Method for determining content of menthol in preparation of traditional Chinese medicine composition

Abstract

A method for determining the content of menthol in a traditional Chinese medicine composition. The traditional Chinese medicine composition consists of the following medicinal materials: Fructus Forsythia, Flos Lonicerae, Radix Isatidis, Semen Armeniacae Amarum, menthol, Herba Houttuyniae, rheum, Herba Pogostemonis, Rhizoma Dryopteris Crassirhizomae, Rhodiola rosea L., Herba Ephedrae, Radix Glycyrrhizae and gypsum. In the method for determining the content, the content of the menthol in the composition is determined by gas chromatography to effectively control the content of menthol in the composition, and the method can save energy and reduce the costs for analysis.

Claims

1. A method for determining the content of menthol in a preparation of a traditional Chinese medicine composition, the preparation of a traditional Chinese medicine composition is prepared from the following raw materials with the following parts by weight: Fructus Forsythia 200-300, Herba Ephedrae 60-100, rheum 40-60, Herba Houttuyniae 200-300, Flos Lonicerae 200-300, Radix Isatidis 200-300, Herba Pogostemonis 60-100, Rhizoma Dryopteris Crassirhizomae 200-300, Rhodiola rosea L. 60-100, menthol 5-9, Semen Armeniacae Amarum 60-100, Radix Glycyrrhizae 60-100 and gypsum 200-300, wherein the content of the menthol is determined according to the method as follows: 1) extracting the preparation of the traditional Chinese medicine composition using a nonpolar solvent to obtain a test solution; 2) preparing a solution of menthol reference substance with a menthol concentration of 4.80 μg/mL or more by a menthol reference substance using the same non-polar solvent as that in step 1); 3) taking the solution of menthol reference substance and the test solution in an equal amount respectively, injecting the solutions into a gas chromatograph, and determining the contents of menthol, wherein the conditions for chromatography are: the column is a capillary chromatographic column containing phenyl-methyl polysiloxane as a stationary phase, the phenyl-methyl polysiloxane has a phenyl content of 1-10% and is a chromatographic column of type HP-5 or type DB-5; the column temperature is increased according to the following temperature programming: initial temperature is 80-100° C., and is kept for 10-15 minutes, increased to 120-160° C. at a rate of 6-10° C. per minute, kept for 1.5-3.5 minutes, then increased to 240-300° C. at a rate of 100-160° C. per minute, kept for 5-20 minutes.

2. The method according to claim 1, wherein the mass-to-volume ratio of the preparation of the traditional Chinese medicine composition to the non-polar solvent as used is (1 g: 400 mL) to (1 g: 50 mL) during the preparation of the test solution.

3. The method according to claim 1, wherein the phenyl-methyl polysiloxane has a phenyl content of 5%.

4. The method according to claim 1, wherein the solution of menthol reference substance and the test solution are injected in an amount of 0.5-2 μL.

5. The method according to claim 1, wherein the solution of menthol reference substance and the test solution are injected by split injection with a split ratio in the range of 50:1 to 10:1 during determination of the menthol content.

6. The method according to claim 5, wherein the solution of menthol reference substance and the test solution are injected by split injection with a split ratio in the range of 30:1 to 20:1 during determination of the menthol content.

7. The method according to claim 5, wherein the solution of menthol reference substance and the test solution are injected by split injection with a split ratio in the range of 25:1 during determination of the menthol content.

8. The method according claim 1, wherein the conditions for chromatography also comprise detector temperature and inlet temperature of 300-400° C.

9. The method according to claim 1, wherein the conditions for chromatography also comprise carried gas and flow rate thereof, the carrier gas is nitrogen with a flow rate of 0.8-1.2 mL/min.

10. The method according to claim 1, wherein the conditions for chromatography also comprises a fuel gas ratio, the fuel gas ratio is the ratio of air to hydrogen being from 8:1 to 12:1.

11. The method according to claim 10, wherein the fuel gas ratio is the ratio of air to hydrogen being 9:1 to 11:1.

12. The method according to claim 10, wherein the fuel gas ratio is the ratio of air to hydrogen being 10:1.

13. The method according to claim 1, wherein the non-polar solvent is selected from the group consisting of non-polar saturated alkanes or halogenated saturated alkanes, and non-polar ester solvents.

