TWI874225B - Method and kit for detecting polyamine, and method for diagnosing cancer in vitro - Google Patents

Abstract

A method for detecting polyamine is used to solve the problem that the conventional method is not suitable for detecting polyamine. The method for detecting polyamine includes providing a sample with polyamine. The sample and a derivatization reagent with an isothiocyanate group are dissolved in an alkaline working solution to form a mixture, while a thiocarbamoylation reaction between the polyamine in the sample and the derivatization reagent, forming a thiourea derivative. The thiourea derivative in a derivatization solution is then detected to obtain a value of polyamine. With such performance, polyamine in the sample can be effectively detected. A kit for detecting polyamine, and a method for diagnosing cancer in vitroare also disclosed.

Description

Polyamine detection method and detection kit, and in vitro cancer diagnosis method

The present invention relates to a detection method, in particular to a detection method for polyamines. The present invention also relates to a detection kit for implementing the detection method for polyamines, and an in vitro diagnosis method for cancer using the detection method for polyamines.

Polyamines are organic compounds with two or more amino groups. The most common examples are spermidine (a triamine), spermine (a tetraamine), and their diamine precursor, putrescine (a diamine).

Polyamines can undergo protonation to form positively charged molecules, which can bind to nucleic acids and chromatin, thereby affecting a series of cell transmission pathways. When polyamine biosynthesis is inhibited in cells, cell growth will be severely blocked or stopped. In actively growing cancer tissues, the synthesis and secretion of polyamines will increase significantly, so the content of polyamines is considered a biomarker for many types of cancer.

In addition, it is known that long-term intake of high levels of polyamines will also increase the polyamine content in the body, thereby increasing the risk of cancer. There is also a method of reducing dietary polyamine intake to treat cancer [called polyamine blocking therapy (PBT)]. Therefore, if the polyamine content in food can be tested, it can also provide a reference for people's dietary intake.

However, since most polyamines are highly polar substances and do not have specific ultraviolet luminescent groups and lack fluorescent properties, polyamines are difficult to detect using conventional detection methods. In view of this, there is still a need to improve a detection method and detection kit for polyamines.

To solve the above problems, the present invention aims to provide a method for detecting polyamines, which can effectively detect polyamines in a sample to be tested.

Another object of the present invention is to provide a method for detecting polyamines, which can shorten the detection time.

Another object of the present invention is to provide a polyamine detection kit for use in implementing the polyamine detection method as described above.

Another object of the present invention is to provide an in vitro cancer diagnosis method using the aforementioned polyamine detection method.

The quantifiers "a" or "an" used in the elements and components described throughout the present invention are only for convenience of use and to provide a general meaning of the scope of the present invention; they should be interpreted in the present invention as including one or at least one, and the single concept also includes the plural case unless it is obvious that it means otherwise.

The polyamine detection method of the present invention may include: providing a sample to be tested, the sample to be tested containing polyamine; dissolving the sample to be tested and a derivatization reagent in a working solvent to form a reaction solution, the derivatization reagent having an isothiocyanate functional group; allowing the polyamine in the reaction solution to undergo a thiocarbamate reaction with the isothiocyanate functional group of the derivatization reagent to obtain a derivatization solution, the derivatization solution containing a thiourea derivative; and detecting the thiourea derivative to obtain a polyamine intensity value.

Accordingly, by allowing the amino groups of the polyamines in the sample to be tested to react with the derivatizing reagent (isothiocyanate having an isothiocyanate functional group) to undergo the thiocarbamate reaction, the thiourea derivative can be obtained. The thiourea derivative can be detected by various known methods (for example, after separation by liquid chromatography, gas chromatography, etc., and then detected by ultraviolet spectroscopy, fluorescence spectroscopy, or mass spectrometry). Therefore, the polyamine detection method of the present invention has good sensitivity, precision and accuracy, and can reduce the usage of the sample to be tested, which is the effect of the present invention.

In the polyamine detection method of the present invention, the derivatization reagent can be 4-dimethylamine-naphthyl isothiocyanate, 1-naphthyl isothiocyanate, benzyl naphthyl isothiocyanate or allyl naphthyl isothiocyanate. Thus, by selecting the derivatization reagent, the content of the thiourea derivative formed by the reaction of the polyamine in the reaction solution with the isothiocyanate functional group of the derivatization reagent can be increased, thereby improving the detection effect of the polyamine.

In the polyamine detection method of the present invention, the working solvent can be acetonitrile, acetone, dimethyl sulfoxide, pyridine, dimethylformamide, dimethyl propylene urea, cyclobutane sulfone, tetrahydrofuran, 1,2-dimethoxyethane, 1,3-dioxolane, dimethyl carbonate or water. Thus, by selecting the working solvent, the dissolution effect of the sample to be tested and the derivatization reagent can be improved, thereby improving the detection effect of polyamines.

The polyamine detection method of the present invention may further include: heating the reaction solution by microwave to drive the thiocarbamate reaction. For example, the reaction solution can be heated by microwave at 100-750 W. In this way, by providing energy by microwave, the thiocarbamate reaction can be accelerated, thereby improving the detection efficiency of polyamine.

The polyamine detection method of the present invention may further include: adding an alkali to the reaction solution, and then subjecting the reaction solution in which the alkali is dissolved to the thiocarbamate reaction. For example, the alkali may be sodium hydroxide, sodium bicarbonate, sodium carbonate, 4-dimethylaminopyridine or triethylamine. Thus, by adding the alkali, the amino group of the polyamine can be deprotonated, the nucleophilicity of the amino group can be increased, the thiocarbamate reaction can be accelerated, and the detection efficiency of the polyamine can be improved.

The polyamine detection method of the present invention may further include: adding a removal reagent to the derivatization solution, so that the removal reagent removes the fat-soluble interferents in the derivatization solution and forms an upper solution and a lower solution, wherein the lower solution contains the thiourea derivative, and the removal reagent is a solvent that is immiscible with the derivatization solution and has a logP between 3.5 and 7.0. For example, the removal reagent may be n-hexane, n-octane, n-decane or dodecane. Thus, by adding the removal reagent, the excess derivatization reagent can be effectively removed to prevent the excess derivatization reagent from affecting the subsequent analysis results, thereby improving the detection accuracy of the polyamine.

The polyamine detection method of the present invention may further include: adding a proton provider and a foaming salt to the derivative solution, allowing the proton provider to react with the foaming salt to form carbon dioxide gas, and forming an upper solution and a lower solution, wherein the upper solution contains the thiourea derivative. For example, the proton provider may be citric acid, ammonium chloride, ammonium sulfate or sodium dihydrogen phosphate, and the foaming salt may be potassium bicarbonate, sodium bicarbonate, ammonium bicarbonate, potassium carbonate or sodium carbonate. Thus, by adding the proton provider and the foaming salt, carbon dioxide gas can be generated, which can promote the dispersion of each molecule in the derivative solution, thereby improving the detection efficiency of polyamines.

The polyamine detection method of the present invention may further include: placing the derivative solution in a cooling bath after adding the proton donor and the foaming salt and before forming the upper solution and the lower solution. In this way, the cooling bath can promote the effective separation of the upper solution and the lower solution, thereby effectively separating the thiourea derivative, thereby improving the detection accuracy of polyamines.

