KR20040035359A - Method of quantitatively analyzing polyethoxlyated ascorbic acid by using high performance liquid chromatograhpy - Google Patents
Method of quantitatively analyzing polyethoxlyated ascorbic acid by using high performance liquid chromatograhpy Download PDFInfo
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Abstract
Description
본 발명은 고성능액체크로마토그래피를 이용한 폴리에톡실레이티드 아스코르브산 유도체의 분석방법에 관한 것이다.The present invention relates to a method for analyzing polyethoxylated ascorbic acid derivatives using high performance liquid chromatography.
아스코르브산은 수용성의 항산화제로 미백효과, 프롤린하이드록실라제의 보조인자로 콜라겐 합성증진의 효과가 있는 것으로 알려져 있다. 그러나, 아스코르브산은 안정성이 낮으므로 장시간의 사용이 요구되는 제품에는 사용에 어려움이 있다. 따라서 아스코르브산의 안정성을 향상시키기 위해서, 화학적으로 불안정의 주요원인인 아스코르브산의 2 또는 3번 탄소 위치를 화학적으로 변화시킨 유도체에 대한 개발이 많이 이루어지고 있다. 현재까지 사용화된 아스코르브산 유도체로는 아스코르브산 6-팔미테이트, 아스코르브산 2,6-디팔미테이트, 아스코르브산 6-스테아레이트, 및 L-아스코르브산-2-인산 에스테르 마그네슘염등이 알려져 있으며(한국특허공고공보 제 1991-8733호, 미국특허 제 4,179,445), 이들 화합물은 항산화 및 미백효과를 가진 화장품의 원료로서 사용되고 있다.Ascorbic acid is a water-soluble antioxidant that is known to have a whitening effect and co-synthesis as a cofactor for proline hydroxylase. However, since ascorbic acid is low in stability, it is difficult to use in a product requiring long time use. Therefore, in order to improve the stability of ascorbic acid, a lot of developments have been made on derivatives that chemically change the carbon positions of ascorbic acid, which are the main causes of chemical instability. Ascorbic acid derivatives used to date are ascorbic acid 6-palmitate, ascorbic acid 2,6-dipalmitate, ascorbic acid 6-stearate, and L-ascorbic acid-2-phosphate ester magnesium salt. (Korean Patent Publication No. 1991-8733, US Patent No. 4,179,445), These compounds are used as raw materials for cosmetics having an antioxidant and whitening effect.
그러나 상기 유도체들은 화학적으로 안정화되었다 하더라도 생체내에서 분해속도가 빨라 지속적인 항산화력을 유지하지 못하는 단점이 있다. 따라서, 한국특허공개공보 제 2001-27446호에는 하기의 화학식 1 및 2을 갖는 폴리에틸렌 글리콜을 폴리에톡실레이티드 아스코르브산의 2번 또는 3번 탄소위치에 도입시켜 기존에 알려진 아스코르브산의 문제점을 해결하였다고 기재하고 있다.However, even though the derivatives are chemically stabilized, they have a disadvantage in that they do not maintain sustained antioxidant capacity due to their rapid degradation rate in vivo. Therefore, Korean Patent Laid-Open Publication No. 2001-27446 solves the problems of known ascorbic acid by introducing polyethylene glycol having the following Chemical Formulas 1 and 2 into carbon positions 2 or 3 of polyethoxylated ascorbic acid. It is described.
상기식에서 PEG는 (CH2CH2O)nR1이며 n은 2 내지 200의 정수이고 R1은 C1-C6 알킬기이다.Wherein PEG is (CH 2 CH 2 O) n R 1, n is an integer from 2 to 200 and R 1 is a C1-C6 alkyl group.
폴리에틸렌 글리콜이 치환된 폴리에톡실레이티드 아스코르브산 유도체를 화장품등에 적용했을 때 그 효과를 유지하기 위해서는 분해되지 않고 존재해야 하는데, 이때 이성분의 정성 및 정량 분석과 안정성 분석이 필수적이다.Polyethylene glycol-substituted polyethoxylated ascorbic acid derivatives should be present without degradation in order to maintain their effects when applied to cosmetics, etc. At this time, qualitative and quantitative analysis and stability analysis of two components are essential.
