KR20130027813A - Method for analyzing vitis vinifera extract - Google Patents

Method for analyzing vitis vinifera extract Download PDF

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KR20130027813A
KR20130027813A KR1020110091271A KR20110091271A KR20130027813A KR 20130027813 A KR20130027813 A KR 20130027813A KR 1020110091271 A KR1020110091271 A KR 1020110091271A KR 20110091271 A KR20110091271 A KR 20110091271A KR 20130027813 A KR20130027813 A KR 20130027813A
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acid
epicatechin
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procyanidin
catechin
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KR101877780B1 (en
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이헌수
신혜승
박용선
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(주)바이오메디앙
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Abstract

PURPOSE: A method for analyzing a Vitis vinifera extract is provided to simultaneously analyze gallic acid, procyanidin-B2, catechin, and epicatechin and to easily quantitate the extract without interference. CONSTITUTION: A method for analyzing bioequivalence of a test drug containing a Vitis vinifera extract comprises: a step of adding acid to plasma collected from an experimental subject who is administered with a test drug and mixing; a step of preparing an organic layer by liquid-liquid extraction using ethyl acetate; a step of drying the organic layer by evaporation and obtaining an analyte sample; and a step of analyzing gallic acid, procyanidin-B2, catechin, and epicatechin by a liquid chromatography-mass spectrometer.

Description

유럽종 포도 추출물의 분석방법 {Method for analyzing Vitis vinifera extract}{Method for analyzing Vitis vinifera extract}

본 발명은 유럽종 포도 추출물의 분석방법에 관한 것으로, 더욱 상세하게는 갈릭산(gallic acid), 프로시아니딘-B2(procyanidin-B2), 카테킨(catechin) 및 에피카테킨(epicatechin)을 지표물질로 이용하여 유럽종 포도(Vitis vinifera) 추출물의 생물학적 동등성을 분석하는 방법에 관한 것이다.
The present invention relates to a method for analyzing European grape extract, and more particularly, using gallic acid, procyanidin-B2, catechin, and epicatechin as an indicator. A method for analyzing the bioequivalence of Vitis vinifera extract.

포도속(Vitis) 식물은 갈매나무목(Rhamnales), 포도과(Vitaceae)에 속하는 덩굴성 식물로 적도부근 및 위도 50°이상 지역을 제외한 지구상 전역에서 자생 혹은 재배되고 있다. 포도과에는 11속 700 여종이 알려져 있으며, 유럽종 포도(Vitis vinifera), 미국종 포도(Vitis labrusca), 강변 포도(Vitis riparia), 사막 포도(Vitis rupestris), 겨울 포도(Vitis berladieri), 머루(Vitis coignetiae), 왕머루(Vitis amurensis) 등이 주로 과실을 이용하기 위하여 재배되고 있다. Vitis is a vine that belongs to Rhamnales and Vitaceae, and grows or grows all over the earth except near the equator and above 50 ° latitude. There are about 11 genera of 700 genera, including Vitis vinifera, Vitis labrusca, Vitis riparia, Vitis rupestris, Winter grapes, Vitis berladieri, and Vitis. coignetiae) and Vitis amurensis are mainly grown for fruit use.

이 중, 유럽종 포도(Vitis vinifera)의 씨로부터 얻어지는 추출물은 (-)에피카테킨, 프로안토시아니딘 B1 및 B2, (+)카테킨, 및 이들의 중합 유도체들의 혼합물들을 포함하며, 이들은 프로시아니돌 또는 플라보놀 올리고머로서 알려져 있다 (GB-A-1541469 및 FR-A-2092743).Among these, extracts obtained from the seeds of the European species Vitis vinifera include (-) epicatechin, proanthocyanidins B1 and B2, (+) catechins, and mixtures of polymerized derivatives thereof, which are procyano Known as stone or flavonol oligomers (GB-A-1541469 and FR-A-2092743).

