CA2727891A1 - Method for measuring total content of natural brassinosteroids using chromatography apparatus - Google Patents
Method for measuring total content of natural brassinosteroids using chromatography apparatus Download PDFInfo
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- CA2727891A1 CA2727891A1 CA 2727891 CA2727891A CA2727891A1 CA 2727891 A1 CA2727891 A1 CA 2727891A1 CA 2727891 CA2727891 CA 2727891 CA 2727891 A CA2727891 A CA 2727891A CA 2727891 A1 CA2727891 A1 CA 2727891A1
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
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Abstract
A method for measuring the total content of brassinosteroids in a sample of extraction from natural resources using chromatography apparatus is disclosed.
The method is characterized in that the total content of natural brassinosteroids is analysed to detect the presence of derivatization reagent residue. The method includes making brassinosteroids derivatives, considering the relationship between the mass of brassinosteroids and the consumption of derivatization reagent, and determining the total content of natural brassinosteroids by using an association factor.
The method is characterized in that the total content of natural brassinosteroids is analysed to detect the presence of derivatization reagent residue. The method includes making brassinosteroids derivatives, considering the relationship between the mass of brassinosteroids and the consumption of derivatization reagent, and determining the total content of natural brassinosteroids by using an association factor.
Description
I I
METHOD FOR MEASURING TOTAL CONTENT OF
NATURAL BRASSINOSTEROIDS USING CHROMATOGRAPHY APPARATUS
FIELD OF INVENTION
The present invention relates to a method for measuring the total content of brassinosteroids in a sample of extraction from natural resources or its preparation by using chromatography apparatus.
BACKGROUND
Brassinosteroids (BRs) are a class of polyhydroxysteroids that have been recognized as a sixth class of plant hormones, including more than 70 compounds ubiquitously occurrence in nature. Brassinosteroids are found in various plant parts, such as pollen, flower buds, fiuits, seeds, vascular cambium, leaves, stem and roots. They play important regulatory roles in numerous plant growth and developmental processes, such as stimulation of cell enlargement and cell division, root and stem elongation, disease resistance, floral initiation, and seed germination. In addition to their practical applications in agriculture, brassinosteroids also show structural similarities to insect, animal and human steroid hormones, and thus some medical applications have also been reported, e.g. anticancer and antiproliferative activity (Jana et al., "Anticancer and antiproliferative activity of natural brassinosteroids", Phytochemistry, 2008, Vol. 69, Pages 418-426). The high biological activity of brassinosteroids has attracted the attention of many specialists in the fields of chemistry, biology, pharmacology and agriculture.
Because plants contain brassinosteroids at such low concentration (10-5-10.11%), it is necessary to develop specific methods for their analysis. Over the past few decades, microanalyses for brassinosteroids have been developed using gas chromatography (GC) and high-performance liquid chromatography (HPLC). One skilled in the art will understand that it is not always possible to isolate each single brassinosteroid in sufficient quantities from natural resources to record all spectral characteristics, since there are various brassinosteroids I I I
with differently alkylated side chains and with very low content coexisting in nature. In such cases, the identification of brassinosteroids has to be done by making derivatives and comparing them with separately synthesized authentic samples.
Since brassinosteroids usually have four hydroxyl groups as two sets of vicinal diols in the A-ring and in the side-chain, it could be derivatized to highly detectable derivatives for GC
analysis or HPLC analysis by using suitable derivatization reagents. The formed derivatives exhibit high sensitivity, specificity, and stability, and thus are suitable for the chromatography analysis.
Let us take brassinolide and methaneboronic acid (a derivatization reagent) as an example, the general scheme of methane-boronate of brassinolide (a derivative) formation is illustrated as follows:
. A le Me8(0ice Mc-8;
eo ch-0 GC in combination with mass spectrometry is employed in most cases for the identification of brassinosteroids. A selected ion monitoring using the GC-CI-MS system is applied to the analysis of natural brassinosteroids in several plants, wherein one or more ions with a certain molecular mass of a known compound are used for registration.
Methaneboronic acid was the derivatization reagent, and then brassinolide, castasterone, 28-norbrassinolide, brassinone, 24-ethylbrassinone and dolichosterone were identified in the immature seeds and sheaths of Chinese cabbage (Ikekawa et al., "Analysis of natural brassinosteroids by gas chromatography and gas chromatography-mass spectrometry", Journal of Chromatography A, 1984, Vol. 290, 289-302).
A good alternative for GC is a method based on HPLC of brassinosteroids derivatives.
Some derivatization reagents (e.g. boronic acid derivatives) have been proved to be satisfactory for use in the derivatization of brassinosteroids with respect to reactivity and I i~ I
sensitivity (Gamoh and Takatsuto, Journal of chromatography A, 1994, 658, 17-25).
Gamoh et al. described a HPLC assay of brassinosteroids in plants as boronate derivatives with UV, fluorimetric and electro-chemical detection ("Liquid chromatographic assay of brassinosteroids in plants", Journal of chromatography A, 1994, 658, 17-25).
Brassinosteroids were readily condensed with the boronic acid derivatives under mild conditions to provide the corresponding boronates. Then the derivatized brassinosteroids were effectively separated by a reversed-phase column and they were monitored by UV, fluorimetric or electrochemical detection depending on the prelabelling reagents. The content of four brassinosteroids (i.e. brassinolide, dolichosterone, 28-norcastasterone and castasterone) in broad bean pollen was then determined respectively.
To date, the above methods are commonly used in the identification and determination of brassinosteroids, especially the HPLC method. However, these methods are all required to synthesize the possible compound and then be compared with the natural compound by chromatography. Due to the large diversity of brassinosteroids as well as the limitation of authentic samples, it is not practical to determine the total content of brassinosteroids in natural materials by indentifying its single compounds.
Therefore, there is a need for developing a convenient and applicable method to measure the total content of natural brassinosteroids.
SUMMARY OF THE INVENTION
A method for measuring the total content of natural brassinosteroids using chromatography apparatus is disclosed. The method includes detecting the residue of derivatization reagent used for brassinosteroids, taking into account both the quantity of brassinosteroids and the consumption of derivatization reagent, and applying an association factor to determine the total content of natural brassinosteroids. The apparatus used for detecting the residue of derivatization reagent may be HPLC or GC depending on the derivatization reagent used.
The method according to the invention may, for instance, be applied to the industrial process for product inspection and quality control, to provide standardization for manufacturing natural brassinosteroids products. It can greatly assist the progress of natural brassinosteroids for broad commercial use.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for measuring the total content of natural brassinosteroids, the method comprising the steps of:
(a) making a standard solution of derivatization reagent;
(b) making a standard solution of brassinosteroid derivative by contacting the brassinosteroid reference standard with said derivatization reagent;
(c) making a test solution of brassinosteroids derivatives by contacting the natural brassinosteroids sample with said derivatization reagent;
(d) analysing the samples of steps (a), (b) and (c) by chromatography apparatus respectively;
(e) recording the areas of derivatization reagent peak; and (f) determining the total content of natural brassinosteroids.
