JP4928247B2 - Method for quantifying iminosaccharides - Google Patents

Description

  The present invention relates to a detection method for accurately detecting iminosaccharides contained in extracts such as plants, pharmaceuticals, health foods and the like.

  1-deoxynojirimycin (hereinafter abbreviated as “DNJ”), 1-deoxymannojirimycin (hereinafter abbreviated as “DMJ”) and 2,5-dideoxy-2,5-imino-D-mannitol (hereinafter abbreviated as “DMDP”) having a pyrrolidine skeleton ) And the like are known to have an action of inhibiting the activity of α-glucosidase to suppress an increase in blood sugar (see Patent Document 1). Many of these iminosaccharides are contained in plants such as Aobana, Mulberry leaves, Oboboshibana, Azalea, etc. These plants are subjected to hot water extraction and the like to obtain and use an extract containing iminosaccharides.

  In Patent Document 1, the content of iminosaccharide in the extract is quantified by high performance liquid chromatography / mass spectrometry (LC / MS), but the mass spectrometer is a very expensive analytical instrument such as a university. However, since it is not widely used, it has been problematic in that it requires cost and time to detect and quantify iminosaccharides.

Therefore, in Patent Document 2, after a sample containing DNJ is separated by liquid chromatography, the content of DNJ is measured with a light scattering detector. However, the light scattering detector is also an expensive detector and is not widely used, and the obtained chromatogram is complicated, and it is difficult to accurately quantify DNJ.
JP 2002-316935 A JP 2004-294384 A

  The present invention has been made in view of the above points, and an object of the present invention is to provide an iminosaccharide quantification method capable of accurately detecting and quantifying iminosaccharide while being an economical and simple method. Is.

  A method for quantifying iminosaccharides according to claim 1 of the present invention is a method for detecting and quantifying iminosaccharides, wherein a sample containing iminosaccharides is introduced into a liquid chromatography and flows out from the column of the liquid chromatography. A sample is introduced into a differential refractometer, and iminosaccharide is detected and quantified.

The method of quantifying imino sugars according to claim 1 of the present invention, after obtaining the extract by immersing the raw material containing imino sugars in the solvent, was passed through the extract to cation exchange resin A sample containing an iminosaccharide is prepared by adsorbing an iminosaccharide on a cation exchange resin and then eluting the iminosaccharide adsorbed on the cation exchange resin with an ammonia solution.

  In the first aspect of the invention, by using an inexpensive differential refractometer, iminosaccharides can be detected and quantified by an economical and simple method, and the iminosaccharide can be easily detected without complicating the chromatogram. The iminosaccharide can be detected and quantified with high accuracy.

Further, in the invention of claim 1 , impurities can be removed from the sample before being subjected to liquid chromatography, the chromatogram is not complicated, and iminosaccharides can be detected and quantified more accurately. Is.

  Hereinafter, the best mode for carrying out the present invention will be described.

  In the present invention, it is possible to detect and quantify iminosaccharides contained in plants such as green mulberry, mulberry, long-horned beetle, and camellia. Examples of iminosaccharides include DNJ, DMJ, and DMDP represented by the following chemical formula. In the present invention, at least one of DNJ, DMJ, and DMDP can be detected and quantified. In the present invention, the “iminosaccharide” refers to a compound in which the ring oxygen atom of a monosaccharide is substituted with an imino group, and a compound or derivative in which the hydroxyl group at the 1-position is a hydrogen atom (a compound having a 5-membered ring) Including).

  The preparation of a sample containing iminosaccharides derived from plants is performed as follows. First, the whole plant plant (various parts such as stems, leaves, roots and flowers) is dried to obtain a raw material containing iminosaccharide. Here, drying conditions can be 12 to 48 hours at room temperature to 60 ° C. The raw material is preferably a powder (dried powder) pulverized after drying. In addition, when detecting or quantifying iminosaccharides contained in pharmaceuticals and health foods, pharmaceuticals and health foods can be used as raw materials instead of the above-mentioned dry powders of plants.

