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WO2008065895A1 - Procédé de séparation/mesure d'un isomère de f2-isoprostane - Google Patents

Procédé de séparation/mesure d'un isomère de f2-isoprostane Download PDF

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Publication number
WO2008065895A1
WO2008065895A1 PCT/JP2007/072146 JP2007072146W WO2008065895A1 WO 2008065895 A1 WO2008065895 A1 WO 2008065895A1 JP 2007072146 W JP2007072146 W JP 2007072146W WO 2008065895 A1 WO2008065895 A1 WO 2008065895A1
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Prior art keywords
isoprostane
phase extraction
sample
solid phase
solid
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PCT/JP2007/072146
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English (en)
Japanese (ja)
Inventor
Zhang Bo
Keijiro Saku
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Zhang Bo
Keijiro Saku
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Publication of WO2008065895A1 publication Critical patent/WO2008065895A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N2030/009—Extraction

Definitions

  • 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.
  • Non-patent Document 1 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.
  • 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.
  • oxidative stress or oxidative stress a state in which the balance is lost due to various factors such as drugs, radiation, and ischemia, and the tendency to oxidize.
  • 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.
  • ROS reactive oxygen species
  • 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.
  • 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.
  • 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).
  • PG prostaglandin
  • 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).
  • 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).
  • comprehensive and specific measurements of the F 2 -isoprostane isomers are required for comprehensive assessment of oxidative stress.
  • 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.
  • 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.
  • 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). ).
  • GC / MS Gas Chromatography / Mass Spectrometry
  • F 2 —isoprostane see, eg, Non-Patent Documents 10 0, 11 and 12.
  • 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.
  • ELISA enzyme immunoassay
  • 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.
  • 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.
  • 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.
  • the measurement substance 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).
  • Urinary F 2 — isoprostane compounds are peroxidation products of arachidonic acid through reactive oxygen species and are reliable markers of oxidative stress.
  • isomers in urinary F 2 -isoprostane compounds 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.
  • this method since ion suppression by the sample matrix is not controlled, stable analysis cannot be performed at present.
  • 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 Document 4 Pratico, D., et al., ⁇ Biol. Chem. 1996, 271: 14916-14924
  • Non-Patent Document 6 Basu, S., FEBS Lett. 1998, 428: 32-36
  • Non-Patent Document 7 Rokach, Tsuji, et al., Chem. Phys. Lipids, 2004, 128: 35-56
  • Non-patent literature 8 Schwedhelm, ⁇ ⁇ , et al., J. Chromatogr. B Biomed. Sc, App, 2000, 744: 99-112
  • Non-Patent Document 9 luliano, L. et al., ⁇ Am. CoH. Cardiol. 2001, 37: 76-80
  • Non-Patent Document 1 1 Reilly, MP "et al., Circulation. 1997, 96: 3314-3320
  • Non-Patent Document 1 2 Mori, T.A., et al., Anal. Biochem. 1999, 268: 117-125
  • Non-Patent Literature 1 3 Pratico, D., et al., Nat. Med. 1998, 4: 1189-1192
  • Non-Patent Document 1 Mallet, C.R., et al., Rapid. Commun. Mass Spectrom. 18: 49-58
  • 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
  • Non-Patent Document 2 0 luliano, L, et al., ⁇ Am. Coll. Cardio 2001, 37: 76-80
  • Non-Patent Document 2 1 Snyder, L.R., et al., ⁇ Chromatogr. A. 2000. 892: 107-121
  • 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.
  • MD-SPE multi-dimensional solid-phase extraction
  • 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.
  • the present invention provides a method for separating and measuring stereoisomers and positional isomers of F 2 -isoprostane compounds.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the first solid phase extraction step is performed by size exclusion chromatography and reverse phase chromatography
  • the second solid phase extraction step is performed by size exclusion chromatography and normal phase chromatography.
  • 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.
  • 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
  • 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
  • the specific particle size that can be separated is 4 ⁇ m or less, and the chromatographic carrier does not have the ability to retain F 2 —isoprostane isomer.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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
  • 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 .
  • Oasis HLB hydrophilic monolipophilic balance
  • MAX reverse phase and anion exchange
  • MCX reverse phase and cation exchange
  • 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.
  • AA means arachidonic acid
  • 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.
  • A is the iPF 2e -lll isomer in the urine sample
  • B is the extracted urine sample
  • 8 o-15 (R) PGF 2a iPF 2tician-ll
  • IPF 2a -lll is the ion channel (m / z 353 ⁇ 193) when 2a is added