As the consumption of derivatization reagent is proportional to the mass of brassinosteroids, an association factor will be obtained by analyzing the samples of steps (a) and (b). This association factor represents the relationship between the mass of brassinosteroid reference standard and the decreased area of derivatization reagent peak (i.e.
the consumption of said derivatization reagent). The total content of natural brassinosteroids will then be determined by applying the association factor to the sample result of test solution.
The method as defined above is therefore characterized in that the total content of natural brassinosteroids is analysed to detect the presence of derivatization reagent residue.
For better understanding of the present invention, numerous specific details are set forth in use of chromatographic techniques, in particular, HPLC analysis and GC
analysis.
An embodiment of the present invention therefore provides a method for measuring the total content of natural brassinosteroids using HPLC. The reagent used for derivatization of brassinosteroids herein may be boronic acid derivatives which give high selectivity and reactivity for the vicinal diols of brassinosteroids, which include naphthaleneboronic acid and its side-chain analogues, ferroceneboronic acid, 1-cyanoisoindole-2-m-phenylboronic acid, phenanthreneboronic acid, dansylaminophenylboronic acid, phenylboronic acid and its side-chain analogues. In one embodiment, the standard solution of derivatization reagent is prepared by dissolving Mo (mg) phenylboronic acid in methanol and then diluting to volume with methanol in a 25 mL volumetric flask.
A subject of the invention is therefore the method as defined above, wherein the brassinosteroid reference standard used may be any highly purified brassinosteroid or synthetic brassinosteroid, such as brassinolide, 24-epibrassinolide, dolicholide, 28-homodolicholide, 28-norbrassinolide, castasterone, 28-norcastasterone, 28-homocastasterone, dolichosterone and 24-epicastasterone. The brassinosteroid derivative is obtained by treating brassinosteroid reference standard with derivatization reagent at a temperature between about 50 C and about 90 C for about 10 minutes to 60 minutes. In a related embodiment, the standard solution of brassinosteroid derivative is prepared by dissolving Mss. (mg) brassinolide (P%, purity) in a small amount of methanol, then adding Mo (mg) phenylboronic acid. The mixture is heated at about 80 C for 30 minutes.
After being cooled, the mixture is then diluted to volume with methanol in a 25 mL
volumetric flask.
The natural brassinosteroids sample to be assayed may be the extraction from natural resources or its preparation, which can be either liquid or solid sample. The brassinosteroids derivatives are obtained by treating natural brassinosteroids sample with derivatization reagent at a temperature between about 50 C and about 90 C for about 10 minutes to 60 minutes.
In a related embodiment, the test solution of brassinosteroids derivatives is prepared by dissolving Mp. (g) analyte in a small amount of methanol, then adding MO (mg) phenylboronic acid. The mixture is heated at about 80 C for 30 minutes. After being cooled, the mixture is then diluted to volume with methanol in a 25 mL volumetric flask.
A subject of the invention is therefore also a method of analysis as defined above by chromatography apparatus, wherein HPLC or GC maybe used. In one embodiment, the apparatus may be any HPLC with a stainless steel column, a suitable UV
detector and a basic integrator. In a related embodiment, a CLC C 18 column having particle size of about 5 un or about 10 m, a column length of between about 10 cm and about 25 cm, and a column diameter of between about 4 mm and about 10 mm may be used. Operating temperature may be room temperature, and pump flow rate may be between 0.4 mL/min and 1.5 mL/min according to the column used.
The HPLC is carried out in a mobile phase which may be a mixture of organic solvent and water in a ratio varying from about 60:40 (v/v) to about 90:10 (v/v). The organic solvent includes, but not limited to the methanol, acetonitrile, and ethyl acetate. In one embodiment, the mobile phase consists of methanol and water in a ratio of 80:20 (v/v), filtered through a 0.5 gm filter membrane before use. Any UV detector that can simultaneously measure absorbance at two or more wavelengths may be used in the invention. In a related embodiment, a UV-visible absorbance detector is used; the wavelengths from about 210 nm to about 270 nm may be chosen as detection.
The injection volume may be from about 5 L to about 20 L. After injecting the samples of the standard solution of derivatization reagent, the standard solution of brassinosteroid derivative and the test solution of brassinosteroids derivatives into a chromatography apparatus respectively, the peak area responses may be recorded and calculated by the integrator.
A subject of the invention is therefore also the method as defined above, wherein the total content of natural brassinosteroids is mathematically determined by using an association factor. The principle of quantification is illustrated as follows:
As mentioned hereinabove, the quantity of derivatization reagent used for all the standard solutions and test solution are fixed as Mo (mg). And it is well known that the peak area is proportional to the quantity of substance injected. In the analysis of the standard solution of derivatization reagent and the standard solution of brassinosteroid derivative respectively, one skilled in the art will understand that an association factor (f) indicating the relationship between the mass of brassinosteroid and the decreased area of derivatization i r - 1 I
reagent peak (i.e. the consumption of derivatization reagent) can be represented as:
_ Area -Areast.
Mst. xP%
Areao is the peak area of derivatization reagent in injection of standard solution of derivatization reagent.
Areast. is the peak area of derivatization reagent residue in injection of standard solution of brassinosteroid derivative.
Mst. is the mass of brassinosteroid reference standard being used for preparing relevant standard solution (in mg).
P.% is the purity of brassinosteroid reference standard.
Therefore, by applying the association factor to the sample result of test solution, the total content of natural brassinosteroids in the sample may be determined as follows:
Mst x (Areao-Areasamp.) Content BRs x P%
Msamp.x (Area -Areast) or Mst; x (Area -Areasam ) %BRs= p' x P% x 100 Msamp.x (Area -Areast) Area,p. is the peak area of derivatization reagent residue in an injection of test solution of brassinosteroids derivatives.
M.p. is the mass of natural brassinosteroids sample being used for preparing relevant test solution (in g).
Another embodiment of the present invention therefore provides a method for measuring the total content of natural brassinosteroids using GC. The derivatization reagent may be lower alkyl-boronic acids or organic acids and its anhydrides, which includes methaneboronic acid (CH5BO2), ethaneboronic acid (C2H7B02) and propylboronic acid (C3H9 B02), acetic acid, acetic anhydride, propionic acid and propionic anhydride.
In one embodiment, the standard solution of derivatization reagent is prepared by dissolving Mo (mg) ethaneboronic acid in dry pyridine and then diluting to volume with dry i I
pyridine in a 10 mL volumetric flask. And then, the standard solution of brassinosteroid derivative is prepared by dissolving MSS. (mg) brassinolide (P%, purity) and Mo (mg) ethaneboronic acid in a small amount of dry pyridine. The mixture is heated at about 80'C for 30 minutes. After being cooled, the mixture is then diluted to volume with dry pyridine in a 10 mL volumetric flask. Finally, the test solution of brassinosteroids derivatives is prepared by dissolving Mp. (g) analyte and Mo (mg) ethaneboronic acid in a small amount of dry pyridine. The mixture is heated at about 80 'C for 30 minutes. After being cooled, the mixture is then diluted to volume dry pyridine in a 10 mL volumetric flask.
