WO2008065895A1 - Method for separation/measurement of f2-isoprostane isomer - Google Patents

Abstract

Disclosed is a method for separation/measurement of a F2-isoprostane isomer, which comprises the steps of: conducting a sample treatment procedure comprising a first solid phase extraction step for passing a sample containing the F2-isoprostane isomer through a first solid phase extraction carrier having a larger solid phase volume to remove contaminants contained in the sample and a second solid phase extraction step for passing a sample eluted from the first solid phase extraction carrier through a second solid phase extraction carrier having a smaller solid phase volume than that of the first solid phase extraction carrier to further remove contaminants from the sample containing F2-isoprostane isomer; applying the eluate obtained in the proceeding step to a chromatography carrier having a specific particle diameter suitable for the separation of the F2-isoprostane isomer and separating the F2-isoprostane isomer from the carrier by chromatography using an eluent comprising an aqueous methanol/acetonitrile solution having a specific concentration ratio to measure the F2-isoprostane isomer. In the method, the specific particle diameter suitable for the separation of the F2-isoprostane isomer is 4 μm or less, and the aqueous methanol/acetonitrile solution has a concentration ratio of 10:90 to 100:0.

Description

 Specification

 F2—Method for separation and measurement of isoprostane isomers

The present invention relates to a method for separating and measuring F ₂ -isoprostane isomers. More specifically, the present invention relates to a method for separating and measuring stereoisomers and positional isomers of F ₂ —isoprostane compounds and a sample processing method for separating and measuring F ₂ —isoprostane isomers. Is. Background

 In recent years, it has been clarified that oxidative stress in a living body is a cause of peroxidation of lipids and phospholipids, and the relationship between oxidative stress and diseases has attracted attention (Non-patent Document 1). The level of oxidation in the living body is regulated by the balance between the reactive oxygen species production system and the consumption system, and is usually kept almost constant. However, a state in which the balance is lost due to various factors such as drugs, radiation, and ischemia, and the tendency to oxidize is called oxidative stress or oxidative stress.

 In other words, oxidative stress is defined as the difference between the oxidative damage potential of reactive oxygen species (ROS) generated in vivo and the antioxidant potential of the in vivo antioxidant system. Reactive oxygen is produced in the body when oxygen is taken from the respiration and used in the mitochondrial electron transport system to generate water, when leukocytes undergo an inflammatory reaction during infection, or during metabolism of arachidonic acid. Is done. Although active oxygen is generally regarded as a bad person, it is originally useful for energy production, invasion of foreign substances, treatment of unwanted cells, and cell information transmission. However, when an excessive amount of reactive oxygen species that cannot be captured by the in vivo antioxidant system is produced, the excess reactive oxygen species are converted to phospholipids, carbohydrates, proteins, Enzyme, nucleic acid, genetic DNA that carries genetic information is damaged by oxidative modification, damages the structure and function of the body, induces disease, promotes aging, causes cancer, lifestyle-related diseases, etc. It is known to be.

In addition, arachidonic acid is a metabolite of linoleic acid, one of the essential n-6 unsaturated fatty acids derived from food, and exists as phospholipids in cell membranes and plasma. It has been reported that when arachidonic acid is peroxidized by reactive oxygen species or free radicals, a series of prostaglandin-like substances with strong biological activity are generated non-enzymatically by radical reactions in the human body (for example, Non-patent document 1). Thus, a series of prostaglandin-like substances generated from arachidonic acid by the reaction mechanism of free radicals are collectively called F ₂ isoprostane compounds.

F ₂ —isoprostane compounds are markers of established oxidative stress (see, eg, Non-Patent Document 2) and are associated with a variety of conditions and diseases including cardiovascular disease and its risk factors (eg, Non-patent document 3). F ₂ -isoprostane has a strong physiological action as a ligand for a prostaglandin (PG) receptor or nuclear receptor bound to a cell membrane (see, for example, Non-Patent Document 4). F ₂ -isoprostane is produced in a state bound to the phospholipid of the cell membrane and lipoprotein (see, for example, Non-Patent Document 5) and dissociates into free F ₂ -isoprostane by the action of phospholipase A _2. It is metabolized in urine (for example, see Non-Patent Document 6).

There are several difficulties in measuring F ₂ -isoprostane in biological samples. First, F ₂ —isoprostan has a number of stereoisomers and 4 series of positional isomers (see, for example, Non-Patent Documents 1 and 7 1 1). In addition, comprehensive and specific measurements of the F ₂ -isoprostane isomers are required for comprehensive assessment of oxidative stress. In addition, F ₂ -isotopes present in biological samples Since the concentration of rostan is low, an analytical method with high analytical sensitivity is required (for example, see Non-Patent Document 8).

Urinary F ₂ —isoprostane has been shown to be a specific and sensitive oxidative stress marker for noninvasive in vivo oxidative stress measurement, which is beginning to be used to assess oxidative stress. Of these F ₂ isoprostane isomers, 8-isoprostane (8-iso-PGF _{2 a} , 8-iso-15 (S) PGF _{2 a} and iPF _{2 a} -lll) is the most studied F ₂ — Isoprostan. iPF _{2 a} -VI is a positional isomer of iPF _{2 a} -lll and is said to be produced in the body more than 8-isoprostan (for example, see Non-Patent Document 9). The F ₂ —isoprostane isomers have different physiological effects and may vary in metabolism in various pathologies and diseases associated with oxidative stress, so specific and comprehensive measurements of the F ₂ —isoprostane isomers Is important.

To diagnose oxidative stress syndrome or disease, for example, brain tissue or cerebrospinal fluid, isoprostane molecular markers one present in plasma or urine (iPF _{2 a} -ll and iPF _{2 a} -VI and 8,12-iso -iPF _{2 a} -VI) is measured by gas chromatography / mass spectrometry (GC / MS) as a method for measuring the degree of lipid peroxidation (for example, see Patent Document 1). ).

Gas Chromatography / Mass Spectrometry (GC / MS) is considered the gold standard for the determination of F ₂ —isoprostane (see, eg, Non-Patent Documents 10 0, 11 and 12). However, GC / MS cannot be used as a routine measurement method due to the lack of measurement specificity as well as troublesome purification and derivatization of Sankare (for example, Non-Patent Documents 1 0 and 1 2). An 8-isoprostane measurement kit based on enzyme immunoassay (ELISA) is also available on the market. However, in addition to problems such as cross-reactivity of antibodies, there is a drawback that exhaustive measurement of F ₂ -isoprostane isomers cannot be performed (for example, see Non-patent Documents 13).

On the other hand, the liquid chroma Bok photography eleven tandem mass spectrometry (LC-MS / MS) F 2 by method - Isopurosu Tan and its metabolites assay, high sensitivity and specificity F ₂ - is a isoprostanes assays (e.g. Non-patent document 1). F ₂ —isoprostane stereoisomers are analyzed by high performance liquid chromatography

(HPLC) and regioisomers can be separated by electrospray (ESI) -MS / MS, allowing specific measurement of F ₂ -isoprostane isomers. Moreover, ESI-MS / MS method in a way that can detect low concentrations of analyte from complex sample matrices can sensitive measurement of F ₂ _ isoprostanes. However, LC-MS / MS methods have significant drawbacks when analyzing biological samples. In other words, the ionization efficiency of the measurement substance by ESI is affected by the sample matrix and HPLC buffer. Since the matrix effect varies from sample to sample (see Non-Patent Documents 14 for example), it is necessary to control the matrix effect in order to perform stable analysis.

(For example, refer nonpatent literature 15).

In such a measurement method, in order to correct measurement data that has fluctuated due to sample injection, sample preparation, instrument parameters, and the matrix effect, the measurement substance is chemically and structurally the same, but only the mass is different, stable. The isotope-labeled analog is used as an internal standard (IS). Even if internal standards such as stable isotopes are used, for example, plasma samples extracted with an organic solvent are separated by HPLC-isolated 8-isoprostane (8-iso-PGF _{2 ( l} -d ₄ ) It has been reported that accurate measurement is not possible due to the presence of contaminants in the peak (for example, see Non-Patent Document 16). Since the ratio to the standard is used to calculate the concentration of the measurement substance, 8-isoprostane cannot be measured accurately, so the HPLC separation gradient time must be reduced in order to separate contaminants from the internal standard. I need to lengthen it (for example, Non-patent document 16). However, the matrix-related compound contaminants that elute together with the analyte will affect the accuracy of the measurement data even when the retention time is the same as the analyte, but the presence of the contaminant cannot be detected. Therefore, in the case of F ₂ -isoprostane measurement by LC-MS / MS, extracting a clean biological sample is the basis for obtaining accurate and reproducible data.

Previously, a method for measuring urinary F ₂ —isoprostane by LC-MS / MS has been reported, but the ion-suppressing effect of the matrix on F ₂ —isoprostane in biological samples has not been investigated (for example, Non-Patent Documents 16, 17, 17, 18 and 19). In addition, contamination of the ion source due to the sample matrix and HPLC buffer reduces the measurement sensitivity and cannot be analyzed stably. Therefore, it must be said that a method for measuring urinary F ₂ -isoprostane by LC-MS / MS has not been established. Control of the ion suppression effect by the sample matrix and HPLC buffer is important in establishing a method for measuring biological samples F ₂ -isoprostane by LC-MS / MS method.

In summary, oxidative stress has been shown to be associated with a variety of conditions and diseases, including cardiovascular disease, and it is an urgent need to measure markers of oxidative stress. Urinary F ₂ — isoprostane compounds are peroxidation products of arachidonic acid through reactive oxygen species and are reliable markers of oxidative stress. However, there are various isomers in urinary F ₂ -isoprostane compounds, and it is important to separate and measure these isomers with high accuracy.

The LC-MS / MS method has been reported to be a specific assay for the F ₂ -isoprostane isomer in biological samples. However, in this method, since ion suppression by the sample matrix is not controlled, stable analysis cannot be performed at present. In addition, fluctuations in the electrospray ionization efficiency due to the sample matrix will affect the accuracy, reproducibility, and sensitivity of the measurement. Therefore, when measuring biological samples by LC-MS / MS, it is very important to control ionization suppression or enhancement by the sample matrix in order to perform stable analysis.

