DE3329936A1 - Method of quantitatively determining alcohols and amines - Google Patents

Abstract

The invention relates to a method of quantitatively determining alcohols or amines in the presence of impurities by reaction with sensitive derivatising reagents in which the substrates of the general formula I R1-Y in which Y denotes an OH or an -NR2H group, where R2 stands for hydrogen or an optionally branched alkyl radical containing 1 to 18 carbon atoms, and R1 denotes an aliphatic, aromatic or heterocyclic radical or a combined radical composed of these constituents, which may be substituted, are reacted with a sensitive derivatising reagent of the general formula II <IMAGE> in which X is a group which reacts as leaving group with respect to the nucleophilic compounds of the formula I, R3 denotes a fluorescent radical of the formula -CHR4R5, R4 denotes an alkyl radical containing 1 to 18 carbon atoms and R5 denotes an aromatic or heterocyclic radical containing 6 to 12 carbon atoms having at least one 6-membered ring combined with R5, with the proviso that the compound R5H derived from R5 has an oscillator strength f of not less than 0.05, to form the derivatives of the general formula III <IMAGE> in which R1 and R3 have the meaning specified above are reacted with the cleavage of the compound IV HX in which X has the meaning specified above and the derivatives of the general formula III produced utilising ... Original abstract incomplete.

Description

Method for the quantitative determination of alcohols or amines Field of invention

The invention relates to a process for the separation of alcohols and / or amines by derivatization with activated acids which carry a fluorescent label.

State of the art

In the case of separation and quantitative determination of compounds that are difficult to separate, e.g. B. chlorine compounds are advantageous sensitive derivatization reagents applied.

Reagents which enable highly sensitive labeling, for example fluorescent labeling, are desirable. From the In peptide chemistry, the end group determination of peptides using l- (Dlmethylamino) -5-naphthalenesulfonyl chloride (dansyl chloride) is known, in which the terminal amino acids react. In the same way, the determination of a large number of primary and secondary Amines, phenols and some alcohols possible after the introduction of the dansyl radical

Fluorescence markers are of great practical importance for the chromatographic application of the substances to be separated, especially chiral compounds. The modern separation processes use thin-layer chromatography and / or high pressure liquid chromatography.

For the isolation of enantiomeric carboxylic acids such as OC-arylpropionic acids the condensation of the carboxy acids to be separated is carried out in the presence of water-soluble carbodiimide with two highly sensitive, chiral derivatization reagents, the D- and L-I-aminoethyl-4-dimethylaminonaphthalene suggested (Anal. Chimica

332993 *

Acta 1980 , (120) 187-192). The diastereomeric amides formed can ζ. B. can be efficiently separated by chromatography on a conventional chromatography column. The visualization was carried out with a fluorescence detector. The detection limit is z. B. 1 ng indicated. The reagents previously used for fluorescent labeling bring a number of disadvantages such. B. instability, including photo instability, formation of by-products during implementation and insufficient fluorescence yield.

task

There was still a need for methods for the quantitative determination of alcohols and / or alcohols that can be carried out in a timely manner Amines in the presence of sometimes considerable amounts of foreign substances.

The focus of interest was the determination of biologically relevant compounds, especially pharmaceuticals in biological Material such as body fluids, excreta, tissue, etc. Because of the very low levels that are usually required Detection limit can only be used with highly sensitive derivatization reagents · The derivatization reagent should therefore have a high Have fluorescence intensity and under as mild conditions as possible quantitatively and specifically with certain functional Groups react: The reaction products should have favorable chromatographic properties so that the Separation of excess reagent, foreign substances and by-products is easy to achieve.

The problem of detection is particularly urgent in the case of applied optically active pharmaceuticals in biological material, as these usually occur in extremely small quantities. If this is optically active compounds do not have strong autofluorescence or strong absorption properties, it appears urgently needed to have a chiral derivatization reagent available, with the help of which the detection limit

BATH ORIGINAL

can be enormously humiliated. In addition to the spectral properties optical purity and optical stability are additional requirements. For the determination of optically active amines In biological material there is currently no suitable fluorescent marker available that meets the requirements of the technology. Up to now, amino acid derivatives have often been used to determine β-blockers used, but generally racemize too quickly. Have optically active reagents from other classes of substances usually too low sensitivity. There was therefore the special task of finding suitable derivatization reagents in the form of optical enantiomers available.

solution

To achieve the stated object, the method according to the claims is proposed.

The derivatizing reagents of the general formula II are characterized by a high reactivity towards the substratum of the general Formula I. The derivatives of the general formula III obtained are - in comparison with derivatization products - general - quite stable products.

Preparation of the derivatizing reagents of the general formula II The carboxylic acids corresponding to the compounds of the formula II

Ro-C-OH (compounds of the formula II A) are known or can can be produced by processes known per se or based on processes known per se.

