JP7801738B2 - Pretreatment method and quantification method - Google Patents

Description

The present invention relates to a method for pretreating a sample containing a substance to be quantified and a method for quantifying the substance.

Foods with functional claims are provided to consumers with a display of functions based on the effects of the ingredients contained in the food. When providing a food with functional claims to consumers, the amount of active ingredient (functional ingredient) contained and its quantification method must be notified. Foods with functional claims may contain plant extracts containing active ingredients. Because plant extracts contain both active ingredients and other ingredients (impurities), it is important that the accuracy of the quantification results for the active ingredients is not reduced by impurities.

A commonly known method for quantifying the above-mentioned active ingredients involves performing liquid chromatography analysis using a standard substance of the active ingredient as the substance to be quantified, creating a calibration curve for the substance to be quantified in advance from the peak areas of the resulting chromatogram, subjecting a plant extract containing the substance to be quantified to liquid chromatography, determining the peak areas from the resulting chromatogram, and quantifying the substance to be quantified using the calibration curve.

Japanese Patent Application Laid-Open No. 2005-221425

In the method of quantifying the target substance from the peak area of the chromatogram obtained by the liquid chromatography process, if the peak of the target substance overlaps with the peak of the impurity, the accuracy of the quantification result of the target substance obtained based on the peak area will be reduced. Therefore, impurities that appear as peaks overlapping with the target substance must be removed as much as possible from the plant extract subjected to the liquid chromatography process.

In view of the above problems, the present invention aims to provide a pretreatment method for quantifying a target substance contained in a plant extract with high accuracy, and a quantification method including the pretreatment method.

To solve the above-mentioned problems, the present invention provides a pretreatment method for quantifying a target substance in a sample containing the target substance and impurities, comprising the steps of: subjecting the sample to alkaline treatment using an alkali; acidifying the sample using an acid; and subjecting the acidified sample to a filtration process using diatomaceous earth as a filter aid, wherein the filter aid contains diatomaceous earth having a water permeability of 85 millidarcy (mDarcy) or less.

The method may include a step of filtering the sample and then eluting the target substance from the diatomaceous earth. The target substance may be eluted from the diatomaceous earth using an alkyl acetate solution. The pH of the sample may be adjusted to 5.0 or less using the acid, and the sample to which the filter aid has been added may be filtered using a filter medium with the filter aid layered on the upstream side. The target substance may be a ceramide, such as glucosylceramide. The impurities may be cellulose, such as hydroxypropyl cellulose and/or hydroxypropylmethyl cellulose.

A method for quantifying a substance to be quantified in a sample containing the substance and impurities, the method comprising the steps of pretreating the sample using the pretreatment method described above, and subjecting the sample after the pretreatment to liquid chromatography analysis.

The present invention provides a pretreatment method for quantifying a target substance contained in a plant extract with high accuracy, and a quantification method that includes this pretreatment method.

FIG. 1 is a flowchart showing a method for quantifying glucosylceramide according to one embodiment of the present invention.

An embodiment of the present invention will now be described in detail.
The quantitative method according to this embodiment includes a pretreatment method (S1) for pretreating a sample containing a substance to be quantified and impurities, and a step (S2) for subjecting the pretreated sample to liquid chromatographic analysis.

In this embodiment, the substance to be quantified may be, for example, a ceramide such as glucosylceramide. The glucosylceramide is preferably obtained from the edible portion of pineapple via extraction using, for example, an 80-100% vol. ethanol aqueous solution. The glucosylceramide may also be obtained from glucosylceramide-containing crops such as rice, konjac, corn, and wheat. The contaminants contained in the sample may be, for example, celluloses such as hydroxypropyl cellulose (HPC) and/or hydroxypropyl methylcellulose (HPMC). Hydroxypropyl cellulose and hydroxypropyl methylcellulose are commonly used as binders in pharmaceutical formulations and are also widely used in the field of food processing. Therefore, the sample containing the substance to be quantified and the contaminants may be an aqueous solution of a composition containing the ceramides and the celluloses, such as an aqueous solution of tablets or capsules containing the ceramides and the celluloses.

