KR20200113384A - Sample preparation method for the analysis of trimethylamine N-oxide and its related compounds in dried blood spots - Google Patents
Sample preparation method for the analysis of trimethylamine N-oxide and its related compounds in dried blood spots Download PDFInfo
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Abstract
Description
본 발명은 건조점적혈액 중 트리메틸아민산화물계 화합물 분석을 위한 전처리 방법에 관한 것으로, 구체적으로는 건조점적혈액 중 트리메틸아민 등 휘발성 화합물의 분석을 위한 전처리 방법에 관한 것이다.The present invention relates to a pretreatment method for analyzing trimethylamine oxide-based compounds in dry drop blood, and specifically, to a pretreatment method for analyzing volatile compounds such as trimethylamine in dry drop blood.
최근 트리메틸아민 N-옥사이드(trimethylamine N-oxide; TMAO)와 죽상동맥경화증 (atherosclerosis)과의 관련성에 대한 연구가 발표된 이후 (2011년) 지금까지 TMAO 및 TMAO 대사에 관련된 다양한 화합물이 질병 기전에 관여하는 것으로 알려져 왔다. 또한, 심근경색 (myocardial infarction), 뇌졸중 (stroke), 혈전증 (thromobosis), 심부전 (heart failure), 심방세동 (atrial fibrillation)과 같은 부정맥 (arrhythmia) 등 다양한 심혈관계 질환이 이들 화합물과 밀접한 관련이 있는 것으로 새로이 밝혀지고 있다.Since a recent study on the relationship between trimethylamine N -oxide (TMAO) and atherosclerosis was published (2011), various compounds related to TMAO and TMAO metabolism have been involved in disease mechanisms. Has been known to do. In addition, various cardiovascular diseases such as myocardial infarction, stroke, thrombosis, heart failure, and arrhythmia such as atrial fibrillation are closely related to these compounds. It is being newly revealed.
따라서 TMAO와 TMAO 대사에 관련된 주변 화합물인 트리메틸아민(trimethylamine; TMA), 카르니틴(carnitine; CAR), 콜린(choline; CHL), 베타인(betaine; BET), γ-부틸로베타인(γ-butyrobetaine; GBB), 트리메틸리신(trimethyllysine; TML), 호모시스테인(homocysteine; HCY), 디메틸글리신(dimethylglycine; DMG) 등이 심혈관계 질환과 관련이 있는 잠재적 바이오마커 (potential biomarker)로 보고되고 있으며, 특히, 이들의 대사에 인체 내 효소뿐만 아니라 장내 미생물 (gut microbiome)이 관여한다는 연구결과가 발표되면서 이들 화합물에 대한 관심도가 높아지고 있다.Therefore, the surrounding compounds involved in TMAO and TMAO metabolism are trimethylamine (TMA), carnitine (Car), choline (CHL), betaine (BET), γ-butyrobetaine. ; GBB), trimethyllysine (TML), homocysteine (HCY), dimethylglycine (DMG), etc. have been reported as potential biomarkers related to cardiovascular diseases. Interest in these compounds is increasing as research results have been published that not only enzymes in the human body but also the gut microbiome are involved in metabolism.
이에, TMAO와 TMAO 대사에 관련된 다양한 화합물과 관련된 진단 및 진단키트의 수요 또한 점차 커질 것이며, 이와 관련된 지속적인 연구가 필요한 실정이다.Accordingly, the demand for diagnosis and diagnostic kits related to various compounds related to TMAO and TMAO metabolism will also increase gradually, and continuous research is required.
건조점적혈액(dried blood spots; DBS)은 규격화된 여과지에 혈액을 소량 점적하여 건조 시킨 형태의 시료로, 상기 건조 과정에서 혈액 중 분석 대상성분과 여과지의 충분한 상호작용을 통해 건조 후에도 대상성분이 여과지 위에 잔류하게 되고, 이 잔류물을 용출하여 분석하는 것이 DBS 전처리의 기본 골격이다. Dried blood spots (DBS) are samples in the form of drying by dropping a small amount of blood on a standardized filter paper.In the drying process, the target component is filtered through sufficient interaction between the analysis target component in the blood and the filter paper. It remains in the stomach, and the basic framework of DBS pretreatment is to elute and analyze this residue.
