JP4594792B2 - Method for measuring the amount of hydroxyhydroquinone - Google Patents
Method for measuring the amount of hydroxyhydroquinone Download PDFInfo
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Description
本発明は、コーヒー飲料又はコーヒー抽出物含有食品中のヒドロキシヒドロキノン量を安定して正確に測定する方法に関する。 The present invention relates to a method for stably and accurately measuring the amount of hydroxyhydroquinone in a coffee beverage or a coffee extract-containing food.
コーヒー飲料やコーヒー抽出物含有食品には、クロロゲン酸、カフェ酸、フェルラ酸等が含まれており、これらの成分は優れた血圧降下作用を示すことが報告されている（特許文献１〜３）。しかしながら、クロロゲン酸類を多量に含むことが知られているコーヒー飲料では、明確な血圧降下作用が認められず、逆に血圧を上昇させるという報告もある（非特許文献１）。 Coffee beverages and coffee extract-containing foods contain chlorogenic acid, caffeic acid, ferulic acid, and the like, and it has been reported that these components exhibit excellent blood pressure lowering action (Patent Documents 1 to 3). . However, coffee beverages known to contain a large amount of chlorogenic acids do not have a clear blood pressure lowering effect, and there is a report that blood pressure is increased (Non-patent Document 1).
一方、コーヒー中に存在するヒドロキシヒドロキノン（ＨＨＱ）は、共存するマトリックスの影響で、大気下に放置しておくだけで酸化、重合又は抱合体を形成し、安定に存在することが困難である。このため、逆相液体クロマトグラフィーや、ヒドロキシル基をトリメチルシリル化した後ガスクロマトグラフィーによる定性的な分析が行なわれているにすぎない（非特許文献２）。
本出願人は、コーヒー飲料がクロロゲン酸を含んでいるにもかかわらず、十分な血圧降下作用を示さないことに着目し、血圧降下作用とコーヒー飲料成分との関係について種々検討した結果、コーヒー飲料に含まれているヒドロキシヒドロキノンがクロロゲン酸類の血圧降下作用を阻害していることを見出した。そして、更に検討した結果、コーヒー飲料中のクロロゲン酸類量を一定範囲に保持し、ヒドロキシヒドロキノン含量を通常含まれる量より十分少ない一定量以下に低下させれば、優れた血圧降下作用を有するコーヒー飲料組成物が得られることを見出した。従って、従来は定量的に測定する必要性のなかった、コーヒー飲料やコーヒー抽出物含有食品中のヒドロキシヒドロキノン量の安定で、正確な測定法の開発が必要となった。 The present applicant pays attention to the fact that coffee drinks do not show sufficient blood pressure lowering action despite containing chlorogenic acid, and as a result of various investigations on the relationship between blood pressure lowering action and coffee drink ingredients, coffee drinks Was found to inhibit the blood pressure lowering action of chlorogenic acids. And as a result of further investigation, if the amount of chlorogenic acids in the coffee beverage is kept within a certain range and the hydroxyhydroquinone content is lowered below a certain amount sufficiently smaller than the amount normally contained, the coffee beverage having an excellent blood pressure lowering effect It was found that a composition was obtained. Therefore, it has been necessary to develop a stable and accurate measuring method for the amount of hydroxyhydroquinone in coffee beverages and coffee extract-containing foods, which has not been conventionally required to be quantitatively measured.
そこで本発明者は、コーヒー飲料組成物等を試料として液体クロマトグラフィーによりヒドロキシヒドロキノン量を測定するに際し、試料中のヒドロキシヒドロキノン経時的変化を抑制し、かつ他の成分との分離性を向上させるべく種々検討した結果、コーヒー飲料等に酸を添加してpHを５以下に調整し、これにキレート剤を添加したものを試料溶液とすれば、ヒドロキシヒドロキノンが液体クロマトグラフィー測定環境で安定に存在することが可能となり、コーヒー飲料等の組成物中のヒドロキシヒドロキノンが正確に測定できることを見出した。 Therefore, when measuring the amount of hydroxyhydroquinone by liquid chromatography using a coffee beverage composition or the like as a sample, the present inventor should suppress the change with time of hydroxyhydroquinone in the sample and improve the separation from other components. As a result of various investigations, if an acid is added to a coffee beverage or the like to adjust the pH to 5 or less and a chelating agent added thereto is used as a sample solution, hydroxyhydroquinone is stably present in a liquid chromatography measurement environment. It has been found that hydroxyhydroquinone in compositions such as coffee beverages can be accurately measured.
