200813426 九、發明說明： ‘ 【發明所屬之技術領域】 本發明係有關-種用於檢測尿液中基因氧化傷害產物之 自動化質譜分析方法，制是指將含有I魏-2，—去氧鳥翼 、呤核酸(8-hydroxy〜2, 一deoxy_nosine 簡稱 8领 . 稱8—0X0dG)之待測尿液樣品中加入穩定同>[立素標定内標準品 後，利用一線上固相萃取裝置配合液相層析串聯質譜儀 (LC-MS/MS)之自錄測綠。财法兼具高鶴性及高敏感度 亚且能快速連續處理大量樣品，大大節省分析之時間與耗材， 可廣泛運用在臨床檢驗分析上。 【先前技術】 生物體内的氧化壓力已被證實是造成老化、慢性疾病及癌 症的原因之一。尿液中基因氧化傷害產物(例如：8—氫氧一2，一 去氧鳥翼嗓呤核酸；8-hydroxy-2, -deoxyguanosine簡稱 ⑩ 8-OHdG，亦可稱8-〇x〇dG)之含量多募，-直是用來評估動物 體内氧化壓力（oxidative stress)的生物指標。尿液中的 8-OHdG係源於細胞内DNA之鹼基在遭受氫氧自由基(hydr〇xyl radical)攻擊後，經體内修復而排至尿液的產物。先前尿液中 基因氧化性傷害產物8-OHdG之檢測方法主要有幾種，包括高 效液相層析儀配合電化學偵測(HPLC—ECD)，氣相層析質譜儀 (GC-MS)分析及酵素連結免疫吸附分析（Enzyme—Ί ink immunosorbent assay，簡稱 ELISA)。 5 200813426 然而尿液中的基質(matrix)非常複雜，極易干擾基因氧化 傷害產物(例如8-0HdG)的分析結果。而上述這些分析方法都 有重大缺點而無法大量應用於例行臨床檢測上或精確地分析 尿液樣品，例如：高效液相層析儀配合電化學偵測(HpLC—ecd) 分析方法需要冗長的淨化前處理程序，以去除尿液中的干擾雜 質；至於氣相層析質譜儀(GC-MS)分析方法也需要冗長的前處 理程序再加上複雜的衍生化反應（chemical derivatization);而酵素連結免疫吸附分析(ELISA)方法是較 為簡易的分析法，但ELISA特異性低，極易受尿液基質影響而 錯估含量。 因此’為求能快速、精確地檢測尿液中基因氧化傷害產物 (例如8-0HdG) ’以利臨床分析的廣泛運用，有必要研發出新 技術。 【發明内容】 本發明之主要目的，在於提供—齡析尿液巾基因氧化傷 吾產物8-氫氧-2，-去氧鳥糞嘌呤核酸 (8-hydroxy-2’ -deoxyguanosine 簡稱 8—〇脇，亦可稱 8—〇x〇dG) 之自動化檢測方法。尿液不需經由前處理可直接分析，大大提 高8-OHdG之分析效率與節她材。因其能快速連續分析大量 尿液樣品，可運用在臨床例行的檢驗分析上。 本發明之次-目的，在於提供—齡析紐巾㈣脇含 里之回準確性檢測方法。彻穩定同位素標定内標準品 6 200813426 (stable isotopically labeled internal standard)^,^^ 能使得尿液巾8-GHdG鮮地被定性蚊量，不㈣尿液基質 變化或檢測儀器感度改變而造成檢測上的誤差。 本發明係提供-觀於檢測尿液巾基因氧化傷害產物之 自動化質譜分析方法，其包括下列步驟： (a) 混合步驟； (b) 固相萃取步驟； (c) 液相層析步驟；以及 (d) 量測步驟。 上述本發明之目的與優點，可由下述所選用實施例之詳細 說明與附圖中獲得深入瞭解。 么么以下列實施例並配合圖示詳細說明本發明於後： 【實施方式】 參閱第一及第二圖，本發明係為一種用於檢測尿液中基因 氧化傷害產物之自動化質譜分析方法，基本上包括下列步驟： [a]混合步驟11:取出一預定量之第一檢體21與一預定 量之穩定同位素標定内標準品z充分混和成一第二檢體22。 首先，準備一待測尿液（即第一檢體21)，其中含有 目標物質X以及雜質Y1〜Y99。本實施例中之目標物質χ係為 一基因氧化傷害產物，例如為8—氫氧—2, _去氧鳥糞嘌呤核酸 ^hydiOxyHoxyguanosine 簡稱 8-OHdG，亦可稱為 8-oxodG)。而雜質Y1〜Y99可包含尿素、尿酸、肌酸酐、氨基 200813426 酸、尿膽元素、維生素、各種鹽類、各種體内的有機代謝物等 等，麵㈣且因人的飲食細職異，在此似γι〜γ99 來代表’以方便說明，實際上雜質騎超過百種。 取出少量之待測尿液(至少為1G μ〇，此被定義為第 -檢體21。紐，將該第—檢體21中加人預定量之穩定同位 素標定内標m8,dG)並充分細，㈣成該第二檢 ㈣。該穩定同位素標定内標準品z之分子結構與目標物質乂 凡全相同，僅其分子結構t所有uN原子崎定同位素15N取代 之故穩疋同位素4^崎準品z的化學特性及質譜碎裂機制 均與目標物質X相同’而穩朗位素標如標準品Z的分子量 比目標物質X還要多5(因為8-〇脇分子中含有5個N原子）。 當然’本範例是以15N取代所有％之方式，_，也可修改為 採用含有VO或％等穩定同位素之8-0HdG内標準品為之。 [b]固相萃取步驟12:利用一自動進樣裝置(磁_細) 32，將第二檢體22導人線上_萃取(on-line solid-phase extraction)裝置33以去除部分雜質； 更詳細的說，此固相萃取步驟12為一種線上固相萃 取步驟(an-line SQlid—細e extraetiQn):其_ —自動進 樣裝置(aut〇sampler)32,將該第二檢體22導入該線上固相萃 取裝置33以去除大部分雜質，而成為—第三檢體23，其係包 括目標物質X、穩定同位素標定内標準品z及雜質Y1-Y2〇(在 200813426 本實施例中，假設雜質由原有的刚種去除為只職種）。 [C]液相層析步驟13:第三檢體23再自動沖提進入一液 相層析裝置34(例如為-液相層析管柱，即加祝 chromatographic column)中進行分離；200813426 IX. Description of the invention: 'Technical field to which the invention pertains» The present invention relates to an automated mass spectrometry method for detecting genetic oxidation damage products in urine, which means that it will contain I Wei-2, a deoxygenated bird. Wing, 呤 nucleic acid (8-hydroxy~2, a deoxy_nosine referred to as 8 collar. Said 8-0X0dG) into the urine sample to be tested with the addition of > [Lisu calibration standard standard, using a line of solid phase extraction device Self-recording green with liquid chromatography tandem mass spectrometry (LC-MS/MS). The financial method has both high crane and high sensitivity. It can process a large number