TWI630387B - METHOD FOR DETECTING A QUATERNARY AMMONIUM COMPOUND HAVING A γ-ACID GROUP, DIAGNOSIS OF CARNITINE DEFICIENCY AND KIT FOR DETECTING THE QUATERNARY AMMONIUM COMPOUND HAVING THE γ-ACID GROUP - Google Patents

Abstract

The present invention provides a method for detecting a quaternary ammonium compound having a γ-carboxylic acid group, which can provide a γ-carboxylic acid quaternary ammonium compound with a fluorophore; a method for diagnosing carnitine deficiency, utilizing The aforementioned method for detecting a quaternary ammonium compound having a γ-carboxylic acid group for detecting a carnitine content in a sample taken from a suspected patient to evaluate whether the suspected patient is suffering from carnitine deficiency; and a method for detecting A kit of quaternary ammonium compounds of a γ-carboxylic acid group capable of detecting a concentration of a quaternary ammonium compound having a γ-carboxylic acid group in a sample to be tested.

Description

 Method for detecting quaternary ammonium compound having γ-carboxylic acid group, method for diagnosing carnitine deficiency, and kit for detecting quaternary ammonium compound having γ-carboxylic acid group  

The present invention relates to a detection method, a diagnostic method and a kit using the same, and particularly to a method for detecting a quaternary ammonium compound having a γ-carboxylic acid group, a method for diagnosing carnitine deficiency, and a method for detecting A kit of quaternary ammonium compounds having a gamma-carboxylic acid group.

A quaternary ammonium compound having a γ-carboxylic acid group (for example, L-carnitine, decylcarnitine, acetaminophen, etc.) is a molecule which is important for the human body, but such a compound has a small molecular weight. Further, since the molecular structure has both a carboxylic acid group and a quaternary ammonium group, the compound has high polarity and is difficult to extract, and the quaternary ammonium compound having a γ-carboxylic acid group lacks a fluorophore. The luminescent group thus makes the quaternary ammonium compound having a γ-carboxylic acid group unsuitable for use in conventional detection methods.

In order to solve the above problems, the present invention provides a method for detecting a quaternary ammonium compound having a γ-carboxylic acid group, which can have a γ-carboxylic acid group quaternary ammonium compound with a fluorophore, and is suitable for fluorescence. The test method is performed by the tester.

The present invention further provides a method for diagnosing carnitine deficiency by detecting a sample taken from a suspected patient by using the aforementioned detection method of a quaternary ammonium compound having a γ-carboxylic acid group. Carnitine content to assess whether the suspected patient is suffering from carnitine deficiency.

The present invention further provides a kit for detecting a quaternary ammonium compound having a γ-carboxylic acid group, which is capable of detecting whether a quaternary ammonium compound having a γ-carboxylic acid group is contained in a sample to be tested.

The method for detecting a quaternary ammonium compound having a γ-carboxylic acid group of the present invention comprises: providing a sample comprising a quaternary ammonium compound having a γ-carboxylic acid group; mixing the sample and having a fluorescent group a fluorescent compound and a polar aprotic solvent to form a reaction solution; the reaction solution is placed at 80 to 120 ° C for 1 to 15 minutes to make the fluorescent compound of the fluorescent compound and the γ-carboxylic acid group The carboxylic acid group of the quaternary ammonium compound is subjected to an S _N 2 nucleophilic substitution reaction to obtain a derivatized solution comprising a derivative which is a fluorophore having a fluorescent compound substituted with the fluorescent compound a quaternary ammonium compound having a γ-carboxylic acid group; adding an extracting agent and a cosolvent to the derivatizing solution, the polarity of the extracting agent is higher than the polarity of the acetonitrile, so that the extracting agent and the cosolvent form an oil together a water-in-water emulsion dispersed in the derivatization solution to extract a derivative in the derivatization solution and obtain a solution to be tested; and detecting a derivative in the solution to be tested, wherein the fluorescent substance is fluorescent The fluorescent compound of the group is 4-bromomethylbiphenyl. The polar aprotic solvent is acetonitrile, the extracting agent is an aqueous solution of ammonium acetate, and the cosolvent is toluene; thus, by adding the fluorescent compound (4-bromomethylbiphenyl) and the polar aprotic solvent (acetonitrile), The quaternary ammonium compound having a γ-carboxylic acid group and the fluorescent compound (4-bromomethylbiphenyl) may be subjected to the S _N 2 nucleophilic substitution reaction, and the γ-carboxylic acid group may be subjected to four stages. The ammonium compound carries the fluorophore, and the quaternary ammonium compound having a γ-carboxylic acid group can be applied not only to the fluorescence detection method but also to reducing the polarity of the quaternary ammonium compound having a γ-carboxylic acid group. Conducive to the subsequent separation and analysis.

The method for detecting a quaternary ammonium compound having a γ-carboxylic acid group according to the present invention, wherein the detection method further comprises: adding an alkali agent to the reaction solution, and continuing to cause the reaction solution in which the alkali agent is dissolved to perform the S _N 2 nucleophilic substitution reaction to obtain the derivatization solution; thus, the time required to shorten the S _N 2 nucleophilic substitution reaction can be achieved.

The method for detecting a quaternary ammonium compound having a γ-carboxylic acid group according to the present invention, wherein the detecting method further comprises: selecting potassium hydroxide, potassium carbonate or potassium hydrogencarbonate as the alkali agent; thus nucleophilic the S _N 2 The substitution reaction provides a suitable reaction environment.

The method for detecting a quaternary ammonium compound having a γ-carboxylic acid group according to the present invention, wherein the γ-carboxylic acid group in the solution to be tested is detected by reverse phase chromatography, fluorescence spectroscopy or mass spectrometry A derivative of the quaternary ammonium compound; thus, the quaternary amine compound having a γ-carboxylic acid group can be easily detected.