그러나 TMA와 같은 휘발성 화합물은 상기와 같이 혈액을 건조하는 과정 중에 여과지에 잔류하지 못하고 휘발될 수 있는 문제점이 있다.However, volatile compounds such as TMA do not remain on the filter paper during the process of drying blood as described above and may be volatilized.
따라서 건조점적혈액 중 휘발성이 높은 화합물의 휘발을 저지하여 물질을 검출할 수 있는 건조점적혈액 여과지의 전처리 방법에 대한 연구가 필요한 실정이다.Therefore, there is a need for a study on the pretreatment method of dry drop blood filter paper that can detect substances by preventing the volatilization of highly volatile compounds in dry drop blood.
본 발명의 목적은 트리메틸아민 N-옥사이드(trimethylamine N-oxide; TMAO) 및 TMAO 대사에 관련된 다양한 화합물 중 휘발성을 가진 아민류 화합물을 분석하기 위한 전처리 방법을 제공하는 데에 있다.An object of the present invention is to provide a pretreatment method for analyzing trimethylamine N -oxide (TMAO) and amine compounds having volatility among various compounds related to TMAO metabolism.
또한, 본 발명의 다른 목적은 상기 전처리 방법에 의해 제조된 염산 처리된 페이퍼 스트립 및 이를 이용한 심혈관계 질환 진단키트를 제공하는 데에 있다.Another object of the present invention is to provide a hydrochloric acid-treated paper strip prepared by the pretreatment method and a cardiovascular disease diagnostic kit using the same.
상기 목적을 달성하기 위하여, 본 발명은 건조점적혈액(dried blood spots; DBS) 여과지를 준비하는 단계; 및 상기 여과지를 염산 처리하는 단계; 를 포함하는 DBS 여과지 전처리 방법을 제공한다.In order to achieve the above object, the present invention comprises the steps of preparing dried blood spots (DBS) filter paper; And hydrochloric acid treatment of the filter paper. It provides a DBS filter paper pre-treatment method including.
또한, 본 발명은 건조점적혈액(DBS) 여과지를 염산 처리한 DBS용 페이퍼 스트립을 제공한다.In addition, the present invention provides a paper strip for DBS treated with hydrochloric acid for dry drop blood (DBS) filter paper.
또한, 본 발명은 상기 염산 처리된 DBS용 페이퍼 스트립을 포함하는 심혈관계 질환 진단키트를 제공한다.In addition, the present invention provides a cardiovascular disease diagnostic kit comprising the hydrochloric acid-treated DBS paper strip.
본 발명에 따른 건조점적혈액(DBS) 여과지 전처리 방법은 건조점적혈액 중 트리메틸아민(trimethylamine; TMA) 등과 같은 휘발성 아민류 화합물의 검출을 가능하게 할 수 있다.The method of pretreatment of dry drop blood (DBS) filter paper according to the present invention may enable detection of volatile amine compounds such as trimethylamine (TMA) in dry drop blood.
또한, 상기 건조점적혈액(DBS) 여과지 전처리 방법은 트리메틸아민(trimethylamine; TMA)을 비롯한 심혈관계 질환 관련 화합물(트리메틸아민 N-옥사이드(trimethylamine N-oxide; TMAO), 카르니틴(carnitine; CAR), 콜린(choline; CHL), 베타인(betaine; BET), γ-부틸로베타인(γ-butyrobetaine; GBB), 트리메틸리신(trimethyllysine; TML), 호모시스테인(homocysteine; HCY), 디메틸글리신(dimethylglycine; DMG))에 대해 부작용 없이 모두 검출 가능하도록 한다.In addition, the method of pretreatment of the dry spot blood (DBS) filter paper includes compounds related to cardiovascular diseases (trimethylamine N -oxide; TMAO), carnitine (Car), choline, including trimethylamine (TMA). (choline; CHL), betaine (BET), γ-butyrobetaine (GBB), trimethyllysine (TML), homocysteine (HCY), dimethylglycine (DMG) ), all can be detected without side effects.