The polyamine detection method of the present invention may further include: adding a removal reagent, a proton donor and a foaming salt to the derivative solution, allowing the removal reagent to remove fat-soluble interferents in the derivative solution, and allowing the proton donor to react with the foaming salt to form carbon dioxide gas, and forming an upper solution, a middle solution and a lower solution, the middle solution contains the thiourea derivative, and the removal reagent is a solvent that is immiscible with the derivative solution and has a logP between 3.5 and 7.0. For example, the removal reagent can be n-hexane, n-octane, n-decane or dodecane, the proton provider can be citric acid, ammonium chloride, ammonium sulfate or sodium dihydrogen phosphate, and the foaming salt can be potassium bicarbonate, sodium bicarbonate, ammonium bicarbonate, potassium carbonate or sodium carbonate. Thus, by adding the removal reagent, the excess derivatization reagent can be effectively removed to prevent the excess derivatization reagent from affecting the subsequent analysis results, thereby improving the detection accuracy of polyamines; and by adding the proton provider and the foaming salt, carbon dioxide gas can be generated, which can promote the dispersion of each molecule in the derivatization solution, thereby improving the detection efficiency of polyamines.

The polyamine detection method of the present invention may further include: placing the derivatization solution in a cooling bath after adding the removal reagent, the proton donor and the foaming salt and before forming the upper solution, the middle solution and the lower solution. In this way, the cooling bath can promote the effective separation of the upper solution, the middle solution and the lower solution, thereby effectively separating the thiourea derivative and improving the detection accuracy of the polyamine.

The polyamine detection kit of the present invention may include: a derivatization reagent having an isothiocyanate functional group; and a working solvent which is a solvent capable of dissolving the derivatization reagent and the polyamine.

Accordingly, the detection kit can be applied to implement the above-mentioned polyamine detection method, and effectively forms a thiourea derivative with good stability together with the polyamine. Therefore, the worker can select various known methods to detect the thiourea derivative according to the needs (for example, after separation by liquid chromatography, gas chromatography, etc., using ultraviolet spectroscopy, fluorescence spectroscopy, or mass spectrometry for detection), which is the effect of the present invention.

In the polyamine detection kit of the present invention, the derivatization reagent can be 4-dimethylamine-naphthyl isothiocyanate, 1-naphthyl isothiocyanate, benzyl naphthyl isothiocyanate or allyl naphthyl isothiocyanate. Thus, by selecting the derivatization reagent, the content of thiourea derivatives formed by the reaction of the polyamine in the reaction solution with the isothiocyanate functional group of the derivatization reagent can be increased, thereby improving the detection effect of polyamine.

The working solvent of the polyamine detection kit of the present invention can be acetonitrile, acetone, dimethyl sulfoxide, pyridine, dimethylformamide, dimethyl propylene urea, cyclobutane sulfone, tetrahydrofuran, 1,2-dimethoxyethane, 1,3-dioxolane, dimethyl carbonate or water. Thus, by selecting the working solvent, the dissolution effect of the sample to be tested and the derivatization reagent can be improved, thereby improving the detection effect of polyamines.

The polyamine detection kit of the present invention may further include: an alkali, which is an alkaline compound that can be dissolved in the working solvent. For example, the alkali may be sodium hydroxide, sodium bicarbonate, sodium carbonate, 4-dimethylaminopyridine or triethylamine. Thus, by adding the alkali, the amino group of the polyamine can be deprotonated, the nucleophilicity of the amino group can be increased, the thiocarbamate reaction can be accelerated, and the detection efficiency of the polyamine can be improved.

The polyamine detection kit of the present invention may further include: a removal reagent, which is a solvent with a logP between 3.5 and 7.0. For example, the removal reagent may be n-hexane, n-octane, n-decane or dodecane. Thus, by adding the removal reagent, the excess derivatization reagent can be effectively removed, thereby preventing the excess derivatization reagent from affecting the subsequent analysis results, thereby improving the detection accuracy of polyamines.

The polyamine detection kit of the present invention may further include: a proton provider for providing hydrogen ions; and a foaming salt for performing an acid-base neutralization reaction with the proton provider to form carbon dioxide gas. For example, the proton provider may be citric acid, ammonium chloride, ammonium sulfate or sodium dihydrogen phosphate, and the foaming salt may be potassium bicarbonate, sodium bicarbonate, ammonium bicarbonate, potassium carbonate or sodium carbonate. Thus, by adding the proton provider and the foaming salt, carbon dioxide gas can be generated, which can enhance the dispersibility of each molecule in the derivative solution and facilitate the salt precipitation.

The polyamine detection kit of the present invention may further include: a removal reagent, which is a solvent with a logP between 3.5 and 7.0; a proton provider, which is used to provide hydrogen ions; and a foaming salt, which is used to perform an acid-base neutralization reaction with the proton provider to form carbon dioxide gas. For example, the removal reagent can be n-hexane, n-octane, n-decane or dodecane, the proton provider can be citric acid, ammonium chloride, ammonium sulfate or sodium dihydrogen phosphate, and the foaming salt can be potassium bicarbonate, sodium bicarbonate, ammonium bicarbonate, potassium carbonate or sodium carbonate. Thus, by adding the removal reagent, the excess derivatization reagent can be effectively removed, thereby preventing the excess derivatization reagent from affecting subsequent analysis results, thereby improving the detection accuracy of polyamines; and by adding the proton donor and the foaming salt, carbon dioxide gas can be generated, which can improve the dispersion of each molecule in the derivatization solution and facilitate the salt precipitation reaction.

The in vitro cancer diagnosis method of the present invention may include: using the polyamine detection method as described above to detect the polyamine content in a biological sample taken from a suspected patient in vitro to obtain a detection value; and comparing the detection value of the biological sample with a reference value, and when the detection value is higher than the reference value, it indicates that the suspected patient suffers from cancer. For example, the cancer may be liver cancer, prostate cancer, pancreatic cancer, colorectal cancer, breast cancer, lymphoma, fibrosarcoma, lung cancer, stomach cancer or oral cancer.

In order to make the above and other purposes, features and advantages of the present invention more clearly understood, the following specifically describes the preferred embodiments of the present invention in detail with reference to the accompanying drawings.

Referring to FIG. 1 , the polyamine detection method of the first embodiment of the present invention may include: a sample providing step S1 , a derivatization step S2 , and an analysis step S3 .

Specifically, in the sample providing step S1, a sample to be tested containing polyamine is provided. For example, the sample to be tested can be from a food sample or from a biological sample (e.g., a blood sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, or an oral tissue sample). When the matrix of the test sample is too complex, for example, it contains macromolecules such as proteins, the worker can first mix the test sample with a protein precipitant, which can be sodium hydroxide (NaOH), acetonitrile, methanol, ethanol, hydrochloric acid (HCl) or ammonium sulfate ((NH ₄ ) ₂ SO ₄ ), etc., so that the protein in the test sample forms a protein precipitate, and then remove the protein precipitate by centrifugation (for example, 15,000 rpm, 2 minutes) to obtain a supernatant, which can be used as the test sample used in the subsequent derivatization step S2.