상기 추출물은 주로 결합조직, 혈관, 림프, 관절 등의 글리코스아미노글리칸(glycosaminoglycan)에 선택적으로 작용하여 콜라겐, 엘라스틴, 피브로넥틴 등의 결합능에 관련하는 섬유들의 합성을 촉진하고 분해를 억제하여, 증가된 모세혈관 투과성을 억제하고 정맥의 신축성을 빠르게 회복시켜 주는 것으로 보고되고 있다[Arteres et veines Vol.5(5), 397～401(1986); Sem-dex Hopitaux 48/47, 2009～2013(1981)]. 또한, 포도씨 추출물은 당뇨병 환자의 모세혈관 취약증, 고혈압에 관련된 망막증, 망막분리후의 잔여 망막부종에 대한 치료효과를 갖는 것이 보고된 바 있으며[Gazette Mde France Vol.88, No.14, 2035～2038(1981)], 망막의 여러 구조를 보호함으로써 시각색소의 재생속도를 빠르게 하며, 임상시험에서 섬광에 노출된 후에 망막을 회복시키는데도 효과가 있는 것으로 보고된 바 있다[Bull. Soc. Opth. France Vol.88(2), 173-4, 177-9(1988)].The extract mainly acts on glycosaminoglycans such as connective tissue, blood vessels, lymph and joints selectively to promote synthesis and inhibit degradation of fibers related to the binding ability of collagen, elastin, fibronectin, etc. It has been reported to inhibit the capillary permeability and quickly restore the elasticity of veins [Arteres et veines Vol. 5 (5), 397-401 (1986); Sem-dex Hopitaux 48/47, 2009-2013 (1981). In addition, grape seed extract has been reported to have a therapeutic effect on capillary vulnerability, hypertension-related retinopathy, and residual retinal edema after retinal detachment in diabetic patients [Gazette Mde France Vol. 88, No. 14, 2035-2038]. (1981), which protects the various structures of the retina, speeds up the regeneration of visual pigment, and has been reported to be effective in restoring the retina after exposure to scintillation in clinical trials [Bull. Soc. Opth. France Vol. 88 (2), 173-4, 177-9 (1988).

현재, 유럽종 포도(Vitis vinifera)의 씨 추출물를 함유한 경구용 정제(엔테론, 한림제약(주), 한국)는 정맥 림프기능부전과 관련된 증상개선, 유방암치료로 인한 림프부종의 보조요법제, 망막 맥락막 순환과 관련된 장애치료 보조제로서 상용화되고 있다.
Currently, oral tablets containing seed extracts of European varieties Vitis vinifera (Enteron, Hallym Pharmaceutical Co., Ltd., Korea) improve symptom associated with venous lymphatic dysfunction, adjuvant therapy of lymphedema due to breast cancer treatment, It is commercially available as a therapeutic aid for disorders associated with retinal choroidal circulation.

한편, 생물학적 동등성(bioequivalence, BE)이란 비슷한 조건 아래에서 같은 용량을 투여했을 경우 각 제제의 흡수의 양과 속도가 유의성 있는 차이를 보이지 않는 경우를 말하는 것이다. 따라서 생물학적으로 동등하다는 것은 같은 정도의 약효를 나타낸다는 뜻은 아니지만 그 전제조건은 된다고 할 수 있다.On the other hand, bioequivalence (BE) refers to a case where there is no significant difference in the amount and rate of absorption of each agent when the same dose is administered under similar conditions. Therefore, biologically equivalent does not mean that they show the same level of efficacy, but it is a precondition.

그러므로 생물학적 동등성 시험(bioequivalence test: BE Test)은 제제학적으로 동등한 두 제제 또는 제제학적으로 대체가능한 제제가 생물학적 이용률에 있어서 통계학적으로 동등하다는 것을 입증하기 위해 실시하는 생체내 실험을 의미한다. 즉, 생물학적 동등성 시험(생동성 시험)이란 제약업체들이 복제약 판매 허가를 받기 전에 실제 사람에게 투여하여 오리지널 약(대조약)과 동일한 성분으로 만들어진 복제 약물(시험약)이 동등한 약효를 나타내는지 여부를 통계학적 방법으로 증명하는 것이다.
Thus, a bioequivalence test (BE Test) refers to an in vivo experiment conducted to demonstrate that two formulations that are pharmaceutically equivalent or agents that are pharmaceutically replaceable are statistically equivalent in bioavailability. In other words, a bioequivalence test (bioequivalence test) is a statistic of whether a drug (test drug) made by the same ingredient as the original drug (control drug), which is administered to a real person before the pharmaceutical company is authorized to sell the drug, has an equivalent effect. It is to prove by scientific method.

