KR20140036849A - Method for detection of drug using liquid-liquid extraction by mixed ethyl acetate and acetonitrile - Google Patents

Abstract

The present invention relates to a method for detecting a drug comprising a liquid-liquid extraction by using an organic solvent mixed solution of ethyl acetate, which is a non-polar organic solvent, and acetonitrile, which is an anodic organic solvent. The method has a higher efficiency of extracting drugs than existing liquid-liquid extraction methods that only use ethyl acetate, thereby enabling a more effective detection of various drugs. [Reference numerals] (AA) (First step) Step of mixing an organic solvent mixed solution of ethyl acetate and acetonitrile in a diluted solution of a drug standard product and performing a liquid-liquid extraction; (BB) (Second step) Step of conducting centrifugation for the liquid-liquid extracted mixed solution in the first step and obtaining a supernatant as an organic solvent layer; (CC) (Third step) Step of drying the supernatant separated in the second step using nitrogen gas and dissolving the dried sample again into a solvent in mobile phase for chromatography; (DD) (Fourth step) Step of quantitatively analyzing the sample dissolved into the solvent in mobile phase of the third step using the chromatography

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting drug including liquid-liquid extraction using mixed liquid of ethyl acetate and acetonitrile,

The present invention relates to a drug detection method or an analytical method including a liquid-liquid extraction method using an organic solvent mixture of ethyl acetate and acetonitrile.

Liquid-liquid extraction is a method of separating component materials in a liquid sample by dissolving them in other types of solvents that are not mixed with the liquid, and is also referred to as solvent extraction. In order to separate the active substance dissolved in the first solvent, a second solvent which is not mixed with the first solvent is added and mixed to extract the effective substance toward the second solvent. . In an ideal situation, the active material is extracted into the second solvent in the mixing process and only the impurities remain in the first solvent. Thereafter, the first solvent is removed from the two-layered solvent layer, and then the active material is separated from the second solvent.

On the other hand, in the conventional liquid-liquid extraction method, since the drug is separated by using a nonpolar organic solvent such as ethyl acetate or n-hexane, the solubility of the polar drug is low to be. In order to extract such a polar drug, it has been mainly extracted by solid-phase extraction. However, since the solid-phase extraction method is more expensive than the liquid-liquid extraction method and consumes a large amount of money, The extraction time is long. Therefore, there is a need for a new extraction method that can extract a wide range of polar drugs while using a liquid-liquid extraction method, and can also increase extraction efficiency.

Accordingly, the inventors of the present invention confirmed that the liquid-liquid extraction method using an organic solvent mixture of ethyl acetate and acetonitrile can more effectively extract various drugs while establishing an effective detection method for the drugs Whereby the present invention can be completed.

On the other hand, a prior art document disclosing a method for extracting a sample or a drug using a liquid-liquid extraction method includes a liquid-liquid extraction method using an additive or a porous film (Korean Patent Publication No. 1989-0001613, Korean Patent Publication No. 1983-0000369 , Korean Patent Publication No. 1993-0001602 and Korean Published Patent Application No. 1993-0003637) have been disclosed. However, the above prior art documents disclose a method for extracting and purifying a drug, including a liquid-liquid extraction method using a mixture of a nonpolar organic solvent and a bipolar organic solvent It can be said that the invention is completely different from the present invention to be analyzed.

Korean Patent Publication No. 1989-0001613 (Separation of Rare Earth Elements by Liquid-Liquid Extraction Using Diluent of Halogenated Diluent or Carboxylic Acid Type, Korean Patent Publication No. 1983-0000369 (Liquid-Liquid Extraction Method, Announcement of Mar. 03, 1983) Korean Patent Publication No. 1993-0001602 (Liquid-Liquid Extraction Method by Microporous Membrane and Its Apparatus, Announced on March 3, 1993) Korean Patent Publication No. 1993-0003637 (Method for recovery of gallium by liquid-liquid extraction, published on May 5, 1993)

It is an object of the present invention to provide a drug detection or analysis method including a liquid-liquid extraction method using an organic solvent mixture of ethyl acetate and acetonitrile.

The present invention relates to a drug detection method including a liquid-liquid extraction method using an organic solvent mixture of ethyl acetate and acetonitrile. Preferably,

(Step 1) Performing liquid-liquid extraction by shaking-mixing an organic solvent mixture of ethyl acetate and acetonitrile with a water dilution of the drug standard;

(Step 2) In the first step, the liquid-liquid extracted mixture is centrifuged to obtain a supernatant which is a nonpolar organic solvent layer;

(Step 3) drying the supernatant separated in the second step using nitrogen gas, and reusing the dried sample as a mobile phase solvent for chromatography; And

(Step 4) Quantitative analysis of the sample re-dissolved in the mobile phase solvent of the third step using chromatography.