크로마토그래프 분석법은 일반적으로 각종 고체 또는 액체를 고정상을 하고 기체 또는 액체를 이동상으로 하여 이동상이 고정상을 통과시켜 사상기 이동상에 시료를 투입하여 시료가 상기 이동상과 고정상 사이에서 흡착성, 분배계수, 이온간 결합, 또는 친화성등의 특성에 의해 시료에 포함된 성분별로 분리하는 방법이다. 다양한 종류의 크로마토그래피가 알려져 있으며 천연물의 분리, 의약품의 정제, 기타 하학물질의 정제등을 포함한 광범위한 분야에서 사용되고 있다.Chromatograph analysis generally involves various solids or liquids as the stationary phase, gas or liquid as the mobile phase, and the mobile phase passes through the stationary phase to inject the sample into the mobile phase of the finishing phase so that the sample is absorbed between the mobile phase and the stationary phase, partition coefficient, and ions. It is a method of separating each component contained in a sample by the characteristics such as binding or affinity. Various types of chromatography are known and are used in a wide range of fields, including separation of natural products, purification of medicines, and purification of other chemicals.
이처럼 다양한 크로마토그래피의 종류가 있고 상용되고 있으나, 분석하고자 하는 시료의 물질적 성질 즉 고정상과 이동상 사이의 분배계수, 분자량 및 이온성 등을 이용한 분리에 기초하고 있기 때문에 목적 시료에 따라 고정상 및 이동상의 선택, 이동상의 흐름속도, 분리되어 용리되는 시료를 검출하는 검출기의 선택 등 여러 분석요소의 상호작용에 의하여 최적화할 필요가 있으며, 이러한 분석방법에서 여러 요소들의 결정은 분석결과를 좌우할 정도로 매우 중요한 요소이다.Although there are various types of chromatography and are commonly used, it is based on the separation using the physical properties of the sample to be analyzed, that is, the partition coefficient between the fixed and mobile phases, molecular weight and ionicity. In this method, the determination of several factors is very important enough to influence the analysis results. .
따라서, 폴리에틸렌 글리콜이 치환된 폴리에톡실레이티드 아스코르브산 유도체를 제품에 적용시, 그 성분의 정량 및 정성 분석을 위한 최적화된 방법이 필요하다.Therefore, when the polyethylene glycol-substituted polyethoxylated ascorbic acid derivative is applied to a product, there is a need for an optimized method for quantitative and qualitative analysis of its components.
상기와 같은 종래의 문제점을 해결하고자, 본 발명은 HPLC를 이용한 폴리에틸렌 글리콜이 치환된 폴리에톡실레이티드 아스코르브산 유도체의 분석방법을 제공하는 것이다.In order to solve the above conventional problems, the present invention is to provide a method for analyzing a polyethoxylated ascorbic acid derivative substituted by polyethylene glycol using HPLC.
도 1은 본 발명의 일실시예에 따라 폴리에톡실레이티드 아스코르브산 유도체를 폴리에틸렌 글리콜의 부가 몰수에 따라 고성능 액체 크로마토그래프의 분석결과를 나타내는 그래프이다.1 is a graph showing the analysis results of a high performance liquid chromatograph of polyethoxylated ascorbic acid derivatives according to the added moles of polyethylene glycol according to an embodiment of the present invention.
도 2는 폴리에틸렌 글리콜의 부가 몰수에 상관없이 폴리에톡실레이티드 아스코르브산 유도체를 고성능 액체 크로마토그래프의 분석결과를 나타내는 그래프이다.2 is a graph showing the results of analysis of high performance liquid chromatographs of polyethoxylated ascorbic acid derivatives regardless of the number of moles of polyethylene glycol added.
상기와 같은 목적을 달성하고자, 본 발명은 비극성 물질이 충진된 분리관을 갖는 고성능액체 크로마토그래피에 아세토니트릴과 물을 포함하는 용출액으로 하기의 화학식 1 또는 2를 갖는 폴리에틸렌클리콜이 치환된 폴리에톡실레이티드 아스코르브산 유도체를 폴리에틸렌 글리콜 부가 몰수에 따른 크로마토그램을 얻고,In order to achieve the above object, the present invention is a high-performance liquid chromatography having a separation tube filled with a non-polar material in an eluate containing acetonitrile and water to a polyethylenyl glycol having the formula (1) or 2 below Toxylated ascorbic acid derivatives were obtained chromatograms according to the number of moles of polyethylene glycol added,
상기 크로마토그램에서 나타난 피크를 합한 후에 검량선을 그려서 폴리에틸렌 글리콜이 치환된 폴리에톡실레이티드 아스코르브산를 정량 분석하는 방법에 관한 것이다.The present invention relates to a method of quantitatively analyzing polyethoxylated ascorbic acid substituted by polyethylene glycol by adding a calibration curve after adding the peaks shown in the chromatogram.