  • C is the ion of the internal standard iPF 2 a -lll-d4 and PGF 2a -d4 added to the urine sample.
  • 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
  • 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,
  • 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.
  • 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.
  • a novel multidimensional (MD) solid-phase extraction (SPE) method was developed in the present invention.
  • 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.
  • 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.
  • the sample 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.
  • 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.
  • acids such as formic acid
  • 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.
  • 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).
  • 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.
  • F 2 -Isopro In addition to removing water remaining on the carrier, F 2 -Isopro It can remove impurities that are less polar than stun.
  • 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.
  • the eluate treated in this way is processed 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 ⁇ 1 ⁇ 2 methanol wash and remove acid.
  • 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.
  • F 2 -isoprostane can be eluted with a small solvent volume, eg 40-50 ⁇ . 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.
  • HLB hydrophilic-lipophilic balance
  • 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.
  • the first solid phase extraction step can be concentrated 1 to 10 times.
  • a solvent with a strong dissolution power such as methanol or acetonitrile
  • the elution volume is small (for example, 0.5 ml)
  • 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.
  • 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.
  • 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.
  • the eluate obtained in the first solid phase extraction step cannot be used in the second solid phase extraction step as it is.
  • 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.
  • 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.
  • 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.
  • 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.
  • the final methanol concentration of the eluted sample must be 20% or less.
  • a carrier with a small solid volume such as an HLB Elution plate
  • 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.
  • the concentration factor when using the Elution plate in the second solid phase extraction step is 25 (from 1000 ⁇ / 50 ⁇ to 250 times.
  • the 40 ⁇ 40 eluate obtained in the first step is Even when diluted with ⁇ , 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.
  • 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.
  • 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.
  • F 2 -isoprostane can be retained using normal phase chromatography mechanisms using a microelution SPE plate.
  • 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.
  • 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.
  • 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.
  • the first solid phase extraction step uses a 1cc / 30 mg solid phase extraction cartridge
  • the second solid phase extraction step uses a small amount of solid phase extraction support (2 mg) ⁇ ⁇ ⁇ plate
  • the retention capacity ratio of 1cc / HLB solid phase extraction cartridge 30 mg and 2_ ⁇ ⁇ ⁇ ution plate 2 mg is 15: 1.
  • a 1 cc / 30 mg HLB solid phase extraction cartridge can hold 2 to 3 ml of urine sample.
  • HLB solid-phase extraction supports are characterized by the ability to retain both hydrophilic and lipophilic compounds. In other words, selectivity is low.
  • 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.
  • sample extraction and solvent exchange for LC-MS / MS analysis can be simultaneously performed in the second solid phase extraction step.
  • sample concentration can be performed at the same time by using a small volume solid phase.
  • 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.
  • the measurement substance 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.
  • the sample elution volume is small (eg, 40 jul)
  • it is possible to easily concentrate a urine sample eg, 2 ml
  • the eluted sample can be used for LC-MS / MS measurement simply by diluting with water.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the novel multidimensional solid phase extraction (MD-SPE) method has significant advantages.
  • 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.
  • extraction of 8 samples is usually possible within 3 hours.
  • this processing method is solid, it is possible to further increase the sample processing efficiency by automatic processing. Conceivable.
  • 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 ⁇ m, 10 ml).
  • 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.
  • 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.
  • Non-patent Document 20 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ODS octadecyl
  • 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.
  • a C8 alkyl group octyl group consisting of 8 carbons
  • a reversed-phase column is required for the analysis of F2-isoprostane.
  • the stationary phase is nonpolar and the mobile phase is polar.
  • 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.
  • F2-isoprostane 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.
  • 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 ⁇ . It should be:
  • 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.
  • the mixing ratio is, for example, 40:60 or less, preferably 50:50 or less.
