KR20010047748A - A method of analyzing a concentration of homocysteine in plasma - Google Patents
A method of analyzing a concentration of homocysteine in plasma Download PDFInfo
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Abstract
Description
본 발명은 호모시스테인을 유도체화하여 혈장 중의 호모시스테인의 농도를 효과적으로 검출하는 방법에 관한 것으로서, 상기 방법은 호모시스테인을 에틸 클로로포르메이트 및 2,2,3,3,3-펜타플루오로프로판올과 반응시켜 호모시스테인을 유도체화한 후 그 농도를 결정하는 것으로 이루어진다.The present invention relates to a method for effectively detecting the concentration of homocysteine in plasma by derivatizing homocysteine, the method comprising reacting homocysteine with ethyl chloroformate and 2,2,3,3,3-pentafluoropropanol to homocysteine After derivatization, the concentration thereof is determined.
본 발명은 또한 상기 유도체화된 호모시스테인을 유기 용매로 추출하여 클린업하고, 기체 크로마토그래피-전자포획 검출기를 사용하여 그 농도를 결정하는 것을 특징으로 한다.The present invention is also characterized in that the derivatized homocysteine is extracted with an organic solvent and cleaned up, and its concentration is determined using a gas chromatography-electron capture detector.
호모시스테인(homocysteine)은 황을 포함하는 아미노산으로서, 그 화학적 구조는 화학식 1과 같다.Homocysteine is an amino acid containing sulfur and its chemical structure is represented by the formula (1).
상기 호모시스테인은 현대인의 성인병 중에서 높은 비율을 차지하는 관상동맥질환과 밀접한 관련이 있으며, 따라서 그에 대한 관심이 날로 증가하고 있는 추세이다. 호모시스테인의 혈중 농도에 영향을 미치는 주요 인자로는 호모시스테인의 대사에 관여하는 효소, 대사과정에서 조인자(cofactor)로 작용하는 비타민 B6(피리독신), B12(코발라민) 및 엽산(folate)이며, 이들 화합물들의 결핍시 호모시스테인의 대사가 불충분해 그 혈중농도를 증가시켜 관상동맥질환을 야기하는 것으로 알려져 있다[Clin.Chem.,39, 263(1993)].The homocysteine is closely related to coronary artery disease, which accounts for a high proportion of the adult diseases of modern people, and thus, interest in it is increasing day by day. The main factors affecting the homocysteine blood concentration are enzymes involved in the metabolism of homocysteine, vitamin B6 (pyridoxine), B12 (cobalamin) and folate, which act as cofactors in the metabolic process. Insufficient metabolism of homocysteine during deficiency increases blood levels and causes coronary artery disease [ Clin . Chem ., 39 , 263 (1993)].
호모시스테인의 혈중 농도를 분석하는 방법은 꾸준히 연구되어 왔으며 구체적으로는 방사성효소 분석(radioenzymatic essay), 아미노산 분석기(amino acid analyzer), 고성능 액체 크로마토그래피(HPLC) 및 기체 가스크로마토그래피(GC) 등이 있다[J.Chromatogr.,579, 55(1992);J.Chromatogr. A,729, 335(1996);J.Chromatogr. B,664, 421(1995)].Methods of analyzing blood levels of homocysteine have been steadily studied, and specific examples include radioenzymatic essay, amino acid analyzer, high performance liquid chromatography (HPLC), and gaseous gas chromatography (GC). [ J. Chromatogr ., 579 , 55 (1992); J. Chromatogr . A, 729 , 335 (1996); J. Chromatogr . B, 664 , 421 (1995).
상기 방법 중 방사성효소 분석은 감도가 좋고 저렴한 방법이기는 하나, 장시간이 소요되고 복잡한 조작과정 및 고가의 시약 등을 사용해야 하는 문제점으로 인해 임상 분석에 적용하기 힘든 문제점이 있었다[Clin.Chem.,39, 1764(1993);J.Chromatogr.,565, 441(1991)].Among the above methods, radioenzyme analysis is a sensitive and inexpensive method, but it is difficult to apply to clinical analysis due to the problem of using a long time, complicated manipulation process, and expensive reagents [ Clin . Chem ., 39 , 1764 (1993); J. Chromatogr ., 565 , 441 (1991).