The method for diagnosing carnitine deficiency of the present invention comprises: obtaining a sample from a suspected patient; detecting the carnitine in the sample in vitro by the above-mentioned method for detecting a quaternary ammonium compound having a γ-carboxylic acid group; a content of the compound to obtain a detection value; and comparing the detected value of the sample with a reference value; wherein the detected value is lower than the reference value to indicate that the suspected patient suffers from carnitine deficiency; - Detection method of a carboxylic acid-based quaternary ammonium compound The sample obtained from the suspected patient can effectively analyze the carnitine content in the sample to effectively diagnose whether the suspected patient suffers from carnitine deficiency.

The method for diagnosing carnitine deficiency according to the present invention, wherein the content of L-carnitine in the sample is detected to obtain the detected value, and the L-carnitine in the sample taken from a healthy individual is detected. The content is obtained to obtain the reference value.

The method for diagnosing carnitine deficiency according to the present invention, wherein the content of L-carnitine and the content of acetylcarnitine in the sample are detected, and the L-carnitine content/acetylcholine content in the sample is calculated. Ratio to obtain the detected value, and detect the content of L-carnitine and the content of acetaminophen in a sample taken from a healthy individual, and calculate the L-carnitine content in the sample of the healthy individual. / ratio of acetaminophen content to obtain the reference value.

The kit for detecting a quaternary ammonium compound having a γ-carboxylic acid group of the present invention comprises: a fluorescent compound having a fluorescent group, and a fluorescent group of the fluorescent compound is used for the γ-carboxy group The carboxylic acid group of the acid-based quaternary ammonium compound undergoes a S _N 2 nucleophilic substitution reaction to form a derivative; a polar aprotic solvent for accelerating the S _N 2 nucleophilic substitution reaction; and an extracting agent for Extracting the derivative, the extracting agent is more polar than the polar aprotic solvent; and a cosolvent for forming a water-in-oil emulsion together with the extracting agent; wherein the fluorescent compound having a fluorescent group Is 4-bromomethylbiphenyl, the polar aprotic solvent is acetonitrile, the extracting agent is an aqueous solution of ammonium acetate, the cosolvent is toluene; thus, it is indeed capable of making a sample containing a quaternary ammonium compound having a γ-carboxylic acid group The S _N 2 nucleophilic substitution reaction is carried out to form the derivative, which is further analyzed by reverse phase chromatography, fluorescence spectroscopy or mass spectrometry.

The kit for detecting a quaternary ammonium compound having a γ-carboxylic acid group, wherein the kit further comprises: an alkali agent which is a basic compound soluble in the polar aprotic solvent, And used to provide an alkaline environment for carrying out the S _N 2 nucleophilic substitution reaction; thus, the time required for the S _N 2 nucleophilic substitution reaction can be shortened.

The kit for detecting a quaternary ammonium compound having a γ-carboxylic acid group, wherein the alkali agent is potassium hydroxide, potassium carbonate or potassium hydrogencarbonate; thus, the S _N 2 nucleophilic substitution reaction can be provided A suitable reaction environment.

Of FIG. 1a: Department of 4-bromomethyl-biphenyl-carnitine for the S _N 2 nucleophilic substitution reaction of the reaction formula.

Of FIG. 1b: 4-bromomethyl-biphenyl-based and acetylcarnitine for S _N 2 nucleophilic substitution reaction of the reaction formula.

Figure 2: The peak area ratio bar graph of the A11~A16 and A21~A26 groups.

Figure 3: Peak area ratio bar graph of groups B11~B16 and B21~B26.

Figure 4: Peak area ratio bar graph of the C10~C13 and C20~C23 groups.

Figure 5: Peak area ratio bar graph of heating in Groups C11 and C21 at 80 °C.

Figure 6: Peak area ratio bar graph of heating in Groups C11 and C21 at 90 °C.

Figure 7: Peak area ratio bar graph of heating in Groups C11 and C21 at 100 °C.

Figure 8: Peak area ratio bar graph of the E11~E16 and E21~E26 groups.

Figure 9: The peak area ratio bar graph of the F11~F16 and F21~F26 groups.

Figure 10: The peak area ratio bar graph of the G10~G14 and G20~G24 groups.

Figure 11: Peak area ratio bar graph of the H10~H14 and H20~H24 groups.

The above and other objects, features and advantages of the present invention will become more <RTIgt; - a quaternary ammonium compound of a carboxylic acid group" means a quaternary ammonium compound having a carbon chain having a carboxylic acid group (i.e., -C(=O)OH) at a γ carbon position, such as a butyl group. Butyrobetaine, L-carnitine, acyl-L-carnitine species, carnitine tartrate, carnitine fumarate or carnitine citrate (Carnitine citrate), etc., wherein mercaptocarnitine includes, but is not limited to, acetyl-L-carnitine, propionyl-L-carnitine, butyryl-L-carnitine , isovaleryl-L-carnitine, hexanoyl-L-carnitine, octanoyl-L-carnitine, decanoyl-L-carnitine, Lauroyl-L-carnitine, myristoyl-L-carnitine, palmitoyl-L-carnitine, and hard Stearoyl-L-carnitine and the like are understood by those of ordinary skill in the art to which the present invention pertains.

The kit for detecting a quaternary ammonium compound having a γ-carboxylic acid group according to an embodiment of the present invention may comprise a fluorescent compound having a fluorophore and a polar aprotic solvent ( Polar aprotic solvent), the kit can be used to detect whether a sample contains a quaternary ammonium compound having a γ-carboxylic acid group.

The sample can be selected from a pharmaceutical sample, a cosmetic sample, a food sample, or a biological sample from a mammalian organism. In detail, before the sample is detected by the kit, appropriate pre-treatment, such as centrifugation or drying, can be performed according to the type of the sample, which is the present invention. It is generally understood by those skilled in the art.