  Next, the iminosaccharide is extracted by immersing the raw material in a solvent. Water or alcohol can be used as a solvent used for extraction, and methanol, ethanol, propanol, butanol, or the like can be used as the alcohol. These water and solvent can be used alone or as a mixed solvent mixed at an arbitrary ratio. When a mixed solvent of water and alcohol is used, the mixing ratio can be 0 to 100% in terms of alcohol concentration. Extraction can be performed by an ultrasonic extraction method or the like, and can be carried out by blending a solvent at a rate of 10 to 50 ml with respect to 1 g of raw material and applying ultrasonic waves thereto. The extraction can be performed at a solution temperature of room temperature to 90 ° C. and an extraction time of 10 to 120 minutes. And the supernatant liquid of the solvent containing the raw material after extraction can be filtered with a filter paper, and an extract can be obtained.

  Next, the extract containing the iminosaccharide is obtained by concentrating the above extract under reduced pressure. Here, the pressure during vacuum concentration can be 50 to 100 Pa, and the temperature can be room temperature to 40 ° C. Further, the concentration ratio is preferably 30 to 100%.

  Next, the concentrate is dissolved in a predetermined amount of a solvent such as water, and the solution is filtered through a filter paper to obtain a filtrate.

Next, a certain amount of this filtrate is accurately weighed and passed through a cation exchange resin to adsorb iminosaccharides to the cation exchange resin. As the cation exchange resin, both weakly acidic cation exchange resins and strongly acidic cation exchange resins can be used. As the weak acid cation exchange resin, for example, Amberlite FPC3500 manufactured by Organo Corporation can be used, and as the strong acid cation exchange resin, for example, Amberlite IR120BHAG manufactured by Organo Corporation can be used. it can. In addition, cartridge-type solid phase extraction may be used for passing the filtrate. In addition, it is preferable to condition the cation exchange resin in advance with 2M sodium hydroxide and 2M hydrochloric acid before passing the filtrate. Moreover, as a strong acid cation exchange resin, the following can be used as said Amberlite 120BHAG alternative.
<Mitsubishi Chemical Corporation>
・ Diaion ^R SK series (SK1B, SK104, SK110, SK112, SK116)
・ Diaion ^R PK series (PK208, PK212, PK216, PK220, PK228)
・ Diaion ^R UBK series (UBK530, UBK550, UBK535, UBK555)
<Nippon Biorad Co., Ltd.>
・ AG-50W
・ AG-MP50
Next, the cation exchange resin adsorbed with the iminosaccharide is washed with water, and then washed with an alcohol such as methanol, and then the ammonia solution is passed through the cation exchange resin, so that the imino adsorbed on the cation exchange resin The saccharide is eluted and collected. As the ammonia solution, an aqueous ammonia solution having a concentration of 0.01 to 28% can be used. If an aqueous ammonia solution other than this concentration is used, iminosaccharides may not be efficiently eluted. Moreover, you may mix | blend an organic solvent in ammonia aqueous solution with a density | concentration of 0.01-28%. In this case, as the organic solvent, an organic solvent having a high compatibility with water (such as methanol, ethanol, isopropyl alcohol, acetone, acetonitrile, tetrahydrofuran) (highly miscible) can be used. It is preferable to blend at 100% (equivalent) or less with respect to the total volume of the aqueous ammonia solution. And by mix | blending an organic solvent with ammonia aqueous solution, compared with the case where it does not mix | blend, imino saccharides can be more efficiently eluted from a cation exchange resin, and imino saccharides can be detected and quantified with high accuracy. Is.

Next, the solvent is distilled off from the ammonia solution from which the iminosaccharide has been eluted to obtain a residue. This solvent can be distilled off in a water bath under a reduced pressure of 50 to 100 Pa. Next, the above residue was dissolved in a mixed solvent of acetonitrile and water, and the residue was dissolved in a solid phase extraction cartridge column having an amino group (such as Sepp Pack NH ₂ manufactured by Waters Co., Ltd.) as necessary. By passing the solution, the sample used in the present invention can be obtained. In this case, the mixing ratio of acetonitrile and water can be set to acetonitrile: water = 4: 6 to 6: 4 by volume ratio. The column is preferably conditioned in advance by passing a mixed solution of 50% (V / V%) acetonitrile and water before passing the sample.