 Non-Patent Document 1 Yin, H., et a, J. Chromatogr. B Analyt. Technol. Biomed. Life Sc, 2005, 827: 157-164

 Non-patent literature 2 Morrow, JD, et al., Arterioscler. Thrornb. Vase. Biol. 2005, 25: 279-286 Non-patent literature 3 Montuschi, P., et al., FASEB J. 2004, 18: 1791-1800

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 Non-Patent Document 9 luliano, L. et al., 丄 Am. CoH. Cardiol. 2001, 37: 76-80

 ^! ^ X ^ 1 0 Tsikas, D., et al., J. Chromatogr. B Analyt. Technol. Biomed. Life Sc 2003, 794: 237-255

 Non-Patent Document 1 1 Reilly, MP "et al., Circulation. 1997, 96: 3314-3320

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Non-Patent Document 1 5 Tiller, PR, et al., Rapid. Commun. Mass Spectrom. 16: 92-98 Non-patent literature 1 6 Taylor, AW, et al., Anal. Biochem. 2006, 350: 41-51 Non-patent literature 1 7 Liang, Y, et al., Free Radic. Biol. Med. 2003, 34: 409- 418

 Non-patent literature 1 8 Li, H "et al., Proc. Natl. Acad. Sci, USA, 1999, 96: 13381-13386 [Non-patent literature 1 9 Bohnstedt, KC, et al., J. Chromatog in B Analyt Technol. Biomed. Life Sci. 2006, 796: 11-19

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Patent Document 1 Special Table 2 0 0 2—5 3 1 8 1 5 gazette Disclosure of Invention Therefore, the present inventor has developed a matrix and one HPLC buffer additive during measurement of F ₂ —isoprostane by LC-MS / MS As a result of intensive investigations on the means to control the ion suppression effect of urine, a new multi-dimensional solid-phase extraction (MD-SPE) urine sample cleanup method and a new buffer-free HPLC method were developed. The present invention was completed by finding that stable measurement of urinary F ₂ -isoprostane without damage by the MS / MS method was possible. In addition, by combining a specific HPLC force ram and solvent composition, 8-isoprostane and a new unknown F ₂ -isoprostane isomer could be separated, enabling specific measurement of 8-isoprostane. . Accordingly, one main object of the present invention is to provide a method for separating and measuring F ₂ -isoprostane isomers.

A preferred embodiment of the present invention is to provide a method for separating and measuring stereoisomers and positional isomers of F ₂ _isoprostane compounds.

Another main object of the present invention is to provide a sample suitable for a method for separating and measuring F ₂ -isoprostane isomers, particularly a method for treating a biological sample such as a urine sample.

In order to achieve the above object of the present invention, the present invention provides a method for separating and measuring stereoisomers and positional isomers of F ₂ -isoprostane compounds.

More specifically, a sample is added to a chromatographic carrier having a specific particle size capable of separating F ₂ -isoprostane isomers, and an elution solution consisting of a methanol noacetonyl aqueous solution having a specific concentration ratio is used. In the method for separating and measuring F ₂ -isoprostane isomers, which consists of separating and measuring F ₂ -isoprostane isomers by chromatography, the specific particle size that can be separated from F ₂ -isoprostane isomers is 4 // Less than m, the chromatographic carrier is not capable of retaining F ₂ —isoprostane isomer, and methanol Z-acetonitrile aqueous solution with a specific concentration ratio is 1 0: 9 0-1 0 0: 0 A method for separating and measuring F ₂ -isoprostane isomers is provided.

A preferred embodiment of the present invention provides a method for separating and measuring F ₂ -isoprostane isomers, wherein the specific particle size capable of separating F ₂ -isoprostane isomers is 3.5 m or less.

Another preferred embodiment of the present invention is a silica carrier in which the chromatographic carrier is C6-C20, preferably C8-C18, more preferably C8, or a C18 long chain alkyl group is chemically bonded to silica gel. A method for separating and measuring F ₂ -isoprostane isomers is provided.

As yet another preferred aspect of the invention, specific concentration ratio of methanol Asetonitoriru aqueous solution 4 0: 6 0-1 0 0: 0, preferably from 5 0: 5 0 - 1 0 0: 0 consists a F ₂ —Methods for separating and measuring isoprostane isomers are provided. Furthermore, the present invention provides a sample processing method suitable for a method for separating and measuring F ₂ -isoprostane isomers. The sample processing method of the present invention is based on the two-step solid-phase extraction using a solid-phase extraction carrier having a different solid-phase capacity, by removing impurities and extracting the F ₂ -isoprostane compound. -It is an epoch-making method that makes it possible to simultaneously perform three things: solvent exchange for MS / MS analysis, concentration to enhance analytical sensitivity, and sample purification at the same time.

More specifically, in the sample processing method according to the present invention, a sample such as a biological sample is passed through a first solid phase extraction carrier having a large solid phase capacity, and F ₂ -isoprostane contained in the sample is passed. A first solid-phase extraction step comprising holding a compound and its isotope on the first solid-phase extraction carrier and removing contaminants contained in the sample, and passing through the first solid-phase extraction carrier The obtained sample is passed through a second solid phase extraction carrier having a smaller solid phase capacity than the solid phase capacity of the first solid phase extraction carrier, and further F ₂ -isopropyl compound and its isotope are extracted into the second solid phase extraction. A second solid-phase extraction step comprising holding the carrier and removing contaminants contained in the sample.

 In a preferred embodiment of the present invention, the first solid phase extraction step is performed by size exclusion chromatography and reverse phase chromatography, and the second solid phase extraction step is performed by size exclusion chromatography and normal phase chromatography. A sample processing method is provided comprising:

Further, according to yet another aspect of the present invention, a sample such as a body sample is passed through a first solid phase extraction carrier having a large solid phase capacity, and the F ₂ -isoprostane compound contained in the sample and its isotope are included. A first solid phase extraction step of holding a body on the first solid phase extraction carrier and removing contaminants contained in the sample, and a sample that has passed through the first solid phase extraction carrier. The F ₂ -isoprostane compound and its isotope are further retained on the second solid phase extraction support through the second solid phase extraction support having a solid phase capacity smaller than the solid phase capacity of the first solid phase extraction support. In addition, a second solid-phase extraction step comprising removing contaminants contained in the sample, a sample treatment method comprising, and an eluate obtained by the treatment by the sample treatment method, the F ₂ _ isoprostane isomers separable specific particle size With an elution solution is added to the chromatographic one carrier consisting of methanol Asetoni Bok Lil aqueous solution of a specific concentration ratios having, chromatography one by F ₂ - becomes the isoprostane isomer from separating measure F ₂ - isoprostane isomer In the method for separating and measuring F ₂ —isoprostane isomer, the specific particle size that can be separated is 4 μm or less, and the chromatographic carrier does not have the ability to retain F ₂ —isoprostane isomer. In addition, the present invention provides a method for separating and measuring F ₂ -isoprostane isomers, wherein the aqueous solution of methanol Z-acetonitrile at a specific concentration ratio is from 10:90 to 100 _: 0: 0.

According to the present invention F ₂ - separation method of measuring Isopurosutan isomers, F ₂ - is cormorants effect has to be able to carry out separation measurement of stereoisomers as well as regioisomers of the isoprostane compounds with high sensitivity and high precision .

Further, the sample processing method according to the present invention has an effect that the method for separating and measuring F ₂ -isoprostane isomer according to the present invention can be performed with high sensitivity and high accuracy.

That is, the present invention is novel sample clean-up method the new F ₂ to the without buffers one by Multidimensional Solid Phase Extraction - by isoprostane separation HPLC method, LC-MS / MS in urine by method F ₂ - exhaustive of I Sopurosutan There is an effect that a target-specific analysis can be performed. In addition, if the novel multidimensional solid phase extraction-LC-MS / MS method according to the present invention is used, the effect of routine analysis of F ₂ -isoprostane in urine samples is also expected. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the structure of an isomer of F ₂ -isoprostane compound and a diuterium-substituted internal standard (IS).

Figure 2 shows (A) 8-iso-PGF _2a , (B) 8-iso-PGF _2a -d4, (C) 2,3-dinor-8-iso-PGF _2a . (D) (±) 5- It is a figure which shows the ion spectrum of iPF _2a -VI, (E) (±) 5-iPF _2a -VI-d11.

Fig. 3 shows the results of liquid chromatography tandem mass (LC-MS / MS) analysis of iPF _2a -ll and iPF _2a -VI isomers detected in MRM mode. In the figure, A and B are m / z353 → 193 ion channels, C is m / z 357 → 197 ion channels, D is m / z 325 → 237 ion channels, and F: m / z364 → 115 ion channels. is there.

Fig. 4 A shows the addition of urine sample with Oasis HLB (hydrophilic monolipophilic balance), MAX (reverse phase and anion exchange), MCX (reverse phase and cation exchange) solid phase extraction cartridge (3cc / 60mg) [ ³ H] A graph showing a retention curve of 8-iso-PGF _2a .

Figure 4B shows the Oasis HLB solid phase of [ ³ H] 8-iso-PGF _2a against methanol concentration under acidic conditions (2% formic acid), neutral side (water), base side (2% ammonia) wash conditions. FIG. 6 is a diagram showing a change in holding characteristics in a cartridge.

 FIG. 4C shows: selective solid phase extraction protocol and multidimensional solid phase extraction method. In the figure, AA means arachidonic acid and EA means ethyl acetate.

FIG. 5 is a diagram showing an MRM chromatogram of F ₂ -isoprostane in a urine sample extracted by a multidimensional solid-phase extraction method. In the figure, A is the iPF _2e -lll isomer in the urine sample, B is the extracted urine sample, 8 o-15 (R) PGF _2a , iPF ₂ „-ll, 15 (R) PGF ₂ and PGF. IPF _2a -lll is the ion channel (m / z 353 → 193) when _2a is added, and C is the ion of the internal standard iPF _{2 a} -lll-d4 and PGF _2a -d4 added to the urine sample. Channel (m / z357 → 197), D indicates the internal standard ion channel (m / z357 → 197) of the blank urine sample, E indicates 2,3-dinor-iPF _2a- lll standard addition

(Dotted line) and 2,3-dinor-iPF _2a -lll ion channel (m / z 325 → 237) in extracted urine without addition, F indicates ((±) 5- iPF _2a -VI added (dotted line) and added The extracted urinary iPF _2a -VI ion channel (m / z 353 → 115) is shown, G: is the urinary internal standard (±) 5-iPF ^ -VI-d "ion channel (m / z 364 → 115).