More specifically, the sample, the fluorescent compound and the polar aprotic solvent may be mixed to form a reaction solution to fluoresce the fluorescent compound with the quaternary ammonium compound having a γ-carboxylic acid group. a carboxylic acid group for S _N 2 nucleophilic substitution reaction (S _N 2 nucleophilic substitution reaction) to form a derivative, the derivative having a carboxylic acid group substituted with a carboxylic γ- fluorophore of the fluorescent compound The acid-based quaternary ammonium compound (for ease of explanation, the reaction solution containing the derivative is referred to as a "derived solution"). Thus, the derivatized solution can be used as a solution to be tested, and the derivative in the solution to be tested can be detected to know whether or not the quaternary ammonium compound having a γ-carboxylic acid group is contained in the sample.

The fluorescence of the fluorophore and the compound comprises a leaving group (leaving group), the S _N 2 nucleophilic substitution reaction, the carboxylic acid groups of the quaternary ammonium compound having the carboxylic acid groups will γ- or neutral Performing a deprotonation reaction in an alkaline environment, attacking the fluorescent compound, and detaching the leaving group from the fluorescent compound, such that the quaternary ammonium compound having a γ-carboxylic acid group and the fluorescent compound The fluorescent group may form the derivative together; for example, the fluorescent compound may be 4-bromomethylbiphenyl, 4-bromoethylbiphenyl, 4 -4-bromomethyl-2'-cyanobiphenyl, (2-biphenyl)diazomethane, 4-aminobiphenyl , biphenyl-4-yl-hydrazine hydrochloride, 4-phenylphenol or 4-biphenyl trifluoromethanesulfonate, This should not be limited to this. In the case where 4-bromomethylbiphenyl is used as the fluorescent compound and carnitine is used as the quaternary ammonium compound having a γ-carboxylic acid group, the reaction formula of the above S _N 2 nucleophilic substitution reaction is as shown in FIG. 1a. Or as shown in the case of 4-bromomethylbiphenyl as the fluorescent compound and acetaminophen as the quaternary ammonium compound having a γ-carboxylic acid group, the above S _N 2 nucleophilic substitution reaction The reaction formula is shown in Figure 1b.

The polar aprotic solvent is used to accelerate the S _N 2 nucleophilic substitution reaction; for example, the polar aprotic solvent can be acetonitrile.

It should be noted that, in forming the reaction solution, the worker may simultaneously mix the sample, the fluorescent compound and the polar aprotic solvent; or the worker may separately dissolve the sample and the fluorescent compound separately. Among the polar aprotic solvents, a fluorescent compound solution and a sample solution are formed to facilitate calculation of the solution concentration and the added amount. For example, mixing the sample with the polar aprotic solvent to form 2 μM of the sample solution, mixing the fluorescent compound and the polar aprotic solvent to form 2 to 35 mM of the fluorescent compound solution, and continuing to mix 20 μl of the sample solution. 5 μl of this fluorescent compound solution was formed to form the reaction solution.

Further, the kit may further comprise a base which promotes deprotonation of the carboxylic acid group of the quaternary ammonium compound having a γ-carboxylic acid group and further promotes the S _N 2 pro The nuclear substitution reaction proceeds. The alkaline agent may be a basic compound soluble in the polar aprotic solvent, such as potassium hydroxide, potassium carbonate or potassium hydrogencarbonate, preferably potassium hydroxide is selected as the alkali agent; further, the alkali agent can The polar aprotic solvent is prepared as a saturated alkaline agent solution to provide a suitable base for optimum reaction efficiency.

Moreover, the worker may directly mix the alkali agent with the sample, the fluorescent compound and the polar aprotic solvent; or, as described above, the worker may first dissolve the alkaline agent in an appropriate amount of the polar aprotic solvent. Forming an alkali solution to continue mixing with the sample solution and the fluorescent compound solution. For example, mixing the sample with the polar aprotic solvent to form 2 μM of the sample solution, mixing the fluorescent compound and the polar aprotic solvent to form 2 to 35 mM of the fluorescent compound solution, mixing the alkali agent and the polarity The protic solvent was used to form the saturated alkaline agent solution, and 20 μl of the sample solution, 5 μl of the fluorescent compound solution and 2 μl of the saturated alkaline agent solution were continuously mixed to form the reaction solution.

When the S _N 2 nucleophilic substitution reaction is carried out, the reaction can be accelerated by additionally supplying heat. In the present embodiment, the reaction solution is placed at 80 to 120 ° C for 1 to 15 minutes to obtain the derivatized solution. For example, the above S _N 2 nucleophilic substitution reaction can be rapidly completed by heating at 80 ° C for 9 minutes or at 90 to 100 ° C for 7 minutes. Moreover, the worker can adjust the heating time according to the heating condition of the container used to avoid evaporation of the polar aprotic solvent.

The derivative solution obtained by the above S _N 2 nucleophilic substitution reaction can be used as the solution to be tested, which can be detected by reverse phase chromatography, fluorescence spectroscopy or mass spectrometry. For example, the reverse phase chromatography can be performed by high pressure liquid chromatography such as narrow-bore liquid chromatography or nano liquid chromatography. The system is going to work. In detail, when the reverse phase chromatography is used, the solution to be tested may be appropriately diluted, or the polarity may be adjusted, and the internal standard may be added, and then the narrow tubular reverse phase chromatograph or the nanometer may be added. In liquid chromatography. In the present embodiment, the solution to be tested is added to methanol, and 9-aminoacridine (as an internal standard) is added, and an appropriate amount is injected into the narrow-tube liquid chromatograph for analysis. Further, in order to match the polarity of the quaternary ammonium compound having a γ-carboxylic acid group, a gradient aqueous solution of an aqueous solution of formic acid (containing 0.2% of formic acid) in combination with methanol was used.