아미노산 분석기를 이용한 방법은 호모시스테인의 총량을 정량하는 전통적 방법으로 시료 준비과정이 간단하지만, 기기 작동에 있어서 숙련되어야 하며, 황을 포함하는 아미노산에 대해 선택적이지 못하고 다른 방법(통상 1 μmol 정도)에 비해 감도가 낮다는 문제점이 있다.The method using the amino acid analyzer is a traditional method of quantifying the total amount of homocysteine, but the sample preparation process is simple, but should be skilled in the operation of the instrument, and is not selective for amino acids containing sulfur, compared to other methods (typically about 1 mol) There is a problem that the sensitivity is low.
고성능 액체 크로마토그래피를 이용한 방법은 가장 널리 사용되는 방법으로, 자외선-가시광선 영역(UV-Visible)에서의 포스트-컬럼 닌히드린(post-column ninhydrin) 유도체화 방법을 이용하거나, 분광 광도 검출기(spectrophotometric detector)와 형광 검출기(fluorescence detector) 그리고 전기화학적 검출기(electrochemical detector) 등의 다른 여러 검출기와 연결하여 사용되어 왔다[J.Chromatogr.,593, 171(1992)]. 그러나 HPLC를 이용하는 방법도 고가이고 불안정한 시약 등의 문제점이 있다.The method using high performance liquid chromatography is the most widely used method using a post-column ninhydrin derivatization method in the ultraviolet-visible region or a spectrophotometric method. It has been used in conjunction with many other detectors, such as detectors, fluorescence detectors and electrochemical detectors [ J. Chromatogr ., 593 , 171 (1992). However, methods using HPLC also have problems such as expensive and unstable reagents.
기체 크로마토그래피를 이용하여 호모시스테인을 분석하는 방법은 단지 소수의 논문들이 발표되었다. GC는 HPLC의 1/3의 비용만으로도 좋은 분리 효율과 빠른 분석시간을 갖는다. 하지만 GC를 이용하여 호모시스테인을 분석하는 가장 큰 문제점은 수용액으로부터 호모시스테인을 추출하는 데 있어서의 곤란성이다. 즉 호모시스테인을 수용액에서 유기층으로 추출할 수 있는 적절한 용매가 없으며, 1시간 또는 그 이상의 다단계의 과정을 거쳐야 한다.How to analyze homocysteine using gas chromatography, only a few papers have been published. GC has good separation efficiency and fast analysis time at only 1/3 the cost of HPLC. However, the biggest problem of analyzing homocysteine using GC is the difficulty in extracting homocysteine from aqueous solution. That is, there is no suitable solvent to extract homocysteine from the aqueous solution into the organic layer, and it must go through a multi-step process for 1 hour or more.
이러한 문제점을 해결하기 위해 호모시스테인을 적절한 화합물과 반응시켜 호모시스테인의 분자 구조에 존재하는 -NH2, -COOH, -SH의 극성기들을 비극성기로 바꾸어 주는 유도체화 방법이 강구되었으며, 그 예로는 호모시스테인을 n-프로필 클로로포메이트와 반응시켜 N(O,S)-프로폭시카보닐 프로필 에스테르 유도체를 합성하여 기체 크로마토그래피-질량 분석기(GC-MS)로 분석하는 방법[J.Chromatogr. A,776, 342(1997)]과 tert-부틸디메틸실릴 유도체를 만들어 GC-MS로 분석하는 방법이 있다[Anal.Biochem.,162, 185(1987)].In order to solve this problem, a derivatization method for converting polar groups of -NH 2 , -COOH, -SH present in the molecular structure of homocysteine into a non-polar group by reacting homocysteine with an appropriate compound has been devised. Reaction with propyl chloroformate to synthesize N (O, S) -propoxycarbonyl propyl ester derivatives and analyze by gas chromatography-mass spectrometry (GC-MS) [ J. Chromatogr . A, 776 , 342 (1997)] and tert-butyldimethylsilyl derivatives are analyzed by GC-MS [ Anal . Biochem ., 162 , 185 (1987)].