Another aspect of the present invention also provides a GC method for detecting the residue of derivatization reagent. In one embodiment, the GC system comprises at least a column (packed column or capillary column), a flame ionization detector (FID) and a basic integrator. In a related embodiment, a packed column coating with 2% OV- 17 on Chromosorb W (80-100 mesh), having a column length of between about 0.5 m and about 2 m, and an internal diameter of about 2 mm may be used. Alternatively, a 25 m x 2 mm (i.d.) glass capillary column coating with OV-101 may be used. Column temperature may be operated from about 200 C to about 350 *C. The carrier gas maybe nitrogen or helium with flow rate between about 1.0 mL/min and about 2.5 mL/min. Injection port temperature and detector temperature are from about 250 C to about 350 'C.
After injecting the standard solution of derivatization reagent, the standard solution of brassinosteroid derivative and the test solution of brassinosteroids derivatives into a chromatography apparatus respectively, the areas of derivatization reagent peak may be calculated by the integrator.
As mentioned hereinabove, the total content of natural brassinosteroids is mathematically determined by using the relationship between the mass of brassinosteroids and the consumption of derivatization reagent. Accordingly, the total content of natural brassinosteroids in a sample is given by:
Mst x (Areao-Areasam ) X
Content BRs Msamp.x (Areao-Areasp) P%
where Areao = Area of derivatization reagent peak in injection of standard solution of derivatization reagent.
Areast. = Area of derivatization reagent peak in injection of standard solution of brassinosteroid derivative.
Areau,,,p. = Area of derivatization reagent peak in an injection of test solution of brassinosteroids derivatives.
Msg. = Mass of brassinosteroid reference standard taken to prepare relevant standard solution (in mg).
Msamp. = Mass of natural brassinosteroids sample taken to prepare relevant test solution (in g or mL).
P% = Purity of brassinosteroid reference standard.
It is to be understood that the phraseology or terminology used herein is only for the purpose of description and not a limitation. Hence, although the present invention has been described hereinabove by way of illustrative embodiments thereof, it can be modified, and applied to other apparatus and other methods without departing from the spirit, scope and nature of the subject invention as defined in the appended claims.
EXAMPLES
The examples below are intended to illustrate the invention. One skilled in the art will understand that the invention is not limited to the embodiments illustrated below.
Measuring the total content of natural brassinosteroids using HPLC
Reagents and chemicals Methanol used was of HPLC grade purity and phenylboronic acid was used of analytical grade. Synthetic brassinolide (90%, purity) was used as the brassinosteroid reference standard.
METHOD FOR MEASURING TOTAL CONTENT OF
NATURAL BRASSINOSTEROIDS USING CHROMATOGRAPHY APPARATUS
FIELD OF INVENTION
The present invention relates to a method for measuring the total content of brassinosteroids in a sample of extraction from natural resources or its preparation by using chromatography apparatus.
BACKGROUND
Brassinosteroids (BRs) are a class of polyhydroxysteroids that have been recognized as a sixth class of plant hormones, including more than 70 compounds ubiquitously occurrence in nature. Brassinosteroids are found in various plant parts, such as pollen, flower buds, fiuits, seeds, vascular cambium, leaves, stem and roots. They play important regulatory roles in numerous plant growth and developmental processes, such as stimulation of cell enlargement and cell division, root and stem elongation, disease resistance, floral initiation, and seed germination. In addition to their practical applications in agriculture, brassinosteroids also show structural similarities to insect, animal and human steroid hormones, and thus some medical applications have also been reported, e.g. anticancer and antiproliferative activity (Jana et al., "Anticancer and antiproliferative activity of natural brassinosteroids", Phytochemistry, 2008, Vol. 69, Pages 418-426). The high biological activity of brassinosteroids has attracted the attention of many specialists in the fields of chemistry, biology, pharmacology and agriculture.
Because plants contain brassinosteroids at such low concentration (10-5-10.11%), it is necessary to develop specific methods for their analysis. Over the past few decades, microanalyses for brassinosteroids have been developed using gas chromatography (GC) and high-performance liquid chromatography (HPLC). One skilled in the art will understand that it is not always possible to isolate each single brassinosteroid in sufficient quantities from natural resources to record all spectral characteristics, since there are various brassinosteroids I I I
with differently alkylated side chains and with very low content coexisting in nature. In such cases, the identification of brassinosteroids has to be done by making derivatives and comparing them with separately synthesized authentic samples.
Since brassinosteroids usually have four hydroxyl groups as two sets of vicinal diols in the A-ring and in the side-chain, it could be derivatized to highly detectable derivatives for GC
analysis or HPLC analysis by using suitable derivatization reagents. The formed derivatives exhibit high sensitivity, specificity, and stability, and thus are suitable for the chromatography analysis.
Let us take brassinolide and methaneboronic acid (a derivatization reagent) as an example, the general scheme of methane-boronate of brassinolide (a derivative) formation is illustrated as follows:
. A le Me8(0ice Mc-8;
eo ch-0 GC in combination with mass spectrometry is employed in most cases for the identification of brassinosteroids. A selected ion monitoring using the GC-CI-MS system is applied to the analysis of natural brassinosteroids in several plants, wherein one or more ions with a certain molecular mass of a known compound are used for registration.
Methaneboronic acid was the derivatization reagent, and then brassinolide, castasterone, 28-norbrassinolide, brassinone, 24-ethylbrassinone and dolichosterone were identified in the immature seeds and sheaths of Chinese cabbage (Ikekawa et al., "Analysis of natural brassinosteroids by gas chromatography and gas chromatography-mass spectrometry", Journal of Chromatography A, 1984, Vol. 290, 289-302).
A good alternative for GC is a method based on HPLC of brassinosteroids derivatives.
Some derivatization reagents (e.g. boronic acid derivatives) have been proved to be satisfactory for use in the derivatization of brassinosteroids with respect to reactivity and I i~ I
sensitivity (Gamoh and Takatsuto, Journal of chromatography A, 1994, 658, 17-25).
Gamoh et al. described a HPLC assay of brassinosteroids in plants as boronate derivatives with UV, fluorimetric and electro-chemical detection ("Liquid chromatographic assay of brassinosteroids in plants", Journal of chromatography A, 1994, 658, 17-25).
Brassinosteroids were readily condensed with the boronic acid derivatives under mild conditions to provide the corresponding boronates. Then the derivatized brassinosteroids were effectively separated by a reversed-phase column and they were monitored by UV, fluorimetric or electrochemical detection depending on the prelabelling reagents. The content of four brassinosteroids (i.e. brassinolide, dolichosterone, 28-norcastasterone and castasterone) in broad bean pollen was then determined respectively.