The mass spectrometry method can be performed with a mass-to-charge ratio detector with low detection limit and high sensitivity; or, since the quaternary ammonium compound having a γ-carboxylic acid group has been nucleophilic substituted by the S _N 2 The reaction has a fluorophore, so the fluorescence spectroscopy can be used, that is, it is detected by a fluorescent detector. When the fluorescence spectrum method is used, the quaternary ammonium compound having a γ-carboxylic acid group has a maximum excitation wavelength of 255 nm and a maximum emission wavelength of 317 nm.

In addition, the kit may further comprise an extractant having a polarity higher than that of the polar aprotic solvent, which may be used to extract the derivative in the derivatized solution, and the extracted extract is also As the solution to be tested, and selecting the reverse phase chromatography, the fluorescence spectroscopy or the mass spectrometry to detect the derivative of the quaternary ammonium compound having a γ-carboxylic acid group in the solution to be tested. .

The extracting agent can select a deionized water or a salt aqueous solution, the salt water The solution may be selected from an aqueous solution of ammonium acetate, an aqueous solution of ammonium monochloride, an aqueous solution of sodium monochloride or an aqueous solution of ammonium hydrogencarbonate. When the extracting agent is an aqueous solution of the salt, the extraction of the derivative can be assisted by the ionic property of the salt after dissociation. The concentration of the aqueous salt solution is preferably 1M. Further, when the sample is a plasma sample, the extractant is preferably a 1 M aqueous solution of ammonium acetate.

Moreover, the kit may further comprise a co-solvent which is an organic solvent having a low density and being immiscible with the extracting agent, so after mixing the derivatizing solution, the extracting agent and the cosolvent, The derivatization solution, the extracting agent and the co-solvent form a cloud state by shaking, in detail, the extracting agent and the co-solvent together form a water-in-oil emulsion and micro droplets A form of a droplet is dispersed in the derivatizing solution to increase a contact area of the extracting agent with the derivatizing solution, whereby extraction efficiency of extracting the derivative in the derivatizing solution by the extracting agent can be improved. For example, the cosolvent can be selected to be toluene.

Further, after the extraction, the extract may be obtained by separation; for example, a mixed solution containing the derivatized solution, the extractant, and the co-solvent may be layered by centrifugation, and the extracted extract may be distributed in the lower layer.

In addition, in the present embodiment, when the derivatization solution (containing acetonitrile as the polar aprotic solvent), the extracting agent and the cosolvent are mixed, the acetonitrile system can help the extracting agent to be dispersed in the derivatizing solution, thereby Further improve the extraction efficiency.

Since the kit is capable of subjecting a sample containing a quaternary ammonium compound having a γ-carboxylic acid group to the S _N 2 nucleophilic substitution reaction to form the derivative, the worker can further utilize the reverse phase chromatography, the firefly. Optical spectrometry or mass spectrometry analysis, therefore, the kit can be applied to a method for detecting a quaternary ammonium compound having a γ-carboxylic acid group. In detail, the detection method comprises providing the sample; mixing the sample, The fluorescent compound and the polar aprotic solvent are formed to form the reaction solution; the reaction solution is placed at 80 to 120 ° C for 1 to 15 minutes to make the fluorescent group of the fluorescent compound and the γ-carboxylic acid group The carboxylic acid group of the quaternary ammonium compound is subjected to the S _N 2 nucleophilic substitution reaction to obtain the derivatized solution, the derivatized solution comprising the derivative; and the derivatized solution is used as the test solution to detect the solution to be tested Derivatives in.

Furthermore, in the method for detecting a quaternary ammonium compound having a γ-carboxylic acid group according to an embodiment of the present invention, the extracting agent may be added to the derivatizing solution to extract a derivative in the derivatizing solution by using the extracting agent. The solution to be tested is obtained.

It is worth noting that the kit can be used to detect biological samples from the mammalian organism, and the kit and the detection method can be further applied to detect the content of carnitine compounds in a suspected patient, thereby evaluating the Suspected patients suffering from carnitine deficiency. In detail, a sample is first obtained from the suspected patient, and a sample of a healthy individual is obtained from a healthy individual, the two samples being the same as a whole blood sample, a serum sample, a plasma sample or a urine sample. And detecting, in vitro , the content of the carnitine compound in the two samples by the above detection method of the quaternary ammonium compound having a γ-carboxylic acid group, to obtain a detection value and a reference value, and comparing The detected value and the reference value, if the detected value is lower than the reference value, indicates that the suspected patient suffers from carnitine deficiency.

For example, Inoue F et al. and Peng M et al. published a plasma L-carnitine content of about 17.51 to 66.14 μM in healthy adults (J Chromatogr B Biomed Sci Appl. 1999 Aug 6; 731(1): 83- 8.; J Chromatogr A.2013 Dec 6; 1319:97-106.), so the aforementioned detection value may be the L-carnitine content value of the suspected patient, and the reference value may be the L- of the healthy individual. The carnitine content value, and when the detection value is the L-carnitine content value in the plasma sample of the suspected patient, the reference value may be 66.14 μM; in addition, Hothi DK et al. and Flanagen JL et al. Taking the ratio of acetaminophen/L-carnitine in the plasma sample as a health indicator, when the ratio of acetaminophen/L-carnitine is higher than 0.25, the individual is suffering from carnitine deficiency (Nephrol Dial Transplant). .2006 Sep;21(9):2637-2641.), so the aforementioned detection value may also be the ratio of the L-carnitine content/acetylcholine content in the plasma sample of the suspected patient, and the reference value is The ratio of L-carnitine content to acetylcholine content in the plasma sample of the healthy individual, and when the detection value is the blood of the suspected patient L-carnitine content in pulp samples / acetyl carnitine The ratio of the quantity can be 0.25.