그러나 상기의 방법도 혈장 속에 존재하는 미량의 호모시스테인의 농도를 분석하기에는 감도가 낮아 그 신뢰도가 떨어지고, 정밀한 측정을 요할 경우에는 불가능할 뿐만 아니라, GC-MS가 고가의 장비라는 문제점이 있다.However, the above method is also low in sensitivity to analyze the concentration of trace amounts of homocysteine present in the plasma, its reliability is low, it is impossible to require precise measurement, there is a problem that GC-MS is expensive equipment.
따라서 호모시스테인의 혈중 농도를 결정하는 데 있어서, 감도의 우수성, 분석 시간의 단축, 저비용 및 편리성 등을 동시에 만족시키는 분석 방법에 대한 요청이 계속되어 왔었다.Therefore, in determining the blood concentration of homocysteine, there has been a continuous demand for an analysis method that satisfies the superiority of sensitivity, shortening of analysis time, low cost and convenience.
따라서 본 발명의 목적은 호모시스테인의 혈중 농도를 측정하는 새로운 분석방법을 제공하는 것이다. 즉 우수한 감도, 분석 시간의 단축, 저비용 및 편리성 등을 동시에 만족시키는 새로운 분석 방법을 제공하는 것을 그 목적으로 한다.It is therefore an object of the present invention to provide a new analytical method for measuring the blood concentration of homocysteine. That is, the object of the present invention is to provide a new analysis method that simultaneously satisfies excellent sensitivity, short analysis time, low cost and convenience.
도 1은 유도체화된 호모시스테인 정량에 사용된 검량 곡선1 is a calibration curve used to quantify derivatized homocysteine
도 2는 본 발명의 유도체화 방법을 사용하여 혈액으로부터 얻은 크로마토그램이다.2 is a chromatogram obtained from blood using the derivatization method of the present invention.
본 발명의 분석 방법은 호모시스테인을 에틸 클로로포르메이트 및 2,2,3,3,3-펜타플루오로프로판올과 반응시켜 호모시스테인을 유도체화하고, 이를 유기 용매를 사용하여 추출한 후, 기체 크로마토그래피-전자포획검출기를 사용하여 호모시스테인의 농도를 분석하는 것으로 이루어진다.The analytical method of the present invention reacts homocysteine with ethyl chloroformate and 2,2,3,3,3-pentafluoropropanol to derivatize homocysteine, extract it with an organic solvent, and then gas chromatography-electron The capture detector is used to analyze the concentration of homocysteine.
호모시스테인을 유도체화하는 방법을 보다 구체적으로 살펴보면, 호모시스테인을 화학식 2를 갖는 에틸 클로로포르메이트 및 화학식 3을 갖는 2,2,3,3,3-펜타플루오로-1-프로판올(PFP-OH)과 반응시키는 것으로 구성되며, 그 반응은 반응식 1과 같다.Looking more specifically at the method of derivatizing homocysteine, homocysteine and ethyl chloroformate having the formula (2) and 2,2,3,3,3-pentafluoro-1-propanol (PFP-OH) having the formula (3) and It consists of reacting, and the reaction is the same as in Scheme 1.
상기 유도체화 방법은 특히 수용액 내에서 바로 유도체화 반응이 진행되므로 생체 시료(혈액 및 뇨)에서의 호모시스테인을 분석하는 데 좋은 방법이라 할 수 있으며, 궁극적으로는 감도 및 선택성이 좋고, 보다 향상된 검출한계를 제공한다. 그리고 상온에서 특별한 조작없이 몇 분 내에 반응이 종결되는 매우 간편한 방법이며, 유기 용매에 대한 추출율을 높여주고, 기존에 사용했던 유도체화 시약의 가격에 비해 저렴하다는 장점을 가지고 있다.The derivatization method is a particularly good method for analyzing homocysteine in biological samples (blood and urine) since the derivatization reaction proceeds in an aqueous solution. Ultimately, the sensitivity and selectivity are excellent and the detection limit is improved. To provide. And it is a very easy way to terminate the reaction in a few minutes at room temperature without any special operation, and has the advantage of increasing the extraction rate for the organic solvent, and compared to the price of the derivatization reagent used previously.