of samples quickly and continuously, greatly saving the analysis time and consumables, and can be widely used in clinical test analysis. [Prior Art] Oxidative stress in living organisms has been proven to be one of the causes of aging, chronic diseases and cancer. Gene oxidative damage products in urine (eg: 8-hydrogen oxy-2, 1-deoxypterin nucleic acid; 8-hydroxy-2, -deoxyguanosine referred to as 10 8-OHdG, also known as 8-〇x〇dG) The amount of the product is raised, and it is a biological indicator used to evaluate the oxidative stress in animals. The 8-OHdG in the urine is derived from the product of DNA in the cell that is subjected to hydr〇xyl radical attack and is repaired by the body to the urine. There are several methods for detecting the oxidative damage product 8-OHdG in the urine, including high performance liquid chromatography with electrochemical detection (HPLC-ECD) and gas chromatography mass spectrometry (GC-MS). And enzyme-linked immunosorbent assay (ELISA). 5 200813426 However, the matrix in urine is very complex and can easily interfere with the analysis of genetic oxidation damage products (eg 8-0HdG). However, these analytical methods have major drawbacks and cannot be applied to routine clinical tests or accurately analyze urine samples. For example, high performance liquid chromatography with electrochemical detection (HpLC-ecd) analysis method requires a long time. Purification pre-treatment procedures to remove interfering impurities in the urine; gas chromatography mass spectrometry (GC-MS) analysis methods also require lengthy pre-treatment procedures plus complex derivatization; The linked immunosorbent assay (ELISA) method is a relatively simple assay, but the ELISA has low specificity and is highly susceptible to the influence of urine matrix. Therefore, in order to quickly and accurately detect gene oxidation damage products (such as 8-0HdG) in urine for the extensive use of clinical analysis, it is necessary to develop new technologies. SUMMARY OF THE INVENTION The main object of the present invention is to provide an 8-year-old urine diarrhea gene oxidative wound product 8-hydrogen oxy-2,-deoxyguanosine nucleic acid (8-hydroxy-2'-deoxyguanosine abbreviated as 8- 〇 Threat, also known as 8-〇x〇dG) automated detection method. Urine can be directly analyzed without pre-treatment, which greatly improves the analytical efficiency of 8-OHdG and its material. Because it can quickly and continuously analyze a large number of urine samples, it can be used in clinical routine analysis. The second purpose of the present invention is to provide a method for detecting the accuracy of the age-inducing towel (4). The stable isotope calibration standard 6 200813426 (stable isotopically labeled internal standard) ^, ^ ^ can make the urine towel 8-GHdG fresh ground by the amount of mosquitoes, not (four) urine matrix changes or the sensitivity of the detection instrument caused by the detection Error. The present invention provides an automated mass spectrometry method for detecting a genetic oxidation damage product of a urine towel, comprising the steps of: (a) a mixing step; (b) a solid