14. The method according to claim 13, wherein the non-polar solvent is selected from the group consisting of n-hexane, dichloromethane, petroleum ether and ethyl acetate.

15. The method according to claim 1, wherein the method for preparing the test solution specifically is as follows: grinding and mixing the preparation of the traditional Chinese medicine composition evenly, placing the obtained substance in a narrow mouth container equipped with a plug, adding the non-polar organic solvent, shaking for 20 seconds or more, filtering then obtaining the test solution.

16. The method according to claim 15, wherein the filtering step is carried out using a 0.2-0.4 μm microporous filter.

17. The method according to claim 15, wherein the filtering step is carried out using a 0.22 μm microporous filter.

18. The method according to claim 15, wherein the method for preparing the test solution further comprises the step of soaking or ultrasonical extraction at room temperature before the shaking step.

19. The method according to claim 1, wherein 2) preparing a solution of menthol reference substance with a menthol concentration of 17.65 μg/mL or more by a menthol reference substance using the same non-polar solvent as that in step 1).

20. The method according to claim 1, wherein 2) preparing a solution of menthol reference substance with a menthol concentration of 0.2-0.3 mg/mL by a menthol reference substance using the same non-polar solvent as that in step 1).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: standard curve of menthol

(2) FIG. 2: the chromatogram of the blank reagent

(3) FIG. 3: the chromatogram of the negative reference substance solution

(4) FIG. 4: the chromatogram of the solution of reference substance

(5) FIG. 5: the chromatogram of the test solution

DETAILED DESCRIPTION

(6) Devices and Reagents

(7) Device: PerKinElmer Clarus 680 Gas Chromatograph, AL204 and AB135-S type Electronic Balances, Agilent J&W Scientific HP-5 Capillary Chromatographic Columns (30 m×0.25 mm, 0.25 μm), Computerized Numerical Control Ultrasonic Cleaner (Model: KQ-500DB, 500 W, 40 KHZ), 0.22 μm microporous membrane (Tianjin Jinteng Experimental Equipment Co., Ltd.).

(8) Reagents: n-hexane (chromatographic grade, Fisher, U.S.), petroleum ether, dichloromethane, ethyl acetate (analytical grade, Beijing Chemical Plant).

(9) Medicine: menthol reference substance (purchased from SIGMA-ALORICH, lot number: M2772-100G-A, purity is 99%).

(10) Each of the following examples was carried out for three times in parallel in accordance with the listed conditions, and the numbers were recorded as 1, 2 and 3, respectively.

EXAMPLE 1

(11) The following materials were weighed according to the ratio below: Fructus Forsythia 200, Flos Lonicerae 300, Radix Isatidis 200, rheum 40, Herba Pogostemonis 60, Rhizoma Dryopteris Crassirhizomae 300, Rhodiola rosea L. 100, menthol 9, Herba Ephedrae 60, Semen Armeniacae Amarum 100, Herba Houttuyniae 200, Radix Glycyrrhizae 100 and gypsum 200, were extracted according to the following process:

(12) (1) the traditional Chinese medicines were weighed according to the weight ratio of raw materials, the medicinal parts were selected and processed into pieces as required;

(13) (2) Herba Pogostemonis was processed into pieces, water was added (the ratio of the volume of water to the weight of medicinal material (L/kg, mL/g) was 10) to extract volatile oil, oil extraction time: 8 hours, the volatile oil was collected for later use; after the extract was filtered, the residue was removed, the filtrate was kept for later use;

(14) (3) Fructus Forsythia, Herba Ephedrae, Herba Houttuyniae and rheum were extracted with 70% ethanol (the ratio of the volume of water to the weight of medicinal material (L/kg, mL/g) was 12) for 3 times, and each extraction took 2.5 hours, the extracts were combined and filtered, the ethanol was recycled, the filtrate was kept for later use;

(15) (4) water was added to Flos Lonicerae, gypsum, Radix Isatidis, Rhizoma Dryopteris Crassirhizomae, Radix Glycyrrhizae and Rhodiola rosea L. (the ratio of the volume of the water to the weight of the medicinal material (L/kg, mL/g) was 12) and the obtained mixture was decocted until the mixture was boiled, Semen Armeniacae Amarum was added, the obtained mixture was decocted for 2 times, each decoction took 1 hour, the extracts were combined and filtered, the obtained filtrate was combined with the filtrate obtained after the oil extraction of Herba Pogostemonis in step (2), the obtained combination was concentrated to obtain a clear paste with a relative density of 1.15 determined at 60° C., ethanol was added, the obtained mixture was adjusted to an alcohol concentration of 70%, refrigerated, filtered, the ethanol was recycled until there's no smell of alcohol, a clear paste was obtained for later use;