The conversion of the compounds of the formula II A into the corresponding Acid chlorides or acid bromides can also be found in themselves

_s _ "■■ · ■■ '" ■■ - S329936

take place in a known manner, for example by reaction with inorganic acid chlorides such as thionyl chloride, in addition also
Phosphorus trichloride, phosphorus pentachloride, phosphorus oxychloride, and the corresponding bromides. In general, a not too great excess of the halide-transferring reagent is sufficient. The reaction can expediently in an inert solvent such as. B. toluene, petroleum ether, chloroform and the like can be carried out, where appropriate, may be mentioned, especially in the last section of the reaction.

The preparation of the compounds of the formula II, in which the compound of the formula II A was reacted with an activating reagent A, can also be carried out on the basis of known reactions of this type (cf. Newer methods of preparation, organic chemistry
Vol. V, 53 (1970), Verlag Chemie, HA Staab, Angew. Chemistry J74,
407 (1962)). So z. B. the compound of formula II A in a suitable inert, suitably dried solvent
such as B. a halogenated hydrocarbon such as dichloromethane, chloroform, an ether such as tetrahydrofuran reacted with 1,1'-carbonyldiimidazole (CDI). Using a
A stoichiometric excess of CDI can be advantageous
be. Excess CDI can be destroyed, for example, by adding glacial acetic acid. Advantageously, the resulting solution of the derivatizing reagent of the formula II
used immediately afterwards for the purposes of the procedure.

Implementation of the procedure

The method according to the invention can be carried out based on to the known acylation reactions of the prior art.

The derivatization reagent is advantageously used in general Formula II, in which X is chlorine or bromine, diluted with a suitable, dry, inert solvent,

for example in a chlorinated hydrocarbon such as dichloromethane, an ether such as dioxane and the like. B. Potassium carbonate or bicarbonate impaired the reaction based on experience does not.

The derivatization reagent is advantageously used to a certain extent Excess, for example 10 to 1000 times, preferably 10 to 50 times the excess over the stoichiometric, Amount related to the substrate I. The implementation of the invention Processed can be done in basically the same way carry out for all substrates of general formula I, but for a better overview in the two substrate types amines and alcohols.

Derivatization of amines

The addition to the amines (substrates of the general formula I, in which Y is -NR ₂ H), can expediently gradually, for. B. done by dropping into the (stirred) solution of the substrate I mainly at room temperature and over a certain period of time. To complete the reaction, the mixture can be stirred for a certain time, for example 3 + 2 hours at an elevated temperature, for example at reflux.

The reaction solution can then expediently be extracted by shaking with water, dried and the solvent removed will.

Quantitative recording

The reaction with (+) - 2- [2- (4-chlorophenyl) -5-benzoxazolyI-] propionic acid chloride is given as an example of the determination of primary and secondary amines (Y in formula I = -NR _{2 H).}

10 ng to 100 μg of the amine are mixed with 500 μΐ of a solution of the (+) - 2- [2- (4-Chlorophenyl) -5-benzoxazolyl] -propionic acid chloride (benoxaprofenic acid chloride) (1 mg / ml) in a dry, inert Solvents such as B. dichloromethane or with a freshly prepared Solution of the reaction product of (+) - 2- [2- (4-chlorophenyl) -5-benzoxazolyl] propionic acid ("Benoxaprofen") with 1,1'-carboxy diimidazole and left to stand for some time (guide value 0.5 hours) usually at room temperature.

The analytical determination

can e.g. B. by means of thin layer chromatography (TLC) or HPLC take place. The following procedures have proven effective:

The solutions are applied to silica gel 60 TLC plates without a fluorescent indicator using the Linomat III (Camag, Switzerland).

Application volume: 10 ul

Band width: 5 mm

Solvent systems: 1. Toluene / dichloromethane /

Tetrahydrofuran (THF) (5/1/1; v / v) NH ₃ atmosphere

2. Toluene / CHCl ₃ / THF

(5/4/1; v / v) NH ₃ atmosphere

3. CHC ^ / MeOH / HCOOH / THF (110/20/5/2; v / v)

4. Cyclohexane / ethyl acetate / MeOH (7/3/2; v / v) NH ₃ atmosphere

Detection: Densitometric measurement of the

Fluorescence intensity with the chromatogram spectrophotometer KM 3 (Zeiss, Oberkochen)

Excitation: Line 313 nm of the Hg medium pressure lamp ST 41 with a slit size of 0.1 \ 6mm Emission: Monochromatic filter M365, amplification 1-10, high voltage on the photoelectron multiplier: 1)

HPLC

Injection volume Pillar:

Length:

Filling material: Mobile phase:

Temperature: flow rate:

Detection:

10 μΐ analytical column from Dupont (Bad Homburg) 250 mm, internal diameter 4.6 mm Zorbax-Sil cyclohexane / dichloromethane / THF (5/1/1, v / v) 25 ⁰ C 1 ml / min (corresponding to a pressure of 8.5 MPa) fluorine! Metric excitation: at wavelength 313 nm emission: at 390 nm

Derivatization of alcohols

The reaction of the alcohols (substrates of the general formula I, in which Y is -OH) with the derivatizing reagents of the general formula II, can, for. B. in a suitable, inert, dry solvent such as acetonitrile, toluene, dioxane, dichloromethane, chloroform and the third. You can work with or without an acid acceptor. In general, when heated, z. B. good results at reflux temperature. As a guide, z. B. given an hour response time. Occasionally, a higher reaction temperature is recommended, for example 110 +10 ^b C.