상기 반응의 생성물인 호모시스테인의 N(O,S)-에틸카보닐 펜타플루오로프로필 에테르 유도체를 클린업하는 공정은 유기 용매를 사용한 추출 단계로 구성된다.상기 추출 단계는 유기화학분야에서 사용되는 일반적 방법을 채용할 수 있다. 추출 용매로 사용될 수 있는 유기 용매로는 클로로포름, 디클로로메탄, 에테르, 에틸아세테이트, 헥산, 펜탄 또는 그들의 혼합물과 같은 무극성 용매이면 특별히 제한되지 아니하나, GC에 직접 주입가능한 추출 용매를 사용하는 것이 보다 바람직하다. 본 발명의 구체예에 따르면, 클로로포름으로 추출한 후 용매를 제거하고, 얻어진 잔류물을 증류수에 용해시킨 후 이를 다시 헥산으로 추출하는 방법이 최상의 결과를 제공하였다.The process of cleaning up the N (O, S) -ethylcarbonyl pentafluoropropyl ether derivative of homocysteine, the product of the reaction, consists of an extraction step using an organic solvent. The extraction step is a general method used in the organic chemistry field. Can be adopted. The organic solvent that can be used as the extraction solvent is not particularly limited as long as it is a nonpolar solvent such as chloroform, dichloromethane, ether, ethyl acetate, hexane, pentane or mixtures thereof, but it is more preferable to use an extraction solvent that can be directly injected into GC. Do. According to an embodiment of the present invention, extraction with chloroform removes the solvent, the resulting residue is dissolved in distilled water and then extracted again with hexane provides the best results.
클린업된 호모시스테인 유도체의 분석은 기체 크로마토그래피-전자포획 검출기(GC-ECD : gas chromatography-electron capture detector)를 사용하여 분석하게 되며, 구체적으로는 GC-ECD에 주입시키고, 산화에 불활성인 기체를 이동상으로 사용하였다. 이동상으로 사용될 수 있는 기체는 GC에 사용되는 통상의 기체이면 족하다. 구체적으로는 산화에 불활성인 질소, 헬륨 및 아르곤 등이 있다. 컬럼의 길이와 내경 등도 호모시스테인의 피크(peak)를 다른 화합물과 구별할 수 있을 정도이면 족하고 특별히 한정되는 것은 아니다.Analysis of the cleaned up homocysteine derivatives is carried out using a gas chromatography-electron capture detector (GC-ECD), specifically, a gas inert to the GC-ECD and an inert gas for oxidation is carried out. Used as. The gas which can be used as the mobile phase is sufficient if it is a conventional gas used for GC. Specific examples include nitrogen, helium, argon, and the like, which are inert to oxidation. The length and internal diameter of the column are also not particularly limited as long as the peak of homocysteine can be distinguished from other compounds.
GC-ECD를 사용하여 호모시스테인의 농도를 결정하기 위해서는 검량 곡선을 작성하여야 하며, 본 발명에서는 혈액 시료에 호모시스테인 표준 용액을 농도별로 첨가하고, 이를 상기 방법에 따라 분석하여 각각에 대한 검량곡선을 얻었다. 구체적인 검량 곡선의 작성 방법은 다음과 같다. 호모시스테인의 혈중 농도가 5, 10, 25 및 50 μmol/L가 되도록 표준물 혼합 용액을 혈액 시료에 첨가하고, 내부 표준 물질인 S-(2-아미노에틸)-L-시스테인·염산 (시그마사 제품)을 10 mmol/L의 농도로 100 ㎕ 첨가하고, 이를 전술한 시료 전처리 방법에 따라 처리한 후, GC-ECD로 분석하여 도 1의 검량 곡선을 얻었다. 도 1의 검량곡선에 도시한 바와 같이, 상기 검량 곡선은 5-50 μmol/L의 농도 범위에서 y=0.0081·x + 0.0204 (R2=0.09979)의 신뢰할 수 있는 결과를 야기하였다. 정량 검출 한계는 S/N 비가 5 이상에서 0.5 μmol/L 이하이며, 따라서 혈장 속에 존재하는 호모시스테인을 검출하기 위한 충분한 감도를 나타내었다.In order to determine the concentration of homocysteine using GC-ECD, a calibration curve should be prepared. In the present invention, a homocysteine standard solution was added to blood samples by concentration, and analyzed according to the above method to obtain a calibration curve for each. The method of preparing a specific calibration curve is as follows. A standard mixed solution is added to the blood sample so that the blood concentration of homocysteine is 5, 10, 25 and 50 μmol / L, and S- (2-aminoethyl) -L-cysteine hydrochloric acid (Sigma) ) Was added at a concentration of 10 mmol / L and treated according to the sample pretreatment method described above, and analyzed by GC-ECD to obtain a calibration curve of FIG. 1. As shown in the calibration curve of FIG. 1, the calibration curve gave a reliable result of y = 0.0081 · x + 0.0204 (R 2 = 0.09979) in the concentration range of 5-50 μmol / L. The limit of quantitative detection is that the S / N ratio is above 0.5 μmol / L at 5 or higher, thus exhibiting sufficient sensitivity to detect homocysteine present in plasma.