[화학식 1][Formula 1]
[화학식 2][Formula 2]
상기 식에서 PEG는 (CH2CH2O)nR1이며 n은 2 내지 200의 정수이고 R1은 C1-C6알킬기이다.Wherein PEG is (CH 2 CH 2 O) n R 1 , n is an integer from 2 to 200 and R 1 is a C 1 -C 6 alkyl group.
이하에서, 본 발명을 자세히 설명하고자 한다.Hereinafter, the present invention will be described in detail.
본 발명에 따라 폴리에틸렌클리콜이 치환된 폴리에톡실레이티드 아스코르브산 유도체를 부가된 폴리에틸렌 글리콜 몰수에 따라 분석하는 방법에서는 고정상으로 비극성 물질을 사용한다. 비극성 물질이 충진된 분리관을 사용하는 이유는 폴리톡실레이티드 아스코르브산 유도체를 폴리에틸렌 글리콜 부가몰수에 따라 분리하기 위해서는 그 유도체의 특성상 비극성 물질이어야 하며, 극성이 높은 아미노기 또는 니트릴기를 갖는 물질을 고정상으로 사용하면 폴리에틸렌 글리콜의 부가몰수에 따라 분리되지 않는다. 상기 비극성 분리관은 사용적으로 구입하여 사용할 수 있으며, 알려진 비극성 분리관은 모두 사용가능하며, 바람직하게는 옥타실란 또는 옥타데실이 충진된 분리관이다.According to the present invention, a non-polar substance is used as a stationary phase in the analysis of polyethyloxylated ascorbic acid derivatives substituted with polyethylene glycol according to the number of moles of polyethylene glycol added. The reason for using a separator tube filled with a non-polar substance is that in order to separate the polytoxylated ascorbic acid derivative according to the polyethylene glycol addition mole number, the non-polar substance must be non-polar substance, and a substance having a high amino group or a nitrile group as a stationary phase is used. If used, it does not separate depending on the number of moles of polyethylene glycol added. The non-polar separator can be purchased and used by use, and all known non-polar separators can be used, and preferably a separator filled with octasilane or octadecyl.
본 발명에서 HPLC에 사용가능한 이동상으로는 아세토니트릴과 물의 혼합용액이 사용되며, 바람직하게는 아세토니트릴 10 내지 30 v/v%와 물 70 내지 90 v/v%의 혼합액인 것이 바람직하다. 용출액에서 아세토니트릴이 10 v/v%미만으로 사용하는경우, 또는 물 90 v/v% 초과하여로 사용하는 경우에는 용매의 극성이 크고 분리관에 체류시간이 커져서 분석기간이 길어지고, 또한 아세토니트릴이 30 v/v% 초과하여 사용하는 경우, 또는 물 70 v/v% 미만으로 사용하는 경우에는 용매의 극성이 작고 분리관에서 체류시간이 짧아서 폴리에틸렌 글리콜 부가몰수에 따라 분리가 되기 어렵다.As the mobile phase usable in HPLC in the present invention, a mixed solution of acetonitrile and water is used, preferably a mixed solution of 10 to 30 v / v% of acetonitrile and 70 to 90 v / v% of water. When the acetonitrile in the eluate is less than 10 v / v% or when the water is used in excess of 90 v / v%, the polarity of the solvent is large and the residence time in the separation tube increases, resulting in a longer analysis period. When the nitrile is used in excess of 30 v / v%, or in the case of using less than 70 v / v% of water, the polarity of the solvent is small and the residence time is short in the separation tube, it is difficult to separate according to the number of moles of polyethylene glycol added.
본 발명에 따라 HPLC로 폴리에톡실레이티드 아스코르브산 유도체를 분석할 때, 이동상 흐름속도는 1.1 내지 2.0 ml/분이 바람직하다. 이동상 흐름속도가 2.0 ml/분을 초과하는 경우에는 이동상의 압력이 2500psi 이상으로 높아져서 장시간 사용시 기기에 무리가 생기고, 흐름속도가 1.0 ml/분 이하인 경우에는 분석기간이 길어지므로, 이동상의 압력이 2000 내지 2500 psi가 되는 1.1 내지 2.0 ml/분이 적절한 분석시간을 제공할 수 있다.When analyzing polyethoxylated ascorbic acid derivatives by HPLC according to the present invention, the mobile phase flow rate is preferably 1.1 to 2.0 ml / min. If the flow rate of the mobile phase exceeds 2.0 ml / min, the pressure of the mobile phase rises to 2500 psi or more, which causes excessive pressure on the instrument. If the flow rate is less than 1.0 ml / min, the analysis period becomes longer. 1.1 to 2.0 ml / min, from 2 to 2500 psi, can provide an appropriate assay time.