In the derivatization step S2, the worker can use an isothiocyanate having an isothiocyanate functional group as a derivatization reagent, dissolve the derivatization reagent in a working solution, and add the sample to be tested therein to prepare a reaction solution. In this way, the derivatization reagent and the polyamine in the sample to be tested undergo a thiocarbamoylation reaction as shown in Figure 2 to obtain a derivatization solution, which contains a thiourea derivative. For example, in the reaction solution, the derivatization reagent may be an isothiocyanate such as 4-dimethylamino-naphthylisothiocyanate (DNITC, having the chemical structure shown in FIG. 3 ), 1-naphthylisothiocyanate (NITC, having the chemical structure shown in FIG. 4 ), benzylisothiocyanate (BITC, having the chemical structure shown in FIG. 5 ) or allylisothiocyanate (AITC, having the chemical structure shown in FIG. 6 ), and the concentration of the derivatization reagent in the reaction solution may be between 5 and 15 mM. Furthermore, the working solvent is a solvent that can dissolve the derivatization reagent and the polyamine in the sample to be tested, and can form the reaction solution together with the derivatization reagent and the polyamine. For example, the working solvent can be acetonitrile (ACN), acetone, dimethyl sulfoxide (DMSO), pyridine, dimethylformamide (DMF), dimethylpropyleneurea (DMPU), sulfolane, tetrahydrofuran (THF), 1,2-dimethoxyethane (DME), 1,3-dioxolane (DOL), dimethyl carbonate (DMCO), dimethyl ether (DMF), dimethyl ether (DMPU), cyclobutane (sulfolane), tetrahydrofuran (THF), 1,2-dimethoxyethane (DME), 1,3-dioxolane (DOL), dimethyl carbonate (DMCO), dimethyl ether (DMF), dimethyl ether (DMPU), dimethyl ether (DMCO), dimethyl ether (DMF), dimethyl ether (DMPU), dimethyl ether (DMCO), dimethyl ether (DMF), dimethyl ether (DMPU), dimethyl ether (DMCO), dimethyl ether (DMF), dimethyl ether (DMPU), dimethyl ether (DMCO), dimethyl ether (DMF), dimethyl ether (DMPU), dimethyl ether (DMCO), dimethyl ether (DM carbonate, abbreviated as DMC) or water and other solvents.

In order to accelerate the thiocarbamate reaction, the worker may add a base to the reaction solution. The base is an alkaline compound that can be dissolved in the working solvent. By adding the base, the amino group of the polyamine can undergo deionization (deprotonation), thereby increasing the nucleophilicity of the amino group, thereby accelerating the thiocarbamate reaction. For example, the alkali may be an alkaline compound such as sodium hydroxide (NaOH), sodium bicarbonate (NaHCO ₃ ), sodium carbonate (Na ₂ CO ₃ ), 4-dimethylaminopyridine (DMAP) or triethylamine (TEA), and the concentration of the alkali in the reaction solution may be between 5 and 75 mM.

Furthermore, in order to accelerate the thiocarbamate reaction, the worker may heat the reaction solution, for example, by placing the reaction solution at a temperature of 20 to 60° C. for 10 minutes to 24 hours, or by heating the reaction solution by microwave, for example, by using a microwave oven with a power of 100 to 750 W to microwave the reaction solution for 0.5 to 10 minutes.

Next, in the analysis step S3, the worker can use the derivatized solution as a test solution and use various known methods to detect the thiourea derivatives in the test solution to obtain a polyamine intensity value, and use the polyamine intensity value to evaluate whether the test sample contains polyamines, or even the amount of polyamines in the test sample. For example, the worker can detect the thiourea derivative in the test solution after separation by liquid chromatography, gas chromatography, etc., and then use ultraviolet spectrometry, fluorescence spectrometry or mass spectrometry; for example, high performance liquid chromatography with ultraviolet detector (HPLC-UV), high performance liquid chromatography with diode-array detector (HPLC-DAD), high performance liquid chromatography with fluorescence detector (HPLC-FLD), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS), etc. The detection methods include chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), gas chromatography-mass spectrometry (GC-MS), and gas chromatography-flame ionization detector (GC-FID).

For example, when performing separation by liquid chromatography, a C18 column (2.1 mm×50 mm; particle size 2.7 μm) can be used as the stationary phase, and a mixed solution as shown in Table 1 can be used as the mobile phase for gradient elution at a flow rate of 180 μL/min.

Table 1: Gradient extraction procedure for separation of the test solution Time (min) Mixed solution Solution A (0.15% formic acid in water) Solution B (Methanol) 0.0→6.0 80% → 60% 20% → 40% 6.0→7.0 60% → 40% 40% → 60% 7.0→11.8 40% → 40% 60% → 60% 11.8→13.3 40% → 0% 60% → 100% 13.3→20.0 0% → 0% 100% → 100%

Please refer to FIG. 7 again. Since the test solution (the derivatization solution) may contain an excessive amount of derivatization reagent (isothiocyanate having an isothiocyanate functional group) when performing the analysis step S3, the polyamine detection method of the second embodiment of the present invention may perform a removal step S4 between the derivatization step S2 and the analysis step S3.

In the removal step S4, a removing reagent is added to the derivatization solution. The removing reagent is a solvent that is immiscible with the derivatization solution and has a logP between 3.5 and 7.0. This can remove fat-soluble interferents such as the derivatization reagent (isothiocyanate with isothiocyanate functional group) in the derivatization solution, and can also separate the derivatization solution into an upper solution and a lower solution. The lower solution contains the thiourea derivative. For example, the removal reagent may be a solvent such as hexane, octane, decane or dodecane, and the amount of the removal reagent added may be between 100 and 1,000 μL for a total volume of about 100 to 500 μL of the derivatization solution. Furthermore, when the derivatization solution contains a large amount of fat-soluble interferents, the worker may also choose to continuously perform the removal step S4 multiple times, that is, after obtaining the lower layer solution, the removal reagent is added again, so that the lower layer solution can be separated into another upper layer solution and another lower layer solution, and at this time, the other lower layer solution contains the thiourea derivative.

After the removal step S4, the worker can take the lower layer solution containing the thiourea derivative as the test solution, and detect the thiourea derivative in the test solution by various known methods as described above, thereby obtaining the polyamine strength value.

Continuing with reference to FIG. 8 , in order to remove undesirable cations and anions in the derivatization solution, the polyamine detection method of the third embodiment of the present invention may further perform a salting out step S5 between the derivatization step S2 and the analysis step S3.

In the salt separation step S5, a proton donor and an effervescent salt are added to the derivatized solution. The proton donor is used to provide hydrogen ions, and the effervescent salt is used to dissociate to form anions and cations, and undergo an acid-base neutralization reaction with the proton donor to form carbon dioxide gas. After the proton donor and the foaming salt are added to the derivatization solution, carbon dioxide gas (in the form of bubbles) can be formed in the derivatization solution, which can improve the dispersion of each molecule in the derivatization solution and help the salting-out process. At this time, under the combined action of the proton donor and the foaming salt, the derivatization solution can be layered to form an upper solution and a lower solution, and the upper solution contains the thiourea derivative. For example, the proton provider can be citric acid, ammonium chloride (NH ₄ Cl), ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) or sodium dihydrogen phosphate (NaH ₂ PO ₄ ), and the foaming salt can be potassium bicarbonate (KHCO ₃ ), sodium bicarbonate (NaHCO ₃ ), ammonium bicarbonate (NH ₄ HCO ₃ ), potassium carbonate (K ₂ CO ₃ ) or sodium carbonate (Na ₂ CO ₃ ), and the amount of the proton provider added can be between 5 and 75 μL for a total volume of about 100 to 500 μL of the derivatization solution. mg, and the amount of the foaming salt added can be between 5 and 75 mg.

It is worth noting that in the salt separation step S5, after adding the proton donor and the foaming salt, and before forming the upper solution and the lower solution, the worker can place the derivatized solution in a cooling bath (with a temperature between -70 and 0°C), thereby promoting the effective separation of the derivatized solution.

After the salt analysis step S5, the worker can take the upper layer solution containing the thiourea derivative as the test solution, and use various known methods as described above to detect the thiourea derivative in the test solution to obtain the polyamine strength value.

Please also refer to FIG. 9 , in the method for detecting polyamines in the fourth embodiment of the present invention, a salt precipitation removal step S6 may be performed between the derivatization step S2 and the analysis step S3.

In the salting out removal step S6, the worker simultaneously adds the removal reagent, the proton provider and the foaming salt to the derivatization reagent. The functions, types and addition amounts of the removal reagent, the proton provider and the foaming salt can be as described above and will not be repeated here. After adding the removal reagent, the proton provider and the foaming salt, the derivatization solution can be layered to form an upper solution, a middle solution and a lower solution, and the middle solution contains the thiourea derivative. Similarly, after adding the removal reagent, the proton donor and the bubbling salt, and between forming the upper solution, the middle solution and the lower solution, the worker may place the derivatization solution in the cooling bath to promote the effective separation of the derivatization solution.