Figure 6 shows (A) iPF _{2 (r} -lll / iPF _2a -lll-d ₄ peak area ratio, (B) iPF _2a -VI / iPFwVI when 1 ml and 2 ml urine samples were extracted from 8 volunteers. -d "peak area ratio, (C) iPF _{2 (l} -lll S / N ratio,

(D) Correlation at the S / N ratio of iPF _2a -VI. The S / N ratios for (C) iPF _2a -ll and (D) iPF _2a -VI are also shown in the box chart. In the figure, the upper and lower borders of the box indicate 75 and 25 percentile values, and the line in the box indicates the median value. The upper and lower error bars indicate the 90th and 10th percentiles. The same amount (2 ng) of iPF _2a -ll d4 and iPF _2a -V d d was added to the 1 ml and 2 ml samples.

Fig. 7 shows the results of examining the resolution of 8-iso-PGF _2a and unknown isomers and the resolution of PGF _2a and unknown isomers by changing the mixing ratio of methanol and acetonitrile. BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a sample matrix during measurement of urinary F ₂ -isoprostane by LC-MS / MS method. In addition, a novel multidimensional solid phase extraction (MD-SPE) method and an HPLC method that can separate F ₂ -isoprostane isomers without using a buffer to control the ion-suppressing effect of an HPLC buffer additive. It is about.

In the case of F ₂ -isoprostane determination by liquid chromatography-tandem mass spectrometry (LC-MS / MS), extracting a clean biological sample is necessary to obtain accurate and reproducible data. As described above, it is basic.

In order to extract a clean biological sample, a novel multidimensional (MD) solid-phase extraction (SPE) method was developed in the present invention. By this method connexion, F to remove contaminants in the urine ₂ - to extract isoprostanes, size exclusion chromatography, by applying the ordinal normal phase chromatography one and reverse phase chromatography machine F ₂ - isoprene Rostand The F ₂ -isoprostane can be selectively extracted by removing the contaminants by the 2D SPE washing method and the selective elution method.

 The novel multidimensional solid-phase extraction method according to the present invention is based on two-step solid-phase extraction using a solid-phase extraction carrier having different solid-phase volumes and selective SPE washing / elution protocol. The two-step solid phase extraction method is composed of a first solid phase extraction step and a second solid phase extraction step.

 That is, the first solid phase extraction step mainly includes a process of removing proteins, salts, etc., a process of removing acidic polar contaminants, a process of removing basic polar contaminants, and a process of removing neutral contaminants. It is configured. These steps should be performed using size exclusion chromatography and reverse phase chromatography.

 In the present invention, before the sample is processed in the first solid phase extraction step, the sample is pretreated so as to be suitable for the subsequent first solid phase extraction step. For example, 10% methanol, heavy water isotopes, and acids such as formic acid may be added to the sample to adjust the sample to an acidity of about pH 3. In addition, it is preferable to remove solid particles present in the sample by filtering this sample. The sample thus treated is used for the next solid phase extraction operation.

 In the process of removing large molecular weight contaminants such as proteins and salts in the first solid phase extraction step, the pretreated sample is added to the first solid phase extraction support and the sample is neutralized, for example with 5% methanol. By washing in this manner, contaminants such as proteins and salts that are not retained on the carrier can be removed. The first solid phase extraction carrier used in this step is adjusted to be acidic before use, for example, by adding an acid such as 5% methanol and formic acid.

 After removing large molecular weight contaminants such as proteins and salts in the above step, remove polar acidic contaminants, for example, by washing the carrier with 5% methanol and 2% ammonia to make it basic. be able to.

 Next, in order to remove the basic polar contaminants, the carrier is washed with, for example, 5% methanol and 2% formic acid and moved to the acidic side. Polar base contaminants are removed by washing with a solvent having a higher concentration of organic solvent on the acidic side, for example, 159% methanol (2% formic acid).

Subsequently, in order to remove neutral contaminants, the neutral contaminants are removed, for example, by washing the carrier with a hexane Z ethyl ether mixture :? ). After removing neutral contaminants in this way, wash the carrier with, for example, hexane-ethyl acetate mixed solution. I. In addition to removing water remaining on the carrier, F ₂ -Isopro It can remove impurities that are less polar than stun. Then, in order to selectively elute F ₂ -isoprostane, for example, polar organic solvent such as jetyl ether and acid elution solvent such as acetic acid are used. A mixture of organic solvent and acetic acid was added to elute the fatty acid fraction containing F ₂ -isoprostane. Prepare the sample so that the eluate thus obtained is diluted in non-polar solvent, eg hexane, and kept in normal phase before the next second solid phase extraction step. The eluate treated in this way is processed in the second solid phase extraction step.

In the second solid phase extraction step, F ₂ -isoprostane is retained in normal phase chromatography to remove contaminants that are less polar than F ₂ -isoprostane. Switch to aqueous solvent with 5 <½ methanol wash and remove acid.

Next, for example, by washing with 5% methanol and 2% ammonia on the base side, contaminants that are weaker than F ₂ -isoprostane on the base side can be further removed when the solid phase capacity is small.

Next, to elute F ₂ -isoprostane, wash the carrier with 5% methanol and 2% formic acid, for example, and move it to the acidic side. The acid is then removed, for example with 5% methanol. Furthermore, increase the concentration of the washing organic solvent to such an extent that F ₂ -isoprostane does not elute. For example, washing with 1596 methanol removes polar impurities and facilitates the elution of F ₂ -isoprostane. Prepare a carrier.

Then, further the concentration of the organic solvent F ₂ - raise enough to elute the isoprostanes, for example, methanol 8096, by selectively F ₂ - eluting the isoprostane F ₂ - isoprostanes by Li held Are removed by leaving solid impurities in the solid phase.

By using a small solid phase, eg 2 mg, in the second solid phase extraction step, F ₂ -isoprostane can be eluted with a small solvent volume, eg 40-50 μΙ. Dilute the eluted sample with water and adjust the organic solvent concentration of the sample to be lower than the initial HPLC organic solvent concentration, eg, 20%. The prepared sample can be directly subjected to LC-MS / MS analysis.

 The first and second solid phase extraction supports used in the first solid phase extraction step and the second solid phase extraction step have a small pore size, for example, hydrophilic-lipophilic balance (HLB). It should be a solid phase polymer of about 7-20. Examples of the solid phase polymer include a copolymer of hydrophilic vinyl pyrrolidone and lipophilic divinyl benzene, and the carrier usable in the present invention is not limited to such a copolymer. Rather, any material that meets the object of the present invention can be used.

 If a 1 cc / 30 mg solid phase extraction cartridge is used in the first solid phase extraction step, the elution volume should be about 1 ml when isoprostane is eluted with diethyl ether (296 acetic acid). If the concentration factor is increased, it is necessary to increase the amount of the sample, and about 3 ml of urine can be added. In the case of a thin urine sample, 4 ml or more may be added. Furthermore, if cell culture medium is used, 10 ml of medium can be used to measure isoprostane. In the case of the family hypercholesterolemic animal model Watanabe Heron, it can be extracted with a 1 ml urine sample. That is, depending on the sample, the first solid phase extraction step can be concentrated 1 to 10 times. When a solvent with a strong dissolution power such as methanol or acetonitrile is used, since the elution volume is small (for example, 0.5 ml), there is an advantage that the concentration multiple can be further increased. Use of a strong solvent with a strong dissolving power requires caution because contaminants will also be eluted.

The first solid-phase extraction step uses the first solid-phase extraction support and uses the F ₂ -isoproteol using size exclusion chromatography, reverse phase chromatography or normal phase chromatography mechanisms depending on the sample type. While holding the stun, contaminants that are not held by the carrier are removed. In other words, when the sample is a urine sample, reverse phase chromatography is used, but the sample is a tissue sample. In some cases, normal phase chromatography can also be used. The first solid-phase extraction step uses an HLB cartridge to retain F ₂ -isoprostane in a size-exclusion chromatography and reverse-phase chromatographic mechanism, and is highly polar with large molecular weight contaminants such as proteins. Small molecular weight contaminants can be removed.

 In the second solid phase extraction step, contaminants that were not removed in the first solid phase extraction step are removed by performing substantially the same operation as the first solid phase extraction step. However, the second solid phase extraction step is operated by a normal phase chromatograph that is different from the reverse phase chromatography used in the first solid phase extraction step. There is a need. In other words, the eluate obtained in the first solid phase extraction step cannot be used in the second solid phase extraction step as it is. For this purpose, the eluate obtained in the first solid phase extraction step is switched to aqueous with a solvent such as hexane and processed in the next second solid phase extraction step.

 The second solid phase extraction step is substantially the same operation as the first solid phase extraction step using a small volume of the solid support, and is a solvent exchange, concentration and further removal of contaminants.

 In other words, the eluate of jetyl ether (added with 2% acetic acid) obtained in the first solid phase extraction step is difficult to use for LC-MS / MS analysis as it is, so it is necessary to exchange and concentrate the solvent. There is. In other words, the internal standard for quantifying isoprostane (heavy water isotope) is not stable under acidic conditions, and the concentration of isoprostane in the eluate is low. Because there is no. Normally, the solvent can be exchanged and concentrated by removing the solvent with nitrogen gas. However, isoprostane may be decomposed if it takes time and effort. On the other hand, if solid phase extraction is used, it is possible to simultaneously perform solvent exchange and concentration without decomposing isoprostane and saving labor and time.

 As the second solid phase extraction carrier used in the second solid phase extraction step, for example, a solid phase carrier having a solid phase capacity smaller than that of the first solid phase extraction carrier, for example, an HLB unit plate or the like is preferably used. In order to analyze with LC-MS / MS, the final methanol concentration of the eluted sample must be 20% or less. However, if a carrier with a small solid volume such as an HLB Elution plate is used, its elution volume Since it is small, there is a feature that the concentration factor can be increased. For example, if a 2 ml urine sample is eluted with 1 ml, the concentration factor is twice, so the concentration of isoprostane contained is too low to provide sufficient measurement sensitivity. Therefore, in order to use such eluate for LC-MS / MS analysis, it is necessary to increase the isoprostane concentration by skipping the solvent.