To confirm that the present invention can surely bond the carboxylic acid group of the quaternary ammonium compound having a γ-carboxylic acid group to the fluorescent group of the fluorescent compound, and extract the quaternary amine compound having a γ-carboxylic acid group, and The quaternary ammonium compound having a γ-carboxylic acid group was analyzed, and the following experiment was carried out:

(A) Effect of Fluorescent Compound Concentration on S _N 2 Nucleophilic Substitution Reaction

In this experiment, carnitine (Groups A11 to A16) and acetaminocarnitine (Groups A21 to A26) were used as the quaternary ammonium compound with γ-carboxylic acid group, and 4-bromomethylbiphenyl was used as the firefly. A light compound to prepare the reaction solution. Among them, the concentrations of carnitine, acetaminophen and 4-bromomethylbiphenyl in each group are shown in Table 1 below.

The above A11~A16 and A21~A26 groups were subjected to S _N 2 nucleophilic substitution reaction, and then toluene and deionized water were continuously added for extraction, and 9-aminoacridine was added as an internal standard in the extracted aqueous phase. Reverse phase chromatography was performed with a narrow tube liquid chromatograph and a fluorescence detector. The peak area ratio of the peak area of each group relative to the peak area of the standard was recorded as shown in Fig. 2. Show.

As can be seen from Fig. 2, each group was able to carry out the S _N 2 nucleophilic substitution reaction and was able to detect it by the reverse phase chromatography and the fluorescence spectrometry. In addition, in the groups A11 to A14 and A21 to A24, as the concentration of 4-bromomethylbiphenyl increases, the efficiency of the reaction also increases. When the concentration of 4-bromomethylbiphenyl reached 3 mM (group A14, group A24), the efficiency of the reaction could not be increased. Therefore, it is preferred that the reaction solution of the present embodiment contains 3 mM of the fluorescent compound.

(B) Effect of the amount of fluorescent compound added on the nucleophilic substitution reaction of S _N 2

In this experiment, carnitine (groups B11 to B16) and acetaminocarnitine (groups B21 to B26) were used as the quaternary ammonium compound with γ-carboxylic acid group, and dissolved in acetonitrile at a concentration of 2 μM in advance. Formulated as an acetonitrile solution containing carnitine and acetaminophen. Further, 4-bromomethylbiphenyl was used as the fluorescent compound, and dissolved in acetonitrile at a concentration of 15 mM to prepare a solution of 4-bromomethylbiphenyl in acetonitrile. The reaction of the B11~B16 group and the B21~B26 group was prepared by adding the amount of carnitine, acetaminophen acetonitrile solution and 4-bromomethylbiphenyl acetonitrile solution as shown in the second table below. Solution.

After the above-mentioned B11~B16 and B21~B26 groups were subjected to the S _N 2 nucleophilic substitution reaction, the toluene and deionized water were continuously added for extraction, and 9-aminoacridine was added as an internal standard in the extracted aqueous phase. Reverse phase chromatography was carried out with a narrow tube liquid chromatograph and a fluorescence detector, and the peak area ratio of the peak area of each group relative to the peak area of the standard was recorded as shown in Fig. 3.

As can be seen from Fig. 3, each group was able to carry out the S _N 2 nucleophilic substitution reaction and was able to detect it by the reverse phase chromatography and the fluorescence spectrometry. Among them, 5 μl of the above 4-bromomethylbiphenyl-containing acetonitrile solution (Groups B12 and B22) was added to provide optimum reaction efficiency. It is speculated that since 5 μl of the above 4-bromomethylbiphenyl-containing acetonitrile solution has provided a sufficient amount of the reaction reagent, the volume of the overall reaction system increases as the volume of the 4-bromomethylbiphenyl-containing acetonitrile solution increases. As a result, the collision efficiency is lowered, and the efficiency of the reaction is lowered.

(C) Effect of the type of alkali agent on the efficiency of nucleophilic substitution reaction of S _N 2

Taking the carnitine-containing acetonitrile solution, the acetonitrile-containing acetonitrile solution and the 4-bromomethylbiphenyl-containing acetonitrile solution in the above (B) experiment, and further preparing the alkaline agent as shown in the following Table 3 A saturated acetonitrile solution of an alkaline agent. 20 μl of the above carnitine-containing acetonitrile solution is mixed with 5 μl of the above 4-bromomethylbiphenyl-containing acetonitrile solution, and 2 μl of the acetonitrile solution containing each alkali agent is added as the C10-C13 group; and 20 μl of the above-mentioned acetaminophen-containing carnitine is taken. The acetonitrile solution was mixed with 5 μl of the above-mentioned acetonitrile solution containing 4-bromomethylbiphenyl, and 2 μl of an acetonitrile solution containing each alkali agent was added as the C20-C23 group, and the alkali agent type of each group is shown in Table 3 below.

After the nucleophilic substitution reaction of S _N 2 in the above C10~C13 and C20~C23 groups, the toluene and deionized water are continuously added for extraction, and 9-aminoacridine is added as an internal standard in the extracted aqueous phase. Reverse phase chromatography was carried out with a narrow tube liquid chromatograph and a fluorescence detector, and the peak area ratio of the peak area of each group relative to the peak area of the standard was recorded as shown in Fig. 4.

As can be seen from Fig. 4, each group was able to carry out the S _N 2 nucleophilic substitution reaction and was able to detect it by the reverse phase chromatography and the fluorescence spectrometry. Among them, the reaction efficiency of Groups C11 and C21 is the best, indicating that the addition of potassium hydroxide as the alkali agent can provide an appropriate alkaline environment to assist the nucleophilic substitution reaction of S _N 2 to provide the best reaction. effectiveness.