또한, 건조점적혈액(DBS) 여과지를 염산 처리하여 제조된 DBS용 페이퍼 스트립은 심혈관계 질환 진단키트에 응용될 수 있을 것이다.In addition, a paper strip for DBS manufactured by treating a dry spot blood (DBS) filter paper with hydrochloric acid may be applied to a cardiovascular disease diagnostic kit.
도 1은 5 가지 다른 조건에서 트리메틸아민(trimethylamine; TMA)에 대한 회수율 비교를 나타낸 도면이다.
도 2는 DBS 건조 시간에 따른 트리메틸아민(TMA) 회수율을 나타낸 도면이다.
도 3은 (A) Whatman 903 protein saver card, (B) Whatman FTA DMPK-A card 및 (C) Whatman FTA DMPK-C card의 3가지 건조혈액점적(DBS) 여과지에서 염산 농도에 따른 트리메틸아민(TMA)의 회수율 효과를 나타낸 도면이다.
도 4는 염산 농도에 따른 심혈관계 질환 화합물의 회수율 효과를 나타낸 도면이다. 1 is a view showing a comparison of the recovery rate for trimethylamine (TMA) under five different conditions.
2 is a diagram showing the recovery rate of trimethylamine (TMA) according to the DBS drying time.
Figure 3 shows trimethylamine (TMA) according to the concentration of hydrochloric acid in three dry blood droplets (DBS) filter papers of (A) Whatman 903 protein saver card, (B) Whatman FTA DMPK-A card and (C) Whatman FTA DMPK-C card. ) Is a diagram showing the effect of the recovery rate.
4 is a diagram showing the effect of recovery rate of a cardiovascular disease compound according to the concentration of hydrochloric acid.
이하에서는 본 발명을 구체적으로 설명한다.Hereinafter, the present invention will be described in detail.
본 발명자들은 건조점적혈액(DBS) 전처리로서 혈액 점적 전 0.5 mol/L 농도의 염산 전처리 통해 휘발성분의 휘발이 저지되어 심혈관계 질환 관련 화합물인 트리메틸아민산화물 계 화합물의 검출이 가능함을 확인하였으며, 이로써 심근경색 (myocardial infarction), 뇌졸중 (stroke), 혈전증 (thromobosis), 심부전 (heart failure), 심방세동 (atrial fibrillation)과 같은 부정맥 (arrhythmia) 등 다양한 심혈관계 질환 진단 및 분석을 위한 진단키트에 유용하게 활용될 수 있음을 밝혀내어 본 발명을 완성하였다.The present inventors have confirmed that the volatilization of volatile components is prevented through pretreatment with hydrochloric acid at a concentration of 0.5 mol/L as a pretreatment for dry drop blood (DBS), thereby enabling the detection of trimethylamine oxide compounds, which are compounds related to cardiovascular diseases. Useful as a diagnostic kit for diagnosing and analyzing various cardiovascular diseases such as myocardial infarction, stroke, thrombosis, heart failure, and arrhythmia such as atrial fibrillation. The present invention was completed by finding that it can be utilized.
본 발명은 건조점적혈액(dried blood spots; DBS) 여과지를 준비하는 단계; 및 상기 여과지를 염산 처리하는 단계; 를 포함하는 DBS 여과지 전처리 방법을 제공한다.The present invention comprises the steps of preparing dry blood spots (DBS) filter paper; And hydrochloric acid treatment of the filter paper. It provides a DBS filter paper pre-treatment method including.
이때, 상기 염산을 0.1 내지 1.0 mol/L의 농도로 처리할 수 있으나, 바람직하게는 0.5 mol/L의 농도로 처리할 수 있다.At this time, the hydrochloric acid may be treated at a concentration of 0.1 to 1.0 mol/L, but preferably at a concentration of 0.5 mol/L.
상기와 같은 DBS 여과지 전처리 방법은 시료 내 휘발성 화합물의 휘발을 저지할 수 있으며, 상기 휘발성 화합물은 트리메틸아민(trimethylamine; TMA)과 같은 아민류 화합물일 수 있다.The DBS filter paper pretreatment method as described above can prevent volatilization of volatile compounds in a sample, and the volatile compounds may be amine compounds such as trimethylamine (TMA).