본 발명의 바람직한 일례에서, 분리관을 통과한 시료를 검출하기 위해서 검출기를 사용가능하며, 220nm 내지 270nm의 파장을 갖는 UV/VIS 검출기를 사용하는 것이 바람직하다. 폴리에톡실레이티드 아스코르브산 유도체는 230 nm-270 nm의 파장을 갖는 빛을 흡수할 수 있으며 270nm이상과 230 nm이하의 파장에서는 빛이 검출되지 않는다. 상기 UV/VIS 검출기는 빛을 흡수할 수 잇는 시료에 일정 파장의 빛을 비추어 흡수가 일어나는 정도에 따라 검출하는 검출기이다.In a preferred embodiment of the present invention, a detector can be used to detect a sample that has passed through the separator, and it is preferable to use a UV / VIS detector having a wavelength of 220 nm to 270 nm. Polyethoxylated ascorbic acid derivatives can absorb light having a wavelength of 230 nm to 270 nm and no light is detected at wavelengths above 270 nm and below 230 nm. The UV / VIS detector is a detector that detects according to the degree of absorption occurs by shining a light of a predetermined wavelength on a sample that can absorb light.
본 발명의 일실시예에서, 폴리에톡실레이티드 아스코르브산 유도체를 폴리에틸렌 글리콜 부가몰수에 따라 분리하는 방법에 있어서, 최적화 HPLC 수행조건을 정하는 단계는, HPLC의 고정상으로서 비극성물질이 충진된 분리관을 연결하는 분리관 설정단계, 폴리에톡실레이티드 아스코르브산 유도체가 포함된 분석시료를 상기 분리관으로 이동시키는 이동상 설정단계, 분리관을 통과한 시료를 검출기로 유입시켜 검출하는 검출단계, 및 이동상의 압력을 2000 내지 2500 psi로 흐르도록 하는 이동상 흐름속도 조절단계로 순차적으로 수행할 수 있다.In one embodiment of the present invention, in the method for separating the polyethoxylated ascorbic acid derivative according to the number of moles of polyethylene glycol, the step of determining the conditions for performing optimized HPLC, the separation of the non-polar material-filled separation tube as a fixed phase of HPLC A separation pipe setting step for connecting, a mobile phase setting step for moving an analytical sample containing a polyethoxylated ascorbic acid derivative to the separation pipe, a detection step for detecting a sample passing through the separation pipe into a detector, and a mobile phase The pressure may be sequentially performed in a mobile phase flow rate adjusting step to flow 2000 to 2500 psi.
본 발명에 따른 HPLC를 이용한 분석방법으로 폴리에톡실레이티드 아스코르브산 유도체 시료가 폴리에틸렌 글리콜의 부가몰수에 따라 분리하여 분석할 수 있으며, 크로마토그램에서 시료의 체류시간은 5.7 내지 18.8 분까지 얻어지는 피크의 합에 의해 표준물질과 시료의 피크를 비교하여 시료내 상기 아스코르브산 유도체를 정량할 수 있다.The analysis method using HPLC according to the present invention can be analyzed by separating the polyethoxylated ascorbic acid derivative samples according to the added mole number of polyethylene glycol, the retention time of the sample in the chromatogram of the peak obtained up to 5.7 to 18.8 minutes The ascorbic acid derivative in the sample can be quantified by comparing the peaks of the standard and the sample by the sum.
다음의 실시예를 들어 본 발명을 더욱 자세히 설명할 것이나, 하기 실시예는 예시적으로 제공될 뿐이며 본 발명의 보호범위에 하기 실시예로 제한되는 의도는 아니다.The present invention will be described in more detail with reference to the following examples, but the following examples are provided by way of example only and are not intended to limit the scope of the present invention to the following examples.