すなわち、本発明は、コーヒー飲料又はコーヒー抽出物含有食品に酸を添加してpHを５以下に調整し、かつキレート剤を添加した試料溶液を液体クロマトグラフィーに付すことを特徴とするコーヒー飲料又はコーヒー抽出物含有食品中のヒドロキシヒドロキノン量の測定法を提供するものである。 That is, the present invention provides a coffee beverage or a coffee beverage characterized in that an acid is added to a coffee beverage or a coffee extract-containing food to adjust the pH to 5 or less, and a sample solution to which a chelating agent is added is subjected to liquid chromatography. A method for measuring the amount of hydroxyhydroquinone in a coffee extract-containing food is provided.
本発明方法によれば、コーヒー飲料又はコーヒー抽出物含有食品中のヒドロキシヒドロキノン含量を安定して、正確に測定できる。従って、クロロゲン酸の持つ高血圧改善効果をコーヒー中で発現させるために、その効果の阻害成分であるヒドロキシヒドロキノンを極力除去したコーヒーを製造し、そのコーヒー中のヒドロキシヒドロキノン量を安定かつ正確に測定できる。また、高血圧改善効果のあるコーヒー及びコーヒー飲料を製造する際に、阻害成分であるヒドロキシヒドロキノンがある一定量以上に存在しないことを確認するために、安定かつ正確にヒドロキシヒドロキノン量を測定することが可能となる。 According to the method of the present invention, the hydroxyhydroquinone content in a coffee beverage or coffee extract-containing food can be measured stably and accurately. Therefore, in order to express the antihypertensive effect of chlorogenic acid in coffee, it is possible to produce coffee from which hydroxyhydroquinone, which is an inhibitor of the effect, is removed as much as possible, and to measure the amount of hydroxyhydroquinone in the coffee stably and accurately. . In addition, when producing coffee and coffee beverages that have an antihypertensive effect, it is possible to measure the amount of hydroxyhydroquinone stably and accurately in order to confirm that there is no more than a certain amount of hydroxyhydroquinone as an inhibitory component. It becomes possible.
本発明方法に用いられるコーヒー飲料又はコーヒー抽出物含有食品としては、焙煎コーヒー抽出液を含有するコーヒー飲料、インスタントコーヒー組成物、容器詰コーヒー飲料、コーヒー抽出物含有チューインガム、コーヒー抽出物含有菓子類等が挙げられる。 The coffee beverage or coffee extract-containing food used in the method of the present invention includes a coffee beverage containing a roasted coffee extract, an instant coffee composition, a packaged coffee beverage, a coffee extract-containing chewing gum, and a coffee extract-containing confectionery. Etc.
本発明においては、まずこれらの試料に酸を添加してpHを５以下に調整する。酸を添加することにより、試料中のヒドロキシヒドロキノンが測定操作中安定に維持される。用いる酸としては、試料溶液のpHを５以下にすることができるものであればよく、リン酸、塩酸、硝酸、硫酸等の鉱酸；ギ酸、酢酸、トリフルオロ酢酸等の有機酸等が挙げられる。また、これらの酸の使用量は、pHを５以下にする量であればよく、特に限定されない。また、好ましいpHは１〜５であり、より好ましいpHは２〜４である。 In the present invention, first, an acid is added to these samples to adjust the pH to 5 or less. By adding the acid, the hydroxyhydroquinone in the sample is kept stable during the measurement operation. The acid to be used is not particularly limited as long as the pH of the sample solution can be adjusted to 5 or less, and includes mineral acids such as phosphoric acid, hydrochloric acid, nitric acid, and sulfuric acid; organic acids such as formic acid, acetic acid, and trifluoroacetic acid. It is done. Moreover, the usage-amount of these acids should just be the quantity which makes pH 5 or less, and is not specifically limited. Moreover, preferable pH is 1-5 and more preferable pH is 2-4.