To date, the above methods are commonly used in the identification and determination of brassinosteroids, especially the HPLC method. However, these methods are all required to synthesize the possible compound and then be compared with the natural compound by chromatography. Due to the large diversity of brassinosteroids as well as the limitation of authentic samples, it is not practical to determine the total content of brassinosteroids in natural materials by indentifying its single compounds.
Therefore, there is a need for developing a convenient and applicable method to measure the total content of natural brassinosteroids.
SUMMARY OF THE INVENTION
A method for measuring the total content of natural brassinosteroids using chromatography apparatus is disclosed. The method includes detecting the residue of derivatization reagent used for brassinosteroids, taking into account both the quantity of brassinosteroids and the consumption of derivatization reagent, and applying an association factor to determine the total content of natural brassinosteroids. The apparatus used for detecting the residue of derivatization reagent may be HPLC or GC depending on the derivatization reagent used.
The method according to the invention may, for instance, be applied to the industrial process for product inspection and quality control, to provide standardization for manufacturing natural brassinosteroids products. It can greatly assist the progress of natural brassinosteroids for broad commercial use.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for measuring the total content of natural brassinosteroids, the method comprising the steps of:
(a) making a standard solution of derivatization reagent;
(b) making a standard solution of brassinosteroid derivative by contacting the brassinosteroid reference standard with said derivatization reagent;
(c) making a test solution of brassinosteroids derivatives by contacting the natural brassinosteroids sample with said derivatization reagent;
(d) analysing the samples of steps (a), (b) and (c) by chromatography apparatus respectively;
(e) recording the areas of derivatization reagent peak; and (f) determining the total content of natural brassinosteroids.
As the consumption of derivatization reagent is proportional to the mass of brassinosteroids, an association factor will be obtained by analyzing the samples of steps (a) and (b). This association factor represents the relationship between the mass of brassinosteroid reference standard and the decreased area of derivatization reagent peak (i.e.
the consumption of said derivatization reagent). The total content of natural brassinosteroids will then be determined by applying the association factor to the sample result of test solution.
The method as defined above is therefore characterized in that the total content of natural brassinosteroids is analysed to detect the presence of derivatization reagent residue.
For better understanding of the present invention, numerous specific details are set forth in use of chromatographic techniques, in particular, HPLC analysis and GC
analysis.
An embodiment of the present invention therefore provides a method for measuring the total content of natural brassinosteroids using HPLC. The reagent used for derivatization of brassinosteroids herein may be boronic acid derivatives which give high selectivity and reactivity for the vicinal diols of brassinosteroids, which include naphthaleneboronic acid and its side-chain analogues, ferroceneboronic acid, 1-cyanoisoindole-2-m-phenylboronic acid, phenanthreneboronic acid, dansylaminophenylboronic acid, phenylboronic acid and its side-chain analogues. In one embodiment, the standard solution of derivatization reagent is prepared by dissolving Mo (mg) phenylboronic acid in methanol and then diluting to volume with methanol in a 25 mL volumetric flask.
A subject of the invention is therefore the method as defined above, wherein the brassinosteroid reference standard used may be any highly purified brassinosteroid or synthetic brassinosteroid, such as brassinolide, 24-epibrassinolide, dolicholide, 28-homodolicholide, 28-norbrassinolide, castasterone, 28-norcastasterone, 28-homocastasterone, dolichosterone and 24-epicastasterone. The brassinosteroid derivative is obtained by treating brassinosteroid reference standard with derivatization reagent at a temperature between about 50 C and about 90 C for about 10 minutes to 60 minutes. In a related embodiment, the standard solution of brassinosteroid derivative is prepared by dissolving Mss. (mg) brassinolide (P%, purity) in a small amount of methanol, then adding Mo (mg) phenylboronic acid. The mixture is heated at about 80 C for 30 minutes.
After being cooled, the mixture is then diluted to volume with methanol in a 25 mL
volumetric flask.
The natural brassinosteroids sample to be assayed may be the extraction from natural resources or its preparation, which can be either liquid or solid sample. The brassinosteroids derivatives are obtained by treating natural brassinosteroids sample with derivatization reagent at a temperature between about 50 C and about 90 C for about 10 minutes to 60 minutes.
In a related embodiment, the test solution of brassinosteroids derivatives is prepared by dissolving Mp. (g) analyte in a small amount of methanol, then adding MO (mg) phenylboronic acid. The mixture is heated at about 80 C for 30 minutes. After being cooled, the mixture is then diluted to volume with methanol in a 25 mL volumetric flask.
A subject of the invention is therefore also a method of analysis as defined above by chromatography apparatus, wherein HPLC or GC maybe used. In one embodiment, the apparatus may be any HPLC with a stainless steel column, a suitable UV
detector and a basic integrator. In a related embodiment, a CLC C 18 column having particle size of about 5 un or about 10 m, a column length of between about 10 cm and about 25 cm, and a column diameter of between about 4 mm and about 10 mm may be used. Operating temperature may be room temperature, and pump flow rate may be between 0.4 mL/min and 1.5 mL/min according to the column used.
The HPLC is carried out in a mobile phase which may be a mixture of organic solvent and water in a ratio varying from about 60:40 (v/v) to about 90:10 (v/v). The organic solvent includes, but not limited to the methanol, acetonitrile, and ethyl acetate. In one embodiment, the mobile phase consists of methanol and water in a ratio of 80:20 (v/v), filtered through a 0.5 gm filter membrane before use. Any UV detector that can simultaneously measure absorbance at two or more wavelengths may be used in the invention. In a related embodiment, a UV-visible absorbance detector is used; the wavelengths from about 210 nm to about 270 nm may be chosen as detection.
The injection volume may be from about 5 L to about 20 L. After injecting the samples of the standard solution of derivatization reagent, the standard solution of brassinosteroid derivative and the test solution of brassinosteroids derivatives into a chromatography apparatus respectively, the peak area responses may be recorded and calculated by the integrator.
A subject of the invention is therefore also the method as defined above, wherein the total content of natural brassinosteroids is mathematically determined by using an association factor. The principle of quantification is illustrated as follows:
As mentioned hereinabove, the quantity of derivatization reagent used for all the standard solutions and test solution are fixed as Mo (mg). And it is well known that the peak area is proportional to the quantity of substance injected. In the analysis of the standard solution of derivatization reagent and the standard solution of brassinosteroid derivative respectively, one skilled in the art will understand that an association factor (f) indicating the relationship between the mass of brassinosteroid and the decreased area of derivatization i r - 1 I
reagent peak (i.e. the consumption of derivatization reagent) can be represented as:
_ Area -Areast.
Mst. xP%
Areao is the peak area of derivatization reagent in injection of standard solution of derivatization reagent.