The pretreatment method in this embodiment includes the steps of subjecting a sample to alkaline treatment (S11), acidifying the sample after alkaline treatment (S12), subjecting the acidified sample to filtration using a filter aid (S13), and eluting the substance to be quantified from the filter aid (S14).

In the step (S11) of subjecting the sample to alkaline treatment, for example, 0.2 to 0.6 mol/L of alkali may be added to the sample. By subjecting the sample to alkaline treatment, it is possible to remove fatty acid esters and the like contained in the sample. Examples of alkali that can be used include aqueous sodium hydroxide, potassium hydroxide, calcium hydroxide, and lithium hydroxide. The amount of alkali added to the sample is not particularly limited and is set appropriately to an amount that can remove the fatty acid esters and the like. Note that if the amount of alkali added is relatively too large, the amount of acid added to acidify the sample in the step (S12) described below will be relatively increased, so it is preferable that the amount of alkali added is the minimum amount necessary to remove the fatty acid esters and the like.

In the step (S12) of acidifying the sample, acid is added to the alkaline-treated sample. By subjecting the sample to alkaline treatment, the sample becomes alkaline. If the alkaline sample is subjected to a filtration process (S13) described below to elute the target substance (S14), the recovery rate of the target substance will decrease. In the quantification method according to this embodiment, the target substance is quantified from the fraction obtained by eluting the target substance. Therefore, if the recovery rate of the target substance decreases, the quantification accuracy will decrease. By adding acid to an alkaline sample to acidify the sample, as in this embodiment, the recovery rate of the target substance can be made substantially 100% (99% or higher).

The acid to be added to the sample is not particularly limited as long as it can make the sample acidic, and examples include hydrochloric acid, sulfuric acid, nitric acid, and citric acid. The amount of acid added to the sample may be, for example, such that the pH of the sample is 5.0 or less, preferably 1.0 to 3.0. Adjusting the sample pH to 5.0 or less can improve the recovery rate of the substance to be quantified during sample pretreatment, thereby improving quantification accuracy.

In the step (S13) of subjecting the acidified sample to a filtration treatment using a filter aid, diatomaceous earth having a water permeability of 85 millidarcy (mDarcy) or less, preferably 20 to 65 millidarcy (mDarcy), can be used as the filter aid. As will be apparent from the test examples described below, using diatomaceous earth having a water permeability of 85 millidarcy (mDarcy) or less as a filter aid can improve the recovery rate of the substance to be quantified in sample pretreatment and can improve quantification accuracy. When a fluid with a viscosity of 1 cP (e.g., pure water at 20°C) flows through a filter aid layer (thickness: 1 cm, cross-sectional area: 1 ^cm2 ) made of diatomaceous earth at a flow rate of 1 ^cm3 /sec with a pressure difference of 1 atmosphere, its permeability is called 1 Darcy (Darcy).

The above-mentioned filtration method can be performed, for example, by placing a filter material such as cellulose filter paper on a perforated tray of a Kiriyama funnel or the like, running pure water or the like through the filter material, and suction-filtering a suspension of a filter aid in pure water or the like to form a filter aid layer (hereinafter sometimes referred to as the "first filter aid layer") on the filter material. Then, a sample adjusted to an acidic condition is suction-filtered (the so-called precoat method). Alternatively, a method in which a slurry of a filter aid suspended in an acidic sample is subjected to filtration (the so-called body feed method) can be used in combination. The first filter aid layer can be made of diatomaceous earth with a water permeability of 85 millidarcy (mDarcy) or less. However, a filter aid layer (hereinafter sometimes referred to as the "second filter aid layer") made of another type of diatomaceous earth or the like may be provided below the first filter aid layer. Examples of diatomaceous earth that can be used for the first filter aid layer include CelPure 65 (manufactured by Advanced Minerals). Using CelPure 65 as the diatomaceous earth constituting the first filter aid layer and subjecting an acidified sample to filtration can improve the recovery rate of the target substance during sample pretreatment, thereby improving quantification accuracy. Furthermore, the filter aid suspended in the acidified sample can be the same as the diatomaceous earth constituting the first filter aid layer. Other types of diatomaceous earth that can be used to form the second filter aid layer include, for example, Celite 545 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and Radiolite Deluxe W-50 (manufactured by Showa Chemical Industry Co., Ltd.). Using CelPure 65 as the diatomaceous earth constituting the first filter aid layer and providing a second filter aid layer below it can improve filterability (filtration rate) during filtration.