また、試料には、キレート剤も添加する。キレート剤の添加により、試料中のヒドロキシヒドロキノンが測定操作中さらに安定に維持される。用いるキレート剤としては、ＥＤＴＡ等のポリアミノカルボン酸類；クエン酸等のヒドロキシカルボン酸類；メタリン酸、ポリリン酸等のリン酸類；１−ヒドロキシエタン−１，１−ジホスホン酸（ＨＥＤＰＯ）、エトリロトリスメチレンホスホン酸（ＮＴＰＯ）等のホスホン酸類が挙げられる。これらキレート剤の使用量は、試料溶液中、０．０１〜１．０ mmol/L以上、さらに０．０２〜０．５ mmol/L、特に０．１ mmol/L程度が好ましい。 A chelating agent is also added to the sample. Addition of a chelating agent keeps the hydroxyhydroquinone in the sample more stable during the measurement operation. Examples of the chelating agent used include polyaminocarboxylic acids such as EDTA; hydroxycarboxylic acids such as citric acid; phosphoric acids such as metaphosphoric acid and polyphosphoric acid; 1-hydroxyethane-1,1-diphosphonic acid (HEDPO), etilotrithmethylene Examples include phosphonic acids such as phosphonic acid (NTPO). The amount of these chelating agents used is preferably 0.01 to 1.0 mmol / L or more, more preferably 0.02 to 0.5 mmol / L, and particularly preferably about 0.1 mmol / L in the sample solution.
さらに、酸の添加によりｐＨ５以下に調整され、かつキレート剤が添加された試料溶液をカチオン交換樹脂処理することが、ヒドロキシヒドロキノンの定量性を向上させるうえで好ましい。カチオン交換樹脂としては、スルホン酸、硫酸等の酸残基を有する強カチオン交換樹脂、カルボン酸等の酸残基を有する弱カチオン交換樹脂が挙げられる。このうち、プロピルベンゼンスルホン酸（ＳＣＸ）、プロピルスルホン酸（ＰＲＳ）、エチルカルボン酸（ＣＢＡ）等のカチオン交換基を有する樹脂が特に好ましい。カチオン交換樹脂による処理は、これらのカチオン交換樹脂を固相化したカラムに試料溶液を通過させるのが好ましい。 Furthermore, it is preferable to treat the sample solution adjusted to pH 5 or less by addition of an acid and to which a chelating agent is added in order to improve the quantitative property of hydroxyhydroquinone. Examples of the cation exchange resin include strong cation exchange resins having acid residues such as sulfonic acid and sulfuric acid, and weak cation exchange resins having acid residues such as carboxylic acid. Among these, resins having a cation exchange group such as propylbenzenesulfonic acid (SCX), propylsulfonic acid (PRS), ethylcarboxylic acid (CBA) and the like are particularly preferable. In the treatment with a cation exchange resin, the sample solution is preferably passed through a column in which these cation exchange resins are solid-phased.
本発明においては、前記の試料溶液を用いて液体クロマトグラフィーによりヒドロキシヒドロキノン量を測定する。本発明の液体クロマトグラフィーに用いるカラムは、逆相液体クロマトグラフィー用カラム、例えばオクタデシルシリル化シリカゲル、オクチルシリル化シリカゲル等を充填したカラムを用いるのが好ましい。また、これらカラムに用いられる充填剤の粒子径は平均粒径３〜５mmが好ましい。 In the present invention, the amount of hydroxyhydroquinone is measured by liquid chromatography using the sample solution. The column used for the liquid chromatography of the present invention is preferably a column for reverse phase liquid chromatography, for example, a column packed with octadecylsilylated silica gel, octylsilylated silica gel or the like. Further, the particle diameter of the filler used in these columns is preferably an average particle diameter of 3 to 5 mm.