Then, the worker can take the middle solution containing the thiourea derivative as the test solution, and use various known methods as mentioned above to detect the thiourea derivative in the test solution, thereby obtaining the polyamine strength value.

According to the above, based on the same technical concept, the polyamine detection kit of the first embodiment of the present invention may include: the derivatization reagent and the working solvent, the derivatization reagent is an isothiocyanate having an isothiocyanate functional group, and the working solvent is a solvent that can dissolve the derivatization reagent and the polyamine in the sample to be tested. In addition, the functions and specific examples of the derivatization reagent and the working solvent can be as described above, and will not be repeated here.

Furthermore, in order to accelerate the thiocarbamate reaction, the polyamine detection kit may further include the alkali, which is an alkaline compound that can be dissolved in the working solvent. Its role and specific examples can also be as described above and will not be repeated here.

Corresponding to the polyamine detection method of the second embodiment as shown in FIG. 7 , the polyamine detection kit of the second embodiment of the present invention may include the removal reagent in addition to the derivatization reagent and the working solvent. The removal reagent is a solvent with a logP between 3.5 and 7.0. Its function and specific examples are as described above and will not be elaborated here.

Corresponding to the polyamine detection method of the third embodiment as shown in FIG. 8 , the polyamine detection kit of the third embodiment of the present invention may include, in addition to the derivatization reagent and the working solvent, the proton provider and the foaming salt. The proton provider is used to provide hydrogen ions, and the foaming salt is used to perform the acid-base neutralization reaction with the proton provider to form carbon dioxide gas. The functions and specific examples of the two can be as described above and will not be repeated here.

Corresponding to the polyamine detection method of the fourth embodiment as shown in Figure 9, the polyamine detection kit of the fourth embodiment of the present invention may include the removal reagent, the proton donor and the foaming salt in addition to the derivatization reagent and the working solvent. The properties, functions and specific examples of the removal reagent, the proton donor and the foaming salt may be as described above and will not be repeated here.

It is worth noting that the aforementioned polyamine detection method (and the detection kit used to implement the polyamine detection method) can be used to detect a biological sample from a mammal, and the polyamine detection method (and the polyamine detection kit) can be further applied to detect the polyamine content in a suspected patient, thereby evaluating whether the suspected patient suffers from cancer. In detail, the biological sample is first obtained from the suspected patient, and the polyamine content in the biological sample is detected in vitro using the aforementioned polyamine detection method to obtain a detection value; and the detection value of the biological sample is compared with a reference value, and when the detection value is higher than the reference value, it indicates that the suspected patient suffers from cancer.

For example, Shirakawa et al. reported that the spermidine in the erythrocytes of healthy adults was 15.04 ± 3.63 ng/g and the spermine was 8.82 ± 3.12 ng/g, while the spermidine in the erythrocytes of pancreatic cancer patients was 47.15 ± 25.97 ng/g and the spermine was 12.27 ± 9.44 ng/g, and the spermidine in the erythrocytes of colorectal cancer patients was 31.97 ± 23.29 ng/g and the spermine was 41.59 ± 37.57 ng/g, indicating that the polyamine detection values in the erythrocytes of pancreatic cancer patients and colorectal cancer patients were higher than the reference value (the detection value of biological samples of healthy adults) (Cancer. 1980 Jan 1;45(1):108-11); Ishikawa et al. also reported that the levels of putrescine and spermidine in the oral tissues of healthy adults were 1048.98 ± 626.75 ng/g and 1260.77 ± 883.12 ng/g, respectively, while the levels of putrescine and spermidine in the oral tissues of oral cancer patients were 7867.86 ± 6073.54 ng/g and 3573.15 ± 3718.40 ng/g, respectively, indicating that the detection values of polyamines in the oral tissues of oral cancer patients were higher than the reference values (the detection values of biological samples of healthy adults) (Sci Rep. 2016 Aug 19:6:31520); in addition, related research reports have also been reported in a variety of cancers, including liver cancer, prostate cancer, breast cancer, lymphoma, fibrosarcoma, lung cancer, or gastric cancer.

In order to verify that the polyamine detection method can be used to detect the content of polyamines in the sample to be tested, the following test was conducted:

(A) Choice of derivatization reagent

In this test, 100 μL of polyamine solution (containing 1 mM putrescine, 1 mM spermidine and 1 mM spermine, respectively, dissolved in water) was added with 200 μL of derivatization reagent solution [containing 10 mM derivatization reagent, dissolved in working solvent (acetonitrile)] and 25 μL of alkaline solution [containing 10 mM alkaline (sodium hydroxide), dissolved in water], and after mixing evenly to form the reaction solution, the reaction solution was heated by microwave (300 W, 5 minutes), and then kept in an ice bath to terminate the thiocarbamate reaction, so as to obtain the derivatization solution of each group. Next, 1 μL of the derivatization solution was taken and observed by thin layer chromatography (TLC) at a wavelength of 254 nm to determine whether the thiourea derivative was generated and to quantify the content of the thiourea derivative.

The derivatization reagents used in each group of this test are shown in Table 2, where the derivatization reagents in Groups A5 and A6 are not isothiocyanate salts having isothiocyanate functional groups.

Table 2. Derivatization reagents selected for each group of this test Group Derivatization reagent Chemical structure A1 4-Dimethylamino-naphthyl isothiocyanate (DNITC) Figure 3 A2 1-Naphthyl isothiocyanate (NITC) Figure 4 A3 Benzylnaphthyl isothiocyanate (BITC) Figure 5 A4 Allyl naphthyl isothiocyanate (AITC) Figure 6 A5 N,N-Dimethyl-1-naphthylamine (DNA) Figure 10 A6 Naphthalene Figure 11

As shown in Figure 12, all of the groups A1 to A4 using isothiocyanate with isothiocyanate functional group as the derivatization reagent have the generation of the thiourea derivative, among which the derivatization reagent (DNITC) of group A1 has the best effect. It is worth noting that by comparing the derivatization reagents of groups A1 and A5, it can be seen that the thiocarbamate reaction is indeed carried out through the isothiocyanate functional group of the derivatization reagent, and the same conclusion can be obtained by comparing the derivatization reagents of groups A2 and A6.

(B) Choice of alkali

In this test, 100 μL of polyamine solution (containing 200 μM putrescine, 200 μM spermidine and 200 μM spermine, respectively, dissolved in water) was added with 200 μL of derivatization reagent solution [containing 10 mM derivatization reagent (DNITC), dissolved in working solvent (acetonitrile)] and 25 μL of alkaline solution (containing 10 mM alkaline, dissolved in water). After mixing well to form the reaction solution, the reaction solution was heated by microwave (300 W, 5 minutes), and then placed in an ice bath to terminate the thiocarbamate reaction, so as to obtain the derivatization solution of each group. Next, 500 μL of a removal reagent (n-octane) and a mixed powder containing the proton donor (citric acid; 25 mg) and the foaming salt (potassium bicarbonate; 10 mg) were added to the derivatization solution, and the solution was incubated at -20°C for 3 minutes. 8 μL of the middle layer solution was taken as the test solution for each group and subsequently analyzed by narrow-bore HPLC-UV.

The alkali used in each group of this test is shown in Table 3, where Group B0 is the group without the addition of the alkali.