The work-up can be carried out in a manner known per se, for example by evaporating the solvent · taking up in a water-immiscible extraction agent such as B. a chlorinated hydrocarbon, extraction with water and work-up of the phase containing the product of the formula III, if appropriate followed by recrystallization from a suitable solvent such as e.g. B. acetonitrile.

For quantitative recording

Exemplary for the determination of alcohols (Y in formula I = OH) the reaction with (+) - 2- [2- (4-chlorophenyl) -5-benzoxazolyl-] propionic acid chloride is indicated.

10 ng - 100 pg of the alcohol with a solution of 500 μΐ Benoxaprofensaurechlorids (1 mg / ml) in acetonitrile or toluene for a period of time to 100 - 110 ⁰ C heated.

The analytical determination can be carried out analogously to that given above for amines.

The substrates of the general formula I.

Suitable substrates of the general formula I are in principle those which also otherwise have acylation reactions with carboxylic acid chlorides or activated carboxylic acids of the formula II type are accessible. Limiting can - as known from analog reactions - At best, steric hindrances, undesirable side reactions, excessive sensitivity and the like act.

Biologically relevant alcohols such as choline, terpenoid alcohols and steroid alcohols, sugar and adrenaline may be mentioned in particular and derivatives, furthermore pharmaceuticals, e.g. B. the alcohols of the phenothiazine type such as perphenazine and of the thioxanthene type such as clopen-

thixol and flupentixol, iminosteal life derivatives such as opipramol, diphenylmethane derivatives such as hydroxyzine, phenylpropanol and pentanol, chloramphenicol, ethynyl carbinols such as methylpentynol, Alkaloids like morphine and derivatives, dibenzazepine derivatives like opipramol, glycerine ethers like guiaphesin, ß-blockers like that Propranolol, oxprenolol, pindolol, alkaloids like atropine, scopolamine, ergometrine, ajmaline, steroids like cortisol, Corticosterone, prednisone, cortexone, digitoxigenin, prostaglandins containing hydroxyl groups such as prostaglandin E ".

Biologically relevant primary or secondary amines which can serve as substrates for the process according to the invention are: Amino acids and similar substrates, the monoamine oxidase inhibitor tranylcypromine, amphetamines with primary and secondary Amino groups, ethylenediamine derivatives such as ethambutol, iminodibenzyl derivatives such as desipraminj dibenzocycloheptadiene and triene compounds such as nortriptyline and protriptyline, dibenzocyclooctadiene derivatives such as benzoctamine, ephedrine, its structural analogues and derivatives such as norpseudoephedrine, phentermine, Propylhexedrine, fenfluramine, phenylethylamine derivatives such as mescaline, Dopa and derivatives such as carbidopa, dopamine, noradrenaline, adrenaline and their breakdown products, sympathomimetics such as Norfenefrine, phenylephrine, isoprenaline, Bamethan, Tyramiri, ephedrine and the like, histamine and relatives such as betazole, penicillamine like ampicillin, epicillin, and poisonous principles like the coniin, muscimol.

Derivatization of chiral substrates

Of particular interest is the derivatization of chiral amines and alcohols of the formula I by the process according to the invention can be done.

The preparation of the optical enantiomers of the general formula II is the example of 2- [2- {4-chlorophenyl) -5-benzoxazolyl] propionic acid chloride ("Benoxaprofene chloride") explained in more detail.

'Λξ> ·

The freshly prepared benoxaprofene chloride is in a suitable Solvents such as B. methylene chloride with the solution of an optically active amine, e.g. B. the solution of (R) - (+) OC-methylbenzylamine in the same solvent, preferably with the slow addition of the amine and thorough stirring. One completes the Reaction, for example by stirring under reflux for several hours, washes, for example, with dilute hydrochloric acid and water and dries the solvent phase. After evaporation of the solvent, a white crystalline product is obtained. The received Diastereomeric mixture of (R, S) - (+) - benoxaprofen- (R) - (+) - otmethylbenzamide can be converted into the two diastereomers (S) - (+) - benoxaprofen- (R) - (+) - OC-methylbenzamide by HPLC and the corresponding R - (-) - compound can be broken.

The free acids can be selected from the diastereomeric amides, e.g. B. obtained by (acid) hydrolysis based on tried and tested Process of acid amide cleavage. You can, for example, the obtained diastereomeric amide in toluene solution with mineral acid, for. B. 45 percent sulfuric acid by refluxing in (R) - (-) - 2- [2- (4-chlorophenyl) -5-benzoxazolyl] propionic acid and (R) - (+) - OC-methylbenzylamine cleave, whereby the acid is predominantly in the aqueous Phase is located. Working up is done in a manner known per se. The (S) - (+) - acid can be represented analogously.