In the first step, the drug reference standard includes naproxen, digoxin, furosemide, acyclovir, desipramine, dexamethasone, nadolol, A drug selected from the group consisting of terbutaline and enalapril may be included.

In the first step, the diluted water of the drug standard is diluted with 1: 100-300 (v: v) to 5 to 50 mM of the drug standard in 100% acetonitrile, : < / RTI > v).

In the first step, the water dilution of the drug standard, and the organic solvent mixture of ethyl acetate and acetonitrile may be mixed at 1: 60-100 (v: v).

In the first step, the organic solvent mixture of ethyl acetate and acetonitrile may be mixed with ethyl acetate and acetonitrile in a ratio of 1: 0.1-0.4 (w: w).

Hereinafter, the present invention will be described in detail.

In the first step, the drug standard may be dissolved in dimethyl sulfoxide (DMSO) to a concentration of 1 to 50 mM. Preferably, the analysis efficiency is best when the concentration of the drug standard is 5 to 15 mM.

In the first step, the water dilution of the drug standard, and the organic solvent mixture of ethyl acetate and acetonitrile may be shaken for 5 to 30 minutes, preferably 5 to 15 minutes.

In the second step, the centrifugation can be centrifuged at 10,000 to 15,000 rpm at 0 to 10 ° C for 5 to 30 minutes.

In the third step, the mobile phase solvent for chromatography may be dissolved in the dried sample by adding 2 to 6 times the volume of the water dilution solution of the drug standard.

In the fourth step, the chromatography includes high performance liquid chromatography, medium pressure liquid chromatography, silica gel vacuum liquid chromatography and gas chromatography Chromatography can be selected, and it is preferable to use high performance liquid chromatography. At this time, when high performance liquid chromatography is used, the mobile phase solvent used in the third step is 95: 5 (v / v) of 0.1% triethylamine (TEA) aqueous solution and acetonitrile organic solvent mixture, v) to 40:60 (v / v) may be used. At this time, 0.1% triethylamine aqueous solution enhances the UV detection sensitivity of the drug. In the above, when high performance liquid chromatography is used, the supernatant can be used by centrifuging the sample re-dissolved in the mobile phase solvent between the third step and the fourth step, and the solution can be used at 10,000 to 15,000 rpm, For 5 to 30 minutes. This is because centrifugation is carried out when impurities and residues remain in the sample even after extraction with an organic solvent because column clogging may occur during liquid chromatography, which may cause damage to the apparatus.

In the fourth step, the drug analysis on the above chromatographic results can be confirmed by using an ultraviolet-visible absorption spectrophotometer, a mass spectrometer, a fluorescence spectrometer or the like.

In the meantime, the present invention can confirm presence or concentration of a drug by using blood, plasma, body fluids, etc., containing the drug instead of the drug standard.

The present invention relates to a drug detection method including a liquid-liquid extraction method using an organic solvent mixture of ethyl acetate as a nonpolar organic solvent and acetonitrile as a bipolar organic solvent. This method can perform various drug detection more effectively since the efficiency of drug extraction is higher than that of the conventional liquid-liquid extraction method using only ethyl acetate.

1 is a flowchart showing a drug detection method including a liquid-liquid extraction method using an organic solvent mixture solution of ethyl acetate and acetonitrile of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

< Example  1. Perform drug detection using liquid-liquid extraction>

Example  1-1. Perform liquid-liquid extraction of drug samples

Liquid - liquid extraction of the drug was carried out using an organic solvent mixture of ethyl acetate as a non - polar solvent and acetonitrile as a bipolar solvent at a ratio of 1: 0.25 (v: v). The drugs used in this liquid-liquid extraction method are naproxen, digoxin, furosemide, acyclovir, desipramine, dexamethasone, nadolol, , Terbutaline, Enalapril, which is dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM, is diluted 20 times (v: v) with 100% acetonitrile, To a concentration of 50 [mu] M,

Thereafter, 50 μl of the drug standard diluted in water and 4 ml of a 1: 0.25 (v: v) mixed solution of ethyl acetate and acetonitrile were added, followed by shaking for 10 minutes to perform liquid-liquid extraction Respectively. Thereafter, the mixture was centrifuged at 12,000 rpm and 4 ° C for 10 minutes, and the organic solvent layer supernatant was transferred to another glass test tube using a pasteur pipet.

100% ethyl acetate was used as a control for the liquid-liquid extraction.

Example  1-2. Of the drug sample The organic solvent layer  Drying using nitrogen gas and re-dissolution of the dried material

The organic solvent layer (supernatant) obtained by performing the liquid-liquid extraction of Example 1-1 was separated, and the organic solvent layer was concentrated and dried at 30 DEG C for 90 minutes using a spray-type test tube concentrator connected to nitrogen gas Then, the dried material was redissolved in 200 mu l of mobile phase solvent of high performance liquid chromatography. The composition of the mobile phase solvent for each drug is shown in Table 1 below.