Table 3. Alkali selected for each group in this test Group Alkali B0 - B1 Sodium hydroxide (NaOH) B2 Sodium bicarbonate (NaHCO ₃ ) B3 Sodium carbonate (Na ₂ CO ₃ ) B4 4-Dimethylaminopyridine (DMAP) B5 Triethylamine (TEA)

As shown in Figure 13, the addition of the alkali agent does help to enhance the progress of the thiocarbamate reaction, thereby forming more thiourea derivatives (groups B1 to B5), and the effect of sodium hydroxide (NaOH) in group B1 is the best.

(C) Choosing microwave wattage

In this test, 100 μL of a polyamine solution (containing 200 μM putrescine, 200 μM spermidine and 200 μM spermine, respectively, dissolved in water) was added with 200 μL of a derivatization reagent solution [containing 10 mM derivatization reagent (DNITC), dissolved in a working solvent (acetonitrile)] and 25 μL of an alkaline solution [containing 10 mM alkaline (sodium hydroxide), dissolved in water], and after mixing well to form the reaction solution, the reaction solution was heated and then placed in an ice bath to terminate the thiocarbamate reaction, thereby obtaining the derivatization solution of each group. Next, 500 μL of a removal reagent (n-octane) and a mixed powder containing the proton donor (citric acid; 25 mg) and the foaming salt (potassium bicarbonate; 10 mg) were added to the derivatization solution, and the solution was incubated at -20°C for 3 minutes. 8 μL of the middle layer solution was taken as the test solution for each group and subsequently analyzed by narrow-bore HPLC-UV.

The heating methods selected by each group in this test are shown in Table 4. Group C0 used a 30°C dry bath for heating, while the other groups adjusted the wattage of the microwave.

Table 4. Heating methods selected for each group in this test Group Heating method C0 30℃ dry bath C1 Microwave (100 W, 5 min) C2 Microwave (300 W, 5 min) C3 Microwave (550 W, 5 minutes) C4 Microwave (750 W, 5 min)

As shown in FIG. 14 , microwave heating does help to promote the progress of the thiocarbamate reaction, thereby forming more thiourea derivatives (groups C1 to C4), and the effect of 300 W in group C2 is the best.

(D) Removal of reagent options

In this test, 100 μL of polyamine solution (containing 200 μM putrescine, 200 μM spermidine and 200 μM spermine, respectively, dissolved in water) was added with 200 μL of derivatization reagent solution [containing 10 mM derivatization reagent (DNITC), dissolved in working solvent (acetonitrile)] and 25 μL of alkaline solution [containing 10 mM alkaline (sodium hydroxide), dissolved in water], and after mixing evenly to form the reaction solution, the reaction solution was heated by microwave (300 W, 5 minutes), and then kept in an ice bath to terminate the thiocarbamate reaction, so as to obtain the derivatization solution of each group. Next, 500 μL of the removal reagent and a mixed powder containing the proton donor (citric acid; 25 mg) and the foaming salt (potassium bicarbonate; 10 mg) were added to the derivatization solution, and the solution was placed at -20°C for 3 minutes. 8 μL of the middle layer solution was taken as the test solution for each group and subsequently analyzed by high performance liquid chromatography (narrow-bore HPLC-UV).

The removal reagents used in each group of this test are shown in Table 5, where Group D0 is the group without the addition of the removal reagent.

Table 5. Removal agents selected for each group in this test Group Reagent removal D0 - D1 n-Hexane D2 n-octane D3 n-Decane D4 Dodecane

As shown in Figure 15, the addition of the removal reagent does help to remove the excess derivatization reagent (residues) (groups D1 to D4), and the effect of n-octane in group D2 is the best.

(E) Number of operations to remove the step

In this test, 100 μL of polyamine solution (containing 200 μM putrescine, 200 μM spermidine and 200 μM spermine, respectively, dissolved in water) was added with 200 μL of derivatization reagent solution [containing 10 mM derivatization reagent (DNITC), dissolved in working solvent (acetonitrile)] and 25 μL of alkaline solution [containing 10 mM alkaline (sodium hydroxide), dissolved in water], and after mixing evenly to form the reaction solution, the reaction solution was heated by microwave (300 W, 5 minutes), and then kept in an ice bath to terminate the thiocarbamate reaction, so as to obtain the derivatization solution of each group.

Next, 500 μL of a removal reagent (n-octane) and a mixed powder containing the proton donor (citric acid; 25 mg) and the foaming salt (potassium bicarbonate; 10 mg) were added to the derivatization solution of group E1, and the mixture was placed at -20°C for 3 minutes. 8 μL of the middle layer solution was taken as the test solution of group E1 and subsequently analyzed by narrow-bore HPLC-UV.

After adding 500 μL of the removal reagent (n-octane) to the derivatization solution of Group E2, the upper layer solution was removed, and then 500 μL of the removal reagent (n-octane) and a mixed powder containing the proton donor (citric acid; 25 mg) and the foaming salt (potassium bicarbonate; 10 mg) were added, and the mixture was placed at -20°C for 3 minutes. 8 μL of the middle layer solution was taken as the test solution of Group E2 for subsequent analysis by high performance liquid chromatography (narrow-bore HPLC-UV).

After adding 500 μL of the removal reagent (n-octane) to the derivatization solution of group E3, the upper layer solution was removed, and after adding 500 μL of the removal reagent (n-octane) again, the upper layer solution was removed. When adding 500 μL of the removal reagent (n-octane) for the third time, a mixed powder containing the proton donor (citric acid; 25 mg) and the foaming salt (potassium bicarbonate; 10 mg) was added at the same time, and the mixture was placed at -20°C for 3 minutes. 8 μL of the middle layer solution was taken as the test solution of group E3, and subsequent analysis was performed by high performance liquid chromatography (narrow-bore HPLC-UV).

In addition, Group E0 of this test is a group to which the removal reagent is not added.

Table 6. Number of removal steps performed in each group of this test Group Number of steps removed E0 0 E1 1 E2 2 E3 3

As shown in Figure 16, performing multiple removal steps does help remove excess derivatization reagents (residues) (Groups E2-E3).

(F) Choice of proton donor

In this test, 100 μL of polyamine solution (containing 200 μM putrescine, 200 μM spermidine and 200 μM spermine, respectively, dissolved in water) was added with 200 μL of derivatization reagent solution [containing 10 mM derivatization reagent (DNITC), dissolved in working solvent (acetonitrile)] and 25 μL of alkaline solution [containing 10 mM alkaline (sodium hydroxide), dissolved in water], and after mixing evenly to form the reaction solution, the reaction solution was heated by microwave (300 W, 5 minutes), and then kept in an ice bath to terminate the thiocarbamate reaction, so as to obtain the derivatization solution of each group. Next, 500 μL of a removal reagent (n-octane) and a mixed powder containing the proton donor (as shown in Table 7; 25 mg) and the foaming salt (potassium bicarbonate; 10 mg) were added to the derivatization solution, and the solution was placed at -20°C for 3 minutes. 8 μL of the middle layer solution was taken as the test solution for each group and subsequently analyzed by high performance liquid chromatography (narrow-bore HPLC-UV).

Table 7. Proton donors selected for each group in this experiment Group Proton Donor F1 Citric Acid F2 Ammonium chloride (NH ₄ Cl) F3 Ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) F4 Sodium dihydrogen phosphate (NaH ₂ PO ₄ )

As shown in FIG. 17 , the thiourea derivatives (Groups F1 to F4) can be separated by using the aforementioned proton donors, and the effect of citric acid in Group F1 is the best.