phase extraction step; (c) a liquid chromatography step; (d) Measurement procedure. The above objects and advantages of the present invention will be apparent from the following detailed description of the embodiments of the invention. BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be described in detail below with reference to the accompanying drawings: [Embodiment] Referring to the first and second figures, the present invention is an automated mass spectrometry method for detecting genetic oxidation damage products in urine. Basically, the following steps are included: [a] Mixing step 11: A predetermined amount of the first sample 21 is taken out and thoroughly mixed with a predetermined amount of stable isotope calibration standard z to form a second sample 22. First, a urine to be tested (i.e., the first sample 21) containing the target substance X and impurities Y1 to Y99 is prepared. The target substance in this embodiment is a genetic oxidation damage product, for example, 8-hydrogen-oxygen-2, _deoxyguanine nucleic acid ^hydiOxyHoxyguanosine (8-OHdG, also known as 8-oxodG). The impurities Y1~Y99 may include urea, uric acid, creatinine, amino 200813426 acid, urinary element, vitamins, various salts, various organic metabolites in the body, etc., and (4) and due to the human diet, This seems to be γι~γ99 to represent 'for convenience of explanation, in fact, impurities ride more than one hundred. Take out a small amount of urine to be tested (at least 1G μ〇, which is defined as the first-sample 21. New, add a predetermined amount of stable isotope to the first sample to the internal standard m8, dG) and fully Fine, (4) into the second inspection (four). The molecular structure of the standard z in the stable isotope calibration is the same as that of the target substance, and only its molecular structure t is substituted by all the uN atoms, the pyrolysis isotope 15N, and the chemical properties and mass spectrometry fragmentation of the stable isotope 4^崎准品z The mechanism is the same as the target substance X. The stable molecular standard, such as the standard Z, has a molecular weight greater than the target substance X (because the 8-oxime molecule contains 5 N atoms). Of course, this example is a way of replacing all % by 15N, _, and can also be modified to use an 8-0HdG internal standard containing stable isotopes such as VO or %. [b] solid phase extraction step 12: using an autosampler (magnetic_fine) 32, the second sample 22 is guided on an on-line solid-phase extraction device 33 to remove some impurities; In detail, the solid phase extraction step 12 is an on-line solid phase extraction step (an-line SQlid-fine e extraetiQn): an_autosampler (autosampler) 32, the second sample 22 is introduced The on-line solid phase extraction device 33 removes most of the impurities to become the third sample 23, which includes the target substance X, the stable isotope calibration standard standard z, and the impurity Y1-Y2〇 (in 200813426, in this embodiment, It is assumed that the impurities are removed from the original species to the only species). [C] liquid chromatography step 13: the third sample 23 is automatically extracted into a liquid phase chromatography device 34 (for example, a liquid chromatography column, that is, a chromatographic column) for separation;