液体クロマトグラフィーに用いる溶離液は、ヒドロキシヒドロキノンを安定的に分析するため、酸及びキレート剤を含むものが好ましい。酸としては、前記試料溶液の調製に用いたものと同じものが挙げられるが、その添加量は溶離液のpHを５以下にする量が好ましい。またキレート剤も、前記試料溶液の調製に用いたものと同じものが挙げられる。キレート剤の使用量も、前記試料溶液と同じ量が好ましい。また溶離液には、試料溶液中の夾雑成分とＨＨＱの分離向上の点から１〜１０容量％の低級アルコール、特にメタノールを含有させるのが好ましい。 The eluent used for liquid chromatography preferably contains an acid and a chelating agent in order to stably analyze hydroxyhydroquinone. Examples of the acid include the same ones used for the preparation of the sample solution, but the addition amount is preferably an amount that makes the pH of the eluent 5 or less. In addition, the same chelating agent as that used for the preparation of the sample solution may be used. The amount of chelating agent used is preferably the same amount as the sample solution. In addition, the eluent preferably contains 1 to 10% by volume of a lower alcohol, particularly methanol, from the viewpoint of improving separation of contaminant components and HHQ in the sample solution.
液体クロマトグラフィーの流速は、液体クロマトグラフィー条件であれば特に制限されないが、０．０５〜１０mL／min、さらに０．２〜１．５mL／minが好ましい。またカラム温度は、２０〜５０℃、さらに３０〜４０℃が好ましい。本発明の液体クロマトグラフィーにおける検出手段としては、ＵＶ検出（ＵＶ／ＶＩＳ多波長検出含む）が一般的であるが、ＦＬ（蛍光）検出、ＣＬ（化学発光）検出、ＥＣ（電気化学）検出、ＭＳ（質量分析）検出等により検出することもできる。 The flow rate of liquid chromatography is not particularly limited as long as it is liquid chromatography conditions, but is preferably 0.05 to 10 mL / min, more preferably 0.2 to 1.5 mL / min. The column temperature is preferably 20 to 50 ° C, more preferably 30 to 40 ° C. As a detection means in the liquid chromatography of the present invention, UV detection (including UV / VIS multi-wavelength detection) is common, but FL (fluorescence) detection, CL (chemiluminescence) detection, EC (electrochemical) detection, It can also be detected by MS (mass spectrometry) detection or the like.
以下の実施例における略語は次のとおりである。
ＨＥＤＰＯ：１−ヒドロキシエタン−１，１−ジホスホン酸水溶液
ＮＴＰＯ：ニトリロトスメチレンホスホン酸三ナトリウム塩
ＳＣＸ：プロピルベンゼンスルホン酸修飾シリカゲル
ＰＲＳ：プロピルスルホン酸修飾シリカゲル
ＣＢＡ：エチルカルボン酸修飾シリカゲル
Abbreviations in the following examples are as follows.
HEDPO: 1-hydroxyethane-1,1-diphosphonic acid aqueous solution NTPO: nitrilotosmethylenephosphonic acid trisodium salt SCX: propylbenzenesulfonic acid modified silica gel PRS: propylsulfonic acid modified silica gel CBA: ethylcarboxylic acid modified silica gel
実施例１、２及び比較例１、２
市販缶コーヒーを開缶直後、１０ｇをスクリュー管に正確にはかりとる。そこに０．１％リン酸、０．１mmol／Ｌ ＨＥＤＰＯ及び５％メタノール溶液１０mLを正確にはかりとり、よく混ぜる。
この溶液を溶液Ａ５mLでコンディショニングしたＳＣＸ固相カラムに、最初２mLを通過させ廃棄した後、再度５mLを通過させ、その溶液（溶液Ｂ）を液体クロマトグラフィーに注入する。
Examples 1 and 2 and Comparative Examples 1 and 2
Immediately after opening canned commercial coffee, weigh 10 g accurately into a screw tube. To this, weigh accurately 10% of 0.1% phosphoric acid, 0.1 mmol / L HEDPO and 5% methanol solution, and mix well.
This solution is first passed through an SCX solid phase column conditioned with 5 mL of solution A and discarded, then 5 mL is passed again, and the solution (solution B) is injected into liquid chromatography.
標準試料溶液は、以下の方法により作成した。
ＨＨＱ １０mgをメスフラスコにはかりとり、０．１％リン酸、０．１mmol／Ｌ ＨＥＤＰＯ及び５％メタノール溶液（溶液Ａ，pH２．６）にて溶解させ正確に１００mLとした。この１００mg／Ｌ溶液を母液として順次希釈し、０．２mg／Ｌ、０．４mg／Ｌ、０．６mg／Ｌ、０．８mg／Ｌ、１．０mg／Ｌ溶液を作成し、標準試料とした。
The standard sample solution was prepared by the following method.