 However, the concentration factor when using the Elution plate in the second solid phase extraction step is 25 (from 1000 μΙ / 50 μ to 250 times. For example, the 40 μ 40 eluate obtained in the first step is Even when diluted with μ, it is possible to obtain a concentration factor of 10 times, which can greatly reduce the time and labor of extraction and greatly increase the efficiency of the sample after dilution with water. If the EIution plate is used in the second solid phase extraction step, the concentration factor will be 5 to 50 times.The optimal range for urinary isoprostane analysis is 10 to 20 times. Therefore, the concentration factor required for LC-MS / MS analysis can be easily achieved.

Since the second solid phase extraction carrier used in the second solid phase extraction step has a small solid phase capacity, it has other conditions than the first solid phase extraction carrier (for example, 10 mg) having a large retention capacity. Even if they are the same, there are few contaminants that can be retained. Therefore, the second solid phase extraction step utilizes another dimension of separation mechanism that utilizes the difference in retention capacity of isoprostane and contaminants relative to the volume of the solid phase. In other words, the second solid-phase extraction step removes contaminants and at the same time provides solvent exchange and analytical sensitivity for LC-MS / MS analysis. It is an epoch-making method that can perform the three processes of concentration for sample collection and sample clean-up simultaneously in a short time.

In this second solid phase extraction step, F ₂ -isoprostane can be retained using normal phase chromatography mechanisms using a microelution SPE plate. In this second solid phase extraction step, as in the first solid phase extraction step, F ₂ -isoprostane is retained on the support using size exclusion chromatography and normal phase chromatography, but not in normal phase chromatography. Low polarity contaminants are removed.

 Also, the first and second solid phase extraction carriers used in the first solid phase extraction step and the second solid phase extraction step may be substantially the same type so that their holding capacities are different. Prepare. In other words, the holding capacity of the first solid phase extraction carrier used in the first solid phase extraction step is prepared to be larger than the holding capacity of the second solid phase extraction carrier used in the second solid phase extraction step. The retention capacity of the solid-phase extraction carrier is proportional to the amount (mg) of the carrier for the same type of carrier. Therefore, in this invention, the ratio between the holding capacity of the first solid phase extraction carrier and the holding capacity of the second solid phase extraction carrier is generally 30: 1 to 50: 1, preferably 1. 0: 1 to 2 0: It should be in the range of about 1. For example, if the first solid phase extraction step uses a 1cc / 30 mg solid phase extraction cartridge, and the second solid phase extraction step uses a small amount of solid phase extraction support (2 mg) μΕΙι ^ οη plate The retention capacity ratio of 1cc / HLB solid phase extraction cartridge 30 mg and 2_Β μ 曰 ution plate 2 mg is 15: 1. In this case, a 1 cc / 30 mg HLB solid phase extraction cartridge can hold 2 to 3 ml of urine sample. In order to reduce the amount of contaminants to the maximum extent, it is better not to add more samples than necessary. In addition, HLB solid-phase extraction supports are characterized by the ability to retain both hydrophilic and lipophilic compounds. In other words, selectivity is low. Therefore, if the amount of sample is increased, the amount of isoprostan will increase, but the amount of contaminants will increase. Most contaminants can be removed by using a 1cc / 30 mg solid phase extraction cartridge in the first solid phase extraction step, so the Elution plate with a small amount of solid phase extraction carrier in the second solid phase extraction step. Will be able to use.

 However, in the conventional solid-phase extraction method, the sample is extracted at room temperature to remove the extraction solvent with nitrogen gas and re-dissolve the sample with the analysis solvent for LC-MS / MS analysis after sample extraction. I had to leave it for hours.

 In the novel multidimensional solid phase extraction method according to the present invention, sample extraction and solvent exchange for LC-MS / MS analysis can be simultaneously performed in the second solid phase extraction step. Furthermore, in the second solid phase extraction step, sample concentration can be performed at the same time by using a small volume solid phase. In addition, when the measurement substance in the sample is concentrated, the contaminants contained in the sample are also concentrated at the same time, which affects the stability of the sample and the stability of the measurement sensitivity. However, in the present invention, if the sample is concentrated using solid phase extraction having a small solid phase capacity, the measurement substance is also concentrated, but there is an advantage that contaminants are removed. This increases the cleanliness of the sample and significantly reduces sample processing time. Furthermore, in the second solid phase extraction step, since the sample elution volume is small (eg, 40 jul), it is possible to easily concentrate a urine sample (eg, 2 ml) 10 times. Furthermore, the eluted sample can be used for LC-MS / MS measurement simply by diluting with water.

In the multidimensional solid phase extraction method of the present invention as described above, a clean sample can be extracted by a two-dimensional selective solid phase extraction washing method and a selective F ₂ -isoprostane elution method. HLB available Polymeric adsorbents can be used in the pH range of 1-14. The two-dimensional cleaning method was developed by taking advantage of the difference in pH and methanol retention characteristics of F ₂ -isoprostane and urinary contaminants. In the case of HLB adsorbents, acidic and basic compounds show opposite retention at low and high pH. At low pH, acidic compounds have strong retention, whereas basic compounds have weak retention. On the other hand, at high pH, acidic compounds have only weak retention, whereas basic compounds have strong retention. Therefore, for example, by adding acid to a urine sample to lower the pH, adding 10% methanol and then injecting it into a solid phase extraction cartridge, the basic compound can be removed. Further, the basic compound can be further removed by washing on the acidic side with 15% methanol / 2% formic acid. On the other hand, washing on the base side using 5% methanol / 2% ammonia can remove acidic contaminants. It has been observed that yellow contaminants are removed by washing on the base side.

Neutral contaminants present in urine can be removed by hexane washing. Hexane is not miscible with methanol and water, so it is recommended to add jetyl ether to the hexane wash. In addition, since ethyl oxalate is miscible with water, the water remaining in the HLB cartridge can be removed by adding a washing step with a mixed solution of ethyl hexane monoacetate (for example, 9 1). Is good. This is because it is necessary to remove the water remaining in the solid phase in order to increase the elution efficiency of the sample with jetyl ether / 2% acetic acid. In the first solid phase extraction step, F ₂ -isoprostane can be selectively eluted by using diethyl ether / 2% acetic acid. Diethyl ether is less polar and more selective than ethyl acetate, so there is less elution of contaminants, and it is observed that yellow contaminants remain in the solid phase cartridge after elution of F ₂ -isoprostane. ing. Therefore, clean sample extraction is possible by selective washing of contaminants and selective elution of F ₂ -isoprostane.

 Small variations in extraction parameters such as organic solvent concentration and pH did not significantly affect sample extraction. This indicates that selective SPE treatment is robust. Other solid phase extraction polymers such as Oasis HLB polymer cartridges, such as Strata X solid phase cartridges, can be used as well, allowing similar sample extraction and recovery. This also indicates that the multidimensional solid-phase extraction method according to the present invention has been established.

 The novel multidimensional solid phase extraction (MD-SPE) method according to the present invention has significant advantages. In other words, sample processing for liquid chromatographic and tandem mass spectrometry (LC-MS / MS) analysis can be performed in a short period of time, and a clean sample extraction can be achieved. According to the inventor's experience, extraction of 8 samples is usually possible within 3 hours. However, since this processing method is solid, it is possible to further increase the sample processing efficiency by automatic processing. Conceivable. In addition, this MD-SPE method can be performed by, for example, cell (endothelial cells, smooth muscle cells, etc.) culture solution (10 ml) or animal ( It is also possible to apply it to urine samples (1 to 2 ml) of magpies, rabbits, etc. Such correction includes, for example, replacing the Captiva Filter cartridge (0.2 jum, 3 ml) with a Captiva Filter cartridge (10 μm, 10 ml).

However, the novel multi-dimensional solid phase extraction method of this invention needs to be modified when preparing plasma samples for analysis of F ₂ -isoprostane using LC-MS / MS. Since F ₂ -isoprostan is bound to plasma phospholipids, to measure total F ₂ -isoprostane, It is necessary to dissociate the isoprostane - the isoprostanes free F ₂ - F ₂ in alkaline water solution. In the case of plasma, free radical scavengers such as ptylhydroxytoluene and cycloxygenase inhibitors such as indomethacin are used to prevent the automatic oxidation of arachidonic acid that may occur during sample separation, storage and processing. Should be added to EDTA-added plasma. It has been reported in the literature that [ ² H ₈ ] arachidonic acid was added to a blood clot sample to detect the production of 8-iso-PGF _{2 a} in vitro (Non-patent Document 20). The present inventors ^{have, [3 HI8-iso-PGF} 2 a of the retention by Oasis HLB cartridge, in plasma samples after alkali treatment with 15% KOH, compared to plasma sample not treated with alkali, it clearly lower Was observed. This means that when preparing clot samples to dissociate not only F ₂ -isoprostanes but also other fatty acids, solid phase cartridges with a larger retention capacity to hold F ₂ _isoprostane (for example, 60 mg or 200 mg Oasis HLB or Strata X solid phase cartridge) is considered necessary.

The F ₂ -isoprostane compound is an isomer of F ₂ -prostaglandin, and there are many stereoisomers (Non-patent Documents 1 and 7). Specific and exhaustive measurements of F ₂ —isoprostane isomers may be used for comprehensive assessment of oxidative stress in various pathologies. An HPLC-specific separation of these isomers is required to make a specific-exhaustive measurement of F ₂ -isoprostanes. Urinary 8-isoprostane (8-iso-PGF _{2 a} ) is one of F ₂ -isoprostanes and is currently measured as a marker of oxidative stress. In literature, 15 (R) 8-iso-PGF _{2 a} and 8-iso-PGF _{2 a} were baseline separated using a Hypersil BDS column (3 〃m, C18, d to 5 cm x 2.1 mm) (Non-patent document 1 8). The inventor has achieved better 15 (R) 8-iso-PGF _{2 a} and 8-iso-PGF _{2 a} using shorter ゝ Hypersil BDS columns (3 um, C18, 15 cm x 2.1 mm id) Separated. There are other reports that the stereoisomers of 15 (R) 8-iso-PGF _{2 α} and 8-iso-PGF _{2 a} can be separated on a C18 column. However, the present inventors have for the first time, 15 (R) 8-iso -PGF 2 and between _{8-iso-PGF 2 a "} , 8-iso-PGF in _{2 a} next new unknown 8-iso-PGF ₂ „We found that an isomer exists (see Fig. A). In other words, the 8-iso-PGF _2a isomer, which had the same retention time as 8-iso-PGF _2a, which could not be separated in previous reports, could be separated on a Hypersil BDS column (3 um C8). This is 8-iso-PGF ₂ which has been separated by the conventional analysis method. In the unknown 8-iso-PGF ₂ . This indicates that isomers were included. Therefore, in the HPLC analysis method of the present invention, the C8 column is considered to have higher selectivity than the C18 column.