更詳細的說，此液相層析步驟13是利用一液相層析 裝置34 ’使得該第三碰23^_柱分離後，因為滯留時間 的至異；使得與該目標物質x及穩妨鱗#^内標準品Z有 相物留時_雜質大幅減少而成為―第四檢體24，該第四 檢體24至少包括該目標物質X、該穩定同位素標定内標準品Z ，二在本實施例中又假定與目標物質χ及該穩定同位素 標疋内標準品ζ有相近滯留時間的雜質降為只剩川。 ^量測步驟…將該第四檢體24導入該串連質譜儀 _ m咖寧t職㈣中，_該細檢體Μ中的 基因乳化傷害產物（亦即目標物fx)及其穩定同位素標定内把 準品Z之各式特定減。此時，_目標物f χ與穩定同錢 標疋内縣品2之各式狀碱比值，轉可讓目機質 確地被定性及定量。 、旱 ”雖耻量測步驟14可在短時間内完成，但為方便理 解，將其分成前後兩個次步驟說明如下： ⑷]串連質譜儀訊麵測步驟14A :將該第四檢體^導 入該串連質譜儀35，並以採用電魏正離子多重反應模式 200813426 (positive ion electrospray multiple reaction monitoring mode)進行虎偵測為較佳’該目標物質x (8-〇jjdG)之監測荷 質比（m/z)採用284(X)»168(X，）作為χ之定量離子 (quantifier ion)且採用 284(Χ) —140 (X”）作為 χ 之定性離 子（qualifier ion);同時該穩定同位素標定内標準品 Z(15N5-8-OHdG)之監測荷質比(m/z)採用289⑵—173(z，）作 為Z之定量離子且採用289(Z)—145(Z”）作為Z之定性離子。 則雜質Y1所產生之荷質比(m/z)YlVn，或γΐνπ，，碰巧等於 28^168、284440、289473 或 289445 的機率非常低，因 此極不易干擾到目標物質X及穩定同位素標定内標準品ζ的定 性及定量。 如第三A、第三Β、第三C及第三D圖所示，係當尿液添 加穩定同位素標定内鮮品後，使財發明之自動化檢測方法 之沖提圖’分別表示。 [d2]定性與定量步驟14B:由於穩定同位素標定内標準 品Zd-S-OHdG)之化學特性及質譜碎裂機制均與目標物質 X(8-0HdG)相同，此係可作為目標物f χ分析定性之用。因此 目標物質X與穩定同位素標定内標準品ζ除了在液相層析裝置 34内滯留時間應相同外’目標物質X之定量離子284⑴— 168(Χ’）和定性離子284⑴）的訊號比值也應與ζ 之疋里離子289(Ζ) —173(Ζ’）和定性離子289(ζ) ^啦，，） 200813426 的訊號比值相近。更詳細地說，萬一某些雜質Y(以雜質一 Y1 為例)碰巧產生干擾目標物質X的訊號，則284(X)a168(X，） 和284(X) ~>140 (X”）的訊號比值會受影響而改變，係可依此 定性方法判斷有無分析的干擾發生。 此外目標物質X之定量離子284(X) il68(X，)訊號與穩 定同位素標定内標準品Z之定量離子289(Z) —173(Z，）訊號 比值係可作為目標物質X分析定量之用。原則上，定量濃度 的方式係採用同位素稀釋法（is〇t〇pe dilution)，主要是將目 標物質X和一預定量之穩定同位素標定内標準品z的訊號強度 比值作為縱轴(Y軸），及以與其相對應的目標物質X濃度為橫 軸(X軸)建立一校正曲線；再將待測尿液中目標物質χ與穩定 同位素疋内標準品ζ的訊號比值帶入校正曲線公式，就可求 出目標物質X的濃度（如第四圖所示，係使用本發明之自動化 檢測方法建立之校正曲線圖）。又，以第三A、第三Β、第三c 及第二D圖所示的尿液樣本為例，其目標物質χ之定量離子 284(Χ)—168(Χ’）訊號(如第三a圖所示）與穩定同位素標定 内標準品Z之定量離子289(Z) —173(Z，）訊號(如第三C圖所 不）比值為0.375，將比值帶入校正曲線公式，就可求出該尿 液樣本之目標物質X濃度為2. 35 ng/mL。 此外，尿液分析過程中該串連質譜儀35的檢測感度若改 變將不會影_目標物質χ的定量，原因是本發明所採用的定In more detail, the liquid chromatography step 13 is to use a liquid chromatography device 34' to separate the third collision 23^_ column, because the residence time is different; and the target substance x and the stability In the scale #^, the standard Z has a phase when it is left _ the impurity is greatly reduced to become the "fourth sample 24", and the fourth sample 24 includes at least the target substance X, the stable isotope calibration standard Z, and two In the examples, it is also assumed that the impurity having a similar residence time with the target substance χ and the standard isotope in the stable isotope standard is reduced to only the remaining. ^Measurement step... The fourth sample 24 is introduced into the tandem mass spectrometer _ m kan Ning t (4), the gene emulsified injury product (ie, target fx) and its stable isotope in the fine sample Within the calibration, the specifics of the standard Z are specifically reduced. At this time, the ratio of the _ target f χ to the stability of the same type of base in the standard 2, the transfer of the target can be qualitatively and quantitatively determined. Although the shame measurement step 14 can be completed in a short time, for the sake of easy understanding, the two steps are