JPS59116544A - Liquid-chromatographic analysis method of enantiomeric mixture of chrysanthemumic acid esters - Google Patents

Abstract

PURPOSE:To separate and analyze an enantiomeric mixture of chrysanthemumic acid esters with an excellent separating efficiency in a short time, by analyzing a specific enantiomeric mixture of chrysanthemumic acid esters, which is a pyrethroid insecticide, by liquid chromatography using a stationary phase wherein an optically active N-acylated amino acid is grafted. CONSTITUTION:As a packing material having a grafted N-acylated amino acid, e.g., a chromatographic packing material wherein an optically active organosilane represented by formula I is grafted onto an inorganic carrier having a hydroxyl group on its surface, is used as a stationary phase. An enantiomeric mixture of chrysanthemumic acid esters represented by formula II is separated by liquid chromatography. In the formulas, R is a lower alkyl group or a halogen atom; A is an alkyl group including optionally substituted aryl, aralkyl, cycloalkyl or heterocyclic group; R1-R3 are each an alkyl group, an alkoxy group, a hydroxyl group, or a halogen atom; R4 is a lower alkyl or phenyl group; (n) is 2-4; and X is an -NHCO- group or the like.

Description

Detailed Description of the Invention The present invention is based on the general formula [■] (wherein k represents a lower alkyl group or a halogen atom, A is an optionally substituted aryl group, an optionally substituted aralkyl group, Represents an optionally substituted cycloalkyl group or an alkyl group containing a heterocycle.

Rice represents asymmetric carbon. The present invention relates to an analytical method characterized in that an enantiomeric mixture of chrysanthemum acid esters shown in the following is analyzed by liquid chromatography using a stationary phase grafted with an optically active N-acylated amino acid.

Chrysanthemum acid esters are highly effective as pyrethroid insecticides, and many insecticides have been developed, including allethrin, 7talsulin, and resmethrin. There are four isomers of chrysanthemum acid: cis, (+)-trans, and H-trans, and the physiological activities of its various esters differ greatly among these isomers, and generally ,
The (t)-trans ester has the greatest insecticidal efficacy. Therefore, various attempts have been made to increase the activity of these esters by optical resolution, and highly effective pyrethroid insecticides containing optically active chrysanthemum acid as the acid moiety are already on the market.

Therefore, analysis of optical isomers of chrysanthemum esters is essential for quality control and efficacy evaluation, and there is a need for the development of accurate analytical methods.

However, until now, there have been no reports on the direct separation of optical isomers of these compounds by chromatography. There is only. However, this method has a low separation ability of the stationary phase, can only be applied to specific compounds, and has some limitations in use, making it impractical. Therefore, at present, the analysis of optical isomers of chrysanthemum acid esters involves first hydrolyzing the esters with an alkali to obtain chrysanthemum acid or its derivatives, and then dehydrating and condensing optically active alcohols or amines to this. Analyze these acids as diastereomers, or treat these acids with amines to form acid amides and separate them using gas chromatography or liquid chromatography using an optically active stationary phase.
This method requires optically active reagents with high optical purity, and requires frequent and long operations.
Moreover, this method is fraught with problems such as isomerization during hydrolysis to esterification or amidation reactions.

Therefore, if it were possible to directly separate and analyze the optical isomers of chrysanthemum acid esters, it would be extremely advantageous.

Under these circumstances, the present inventors conducted extensive studies and found that when a packing material grafted with an optically active N-acylated amino acid was used as a stationary phase in liquid chromatography, mirror images of chrysanthemum esters The inventors have now completed the present invention by discovering that isomer mixtures can be directly and well separated and that analysis of the mixing ratio of isomers, that is, analysis of optical purity, can be carried out easily and accurately.

The filler grafted with an optically active N-acylated amino acid used in the method of the present invention is, for example, one represented by the general formula [1] representing a loxyl group or a halogen atom, and having at least one
One is an alkoxyl group or a halogen atom. R4 represents a lower alkyl group or a phenyl group, and n is an integer from 2 to 4. X is -NHCO- group or -NH
,0 represents a CO- group, and the latter represents an asymmetric carbon. ) N-(
Examples include silica gel to which 3,5-dinitrobenzoyl)-D-7enylglycine or N-(3,5-dinitrobenzoyl)-L-valine is grafted via ω-aminopropylsilane.