In addition, as a solid phase extraction cartridge column having an amino group, the following can be used as a substitute for Ceppack NH ₂ .
<Made by GL Sciences Inc.>
Bond Elute ^R Regular [ion exchange] NH ₂ aminopropyl Bond Elute LRC ^R type ion-exchange phase NH ₂ aminopropyl Mega Bond Elute ^R ion-exchange phase NH ₂ aminopropyl Bond Elute High Flow ^R Regular Ion Exchange] NH ₂ HF Aminopropyl Megabond Elut High Flow ^R Ion Exchange Phase NH ₂ Aminopropyl Bond Elut LRC High Flow ^R Type Ion Exchange Phase NH ₂ HF Aminopropyl Bond Elut Junior ^R Ion Exchange Phase NH ₂ Aminopropyl -Solid phase extraction filler-Bondecyl-Ion exchange NH ₂ aminopropyl-Bond elute matrix ^R NH ₂ aminopropyl-Versaplate ^TM NH ₂
<Phenomenex ^R company>
・ Strata NH ₂
Strata 96 well plate NH ₂
SEPRA ^™ bulk filler ion exchange NH ₂
<Oltech's product>
・ SPE Solid phase extraction filler Normal phase Aminopropyl NH ₂
・ NH ₂ Extract Clean Ultra Clean SPE Solid Phase Extraction ・ Extract Clean EV for NH ₂ Robot Solid Phase Extraction ・ NH ₂ Maxi Clean SPE Solid Phase Extraction Cartridge ・ Solid Phase Extraction Bulk Filler Polar Adsorbent NH ₂
<Merck Co.>
・ Rechlorute ^R extraction column NH ₂
In the present invention, the sample prepared as described above is introduced into liquid chromatography. High-performance liquid chromatography can be used as the liquid chromatography, and the column (stationary phase) is obtained by chemically bonding a polar group such as an amide group to a carrier such as silica gel from the viewpoint of separation performance of iminosaccharide. However, it is not limited to this.

  As a mobile phase used in liquid chromatography, a mixed solution of water and an organic solvent in which iminosaccharide can be dissolved can be used. The organic solvent is not particularly limited as long as it is miscible with water, and examples thereof include lower alcohols such as methanol, ethanol and isopropyl alcohol, acetonitrile, tetrahydrofuran and the like. In the present invention, it is preferable to use a polar solvent such as acetonitrile, methanol, ethanol or the like, and it is particularly preferable to use a mixed solution of water and acetonitrile. The mixing ratio of water and organic solvent can be 1: 9 to 9: 1, preferably 5: 5 to 1: 9. Moreover, it is preferable not to use a salt for the mobile phase and to perform the separation near neutrality (pH 5 to 7). As a result, there is no salt precipitation in liquid chromatography, and there is no need to add acidic substances to the mobile phase, so the burden on the column and liquid chromatography equipment is low, and maintenance after use is easy. Can be done. The column temperature can be from room temperature to 50 ° C., and the flow rate can be from 0.5 to 1.5 ml / min, but is not limited thereto.

  In the present invention, the sample introduced into the liquid chromatography as described above is introduced into the differential refractometer (RI detector) while flowing out of the column. As the differential refractometer, those conventionally used for chemical analysis can be used. For example, shodex RI-101 manufactured by Showa Denko KK can be used. Then, a chromatogram is obtained with a differential refractometer, and the various iminosaccharides can be identified and detected by the position of the peak (retention time on the chromatogram). Moreover, various iminosaccharides contained in the sample can be quantified by calculating the area of each peak. In this case, the identification and quantification of the various iminosaccharides in the sample can be performed by comparing the data of known chromatograms of the various iminosaccharides with the data of the chromatogram obtained by measuring the sample. In other words, apart from the sample, a standard solution in which the content of various iminosaccharides is known is prepared, and this is analyzed by liquid chromatography and a differential refractometer in the same manner as the sample to obtain a chromatogram. By comparing the chromatogram of the standard solution with the chromatogram of the sample, various iminosaccharides in the sample can be identified and quantified.

  Hereinafter, the present invention will be described specifically by way of examples.

[Preparation of Sample 1]
Aobana crude drug (Aobana whole grass (stems, leaves, roots, flowers) cultivated in Kusatsu City, Shiga Prefecture) was dried at 60 ° C. for 24 hours and pulverized to obtain a dried Aobana raw material. Next, 17 g of this raw material was precisely weighed, then immersed in 300 ml of water, and subjected to ultrasonic extraction for 10 minutes at room temperature. This liquid was filtered with a filter paper to obtain an extract. Next, the filtered residue was immersed in 200 ml of water and subjected to ultrasonic extraction as described above, and this liquid was filtered with a filter paper to obtain an extract. Next, the filtered residue was immersed in 300 ml of water and subjected to ultrasonic extraction as described above, and this liquid was filtered with a filter paper to obtain an extract. In the above extraction, 100% water was used, but methanol of 50% concentration could be used instead.