divided into the following two steps: (4)] tandem mass spectrometer information surface test step 14A: the fourth sample body ^Importing the tandem mass spectrometer 35, and using the positive ion electrospray multiple reaction monitoring mode 200813426 (the positive ion electrospray multiple reaction monitoring mode) for the detection of the tiger's target substance x (8-〇jjdG) The mass ratio (m/z) is 284(X)»168(X,) as the quantitation ion of χ and 284(Χ)-140(X") is used as the qualifier ion of χ; The monitoring charge-to-mass ratio (m/z) of the standard Z (15N5-8-OHdG) in the stable isotope calibration is 289(2)-173(z,) as the quantitative ion of Z and 289(Z)-145(Z") As a qualitative ion of Z. The impurity-to-mass ratio (m/z) YlVn, or γΐνπ, produced by the impurity Y1 is very low, which happens to be equal to 28^168, 284440, 289473 or 289445, so it is extremely difficult to interfere with the target substance X. And the qualitative and quantitative determination of the standard product within the stable isotope calibration. For example, the third A, the third, the third C and the third D diagram show that when the urine is added with stable isotope calibration inside the fresh product, the chart of the automated detection method of the invention is shown separately. [d2] Qualitative and quantitative step 14B: calibration due to stable isotope The chemical characteristics and mass spectrometry fragmentation mechanism of the internal standard Zd-S-OHdG are the same as the target substance X(8-0HdG), which can be used as the target f χ for qualitative analysis. Therefore, the target substance X and stable isotope calibration The internal standard product should have the same signal retention ratio as the quantitative ion 284(1)-168(Χ') and the qualifier ion 284(1) of the target substance X except for the retention time in the liquid chromatography device 34. Ζ) —173(Ζ') and qualifier ion 289(ζ) ^啦,,) The signal ratio of 200813426 is similar. In more detail, in case some impurity Y (taking impurity-Y1 as an example) happens to produce interference with the target substance For the signal of X, the signal ratio of 284(X)a168(X,) and 284(X) ~>140(X") will be affected, and the qualitative interference can be judged according to the qualitative method. In addition, the quantitative ion 284(X) il68(X,) signal of the target substance X and the quantitative ion 289(Z)-173(Z,) signal ratio of the standard Z in the stable isotope calibration can be used as the target substance X for analysis and quantification. . In principle, the method of quantitative concentration is the isotope dilution method (is〇t〇pe dilution), which mainly uses the target material X and a predetermined amount of stable isotope to calibrate the signal intensity ratio of the standard product z as the vertical axis (Y axis). And establishing a calibration curve with the X concentration of the target substance X corresponding thereto as the horizontal axis (X axis); and then introducing the signal ratio of the target substance χ in the urine to be tested and the standard ζ in the stable isotope 带 into the calibration curve formula, The concentration