After the filtration process (S13), in the step (S14) of eluting the substance to be quantified from the filter aid, the substance to be quantified captured in the filter aid is eluted using an eluent capable of eluting the substance to be quantified, and a fraction containing the substance to be quantified is obtained.

The eluent is not particularly limited as long as it can elute the substance to be quantified. For example, alkyl acetates such as ethyl acetate and butyl acetate; chloroform, methanol, ethanol, isopropyl alcohol, acetone, diethyl ether, a chloroform-methanol mixed solvent, or a chloroform-ethanol mixed solvent can be used. In particular, using alkyl acetates such as ethyl acetate as the eluent can significantly improve the recovery rate of the substance to be quantified.

The fraction containing the substance to be quantified obtained by elution as described above may be purified by conventional methods. For example, the fraction may be dissolved in a developing solvent such as a chloroform/methanol mixed solvent, and subjected to column chromatography using a porous material such as silica gel as a packing material.

In addition, before eluting the substance to be quantified from the filter aid, the filter aid can be thoroughly washed with water to remove any impurities captured by the filter aid. Celluloses such as hydroxypropyl methylcellulose, which are used as impurities, readily dissolve in cold water (approximately 1 to 10°C). Therefore, washing the filter aid with cold water can remove the impurities and improve the recovery rate of the substance to be quantified.

The fraction containing the substance to be quantified obtained as described above is used as a quantitative sample solution and subjected to liquid chromatography to obtain a quantitative chromatogram (S2). The peak area of the substance to be quantified in the quantitative chromatogram is then determined, and the substance to be quantified is quantified based on this peak area. Quantification of the substance to be quantified can be performed based on the peak area determined from the quantitative chromatogram and the calibration curve, which is prepared in advance by liquid chromatography using a standard sample of the substance to be quantified. Note that the processing conditions (e.g., liquid chromatography apparatus, type of stationary phase, type of mobile phase, etc.) of the liquid chromatography process for obtaining the quantitative chromatogram are the same as the processing conditions (e.g., liquid chromatography apparatus, type of stationary phase, type of mobile phase, etc.) of the liquid chromatography process for preparing the calibration curve.

In this embodiment, the fraction containing the target substance to be quantified and subjected to the liquid chromatography process is substantially free of impurities whose peaks overlap with those of the target substance. Therefore, the target substance can be quantified with high accuracy from the peak area of the target substance in the quantitative chromatogram obtained by the liquid chromatography process and the calibration curve.

The above-described embodiments have been described to facilitate understanding of the present invention, and are not intended to limit the scope of the present invention. Therefore, each element disclosed in the above embodiments is intended to include all design modifications and equivalents that fall within the technical scope of the present invention.

The present invention will be explained in more detail below using test examples, but the present invention is not limited to the test examples below.

Test Example 1
0.56 g of a compound tablet (glucosylceramide content: 1.44 mg) containing pineapple ceramide (Pinecera, manufactured by Maruzen Pharmaceutical Co., Ltd.), containing glucosylceramide as the substance to be quantified and hydroxypropyl methylcellulose (HPMC) as an impurity, was prepared as a sample. The tablet was dissolved in 20 mL of pure water to prepare a sample solution. 1 mL of 48% by mass potassium hydroxide aqueous solution was added to the 20 mL of sample solution, and the mixture was stirred at 40 ° C for 30 minutes to perform an alkali treatment (S11). 1.5 mL of concentrated hydrochloric acid was added to the sample solution after the alkali treatment (S12). The pH of the sample solution after the addition of concentrated hydrochloric acid was 1.02. 1 g of diatomaceous earth (Celpure 65, manufactured by Advanced Minerals, water permeability: 65 mDarcy) was added to the sample solution after the addition of concentrated hydrochloric acid, and the mixture was stirred. Filtration (S13) was performed using a Kiriyama funnel (φ60 mm). In addition, in the Kiriyama funnel, a filter paper (No. 3) was attached to the perforated plate, and a second filter aid layer was provided on the filter paper using 1 g of diatomaceous earth (Celite 545, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., water permeability: 4000 mDarcy), and a first filter aid layer was provided on the second filter aid layer using 2 g of diatomaceous earth (Celpure 65, manufactured by Advanced Minerals).