상기 TMA는 분자 그 자체로는 염기성인 휘발성 성분이지만 염산인 산과 반응하게 되면 염을 생성하여 상온에서도 고상으로 존재할 수 있어 휘발성이 저지되는 것이다.The TMA molecule itself is a basic volatile component, but when it reacts with an acid, which is hydrochloric acid, it generates a salt and can exist in a solid state even at room temperature, thereby preventing volatility.
이때, 상기와 같은 여과지 전처리 조건을 벗어나면 건조점적혈액(dried blood spots; DBS) 중 휘발성 화합물이 휘발되어 회수율이 떨어지거나, 사용한 전처리 화합물 대비 회수율의 효과가 좋지 않아 경제적이지 못한 문제가 야기될 수 있다. At this time, if the filter paper pretreatment conditions are out of the above, volatile compounds in the dried blood spots (DBS) volatilize and the recovery rate decreases, or the effect of the recovery rate is poor compared to the used pretreatment compound, which may lead to economic problems. have.
또한, 본 발명은 건조점적혈액(DBS) 여과지를 염산 처리한 DBS용 페이퍼 스트립을 제공한다.In addition, the present invention provides a paper strip for DBS treated with hydrochloric acid for dry drop blood (DBS) filter paper.
이때, 상기 염산을 0.1 내지 1.0 mol/L의 농도로 처리할 수 있으나, 바람직하게는 0.5 mol/L의 농도로 처리할 수 있다.At this time, the hydrochloric acid may be treated at a concentration of 0.1 to 1.0 mol/L, but preferably at a concentration of 0.5 mol/L.
상기와 같이 염산 처리된 DBS용 페이퍼 스트립은 시료 내 휘발성 화합물의 휘발을 저지할 수 있으며, 상기 휘발성 화합물은 트리메틸아민(trimethylamine; TMA)과 같은 아민류 화합물일 수 있다.The DBS paper strip treated with hydrochloric acid as described above may prevent volatilization of volatile compounds in a sample, and the volatile compounds may be amine compounds such as trimethylamine (TMA).
이때, 상기와 같은 여과지 전처리 조건을 벗어나면 건조점적혈액(dried blood spots; DBS) 중 휘발성 화합물이 휘발 되어 회수율이 떨어지거나, 사용한 전처리 화합물 대비 회수율의 효과가 좋지 않아 경제적이지 못한 문제가 야기될 수 있다. At this time, if the filter paper pretreatment conditions are out of the above, volatile compounds in the dried blood spots (DBS) volatilize and the recovery rate decreases, or the effect of the recovery rate is poor compared to the used pretreatment compound, which may lead to economic problems. have.
또한, 본 발명은 상기와 같이 염산 처리된 DBS용 페이퍼 스트립을 포함하는 심혈관계 질환 진단키트를 제공한다.In addition, the present invention provides a cardiovascular disease diagnostic kit comprising a paper strip for DBS treated with hydrochloric acid as described above.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention, to those of ordinary skill in the art to which the present invention pertains. It will be self-evident.
<< 실시예Example 1> DBS 여과지에서 1> In DBS filter paper TMATMA 화합물의 휘발성 측정 Measurement of the volatility of compounds
TMA 표준용액(standard; STD)과 TMA에 안정동위원소를 표지한 화합물 TMA-d9을 첨가한 혈액 각각을 DBS 여과지 3종 (Whatman 903 protein saver card, Whatman FTA DMPK-A card, Whatman FTA DMPK-C card)에 점적하고 건조하여 시료 전처리 및 LC-HRMS(Liquid chromatography-high resolution mass spectrometry) 분석을 수행하였다. TMA standard solution (standard; STD) and the addition of a compound TMA-d 9 cover the stable isotope in the blood TMA each DBS three kinds of filter paper (Whatman 903 protein saver card, Whatman FTA DMPK-A card, Whatman FTA DMPK- C card) and dried to perform sample preparation and LC-HRMS (Liquid chromatography-high resolution mass spectrometry) analysis.