of the target substance X can be determined (as shown in the fourth figure, the calibration curve established using the automated detection method of the present invention). Further, taking the urine sample shown in the third A, third, third c, and second D diagrams as an example, the target substance 定量 quantitative ion 284 (Χ) - 168 (Χ ') signal (such as the third Figure a) The ratio of the quantitative ion 289(Z)-173(Z,) signal (such as the third C figure) of the standard Z in the stable isotope calibration is 0.375, and the ratio is brought into the calibration curve formula. The target substance X concentration of the urine sample was 2.35 ng/mL. In addition, if the detection sensitivity of the tandem mass spectrometer 35 is changed during the urine analysis, the amount of the target substance χ will not be affected, because the invention adopts
11 200813426 量方式係為目標物質χ的定量離子284(χ) —168(X，）訊號與 穩定同位素標定内標準品Z的定量離子289(Z) ~>173(Z，）訊 號比值。當該串連質譜儀35的檢測感度上升時兩者之定量離 子感度皆上升，其比值將依然維持不變，而感度下降時亦同。 實務上，如第二圖所示，該線上固相萃取裝置33包含一 固相萃取卡匣331及一切換單元332，係置於液相層析串聯質 邊儀（LC-MS/MS)(此即該液相層析裝置34與該串連質譜儀35 串連後之稱呼)之前，作為線上樣品前處理之用。 其次，關於該固相萃取卡匣331係為可填充C18之卡匣； 且該切換單元332係為一閥門，係用於轉換流體的沖提方向， 使流體將預定檢體沖提至該固相萃取卡匣331或是液相層析 管柱(也可以講是該液相層析裝置34)内。 再者，該液相層析裝置34係選用逆相層析管柱(reverse Phase column)或正相層析管柱(normai phase c〇lumn)。又， 該串連質譜儀35係為電喷灑離子化串連質譜儀。 综上所述，本明之優點及功效可歸納為： [1]檢測效率提高並節省分析耗材。本發明所採用之自動 檢法，尿液不需經由前處理可直接分析，其所需時間大幅 ^—— -----------------------—一- ~ 縮短。不僅大大提高尿液中基因氧化傷害產物（8一氫氧-2，一 去氧鳥糞嘌呤核酸，即8-OHdG)之檢測效率，同時節省尿液前 處理所需耗材。因其能快速連續分析大量不同的檢體（即待測 200813426 尿液），可運用在例行的臨床檢驗分析上。 ⑵準確性高。利用穩定同位素標定内標準品(stable isotopically labeled internal standard)的添加使得尿液 中8-OHdG祕準地被定性與定量，不易因尿液基質變化或檢 測儀器感度改變而造成檢測上的誤差。 以上僅是藉由較佳實施例詳細說明本發明，對於該實施例 所做的任何鮮修改與變化，皆不麟本發明之精神與範圍。 由以上詳細說明，可使熟知本項技藝者明瞭本發明的確可 達成月ίΐ述目的，實已符合專利法之規定，爰提出發明專利申請。 200813426 【圖式簡單說明】 第一圖係本發明之流程圖 第二圖係本發明之運作過程之示意圖 第三A、第三B、第三C及第三D圖係本發明之自動化檢測方 法之尿液中基因氧化傷害產物之定量離子及定性離子 以及其穩定同位素標定内標準品之定量離子及定性離 子之檢測訊號沖提示意圖11 200813426 The quantity method is the ratio of the quantitative ion 284(χ)-168(X,) signal of the target substance 定量 to the quantitative ion 289(Z) ~>173(Z,) signal of the standard Z in the stable isotope calibration. When the detection sensitivity of the tandem mass spectrometer 35 rises, the quantitative ion sensitivity of both increases, and the ratio remains unchanged, and the sensitivity decreases. In practice, as shown in the second figure, the on-line solid phase extraction device 33 comprises a solid phase extraction cassette 331 and a switching unit 332, which are placed in a liquid chromatography serial mass spectrometer (LC-MS/MS) ( That is, before the liquid chromatography device 34 is referred to in series with the tandem mass spectrometer 35, it is used as an online sample pretreatment. Secondly, the solid phase extraction cassette 331 is a cassette capable of filling C18; and the switching unit 332 is a valve for switching the direction of the fluid to be flushed, so that the fluid can flush the predetermined sample to the solid. The phase extraction cassette 331 or the liquid chromatography column (also referred to as the liquid chromatography apparatus 34). Furthermore, the liquid chromatography apparatus 34 is