(16) (5) the clear paste obtained in step (4) was combined with the ethanol extract in step (3), the obtained combination was concentrated to obtain a clear paste with a relative density of 1.20 determined at 60° C., the clear paste was dried to obtain dry paste powders for later use;

(17) (6) the dry paste powders obtained in step (5) was added to a suitable pharmaceutically acceptable auxiliary material for granulation; the auxiliary material can be 35 g of starch;

(18) (7) menthol and the volatile oil obtained in step (2) were added to ethanol and allowing the menthol and volatile oil dissolved in ethanol, the granules obtained in step (6) were sprayed into the obtained mixture, the product was obtained after encapsulating.

(19) The method for determining the content of menthol:

(20) test solution preparation: the substance contained in the composition from the samples of loading difference test was taken, ground finely, mixed evenly, 0.3 g was taken, weighed accurately, placed in a conical flask equipped with a plug, 25 mL of n-hexane was added accurately, the flask was shaken clockwise and counterclockwise for about 30 seconds, a suitable amount of solution was taken, filtered through a 0.22 μm microporous membrane to obtain the test solution;

(21) preparation of the solution of reference substance: a suitable amount of menthol reference substance was taken, weighed accurately, n-hexane was added to prepare a menthol solution with a menthol concentration of 0.23 mg per 1 mL to obtain the solution of menthol reference substance;

(22) conditions for chromatography: chromatographic column: Agilent J&W Scientific HP-5 capillary chromatographic column (30 m×0.25 mm, 0.25 μm); the column temperature was increased according to the following temperature programming: initial temperature of 98° C., the temperature was kept for 12 minutes, increased to 140° C. at a rate of 8° C. per minute, kept for 2.5 minutes, then increased to 280° C. at a rate of 140° C. per minute, kept for 10 minutes; detector temperature was 300° C.; inlet temperature was 300° C.; carrier gas was nitrogen, flow rate: 1 mL/min; injected by split injection, split ratio: 25:1; the volume of injected sample: 1 μL; fuel gas ratio: air-hydrogen (450:45);

(23) determination method: 1 μL of the solution of menthol reference substance and the test solution were accurately taken, respectively, and were injected into a gas chromatograph, were determined to obtain the results.

(24) Results of the Determination of Menthol Content in the Composition

(25) TABLE-US-00015 No. Menthol contents (%) 1 1.98 2 1.92 3 1.95

(26) Conclusion: the result is that the menthol was well separated and can be used to control the quality of the traditional Chinese medicine composition.

EXAMPLE 2

(27) The following materials were weighed according to the ratio below: Fructus Forsythia 300, Flos Lonicerae 200, Radix Isatidis 300, rheum 60, Herba Pogostemonis 100, Rhizoma Dryopteris Crassirhizomae 200, Rhodiola rosea L. 60, menthol 5, Herba Ephedrae 100, Semen Armeniacae Amarum 60, Herba Houttuyniae 300, Radix Glycyrrhizae 60 and gypsum 300, were extracted according to the following process:

(28) (1) the traditional Chinese medicines were weighed according to the weight ratio of raw materials, the medicinal parts were selected and processed into pieces as required;

(29) (2) Herba Pogostemonis was processed into pieces, water was added (the ratio of the volume of water to the weight of medicinal material (L/kg, mL/g) was 10) to extract volatile oil, oil extraction time: 8 hours, the volatile oil was collected for later use; after the extract was filtered, the residue was removed, the filtrate was kept for later use;

(30) (3) Fructus Forsythia, Herba Ephedrae, Herba Houttuyniae and rheum were extracted with 70% ethanol (the ratio of the volume of water to the weight of medicinal material (L/kg, mL/g) was 12) for 3 times and each extraction took 2.5 hours, the extracts were combined and filtered, the ethanol was recycled, the filtrate was kept for later use;