Areast. is the peak area of derivatization reagent residue in injection of standard solution of brassinosteroid derivative.
Mst. is the mass of brassinosteroid reference standard being used for preparing relevant standard solution (in mg).
P.% is the purity of brassinosteroid reference standard.
Therefore, by applying the association factor to the sample result of test solution, the total content of natural brassinosteroids in the sample may be determined as follows:
Mst x (Areao-Areasamp.) Content BRs x P%
Msamp.x (Area -Areast) or Mst; x (Area -Areasam ) %BRs= p' x P% x 100 Msamp.x (Area -Areast) Area,p. is the peak area of derivatization reagent residue in an injection of test solution of brassinosteroids derivatives.
M.p. is the mass of natural brassinosteroids sample being used for preparing relevant test solution (in g).
Another embodiment of the present invention therefore provides a method for measuring the total content of natural brassinosteroids using GC. The derivatization reagent may be lower alkyl-boronic acids or organic acids and its anhydrides, which includes methaneboronic acid (CH5BO2), ethaneboronic acid (C2H7B02) and propylboronic acid (C3H9 B02), acetic acid, acetic anhydride, propionic acid and propionic anhydride.
In one embodiment, the standard solution of derivatization reagent is prepared by dissolving Mo (mg) ethaneboronic acid in dry pyridine and then diluting to volume with dry i I
pyridine in a 10 mL volumetric flask. And then, the standard solution of brassinosteroid derivative is prepared by dissolving MSS. (mg) brassinolide (P%, purity) and Mo (mg) ethaneboronic acid in a small amount of dry pyridine. The mixture is heated at about 80'C for 30 minutes. After being cooled, the mixture is then diluted to volume with dry pyridine in a 10 mL volumetric flask. Finally, the test solution of brassinosteroids derivatives is prepared by dissolving Mp. (g) analyte and Mo (mg) ethaneboronic acid in a small amount of dry pyridine. The mixture is heated at about 80 'C for 30 minutes. After being cooled, the mixture is then diluted to volume dry pyridine in a 10 mL volumetric flask.
Another aspect of the present invention also provides a GC method for detecting the residue of derivatization reagent. In one embodiment, the GC system comprises at least a column (packed column or capillary column), a flame ionization detector (FID) and a basic integrator. In a related embodiment, a packed column coating with 2% OV- 17 on Chromosorb W (80-100 mesh), having a column length of between about 0.5 m and about 2 m, and an internal diameter of about 2 mm may be used. Alternatively, a 25 m x 2 mm (i.d.) glass capillary column coating with OV-101 may be used. Column temperature may be operated from about 200 C to about 350 *C. The carrier gas maybe nitrogen or helium with flow rate between about 1.0 mL/min and about 2.5 mL/min. Injection port temperature and detector temperature are from about 250 C to about 350 'C.
After injecting the standard solution of derivatization reagent, the standard solution of brassinosteroid derivative and the test solution of brassinosteroids derivatives into a chromatography apparatus respectively, the areas of derivatization reagent peak may be calculated by the integrator.
As mentioned hereinabove, the total content of natural brassinosteroids is mathematically determined by using the relationship between the mass of brassinosteroids and the consumption of derivatization reagent. Accordingly, the total content of natural brassinosteroids in a sample is given by:
Mst x (Areao-Areasam ) X
Content BRs Msamp.x (Areao-Areasp) P%
where Areao = Area of derivatization reagent peak in injection of standard solution of derivatization reagent.
Areast. = Area of derivatization reagent peak in injection of standard solution of brassinosteroid derivative.
Areau,,,p. = Area of derivatization reagent peak in an injection of test solution of brassinosteroids derivatives.
Msg. = Mass of brassinosteroid reference standard taken to prepare relevant standard solution (in mg).
Msamp. = Mass of natural brassinosteroids sample taken to prepare relevant test solution (in g or mL).
P% = Purity of brassinosteroid reference standard.
It is to be understood that the phraseology or terminology used herein is only for the purpose of description and not a limitation. Hence, although the present invention has been described hereinabove by way of illustrative embodiments thereof, it can be modified, and applied to other apparatus and other methods without departing from the spirit, scope and nature of the subject invention as defined in the appended claims.
EXAMPLES
The examples below are intended to illustrate the invention. One skilled in the art will understand that the invention is not limited to the embodiments illustrated below.
Measuring the total content of natural brassinosteroids using HPLC
Reagents and chemicals Methanol used was of HPLC grade purity and phenylboronic acid was used of analytical grade. Synthetic brassinolide (90%, purity) was used as the brassinosteroid reference standard.
Natural brassinosteroids sample was obtained from the extract of rape pollen.
Preparation of solutions a) Standard solution of phenylboronic acid Accurately weighed 10.0 mg (Mo) phenylboronic acid was dissolved by using small amount of methanol in a 25 mL volumetric flask. Then the volume was made up to the mark with methanol. 0.5 mL of this solution was further diluted to volume in a 10 mL volumetric flask with methanol.
b) Standard solution of brassinolide derivative Accurately weighed 15.0 mg (Mst.) synthetic brassinolide and 10.0 mg (Mo) phenylboronic acid were dissolved by using suitable amount of methanol in a 25 mL
volumetric flask. The mixture was heated at 80 C for 30 minutes in water bath. After being cooled down to ambient temperature, the solution was diluted to volume with methanol.
0.5 mL of this solution was further diluted to volume in a 10 mL volumetric flask with methanol.
c) Test solution of brassinosteroids derivatives Accurately weighed 12.0 g (Msamp) brassinosteroids solid sample and 10.0 mg (Mo) phenylboronic acid were dissolved by using suitable amount of methanol in a 25 mL
volumetric flask. The mixture was then heated at 80 C for 30 minutes in water bath. After being cooled down to ambient temperature, the solution was diluted to volume with methanol.
0.5 mL of this solution was further diluted to volume in a 10 mL volumetric flask with methanol.
Chromatographic conditions C18 column (15 cm x 6.0 mm i.d.). Mobile phase: methanol-water 80:20 (v/v).
Flow-rate: 0.8 mL/min. Column temperature: room temperature. Detection: UV at 222 nm.
Injection volume: 10 L.
Preparation of solutions a) Standard solution of phenylboronic acid Accurately weighed 10.0 mg (Mo) phenylboronic acid was dissolved by using small amount of methanol in a 25 mL volumetric flask. Then the volume was made up to the mark with methanol. 0.5 mL of this solution was further diluted to volume in a 10 mL volumetric flask with methanol.
b) Standard solution of brassinolide derivative Accurately weighed 15.0 mg (Mst.) synthetic brassinolide and 10.0 mg (Mo) phenylboronic acid were dissolved by using suitable amount of methanol in a 25 mL
volumetric flask. The mixture was heated at 80 C for 30 minutes in water bath. After being cooled down to ambient temperature, the solution was diluted to volume with methanol.