a reverse phase column or a normai phase c〇lumn. Further, the tandem mass spectrometer 35 is an electrospray ionization tandem mass spectrometer. In summary, the advantages and effects of the present invention can be summarized as follows: [1] The detection efficiency is improved and the analytical consumables are saved. According to the automatic detection method adopted by the invention, the urine can be directly analyzed without pre-treatment, and the time required is greatly ^^ ---------------------- -—一-~ Shorten. It not only greatly improves the detection efficiency of the gene oxidative damage product (8-hydrogen oxy-2, a deoxyguanosene nucleic acid, ie 8-OHdG) in the urine, but also saves the consumables required for pretreatment of urine. Because it can quickly and continuously analyze a large number of different specimens (ie, 200813426 urine to be tested), it can be used in routine clinical testing analysis. (2) High accuracy. The addition of the stable isotopically labeled internal standard makes the 8-OHdG in the urine qualitative and quantitative, which is not easy to cause errors in detection due to changes in the urine matrix or changes in the sensitivity of the test instrument. The present invention has been described in detail by the preferred embodiments thereof, and the invention is not limited to the scope of the invention. From the above detailed description, those skilled in the art can understand that the present invention can indeed achieve the purpose of the month, and has already met the requirements of the patent law, and has filed an invention patent application. The first diagram is a flow chart of the present invention. The second diagram is a schematic diagram of the operation process of the present invention. The third embodiment, the third B, the third C and the third D diagram are the automatic detection methods of the present invention. Quantitative Ions and Qualitative Ions of Gene Oxidative Damage Products in Urine and Detected Signals of Quantitative Ions and Qualitative Ions in Standards of Stable Isotope Calibration
第四圖係使用本發明之自動化檢測方法建立之校正曲線圖 12固相萃取步驟 14量測步驟The fourth figure is a calibration curve established using the automated detection method of the present invention. 12 Solid phase extraction step 14 Measurement step
【主要元件符號說明】 11混合步驟 13液相層析步驟 14A串連質譜儀訊號偵娜步驟 21第一檢體 23弟三檢體 犯自動進樣裝置 331固相萃取卡匣 34液相層析裝置 X目標物質 X” X之定性離子 ΥΓ 、Y1” Y1之碎片離子 ζ ζ之定量離子 14Β定性與定量步驟 22第二檢體 24第四檢體 33線上固相萃取裝置 332切換單元 35串連質譜儀 X’ X之定量離子 Υ1〜Υ99雜質 Ζ穩定同位素標定内標準品 ζ” ζ之定性離子 14 ⑤[Main component symbol description] 11 mixing step 13 liquid chromatography step 14A serial mass spectrometer signal detection step 21 first sample 23 brother three test body automatic sample introduction device 331 solid phase extraction card 匣 34 liquid chromatography Device X target substance X" X Qualitative ion ΥΓ, Y1" Y1 fragment ion ζ ζ Quantitative ion 14 Β Qualitative and quantitative step 22 Second sample 24 Fourth sample 33 Online solid phase extraction device 332 Switching unit 35 in series Mass spectrometer X' X quantitative ion Υ1~Υ99 impurity Ζ stable isotope calibration internal standard ζ” ζ Qualitative ion 14 5