그런데, 천연추출물을 원료로 한 의약은 대사체의 종류가 많고 복잡할 뿐만 아니라, 내인성 물질과 간섭하거나 농도가 낮아 정량이 어려운 문제 등으로 인하여 생물학적 동등성 시험을 수행하기 어려운 문제점이 있었다. 특히, 엔테론의 경우 원료물질인 유럽종 포도 추출물이 다당류로서 체내에 투여된 후 다양한 형태의 구조를 가진 여러가지 플라보노이드 대사체들로 분해되고, 대사체들을 정량하기 어려운 문제가 있었다.
By the way, the drug using a natural extract as a raw material has a problem that it is difficult to carry out a bioequivalence test due to a lot of complex metabolites and complex, as well as difficult to quantify due to interference with low endogenous substances or low concentration. In particular, in the case of enteron, the European grape extract, which is a raw material, is administered into the body as a polysaccharide, and then degraded into various flavonoid metabolites having various forms of structure, and it is difficult to quantify the metabolites.

본 발명자들은 유럽종 포도 추출물의 생물학적 동등성 분석방법을 개발하고자 예의 연구 검토한 결과, 갈릭산, 프로시아니딘-B2, 카테킨 및 에피카테킨을 지표물질로 이용하여 유럽종 포도 추출물의 생물학적 동등성을 간단하고 용이하게 분석할 수 있음을 발견하고 본 발명을 완성하게 되었다. The present inventors conducted a thorough study to develop a method for analyzing the bioequivalence of European grape extracts, and as a result, the biological equivalence of European grape extracts was simply and easily analyzed using gallic acid, procyanidin-B2, catechin and epicatechin as indicators. It was found that the present invention can be completed.

따라서, 본 발명의 목적은 갈릭산, 프로시아니딘-B2, 카테킨 및 에피카테킨을 지표물질로 이용하여 유럽종 포도 추출물의 생물학적 동등성을 분석하는 방법을 제공하는 것이다.
Accordingly, it is an object of the present invention to provide a method for analyzing the bioequivalence of European grape extract using gallic acid, procyanidin-B2, catechin and epicatechin as indicators.

본 발명은 갈릭산(gallic acid), 프로시아니딘-B2(procyanidin-B2), 카테킨(catechin) 및 에피카테킨(epicatechin)을 지표물질로 이용하여 유럽종 포도(Vitis vinifera) 추출물을 포함하는 시험약의 생물학적 동등성을 분석하는 방법에 관한 것이다.
The present invention relates to the biological equivalence of a test drug comprising an extract of Vitis vinifera using gallic acid, procyanidin-B2, catechin, and epicatechin as indicators. It is about how to analyze.

다른 태양으로, 본 발명은 유럽종 포도(Vitis vinifera) 추출물을 포함하는 시험약의 생물학적 동등성을 분석하는 방법으로서,In another aspect, the present invention provides a method for analyzing the bioequivalence of a test drug comprising a European Vitis vinifera extract,

(i) 상기 시험약을 투여한 피험자로부터 채취한 혈장에 산을 가하고 혼합하는 단계;(i) adding acid to the plasma collected from the subject to which the test drug is administered and mixing;

(ii) 생성된 혼합물을 에틸아세테이트로 액체-액체 추출법에 의해 추출하여 유기층을 수득하는 단계;(ii) extracting the resulting mixture by liquid-liquid extraction with ethyl acetate to obtain an organic layer;

(iii) 수득한 유기층을 증발 건조하여 분석 시료를 수득하는 단계; 및(iii) evaporating to dryness the obtained organic layer to obtain an analytical sample; And

(iv) 상기 분석 시료로부터 갈릭산(gallic acid), 프로시아니딘-B2(procyanidin-B2), 카테킨(catechin) 및 에피카테킨(epicatechin)을 액체 크로마토그래피(LC)-질량분석기(MS/MS)로 분석하는 단계를 포함하는 것을 특징으로 하는 방법에 관한 것이다. (iv) analyzing the gallic acid, procyanidin-B2, catechin and epicatechin from the assay sample by liquid chromatography (LC) -mass spectrometry (MS / MS) It relates to a method characterized in that it comprises a step.