0.5 mL of this solution was further diluted to volume in a 10 mL volumetric flask with methanol.
c) Test solution of brassinosteroids derivatives Accurately weighed 12.0 g (Msamp) brassinosteroids solid sample and 10.0 mg (Mo) phenylboronic acid were dissolved by using suitable amount of methanol in a 25 mL
volumetric flask. The mixture was then heated at 80 C for 30 minutes in water bath. After being cooled down to ambient temperature, the solution was diluted to volume with methanol.
0.5 mL of this solution was further diluted to volume in a 10 mL volumetric flask with methanol.
Chromatographic conditions C18 column (15 cm x 6.0 mm i.d.). Mobile phase: methanol-water 80:20 (v/v).
Flow-rate: 0.8 mL/min. Column temperature: room temperature. Detection: UV at 222 nm.
Injection volume: 10 L.
Quantitative analysis gL of the standard phenylboronic acid solution was injected and the chromatogram was recorded. Under the same chromatographic conditions, the procedure was repeated for the standard solution of brassinolide derivative and the test solution respectively. Table 1 showed 5 the areas of phenylboronic acid peak.
Table 1 Mass Solution Phenylboronic Area of phenylboronic acid Brassinosteroid(s) acid peak Std. Phenylboronic acid 10.0 mg 0 1417989.9 Std. brassinolide (90%) 10.0 mg 15.0 mg 1007867.0 derivative Natural brassinosteroids 10.0 mg 12.0 g 983630.6 derivatives The total content of brassinosteroids in the sample was obtained by:
Mst x (Areao-Areasamp.) X P% = 15.0 x (1417989.9 - 983630.6) X 90%
Content BRs = Msamp.x (Areao-Areast) 12.0 x (1417989.9 -1007867.0) =1.19 mg/g 10 or Mst. X (Areao-Areasamp.) % BRs= xP%x100 Msamp.x (Areao-Areast) x (1417989.9 - 983630.6) x 90% x 100 12000 x (1417989.9 - 1007867.0) = 0.119 Measuring the total content of natural brassinosteroids using HPLC
15 Example 1 was repeated in the same manner as described except that the natural brassinosteroids preparation was from a liquid sample. In some cases, the total content of brassinosteroids may be presented in mg/mL, thus the preparation of test solution is illustrated as the following.
Accurately aspirated 1.5 mL liquid sample was delivered in a beaker, and evaporated to dryness by soaking in a water bath at a temperature of about 80 C to about 90 C .
Then accurately weighted 10.0 mg (Mo) phenylboronic acid and suitable amount of methanol were added. The mixture was transferred in a 25 mL volumetric flask and then was heated at 80 C for 30 minutes in water bath. After being cooled down to ambient temperature, the solution was diluted to volume with methanol. 0.5 mL of this solution was further diluted to volume in a 10 mL volumetric flask with methanol.
After injection, the results were summarized in Table 2.
Table 2 Phenylboronic Area of phenylboronic Solution acid Brassinosteroid(s) acid peak Std. Phenylboronic acid 10.0 mg 0 1417635.2 Std. brassinolide (90%) 10.0 mg 15.0 mg 1005934.2 derivative Natural brassinosteroids derivatives 10.0 mg 1.5 mL 1062543.5 The total content of brassinosteroids in the sample was obtained by:
Content Mst x (Areao-Areasamp,) X P% = 15,O x (1417635.2 -1062543.5) X 90%
BR, = M -Area camp.x (Area o st.) 1.5 x (1417635.2 -1005934.2) =7.76 mg/mL
Measuring the total content of natural brassinosteroids using GC
Chromatographic conditions A 0.5 m X 2 mm (i.d.) stainless steel column was packed with 2% OV-17 on Chromosorb W 80/100 mesh. The column temperature was 250 C. Injection port temperature and detector temperature were 290 'C. Helium (ultra carrier grade) was used as the carrier gas for the GC/FID analysis, and its flow rate was controlled at 1.5 mL/min.
Reagents Dry pyridine used was of GC grade purity and ethaneboronic acid was used of analytical grade. Synthetic brassinolide (90%, purity) was used as the brassinosteroid reference standard.
Natural brassinosteroids sample was obtained from the extract of rape pollen.
Preparation of solutions a) Standard solution of ethaneboronic acid Accurately weighed 3.00 mg (Mo) ethaneboronic acid was dissolved by using small amount of dry pyridine in a 10 mL volumetric flask. Then the volume was made up to the mark with dry pyridine. 1.0 mL of this solution was further diluted to volume in a 10 mL
volumetric flask with dry pyridine.
b) Standard solution of brassinolide derivative Accurately weighed 1.00 mg (Mst) synthetic brassinolide and 3.00 mg (Mo) ethaneboronic acid were dissolved by using suitable amount of dry pyridine in a 10 mL
volumetric flask. The mixture was heated at 80 C for 30 minutes in water bath. After being cooled down to ambient temperature, the solution was diluted to the mark with dry pyridine.
1.0 mL of the solution was transferred to a 10 mL volumetric flask and diluted to the mark with dry pyridine.
c) Test solution of brassinosteroids derivatives Accurately weighed 5.00 g (Mp) brassinosteroids solid sample and 3.00 mg (Mo) ethaneboronic acid were dissolved by using suitable amount of dry pyridine in a 10 mL
volumetric flask. The mixture was heated at 80 C for 30 minutes in water bath. After being cooled down to ambient temperature, the solution was diluted to the mark with dry pyridine.
1.0 mL of the solution was transferred to a 10 mL volumetric flask and diluted to the mark with dry pyridine.
Table 1 Mass Solution Phenylboronic Area of phenylboronic acid Brassinosteroid(s) acid peak Std. Phenylboronic acid 10.0 mg 0 1417989.9 Std. brassinolide (90%) 10.0 mg 15.0 mg 1007867.0 derivative Natural brassinosteroids 10.0 mg 12.0 g 983630.6 derivatives The total content of brassinosteroids in the sample was obtained by:
Mst x (Areao-Areasamp.) X P% = 15.0 x (1417989.9 - 983630.6) X 90%
Content BRs = Msamp.x (Areao-Areast) 12.0 x (1417989.9 -1007867.0) =1.19 mg/g 10 or Mst. X (Areao-Areasamp.) % BRs= xP%x100 Msamp.x (Areao-Areast) x (1417989.9 - 983630.6) x 90% x 100 12000 x (1417989.9 - 1007867.0) = 0.119 Measuring the total content of natural brassinosteroids using HPLC
15 Example 1 was repeated in the same manner as described except that the natural brassinosteroids preparation was from a liquid sample. In some cases, the total content of brassinosteroids may be presented in mg/mL, thus the preparation of test solution is illustrated as the following.
Accurately aspirated 1.5 mL liquid sample was delivered in a beaker, and evaporated to dryness by soaking in a water bath at a temperature of about 80 C to about 90 C .