그 결과 도 1과 같이 TMA 표준용액(STD)을 점적한 시료는 양호한 회수율을 보였지만, TMA-d9를 첨가한 DBS는 거의 모든 성분이 회수되지 않았다. 또한, 여과지에 따라 회수율 편차를 보일 수 있으므로 3종의 여과지를 비교 시험해 보았지만, 모든 여과지(Whatman 903 protein saver card, Whatman FTA DMPK-A card, Whatman FTA DMPK-C card)에서 5% 미만의 회수율을 나타냈다. As a result, even a sample dropwise to TMA standard solution (STD), such as 1, showed a good recovery rate, DBS was added to TMA-d 9 is not nearly of the components are recovered. In addition, since the recovery rate may vary depending on the filter paper, we compared and tested the three filter papers, but all filter papers (Whatman 903 protein saver card, Whatman FTA DMPK-A card, Whatman FTA DMPK-C card) showed a recovery rate of less than 5%. Showed.
게다가, 혈액 중 단백질 등 다른 간섭성분과 결합하여 회수가 안될 가능성도 고려하여, 별도로 TMA-d9가 첨가된 혈액(Whole blood)(여과지에 점적하지 않은 혈액 시료 자체)을 분석한 결과, 혈액 시료에서는 표준용액과 유사하게 양호한 회수율을 보였다. In addition, the protein in the blood, such as by combination with other interference component count is integral likely also be considered, a separate added TMA-d 9 Blood (Whole blood) The analysis of the (blood sample itself is not drip on the filter paper), blood samples The recovery rate was similar to that of the standard solution.
따라서 TMA의 회수율 저하 문제는 DBS에서만 특징적으로 나타나며, 이는 여과지에서 혈액을 건조할 때 TMA도 같이 휘발되기 때문인 것으로 가정하였다. 다만, 여과지에 혈액을 점적했을 때와 달리 표준용액을 점적했을 때는 양호한 회수율을 나타냈는데, 이는 표준용액 중에는 대상성분만 존재하므로 여과지와 목적성분이 원활하게 상호작용하여 여과지에 TMA가 잔류할 수 있기 때문에 나타난 결과이다. Therefore, the problem of lowering the recovery rate of TMA appears characteristically only in DBS, and it is assumed that this is because TMA is also volatilized when blood is dried on the filter paper. However, unlike in the case of dropping blood on the filter paper, when the standard solution was dropped, it showed a good recovery rate.This is because only the target component is present in the standard solution, so that the filter paper and the target component interact smoothly, so that TMA can remain on the filter paper. This is the result that appeared.
하지만, DBS는 목적성분뿐만 아니라 혈액 중 다양한 간섭성분들도 여과지와 경쟁적으로 상호작용하게 되고, 이런 간섭성분으로 인해 여과지의 수용력이 포화되어 휘발성이 큰 TMA는 건조 과정 중에 휘발·손실되는 것으로 판단하였다. However, DBS determined that not only the target component but also various interfering components in the blood competitively interact with the filter paper, and due to this interference component, the capacity of the filter paper was saturated, and the high volatile TMA was determined to be volatilized and lost during the drying process. .
이를 실험적으로 확인하고자 DBS 건조 시간에 따른 TMA 회수율을 측정해보았다. TMA-d9를 첨가한 혈액을 여과지에 점적하고 5, 10, 15, 20, 30, 45, 60분마다 각 DBS 디스크(disc)를 펀치(punch)하여 전처리한 후 분석하였다. To confirm this experimentally, the TMA recovery rate according to the DBS drying time was measured. Blood to which TMA-d 9 was added was instilled on a filter paper, and each DBS disk was punched every 5, 10, 15, 20, 30, 45, and 60 minutes, followed by pretreatment and analysis.