After the filtration process (S13), the first and second filter aid layers were washed with pure water and collected in a weighing vessel. 50 mL of eluent (chloroform/methanol solvent, volume ratio = 2:1) was added and ultrasonic waves were applied for 5 minutes, followed by filtration to collect the filtrate. The same procedure was repeated once more, followed by filtration to collect the filtrate. The filtrate was concentrated under reduced pressure at 40°C or below, dissolved in 20 mL of solvent (chloroform/methanol solvent, volume ratio = 99:1), and passed through a silica gel minicolumn (Sep-Pak plus Silica, manufactured by Waters, 690 mg). 10 mL of solvent (chloroform/methanol solvent, volume ratio = 99:1) was passed twice, followed by 10 mL of solvent (chloroform/methanol solvent, volume ratio = 50:1) twice, and the passed-through liquid was discarded. Then, 20 mL of solvent (chloroform/methanol solvent, volume ratio = 2:1) was passed through the silica gel mini column once, and the eluate was collected.

The resulting eluate was concentrated under reduced pressure at 40°C or below. 10 mL of 85% by volume aqueous methanol solution was added and sonicated to dissolve the concentrate, which was then passed through an octadecylsilylated silica gel minicolumn (Sep-Pak Vac tC18, Waters, 500 mg). 10 mL of 85% by volume aqueous methanol solution was then passed through the octadecylsilylated silica gel minicolumn once, and the passed-through liquid was discarded. Then, 20 mL of methanol was passed through, and the recovered eluate was concentrated under reduced pressure at 40°C or below using a rotary evaporator. A solvent (chloroform/methanol solvent, volume ratio = 2:1) was added to the resulting concentrate, which was then filtered through a membrane filter (pore size: 0.45 μm). The initial flow was discarded, and the filtrate was prepared as a sample solution for quantification.

The quantitative sample solution obtained as described above was subjected to liquid chromatography under the following conditions to obtain a quantitative chromatogram.
<Liquid chromatography conditions>
Liquid chromatograph: Agilent 1260 Infinity (Agilent Technologies)
Detector: Evaporative light scattering detector (G4260B, Agilent Technologies)
Injection volume: 10μL
Column: InertsilSil 100A 5 μm (150 mm × 4.6 mm, manufactured by GL Sciences)
Column temperature: 40°C
Flow rate: 1.0mL/min
Mobile phase A: chloroform Mobile phase B: methanol/water (volume ratio = 95:5)
(Gradient conditions)
0-15min: Mobile phase B (1-25%)
15-20 min: Mobile phase B (25-90%)
20-21 min: Mobile phase B (90-100%)
21-26min: Mobile phase B (100%)
Post time: 10 min

The peak area of glucosylceramide in the resulting quantitative chromatogram was calculated, and glucosylceramide was quantified based on a calibration curve previously prepared using a glucosylceramide standard. The recovery rate (%) of glucosylceramide was calculated from the quantitative results. The glucosylceramide standard was dissolved in a chloroform/methanol solvent (volume ratio = 2:1), filtered through a membrane filter (pore size: 0.45 μm), and the initial flow was discarded. This filtrate was used as the standard solution, and the calibration curve was prepared by subjecting this standard solution to liquid chromatography under the conditions described above. The results are shown in Table 1.

[Test Example 2]
After the filtration treatment (S13), the first filter aid layer and the second filter aid layer were dried, and the eluate was recovered by filtering 50 mL of ethyl acetate three times. Except for this, glucosylceramide was quantified in the same manner as in Test Example 1, and the recovery rate (%) of glucosylceramide was calculated from the quantification results. The results are shown in Table 1.