실시예Example
실시예 1Example 1
인체로부터 채취한 혈장 500 ㎕에 증류수 500 ㎕를 혼합하고, 내부 표준 물질인 S-(2-아미노에틸)-L-시스테인·염산(시그마사 제품)을 10 mmol/L 농도로 100 ㎕를 첨가한 후, S-S 결합(디설파이드 결합)을 끊기 위하여 환원제인 12.5(w/v) 디트레이톨 25 ㎕를 함께 넣어 40℃에서 30분 동안 열진탕하였다. 그 후, 72(w/v) 트리클로로아세트산 100 ㎕를 첨가하고 10분간 3000 rpm으로 원심분리하여 단백질을 침전시켰다. 상층 600 ㎕를 취하여 PFP-OH와 피리딘을 4:1로 혼합한 용액 400 ㎕를 가하고, 여기에 에틸 클로로포르메이트 50 ㎕를 넣어 보르텍스 혼합기로 혼합한 뒤, 실온에서 5분간 방치하여 혈장속에 존재하는 호모시스테인의 유도체화 반응을 완결시켰다.500 µl of distilled water was mixed with 500 µl of plasma collected from the human body, and 100 µl of S- (2-aminoethyl) -L-cysteine hydrochloric acid (Sigma) was added at a concentration of 10 mmol / L. After that, 25 µl of 12.5 (w / v) dethritol, which is a reducing agent, was put together to break the SS bond (disulfide bond), followed by thermal shaking at 40 ° C. for 30 minutes. Thereafter, 100 μl of 72 (w / v) trichloroacetic acid was added and centrifuged at 3000 rpm for 10 minutes to precipitate the protein. 400 µl of the upper layer was taken and 400 µl of a mixture of PFP-OH and pyridine was added 4: 1, 50 µl of ethyl chloroformate was added thereto, mixed with a vortex mixer, and allowed to stand at room temperature for 5 minutes to be present in plasma. The derivatization reaction of homocysteine was completed.
실시예 2Example 2
유도체화 반응이 끝난 시료 용액에 클로로포름 1 ml를 가하여 진탕시키고, 유기층과 수층을 분리한 다음 유기층 1.2 ml를 취하여 질소 기체로 용매를 말렸다. 이를 다시 증류수 200 ㎕에 용해시키고, 헥산 200 ㎕로 추출하여 GC-ECD 주입용 시약을 제조하였다.1 ml of chloroform was added to the sample solution after the derivatization reaction was shaken, the organic layer and the aqueous layer were separated, 1.2 ml of the organic layer was taken, and the solvent was dried with nitrogen gas. This was again dissolved in 200 μl of distilled water and extracted with 200 μl of hexane to prepare a reagent for injection of GC-ECD.