그 결과 도 2와 같이 혈액을 건조한지 채 5분도 되지 않아 40% 미만의 회수율을 보였으며, 약 20분 이후로는 거의 모든 TMA-d9이 회수되지 않는 양상을 보였다. 이를 통해 실제로 여과지 중 혈액 건조 과정에서 TMA가 휘발됨을 확인하였다.As a result, as shown in FIG. 2, the blood was dried for less than 5 minutes, showing a recovery rate of less than 40%, and almost all TMA-d 9 was not recovered after about 20 minutes. Through this, it was confirmed that TMA was actually volatilized during the blood drying process in the filter paper.
<< 실시예Example 2> DBS 여과지 전처리 2> DBS filter paper pretreatment
본 발명에서는 혈액을 점적하기 전 여과지에 염산(hydrochloric acid)을 소량 도포하여 TMA의 휘발을 방지할 수 있는지 확인하였으며, 서로 다른 농도의 0.001, 0.05, 0.01, 0.05, 0.1, 0.5, 1.0 mol/L 염산(hydrochloric acid) 용액을 제조하여 비교 시험하였다.In the present invention, it was confirmed that the volatilization of TMA could be prevented by applying a small amount of hydrochloric acid to the filter paper before dropping blood, and different concentrations of 0.001, 0.05, 0.01, 0.05, 0.1, 0.5, 1.0 mol/L A hydrochloric acid solution was prepared and tested for comparison.
먼저, 혈액 중 TMA-d9의 농도가 100 μmol/L가 되도록 제조하였다. DBS 여과지인 와트만 903 프로틴 세이버 카드(Whatman 903 protein saver card)의 홀 (hole)마다 0.001, 0.05, 0.01, 0.05, 0.1, 0.5, 1.0 mol/L의 염산 용액 40 μL를 점적하고 상온에서 2시간 건조한 후, 혈액 30 μL를 점적하고 다시 상온에서 2시간 건조하였다. 이후, 직경 6 mm 규격의 펀처(puncher)로 펀칭(punching)하여 만든 DBS 디스크(disc)를 1.5 mL 마이크로튜브(microtube)에 넣었다. 10 mmol/L 아세트산 암모늄(ammonium acetate) (pH 9.0) 380 μL와 5 mmol/L 다이티오트레이톨(dithiothreitol; DTT) 20 μL를 가하여 30분간 교반하고 상온에서 20분간 방치한 후 13,000 rpm으로 3분간 원심분리하여 상징액(supernatant) 200 μL를 다른 1.5 mL 마이크로튜브(microtube)로 옮겼다. 여기에 1.0 mol/L 에틸 브로모아세테이트(ethyl bromoacetate;EtBAc) 용액 100 μL를 각각 가하여 5분간 교반하고 60˚C의 항온수조(water-bath)에서 1시간 동안 유도체화 반응을 진행한 후 마이크로튜브(microtube)를 항온수조(water-bath)에서 꺼냈다. 반응이 끝난 용액에 20 mmol/L 포름산(formic acid) (아세토니트릴(acetonitrile)에 용해) 200 μL를 가하고 5분간 교반한 후 13,000 rpm에서 5분간 원심분리하였다. 상징액을 취하여 실린지 필터(syringe filter)(0.2 μm, PTFE)로 여과한 후 LC-HRMS로 주입하여 분석하였다. 염산의 각 농도별로 3회 반복 시험하여 도출된 회수율을 비교 검토하여 TMA 회수 여부 및 최적의 염산 농도를 확인하였다.First, it was prepared so that the concentration of TMA-d 9 in blood was 100 μmol/L. In each hole of the
<< 실험예Experimental example 1> 최적의 전처리 농도 1> Optimal pretreatment concentration
상기 실시예 1에서와 같이 DBS에서 특이적으로 TMA의 회수율이 낮아지는 현상을 해결하고자 상기 실시예 2와 같이 여과지에 혈액을 점적하기 전에 염산 용액을 미리 처리하였다. 이는, 혈액을 점적했을 때 여과지 표면에 잔류하는 염산이 혈중 TMA와의 중화반응 (on-spot reaction)을 통해 TMA 염산염(hydrochloride)을 생성할 수 있고, 생성된 염이 상온에서 고체상태로 존재하여 TMA의 휘발을 방지할 수 있을 것이라 가정하여 최적의 염산 농도를 확인하고자 서로 다른 농도인 0.001, 0.05, 0.01, 0.05, 0.1, 0.5, 1.0 mol/L의 염산 수용액을 제조하여 비교 시험하였다. DBS 여과지로는 와트만 903 프로틴 세이버 카드(Whatman 903 protein saver card)를 사용하였다.As in Example 1, in order to solve the problem that the recovery rate of TMA was specifically lowered in DBS, as in Example 2, before dropping blood on the filter paper, a hydrochloric acid solution was previously treated. When blood is instilled, the hydrochloric acid remaining on the surface of the filter paper can generate TMA hydrochloride through an on-spot reaction with TMA in the blood, and the resulting salt exists in a solid state at room temperature. In order to check the optimal hydrochloric acid concentration, assuming that it would be possible to prevent volatilization of, hydrochloric acid aqueous solutions of different concentrations of 0.001, 0.05, 0.01, 0.05, 0.1, 0.5, and 1.0 mol/L were prepared and tested for comparison.