10 mg of HHQ was weighed into a volumetric flask and dissolved in 0.1% phosphoric acid, 0.1 mmol / L HEDPO and 5% methanol solution (solution A, pH 2.6) to make exactly 100 mL. This 100 mg / L solution was sequentially diluted as a mother liquor to prepare 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, and 1.0 mg / L solutions as standard samples. .
液体クロマトグラフィー条件は以下のとおりである。
装置：ＨＰ１１００
カラム：Ｉｎｅｒｔｓｉｌ ＯＤＳ−３ ４．６mmi.d. ×２５０mm（粒子径５μm）
検出器：ＵＶ検出器 ２８８nm
溶離液：０．１％リン酸、０．１mmol／Ｌ ＨＥＤＰＯ５％メタノール溶液（pH２．６）
流速：１．０mL／min
注入量：２０μＬ
カラム温度：３５℃
The liquid chromatography conditions are as follows.
Device: HP1100
Column: Inertsil ODS-3 4.6 mm i.d. × 250 mm (particle diameter 5 μm)
Detector: UV detector 288nm
Eluent: 0.1% phosphoric acid, 0.1 mmol / L HEDPO 5% methanol solution (pH 2.6)
Flow rate: 1.0 mL / min
Injection volume: 20 μL
Column temperature: 35 ° C
上記溶液Ａを直接液体クロマトグラフィーに注入した場合（実施例１）の結果を図１に示す。その結果、リテンショタイム７．３分にヒドロキシヒドロキノンのピークが生じた。
また、上記のように溶液Ａをカチオン交換樹脂であるＳＣＸ固相カラム処理した試料溶液Ｂを高速液体クロマトグラフィーに注入した場合（実施例２）の結果を図２に示す。その結果、リテンショタイム７．３分にヒドロキシヒドロキノンのピークが生じ、８．２mg／Ｌのヒドロキシヒドロキノンと定量された。本試料溶液における標準ヒドロキシヒドロキノンのスパイクテストにおいて、その添加回収率は９８％であった。
FIG. 1 shows the results when the solution A was directly injected into liquid chromatography (Example 1). As a result, a hydroxyhydroquinone peak occurred at a retention time of 7.3 minutes.
Further, FIG. 2 shows the results when the sample solution B obtained by treating the solution A with the SCX solid phase column as a cation exchange resin as described above was injected into high performance liquid chromatography (Example 2). As a result, a hydroxyhydroquinone peak was produced at a retention time of 7.3 minutes, and the amount was determined to be 8.2 mg / L of hydroxyhydroquinone. In the spike test of standard hydroxyhydroquinone in this sample solution, the recovery rate was 98%.
一方、０．１％リン酸を添加しないこと以外は、前記溶液Ａと同様にして調製された溶液を、直接液体クロマトグラムに注入した場合（比較例１）の結果を図３に示す。その結果、リテンショタイム７．３分に、図１及び図２に示されるようなヒドロキシヒドロキノンのピークが認められなかった。
また、０．１％リン酸及びＨＥＤＰＯの両者を添加しないこと以外は溶液Ａと同様にして調製された溶液を直接液体クロマトグラムに注入した場合（比較例２）も、比較例１の場合と同様にリテンショタイム７．３分に、図１及び図２に示されるようなヒドロキシヒドロキノンのピークが認められなかった。
On the other hand, FIG. 3 shows the results when a solution prepared in the same manner as the solution A except that 0.1% phosphoric acid was not added was directly injected into the liquid chromatogram (Comparative Example 1). As a result, no hydroxyhydroquinone peak as shown in FIGS. 1 and 2 was observed at a retention time of 7.3 minutes.
Further, the case where a solution prepared in the same manner as Solution A except that both 0.1% phosphoric acid and HEDPO are not added is directly injected into the liquid chromatogram (Comparative Example 2) is different from the case of Comparative Example 1. Similarly, no hydroxyhydroquinone peak as shown in FIGS. 1 and 2 was observed at a retention time of 7.3 minutes.