상기 단계 (i)에서 산으로는 염산, 황산아연, 과염소산(perchloric acid), 옥살산 등을 사용할 수 있으나, 이에 한정되는 것은 아니다. 단계 (i)에서 산을 부가함으로써 단계 (ii)에서의 추출 효율을 증가시켜 지표물질의 정량을 용이하게 할 수 있다. The acid in step (i) may be hydrochloric acid, zinc sulfate, perchloric acid, oxalic acid, and the like, but is not limited thereto. By adding an acid in step (i), the extraction efficiency in step (ii) can be increased to facilitate quantification of the indicator.

상기 단계 (ii)에서는 생성된 혼합물에 에틸아세테이트를 가하고 진탕한 후, 원심분리하여 유기층을 수득할 수 있다. In step (ii), ethyl acetate may be added to the resulting mixture, shaken, and centrifuged to obtain an organic layer.

상기 단계 (iv)에서는 분석 시료를 바람직하게는 메탄올에 용해시킨 다음, 일정량, 예를 들어 5-10 ul를 취하여 LC-MS/MS에 주입하고 갈릭산, 프로시아니딘-B2, 카테킨 및 에피카테킨의 농도를 분석한다. 이때 액체 크로마토그래피는 역상 컬럼을 사용하고, 0.1% 포름산을 함유한 물과 0.1% 포름산을 함유한 메탄올을 이용한 경사 용리(gradient elution) 방식에 의해 수행하는 것이 바람직하다. 또한, 질량분석은 전구 이온(precursor ion)과 생성 이온(product ion)을 동시에 측정하는 다중 반응 모니터링(multiple reaction monitoring: MRM) 방식으로 수행하는 것이 바람직하다.
In the above step (iv), the analytical sample is preferably dissolved in methanol, and then injected into LC-MS / MS by taking a predetermined amount, for example, 5-10 ul, and the concentrations of gallic acid, procyanidin-B2, catechin and epicatechin are determined. Analyze At this time, the liquid chromatography is preferably performed by a gradient elution method using a reverse phase column, using water containing 0.1% formic acid and methanol containing 0.1% formic acid. In addition, mass spectrometry is preferably performed in a multiple reaction monitoring (MRM) manner in which both precursor and product ions are measured simultaneously.

본 발명의 분석방법에 따르면, 시험약을 피험자에 투여한 후 일정 시간 간격으로 채취한 혈장에 대해 상기한 (i) 내지 (iv)의 단계를 반복 수행함으로써, 지표물질들의 시간에 따른 혈중 농도를 측정할 수 있다. 그리고 이를 대조약인 엔테론과 비교하여 생물학적 동등성을 확인할 수 있다.
According to the analytical method of the present invention, by repeating the steps (i) to (iv) described above for the plasma collected at regular time intervals after administering the test drug to the subject, blood concentrations over time of the indicators are determined. It can be measured. The bioequivalence can be confirmed by comparing it with the reference drug enterrone.

본 발명의 분석방법에 따르면, 갈릭산, 프로시아니딘-B2, 카테킨 및 에피카테킨을 동시에 분석할 수 있으며, 내인성 물질이나 다른 대사체와의 간섭 없이 용이하게 정량할 수 있다.According to the analytical method of the present invention, gallic acid, procyanidin-B2, catechin and epicatechin can be analyzed simultaneously, and easily quantified without interference with endogenous substances or other metabolites.