그 결과 도 3A와 같이 염산 수용액의 농도가 증가할수록 TMA 회수율이 점차 개선되었으며, 0.5 mol/L의 농도를 처리했을 때 약 100%에 가까운 회수율을 보였다. 추가로 2종의 여과지 (Whatman FTA DMPK-A card, Whatman FTA DMPK-C card)에도 시험한 결과, 모두 동일한 경향을 확인할 수 있었다 (도 3B, C). As a result, as the concentration of the aqueous hydrochloric acid solution increased as shown in FIG. 3A, the TMA recovery rate was gradually improved, and when the concentration of 0.5 mol/L was treated, the recovery rate was close to about 100%. In addition, two kinds of filter papers (Whatman FTA DMPK-A card, Whatman FTA DMPK-C card) were also tested, and the same trend was found in both (FIGS. 3B and C).
결론적으로 TMA-염산 중화반응을 통해 DBS 중 TMA의 손실을 방지할 수 있었으며, 염산 0.5 mol/L 농도에서 최적의 효율을 나타냄을 알 수 있었다.In conclusion, the loss of TMA in DBS could be prevented through the TMA-hydrochloric acid neutralization reaction, and it was found that the optimum efficiency was achieved at a concentration of 0.5 mol/L of hydrochloric acid.
<< 실험예Experimental example 2> 심혈관계 질환 관련 화합물의 검출 2> Detection of compounds related to cardiovascular diseases
염산은 강산으로서 여과지 표면의 성질을 변화시킬 수 있으며, 이를 통해 여과지와 목적성분의 상호작용을 방해하여 목적성분이 여과지 표면에 잔류하지 못하게 할 수도 있으며, 산에 불안정한 물질의 분해를 촉진하는 등의 부작용도 유발할 수 있을 것이다. 이를 확인하고자 TMA-d9외에 TMAO 및 TMAO 대사에 관련된 나머지 분석 대상성분 8종 (HCY(호모시스테인;homocysteine)-d4, DMG(디메틸글리신;dimethylglycine)-d6, BET(베타인;betaine)-d9, GBB(γ-부틸로베타인;γ-butyrobetaine)-d9, CAR(카르니틴;carnitine)-d3, CHL(콜린;choline)-d9, TML(트리메틸리신;trimethyllysine)-d9,T MAO(트리메틸아민 N-옥사이드;trimethylamine N-oxide)-d9)도 같이 시험하였다. Hydrochloric acid, as a strong acid, can change the properties of the filter paper surface. Through this, it can interfere with the interaction between the filter paper and the target component to prevent the target component from remaining on the filter paper surface, and promote the decomposition of acid-unstable substances. It can also cause side effects. To confirm this, in addition to TMA-d 9 , 8 other components to be analyzed related to TMAO and TMAO metabolism (HCY (homocysteine)-d 4 , DMG (dimethylglycine)-d 6 , BET (betaine)- d 9 , GBB (γ-butyrobetaine)-d 9 , CAR (carnitine)-d 3 , CHL (choline)-d 9 , TML (trimethyllysine; trimethyllysine)-d 9 , T MAO (trimethylamine N- oxide; trimethylamine N -oxide) -d 9) were also tested as shown.