実施例３
実施例２において、０．１％リン酸（pH２．６）を０．１％リン酸緩衝液pH２．０〜４．０に代えること以外は同様にして試料溶液を調製し、ヒドロキシヒドロキノン量を１２時間連続して（１時間に１回）測定した。その結果、試料溶液をpH２．０〜４．０に調製した場合、ヒドロキシヒドロキノンの定量値は、１２時間後でも初期（０時間）の９０％以上の値を示した。
Example 3
In Example 2, a sample solution was prepared in the same manner except that 0.1% phosphate (pH 2.6) was replaced with 0.1% phosphate buffer pH 2.0 to 4.0, and the amount of hydroxyhydroquinone was adjusted. Measurements were taken continuously for 12 hours (once every hour). As a result, when the sample solution was adjusted to pH 2.0 to 4.0, the quantitative value of hydroxyhydroquinone showed 90% or more of the initial value (0 hour) even after 12 hours.
実施例４
実施例２において、０．１％リン酸（pH２．６）を、酢酸５０mmol／Ｌ（pH２．９）、ギ酸１００mmol／Ｌ（pH２．５）、トリフルオロ酢酸１０mmol／Ｌ（pH２．２）、又は塩酸１mmol／Ｌ（pH２．９）に代えること以外は同様にして試料溶液を調製し、ヒドロキシヒドロキノン量を２４時間連続して（２時間に１回）測定した。その結果、いずれの酸を用いた場合でもヒドロキシヒドロキノンの定量値は２４時間後でも初期（０時間）の９０％以上の値を示した。これらの酸のうち、リン酸、酢酸、ギ酸、トリフルオロ酢酸が特に良好であった。
Example 4
In Example 2, 0.1% phosphoric acid (pH 2.6) was added to acetic acid 50 mmol / L (pH 2.9), formic acid 100 mmol / L (pH 2.5), trifluoroacetic acid 10 mmol / L (pH 2.2), Alternatively, a sample solution was prepared in the same manner except that hydrochloric acid was changed to 1 mmol / L (pH 2.9), and the amount of hydroxyhydroquinone was measured continuously for 24 hours (once every 2 hours). As a result, when any acid was used, the quantitative value of hydroxyhydroquinone was 90% or more of the initial value (0 hour) even after 24 hours. Of these acids, phosphoric acid, acetic acid, formic acid, and trifluoroacetic acid were particularly good.
実施例５
実施例２において、キレート剤である０．１mmol／Ｌ ＨＥＤＰＯを、０．１mmol／ＬＮＴＰＯ、０．１％メタリン酸ナトリウム又は０．１mmol／Ｌ ＥＤＴＡ−２Ｎａに代えること以外は同様にして試料溶液を調製し、ヒドロキシヒドロキノン量を２４時間連続（２時間に１回）測定した。その結果、いずれのキレート剤を用いた場合でもヒドロキシヒドロキノンの定量値は２４時間後でも初期（０時間）の９０％以上の値を示した。
Example 5
In Example 2, a sample solution was prepared in the same manner except that 0.1 mmol / L HEDPO as a chelating agent was replaced with 0.1 mmol / LNTPO, 0.1% sodium metaphosphate or 0.1 mmol / L EDTA-2Na. The amount of hydroxyhydroquinone was measured continuously for 24 hours (once every 2 hours). As a result, even when any chelating agent was used, the quantitative value of hydroxyhydroquinone was 90% or more of the initial value (0 hour) even after 24 hours.
実施例６
実施例２において、溶離液中の０．１％リン酸に代えてpH２．０〜６．０のリン酸緩衝液を用いること以外は実施例２と同様にしてヒドロキシヒドロキノン量を２４時間連続して（２時間に１回）測定した。その結果、溶離液のpHが２．０〜５．０の範囲内では、ヒドロキシヒドロキノンの定量値は２４時間後でも初期（０時間）の９０％以上の値を示した。
Example 6
In Example 2, the amount of hydroxyhydroquinone was continuously increased for 24 hours in the same manner as in Example 2 except that a phosphate buffer having a pH of 2.0 to 6.0 was used instead of 0.1% phosphoric acid in the eluent. (Once every 2 hours). As a result, when the pH of the eluent was within the range of 2.0 to 5.0, the quantitative value of hydroxyhydroquinone was 90% or more of the initial value (0 hour) even after 24 hours.
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