(G) Choice of foaming salt

In this test, 100 μL of polyamine solution (containing 200 μM putrescine, 200 μM spermidine and 200 μM spermine, respectively, dissolved in water) was added with 200 μL of derivatization reagent solution [containing 10 mM derivatization reagent (DNITC), dissolved in working solvent (acetonitrile)] and 25 μL of alkaline solution [containing 10 mM alkaline (sodium hydroxide), dissolved in water], and after mixing evenly to form the reaction solution, the reaction solution was heated by microwave (300 W, 5 minutes), and then kept in an ice bath to terminate the thiocarbamate reaction, so as to obtain the derivatization solution of each group. Next, 500 μL of a removal reagent (n-octane) and a mixed powder containing the proton donor (citric acid; 25 mg) and the foaming salt (as shown in Table 8; 10 mg) were added to the derivatization solution, and the solution was incubated at -20°C for 3 minutes. 8 μL of the middle layer solution was taken as the test solution for each group and subsequently analyzed by high performance liquid chromatography (narrow-bore HPLC-UV).

Table 8. Foaming salts selected for this test Group Foaming salt G1 Potassium bicarbonate (KHCO ₃ ) G2 Sodium bicarbonate (NaHCO ₃ ) G3 Ammonium bicarbonate (NH ₄ HCO ₃ ) G4 Potassium carbonate (K ₂ CO ₃ ) G5 Sodium carbonate (Na ₂ CO ₃ )

As shown in FIG. 18 , the thiourea derivatives (groups G1 to G5) can be separated by using the aforementioned foaming salts, and the potassium bicarbonate (KHCO ₃ ) of group G1 has the best effect.

(H) Effects of cold bath

In this test, 100 μL of polyamine solution (containing 200 μM putrescine, 200 μM spermidine and 200 μM spermine, respectively, dissolved in water) was added with 200 μL of derivatization reagent solution [containing 10 mM derivatization reagent (DNITC), dissolved in working solvent (acetonitrile)] and 25 μL of alkaline solution [containing 10 mM alkaline (sodium hydroxide), dissolved in water], and after mixing evenly to form the reaction solution, the reaction solution was heated by microwave (300 W, 5 minutes), and then kept in an ice bath to terminate the thiocarbamate reaction, so as to obtain the derivatization solution of each group.

Next, 500 μL of a removal reagent (n-octane) and a mixed powder containing the proton donor (citric acid; 25 mg) and the foaming salt (potassium bicarbonate; 10 mg) were added to the derivatization solution, and the solution was allowed to stand at -20°C for 3 minutes. 8 μL of the middle layer solution was taken as the test solution of group H1 for subsequent analysis by narrow-bore HPLC-UV.

The test solution of group H2 was obtained by adding 500 μL of the removal reagent (n-octane) and a mixed powder containing the proton donor (citric acid; 25 mg) and the foaming salt (potassium bicarbonate; 10 mg) to the derivatization solution after obtaining the derivatization solution, and standing the solution at room temperature for 3 minutes. 8 μL of the obtained intermediate solution was also taken for subsequent analysis by narrow-bore HPLC-UV.

Table 9. Reaction temperature of each group in this test Group temperature H0 About 25℃ H1 -20℃

As shown in FIG. 19 , the cooling bath can effectively separate the derivatization solution, thereby enhancing the separation effect of the thiourea derivative (Group H1).

（I）HPLC chromatogram

Group I1 test samples: 100 μL of polyamine solution (containing 200 μM putrescine, 200 μM spermidine and 200 μM spermine, respectively, dissolved in water) was taken, 200 μL of derivatization reagent solution [containing 10 mM derivatization reagent (DNITC), dissolved in working solvent (acetonitrile)] and 25 μL of alkaline solution [containing 10 mM alkaline (sodium hydroxide), dissolved in water] was added, and after mixing evenly to form the reaction solution, the reaction solution was heated by microwave (300 W, 5 minutes), and then kept in an ice bath to terminate the thiocarbamate reaction, so as to obtain the derivatization solution as Group I1 test samples.

Group I2 test samples: 100 μL of water (without putrescine, spermidine and spermine) was taken, and 200 μL of a derivatization reagent solution [containing a derivatization reagent (DNITC) at a concentration of 10 mM, dissolved in a working solvent (acetonitrile)] and 25 μL of an alkaline solution [containing an alkaline (sodium hydroxide) at a concentration of 10 mM, dissolved in water] were added. After being mixed evenly to form the reaction solution, the reaction solution was heated by microwave (300 W, 5 minutes), and then kept in an ice bath to terminate the thiocarbamate reaction, so as to obtain the derivatization solution. Next, after adding 500 μL of a removal reagent (n-octane) to the derivatization solution, the upper layer solution was removed, and after adding 500 μL of a removal reagent (n-octane) again, the upper layer solution was removed. When 500 μL of a removal reagent (n-octane) was added for the third time, a mixed powder containing the proton donor (citric acid; 25 mg) and the foaming salt (potassium bicarbonate; 10 mg) was added at the same time, and the solution was allowed to stand at -20°C for 3 minutes to obtain a middle layer solution as the I2 group of test samples.

Group I3 test samples: 100 μL of polyamine solution (containing 200 μM putrescine, 200 μM spermidine and 200 μM spermine, respectively, dissolved in water) was taken, 200 μL of derivatization reagent solution [containing 10 mM derivatization reagent (DNITC), dissolved in working solvent (acetonitrile)] and 25 μL of alkaline solution [containing 10 mM alkaline (sodium hydroxide), dissolved in water] was added, after mixing evenly to form the reaction solution, the reaction solution was heated by microwave (300 W, 5 minutes), and then kept in an ice bath to terminate the thiocarbamate reaction, so as to obtain the derivatization solution. Next, after adding 500 μL of a removal reagent (n-octane) to the derivatization solution, the upper layer solution was removed, and after adding 500 μL of a removal reagent (n-octane) again, the upper layer solution was removed. When 500 μL of a removal reagent (n-octane) was added for the third time, a mixed powder containing the proton donor (citric acid; 25 mg) and the foaming salt (potassium bicarbonate; 10 mg) was added at the same time, and the solution was allowed to stand at -20°C for 3 minutes to obtain a middle layer solution as the test sample of group I3.

8 μL of each test sample was taken and analyzed by narrow-bore HPLC-UV. The results are shown in Figure 20. Although the thiourea derivatives of each polyamine were present in the test sample of group I1 (as shown by peaks 1, 2, and 3), it contained more derivatization reagents (DNITC). In the treated test sample of group I3, most of the derivatization reagents (DNITC) were removed, and there were still clear peaks representing the thiourea derivatives of each polyamine.

(J) Stability test of thiourea derivatives

In this test, 100 μL of polyamine solution (containing 200 μM putrescine, 200 μM spermidine and 200 μM spermine, respectively, dissolved in water) was added with 200 μL of derivatization reagent solution [containing 10 mM derivatization reagent (DNITC), dissolved in working solvent (acetonitrile)] and 25 μL of alkaline solution [containing 10 mM alkaline (sodium hydroxide), dissolved in water], and after mixing evenly to form the reaction solution, the reaction solution was heated by microwave (300 W, 5 minutes), and then kept in an ice bath to terminate the thiocarbamate reaction, so as to obtain the derivatization solution of each group. Next, 500 μL of a removal reagent (n-octane) and a mixed powder containing the proton donor (citric acid; 25 mg) and the foaming salt (potassium bicarbonate; 10 mg) were added to the derivatization solution, and the mixture was incubated at -20°C for 3 minutes to collect the middle layer solution.

8 μL of the middle layer solution was taken as the test solution every 3 hours and subsequently analyzed by narrow-bore HPLC-UV to compare the changes in the area under a peak of the thiourea derivatives representing each polyamine within 24 hours. The results are shown in Figure 21. Within 24 hours, the concentration of the thiourea derivatives representing each polyamine did not change significantly, indicating that the thiourea derivatives have good stability.

(J) Effectiveness evaluation

This test was conducted using polyamine solutions with concentrations of 1, 5, 25, 100 and 200 μM. After obtaining the test solution, subsequent analysis was performed using narrow-bore HPLC-UV. The linear regression equation and coefficient of determination (R ² ) obtained by linear regression calculation are shown in Table 10.