[Test Example 3]
The glucosylceramide was quantified in the same manner as in Test Example 2, except that the amount of concentrated hydrochloric acid added was changed and the pH of the sample solution after the addition of concentrated hydrochloric acid was adjusted to 3.0. The recovery rate (%) of glucosylceramide was calculated from the quantification results. The results are shown in Table 1.

[Test Example 4]
Except for changing the amount of concentrated hydrochloric acid added and adjusting the pH of the sample solution after the addition of concentrated hydrochloric acid to 4.74, glucosylceramide was quantified in the same manner as in Test Example 2, and the recovery rate (%) of glucosylceramide was calculated from the quantification results. The results are shown in Table 1.

[Test Example 5]
Except for changing the amount of concentrated hydrochloric acid added and adjusting the pH of the sample solution after the addition of concentrated hydrochloric acid to 5.47, glucosylceramide was quantified in the same manner as in Test Example 2, and the recovery rate (%) of glucosylceramide was calculated from the quantification results. The results are shown in Table 1.

[Test Example 6]
Except for changing the amount of concentrated hydrochloric acid added and adjusting the pH of the sample solution after the addition of concentrated hydrochloric acid to 6.51, glucosylceramide was quantified in the same manner as in Test Example 5, and the recovery rate (%) of glucosylceramide was calculated from the quantification results. The results are shown in Table 1.

[Test Example 7]
The glucosylceramide was quantified in the same manner as in Test Example 5, except that the amount of concentrated hydrochloric acid added was changed and the pH of the sample solution after the addition of concentrated hydrochloric acid was adjusted to 8.32. The recovery rate (%) of glucosylceramide was calculated from the quantification results. The results are shown in Table 1.

[Test Example 8]
Except for changing the amount of concentrated hydrochloric acid added and adjusting the pH of the sample solution to 9.88 after adding concentrated hydrochloric acid, glucosylceramide was quantified in the same manner as in Test Example 1, and the recovery rate (%) of glucosylceramide was calculated from the quantification results. The results are shown in Table 1.

As is clear from the results shown in Table 1, it was confirmed that the recovery rate of glucosylceramide can be improved by acidifying the pH of the sample solution after alkali treatment and eluting it from the filter aid using ethyl acetate. In particular, it was found that the recovery rate of glucosylceramide can be significantly improved by adjusting the pH of the sample solution to 5.0 or less. These results confirmed that glucosylceramide can be quantified with high accuracy by acidifying the pH of the sample solution after alkali treatment and subjecting it to filtration using a specified filter aid. Furthermore, when a chloroform/methanol solvent is used as the eluent to elute glucosylceramide from the filter aid, as in Test Example 1, hydroxypropyl methylcellulose is eluted along with glucosylceramide, and the two peaks overlap in the chromatogram, which is thought to be why the recovery rate exceeded 100%.

[Test Example 9]
The pH of the sample solution after the addition of concentrated hydrochloric acid was adjusted to 1.38, and 1 g of diatomaceous earth (Cellpure 300, Advanced Minerals, water permeability: 300 mDarcy) was added to the sample solution after the addition of concentrated hydrochloric acid. Except for the first filter aid layer being provided using 2 g of diatomaceous earth (Cellpure 300, Advanced Minerals), the glucosylceramide was quantified in the same manner as in Test Example 2, and the recovery rate (%) of glucosylceramide was calculated from the quantification results. The results are shown in Table 2.

[Test Example 10]
The pH of the sample solution after the addition of concentrated hydrochloric acid was adjusted to 1.36, and 1 g of diatomaceous earth (Cellpure 1000, Advanced Minerals, water permeability: 1000 mDarcy) was added to the sample solution after the addition of concentrated hydrochloric acid. Except for the first filter aid layer being provided using 2 g of diatomaceous earth (Cellpure 1000, Advanced Minerals), the glucosylceramide was quantified in the same manner as in Test Example 2, and the recovery rate (%) of glucosylceramide was calculated from the quantification results. The results are shown in Table 2.