  After the total extracts obtained by the above three extractions were combined, the extract was concentrated under reduced pressure under the conditions of a pressure of 50 to 100 Pa, a room temperature to 40 ° C., and a concentration ratio of 80 to 100% to obtain a concentrate. . Next, the concentrate was made up to a volume of 50 ml with water, and the solution was filtered with a filter paper to obtain a filtrate. Next, 10 ml of the filtrate was accurately weighed and passed through a 50 cc strongly acidic cation exchange resin (Amberlite IR120BHAG manufactured by Organo Corporation). Before passing through the filtrate, the strongly acidic cation exchange resin was conditioned in advance with 2M sodium hydroxide and 2M hydrochloric acid.

The strongly acidic cation exchange resin was washed with 150 ml of water, washed with 30 ml of methanol, and further washed with 100 ml of water. 80 ml of an aqueous ammonia solution having a concentration of 2% was passed through this strongly acidic cation exchange resin to collect iminosaccharides. Next, the aqueous ammonia was distilled off from the aqueous ammonia solution after passing through to obtain 1 ml of a residue. Ammonia water was distilled off in a water bath under reduced pressure of 50 to 100 Pa. Next, the above residue is dissolved in a mixed solvent of acetonitrile and water (mixing ratio is acetonitrile: water = 5: 5 in a volume ratio), and a solid phase extraction cartridge column having an amino group (Ceppack manufactured by Waters Co., Ltd.) NH ₂ etc.) was passed through a solution in which the residue was dissolved, the passing liquid was collected, and the solvent was distilled off on a water bath under reduced pressure. Sample 1 was obtained by adding a mixed solvent of acetonitrile and water to the residue (mixing ratio of acetonitrile: water = 4: 6 in volume ratio).

[Preparation of Sample 2]
Sample 2 was obtained in the same manner as Sample 1, except that mulberry leaves (mulberry leaves cultivated in Ikeda City, Tokushima Prefecture) were used instead of Aobana.

[Preparation of Sample 3]
Sample 3 was obtained in the same manner as Sample 1 except that Tsuyukusa (whole plant of Tsuyukusa cultivated in Ikeda City, Tokushima Prefecture) was used instead of Aobana.

Example 1
Chromatograms of Samples 1 to 3 were obtained using high performance liquid chromatography (LC10AD manufactured by Shimadzu Corporation) and a differential refractometer (shodex RI-101 manufactured by Showa Denko KK). 1A shows the chromatogram of sample 1, FIG. 1B shows the chromatogram of sample 2, and FIG. The vertical axis of this chromatogram is the signal intensity.

The conditions of high performance liquid chromatography used in the examples are as follows.
Column: Aminopropylsilylated silica gel (Cosmosil Packed ColumnSUGAR-D (4.6 × 250mm), same guard column)
Mobile phase: acetonitrile: water = 75: 25 (w / w%) mixed solvent was filtered through a membrane filter (for water resistance, 0.45 μm) and then degassed Column temperature: 35 ° C.
Flow rate: 0.8 ml / min (Adjust so that the DNJ retention time is about 19 minutes)
(Comparative Example 1)
Chromatograms of Samples 1 to 3 were obtained in the same manner as described above except that an ultraviolet-visible spectrophotometer (UV, VIS detector) (SPD20A manufactured by Shimadzu Corporation) was used instead of the differential refractometer. 2A shows the chromatogram of sample 1, FIG. 2B shows the chromatogram of sample 2, and FIG. The vertical axis of this chromatogram is the signal intensity.

(Comparative Example 2)
A chromatogram and a mass spectrum of Sample 1 were obtained in the same manner as described above except that a mass spectrometer (M8000 LC / 3DQMS manufactured by Hitachi, Ltd.) was used instead of the differential refractometer. 3A is a chromatogram for molecular weight (m / z) 50 to 200, and FIG. 3B is a chromatogram for molecular weight (m / z) 164. FIG. 4A is a mass spectrum for identifying DNJ, and FIG. 4B is a mass spectrum for identifying DMDP.