(31) (4) water was added to Flos Lonicerae, gypsum, Radix Isatidis, Rhizoma Dryopteris Crassirhizomae, Radix Glycyrrhizae and Rhodiola rosea L. (the ratio of the volume of the water to the weight of the medicinal material (L/kg, mL/g) was 12) and the obtained mixture was decocted until the mixture was boiled, Semen Armeniacae Amarum was added, the obtained mixture was decocted for 2 times, each decoction took 1 hour, the extracts were combined and filtered, the obtained filtrate was combined with the filtrate obtained after the oil extraction of Herba Pogostemonis in step (2), the obtained combination was concentrated to obtain a clear paste with a relative density of 1.10 determined at 60° C., ethanol was added, the obtained mixture was adjusted to an alcohol concentration of 70%, refrigerated, filtered, the ethanol was recycled until there's no smell of alcohol, a clear paste was obtained for later use;

(32) (5) the clear paste obtained in step (4) was combined with the ethanol extract in step (3), the obtained combination was concentrated to obtain a clear paste with a relative density of 1.15 determined at 60° C., the clear paste was dried to obtain dry paste powders for later use;

(33) (6) the dry paste powders obtained in step (5) was added to a suitable pharmaceutically acceptable auxiliary material for granulation; the auxiliary material can be 35 g of starch;

(34) (7) menthol and the volatile oil obtained in step (2) were added to ethanol and allowing the menthol and volatile oil dissolved in ethanol, the granules obtained in step (6) were sprayed into the obtained mixture, the product was obtained after tabletting.

(35) The method for determining the content of menthol:

(36) test solution preparation: the substance contained in the composition from the samples of loading difference test was taken, ground finely, mixed evenly, 0.2 g was taken, weighed accurately, placed in a conical flask equipped with a plug, 20 mL of n-hexane was added accurately, the flask was shaken clockwise and counterclockwise for about 20 seconds, a suitable amount of solution was taken, filtered through a 0.22 μm microporous membrane to obtain the test solution;

(37) preparation of the solution of reference substance: a suitable amount of menthol reference substance was taken, weighed accurately, n-hexane was added to prepare a menthol solution with a menthol concentration of 0.23 mg per 1 mL of n-hexane to obtain the solution of menthol reference substance;

(38) Conditions for chromatography: chromatographic column: Agilent J&W Scientific HP-5 capillary chromatographic column (30 m×0.25 mm, 0.25 μm); the column temperature was increased according to the following temperature programming: initial temperature was 98° C., the temperature was kept for 12 minutes, increased to 140° C. at a rate of 8° C. per minute, kept for 2.5 minutes, then increased to 280° C. at a rate of 140° C. per minute, kept for 5-20 minutes; detector temperature was 350° C.; inlet temperature was 350° C.; carrier gas was nitrogen, flow rate: 0.8 mL/min; injected by split injection, split ratio: 25:1; the volume of injected sample: 0.5 μL; fuel gas ratio: air-hydrogen (450:45);

(39) determination method: 0.5 μL of the solution of menthol reference substance and the test solution were accurately taken, respectively, and were injected into a gas chromatograph, were determined to obtain the results.

(40) Results of the Determination of Menthol Content in the Composition

(41) TABLE-US-00016 No. Menthol contents (%) 1 1.28 2 1.36 3 1.24

(42) Conclusion: the result is that the menthol was well separated and can be used to control the quality of the traditional Chinese medicine composition.

EXAMPLE 3

(43) The following materials were weighed according to the ratio below: Fructus Forsythia 278, Flos Lonicerae 294, Radix Isatidis 285, rheum 55, Herba Pogostemonis 95, Rhizoma Dryopteris Crassirhizomae 290, Rhodiola rosea L. 87, menthol 8.5, Herba Ephedrae 88, Semen Armeniacae Amarum 80, Herba Houttuyniae 284, Radix Glycyrrhizae 95 and gypsum 277, were extracted according to the following process:

(44) (1) the traditional Chinese medicines were weighed according to the weight ratio of raw materials, the medicinal parts were selected and processed into pieces as required;

(45) (2) Herba Pogostemonis was processed into pieces, water was added (the ratio of the volume of water to the weight of medicinal material (L/kg, mL/g) was 10) to extract volatile oil, oil extraction time: 8 hours, the volatile oil was collected for later use; after the extract was filtered, the residue was removed, the filtrate was kept for later use;