The present inventor has buffer one additive F ₂ - results the finding that separation of iso prostanoic was not affected even by the addition of acetic acid to extract urine sample - isoproterenol induced vital to Ion suppression stun F ₂ An HPLC method that does not use a buffer for pH control was developed. LC-MS grade water should be used for LC-MS / MS analysis.

In the conventional 8-iso-PGF _2a separation method using HPLC, a buffer is added to an organic solvent to control the pH within a certain range. The present inventor has observed that the ionization efficiency of F ₂ -isoprostane clearly decreases when a buffer additive such as ammonia oxalate or formic acid is added by a post column. In addition, the ionization efficiency of F ₂ -isoprostane, an ammonia commonly used as a buffer additive, was examined, but stable results were not obtained. The cause of this is thought to be that ammonia reacts with carbon dioxide in the air to produce ammonia acetate. This means that ammonia must be freshly made to use it as a buffer additive. Further, acetic acid F ₂ - suppressing the ionization of Isopurosutan also, F ₂ - of isoprostanes separation Also had no effect. In other words, it was found that pH did not affect the separation of F ₂ -isoprostane stereoisomers. This indicates that it is not necessary to control the pH with a single buffer for the separation of F ₂ -isoprostane stereoisomers. Therefore, the present inventor decided to develop an HPLC separation method that does not use a buffer.

In this invention, instead of using a buffer, commercially available high purity (LC-MS grade) water is used. We also screened HPLC columns that do not tail peak under neutral conditions. When the F ₂ —isoprostan standard is analyzed under neutral conditions, the columns (3 01, 08 and 8) have a good separation of F ₂ —isoprostane and a good balance. It was found that it has characteristics such as good symmetry, low column back pressure, and good column reproducibility. Therefore, in the present invention, the column (C8) is less selective than the column (3 μηι, C18) for the separation of F ₂ —isoprostane during urine sample analysis. Was used as the analytical column.

From this analysis result, it is preferable to use, for example, a silica support as a packing material for liquid chromatography (HPLC) that can be used in the LC-MS or LC-MS / MS method of the present invention. That is, since the molecular weight of F ₂ -isoprostane is small, a silica carrier generally selected for low molecular weight analysis is preferred. Silica gel polymers are synthesized as fillers in various separation modes by introducing various functional groups into silica gel. The packing material used for reversed phase columns is often C6—C20, preferably C8—C18, more preferably C8 long chain alkyl group chemically bonded to silica gel. The C18 force ram is a filler in which a C18 alkyl group (octadecyl group consisting of 18 carbons) is bound with silica gel, that is, a filler synthesized by reacting octadecyl (ODS) silane with a silanol group on the silica gel surface. The column is filled with, and is also called Octadecyl (ODS) column. The C8 column is a column packed with a filler in which a C8 alkyl group (octyl group consisting of 8 carbons) is bonded by silica gel, that is, a filler synthesized by reacting octylsilane with a silanol group on the surface of the silica gel.

 Also, a reversed-phase column is required for the analysis of F2-isoprostane. In reverse phase chromatography, the stationary phase is nonpolar and the mobile phase is polar. As such a filler, in general, a type in which a functional group of an aliphatic hydrocarbon is bonded to a silica-based carrier is often used.

 Note that when F2-isoprostane is analyzed under neutral conditions without using a buffer, F2-isoprostane is considered to exist as an ion, which is advantageous for ionization, that is, sensitivity of analysis, but peak tailing Therefore, it is necessary that the silica filler be completely end-capped (inactivated active silanol groups) with high-purity silica.

 Furthermore, the packing material for liquid chromatography (HPLC) that can be used in the present invention has a spherical shape with a pore size of 10 nm (130 A) or less and a particle size of, for example, 4 im or less, preferably 3.5 μηι. It should be:

In the conventional LC-MS / MS separation method, acetonitrile is the main component and a mixed solvent with 5% methanol is used as the HPLC solvent. However, when the solvent ratio of acetonitrile and methanol is adjusted to 95: 5 as in the literature, even if a C8 column is used, it exists between 15 (R) 8-iso-PGF _{2 a} and 8-iso-PGF ₂ „. New unknown isomers could not be separated.

Therefore, when the solvent composition used in the liquid chromatography (HPLC) in the present invention was changed and the mixed ratio of the mixed solvent with acetonitrile or methanol was changed, a new unknown isomer was 8-iso-PGF. It was found that it can be separated from ₂ „. LC-MS / MS shows that specific measurement of 8-iso-PGF _{2 a} is not possible and uses a combination of selectivity of specific force and organic solvent composition. This shows that the novel HPLC method of the invention has enabled the specific analysis of 8-iso-PGF _{2 a} for the first time.

Therefore, the solvent composition that can be used in the present invention should be acetonitrile or a mixed solvent of acetonitrile and methanol having a specific mixing ratio. For 8-iso-PGF _2a and unknown isomers, The mixing ratio is, for example, 40:60 or less, preferably 50:50 or less. In this case, 8-iso-PGF _2a and the unknown isomer can be separated even with acetonitrile alone. The buffer-free HPLC method used in this invention makes it possible to minimize the maintenance of LC-MS / MS analytical instruments and reduce the time required for routine buffer preparation. This buffer-free HPLC method uses a three-solvent system and can perform analysis by simply supplementing water, methanol, and acetonitrile with occasional replenishment. In addition, according to this LC-MS / MS method, F ₂ —isoprostan standard is used for slight fluctuations in analysis temperature, mobile phase composition, gradient gradient, pH, sample injection volume, etc. The separation of (see Figure 1) had no significant effect. This indicates that the newly developed HPLC method is a solid F ₂ -isoprostane isomer separation method.

Further, in order to show that this novel HPLC separation method is firmly established, the present inventors have extracted and F ₂ one isoprostane standards on different HPLC instruments of the system volume (Waters Alliance 2796 with 2695 separation module) Urine samples were analyzed. The results showed that the same F ₂ -isoprostane separation can be achieved simply by adjusting the prevolume parameter and gradient gradient. This result supports the report that the separation of stereoisomers can be predicted by computer simulation using DryLab as a function of temperature and gradient (26 Non-Patent Literature). As a result, the inventor has developed an efficient and robust HPLC method for the first time using computer simulation software.

The novel multi-dimensional solid phase extraction (MD-SPE) and the puffer-free HPLC method according to the present invention can eliminate the trouble of the HPLC column and reduce the contamination of the ion source of the mass spectrometer. Even when the urine sample extracted by this multi-dimensional solid-phase extraction method was injected 400 times, the HPLC column back pressure was not clearly increased. In addition, there was no need to use a guard column and pre-filter as used in conventional HPLC analysis. Even after the extracted urine sample was injected more than 40 times into the HPLC column, the sample cone of the mass spectrometer detector had no visible contamination. For this reason, the method of the present invention not only greatly reduces the labor for maintenance of the mass spectrometer, but also stabilizes the detection sensitivity. In addition, this new MD-SPE-buffer-free HPLC method can extract iPF ₂ „-llll and iPF ₂ „ -VI internal standards added in urine with a stable recovery rate, and iPF _{2 The} matrix ion controls the ionization efficiency of _tt -lll and iPF _{2 a} -VI (see Table 1). The above description specifically and exemplarily describes the best mode for carrying out the present invention, and the present invention is not limited to the above description at all. It should be understood that all improvements and modifications that can be easily inferred are included in the scope of the present invention. Similarly, the following examples are set forth in order to explain specific embodiments of the present invention in more detail. Of course, the present invention is not limited to the following examples. Should be understood.

[Example 1] (Tandem mass spectrometry)

Detection of F ₂ —isoprostane was performed with a Quattro Premier tandem mass spectrometer controlled with MassLynx (version 4.1). Ionization was achieved by negative ion mode electrospray ionization (ESI). The position of the ESI probe and single mass detection (MS) and tandem mass detection (MS / MS) parameters are set so that a 200 ng / ml 8-iso-PGF _{2 a} standard solution infused with a microsyringe can achieve maximum sensitivity. Optimized for. Optimum tuning conditions for electrospray ionization are as follows: Cavity voltage, 3 kV; Ion S block temperature, 120 ° C; Extractor, 3.0 kV; High frequency lens, 0.1 V; Desolvation gas (nitrogen) Gas) Temperature, 400 ° C; Flow rate, 1200 l / h; Cone gas flow rate, 50 l / h; Ion energy, 1.0; Multiplier, 650 V; Low and high mass resolution, 13. MS / MS tuning inlet voltage, outlet voltage and collision gas (argon gas) flow rates were set to -2V, 2V and 0.35ml / min, respectively.

 Mass calibration, including static calibration, scan calibration, and scan speed calibration, was performed using the S calibration reference file for NA in positive ion ESI mode with MassLynx software.

MS tuning parameters are as follows: Capillary voltage, 3 kV; cone voltage, 40 V; Ion source block temperature, 80 ° C; Ex small lacta, 3.0 kV: High frequency lens, 0.1 V; Desolvation Gas (nitrogen gas) temperature: 150 ° C, flow rate: 350 l / h. Atmospheric pressure ionization calibration solution (NaCsl) (Waters Corp., USA) was introduced by syringe pump at a flow rate of 10 (1 / min).