Then accurately weighted 10.0 mg (Mo) phenylboronic acid and suitable amount of methanol were added. The mixture was transferred in a 25 mL volumetric flask and then was heated at 80 C for 30 minutes in water bath. After being cooled down to ambient temperature, the solution was diluted to volume with methanol. 0.5 mL of this solution was further diluted to volume in a 10 mL volumetric flask with methanol.
After injection, the results were summarized in Table 2.
Table 2 Phenylboronic Area of phenylboronic Solution acid Brassinosteroid(s) acid peak Std. Phenylboronic acid 10.0 mg 0 1417635.2 Std. brassinolide (90%) 10.0 mg 15.0 mg 1005934.2 derivative Natural brassinosteroids derivatives 10.0 mg 1.5 mL 1062543.5 The total content of brassinosteroids in the sample was obtained by:
Content Mst x (Areao-Areasamp,) X P% = 15,O x (1417635.2 -1062543.5) X 90%
BR, = M -Area camp.x (Area o st.) 1.5 x (1417635.2 -1005934.2) =7.76 mg/mL
Measuring the total content of natural brassinosteroids using GC
Chromatographic conditions A 0.5 m X 2 mm (i.d.) stainless steel column was packed with 2% OV-17 on Chromosorb W 80/100 mesh. The column temperature was 250 C. Injection port temperature and detector temperature were 290 'C. Helium (ultra carrier grade) was used as the carrier gas for the GC/FID analysis, and its flow rate was controlled at 1.5 mL/min.
Reagents Dry pyridine used was of GC grade purity and ethaneboronic acid was used of analytical grade. Synthetic brassinolide (90%, purity) was used as the brassinosteroid reference standard.
Natural brassinosteroids sample was obtained from the extract of rape pollen.
Preparation of solutions a) Standard solution of ethaneboronic acid Accurately weighed 3.00 mg (Mo) ethaneboronic acid was dissolved by using small amount of dry pyridine in a 10 mL volumetric flask. Then the volume was made up to the mark with dry pyridine. 1.0 mL of this solution was further diluted to volume in a 10 mL
volumetric flask with dry pyridine.
b) Standard solution of brassinolide derivative Accurately weighed 1.00 mg (Mst) synthetic brassinolide and 3.00 mg (Mo) ethaneboronic acid were dissolved by using suitable amount of dry pyridine in a 10 mL
volumetric flask. The mixture was heated at 80 C for 30 minutes in water bath. After being cooled down to ambient temperature, the solution was diluted to the mark with dry pyridine.
1.0 mL of the solution was transferred to a 10 mL volumetric flask and diluted to the mark with dry pyridine.
c) Test solution of brassinosteroids derivatives Accurately weighed 5.00 g (Mp) brassinosteroids solid sample and 3.00 mg (Mo) ethaneboronic acid were dissolved by using suitable amount of dry pyridine in a 10 mL
volumetric flask. The mixture was heated at 80 C for 30 minutes in water bath. After being cooled down to ambient temperature, the solution was diluted to the mark with dry pyridine.
1.0 mL of the solution was transferred to a 10 mL volumetric flask and diluted to the mark with dry pyridine.
t i Quantitative analysis gL samples of steps (a), (b) and (c) were injected into GC, respectively. The chromatograms were recorded and the areas of ethaneboronic acid peak were shown in Table 5 3.
Table 3 Mass Solution Phenylboronic Area of phenylboronic acid Brassinosteroid(s) acid peak Std. Phenylboronic acid 3.00 mg 0 6275883.6 Std. brassinolide (90%) 3.00 mg 1.00 mg 5845690.2 derivative Natural brassinosteroids derivatives 3.00 mg 5.00 g 3811864.8 The total content of brassinosteroids in the sample was obtained by:
Mst. x (Areao-Areasamp.) 1.00 x (6275883.6-3811864.8) Content BRs= x P% = x 90%
Msamp.x (Areao-Areast.) 5.00 x (6275883.6-5845690.2) = 1.03 mglg
Table 3 Mass Solution Phenylboronic Area of phenylboronic acid Brassinosteroid(s) acid peak Std. Phenylboronic acid 3.00 mg 0 6275883.6 Std. brassinolide (90%) 3.00 mg 1.00 mg 5845690.2 derivative Natural brassinosteroids derivatives 3.00 mg 5.00 g 3811864.8 The total content of brassinosteroids in the sample was obtained by:
Mst. x (Areao-Areasamp.) 1.00 x (6275883.6-3811864.8) Content BRs= x P% = x 90%
Msamp.x (Areao-Areast.) 5.00 x (6275883.6-5845690.2) = 1.03 mglg
Claims (27)
1. A method for measuring the total content of brassinosteroids in a sample of extraction from natural resources or its preparation, comprising:
a) making brassinosteroids derivatives by using derivatization reagent;
b) detecting the residue of said derivatization reagent by a chromatography apparatus;
and c) determining the total content of brassinosteroids in said sample by using the relationship between the mass of brassinosteroids and the consumption of said derivatization reagent.
a) making brassinosteroids derivatives by using derivatization reagent;
b) detecting the residue of said derivatization reagent by a chromatography apparatus;
and c) determining the total content of brassinosteroids in said sample by using the relationship between the mass of brassinosteroids and the consumption of said derivatization reagent.
2. The method of claim 1, wherein said chromatography apparatus includes high-performance liquid chromatography and gas chromatography.
3. The method of claim 1, wherein said derivatization reagent includes boronic acid derivatives for high-performance liquid chromatography analysis.
4. The method of claim 1, wherein said derivatization reagent includes lower alkyl-boronic acids, organic acids and its anhydrides for gas chromatography analysis.
5. The method of claim 3, wherein the boronic acid derivatives include 1-cyanoisoindole-2-m-phenylboronic acid, phenanthreneboronic acid, dansylaminophenylboronic acid, ferroceneboronic acid, naphthaleneboronic acid and its side-chain analogues, phenylboronic acid and its side-chain analogues.
6. The method of claim 4, wherein the lower alkyl-boronic acids include methaneboronic acid (CH5BO2), ethaneboronic acid (C2H7BO2) and propylboronic acid (C3H9 BO2).
7. The method of claim 4, wherein the organic acids and its anhydrides include acetic acid, acetic anhydride, propionic acid and propionic anhydride.
8. The method of claim 1, further comprising:
a) making a standard solution of derivatization reagent;
b) making a standard solution of brassinosteroid derivative by contacting the brassinosteroid reference standard with said derivatization reagent; and c) making a test solution of brassinosteroids derivatives by contacting the natural brassinosteroids sample with said derivatization reagent.
a) making a standard solution of derivatization reagent;
b) making a standard solution of brassinosteroid derivative by contacting the brassinosteroid reference standard with said derivatization reagent; and c) making a test solution of brassinosteroids derivatives by contacting the natural brassinosteroids sample with said derivatization reagent.