(46) (3) Fructus Forsythia, Herba Ephedrae, Herba Houttuyniae and rheum were extracted with 70% ethanol (the ratio of the volume of water to the weight of medicinal material (L/kg, mL/g) was 12) for 3 times, and each extraction took 2.5 hours, the extracts were combined and filtered, the ethanol was recycled, the filtrate was kept for later use;

(47) (4) water was added to Flos Lonicerae, gypsum, Radix Isatidis, Rhizoma Dryopteris Crassirhizomae, Radix Glycyrrhizae and Rhodiola rosea L. (the ratio of the volume of the water to the weight of the medicinal material (L/kg, mL/g) was 12) and the obtained mixture was decocted until the mixture was boiled, Semen Armeniacae Amarum was added, the obtained mixture was decocted for 2 times, each decoction took 1 hour, the extracts were combined and filtered, the obtained filtrate was combined with the filtrate obtained after the oil extraction of Herba Pogostemonis in step (2), the obtained combination was concentrated to obtain a clear paste with a relative density of 1.13 determined at 60° C., ethanol was added, the obtained mixture was adjusted to an alcohol concentration of 70%, refrigerated, filtered, the ethanol was recycled until there's no smell of alcohol, a clear paste was obtained for later use;

(48) (5) the clear paste obtained in step (4) was combined with the ethanol extract in step (3), the obtained combination was concentrated to obtain a clear paste with a relative density of 1.18 determined at 60° C., the clear paste was dried to obtain dry paste powders for later use;

(49) (6) the dry paste powders obtained in step (5) was added to a suitable pharmaceutically acceptable auxiliary material for granulation; the auxiliary material can be 35 g of starch;

(50) (7) menthol and the volatile oil obtained in step (2) were added to ethanol and allowing the menthol and volatile oil dissolved in ethanol, the granules obtained in step (6) were sprayed into the obtained mixture, the product was obtained after bagging.

(51) The method for determining the content of menthol:

(52) test solution preparation: the substance contained in the composition from the samples of loading difference test was taken, ground finely, mixed evenly, 0.5 g was taken, weighed accurately, placed in a conical flask equipped with a plug, 30 mL of n-hexane was added accurately, the flask was shaken clockwise and counterclockwise for about 50 seconds, a suitable amount of solution was taken, filtered through a 0.22 μm microporous membrane to obtain the test solution;

(53) preparation of the solution of reference substance: a suitable amount of menthol reference substance was taken, weighed accurately, n-hexane was added to prepare a menthol solution with a menthol concentration of 0.23 mg per 1 mL of n-hexane to obtain the solution of menthol reference substance;

(54) Conditions for chromatography: chromatographic column: Agilent J&W Scientific HP-5 capillary chromatographic column (30 m×0.25 mm, 0.25 μm); the column temperature was increased according to the following temperature programming: initial temperature 98° C., the temperature was kept for 12 minutes, increased to 140° C. at a rate of 8° C. per minute, kept for 2.5 minutes, then increased to 280° C. at a rate of 140° C. per minute, kept for 5-20 minutes; detector temperature was 400° C.; inlet temperature was 400° C.; carrier gas was nitrogen, flow rate: 1.2 mL/min; injected by split injection, split ratio: 25:1; the volume of injected sample: 2 μL; fuel gas ratio: air-hydrogen (450:45);

(55) determination method: 2 μL of the solution of menthol reference substance and the test solution were accurately taken, respectively, and were injected into a gas chromatograph, were determined to obtain the results.

(56) Results of the Determination of Menthol Content in the Composition

(57) TABLE-US-00017 No. Menthol contents (%) 1 1.85 2 1.78 3 1.72

(58) Conclusion: the result is that the menthol was well separated and can be used to control the quality of the traditional Chinese medicine composition.