The preparation of the acid chlorides from the diastereomeric acids takes place as described above for the racemic compounds.

The derivatization

Be based on the detection of various amines in biologically active Material explained in more detail.

You can, for example, make an amine determination from plasma by adding a certain amount of plasma, for. B. 1 ml, with diluted

-χ-

Alkali and with an organic extractant such as ζ. B. η-hexane extracted. The amine becomes the organic phase preferably obtained by evaporation and treated with a solution of the optically active derivatization reagent of the general formula TI reacted in a dry, inert solvent such as dichloromethane.

The derivatization with the optically active benoxaprofenic acid chlorides mentioned above leads to derivatives of the formula III with a remarkably low tendency to racemize.

The following examples serve to illustrate the invention. The following abbreviations are used:

2- [2- (4-chlorophenyl) -5-benzoxazolyl-] propionic acid = benoxaprofen or BOP

2- [2- (4-chlorophenyi) -5-benzoxazolyl-] propionic acid chloride = benoxaprofen chloride or BOP-Cl

2- [2- (4-chlorophenyl) -5-benzoxazolyl-] propionic acid-Ot-methylbenzylamide = Benoxaprofen-Ot-methylbenzylamide = BOP-MBA

stating the chiralities associated with the individual compounds in the usual notation.

Devices used:

IR spectra are recorded in KBr pellets with a Beckman Acculab 2

Spectrophotometer .. recorded ...

Chromatogram spectrophotometer KM3 (Carl Zeiss, Oberkochen,

G. F. R.) with Recorder 56 (Perkin Eimer), Linomat III (Camag, Muttenz, Switzerland) HPLC LC 601 with fluorescence detector 605-1OS (Perkin Elmer, Überlingen, G. F. R.) with an analytical silica gel column from Dupont (Bad Homburg).

Preparation of the derivatizing reagents of the general formula II

1. Activation of benoxaprofen

a) conversion to the acid chloride

600 mg benoxaprofen (approx. 2 mmol) are dissolved in 50 ml toluene, 5 ml of thionyl chloride (approx. 20 times the amount) were slowly added dropwise and then refluxed for 30 minutes. The solution is evaporated in a water jet vacuum and the crystalline residue is recrystallized with dichloromethane if necessary

b) conversion to imidazolide

100 mg benoxaprofen are mixed with a solution of 500 mg Carbonyldiimidazole in 50 ml of dried dichloromethane are added. The solution is left to stand at room temperature for 30 minutes and occasionally shaken. To destroy excess carbonyldiimidazole, 5 ml of glacial acetic acid is added. The reagent solution thus obtained must be used immediately.

Characterization of the physical and spectral properties of benoxaprofen or benoxaprofen acid chloride

IR (cm " ¹ ): BOP 1700 (C = O, acid); BOP-Cl 1775 (C = O, acid chloride)

Elemental analysis: BOP (C ₁₆ H _{12 ClNO 3} )

Calcd .: C 63.69 H 4.01 N 4.64

Found: C 63.49 H 4.05 N 4.59

BOP-Cl (C ₁₆ H ₁₁ Cl ₂ NO ₂ )

Calcd .: C 60.02 H 3.46 N 4.37

Found: C 60.32 H 3.40 N 4.33 Melting points: BOP 191 °

BOP-Cl 91.5 °
Emission spectra: BOP λ 350 nm and 365 nm

IHSX

BOP-Cl λ 350 nm and 365 nm max

Excitation spectra: BOP Λ 312 nm

Max

BOP-Cl \. 312 nm
Max

2. Derivatization of amines and alcohols of the general formula I.

a) with OC-methylbenzylamine

600 mg of benoxyprofenic acid chloride are dissolved in 50 ml of dry dichloromethane. 10 ml of a solution of OC-methylbenzylamine in dichloromethane (1.5 ml of MBA and 8.5 ml of dichloromethane) are slowly added dropwise with stirring at room temperature. It is then refluxed for 3 hours. The solution is washed first with 0.2 N HCl and then with water and then dried over Na SO. After evaporation of the solvent a white crystalline residue is obtained. When tested by thin-layer chromatography, the material shows a fluorescent spot.
M.p .: 169 °

Solvent system: chloroform / methanol / water / ammonia 25 percent. (70/30/5/1; v / v)

Rf values: 0.89 (amide), 0.35 (BOP)
IR (cm " ¹ ): 1640 (C = O, amide)

b) with tranylcypromine

The amide of tranylcypromine is analogous to this with benoxaprofen synthesized, which precipitates during the reaction due to its lack of solubility in dichloromethane and thus is easy to isolate.