그 결과 도 4와 같이 TMAO 및 TMAO 대사에 관련된 나머지 분석 대상 성분 8종도 높은 회수율을 나타내었으며, 1.0 mol/L의 염산 용액을 처리하더라도 다른 성분의 분석에는 큰 영향을 미치지 않음을 확인하였다. As a result, as shown in FIG. 4, it was confirmed that the remaining 8 components to be analyzed related to TMAO and TMAO metabolism also exhibited a high recovery rate, and even if a 1.0 mol/L hydrochloric acid solution was treated, the analysis of other components was not significantly affected.
따라서 최적의 효율을 나타낸 0.5 mol/L를 염산의 최종 농도로 선정하였다. Therefore, 0.5 mol/L, which showed the optimum efficiency, was selected as the final concentration of hydrochloric acid.
결과적으로 본 시험에서는 DBS 제작 시 먼저 여과지에 0.5 mol/L 염산 용액을 처리한 다음 혈액을 점적한 결과, 휘발성 아민 화합물의 휘발이 저지되어 심혈관계 질환 관련 화합물의 검출이 가능함을 확인하였다. 따라서 여과지 표면에서 간단한 산-염기 중화반응을 이용하여 휘발성 성분의 손실을 방지할 수 있을 것이다.As a result, in this test, when preparing DBS, 0.5 mol/L hydrochloric acid solution was first treated on a filter paper, and then blood was instilled. As a result, it was confirmed that the volatilization of volatile amine compounds was prevented, and thus cardiovascular disease-related compounds could be detected. Therefore, the loss of volatile components can be prevented by using a simple acid-base neutralization reaction on the surface of the filter paper.
Claims (9)
상기 여과지를 염산 처리하는 단계; 를 포함하는 DBS 여과지 전처리 방법.Preparing a dry blood spots (DBS) filter paper; And
Hydrochloric acid treatment of the filter paper; DBS filter paper pretreatment method comprising a.
여과지를 염산 처리하는 단계는,
염산을 0.1 내지 1.0 mol/L의 농도로 처리하는 것을 특징으로 하는 DBS 여과지 전처리 방법.The method of claim 1,
The step of treating the filter paper with hydrochloric acid,
DBS filter paper pretreatment method, characterized in that the hydrochloric acid is treated at a concentration of 0.1 to 1.0 mol/L.
DBS 여과지 전처리 방법은 시료 내 휘발성 화합물의 휘발을 저지하는 것을 특징으로 하는 DBS 여과지 전처리 방법.The method of claim 1,
The DBS filter paper pretreatment method is a DBS filter paper pretreatment method, characterized in that the volatilization of volatile compounds in the sample is prevented.
휘발성 화합물은 트리메틸아민(trimethylamine; TMA)인 것을 특징으로 하는 DBS 여과지 전처리 방법.The method of claim 3,
DBS filter paper pretreatment method, characterized in that the volatile compound is trimethylamine (TMA).
DBS 여과지 염산 처리는,
염산을 0.1 내지 1.0 mol/L의 농도로 처리하는 것을 특징으로 하는 DBS용 페이퍼 스트립.The method of claim 5,
DBS filter paper hydrochloric acid treatment,
Paper strip for DBS, characterized in that treating hydrochloric acid at a concentration of 0.1 to 1.0 mol/L.
DBS용 페이퍼 스트립은 시료 내 휘발성 화합물의 휘발을 저지하는 것을 특징으로 하는 DBS용 페이퍼 스트립.The method of claim 5,
The paper strip for DBS is a paper strip for DBS, characterized in that it blocks volatilization of volatile compounds in a sample.
휘발성 화합물은 트리메틸아민(trimethylamine; TMA)인 것을 특징으로 하는 DBS용 페이퍼 스트립.The method of claim 7,
The volatile compound is trimethylamine (TMA), characterized in that the DBS paper strip.
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| CN113686998A (en) * | 2021-09-28 | 2021-11-23 | 江苏晗睿达精准医学科技有限公司 | Method for determining trimethylamine oxide in dried blood spots by LC-MS/MS technology |
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