EXAMPLE 4

(59) The following materials were weighed according to the ratio below: Fructus Forsythia 255, Flos Lonicerae 255, Radix Isatidis 255, rheum 51, Herba Pogostemonis 85, Rhizoma Dryopteris Crassirhizomae 255, Rhodiola rosea L. 85, menthol 7.5, Herba Ephedrae 85, Semen Armeniacae Amarum 85, Herba Houttuyniae 255, Radix Glycyrrhizae 85 and gypsum 255, were extracted according to the following process:

(60) (1) the traditional Chinese medicines were weighed according to the weight ratio of raw materials, the medicinal parts were selected and processed into pieces as required; (2) Herba Pogostemonis was processed into pieces, water was added (the ratio of the volume of water to the weight of medicinal material (L/kg, mL/g) was 10) to extract volatile oil, oil extraction time: 8 hours, the volatile oil was collected for later use; after the extract was filtered, the residue was removed, the filtrate was kept for later use;

(61) (3) Fructus Forsythia, Herba Ephedrae, Herba Houttuyniae and rheum were extracted with 70% ethanol (the ratio of the volume of water to the weight of medicinal material (L/kg, mL/g) was 12) for 3 times, and each extraction took 2.5 hours, the extracts were combined and filtered, the ethanol was recycled, the filtrate was kept for later use;

(62) (4) water was added to Flos Lonicerae, gypsum, Radix Isatidis, Rhizoma Dryopteris Crassirhizomae, Radix Glycyrrhizae and Rhodiola rosea L. (the ratio of the volume of the water to the weight of the medicinal material (L/kg, mL/g) was 12) and the obtained mixture was decocted until the mixture was boiled, Semen Armeniacae Amarum was added, the obtained mixture was decocted for 2 times, each decoction took 1 hour, the extracts were combined and filtered, the obtained filtrate was combined with the filtrate obtained after the oil extraction of Herba Pogostemonis in step (2), the obtained combination was concentrated to obtain a clear paste with a relative density of 1.14 determined at 60° C., ethanol was added, the obtained mixture was adjusted to an alcohol concentration of 70%, refrigerated, filtered, the ethanol was recycled until there's no smell of alcohol, a clear paste was obtained for later use;

(63) (5) the clear paste obtained in step (4) was combined with the ethanol extract in step (3), the obtained combination was concentrated to obtain a clear paste with a relative density of 1.19 determined at 60° C., the clear paste was dried to obtain dry paste powders for later use;

(64) (6) the dry paste powders obtained in step (5) was added to a suitable pharmaceutically acceptable auxiliary material for granulation; the auxiliary material can be 35 g of starch;

(65) (7) menthol and the volatile oil obtained in step (2) were added to ethanol and allowing the menthol and volatile oil dissolved in ethanol, the granules obtained in step (6) were sprayed into the obtained mixture, the product is obtained after encapsulating.

(66) The method for determining the content of menthol:

(67) test solution preparation: the substance contained in the composition from the samples of loading difference test was taken, ground finely, mixed evenly, 0.4 g was taken, weighed accurately, placed in a conical flask equipped with a plug, 20 mL of n-hexane was added accurately, the flask was shaken clockwise and counterclockwise for about 40 seconds, a suitable amount of solution was taken, filtered through a 0.22 μm microporous membrane to obtain the test solution;

(68) preparation of the solution of reference substance: a suitable amount of menthol reference substance was taken, weighed accurately, n-hexane was added to prepare a menthol solution with a menthol concentration of 0.23 mg per 1 mL to obtain the solution of menthol reference substance;

(69) conditions for chromatography: chromatographic column: Agilent J&W Scientific HP-5 capillary chromatographic column (30 m×0.25 mm, 0.25 μm); the column temperature was increased according to the following temperature programming: initial temperature 98° C., the temperature was kept for 12 minutes, increased to 140° C. at a rate of 8° C. per minute, kept for 2.5 minutes, then increased to 280° C. at a rate of 140° C. per minute, kept for 5-20 minutes; detector temperature was 350° C.; inlet temperature was 350° C.; carrier gas was nitrogen, flow rate: 1.1 mL/min; injected by split injection, split ratio: 25:1; the volume of injected sample: 1 μL; fuel gas ratio: air-hydrogen (450:45);

(70) determination method: 1 μL of the solution of menthol reference substance and the test solution were accurately taken, respectively, and were injected into a gas chromatograph, were determined to obtain the results.

(71) Results of the Determination of Menthol Content in the Composition

(72) TABLE-US-00018 No. Menthol contents (%) 1 1.63 2 1.61 3 1.57

(73) Conclusion: the result is that the menthol was well separated and can be used to control the quality of the traditional Chinese medicine composition.