Mp .: 204 ⁰ C

Solvent system: cyclohexane / ethyl acetate / methanol

(70/30/20; v / v)! ^ - Atmosphere RF values: 0.43 (amide), O.10 (BOP)
IR (cm " ¹ ): 1640 (C = O, amide)

c) with choline

300 mg benoxaprofenic acid chloride (approx. 1 mmol) and 420 mg Choline chloride (about 4 mmol) are refluxed in acetonitrile for one hour. Excess choline chloride crystallizes out almost quantitatively on cooling the reaction mixture. Acetonitrile is concentrated to dryness, the The residue was taken up in water and the aqueous phase was extracted with dichloromethane. The aqueous phase is in turn to Concentrated to dryness and the residue recrystallized from acetonitrile.

Mp .: 205 ⁰ C

Solvent system: acetone / methanol / formic acid (2/2/1; ν / ν) RF values: 0.36 (ester), 0.86 (BOP)

IR (cm " ¹ ): 1725 (C = O, ester)

3. Quantitative recording of amines and alcohols in general

Formula I.

a) primary and secondary amines

Reaction with the acid chloride and with benoxaprofen imidazolide

Reaction conditions:

10 ng - 100 μg of the corresponding amine are 500 μΐ a solution of benoxaprofenic acid chloride (1 mg / ml) in dry dichloromethane or that described above fresh prepared benoxaprofen imidazolide solution left to stand for half an hour at room temperature.

Chromatographic conditions:

TLC: The solutions are applied to silica gel 60 TLC plates without a fluorescent indicator using the Linomat III.

Order volume: 10 μΐ

Band width: 5 mm

Solvent systems: 1. Toluene / dichloromethane / THF

(5/1/1; v / v) NH ₃ atmosphere

2. Toluene / CHCl ₃ / THF

(5/4/1; v / v) NH ₃ atmosphere

3. CHCl ₃ / methanol / formic acid / THF (110/20/5/2; v / v)

4. Cyclohexane / ethyl acetate / methanol (7/3/2; v / v) NH ₃ atmosphere

Detection:

- Ar-

Densitometric measurement of the fluorescence intensity with the Chromatogram spectrophotometer K 3

Excitation: line 313 nm of the Hg medium pressure lamp ST 41 at a gap size of 0.1 χ 6mm emission: monochromatic filter M 365, amplification 1-10, high voltage at the photoelectron multiplier 1

HPLC injection volume: 10 μΐ Column: analytical column of the

Dupont company

Length: 250 mm, inner diameter 4.6 mm

Filling material: Zorbax-Sil Mobile phase: cyclohexane / dichloromethane / THF (5/1/1; v / v) Temperature: 25 ⁰ C
Flow rate: 1 ml / min (corresponding to a pressure of 8.5 MPa)
Detection: fluorometric

Exitation: at a wavelength of 313 nm, emission at 390 nm

b) alcohols:

Reaction conditions for the reaction with benoxaprofen

acid chloride

10 ng - 100 μg of the corresponding alcohol are with 500 μΐ a solution of benoxaprofen acid chloride (1 mg / ml) in Acetonitrile or toluene heated to 100-110 ° C for one hour.

- IA-

The alcohols were usually determined by thin-layer chromatography, and the conditions correspond to those given above.

4. Representation of the benoxaprofen enantiomers

a) Conversion of benoxaprof en to OC-methylbenz ylamid '. 600 mg of benoxaprofen (approx. 2 mmol) are dissolved in 50 ml of toluene dissolved, 5 ml of thionyl chloride (approx. 20 times the amount) slowly added dropwise and then refluxed for 30 minutes. The solution has evaporated in a water jet vacuum and the crystalline residue is recrystallized with dichloromethane if necessary. The BOP-Cl is then in Dissolved 50 ml of methylene chloride and 10 ml of a solution of (R) - (+) - OC-methylbenzylamine in methylene chloride (1.5 ml Methylbenzylamine and 8.5 ml of methylene chloride) were slowly added with stirring. After three hours of cooking on At reflux, the solution is washed first with 0.2 N HCl and then with water and dried over Na SO. Man the solvent evaporates and a white crystalline residue is obtained.

M.p .: 169 °
Elemental analysis: C-ZH _{21 ClN 9} O ₅

Calcd .: C 71.20 H 5.23 N 6.92 found: C 71.31 H 5.44 N 6.68

The solid gives a fluorescent spot in thin layer chromatography in the solvent system chloroform / methanol / water / ammonia 25 percent. (70/30/5/1; v / v) with an Rf value of 0.89 and two spots in the solvent system toluene / dichloromethane / THF (5/1/1; v / v) NH ₃ atmosphere with the Rf Values of 0.16 and 0.28.

b) Separation of the mixture of diastereomers by preparative HPLC. The mixture of distereomers (RS) - (+/-) - benoxaprofen- (R) - (+) - 0C Methylbenzylamide is converted into the two diastereomers (S) - (+) - BOP- (R) - (+) OU-MBA and (R) - (-) - B0P- (R) - (+) OC-MBA separated.