2,3-dinor-8-iso-PGF _2a (2,3 "dinor-iPF _2a -lll), iPF _2a -llk iPF _2a -ll 卜 d4, iPF _2a -VI and iPF _{2 a} -VI-d11 The mass data acquisition parameters were determined by injecting a standard (1000 ng / ml) standard for each compound by flow injection (without HPLC column connected). The mass-to-charge ratio (m / z) of the molecular weight-related ions (m / z) was determined in the MS scan mode, and the sample cone voltage that maximizes the ion intensity of the deprotonated ion [MH] was determined in the selective reaction monitoring mode. Fragment ions generated from deprotonated molecules were detected by MS / MS tuning F2—Daughter ion (fragment ion) with the highest ionic strength of isoprostane and internal standard was the fragment ion (fragment ion) scan mode The collision energy that maximizes the precursor product ion intensity in the multi-reaction monitoring (MRM) mode was determined, the delay time between data acquisition channels was 0.01 seconds, the delay time between scans was 0.01 seconds, and the dwell time (detection) The time to acquire the ion mass data from the chamber was 0.1 s) Figure 2 shows the optimized cone voltage and collision energy for each compound.

The F ₂ -isoprostane isomer detected by tandem mass spectrometry is explained.

Oxidation of arachidonic acid produces four groups of F ₂ -isoprostane positional isomers. The four groups consist of Group III (15 Series), Group IV (8 Series), Group V (12 Series), and Group VI (5 Series). Group III (iPF _2a -lll) and Group VI (iPF _2a -VI) F ₂ - isoprostanes is the most abundant F ₂ - is a isoprostanes.

Figure 1 shows commercially available group III F ₂ —isoprostane (A: 8-iso-PGF _2a G: 8-iso-PGF _2i ; J: 8-iso-15 (R) PGF _2a ), group VI F ₂ — Prostaglandins (N: (Sat) 5-iPF _2a -VI), (B: PGF _2α ; C: 11i8-PGF _2a ; F: 5-trans-PGF _2a ; H: PGF ₂ I, 5-trans-PGF _2i ; K: 15 (R) PGF _2a ) and metabolites of 8-iso-PGF _2a and PGF _2a (M: 2, 3-dinor-8-iso-PGF _2a N: 2, 3-dinor-1ip- The chemical structure of PGF _2a ) is shown. Isotopes of internal standard products iPF _2a -lll, PGF _2a and iPF _2a -VI (D; 8-iso-PGF _{The structure of 2 a} -d ₄ ; E: PGF _{2 a} -d ₄ ; ± ±) 5-iPF _{2 a} -VI -d ") is also shown in FIG.

Figure 2 shows the spectrum of the product (product) ion of F ₂ —isoprostane. “The molecular weight related ions produced by monoisoprostane in negative ion electrospray (ES) mode are as follows: 8-iso-PGF _{2 a} (Fig. 2 A) and (Sat) 5-iPF _{2 a} -VI ( Fig. 2 D), m / z 353; 8-iso-PGF _{2 a} -d ₄ (Fig. 2 B), m / z 357; 2, 3-dinor-8-iso-PG ^ _a (Fig. 2 C), m / z 325; (Sat) 5-iPF _{2 a} -V 卜 d "(Fig. 2 E), m / z 364, 8-iso-PGF _{2 (I} and (Sat) 5-iPF _{2 a} -VI, Specifically, product ions of m / z 193 and m / z 115 are generated 8-iso-PGF _{2 a} -d ₄ (Fig. 2 B), 2, 3-dinor-8-iso-PGF _{2 a} The product ions for (Fig. 2 C) and (Sat) 5-iPF _{2 a} -V 卜 d (Fig. 2 E) were m / z 197, m / z 237 and m / z 115, respectively. The precursor / product ion pairs used to detect F ₂ —isoprostan in the mode are: 8-iso-PGF _{2 (l} , m / z 353/193; 8-iso-PGF _{2 a} -d ₄ M / z 357/197; 2,3-dinor-8-iso-PGF _{2 a} , m / z 325/237; iPF ₂ -V and m / z 353 / 115; iPF _{2 a} -VI -d ", m / z 364/115.

Since 8-iso-PGF ₂ „and PGF _2a show the same fragmentation pattern, they cannot be separated by tandem mass spectrometry. Therefore, 8-iso-PGF _{2 (r} is specifically measured. To achieve this, 8-iso-PGF _2a and its isomer PGF _2a must be separated by HPLC.

 [Example 2]

 (Sample pretreatment)

 The pH of the sample was adjusted to about 3 by adding 0.4 ml of 10% methanol, internal isotopes, and 1-3 ml of 1% formic acid to 2 ml of the urine sample. The sample was passed through a Captive filter cartridge (0.2 μηι) to remove solid particles. The sample thus treated was used for the next solid phase extraction. (Selective solid phase extraction method)

F ₂ —Isoprostan is a weakly acidic, weakly polar and lipophilic compound, so three polymer solid-phase adsorbents for F ₂ —Isoprostane in urine samples, HSL (hydrophilic-lipophilic balance) MAX (mixed -mode anion exchange) and MCX (mixed-mode cation exchange) retention capacity were screened. Figure 4 A, shows the retention volume curve in and added to the urine sample _{^{[3 HI8-iso-PGF 2 Oasis}} HLB of _alpha, MAX and MCX solid phase cartridge (3 cc 60 mg). As shown in the figure, the Oasis HLB solid phase cartridge stably held samples up to 10 ml, while the Oasis MAX and MCX solid phase cartridges were compatible with [ ³ H] 8-iso-PGF _{2 a.} Compared to the above, it showed weak holding power. This result showed that the Oasis HLB solid phase cartridge had the largest retention capacity for urinary F ₂ -isoprostane. Therefore, the Oasis HLB solid phase was used for the development of the next solid phase extraction method.

F ₂ urinary - to remove contaminating substances isoprostanes selectively extracted and acidic pH (2% formic acid), washing with a neutral pH (water) and alkaline pH (2. / ₀ ammonia) Under the conditions, the retention characteristics of [ ³ H] 8-iso-PGF _{2 a} when methanol was increased by 10% from 00 were investigated. As shown in Figure 4B, [ ³ HI8-iso-PGF _{2 a} was maintained in methanol wash up to 50% under neutral and acidic pH wash conditions, but under basic pH conditions, 20 When it was washed with methanol at a concentration of more than%, it was not retained. The result is [ ³ H] 8-iso-PGF ₂ . It shows that the elution characteristics with respect to methanol concentration differ between low pH and high pH conditions. Based on these results, a selective solid phase extraction washing method was incorporated into the next multidimensional solid phase extraction method.

In order to efficiently and selectively extract urinary F ₂ —isoprostane by solid phase extraction, [ ³ H] 8-iso-PGF _{2 a} is added to the pH-adjusted urine sample and 8-iso ^ PGF ₂ „and Extracted together Oasis solid phase cartridge (3∞ / 60 mg) was treated with 2 ml methanol and 2% formic acid to adjust the solid phase to facilitate sample retention. Solid phase extraction was performed using vacuum hold under vacuum. Different types of Oasis solid phase adsorbents

Comparison of the retention capacities of (HLB. MAX and MCX) was performed by adding the urine sample (1 ml to 10 ml each) to the solid phase cartridge and measuring the radiation dose of the liquid passed through. 200 μΙ of waste solution and eluate were mixed with 3 ml of scintillation solution (light emitted when radiation collides with fluorescent material), and the radiation dose was measured using a liquid scintillation counter.

This selective solid phase extraction washing method was developed by taking advantage of the fact that the retention characteristics of F ₂ -isoprostane and urine sample matrix differed when the pH of the contaminants and the concentration of the organic solvent were changed. Under washing conditions on the acidic side (29 formic acid), neutral side (water) and base side (2% ammonia), the concentration of methanol (10% —100Q6) was changed, the waste liquid was recovered and the radiation dose was recovered. Was used to determine the concentration of methanol used in the cleaning method to remove contaminants. The elution volume was determined by collecting 0.5 ml each of the eluate of jetyl ether acetic acid (100: 2) and measuring the radiation dose.

 [Example 3]

 (Multidimensional solid phase extraction method)

 Oasis HLB cartridge (1cc 30 mg) and Oasis HLB μ 曰 ution plate (750 μΙ / 2 mg) were used for the two-step multidimensional solid-phase extraction method.

Mix 2 ml of the urine sample after centrifugation and 1/5 of the amount of methanol, and mix the internal standard products 8-iso-PGF _{2 ff} -d4, PGF _{2 a} -d4, and iPF _{2 a} -V 卜 d11 The mixture was added and left on ice for 30 minutes before sample extraction. The pH of the sample was adjusted to about 3 with 1-2 ml of 1% formic acid, and the Oasis HLB cartridge was treated with 1 ml of methanol and 1 ml of 596 methanol / 2% formic acid before use. Next, the treated urine sample was added to a 0.2 m Captiva filter directly connected to the top of the Oasis HLB cartridge using a vacuum manifold at a flow rate of 3 ml / min or less. After sample addition, the filter cartridge was discarded and the solid phase extraction cartridge was washed with 1 ml of 5% methanol. The next extraction was performed using a pressurized manifold (Cerex system 48) with nitrogen gas.

 The eluate from the Oasis HLB solid-phase force was applied to the Oasis HLB μ Elution plate.

Solid phase extraction on Oasis HLB® plates was performed using a plate solid phase extraction manifold.

The eluate from the Oasis HLB® ution plate was diluted with water and directly used for LC-MS / MS analysis.

 A urine sample processing method for LC-MS / MS analysis among multidimensional solid-phase extraction methods is described with reference to FIG. 4C.

 First, an acidified urine sample containing 10% methanol is added to the HLB solid phase cartridge, and then washed with 5% methanol. In this sample addition and cartridge washing step 1, high molecular weight contaminants (eg, proteins) were removed by a size exclusion chromatography method using a small pore size (8 nm) HLB solid phase adsorbent. Polar contaminants such as salts and other carbohydrate compounds are removed because they are not retained by reversed-phase chromatography.

 In cartridge wash step 2 (base wash), acidic, medium polarity and hydrophobic contaminants are removed by washing with 2% ammonia containing 5% methanol. In this washing step, elution of yellow contaminants in the urine sample was observed.

In Cartridge Cleaning Step 3, the pH is switched to acidic pH by washing with 2% formic acid containing 5% methanol. Rabbit and hydrophobic contaminants are removed by washing with 2% formic acid containing 15% methanol.

 In the cartridge washing steps 5 and 6, neutral contaminants are removed by washing with a hexane jetyl ether (9: 1) mixture and a hexane / ethyl acetate (9: 1) mixture.

 In elution step A, the fatty acid fraction was selectively eluted with jetyl ether containing 2% acetic acid, but yellow contaminants remained on the solid phase cartridge.