따라서, 이들을 지표물질로 사용하여 시험약을 피험자에 투여한 후 시간에 따른 혈중 농도를 분석함으로써, 유럽종 포도 추출물을 포함하는 시험약의 생물학적 동등성을 확인할 수 있다.Therefore, by using these as indicators, the test drug is administered to the subject, and the blood concentrations of the test drug including the European grape extract can be confirmed by analyzing the blood concentration over time.

특히, 본 발명의 분석방법에 따르면 우수한 감도, 분석시간의 단축, 간편성 및 경제성을 동시에 얻을 수 있다.
In particular, according to the analysis method of the present invention, excellent sensitivity, shortening of analysis time, simplicity, and economical efficiency can be simultaneously obtained.

도 1은 시험약을 피험자에 투여한 후 시간에 따른 갈릭산의 혈중 농도를 나타낸 그래프이다.
도 2는 시험약을 피험자에 투여한 후 시간에 따른 프로시아니딘-B2의 혈중 농도를 나타낸 그래프이다.
도 3은 시험약을 피험자에 투여한 후 시간에 따른 카테킨의 혈중 농도를 나타낸 그래프이다.
도 4는 시험약을 피험자에 투여한 후 시간에 따른 에피카테킨의 혈중 농도를 나타낸 그래프이다. 1 is a graph showing the blood concentration of gallic acid over time after administering a test drug to a subject.
2 is a graph showing the blood concentration of procyanidin-B2 over time after administering a test drug to a subject.
3 is a graph showing the blood concentration of catechin over time after administering the test drug to the subject.
Figure 4 is a graph showing the blood concentration of epicatechin over time after administering the test drug to the subject.

이하, 실시예에 의해 본 발명을 보다 구체적으로 설명하고자 한다. 이들 실시예는 오직 본 발명을 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업자에게 있어서 자명하다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are for illustrative purpose only and that the scope of the present invention is not limited to these embodiments.

실시예 1: 분석 시료의 제조Example 1 Preparation of Analytical Samples

엔테론 300 mg을 피험자에 경구투여하고, 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8 및 10시간 후에 각각 혈액을 채취하여 혈장을 수득하였다.300 mg of enterone was orally administered to the subject, and blood was collected after 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8 and 10 hours respectively to obtain plasma.

수득한 혈장 500 ul에 0.5 M HCl을 100 ul 첨가하고 30초간 혼합하였다. 혼합물에 에틸아세테이트 5.0 mL를 가하고 15분간 진탕한 후, 4000 rpm에서 5분간 원심분리하였다. 그 후 상등액을 튜브에 옮기고 40 ℃로 맞춰진 질소 농축기에서 증발 건조시켜 분석 시료를 제조하였다.
100 ul of 0.5 M HCl was added to 500 ul of the obtained plasma and mixed for 30 seconds. 5.0 mL of ethyl acetate was added to the mixture, followed by shaking for 15 minutes, followed by centrifugation at 4000 rpm for 5 minutes. The supernatant was then transferred to tubes and evaporated to dryness in a nitrogen concentrator set at 40 ° C. to prepare analytical samples.

실시예 2: 지표물질의 정량Example 2: Quantification of Indicators

실시예 1에서 수득한 분석 시료를 50% 메탄올 100 ul에 용해시킨 다음, 10 ul를 취하여 LC-MS/MS에 주입하고, 하기와 같은 분석 조건하에서 갈릭산, 프로시아니딘-B2, 카테킨 및 에피카테킨의 농도를 분석하였다.The analytical sample obtained in Example 1 was dissolved in 100 ul of 50% methanol, then 10 ul was taken into LC-MS / MS, and the concentrations of gallic acid, procyanidin-B2, catechin and epicatechin under the following analysis conditions. Was analyzed.