(D) Effect of heating temperature and time on the efficiency of nucleophilic substitution reaction of S _N 2

Taking the above-mentioned mixed carnitine-containing acetonitrile solution, 4-bromomethylbiphenyl-containing acetonitrile solution, and potassium hydroxide-containing saturated acetonitrile solution, group C11 (hereinafter referred to as group C11), and mixing acetaminophen-containing The acetonitrile solution, the acetonitrile solution containing 4-bromomethylbiphenyl and the C21 group (hereinafter referred to as the C21 group) containing the saturated acetonitrile solution of potassium hydroxide are respectively reacted at different heating temperatures and times, and then toluene is continuously added. Deionized water was used for extraction, and 9-aminoacridine was added as an internal standard in the extracted aqueous phase, and reverse phase chromatography was performed with a narrow tube liquid chromatograph and a fluorescent detector, and the peaks of each group were obtained. The area ratio of the area calculated from the peak area of the standard is recorded as shown in Figures 5-7: heating at 80 °C for 3, 5, 7, 9, 11, and 13 minutes, recorded as Figure 5; The mixture was heated at ° C for 3, 5, 7, 9, 11, and 13 minutes and recorded as Fig. 6; and heated at 100 ° C for 3, 5, 7, 9, 11, and 13 minutes, and recorded as Fig. 7.

It can be seen from Figures 5 to 7 that each group is capable of performing a S _N 2 nucleophilic substitution reaction and is capable of being detected by the reverse phase chromatography and the fluorescence spectrometry. Among them, the optimum reaction efficiency can be achieved by heating at 80 ° C for 9 minutes or more; and the optimum reaction efficiency can be achieved by heating at 90 to 100 ° C for 7 minutes or more.

(E) Effect of cosolvent volume on extraction

The above C11 and C21 groups were taken as the reaction solution for extraction. As described above, the volume of the C11 and C21 groups was 27 μl. Group C11 comprises carnitine with a methylbiphenyl group, and Group C21 comprises acetaminophen with a methylbiphenyl group as a fluorophore on which a fluorochemical group is substituted with the fluorescent compound. The quaternary ammonium compound having a γ-carboxylic acid group. Toluene was used as the co-solvent. The same amount of deionized water and different volumes of toluene were added to the C11 group as the E11~E16 group. In addition, the same amount of deionized water and different volumes of toluene were added to the C21 group as the E21~ In the E26 group, the amounts of deionized water and toluene added to each group are shown in Table 4 below.

After the E11~E16 and E21~E26 groups were shaken and atomized separately, the aqueous phase was extracted by centrifugation and extracted, and 9-aminoacridine was added as an internal standard. The narrow-tube liquid chromatography was combined with fluorescence detection. The reverse phase chromatography was carried out, and the peak area ratio of the peak area of each group with respect to the peak area of the standard was recorded as shown in Fig. 8.

As can be seen from Fig. 8, each group is capable of extraction and can be detected by the reverse phase chromatography and the fluorescence spectrometry. In the groups E11 to E14 and E21 to E24, as the volume of toluene increased, the extraction efficiency was improved because the extractant was well dispersed in the extraction system. However, when the volume of toluene is more than 85 μl, the extraction efficiency is no longer affected, so that it is preferable to mix 5 μl of water and 85 μl of toluene per 27 μl of the reaction solution.

(F) Effect of the amount of extractant added on the extraction

The above C11 and C21 groups were taken as the reaction solution for extraction. As described above, the volume of the C11 and C21 groups was 27 μl. Group C11 comprises carnitine with a methylbiphenyl group, and Group C21 comprises acetaminophen with a methylbiphenyl group as a fluorophore on which a fluorochemical group is substituted with the fluorescent compound. The quaternary ammonium compound having a γ-carboxylic acid group. Toluene was used as the co-solvent, and deionized water was used as the extractant. The same amount of toluene and different volumes of deionized water were added to the C11 group as the F11~F16 group; and the same amount of toluene was added to the C21 group. The volume of deionized water was used as the F21 to F26 group, and the deionized water and toluene addition amounts of each group are shown in Table 5 below.

After the vortexing and atomization of the F11~F16 and F21~F26 groups respectively, the centrifugally extracting and extracting the extracted aqueous phase, adding 9-aminoacridine as the internal standard, and narrow-tube liquid chromatography with fluorescence detection Reverse phase chromatography was performed, and the peak area of each group was calculated relative to the peak area of the standard. The peak area ratio is recorded as shown in Fig. 9.

As can be seen from Fig. 9, each group is capable of extraction and can be detected by the reverse phase chromatography and the fluorescence spectrometry. When the amount of the extracting agent is 5 to 15 μl, atomization can occur; however, the smaller the amount of the extracting agent, the higher the concentration efficiency and the higher the peak area ratio. However, when the amount of the extracting agent added is less than 5 μl, the aqueous phase is difficult to extract, so that 5 μl of the extracting agent and 85 μl of the cosolvent are preferably mixed per 27 μl of the reaction solution.

(G) Effect of the type of the salt aqueous solution on the extraction

The above C11 and C21 groups were taken as the reaction solution for extraction. As described above, the volume of the C11 and C21 groups was 27 μl. Group C11 comprises carnitine with a methylbiphenyl group, and Group C21 comprises acetaminophen with a methylbiphenyl group as a fluorophore on which a fluorochemical group is substituted with the fluorescent compound. The quaternary ammonium compound having a γ-carboxylic acid group. Toluene was used as the co-solvent, and the salt aqueous solution was used as the extractant. In the C11 group, 85 μl of toluene and 1 M salt aqueous solution 5 μl were added as the G10-G14 group; and in the C21 group, 85 μl of toluene and 1 M were added. 5 μl of a salt aqueous solution was used as the G20 to G24 group, and the salt types of each group are shown in Table 6 below. Among them, the G10 and G20 groups were replaced with deionized water (no salt added) to replace the salt aqueous solution.