Fraction A eluted from the HLB solid phase cartridge is diluted with hexane and added to the HLB // 曰 ution plate. In this step, F ₂ -isoprostane was retained on the SPE adsorbent by a normal phase chromatography mechanism. In plate washing step 1, acetic acid in the eluted sample was removed by washing with 5% methanol. In the plate washing step 2 (base side washing), the remaining slight amount of yellow contaminants was removed by washing with 2% ammonia containing 5% methanol. In plate washing steps 3 and 4, the pH was switched to acidic side with 2% formic acid containing 5% methanol and then formic acid was removed by washing with 5 <½ methanol. In plate washing step 5, neutral and hydrophobic Sexual contaminants were removed by washing with 15% methanol.

In elution fraction step B, the F ₂ -isoprostane fraction was washed with 80% methanol and eluted. This eluate was clear and diluted directly with water and used directly for LC-MS / MS analysis.

 [Example 4]

(Reversed-phase HPLC method for F ₂ -isoprostane stereoisomer separation)

As the first step in developing a new HPLC method, the ACE column (3 im, C8, 50 x 2.1 mm d.) Is used to separate the 8-iso-PGF _{2 a} isomers in the organic solvent composition of the mobile phase. The effect on the environment was examined. When the mobile phase is methanol, 8-ion-PGF _{2 a} is more efficiently ionized than methanol, so the mobile phase used for the F ₂ —isoprostane isomer separation is methanol. A mixed solvent in which acetonitrile was added was used. When the ratio of acetonitrile to methanol was increased, the separation of 15 (R) -iPF _{2 a} -lll, iPF _{2 a} -lll, 15 (R) PGF _{2 a} and PGF _{2 a} improved. Also, sufficient separation was obtained when the ratio of acetonitrile to methanol was 1: 2. However, under the same separation conditions, the ACE column (3 〃m, C18, 50 x 2.1 mm id) did not provide sufficient isomer separation. This indicates that the C8 column has better separation selectivity for all 8-iso-PGF _2a stereoisomers than the C18 column.

Ammonia is _a commonly used buffer additive for 8-iso-PGF _2a analysis by LC-MS / MS. In the present invention, by the addition of ammonium acetate 15 (R) iPF _{2 a} -ll and iPF _{2 a} -LLL, the 15 (R) PGF _{2 a} Oyo standard solution of beauty PGF _{2 a,} the ionization of these isomers The effect on efficiency was examined. Surprisingly, even at low concentrations of ammonia acetate, the ionic strength of the 8-iso-PGF ₂ „isomer was significantly reduced. Ammonium acetate reduced the retention time of the 8-iso-PGF _{2 a} isomer. However, it did not affect the separation of isomers.

The effect of pH on the separation of 8-iso-PGF _2a isomers was examined with standard solutions made with 0.0001%, 0.01% and 0.1% acetic acid. Acetic acid increased the retention time of the 8-iso-PGF _2a isomer, but did not affect the separation. Therefore, it was determined that the F ₂ -isoprostane isomer could be separated under neutral conditions without the need to control the pH with a buffer, and a buffer-free HPLC method, the so-called buffer-free HPLC method, was developed. .

8-iso-PGF ₂ . In order to understand the reverse phase separation characteristics of the isomers, the optimal temperature and gradient conditions for the 8-iso-PGF _2a isomer separation were examined using the computer simulation software DryLab 2000 Plus. Temperature (20 ° C and 40 ° C) and gradient slope (gradient time, 6 minutes and 1 The experiment was performed by computer simulation with 8 minutes, gradient range, 40-90% MeOH / ACN (2: 1)) changed at the same time. As a result, 8-iso-PGF ₂ under low separation temperature and shallow gradient conditions. It was found that the separation of isomers was good.

Further, in order to optimize the 8-iso-PGF _2a isomer peak shape and separation conditions under neutral conditions, using a number of C8 column different types, six 8-iso-PGF _2a Screening for separation ability against isomers (8-iso-15 (R) PGF _2a , 8-iso-PGF _2a , 11 (-PGF _2a , 15 (R) PGF _2a , 5-trans-PGF _2a , PGF _2a ) As a result, the HypersilBDSC8 column (3 μηι, 50x2.1 mm id) is 8-iso-PGF2c (good separation for isomers, good peak symmetry, and ACE C8 column) It was chosen as the analytical column because it had a stronger retention.

 Figure 3 shows the results of standard analysis of F 2 -isoprostane and F 2 -prostaglandin isomers by LC-MS / MS. As shown in Figure 3A, iPF2a-lll (8-iso-PGF 2 a) is 8-iso-15 (R) PGF2 a, 11 (-PGF2a, 15 (R) PGF2a, 5-trans-PGF2a, PGF2a As shown in Figure 3B, 8-iso-PGF 2a and PGF2 (have the same retention time as iPF2a-ll, and 5-trans-PGF 2α has 11 (same as -PGF2a Figure 3C—F shows another stereoisomer pair, i.e. iPF 2 al Il "d4 (8-iso-PGF 2 α Ι4) and PGF2a-d4, 2 , 3-dinor-8-iso-PGF 2a and 2, 3-dinor-PGF2a, iPF2a-VI and 5-epi-iPF 2 a-VU and iPF2a-V 卜 d11 and 5-epi-iPF2 a-VI-d11 As shown in Figure 3, the Hypersil BDS (3 μπη. C8) column not only separated the F 2 -isoprostane stereoisomer into the baseline, as shown in Figure 3. A peak with a sharp peak shape and good symmetry was obtained.

 [Example 5]

 Reversed phase HPLC was performed on a Waters Alliance 2796 and 2695 separation module connected to a mass spectrometer. Instrument control and data acquisition were performed using MassLynx (version 4.1) software. A gradient was created with a 2 pump Z4 solvent system. Solvent A used water, solvent B used methanol, and solvent C used acetonitrile. ACE (3 〃m, C8, 50x2.1 mm) ID and Hypersil BDS (3 〃m, C8, 50x2.1 mm) columns were used for HPLC separation and analysis of urine samples. Initial HPLC separation conditions were examined using the computer simulation software DryLab 2000 Plus. The column temperature was set to 24 ° C and the mobile phase flow rate was set to 0.2 ml / min. The gradient program is as follows: 0-6 min, 21% to 40.5% B / C (B: C = 2: 1 or 7: 3) linear (linear) gradient (curve 1), 6 -17 minutes, 40.5% to 43.5% B / C (B: C = 2: 1 or 7: 3) linear gradient (curve 6), 17-22 minutes, 43.5% to 100% B / C ( B: C = 2: 1 or 7: 3) linear one (gradient (curve 1), 22-27 minutes, 100. / c ^, 21% B / C (B: C = 2: 1 or 7: 3) Linear gradient (Curve 1).

 (Analysis of urine extraction sample by LC-MS / MS)

Figure 5 shows the results of urine sample analysis by multi-dimensional solid-phase extraction LC-MS / MS detected in MRM mode. As shown in Figure 5A, 8-iso-15 (R) PGF _2a and 8-iso-PGF _2a were baseline separated from PGF _2a and other unknown isomers. The peaks of 8-iso-15 (R) PGF _2a , 8-iso-PGF _2a , 15 (R) PGF _2α and PGF _2a in urine samples were identified by adding these compounds to the extracted sample. (Figure 5B). Figure 5C shows the MRM chromatogram of the stable isotopes of hydrogen H (deuterium) labeled 8-iso-PGF ₂ „and PGF _2a analyzed in the extracted urine sample. Figure 5D shows the 8-iso-PGF _{2a (8-iso- PGF 2a -d 4} ) and a blank sample with no added stable isotopes PGF _2a (PGF _2a -d ₄₎ (internal _{standard), 8-iso-PGF 2i} d 4 and PGF _2a - d Shows no contaminants at a retention time of _4. Figure 5 E shows 2,3-dinor-iPF _{2 a} -lll is baseline separated from other unknown isomers. Increasing the gradient time and using a longer column could also separate other isomers. . As shown in FIG. 5 F, iPF _{2 a} -VI and 5-epi-iPF ₂ "-VI were other isomers and baseline resolution FIG 5 G is, iPF _{2 a} extracted from the urine sample - VI-d "and S-epi-iPFwVI-c ^ are separated from each other. In the blank sample, there were no contaminants with the same retention time as iPF ^ -VI-d and S-epi-iPF ^ -VI-d.

 [Example 6]

Figure 7 shows the results of examining the resolution of 8-iso-PGF _2a and unknown isomers by changing the mixing ratio of methanol and acetonitrile. As is clear from the results shown in the figure, 8-iso-PGF _2a and the unknown isomers began to separate when the mixing ratio of methanol and acetonitrile was 40:60, and methanol and acetonitrile A clear separation was observed when the mixing ratio was 50:50. In addition, it was separated even if the acetonitrile was 100%.

 [Example 7]

To examine whether iPF _{2 a} -lll and iPF _{2 a} -VI recoveries are proportional to urine sample volume, the same amount of iPF ₂ was collected in 1 ml and 2 ml urine samples collected from 8 volunteers. _a -lll-d ₄ and iPF ^ -VI-d

(2 ng) was added and the sample was extracted by multidimensional solid phase extraction. As shown in Figure 6, the peak area ratio of iPF _{2 ff} -lll and iPF _{2 a} -IIUd ₄ extracted from 1 ml and 2 ml urine samples (Figure 6 and iPF _{2 a} -VI and iPF -VI-d " The iPF _{2 a} -lll and iPF _{2 a} -VI signal / noise (S / N) ratio ranges were 16-118 and 143-355, respectively. As shown in Figure 6, the S / N ratio of iPF _{2 a} -lll and iPF _{2 a} -VI extracted from a ₂ ml urine sample was found to be greater than when extracted from a 1 ml urine sample. These results indicate that a peak-to-peak S / N of 20 or more was obtained by extracting 2 ml of a urine sample from 8 volunteers selected arbitrarily. It was shown that sufficient analytical sensitivity was obtained from a 2 ml urine sample.

 [Example 8]

 (Evaluation of sample extraction recovery rate)

The recovery rate of urine samples by the two-step multidimensional solid-phase extraction method is determined by adding the internal standard mixture (8-iso-PGF _{2 a} -d ₄ and iPF ^ -VI-d) before and after sample extraction. evaluated. The recovery rate (%) was calculated by multiplying the ratio of the peak area of the internal standard added to the urine sample before extraction to the peak area of the internal standard added to the urine sample after extraction by 100. The solid phase extraction recovery rate was calculated as follows by the following data analysis.