Table 10. Linear regression equation and coefficient of determination between different batches and the same batch Samples to be tested Linear regression equation Determination coefficient Same batch Putrescine y＝0.0058x＋0.0060 0.9999 Spermidine y＝0.114x＋0.0162 0.9999 Spermine y＝0.0119x＋0.0023 0.9998 Different batches Putrescine y＝0.0059x＋0.0058 0.9999 Spermidine y＝0.0107x＋0.0190 0.9998 Spermine y＝0.0121x－0.0010 0.9999

Please refer to Table 10. Whether it is the analysis between the same batch or the analysis between different batches, the coefficient of determination is greater than 0.9998, indicating that the polyamine detection method has a good linear relationship in the concentration range of 1 to 200 μM.

Next, polyamine solutions with concentrations of 3, 20, and 120 μM were tested. After obtaining the test solution, subsequent analysis was performed using narrow-bore HPLC-UV. The relative standard deviation (RSD) and relative error (RE) of the analysis results between different batches and within the same batch were calculated and recorded in Table 11.

Table 11. Relative standard deviation and relative error between the same batch and different batches Concentration (μM) Same batch Different batches RSD（%) RE（%) RSD（%) RE（%) Putrescine 3 2.96 -1.80 3.84 1.21 20 1.04 0.15 4.72 -3.11 120 0.53 0.53 1.13 -0.91 Spermidine 3 5.41 -1.21 2.30 -6.82 20 1.08 3.65 5.92 0.72 120 2.01 3.92 3.23 -0.42 Spermine 3 3.14 -4.15 4.79 5.59 20 1.84 -2.20 4.58 1.39 120 3.10 3.23 2.02 -10.9

Please refer to Table 11. In the analysis of the same batch, the relative standard deviation is less than 5.41% and the relative error is less than -4.15%. In the analysis of different batches, the relative standard deviation is less than 5.92% and the relative error is less than -6.82%, indicating that the polyamine detection method has good accuracy and precision, that is, it has good reliability.

(K) Test results of food samples

Commercially available brown rice, wheat, buckwheat, oats, soybeans and red wine were subjected to pre-treatment such as grinding and protein precipitation, and then analyzed using the polyamine detection method described above. The results are shown in Table 12.

Table 12. Test results of food samples Food samples Polyamine Concentration (mg/kg) RSD（%) brown rice Putrescine ND - Spermidine 2.55 7.49 Spermine 9.96 2.00 Wheat Putrescine 6.40 2.26 Spermidine 11.62 3.63 Spermine 12.31 1.59 Buckwheat Putrescine 4.07 2.08 Spermidine 17.45 2.22 Spermine 20.78 4.46 Oats Putrescine 3.47 4.85 Spermidine 6.43 0.62 Spermine 4.01 3.12 Soybean Putrescine 12.79 5.28 Spermidine 83.95 1.85 Spermine 24.35 3.82 Red wine 1 Putrescine 1.22 17.29 Spermidine 2.05 1.47 Spermine 4.19 2.29 Red wine 2 Putrescine 7.91 2.34 Spermidine 4.27 0.72 Spermine 5.91 7.65

As shown in Table 12, the polyamine detection method can indeed be applied to detect the polyamine content in food samples.

(L) Test results of cell line samples

This test selected cancer cell lines such as DU145 (prostate cancer), T24 (bladder cancer), Hep3B (liver cancer), Huh7 (liver cancer), HA22T (liver cancer), and Mahlavu (liver cancer). After the aforementioned cells were lysed, they were analyzed using the aforementioned polyamine detection method. The results are shown in Table 13.

Table 13. Test results of cell line samples Cell line samples Polyamine Concentration (ng/10 ⁷ cell) RSD（%) DU145 Putrescine 1202.19 2.45 Spermidine 1645.28 2.79 Spermine 1436.08 3.89 T24 Putrescine 542.30 9.53 Spermidine 450.88 5.67 Spermine ND - Hep3B Putrescine 2045.40 0.97 Spermidine 1436.79 1.65 Spermine 2881.44 5.36 Huh7 Putrescine 3732.80 5.48 Spermidine 630.65 7.61 Spermine 2701.00 6.10 HA22T Putrescine 3298.51 2.06 Spermidine 787.20 3.07 Spermine 2243.30 4.63 Mahlavu Putrescine 373.00 0.36 Spermidine 89.52 5.11 Spermine 531.39 1.78

As shown in Table 13, the polyamine detection method can indeed be applied to detect the polyamine content in cell line samples.

(K) Whole blood sample test results

This test used whole blood samples from five healthy subjects. After pre-treatment such as protein precipitation, the samples were analyzed using the polyamine detection method described above. The results are shown in Table 14.

Table 14. Test results of whole blood samples Whole blood samples Polyamine Concentration (μM) Subject 1 Spermidine 12.82±0.44 Spermine 2.16±0.31 Subject 2 Spermidine 12.52±0.24 Spermine 6.83±0.53 Subject 3 Spermidine 12.62±0.14 Spermine 5.74±0.13 Subject 4 Spermidine 10.56±0.35 Spermine 4.48±0.47 Subject 5 Spermidine 16.87±0.15 Spermine 6.31±0.86

As shown in Table 14, the polyamine detection method can indeed be applied to detect the polyamine content in whole blood samples.

In summary, the thiourea derivative can be obtained by reacting the amino group of the polyamine in the sample to be tested with the derivatization reagent (isothiocyanate having an isothiocyanate functional group). The thiourea derivative can be detected by various known methods (for example, after separation by liquid chromatography, gas chromatography, etc., and then detected by ultraviolet spectroscopy, fluorescence spectroscopy, or mass spectrometry). Therefore, the polyamine detection method of the present invention has good sensitivity, precision and accuracy, and can reduce the usage of the sample to be tested, which is the effect of the present invention.

Furthermore, the detection kit can be applied to implement the aforementioned polyamine detection method, and effectively forms a thiourea derivative with good stability together with the polyamine. Therefore, the worker can select various known methods to detect the thiourea derivative according to the needs (for example, after separation by liquid chromatography, gas chromatography, etc., detection by ultraviolet spectroscopy, fluorescence spectroscopy, or mass spectrometry), which is the effect of the present invention.

Although the present invention has been disclosed using the above preferred embodiments, they are not intended to limit the present invention. Any person skilled in the art can make various changes and modifications to the above embodiments without departing from the spirit and scope of the present invention, and the scope of protection of the present invention includes all changes within the meaning and equivalent scope of the attached patent application. In addition, when the above several embodiments can be combined, the present invention includes any combination of implementations.