Chromatographic conditions:
Injection volume: 2 ml
Column: prep. Column from Dupont
Length: 25 cm, inner diameter 21.2 mm Filling material: Zorbax-Sil

Mobile phase: CyclohexanZDichloromethanZTHF (10/10/1; v / v) Temperature: 25 ⁰ C

Flow rate: 15 ml (corresponding to a pressure of 5.0 MPa) Detection: UV absorption at 309 mn Under these conditions, (S) - (-) - BOP - (+) OC-MBA has a Retention time of 26 minutes and (R) - (+) - BOP - (+) Ot-MBA of 38 minutes.

After the two distereomers have been collected separately, the solvent is evaporated off and the substances over P ₉ O ₁ . dried. If necessary, they are recrystallized from acetone.

c) hydrolysis of the amides

400 mg (R) - (-) - benoxaprofen- (R) - (+) - Ot-methylbenaylamide are dissolved in 40 ml of toluene, mixed with 40 ml of 45 percent sulfuric acid and refluxed for about an hour cooked until the amide is completely in (R) - (-) - benoxaprofen and (R) - (+) 6 · -methylbenzylamine. is split. The toluene phase, which contains only a little benoxaprofen, is separated off and evaporated to dryness; you get a white one, crystalline residue. The (R) - (-) - benoxaprofen is extracted from the aqueous phase with dichloromethane at pH 2-3, evaporated to dryness and combined with the residue from the toluene phase over Na — SO, dried. Likewise, (S) - (+) - Benoxaprofen- (R) - (+) - OC-methylbenzylamide hydrolyzed. Optionally, the two benoxaprofen enantiomers recrystallized from methanol. Optical rotation:

lproc. Solution of (+) (R, S) -BOP in CHCl ₃ ZMeOH (1/1; v / v) M? = 0

lproc. Solution of (-) - (R) -B0P in CHCl ₃ ZMeOH (1/1; v / v) 00 J jj = - Zo

lproc. Solution of (+) - (S) -BOP in CHCLj / MeOH (1/1; v / v) ? - + 27.5 °

η-

d) Activation of the benoxaprofen enantiomers to acid chlorides: The two enantiomers are activated to the acid chloride as described under 1 a.

Determination of the optical purity

In order to determine the optical purity of the benoxaprofen enantiomers, the optical purity of the Be known amines with which benoxaprofen is reacted. The optical purity of these amines is determined by gas chromatography using activated OC-methoxy-00-trifluoro-methy! Phenylacetic acid, a reagent for determining the optical purity of alcohols and amines (10. J. A. Dale, D. L. Dull, H. S. Mosher, J. of Org. Chem., 34_, 2543, 1969).

0> 1 g (+) Oi-methoxy-OC-trifluoromethylphenylacetic acid and 1 ml thionyl chloride were refluxed for 40 h and then evaporated to dryness at 50 ° with dried nitrogen. The residue is taken up in 10 ml of toluene. Approx. 10 mg of amine in each case are mixed with 1 ml of this solution and ^{heated at 80 °} C. for one hour. The amide formed is immediately determined by gas chromatography. Chromatographic conditions:
Injection volume: 1 μΐ
Column: OV 225 glass capillary, 25 m
Carrier gas: H ₂ ,
Temperature: injection block 230 °

Oven 210 °

Detector 250 °

Detection: flame ionization detector Under these conditions the optical purity was from (S) - (+) - Amphetamine: 95.27%

OS) - (-) - Tranylcypromine: 98.06%

CR) - (+) - Tranylcypromine: 97.81%

(S) - (-) 0C-methylbenzylamine: 97.12%

(R) - (+) 0C-methylbenzylamine: 96.99%

Subsequently, all amines with (S) - (+) - Benoxaprofen-

acid chloride reacted and the corresponding amides determined quantitatively by HPLC. This resulted in an optical one Purity for (S) - (+) - benoxaprofen of 95.34% and for (R) - (-) - benoxaprofen of 97.31%.

5. Quantitative recording of various amines in biological

material

Extraction and derivatization

In a sealable centrifuge tube, 1 ml of plasma is made alkaline with 1 ml of 0.1 N NaOH and then with 3 ml for 20 minutes η-hexane shaken by machine- After centrifuging for 10 min, 2 ml of the org. Phase into a second centrifuge tube transferred and evaporated to dryness in the vacuum centrifuge. 0 ~ 5 ml of reagent solution (1%) are added to the residue. Solution of (S) - (+) - benoxaprofenic acid chloride in dry dichloromethane) and stand for 30 min at room temperature calmly.