Before and after extraction, urine samples (1 ml and 2 ml) were added with internal standard products 8-iso-PGF _{2 a} -d ₄ and iPF _{2 a} -VI-d "to examine the recovery rate of sample extraction ( Table 1) As shown in Table 1, the recovery rate of the internal standard added from 1 ml and 2 ml samples of 4 volunteers was about 50-70% in 2 ml urine samples from 4 people. Four-time repeated extraction (extraction 1 "4) gave a stable recovery. The recovery rates of iPF _{2 a} -lll-d ₄ , iPF ₂ „-VI-d” and S-epi-iPF ^ -VI-d were 62 soil 8%, 60 soil 10% and 61 soil 8%, respectively. .

【table 1】 TABLE 1. Recovery in solid-phase extraction of tiiine samples from four volunteers

 Extraction 1 Extra don 2 Extraction 3 Extraction 4

1 ml of Urine 2 mt of Unnc 2 Urine 2 ml of Urine 2 ml of Urine Average

 Isopnwtane (n = 4) in = 4) ("= 4) (n-4) (n = 4) Rctxwcry of Variation

63 ± 10 72 13 57 68 ± 7 51 ± 16 62 ± 8 Ϊ3 iPF-\ T ^ _U 54 ± 4 62 ± 8 60 ± 12 68 ± 3 S6 ± 15 60 ± 6 10 δ-Epi-iPF ^ -VI- du 53 ± 8 58 ± 9 62 ± 8 74 ± 5 57 ± 9 61 ± 8 13 iP, prostane.All values shown are percentages.

(Evaluation of ion suppression by matrix)

 0 0

Ion ot down inhibition sample matrices, standard (23-dinor-8-iso -PGF 2a 8-iso- PGF 2 a and (Sat) 5-iPF _2a -VI) and an internal standard (8-an iso- PGF _2tr l ₄ and (soil) S-iPF ^ -VI-d ") were investigated by adding the solution to the extracted urine sample with water. Calculated by multiplying the ratio of the peak area of the internal standard to the peak area of the standard or internal standard added to water by 100.

Urine samples matrix F ₂ - effect on the ionization efficiency of isoprostanes, F ₂ in urine samples extracted with water - have been conducted under consider adding standard solution and internal standard solution isoprostanes. As shown in Table 2, when extracted from urine samples (1 ml and 2 ml) or repeatedly extracted from urine samples (2 ml), 2,3-dinor-iPF _2a -lll is approximately 12 -23% ion suppression (Matrix action, 77-88%) o Matrix effects on other F ₂ -isoprostanes were 88% to 127%, 15 (R) iPF _2a -lll iPF _2a -lll iPF _2a -lll-d ₄ .iPF _2a -V and 5-epi-iPF _2a -V and iPF _2a -VI-d ₁₁ and S-epi-iPFwVI-d "were each subjected to a matrix action of 95 soil 6% 100 ± 6% 103 ± 5% 96 ± 3% 100 ± 3% 109 ± 15% and 104 ± 16% These results show that the ion suppression effect of the sample matrix extracted by multidimensional solid-phase extraction is F _This shows that there was no significant effect on the ionization efficiency of 2_isoprostane.

 [Table 2]

TABLE 2. Matrix-related ion suppression effects

 Extraction Extraction 2 Exiractimi 3 tx traction 4

 1 ml of Urine 2 ml of Urine 2 inl of Urine 2 ml of Urine 2 ml of Urine Av ragi- Matrix t-oeificicnt of

 = 4 in = 4i ίπ = 41 Effit Variation

2, S-DinoHPF _2u -in 85 ± 11 77 80 ± 8 82 ± 10 88 ± 13 82 ± 4

 I3 (R) iPF '-m 100 ± 12 87 ± 3 91 ± 3 10] ± 5 95 ± 6

_{iPF 2ft -in 106 ± 15 101 91} ± 6 96 ± 30 106 ± 6 100 ± 6

iPF _2a -III-d ₄ IOI 10 97 702 ± 2 304 ± 8 M0i 4 s ±

 98 ή 97 ± 6 100 U 98: 5 96 ± 3 4

5-Epi-iPF _2a -VI 102 ± 4

101 ± 6 104 9 98 3 100 ± 3 3 iPF 2a -! \ Ld u 108 is 127 ± 4 103 119 ± 24 88 ± 25 109 i 15 14

 91 ± 9 7 1 J2 9 126 ± 5 103 ± 2] 04 ± 16 13

All values shown are percentages

Data analysis was performed as follows. The signal-to-peak signal-to-peak ratio (S / N ratio) of iPF _2a -lll and 5-iPF _2a -VI in the mask mouth matogram was obtained after smoothing the peak using the Moving Mean method with MassLynx (version 4.1) software. Significant differences in the S / N ratio between iPF _2a -lll and 5-iPF _2a -VI in urine samples (1 ml and 2 ml) were tested by the Wilcoxon signed test using the SAS software package. Multiple reaction monitoring (MRM) Automatic peak identification and area calculation in chromatograms were performed with QuanLynx software. The calculated area of the analysis sample was exported as a text file, combined into one file using a self-made Excel macro, and the recovery rate and matrix effect were calculated using a SAS software package. F ₂ according to the availability to the invention on the industrial - separation method for measuring isoprostane isomers, F is isolated by oxidative stress marker one ₂ - and Isopurosutan isomer with high accuracy to measure the separation at high efficiency Therefore, F ₂ -isoprostane isomers caused by oxidative stress can be identified, so that the pathological condition and the cause of the disease can be accurately diagnosed. Therefore, the method for separating and measuring F ₂ -isoprostane isomers according to the present invention can be effectively applied particularly to the field of disease diagnosis.

Claims

The scope of the claims

1 Chromatographic analysis of F ₂ -isoprostane isomers with a specific particle size capable of separating F ₂ -isoprostane isomers and using an eluate consisting of an aqueous solution of methanol acetonitrile with a specific concentration ratio. _{In the method} for separating and measuring the F ₂ —isoprostan isomer, which consists of separating and measuring the ₂ —isoprostane isomer, the specific particle size that can be separated by the F ₂ —isoprostan isomer is 4 / m or less. use carrier F ₂ - without a holding ability isoprostane isomers, and methanol / Asetonitoriru solution of a particular concentration ratio of 1 _0: 9 0-1 0 0: 0 consists a F ₂ - isoproterenol Separation measurement method of stan isomers.

2 Separation of the F ₂ -isoprostane isomer according to claim 1; In the II determination method, the specific particle size capable of separating the F ₂ -isoprostane isomer is 3.5 μm or less A method for separating and measuring F ₂ monoisoprostane isomers.

3 In the method for separating and measuring the F ₂ -isoprostane isomer according to claim 1 or 2, the chromatographic carrier force << C6-C20 long chain alkyl group is chemically bonded to silica gel. A method for separating and measuring F ₂ -isoprostane isomers comprising a silica support.

4 F ₂ ranges described in the third preceding claims - isoprostane in isomer separation "measuring method, the Can chromatography one carrier is a silica support C8 alkyl group is chemically bonded to silica gel Ranaru F ₂ - Separation measurement method of isoprostane isomers.

5 F as claimed in any one Claims first term to the fourth term ₂ - in the separation method of measuring isoprostane isomers, specific澹度ratio of the methanol / Asetonitoriru aqueous solution 4 _0: 6 0-1 A method for separating and measuring F _z —isoprostane isomers consisting of 0 0: 0.

6. The method for separating and measuring F ₂ -isoprostane isomer according to any one of claims 1 to 5, wherein the specific concentration ratio of the methanolic acetonitrile aqueous solution is 50:50 to 1 0 0: Method for measuring the fraction of F ₂ -isoprostane isomers consisting of 0.

7 When a sample such as a biological sample is passed through a first solid phase extraction carrier having a large solid phase capacity, the F ₂ -isoprostane compound and its isotope contained in the sample are retained on the first solid phase extraction carrier. A first solid-phase extraction step comprising removing contaminants contained in the sample;

The sample that has passed through the first solid-phase extraction carrier passes through the second solid-phase extraction carrier having a solid volume smaller than the solid-phase capacity of the first solid-phase extraction carrier, and further passes through the F ₂ -isoprostan compound and its isotope. A second solid phase extraction step comprising holding a body on the second solid phase extraction carrier, and removing contaminants contained in the sample;

A sample processing method comprising:

8. The sample processing method according to claim 7, wherein the first solid-phase extraction step is performed by size-exclusion chromatography and reverse-phase chromatography, and the second solid-phase extraction step is sized. Exclusion chromatography and normal phase chromatography

^ Replacement form (Rule 26) Sample processing method.

9 When a sample such as a biological sample is passed through a first solid phase extraction carrier having a large solid phase capacity, the F ₂ -isoprostane compound and its isotope contained in the sample are retained on the first solid phase extraction carrier. In addition, a first solid phase extraction step consisting of removing contaminants contained in the sample, and a sample that has passed through the first solid phase extraction carrier are compared with the solid phase capacity of the first solid phase extraction carrier. In addition, the F ₂ -isoprostane compound and its isotope are retained on the second solid-phase extraction support through the second solid-phase extraction support with a small solid-phase capacity, and contaminants contained in the sample are removed. A second solid phase extraction step comprising:

A sample processing method comprising:

The eluate obtained by the above-mentioned sample processing method is added to a carrier for a chromatography matrix having a specific particle size capable of separating F ₂ -isoprostan isomer, and methanol Z having a specific concentration ratio is added. with an elution solution comprising § Se Tonitoriru solution, F ₂ by chromatography one - in the separation渊定method isoprostane isomer, F ₂ - - Isopurosuta consists of a down isomer separating measure F ₂ isoprostane isomers The specific particle size that can be separated is 4 // m or less, the chromatographic support is not capable of retaining the F ₂ -isoprostane isomer, and a methanol / acetonitrile aqueous solution with a specific concentration ratio is 10 : 9 0 to 1 0 0: Method for separating and determining F ₂ — ^ T soprostan isomers consisting of 0.

Replacement invitation (summary y26)