분석 조건Analysis condition

컬럼: Gemini C18 (2.0 X 150 mm, 5 um, Phenomenex)Column: Gemini C 18 (2.0 X 150 mm, 5 um, Phenomenex)

이동상 용매: 증류수(0.1% 포름산)와 메탄올(0.1% 포름산)의 혼합 비율을 90: 10에서 10:90으로 시간대별로 조절하는 경사 프로그램(gradient program)Mobile phase solvent: gradient program to adjust the mixing ratio of distilled water (0.1% formic acid) and methanol (0.1% formic acid) from 90: 10 to 10:90

유속: 200 ul/minFlow rate: 200 ul / min

시료 주입량: 10 ulSample injection volume: 10 ul

LC 펌프: Nanospace SI-23201 (Shiseido, Japan)LC Pump: Nanospace SI-23201 (Shiseido, Japan)

오토샘플러(autosampler): Nanospace SI-23133 (Shiseido, Japan)Autosampler: Nanospace SI-23133 (Shiseido, Japan)

검출기: API 5000TM (Sciex Division of MDS Inc., Canada)Detector: API 5000 TM (Sciex Division of MDS Inc., Canada)

분무가스(nebulizing gas; GS1)와 가열가스(heated gas; GS2)의 플로우: 각각 50 psiNebulizing gas (GS1) and heated gas (GS2) flow: 50 psi each

커튼가스(curtain gas)의 플로우: 20 psiCurtain gas flow: 20 psi

소스(source) 온도: 500 ℃Source temperature: 500 ° C

이온 분무(IS) 전압: -4,500 V (negative mode)와 4,500 V (positive mode)
Ion Spray (IS) voltage: -4,500 V (negative mode) and 4,500 V (positive mode)

갈릭산, 프로시아니딘-B2, 카테킨 및 에피카테킨의 정량을 위해 전구 이온(precursor ion)과 생성 이온(product ion)을 동시에 측정하는 다중 반응 모니터링(multiple reaction monitoring: MRM)을 수행하였으며, 갈릭산의 [M-H]- 이온 질량 전이(mass transition)는 169.0 → 125.0이고, 카테킨과 에피카테킨의 질량 전이는 289.0 → 109.1로 동일하였고, 프로시아니딘-B2의 질량 전이는 577.1 → 289.2 이었다.
To quantify gallic acid, procyanidin-B2, catechin and epicatechin, multiple reaction monitoring (MRM) was performed to simultaneously measure precursor and product ions. ] -The mass transition was 169.0 → 125.0, the mass transition of catechin and epicatechin was 289.0 → 109.1, and the mass transition of procyanidin-B2 was 577.1 → 289.2.

상기한 분석방법으로 측정한 갈릭산, 프로시아니딘-B2, 카테킨 및 에피카테킨의 시간에 따른 혈중 농도를 각각 도 1 내지 도 4에 나타내었다. The blood concentrations over time of gallic acid, procyanidin-B2, catechin and epicatechin measured by the analytical method described above are shown in FIGS. 1 to 4, respectively.

최고 혈중 농도가 나타나는 Tmax는 갈릭산, 카테킨 및 에피카테킨의 경우에는 3 시간에 나타났으며, 프로시아니딘-B2(Pro-B2)의 경우에는 3.5 시간에 나타났다. 최고 혈중 농도는 갈릭산은 6.68 ± 0.8 ng/mL, 프로시아니딘-B2(Pro-B2)는 10.32 ± 1.20 ng/mL, 카테킨은 11.12 ± 1.19 ng/mL, 에피카테킨은 5.21 ± 0.6 ng/mL으로 나타났다. Tmax with peak blood levels was seen at 3 hours for gallic acid, catechin and epicatechin, and at 3.5 hours for procyanidin-B2 (Pro-B2). The highest blood levels were 6.68 ± 0.8 ng / mL for gallic acid, 10.32 ± 1.20 ng / mL for procyanidin-B2 (Pro-B2), 11.12 ± 1.19 ng / mL for catechin, and 5.21 ± 0.6 ng / mL for epicatechin.

따라서, 본 발명의 분석방법에 따라 갈릭산, 프로시아니딘-B2, 카테킨 및 에피카테킨의 농도를 분석함으로써, 유럽종 포도 추출물의 생물학적 동등성을 확인할 수 있음을 알 수 있다.Thus, by analyzing the concentrations of gallic acid, procyanidin-B2, catechin and epicatechin according to the analysis method of the present invention, it can be seen that the bioequivalence of European grape extract.