Chromatographic Conditions

TLC: The solutions are applied to silica gel 60 TLC plates without a fluorescent indicator using the Linomat III. Application volume: 10 μΐ
Band width: 5 mm
Solvent systems: 1. Toluene / dichloromethane / THF

(5/1/1; v / v) 2. Toluene / CHCl ₃ / THF

(5/4/1; v / v) NH ₃ atmosphere

Detection: Densitometric measurement of the fluorescence intensity with the chromatogram spectrophotometer KM 3

Excitation: line 313 nm of the Hg medium pressure lamp ST 41 with a gap size of 0.1 χ 6 mm

Emission: monochromatic filter M 365, amplification 1–10, high voltage at the photoelectron multiplier 1

HPLC: injection volume: column: length:

Filling material: Mobile phase:

Temperature: flow rate:

10 μΐ analytical column from Dupont 250 mm, internal diameter 4.6 mm Zorbax-Sil cyclohexane / dichloromethane / THF (5/1/1; v / v) 25 ⁰ C 1 ml / min (corresponding to a pressure of 8.5 MPa) detection: fluorometric excitation: at wavelength 313 mn emission: at 390 nm

Results

The maximum of the excitation spectrum for Benoxaprofen at 310 nm, the maximum of the emission spectrum at 365 nm. The spectra recorded during the HPLC separation show an excitation maximum of 312 nm; in the emission spectrum two maxima occur, namely at the wavelengths 350 and 365 nm. The spectra of the column chloride are identical to those identical to benoxaprofen itself. These spectra were measured uncorrected.

Examples of substances (partly pharmaceuticals) that contain benoxaprofenic acid chloride or have been reacted with BOP imidazolide, and the chromatographic behavior of the reaction products are listed in Table 1.

All reaction products show fluorescence properties similar to those of benoxaprofen. In solution d. H. after HPLC separation, they are correct Excitation and emission spectra of all investigated spectra with

-Ib ..

match the Benoxaprof spectra. On the TLC plate there are various but only minor both hypsochromic and determine bathochromic shifts that are not different pH values of the superplasticizers can be traced back.

Because of the strong fluorescence intensity of benoxaprofen it is the detection limit is very low, namely 50-100 pg BOP / spot, corresponding to 1/6 to 1/3 pmol benoxaprofen or reaction product. This makes it possible to quantitatively record substances - even in biological material - in the smallest concentrations not converted or after derivatization with other conversion reagents not at all or only in correspondingly higher concentrations can be determined. The detection limit can even be lowered by immersing it in, for example, paraffin-cyclohexane.

The advantage of this fluorescent marker is also its chemical stability, so that by-products and decomposition products, which could interfere with the fluorescence measurements do not occur. Benoxaprofen activated to acid chloride or imidazolide is characterized by good reactivity; reaction at room temperature is usually possible.

The reaction products are so stable in solutions that they too can be quantified even after days. Chromatography (TLC, HPLC) can also be used in neutral, but also in acidic and basic eluents can be carried out without decomposition occurring.

When developing analytical methods, it is advantageous if Highly pure reference substances (reaction products) are available in sufficient quantities. In general, the Represent reaction products of benoxaprofen with amines and alcohols simply and in sufficient quantities. Amino acids and amino acid-like substances can also be detected in the lowest concentrations.

Even problem substances such as quaternary compounds with a derivatisable one functional group (e.g. choline, scopolamine butyl bromide), can be easily converted into fluorescent and easily detectable Transfer connections.

The results of the investigations carried out make it clear that benoxaprofen enantiomers are suitable for the stereoselective determination optically active compounds are well suited. The method described here for the preparation of an optically active conversion reagent is easy to carry out and leads to good results. The rate of racemization is low.

"η he lie I: Rf values and retention times C'ti of some reaction products for different

chroraatoftraphic conditions.

Rf values on DC plaques

HPLC retention times (min)

• itatioiare phase I. Silica gel 60 III RP-18 Zorbax-Sil , 7
,8th V ibt le phase II : .v I. , 0
, 7 link 0.16
0.28 0.81 , 0
, 5 "-0P-OC -Methylbenzylamine 0.16
0.21 0.45
0.56 0.76 0.31 6th
10 , 5
ς :, 0P-tranylcypromine 0.14
0.21 0.39
0.45 0.81 0.32 9
10 ,8th ~; 0 P-Amphe tamiη 0.27
0.33 0.53
0.59 0.79 η, 29 8th
9 , 9 ^! iOP-metharaphetamine 0.16 0.65 0.79 0.23 10
11 , 3 -OP-B-phenylethylamine 0.18 0.49 0.79 0.33 11 , 3 • IOP-rolylethylamine 0.18 0.49 0.80 0.30 10 , 6 iOP-phenylbutylamine 0.19 0.48 0.77 0.26 11 , 5 BOP benzylarain 0.25 0.44 0.82 0.37 10 , 4 30P-maprotiline 0.08 0.69 0.30 0.13 8th , 9 BOP procaine 0.69 0.14 0.04 8th , 5 «OP-methanol 0.73 0.44 .0.32 11 , 3 BOP ethanol 0.79 0.48 0.29 9 , 7 BOP-propanol-1 0.81 0.51 0.25 8th , 3 : 30P-Bu'canol-1 0.83 0.53 0.22 7th , 3 BOP-3-methylbutanol-l 0.09 0.54 0.68 0.20 6th 3enoxaprofen (BOP) 0.64 0.05 0.85 0.43 4th Benoxaprofenic acid chloride 0.84 4th

(BOP-Cl)

BATH

Mobile phases: I toluene / dichloromethane / tetrahydrofuran (5/1/1; v / v)