실시예 1 내지 3Examples 1 to 3
세가지 다른 배합을 갖는 화장품을 메탄올에 녹이고 0.45 ㎛ 여과막으로 여과하여 시료로 사용하였다. HPLC 장치(휴렛팩커드 HP1050 시리즈)에 아세토니트릴 10 내지 30 v/v% 와 물 70 내지 90 v/v%을 포함하는 용액으로 평형화된 옥타데실 실란기가 충진된 Zorbax C18 분리관(150 x 4.6 mm, 휴렛펙커드, 미국)에 이동상의 흐름속도를 1.5 ml/분으로 하고 시료의 검출을 위한 245nm의 UV/VIS 검출기를 사용하고 시료를 적당량 투여한 후에 용출시켜 얻은 크로마토그램을 도 1에 표시하였다.Cosmetics with three different formulations were dissolved in methanol and filtered through a 0.45 μm filtration membrane to use as a sample. Zorbax C18 separator (150 x 4.6 mm, Hewlett-Packard) filled with octadecyl silane equilibrated in a solution containing 10-30 v / v% acetonitrile and 70-90 v / v% water in an HPLC apparatus (Hewlett-Packard HP1050 series) The chromatogram obtained by using a 245 nm UV / VIS detector for the detection of a sample and an appropriate amount of the sample was eluted with a flow rate of 1.5 ml / min for a mobile phase in Peckard, USA) is shown in FIG. 1.
도 1의 그래프에 나타난 바와 같이, 폴리에틸렌 글리콜의 부가물수에 따라 분리되어 분석되었음을 확인할 수 있었다. 상기 크로마토그램에서 나타난 피크를 합한 후에 검량선을 그려서 폴리에틸렌 글리콜이 치환된 폴리에톡실레이티드 아스코르브산의 정량분석을 적절하게 수행할 수 있었다.As shown in the graph of Figure 1, it was confirmed that the separation was analyzed according to the number of adducts of polyethylene glycol. After summing the peaks shown in the chromatogram, a calibration curve was drawn to properly perform quantitative analysis of polyethoxylated ascorbic acid substituted with polyethylene glycol.
비교예 1Comparative Example 1
HPLC의 이동상의 흐름속도는 1.0 ml/분으로 실험을 수행한 것을 제외하고는 상기 실시예 1 내지 3과 동일하게 실험을 수행하였다.The flow rate of the mobile phase of HPLC was performed in the same manner as in Examples 1 to 3 except that the experiment was performed at 1.0 ml / min.
실험결과를 도 1에 나타냈으며, 이로부터 이동상의 흐름 속도가 1.0ml/분인 경우에는 폴리에틴렌 글리콜의 부가몰수에 따라 분리되지 않음을 알 수 있었다.The experimental results are shown in FIG. 1, and it can be seen that when the flow velocity of the mobile phase was 1.0 ml / min, the separation was not performed according to the added mole number of the polyethylene glycol.
본 발명은 고성능액체크로마토그래피를 이용한 폴리에틸렌 글리콜이 결합된 폴리에톡실레이티드 아스코르브산 유도체를 폴리에틸렌 글리콜의 부가몰수에 따라 분석하는 방법을 제공하여, 시료에 포함된 폴리에톡실레이티드 아스코르브산 유도체를 정확하고 용이하게 정량할 수 있다.The present invention provides a method for analyzing a polyethoxylated ascorbic acid derivative in which polyethylene glycol is bound using high performance liquid chromatography according to the added mole number of polyethylene glycol, thereby determining a polyethoxylated ascorbic acid derivative contained in a sample. It can be accurately and easily quantified.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012129847A1 (en) * | 2011-03-25 | 2012-10-04 | 湖南汉森制药股份有限公司 | Methods for simultaneously determinating multi-component of simotang and fingerprint building method thereof |
| CN102749394A (en) * | 2012-06-06 | 2012-10-24 | 南京农业大学 | Separation and measurement method for reduction-type ascorbic acid and erythorbic acid in fruit and vegetable tissues and related products |
| CN104991016A (en) * | 2015-06-25 | 2015-10-21 | 吉林大学 | Quantitative assay method of PEG-modified medicine in biological sample |
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2002
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012129847A1 (en) * | 2011-03-25 | 2012-10-04 | 湖南汉森制药股份有限公司 | Methods for simultaneously determinating multi-component of simotang and fingerprint building method thereof |
| CN102749394A (en) * | 2012-06-06 | 2012-10-24 | 南京农业大学 | Separation and measurement method for reduction-type ascorbic acid and erythorbic acid in fruit and vegetable tissues and related products |
| CN104991016A (en) * | 2015-06-25 | 2015-10-21 | 吉林大学 | Quantitative assay method of PEG-modified medicine in biological sample |
| CN104991016B (en) * | 2015-06-25 | 2017-04-05 | 吉林大学 | The method for quantitatively determining of PEG chemical medicine thing in a kind of biological specimen |
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