After the G10~G14 and G20~G24 groups were shaken and atomized separately, the aqueous phase was extracted by centrifugation and extracted, and 9-aminoacridine was added as an internal standard. The narrow-tube liquid chromatography was combined with fluorescence detection. The reverse phase chromatography was carried out, and the peak area ratio of the peak area of each group with respect to the peak area of the standard was recorded as shown in Fig. 10.

As can be seen from Fig. 10, each group is capable of extraction and can be detected by the reverse phase chromatography and the fluorescence spectrometry. Among them, the deionized water (no salt added) of the G10 and G20 groups is the best, and the ammonium acetate aqueous solution of the G11 and G21 groups is the second.

(H) Effect of the type of salt aqueous solution on plasma sample extraction

Take human plasma samples, add protein precipitant to remove protein, dissolve in acetonitrile and add carnitine (H10~H14 group), acetyl carnitine (H20~H24 group) and butyl betaine (H30~) In group H34), an S _N 2 affinity substitution reaction was carried out using 4-bromomethylbiphenyl as the fluorescent compound. The same amount of the plasma sample after the reaction was taken, and an equal amount of toluene was added as the co-solvent, and an equal amount of different kinds of 1 M salt aqueous solution was used as the extracting agent, and the salt types of each group were as shown in the seventh table below. .

After the H10~H14, H20~H24 and H30~H34 groups were shaken and atomized respectively, the aqueous phase was extracted by centrifugation and extracted, and 9-aminoacridine was added as an internal standard, and the narrow-tube liquid chromatography was used. The fluorescence detector was subjected to reverse phase chromatography, and the peak area ratio of the peak area of each group relative to the peak area of the standard was recorded as shown in Fig. 11.

As can be seen from Fig. 11, each group is capable of extraction and can be detected by the reverse phase chromatography and the fluorescence spectrometry. Among them, for the plasma sample, the ammonium acetate aqueous solution of the H11, H21, and H31 groups was the best, and the ammonium chloride aqueous solution of the H12, H22, and H32 groups was next.

(I) Analysis of human plasma samples by mass spectrometry

1 μl of the plasma sample was taken, and pretreatment, S _N 2 affinity substitution reaction and extraction were carried out according to the above, and deionized water was used as the extractant. After the extraction, reverse phase chromatography was continued, and analyzed by mass spectrometry, and the eighth table below was recorded.

As is apparent from the eighth table, the detection method of the quaternary ammonium compound having a γ-carboxylic acid group of the present example can surely use mass spectrometry and simultaneously detect up to 13 carnitine compounds in the human body.

(J) Analysis of commercial tablets

After the carnitine-containing tablet is pulverized into a powder, the powder is dissolved in acetonitrile, and the supernatant is taken after shaking and centrifugation, and subjected to S _N 2 nucleophilic substitution reaction and extraction according to the above, and then detected by chromatography. The measured carnitine content was recorded in Table 9 below. The tablet is indicated to contain 1 g of carnitine per ingot.

Table 9: Analysis results of lozenges containing carnitine

As is apparent from the ninth table, the detection method of the quaternary ammonium compound having a γ-carboxylic acid group of the present example can surely detect the carnitine content in the tablet. Further, according to the analysis results provided by the detection method of the quaternary ammonium compound having a γ-carboxylic acid group of the present embodiment, it is possible to judge whether the analyzed drug sample meets the specification of the ingredient label, for example, the US Pharmacopoeia specification carnitine lozenge The actual content must be between 90.0% and 110.0% of the indicated content.

(K) Analysis of commercially available injections

Taking a carnitine-containing injection, mixing the injection with acetonitrile, shaking and centrifuging, taking the supernatant, and performing S _N 2 nucleophilic substitution reaction and extraction according to the above, and detecting by chromatography, the measured Carnitine content is reported in Table 10 below. The injection is indicated to contain 1 g of carnitine per 5 ml.

As is apparent from the tenth table, the detection method of the quaternary ammonium compound having a γ-carboxylic acid group of the present example can surely detect the carnitine content in the injection. Similarly, it is possible to judge whether or not the analyzed drug sample conforms to the specification of the component labeling based on the analysis result provided by the detection method of the quaternary ammonium compound having a γ-carboxylic acid group of the present embodiment. For example, the actual content of the US Pharmacopoeia specification for carnitine injections must be between 90.0% and 110.0% of the indicated content.

(L) Analysis of commercially available foods and cosmetics

Take the carnitine-containing foods (No. A~D) and cosmetics (No. E~L), respectively, after sample pretreatment, and carry out S _N 2 nucleophilic substitution reaction and extraction according to the above, and then detect by chromatography. The measured carnitine content is recorded in Table 11 below. Among them, the number A indicates that the carnitine content is 500 mg, the number B indicates that the carnitine content is 250 mg, and the number C indicates that the carnitine content is 20 mg.

As is apparent from the eleventh table, the detection method of the quaternary ammonium compound having a γ-carboxylic acid group of the present example can surely detect the carnitine content in different kinds of samples such as foods or cosmetics, that is, the present invention has "applicable to Type of sample to be tested" efficacy.

According to the above experiment, the detection method of the quaternary ammonium compound having a γ-carboxylic acid group of the present invention can be confirmed, and the addition of the fluorescent compound and the polar aprotic solvent can surely make the γ-carboxylic acid group have four stages. The ammonium compound and the fluorescent compound undergo the nucleophilic substitution reaction of the S _N 2 , and the quaternary ammonium compound having a γ-carboxylic acid group is brought to the fluorophore to achieve “the fourth γ-carboxylic acid group Grade ammonium compounds are suitable for use in fluorescence spectroscopy. Meanwhile, by bonding the quaternary ammonium compound having a γ-carboxylic acid group to the fluorophore of the fluorescent compound, the polarity of the quaternary ammonium compound having a γ-carboxylic acid group can be lowered to facilitate subsequent separation and For analysis, for example, when the quaternary ammonium compound having a γ-carboxylic acid group is separated by reverse phase chromatography, the residence time of the reverse phase chromatograph can be prolonged, and the effect of "lifting separation effect" can be achieved.