[The present invention] S1: Sample providing step S2: Derivatization step S3: Analysis step S4: Removal step S5: Salting step S6: Salting removal step

[Figure 1] Flow chart of the method for detecting polyamines according to the first embodiment of the present invention. [Figure 2] Chemical equation of the thiocarbamate reaction of polyamines and a derivatization reagent (isothiocyanate). [Figure 3] Chemical structure of the derivatization reagent 4-dimethylamine-naphthyl isothiocyanate. [Figure 4] Chemical structure of the derivatization reagent 1-naphthyl isothiocyanate. [Figure 5] Chemical structure of the derivatization reagent benzylnaphthyl isothiocyanate. [Figure 6] Chemical structure of the derivatization reagent allylnaphthyl isothiocyanate. [Figure 7] Flow chart of the method for detecting polyamines according to the second embodiment of the present invention. [Figure 8] Flow chart of the method for detecting polyamines according to the third embodiment of the present invention. [Figure 9] Flow chart of the method for detecting polyamines according to the fourth embodiment of the present invention. [Figure 10]    Chemical structure of N,N-dimethyl-1-naphthylamine. [Figure 11]    Chemical structure of naphthalene. [Figure 12]    Bar graph of relative intensity of thiourea derivatives in each group of derivative solutions in test (A). [Figure 13]    Bar graph of relative intensity of thiourea derivatives in each group of test solutions in test (B). [Figure 14]    Bar graph of relative intensity of thiourea derivatives in each group of test solutions in test (C). [Figure 15]    Bar graph of relative intensity of thiourea derivatives in each group of test solutions in test (D). [Figure 16]    Bar graph of relative intensity of thiourea derivatives in each test solution in test (E). [Figure 17]    Bar graph of relative intensity of thiourea derivatives in each test solution in test (F). [Figure 18]    Bar graph of relative intensity of thiourea derivatives in each test solution in test (G). [Figure 19]    Bar graph of relative intensity of thiourea derivatives in each test solution in test (H). [Figure 20]    HPLC chromatogram of each test solution in test (I). Peaks 1, 2, and 3 refer to thiourea derivatives formed by putrescine, spermidine, and spermine, respectively; peak DNITC is the peak formed by the derivatization reagent (DNITC). [Figure 21]    Line graph of the relative strength changes of thiourea derivatives in each group of test solutions in test (J).

S1: Sample provision step S2: Derivatization step S3: Analysis step

Claims (28)

A method for detecting polyamines comprises: providing a sample to be tested, the sample to be tested containing polyamines; dissolving the sample to be tested and a derivatization reagent in a working solvent to form a reaction solution, the derivatization reagent having an isothiocyanate functional group; subjecting the polyamine in the reaction solution to a thiocarbamate reaction with the isothiocyanate functional group of the derivatization reagent to obtain a derivatization solution, the derivatization solution containing a thiourea derivative; and detecting the thiourea derivative to obtain a polyamine intensity value. The method for detecting polyamines according to claim 1, wherein the derivatization reagent is 4-dimethylamine-naphthyl isothiocyanate, 1-naphthyl isothiocyanate, benzyl naphthyl isothiocyanate or allyl naphthyl isothiocyanate. The method for detecting polyamines according to claim 1, wherein the working solvent is acetonitrile, acetone, dimethyl sulfoxide, pyridine, dimethylformamide, dimethylpropylene urea, cyclobutane sulfone, tetrahydrofuran, 1,2-dimethoxyethane, 1,3-dioxolane, dimethyl carbonate or water. The polyamine detection method of claim 1 further comprises: heating the reaction solution by microwave to drive the thiocarbamate reaction. The method for detecting polyamines according to claim 4, wherein the reaction liquid is microwaved at a wattage of 100 to 750 W. The polyamine detection method of claim 1 further comprises: adding an alkali to the reaction solution, and then subjecting the reaction solution containing the alkali to the thiocarbamate reaction. The method for detecting polyamines of claim 6, wherein the alkali is sodium hydroxide, sodium bicarbonate, sodium carbonate, 4-dimethylaminopyridine or triethylamine. The method for detecting polyamines as claimed in claim 1 further comprises: adding a removal reagent to the derivatization solution, allowing the removal reagent to remove fat-soluble interferents in the derivatization solution and form an upper solution and a lower solution, wherein the lower solution contains the thiourea derivative, and the removal reagent is a solvent that is immiscible with the derivatization solution and has a logP between 3.5 and 7.0. The method for detecting polyamines as claimed in claim 1 further comprises: adding a proton donor and a foaming salt to the derivative solution, allowing the proton donor to react with the foaming salt to form carbon dioxide gas, and forming an upper solution and a lower solution, wherein the upper solution contains the thiourea derivative. The method for detecting polyamines according to claim 9 further comprises: placing the derivatized solution in a cooling bath after adding the proton donor and the foaming salt and before forming the upper solution and the lower solution. The method for detecting polyamines as claimed in claim 1 further comprises: adding a removal reagent, a proton donor and a foaming salt to the derivatization solution, allowing the removal reagent to remove fat-soluble interferents in the derivatization solution, and allowing the proton donor to react with the foaming salt to form carbon dioxide gas, and to form an upper solution, a middle solution and a lower solution, wherein the middle solution contains the thiourea derivative, and the removal reagent is a solvent that is immiscible with the derivatization solution and has a logP between 3.5 and 7.0. The method for detecting polyamines according to claim 11 further comprises: placing the derivatization solution in a cooling bath after adding the removal reagent, the proton donor and the foaming salt and before forming the upper solution, the middle solution and the lower solution. The method for detecting polyamines according to claim 8 or 11, wherein the removal reagent is n-hexane, n-octane, n-decane or dodecane. The method for detecting polyamines according to claim 9 or 11, wherein the proton donor is citric acid, ammonium chloride, ammonium sulfate or sodium dihydrogen phosphate. The method for detecting polyamines according to claim 9 or 11, wherein the foaming salt is potassium bicarbonate, sodium bicarbonate, ammonium bicarbonate, potassium carbonate or sodium carbonate. A polyamine detection kit comprises: a derivatization reagent having an isothiocyanate functional group; and a working solvent which is a solvent capable of dissolving the derivatization reagent and polyamine. A polyamine detection kit as claimed in claim 16, wherein the derivatization reagent is 4-dimethylamine-naphthyl isothiocyanate, 1-naphthyl isothiocyanate, benzyl naphthyl isothiocyanate or allyl naphthyl isothiocyanate. A polyamine detection kit as claimed in claim 16, wherein the working solvent is acetonitrile, acetone, dimethyl sulfoxide, pyridine, dimethylformamide, dimethylpropylene urea, cyclobutane sulfone, tetrahydrofuran, 1,2-dimethoxyethane, 1,3-dioxolane, dimethyl carbonate or water. The polyamine detection kit of claim 16 further comprises: an alkali, which is an alkaline compound that can be dissolved in the working solvent. As in the polyamine detection kit of claim 19, wherein the alkali is sodium hydroxide, sodium bicarbonate, sodium carbonate, 4-dimethylaminopyridine or triethylamine. The polyamine detection kit of claim 16 further comprises: a removal reagent, which is a solvent with a logP between 3.5 and 7.0. The polyamine detection kit of claim 16 further comprises: a proton donor for providing hydrogen ions; and a foaming salt for reacting with the proton donor to undergo an acid-base neutralization reaction to form carbon dioxide gas. The polyamine detection kit of claim 16 further comprises: A removal reagent, which is a solvent with a logP between 3.5 and 7.0; A proton donor, which is used to provide hydrogen ions; and A foaming salt, which is used to undergo an acid-base neutralization reaction with the proton donor to form carbon dioxide gas. A polyamine detection kit as claimed in claim 21 or 23, wherein the removal reagent is n-hexane, n-octane, n-decane or dodecane. The polyamine detection kit of claim 22 or 23, wherein the proton donor is citric acid, ammonium chloride, ammonium sulfate or sodium dihydrogen phosphate. A polyamine detection kit as claimed in claim 22 or 23, wherein the foaming salt is potassium bicarbonate, sodium bicarbonate, ammonium bicarbonate, potassium carbonate or sodium carbonate. An in vitro method for diagnosing cancer, comprising: Using the polyamine detection method of any one of claims 1 to 15, in vitro detection of the polyamine content in a biological sample taken from a suspected patient to obtain a detection value; and Comparing the detection value of the biological sample with a reference value, when the detection value is higher than the reference value, it indicates that the suspected patient suffers from cancer. The in vitro diagnosis method of cancer as claimed in claim 27, wherein the cancer is liver cancer, prostate cancer, pancreatic cancer, colorectal cancer, breast cancer, lymphoma, fibrosarcoma, lung cancer, gastric cancer or oral cancer.