation of chromatographic peaks, and technical requirements. When substitute reference substance method is used for fingerprint identification or multiple components assay in traditional Chinese medicines, the analytical method can be validated following the guideline.

Preparation of naringin and neohesperidin reference substances from Citrus aurantium by medium-low-pressure preparative chromatography / 中草药

Objective: To establish a separation method for naringin and neohesperidin reference substances from Citrus aurantium. Methods: Naringin and neohesperidin monomers in C. aurantium were isolated and purified by macroporous resin and medium-low- pressure preparative chromatography. Results: The contents of the prepared naringin and neohesperidin reached to 98.76% and 99.50%, respectively. Conclusion: This method is effective for the preparation of naringin and neohesperidin with high purity. It could be used to prepare the reference substances for the qualitative and quantitative analyses of Chinese materia medica.

Preparation of tricin from Bambusa textilis by preparative HPLC / 中草药

Objective: To establish a separation method of tricin reference substance from Bambusa textilis. Methods: After extracted with ethyl acetate, the extract of B. textilis was isolated and purified by silica gel column chromatography and preparative HPLC. Tricins were identified by melting point, UV, and IR spectroscopy. Results: The content of tricin was over 98% by normalization method of HPLC. Conclusion: The developed method is simple with lower cost, by which tricin can be used as reference substances for the qualitative and quantitative analyses of Chinese herbal medicine.

Preparation of loganin and morroniside reference substances from Fructus Corni / 中草药

Objective To establish a separation method of loganin and morroniside reference substance from Fructus Corni. Methods After extracted with hot water and precipitated with alcohol, the extract of Fructus Corni was isolated and purified by macroporous resin, silica gel column chromatography, and preparative HPLC. Loganin and morroniside were identified by UV, 1H-NMR, 13C-NMR, and MS. Results The contents of loganin and morroniside was higher than 98% by normalization method of HPLC. Conclusion The developed method is simple with lower cost, by which loganin and morroniside can be used as reference substances for the qualitative and quantitative analysis of Chinese herbal medicine.

Preparation of neoeriocitrin and naringin reference substances from Drynaria fortunei / 中草药

Objective To establish a separation method for neoeriocitrin and naringin reference sub- stances from Drynaria fortunei.Methods The extract of D.fortunei was isolated and purified by macro porous resin,silica gel column chromatography,neutro-aluminum oxide adsorbing,Sephadex LH-20 co- lumn chromatography,and recrystallizations.The structures of neoeriocitrin and naringin were identified by MS,IR,UV,~1H-NMR,and ~(13)C-NMR.Results The contents of neoeriocitrin and naringin were 99.5% and 99.3%,respectively.Conclusion The developed method is simple with lower cost,by which neoeriocitrin and naringin can be used as reference substances for the qualitative and quantitative analysis of Chinese herbal medicine.

Preparation of acteoside and forsythoside B reference substances from Lamiophlomis rotata / 中草药

Objective To establish a method for the isolation and preparation of forsythoside B and acteoside reference substances from Lamiophlomis rotata.Methods Forsythoside B and acteoside in L.rotata were isolated and purified by macroporous resin,Sephadex column,and preparative HPLC.Results Analysis with HPLC showed the content of the prepared acteoside and forsythoside B reached to 98.93 and 99.91%,respectively.Conclusion This method is effective for the high purity of prepared acteoside and forsythoside B.It can be used as reference substances for the qualitative and quantitative analyses of Chinese herbal medicine.

Preparation Method for Reference Substances of Hyperin and Isorhamnetin-3-O-galactoside / 中药新药与临床药理

Objective To establish a method for preparing the reference substances of hyperin and isorhamnetin- 3- O- galactoside. Methods Methanol- extract of Fructus Evodia was isolated and purified by combining solvent extraction with column chromatography and recrystallization. Hyperin and isorhamnetin- 3- O- galactoside were identified by mass spectrometry (MS), 1H nuclear magnetic resonance (1HNMR ) and 13C nuclear magnetic resonance (13CNMR) and their content was determined by HPLC. Results The purity was 99.6 % and 98.0 % for hyperin and isorhamnetin- 3- O- galactoside respectively. Conclusion This method is simple cancl effective to yield high- purity products. And these high- purity products can be used as reference substances for the research of herbal medicine.

Prepartion of reference substance of flavonoid in Desmodium styracifolium by RP-HPLC / 中成药

AIM: To develop an RP-HPLC method for preparing the reference substanc of vicenin-2 in Desmodium styracifolium. METHODS: Ethanol-extract of desmodium styracifolium was isolated and purified by RP-HPLC combining solvent extraction with column chromatography and recrystalliztion.The purity and content of vicenin-2 were identified by HPLC. RESULTS: The flvonoids were completely separated under this chromatographic condition.The purity of the reference substance was 99.0% or above. CONCLUSION: The method is simple,accurate,better on repeatability,and effective to yield high-purity product.It can be used as reference substance for the research of herbal medicine.