The method for detecting a quaternary ammonium compound having a γ-carboxylic acid group of the present invention can further enhance the fluorination group by extracting the quaternary ammonium compound having a γ-carboxylic acid group with a fluorescent group. The concentration of the quaternary ammonium compound having a γ-carboxylic acid group further achieves the effect of "increasing the detection sensitivity of the quaternary ammonium compound having a γ-carboxylic acid group".

In the method for diagnosing carnitine deficiency according to the present invention, the above-mentioned sample obtained by a suspected patient can be detected by using the above-mentioned detection method of a quaternary ammonium compound having a γ-carboxylic acid group, and the carnitine content in the sample can be effectively analyzed. "Effective diagnosis of carnitine deficiency".

The kit for detecting a quaternary ammonium compound having a γ-carboxylic acid group of the present invention can surely perform a S _N 2 nucleophilic substitution reaction on a sample containing a quaternary ammonium compound having a γ-carboxylic acid group to form a derivative. Further, the derivative can be further analyzed by an analytical method such as reverse phase chromatography, fluorescence spectroscopy or mass spectrometry to determine whether or not the sample contains a quaternary ammonium compound having a γ-carboxylic acid group. efficacy.

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

Claims (10)

A method for detecting a quaternary ammonium compound having a γ-carboxylic acid group, comprising: providing a sample comprising a quaternary ammonium compound having a γ-carboxylic acid group; mixing the sample, having a fluorescent group a photo compound and a polar aprotic solvent to form a reaction solution; the reaction solution is placed at 80 to 120 ° C for 1 to 15 minutes to make the fluorescent compound of the fluorescent compound and the γ-carboxylic acid group The carboxylic acid group of the ammonium compound is subjected to an S _N 2 nucleophilic substitution reaction to obtain a derivatized solution comprising a derivative which is a fluorophore on which a fluorophore is substituted with the fluorescent compound. a quaternary ammonium compound having a γ-carboxylic acid group; adding an extracting agent and a cosolvent to the derivatizing solution, the polarity of the extracting agent is higher than the polarity of the acetonitrile, so that the extracting agent and the cosolvent form a water-in-oil And the emulsion is dispersed in the derivatization solution to extract the derivative in the derivatization solution and obtain a solution to be tested; and detect the derivative in the solution to be tested; wherein the fluorophore is The fluorescent compound is 4-bromomethylbiphenyl, the pole The aprotic solvent is acetonitrile, the extraction agent is an aqueous ammonium acetate solution, the co-solvent is toluene. The method for detecting a quaternary ammonium compound having a γ-carboxylic acid group according to claim 1, wherein the detecting method further comprises: adding an alkali agent to the reaction solution, and continuously dissolving the alkali agent; The reaction solution is subjected to the S _N 2 nucleophilic substitution reaction to obtain the derivatized solution. The method for detecting a quaternary ammonium compound having a γ-carboxylic acid group as described in claim 2, wherein the detecting method further comprises: selecting potassium hydroxide, potassium carbonate or potassium hydrogencarbonate as the alkali agent. The method for detecting a quaternary ammonium compound having a γ-carboxylic acid group as described in claim 1, wherein the method for detecting the solution is detected by reverse phase chromatography, fluorescence spectroscopy or mass spectrometry. A derivative of a quaternary ammonium compound having a γ-carboxylic acid group. A method for diagnosing carnitine deficiency includes: Obtaining a sample from a suspected patient; detecting the amount of carnitine compound in the sample in vitro to obtain a detection value by using the detection method as described in claim 1 of the patent application; and comparing the sample The measured value and a reference value; wherein the detected value is lower than the reference value to indicate that the suspected patient suffers from carnitine deficiency. The method for diagnosing carnitine deficiency according to claim 5, wherein the content of L-carnitine in the sample is detected to obtain the detection value, and the sample taken from a healthy individual is detected. The content of L-carnitine in the middle to obtain the reference value. The method for diagnosing carnitine deficiency according to claim 5, wherein the content of L-carnitine and the content of acetaminophen in the sample are detected, and L-carnitine in the sample is calculated. The ratio of the content / acetaminophen content to obtain the detected value, and detecting the content of L-carnitine and the content of acetaminophen in a sample taken from a healthy individual, and calculating the sample of the healthy individual The ratio of L-carnitine content / acetaminophen content in the middle to obtain the reference value. A kit for detecting a quaternary ammonium compound having a γ-carboxylic acid group, comprising: a fluorescent compound having a fluorescent group, the fluorescent group of the fluorescent compound being used for the γ-carboxylic acid group a carboxylic acid group of the quaternary ammonium compound undergoes a S _N 2 nucleophilic substitution reaction to form a derivative; a polar aprotic solvent for accelerating the S _N 2 nucleophilic substitution reaction; and an extracting agent for extracting the a derivative having a polarity higher than that of the polar aprotic solvent; and a cosolvent for forming a water-in-oil emulsion together with the extracting agent; wherein the fluorescent compound having a fluorescent group is 4 Bromomethylbiphenyl, the polar aprotic solvent is acetonitrile, the extractant is an aqueous solution of ammonium acetate, and the cosolvent is toluene. The kit for detecting a quaternary ammonium compound having a γ-carboxylic acid group, as described in claim 8, wherein the kit further comprises: an alkali agent which is soluble in the polarity a basic compound of a protic solvent and used to provide an alkaline environment for carrying out the S _N 2 nucleophilic substitution reaction. The kit for detecting a quaternary ammonium compound having a γ-carboxylic acid group as described in claim 9 wherein the alkali agent is potassium hydroxide, potassium carbonate or potassium hydrogencarbonate.