Drug name 0.1% TEA pH 3 (%) ACN (%) Naproxen 40 60 Digoxin 65 35 Furosemide 55 45 Acyclovir 95 5 Desipramine 65 35 Dexamethasone 55 45 Nadolol 80 20 Terbutaline 95 5 Enalapril 95 5

Example  1-3. Performing high performance liquid chromatography

200 占 퐇 of the sample redissolved in the mobile phase solvent obtained in Example 1-2 was centrifuged at 13,000 rpm for 10 minutes, and 100 占 퐇 of the supernatant was taken and detected using a high performance liquid chromatography-ultraviolet spectrophotometer .

The instrument used was a high performance liquid chromatography Waters 2695 (Waters Co., Massachusetts, USA) and a Photodiode Array detector Waters W2996 (Waters Co., Massachusetts, USA). The analytical column used was a Zorbax column (300SB C18, 250 mm x 4.6 mm) from Agilent Co., Palo Alto, USA, having a length of 250 mm, an inner diameter of 4.6 mm and a particle size of 5 μm Respectively.

As the mobile phase solvent of the high performance liquid chromatography, the mobile phase solvent of Table 1 in which the drug sample was re-dissolved was used and the flow rate was set at 1 ml / min.

Thereafter, a sample passed through an analytical column of a high-performance liquid chromatography was detected by applying to an ultraviolet-visible absorptiometer, and the maximum absorption wavelength of each drug with respect to the solvent of the liquid-liquid extraction method of the present invention as compared with the control using only ethyl acetate was The results are shown in Table 2 below.

Drug name Absorption maximum wavelength-lambda max (nm) Naproxen 274 Digoxin 242 Furosemide 276 Acyclovir 254 Desipramine 251 Dexamethasone 242 Nadolol 205 Terbutaline 231 Enalapril 224

< Example  2. Drug analysis>

(n = 3), which is the standard of the extraction rate for each drug (control - sample for standard concentration confirmation of drug concentration when blood sample is used), 100% ethyl acetate The extraction efficiency of the analyzed samples was confirmed by the method using an organic solvent mixture of ethyl acetate and acetonitrile of the present invention and the results were analyzed using Empower Pro (Waters Co., Massachusetts, USA) Respectively. The results of the above steps are shown in Table 3 below.

Drug name Contrast to ethyl acetate
The extraction efficiency (fold) of ethyl acetate and acetonitrile mixture (1: 0.25 [v: v]) Naproxen 1.38 Digoxin 1.28 Furosemide 1.39 Acyclovir 1.30 Desipramine 1.47 Dexamethasone 1.49 Nadolol 1.32 Terbutaline 1.36 Enalapril 13.09

As shown in Table 3, when the liquid-liquid extraction was performed by mixing acetonitrile with ethyl acetate at a ratio of 1: 0.25 (v: v) under the conditions of the present invention, It can be seen that the extraction efficiency of the drug is remarkably increased, and it can be confirmed that the present invention is a more suitable method for extracting the above drugs.

Claims (5)

(First step) performing a liquid-liquid extraction by shaking-mixing an organic solvent mixture of ethyl acetate and acetonitrile in a water diluent of the drug standard product;
(Step 2) In the first step, the liquid-liquid extracted mixture is centrifuged to obtain a supernatant which is a nonpolar organic solvent layer;
(Step 3) drying the supernatant separated in the second step using nitrogen gas, and reusing the dried sample as a mobile phase solvent for chromatography; And
(Step 4) quantitatively analyzing the redissolved sample by the mobile phase solvent of the third step using chromatography;
&Lt; / RTI &gt; The method of claim 1,
In the first step, the drug standard, naproxen, Digoxin, Purosemide, Acyclovir, Desipramine, Dexamethasone, Naxalol , Terbutaline (Terbutaline) and enalapril (Enalapril) drug detection method comprising a drug selected from the group consisting of. The method of claim 1,
In the first step, the diluted water of the drug standard is diluted with 1: 100-300 (v: v) to 1 to 50 mM of the drug standard in 100% acetonitrile, : v). &lt; / RTI &gt; The method of claim 1,
In the first step, the water dilution of the drug standard and the organic solvent mixture of ethyl acetate and acetonitrile are mixed in a ratio of 1:60 to 100 (v: v). The method of claim 1,
In the first step, the organic solvent mixture of ethyl acetate and acetonitrile is mixed with ethyl acetate and acetonitrile in a ratio of 1: 0.1 to 0.4 (v: v).

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