When preparing the filler, the method of Pirkle et al. (J
6Chromatogr, , 192, 143
(1980), J. Org.

Chem, 48, 2935 (1981),
J., Am., ChemJoc.

For example, a method of grafting an organosilane obtained by the reaction of an optically active N-acylated amino acid with an ω-aminoalkylsilane onto an inorganic support such as silica gel, Either ω-aminoalkylsilane is grafted onto an inorganic carrier such as silica gel in advance, and an optically active N-acylated amino acid is bonded thereto (by dehydration condensation), or
Alternatively, it can be prepared by a method of ionic bonding.

The packing material grafted with an optically active N-acylated amino acid used in the method of the present invention is packed into a chromatographic column according to a conventional method and used as a stationary phase in liquid chromatography. The stationary phase is prepared by filling a chromatographic column with a packing material grafted with ω-aminoalkylsilane in advance in accordance with a conventional method, and then flowing a solution of an optically active N-acylated amino acid through the column. It can also be prepared by a method of immobilizing optically active N-acylated amino acids through ionic bonding.

In liquid chromatography using the stationary phase of the present invention, appropriate elution conditions, especially the normally used normal phase partition conditions, can be selected.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples.

Example I D phenylglycine 20y and 3,5-dinitrobenzoyl chloride 30F to dehydrated tetrahydro 7ran 850
rnl, and stirred at room temperature for 10 days. After filtering out insoluble matter, the solvent was distilled off under reduced pressure. To the residue was added 200 g of an aqueous solution of 11 amium carbonate, and after stirring occasionally, the mixture was left in the apparatus overnight. After filtering off the insoluble matter, the filtrate was washed twice with 50% ether, and then 10% polyhydrochloric acid was added dropwise.
The pH was adjusted to 5.0.

Dissolve the precipitated crystals in 200ml of ethyl acetate, wash the ethyl acetate layer three times with 50ml of water, and then add anhydrous sulfuric acid.)
The mixture was dried over IJum and the solvent was distilled off under reduced pressure. The residue was recrystallized from a mixed solvent of tetrahydrofuran-〇-hexane (5:I) to give h+-(3,5-dinitrobenzoyl).
-10 g of phenylglycine was obtained.

Melting point = 215 to 217°C [α) D: -89.6° (c = Q, 93%, tetrahydro 7 run) Elemental analysis C (chi) Hf penalty N (correspondence) Actual value -51°97 3.12 12゜29 Calculated value 52
゜183゜21 12.17 (C1δH1IN307
Next, ω-aminopropyl silylated silica gel for high performance liquid chromatography (particle size 5μ, pore size 60X) 3F
After adding dehydrated tetrahydro 7ran 50- to the solution and degassing under reduced pressure, N-(3,5-dinitrobenzoyl)D-phenylglycine 31 and N-ethoxycarbonyl-2-
Ethoxy-1,2-dihydroquinoline 21 was added and stirred at room temperature for 2 hours.

The reaction solution was filtered through a glass filter, and the obtained silica gel carrier was washed on the filter with tetrahydrofuran, methanol, acetone, and ether in this order, and dried under reduced pressure to obtain N (3t5-dinitrobenzoyl) D 7. The desired filler in which nylglycine was grafted via the ω-aminopropylsilyl group was obtained.

The elemental analysis values for this are N: 2.43%, C: 9.02
%Met.

The filler thus obtained had an inner diameter of 4 ynm.

Fill the slurry into a stainless steel column with a length of 3001 mm,
These two packed columns were connected in series and under the following conditions (
Analyzing 5-benzyl-2-furyl)methyl (to)-cis/trans-chrysampmate (resmethrin),
The chromatogram shown in Figure 1 was obtained.

In Figure 1, peak number (1) is (5-benzyl-3-furyl)methyl (tho)-cis-chrysanthemate (f+
It is abbreviated as 1-cis body. Hereinafter, other isomers will be similarly abbreviated. ), (2) are the peaks of the (−)-cis form, (3) are the peaks of the t)-trans form, and (4) are the peaks of the ←)-trans form. The time required for the peak (1) to elute is approximately 26 minutes, the separation coefficient between the cis-isomer peaks of (1) and (2) is 1°05, and the area ratio of the peaks is 47:53.
The separation factor between the peaks of +31 and trans isomer (4) is 1.
04, the peak area ratio was 50:50.