II toluene / chloroform / tetrahydrofuran (5/4/1; v / v) NH atmosphere

III chloroform / methanol / tetrahydrofuran / formic acid (110/20/2/5; v / v)

IV methanol / water (9/1; v / v)

GOPY

Claims (19)

Method for the quantitative determination of alcohols and amines Claims / l) Method for the quantitative determination of alcohols or of Amines in the presence of foreign substances through reaction with sensitive derivatization reagents characterized in that the substrates of general formula I R ₁ -Y wherein Y is an OH or a -NR H group, where R is hydrogen or an optionally branched alkyl radical having 1 to 18 carbon atoms, and R is an aliphalic, aromatic or heterocyclic radical or a radical combined from these components which are substituted can, means with a sensitive derivatization reagent of the general formula II, 0
H R-CX II wherein X is a group which reacts as a leaving group with respect to the nucleophilic compounds of the formula I Represents group and R represents a fluorescence-active residue of Formula -CHR. R ₁ . wherein R, is an alkyl radical with 1 to 18 4 5 4 Carbon atoms and R an aromatic or heterocyclic one Remainder with 6 to 12 carbon atoms with at least represents a 6-ring in the association of R, with the proviso that the compound derived from R represents BATH ORIGINAL ■ ι. R H has an oscillator strength f of at least 0.05, to the derivatives of the general formula Hi 0
Ii R-Y-C-R III wherein R and R have the meaning given above with splitting off of the compound IV HX IV wherein X has the meaning indicated above, and the resulting derivatives of the general formula III under Use of the fluorescent marking separates from the foreign matter. 2. The method according to claim 1, characterized in that R a radical in the derivatization reagents of the general formula II the formula which can optionally also be substituted, or a radical which can optionally also be substituted, or a radical III wherein Q is -CH 1 -, "C ^ -," ^ H ^, -C ₅ H ₄ -, -Ο-, -NR ^, -S-, where R 1 is hydrogen or an alkyl or aryl radical with 1 to 12 carbon atoms, or a radical where Z is -0-, -NR- or -S- and P is CH or N, where R _{fi has} the meaning of R 1, which can optionally also be substituted, or a radical where II is a heterocyclic, optionally alkyl-substituted radical and R ₇ and Rg are hydrogen, an alkyl radical having 1 to 4 carbon atoms or an optionally substituted aromatic radical, with the proviso that the linkage of the radicals R _ to R, - via any possible Binding site can take place in the radical R _{5, means.} 3. The method according to claim 2, characterized in that the radicals R and / or R _g stand for a phenyl radical. 4. The method according to claims 2 and 3, characterized in that that the aromatic radicals in the formulas R, - to V
R c are substituted by one or more halogen atoms from the group consisting of fluorine, chlorine or bromine. 5. The method according to claims 1-4, characterized in that the leaky group X in the general formula II a chlorine or bromine group or an OH group (compound of formula II A) in combination with one that activates them Represents reagent A. 6. The method according to claim 5, characterized in that the activating reagent A is a 1,1-carbonyldiimidazole or Represents dicyclohexylcarbodiimide. 7. The method according to claims 1 to 5, characterized in that that as a sensitive derivatization reagent of the general formula II (+) - 2- [2- (4-chlorophenyl) -5-benzoxazolyl-] propionic acid is used. 8. The method according to claims 1-7, characterized in that the compound R ^ -Y of the general formula I. has at least one chiral center. 9. The method according to claims 1 to 8, characterized in that that the separation of the derivatives of the general formula III takes place by means of chromatography. 10. The method according to claims 1 to 8, characterized in that that the alcohols and / or the amines of the formula I are present in the biological material. 11. The method according to claim 10, characterized in that the alcohols and / or the amines of the formula I in body fluids or excreta are present. 12. (+) - 2- [2- (4-Chlorophenyl-5-benzoxazolyl] propionic acid chloride. 13. R - (-) - [2- (4-Chlorophenyl-5-benzoxazolyl] propionic acid chloride. 14. S - (+) - [2- (4-chlorophenyl-5-benzoxazolyl] propionic acid chloride. 15. R - (-) - [2- (4-Chlorophenyl-5-benzoxazolyl] -propionic acid- R - (+) - OC-methylbenzamide . 16. S - (+) - [2- (4-Chlorophenyl-5-benzoxazolyl] propionic acid - R - (+) - OC-methylbenzamide. - * - ■ 332993 ( 17. R - (-) - [2- (4-Chlorophenyl-5-benzoxazolyl] propionic acid. 18. S - (+) - [2- (4-Chlorophenyl-5-benzoxazolyl] propionic acid. 19. The method according to claims 1, 8, 13 and 14, characterized in that that the racemates of the chiral substrates of the general formula I with the R - (-) or the S - (+) - [2- (4-chlorophenyl-5-benzoxazolyl] -propionic acid chloride and the resulting derivatives of the general formula III separates by means of thin-layer or HPLC chromatography, the detection being carried out by the fluorescent marking.