  When FIG. 1 is compared with FIG. 2, there are few noise peaks in FIG. 1 and the peaks of DNJ, DMJ, and DMDP are clear and easy to distinguish, whereas in FIG. 2, there are many noise peaks and DNJ, DMJ and DMDP peaks are unclear and difficult to distinguish. This is probably because the sample obtained from the plant contains a large amount of organic matter that is absorbed in the ultraviolet and visible light regions, so that Comparative Example 1 is a more complex chromatogram than the Example. In addition, in the ultraviolet visible spectrophotometer, it cannot detect unless the target compound is a compound having absorption in the UV region. Since iminosaccharide has no absorption in the UV region, it can be detected by derivatization to a compound having absorption in the UV region using FMOC (9-fluorenyl chloroformate), etc. In addition, the operation is complicated and the chromatogram is often complicated. In such a case, the quantitative property is often lacking. In addition, when analyzing iminosaccharides on the lower wavelength side, peaks often overlap with other compounds.

  Further, in FIG. 3, there are few noise peaks, and the DNJ, DMJ, and DMDP peaks can be discriminated. In particular, in FIG. 3B, the DNJ, DMJ, and DMDP peaks can be clearly and easily discriminated. However, mass spectrometers are expensive and are not widely used. In addition, when analyzing in the presence of contaminants and a highly pure standard product, ionization conditions may be different, and skill is required for analysis. In addition, it is difficult to continuously analyze multiple samples.

  FIG. 5 shows FIG. 1 of Patent Document 2. Although this is a chromatogram using a light scattering detector, there are many noise peaks and the peaks of DNJ, DMJ and DMDP are unclear and difficult to discriminate. Further, the light scattering detector is more expensive than the differential refractometer and is not widely used.

[Preparation of sample solution for determination of DNJ in Aobana bulk drug]
A sample solution was prepared in the same manner as Sample 1 above. That is, after the dried Aobana crude drug substance was pulverized, 17 g was accurately weighed and extracted with 300 ml of hot water of about 90 ° C. or higher for 120 minutes and filtered through filter paper. The filtrate is distilled off in a water bath under reduced pressure, and then made up to 50 ml with water. 10 ml of this solution was accurately weighed, passed through a strongly acidic cation exchange resin (Amberlite IR120BHAG manufactured by Organo Corporation), washed with water, washed with methanol, and then the iminosaccharide fraction with 2% aqueous ammonia solution. Was collected.

Next, the solvent obtained by collecting the fraction of iminosaccharide was distilled off on a water bath under reduced pressure, and then dissolved in a mixed solvent of acetonitrile and water (mixing ratio is 5: 5 by volume), The solution is passed through a pre-conditioned solid phase extraction cartridge (such as Ceppack NH ₂ manufactured by Waters Co., Ltd.) to collect the passing solution. Next, after distilling off the solvent in a water bath under reduced pressure, 1 ml of a mixed solvent of acetonitrile and water (a mixing ratio of 4: 6 by volume) was accurately added to prepare a sample solution. The sample solution was prepared in the same manner to prepare 5 types (raw drug samples 1 to 5).

[Preparation of sample solution for quantification of DNJ in aobana extract powder]
Weigh accurately 2.5 g of Aoba na extract powder, add water and mix well to make exactly 50 ml. After filtering this liquid through a filter paper, weigh exactly 10 ml, and pass it through a strongly acidic cation exchange resin (Amberlite IR120BHAG manufactured by Organo Corporation), followed by washing with methanol, followed by 2% ammonia aqueous solution. The iminosaccharide fraction was collected at

Next, the solvent was distilled off from the liquid in which the fraction of iminosaccharide was collected on a water bath under reduced pressure, and then dissolved in a mixed solvent of acetonitrile and water (mixing ratio is 5: 5 by volume). Then, the solution is passed through a pre-conditioned solid phase extraction cartridge (such as Ceppack NH ₂ manufactured by Waters Co., Ltd.) to collect the passing solution. Next, after distilling off the solvent in a water bath under reduced pressure, 1 ml of a mixed solvent of acetonitrile and water (a mixing ratio of 4: 6 by volume) was accurately added to prepare a sample solution.

  In addition, the sample solution performed the same operation and produced three types (extract powder samples 1-3).