[Test Example 11]
The pH of the sample solution after the addition of concentrated hydrochloric acid was adjusted to 1.32, 1 g of diatomaceous earth (Radiolite Deluxe P-5, manufactured by Showa Chemical Industry Co., Ltd., water permeability: 90 mDarcy) was added to the sample solution after the addition of concentrated hydrochloric acid, and 2 g of diatomaceous earth (Radiolite Deluxe P-5, manufactured by Showa Chemical Industry Co., Ltd.) was used to form a first filter aid layer. Except for this, glucosylceramide was quantified in the same manner as in Test Example 2, and the recovery rate (%) of glucosylceramide was calculated from the quantification results. The results are shown in Table 2.

[Test Example 12]
The pH of the sample solution after the addition of concentrated hydrochloric acid was adjusted to 1.32, and 1 g of diatomaceous earth (Radiolite Deluxe W-50, manufactured by Showa Chemical Industry Co., Ltd., water permeability: 2100 mDarcy) was added to the sample solution after the addition of concentrated hydrochloric acid. Except for the addition of 2 g of diatomaceous earth (Radiolite Deluxe W-50, manufactured by Showa Chemical Industry Co., Ltd.) to form a first filter aid layer, glucosylceramide was quantified in the same manner as in Test Example 2, and the recovery rate (%) of glucosylceramide was calculated from the quantification results. The results are shown in Table 2.

[Test Example 13]
1 g of diatomaceous earth (Hyflo Super Cel, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., water permeability: 1100 mDarcy) was added to the sample solution (pH = 13.16) after alkali treatment, and 3 g of diatomaceous earth (Hyflo Super Cel, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used instead of the first and second filter aid layers to form a filter aid layer. Except for this, glucosylceramide was quantified in the same manner as in Test Example 8, and the recovery rate (%) of glucosylceramide was calculated from the quantification results. The results are shown in Table 2.

[Test Example 14]
The pH of the sample solution after the addition of concentrated hydrochloric acid was adjusted to 1.13, and 1 g of diatomaceous earth (Hyflo Super Cel, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., water permeability: 1100 mDarcy) was added to the sample solution after the addition of concentrated hydrochloric acid. Except for the first filter aid layer and the second filter aid layer, 2 g of diatomaceous earth (Hyflo Super Cel, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used to provide a filter aid layer, the glucosylceramide was quantified in the same manner as in Test Example 1, and the recovery rate (%) of glucosylceramide was calculated from the quantification results. The results are shown in Table 2.

As is clear from the results shown in Table 2, it was confirmed that the recovery rate of glucosylceramide can be improved by subjecting an acidic sample solution to a filtration process using diatomaceous earth with a water permeability of 85 mDarcy or less as a filter aid. These results confirm that glucosylceramide can be quantified with high accuracy by acidifying the pH of the sample solution after alkaline treatment and subjecting it to a filtration process using diatomaceous earth with a water permeability of 85 mDarcy or less as a filter aid.

The present invention is useful as a method for quantifying target substances such as glucosylceramide that are added to foods with functional claims, etc.

Claims (6)

A pretreatment method for quantifying a substance to be quantified in a sample containing the substance to be quantified and impurities, comprising:
subjecting the sample to an alkaline treatment using an alkali;
acidifying the sample with an acid;
and subjecting the acidified sample to a filtration process using diatomaceous earth as a filter aid;
the substance to be quantified is glucosylceramide,
the impurities are hydroxypropyl cellulose and/or hydroxypropyl methylcellulose,
The pretreatment method, wherein the filter aid comprises the diatomaceous earth having a water permeability of 85 millidarcy (mDarcy) or less. The pretreatment method described in claim 1 further comprises a step of eluting the substance to be quantified from the diatomaceous earth after subjecting the sample to a filtration process. The pretreatment method described in claim 2, characterized in that the substance to be quantified is eluted from the diatomaceous earth using an alkyl acetate solution. A pretreatment method according to any one of claims 1 to 3, characterized in that the pH of the sample is adjusted to 5.0 or less using the acid. A pretreatment method according to any one of claims 1 to 4, characterized in that the sample to which the filter aid has been added is subjected to a filtration process using a filter medium on which the filter aid is layered upstream. A method for quantifying a substance to be quantified in a sample containing the substance to be quantified and impurities, comprising:
A step of pretreating the sample by the pretreatment method according to any one of claims 1 to 5 ;
and subjecting the sample after the pretreatment to liquid chromatography analysis.