실시예 3Example 3
실시예 2에서 얻은 추출용액 2 ㎕를 취하고, GC-ECD에 주입하여 도 2와 같이 미량의 호모시스테인 피크가 깨끗하게 분리된 크로마토그램을 얻었다. 호모시스테인의 농도를 결정하기 위해 사용된 GC 및 ECD는 휴렉 패커드 사의 HP5890 II 기체크로마토그래프와 HP3365 전자포획검출기였다. 분석을 위한 기기조건은 다음과 같다. GC-ECD에 사용된 모세관 컬럼은 HP-1(가교된 메틸 실리콘 수지)이였고, 그 길이 및 내경은 25 m 및 0.2 mm였으며, 필름 두께는 0.33 ㎛이였다. 이동상 기체는 질소를 사용하였고, 그 유속은 0.8 ml/min이었고, 주입 방식은 1:20의 분할 형식(split mode)을 이용하였다. 컬럼의 온도는 150℃에서 10℃/min씩 올려 300℃까지 올려주었으며, 300℃에서 5분간 머물게 하였고, 주입구와 검출기의 온도는 300℃로 하였다.2 μl of the extract solution obtained in Example 2 was taken and injected into GC-ECD to obtain a chromatogram in which trace homocysteine peaks were cleanly separated as shown in FIG. 2. The GCs and ECDs used to determine the concentration of homocysteine were HP5890 II gas chromatograph and HP3365 electron capture detector from Hurek Packard. The instrument conditions for the analysis are as follows. The capillary column used for GC-ECD was HP-1 (crosslinked methyl silicone resin), its length and inner diameter were 25 m and 0.2 mm, and the film thickness was 0.33 μm. Nitrogen was used as the mobile phase gas, and the flow rate was 0.8 ml / min, and the injection mode was a split mode of 1:20. The temperature of the column was raised to 300 ℃ by 10 ℃ / min at 150 ℃, stayed for 5 minutes at 300 ℃, the temperature of the inlet and detector was 300 ℃.
실시예 4Example 4
본 발명의 분석 방법을 5명의 자원자를 대상으로 실시하여, 호모시스테인의 혈중 농도를 측정하여 아래의 표 1의 결과를 얻었다. 표 1에서 Hcy는 호모시스테인이고, Cys는 시스테인이며, Met는 메티오닌을 말한다. 본 발명의 방법에 따르면, 호모시스테인의 대사과정에 관여하는 메티오닌과 시스테인도 동시에 검출되었다.The analysis method of the present invention was carried out for five volunteers, and the blood concentration of homocysteine was measured to obtain the results shown in Table 1 below. In Table 1 Hcy is homocysteine, Cys is cysteine and Met refers to methionine. According to the method of the present invention, methionine and cysteine, which are involved in the metabolic process of homocysteine, were also detected simultaneously.
본 발명의 유도체화 방법은 혈장으로부터 호모시스테인을 효과적으로 추출할 수 있도록 하며, 선택적이고 우수한 감도를 가지며, 물질의 분석에 일반적으로 사용되는 GC-ECD로 분석하는 최적의 분석 조건을 제공한다. 따라서 본 발명의 유도체화 방법과 클린업방법을 이용할 경우, 우수한 감도, 재현성의 향상, 정확성, 분석 시간의 단축, 저비용 및 편리성 등을 동시에 만족시킬 수 있으며, 이로 인해 심장질환의 진단 및 치료에 널리 응용될 수 있다.The derivatization method of the present invention enables efficient extraction of homocysteine from plasma, has selective and excellent sensitivity, and provides optimal analytical conditions for analysis by GC-ECD generally used for the analysis of substances. Therefore, when the derivatization method and the clean-up method of the present invention are used, it is possible to satisfy excellent sensitivity, reproducibility, accuracy, shorten analysis time, low cost and convenience at the same time, and thus it is widely used for diagnosis and treatment of heart disease. Can be applied.
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| CN109425666A (en) * | 2017-08-28 | 2019-03-05 | 广东东阳光药业有限公司 | A kind of LC-MS analysis method of chloride derivative |
| KR102120822B1 (en) * | 2019-03-25 | 2020-06-09 | 중앙대학교 산학협력단 | Analytical method for the simultaneous determination of trimethylamine N-oxide and its related compounds in dried blood spots |
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| KR101712283B1 (en) * | 2015-08-31 | 2017-03-03 | 쌍용자동차 주식회사 | Battery tray for automobile |
| CN109425666A (en) * | 2017-08-28 | 2019-03-05 | 广东东阳光药业有限公司 | A kind of LC-MS analysis method of chloride derivative |
| KR102120822B1 (en) * | 2019-03-25 | 2020-06-09 | 중앙대학교 산학협력단 | Analytical method for the simultaneous determination of trimethylamine N-oxide and its related compounds in dried blood spots |
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