[Preparation of standard solution for quantification of DNJ]
Weigh 5 milligrams of DNJ standard product (1-deoxynojirimycin manufactured by Wako Pure Chemical Industries, Ltd.) and add 10 ml of a mixed solvent of acetonitrile and water (mixing ratio is 4: 6 by volume) to this standard. It was set as the solution.

(Example 2)
Weigh 20 microliters of the sample solution and standard solution obtained from Aobana drug substance, and obtain the chromatogram of the sample solution and standard solution using liquid chromatography and differential refractometer in the same manner as in Example 1 above. Then, the peak area of DNJ in the chromatogram of the sample solution relative to the peak area of DNJ in the chromatogram of this standard solution was determined, and the content of DNJ in the Aobana drug substance was measured. In this measurement, the following formulas (1) and (2) were used.
DNJ detection amount (mg) = DNJ standard product use amount × (DNJ peak area of sample solution chromatogram) / (DNJ peak area of standard solution chromatogram) (1)
DNJ content (%) = DNJ detection amount / Aobana drug substance use amount × 100 (2)
(Example 3)
Except that the sample solution obtained from Aobana extract powder was used, the DNJ content in the Aobana extract powder was measured in the same manner as in Example 2. In the above formulas (1) and (2), “Aobana raw drug” was used as “Aobana extract powder”.

(Comparative Example 3)
In the same manner as in Example 2, except that the chromatograms of the sample solution and the standard solution obtained from Aobana drug substance were obtained using liquid chromatography and a mass spectrometer in the same manner as in Comparative Example 2. The DNJ content was measured.

(Comparative Example 4)
The DNJ content in the Aoba na extract powder was measured in the same manner as in Comparative Example 3 except that the sample solution obtained from the Aoba na extract powder was used.

  The results of Examples 2 and 3 and Comparative Examples 3 and 4 are shown in Table 1.

  As is clear from Table 1, the quantitative value of the DNJ content according to Examples 2 and 3 using a differential refractometer, and the quantitative value of the DNJ content according to Comparative Examples 3 and 4 using a mass spectrometer, and Show almost equivalent results. Therefore, the present invention has a performance equivalent to that when using a mass spectrometer that can quantify most accurately at present. In addition, the variation of the quantitative value (n = 3) of this invention is CV = 2 to 3%, and is favorable.

[Preparation of sample solution for preparing calibration curve]
10 milligrams of 1-deoxynojirimycin (manufactured by Wako Pure Chemical Industries, Ltd.) is accurately weighed and adjusted to a volume of 10 milliliters with a mixed solvent of acetonitrile and water (formulation ratio is 4: 6 by volume). This solution was appropriately diluted with the above solvent, and then a chromatogram was obtained using the same liquid chromatography and differential refractometer as in Example 1. The peak area of DNJ was obtained from this chromatogram, and the horizontal axis The concentration and the peak area value were plotted on the vertical axis, and regression analysis was performed to obtain a calibration curve. The result is shown in FIG. This calibration curve was a regression equation <y = 679815x-16109> and a correlation coefficient <r ² = 0.9992>, and a good straight line was obtained.

The chromatogram obtained by this invention is shown, (a) shows sample 1, (b) shows sample 2, and (c) shows the chromatogram of sample 3. The chromatogram at the time of using an ultraviolet visible spectrophotometer is shown, (a) shows sample 1, (b) shows sample 2, (c) shows the chromatogram of sample 3, respectively. The chromatogram at the time of using a mass spectrometer is shown, (a) is a chromatogram about the molecular weight (m / z) 50-200 of the sample 1, (b) is a chromatogram about the molecular weight (m / z) 164 of the sample 1. Respectively. (A) (b) shows the mass spectrum at the time of using the mass spectrometer about the sample 1. FIG. The chromatogram at the time of using a light-scattering detector is shown. It is a graph which shows the calibration curve of DNJ obtained by the present invention.

Claims (1)

A method for detecting and quantifying iminosaccharides, wherein an extract is obtained by immersing a raw material containing iminosaccharides in a solvent, and then the extract is passed through a cation exchange resin to obtain a cation exchange resin. A sample containing iminosaccharide is prepared by adsorbing iminosaccharide, and then eluting the iminosaccharide adsorbed on the cation exchange resin with an ammonia solution, and this sample is introduced into liquid chromatography. A method for quantifying iminosaccharides, comprising introducing a sample flowing out from the column into a differential refractometer and detecting and quantifying iminosaccharides.

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