Example 2 Dehydrated tetrahydrofuran 50- was added to 3F of ω-aminopropylated silica gel for high performance liquid chromatography (particle size 5μ, pore size 60K) and degassed under reduced pressure. 3,5-dinitrobenzoyl)D-phenylglycine was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was filtered through a glass filter, and the resulting silica gel carrier was mixed with tetrahydrofuran,
Washing with methanol, acetone, and ether in this order, and drying under reduced pressure, the desired product to which N-(3,5-dinitrobenzoyl)-D-phenylglycine was grafted via the ω-aminopropylsilyl group by an ionic bond was obtained. A filler was obtained.

The elemental analysis values of this product were N: 2.21%, C: 8.
It was 16%.

The filler thus obtained had an inner diameter of 4 mm.

The slurry was packed into a stainless steel column with a length of 30 ctn, and (t)-3-allyl-2-methyl-4-
, 11-2-cyclopentenyl(t)-trans-chrysaditemate (allethrin) was analyzed, and the chromatogram shown in Figure 2 was obtained.

In Figure-(2), peak number (1) is (+-)-3-allyl-2-methyl-4-oxo-2-cyclopentenyl (
-1-1-transucrysanthemate ((-1-)-
(g) Abbreviated as body. Hereinafter, other isomers will be similarly abbreviated. ), (2) is (c) - mortar, (3) is (c) = h +
and (4) are the peaks of the (−))−←) body.

The time required for peak (1) to elute is approximately 29 minutes, the separation coefficient for both peaks (1,) and (2) is 1°05,
The area ratio was 52:48, the separation factor for both peaks (31 and (4)) was 1°02, and the area ratio was 50:50.

Example 3 Slurry filled with filler to which N-(3,5-dinitrobenzoyl)-D-phenylglycine obtained in Example 1 was grafted via an ω-aminopropylsilyl group ← Inner diameter, length σ-Bromo β-naphthyl (to)-cis/trans-chrysampmate was analyzed using a stainless steel column with a diameter of 30 nm under the following conditions, and the interim chromatogram shown in Figure 3 was obtained.

In Figure 3, peak number (1) is α-promo β-naphthyl (+1-cis-chrysanthemate (abbreviated as -1-1-cis form.Hereinafter, other isomers are also abbreviated in the same way.

), f2) are the peaks of the ←)-cis form, (3) is the (t)-trans form, and (4) is the peak of the H-1 lance form.

The time required for the peak of (1) to elute is approximately 14 minutes, and the separation coefficient between the peaks of the cis form of (1) and (2) is 1.1.
8. The area ratio is 50:50, the separation coefficient between the peaks of +31 and (4) trans isomer is 1°22, and the area ratio is 51.
:, 49.

Example 4 Slurry filled with a filler in which N-(3,5-dinitrobenzoyl)-D-phenylglycine obtained in Example 1 was grafted via an ω-aminopropylsilyl group, inner diameter 4 mm, length Using one 301 stainless steel column, the following compounds were analyzed under the following conditions, and the separation coefficients were determined.

The results are shown in the table below.

[Brief explanation of the drawing]

Figures 1, 2 and 3 are chromatograms obtained in Examples 1.2 and 3, respectively, where the vertical axis represents intensity and the horizontal axis represents retention time. Figure-1 (3) 20 30 (min) Figure-
2 (1) Figure-3 (3)

Claims (2)

[Claims] (1) General formula (wherein k represents a lower alkyl group or a halogen atom, A is an optionally substituted aryl group, an optionally substituted aralkyl group, an optionally substituted cycloalkyl group, or The enantiomeric mixture of chrysanthemum esters represented by An analysis method characterized by analyzing (2) A stationary phase grafted with an optically active N-acylated amino acid has a general formula (wherein, R2 and R3 are the same or different, alkyl group, alkoxyl hydroxyl group, or) 10 gene atoms. and at least one is an alkoxyl group or a halogen atom. R4 represents a lower alkyl group or a phenyl group,
n is an integer from 2 to 4. According to claim 1, wherein Analysis method.