Claims (7)

갈릭산(gallic acid), 프로시아니딘-B2(procyanidin-B2), 카테킨(catechin) 및 에피카테킨(epicatechin)을 지표물질로 이용하여 유럽종 포도(Vitis vinifera) 추출물을 포함하는 시험약의 생물학적 동등성을 분석하는 방법.Analyzing the bioequivalence of test drugs including the extract of Vitis vinifera using gallic acid, procyanidin-B2, catechin and epicatechin as indicators Way. 제1항에 있어서, 유럽종 포도(Vitis vinifera) 추출물을 포함하는 시험약의 생물학적 동등성을 분석하는 방법으로서,
(i) 상기 시험약을 투여한 피험자로부터 채취한 혈장에 산을 가하고 혼합하는 단계;
(ii) 생성된 혼합물을 에틸아세테이트로 액체-액체 추출법에 의해 추출하여 유기층을 수득하는 단계;
(iii) 수득한 유기층을 증발 건조하여 분석 시료를 수득하는 단계; 및
(iv) 상기 분석 시료로부터 갈릭산(gallic acid), 프로시아니딘-B2(procyanidin-B2), 카테킨(catechin) 및 에피카테킨(epicatechin)을 액체 크로마토그래피(LC)-질량분석기(MS/MS)로 분석하는 단계를 포함하는 것을 특징으로 하는 방법.The method according to claim 1, wherein the bioequivalence of the test drug containing the European Vitis vinifera extract is analyzed.
(i) adding acid to the plasma collected from the subject to which the test drug is administered and mixing;
(ii) extracting the resulting mixture by liquid-liquid extraction with ethyl acetate to obtain an organic layer;
(iii) evaporating to dryness the obtained organic layer to obtain an analytical sample; And
(iv) analyzing the gallic acid, procyanidin-B2, catechin and epicatechin from the assay sample by liquid chromatography (LC) -mass spectrometry (MS / MS) And comprising a step. 제2항에 있어서, 단계 (i)에서 산이 염산인 것을 특징으로 하는 방법.The process according to claim 2, wherein the acid in step (i) is hydrochloric acid. 제2항에 있어서, 단계 (ii)에서, 생성된 혼합물에 에틸아세테이트를 가하고 진탕한 후, 원심분리하여 유기층을 수득하는 것을 특징으로 하는 방법.The method according to claim 2, wherein in step (ii), ethyl acetate is added to the resulting mixture, shaken, and centrifuged to obtain an organic layer. 제2항에 있어서, 단계 (iv)에서 분석 시료를 메탄올에 용해시킨 다음, 일정량을 취하여 LC-MS/MS에 주입하고 갈릭산, 프로시아니딘-B2, 카테킨 및 에피카테킨의 농도를 분석하는 것을 특징으로 하는 방법.The method according to claim 2, characterized in that the analytical sample is dissolved in methanol in step (iv), then a predetermined amount is injected into LC-MS / MS and the concentrations of gallic acid, procyanidin-B2, catechin and epicatechin are analyzed. Way. 제2항에 있어서, 단계 (iv)에서 액체 크로마토그래피가 역상 컬럼을 사용하고, 0.1% 포름산을 함유한 물과 0.1% 포름산을 함유한 메탄올을 이용한 경사 용리(gradient elution) 방식에 의해 수행되는 것을 특징으로 하는 방법.The liquid chromatography of claim 2, wherein the liquid chromatography in step (iv) is performed by using a reversed phase column and a gradient elution method using water containing 0.1% formic acid and methanol containing 0.1% formic acid. How to feature. 제2항에 있어서, 단계 (iv)에서 질량분석이 전구 이온(precursor ion)과 생성 이온(product ion)을 동시에 측정하는 다중 반응 모니터링(multiple reaction monitoring: MRM) 방식으로 수행되는 것을 특징으로 하는 방법.The method of claim 2, wherein in step (iv) the mass spectrometry is carried out in a multiple reaction monitoring (MRM) manner in which the precursor ions and the product ions are measured simultaneously. .
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