9. The method of claim 8, wherein the brassinosteroid reference standard is selected from any one of the highly purified brassinosteroid or synthetic brassinosteroid.
10. The method of claim 8, wherein said contacting the brassinosteroid reference standard with said derivatization reagent is performed at a temperature between about 50 °C and about 90 °C for about 10 minutes to 60 minutes.
11. The method of claim 8, wherein said contacting the natural brassinosteroids sample with said derivatization reagent is performed at a temperature between about 50 °C and about 90 °C for about 10 minutes to 60 minutes.
12. The method of claim 1, wherein said step of detecting the residue of derivatization reagent further comprising:
a) injecting the standard solution of derivatization reagent, the standard solution of brassinosteroid derivative and the test solution of brassinosteroids derivatives into a chromatography apparatus, respectively; and b) recording the areas of said derivatization reagent peak.
a) injecting the standard solution of derivatization reagent, the standard solution of brassinosteroid derivative and the test solution of brassinosteroids derivatives into a chromatography apparatus, respectively; and b) recording the areas of said derivatization reagent peak.
13. The method of claim 12, wherein the injection volume is from about 5 µL
to about 20 µL
to about 20 µL
14. The method of claim 1, wherein said step of determining the total content of brassinosteroids in said sample further comprising:
a) obtaining an association factor by detecting the residue of derivatization reagent in the samples of the standard solution of derivatization reagent and the standard solution of brassinosteroid derivative respectively; and b) determining the total content of brassinosteroids by applying said association factor to the sample result of test solution.
a) obtaining an association factor by detecting the residue of derivatization reagent in the samples of the standard solution of derivatization reagent and the standard solution of brassinosteroid derivative respectively; and b) determining the total content of brassinosteroids by applying said association factor to the sample result of test solution.
15. The method of claim 14, wherein said association factor is generated by using the mass of brassinosteroids reference standard and the decreased area of said derivatization reagent peak (i.e. the consumption of said derivatization reagent).
16. The method of claim 14, wherein said sample result of test solution is the peak area of derivatization reagent residue in an injection of test solution of brassinosteroids derivatives.
17. The method of claim 14, wherein the total content of brassinosteroids in said sample is determined by:
Content BRs x P%
where Content BRs represents the total content of brassinosteroids in the sample (in mg/g or mg/mL).
Area0 = Area of derivatization reagent peak in injection of standard solution of derivatization reagent Are st. = Area of derivatization reagent peak in injection of standard solution of brassinosteroid derivative Area samp. = Area of derivatization reagent peak in an injection of test solution of brassinosteroids derivatives M st. = Mass of brassinosteroid reference standard taken to prepare relevant standard solution (in mg) M samp. = Mass of natural brassinosteroids sample taken to prepare relevant test solution (in g or mL) P% = Purity of brassinosteroid reference standard
Content BRs x P%
where Content BRs represents the total content of brassinosteroids in the sample (in mg/g or mg/mL).
Area0 = Area of derivatization reagent peak in injection of standard solution of derivatization reagent Are st. = Area of derivatization reagent peak in injection of standard solution of brassinosteroid derivative Area samp. = Area of derivatization reagent peak in an injection of test solution of brassinosteroids derivatives M st. = Mass of brassinosteroid reference standard taken to prepare relevant standard solution (in mg) M samp. = Mass of natural brassinosteroids sample taken to prepare relevant test solution (in g or mL) P% = Purity of brassinosteroid reference standard
18. A method for detecting the residue of derivatization reagent, said method comprising the steps of:
a) providing a high-performance liquid chromatography system comprising at least a stainless steel column, a UV detector and an integrator;
b) using a mobile phase which is a mixture of organic solvent and water in a ratio of about 60:40 (v/v) to about 90:10 (v/v);
c) controlling the mobile phase flow rate between about 0.4 mL/min to about 1.5mL/min; and d) choosing detection wavelengths from about 210 nm to about 270 nm.
a) providing a high-performance liquid chromatography system comprising at least a stainless steel column, a UV detector and an integrator;
b) using a mobile phase which is a mixture of organic solvent and water in a ratio of about 60:40 (v/v) to about 90:10 (v/v);
c) controlling the mobile phase flow rate between about 0.4 mL/min to about 1.5mL/min; and d) choosing detection wavelengths from about 210 nm to about 270 nm.
19. The method of claim 18, wherein said stainless steel column is C18 column having particle size about 5 µm or about 10 µm.
20. The method of claim 19, wherein the length of said C18 column is between about 10 cm and about 25 cm.
21. The method of claim 19, wherein the diameter of said C18 column is between about 4 mm and about 10 mm.
22. The method of claim 18, wherein said organic solvent is selected from the group consisting of methanol, acetonitrile, and ethyl acetate.
23. A method for detecting the residue of derivatization reagent, said method comprising the steps of:
a) providing a gas chromatography system comprising at least a packed column or a glass capillary column, a flame ionization detector and a basic integrator;
and b) controlling the carrier gas flow rate between about 1.0 mL/min to about 2.5 mL/min.
a) providing a gas chromatography system comprising at least a packed column or a glass capillary column, a flame ionization detector and a basic integrator;
and b) controlling the carrier gas flow rate between about 1.0 mL/min to about 2.5 mL/min.
24. The method of claim 23, wherein said carrier gas is selected from the group consisting of nitrogen and helium.
25. The method of claim 23, wherein the gas chromatography is carried out at a column temperature of about 200 °C to about 350 °C.
26. The method of claim 23, wherein the gas chromatography is carried out at an injection port temperature of about 250 °C to about 350 °C.
27. The method of claim 23, wherein the gas chromatography is carried out at a detector temperature of about 250 °C to about 350 °C.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103743834A (en) * | 2013-12-23 | 2014-04-23 | 广西科技大学 | Detection method for food preservative, propionic acid |
| CN115047119A (en) * | 2022-05-31 | 2022-09-13 | 浙江皇马科技股份有限公司 | Method for detecting trace acetic anhydride in acetyl terminated polyether |
| WO2022250758A1 (en) * | 2021-05-27 | 2022-12-01 | Suntton Company Limited | A method of and composition for protecting plants against cercospora leaf spot |
-
2011
- 2011-01-14 CA CA 2727891 patent/CA2727891A1/en not_active Abandoned
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103743834A (en) * | 2013-12-23 | 2014-04-23 | 广西科技大学 | Detection method for food preservative, propionic acid |
| WO2022250758A1 (en) * | 2021-05-27 | 2022-12-01 | Suntton Company Limited | A method of and composition for protecting plants against cercospora leaf spot |
| US20220386604A1 (en) * | 2021-05-27 | 2022-12-08 | Suntton Company Limited | Method of and composition for protecting plants against cercospora leaf spot |
| CN115047119A (en) * | 2022-05-31 | 2022-09-13 | 浙江皇马科技股份有限公司 | Method for detecting trace acetic anhydride in acetyl terminated polyether |
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