  • 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.
  • 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.
  • 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.
  • this new MD-SPE-buffer-free HPLC method can extract iPF 2 till-llll and iPF 2 till -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).
  • Detection of F 2 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).
  • 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.
  • 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 2 ⁇ ; 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 ⁇ d "(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-is
  • 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.
  • 8-iso-PGF 2 Since 8-iso-PGF 2 complicatand 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.
  • 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 ⁇ ) to remove solid particles. The sample thus treated was used for the next solid phase extraction. (Selective solid phase extraction method)
  • F 2 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).
  • 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.
  • 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 ⁇ 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.
  • Oasis HLB cartridge (1cc 30 mg) and Oasis HLB ⁇ ⁇ ution plate (750 ⁇ / 2 mg) were used for the two-step multidimensional solid-phase extraction method.
  • 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.
  • 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 ⁇ Elution plate.
  • 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.
  • an acidified urine sample containing 10% methanol is added to the HLB solid phase cartridge, and then washed with 5% methanol.
  • high molecular weight contaminants eg, proteins
  • 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.
  • cartridge wash step 2 base wash
  • acidic, medium polarity and hydrophobic contaminants are removed by washing with 2% ammonia containing 5% methanol.
  • this washing step elution of yellow contaminants in the urine sample was observed.
  • 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.
  • 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 // ⁇ ution plate.
  • F 2 -isoprostane was retained on the SPE adsorbent by a normal phase chromatography mechanism.
  • plate washing step 1 acetic acid in the eluted sample was removed by washing with 5% methanol.
  • 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.
  • 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 ⁇ 1 ⁇ 2 methanol.
  • plate washing step 5 neutral and hydrophobic Sexual contaminants were removed by washing with 15% methanol.
  • 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.
  • 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.
  • 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.
  • Ammonia is a commonly used buffer additive for 8-iso-PGF 2a analysis by LC-MS / MS.
  • the 15 (R) PGF 2 a Oyo standard solution of beauty PGF 2 a 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 paragraphisomer 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.
  • 8-iso-PGF 2 8-iso-PGF 2 .
  • 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.
  • FIG 3 shows the results of standard analysis of F 2 -isoprostane and F 2 -prostaglandin isomers by LC-MS / MS.
  • iPF2a-lll 8-iso-PGF 2 a
  • iPF2a-lll 8-iso-PGF 2 a
  • R PGF2 a
  • 11 -PGF2a
  • 15 R
  • PGF2a As shown in Figure 3B, 8-iso-PGF 2a and PGF2 (have the same retention time as iPF2a-ll, and 5-trans-PGF 2 ⁇ has 11 (same as -PGF2a
  • the Hypersil BDS (3 ⁇ . 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.
  • 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.
  • Figure 5 shows the results of urine sample analysis by multi-dimensional solid-phase extraction LC-MS / MS detected in MRM mode.
  • 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 2 ⁇ and PGF 2a in urine samples were identified by adding these compounds to the extracted sample.
  • Figure 5B shows the MRM chromatogram of the stable isotopes of hydrogen H (deuterium) labeled 8-iso-PGF 2 hurryand 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.
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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
  • 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.

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Abstract

L'invention concerne un procédé de séparation/mesure d'un isomère de F2-isoprostane, qui comprend les étapes consistant à conduire une procédure de traitement d'échantillon comprenant une première étape d'extraction de phase solide, pour faire passer un échantillon contenant l'isomère de F2-isoprostane à travers un premier support d'extraction de phase solide ayant un volume de phase solide plus important pour éliminer les contaminants contenus dans l'échantillon, et une seconde étape d'extraction de phase solide pour faire passer un échantillon élué du premier support d'extraction de phase solide à travers un second support d'extraction de phase solide ayant un volume de phase solide inférieur à celui du premier support d'extraction de phase solide pour encore éliminer les contaminants de l'échantillon contenant l'isomère de F2-isoprostane ; appliquer l'éluat obtenu à l'étape précédente à un support de chromatographie ayant un diamètre de particule spécifique approprié pour la séparation de l'isomère de F2-isoprostane et séparer l'isomère de F2-isoprostane du support par chromatographie à l'aide d'un éluant comprenant une solution aqueuse de méthanol/acétonitrile ayant un rapport de concentration spécifique pour mesurer l'isomère de F2-isoprostane. Dans le procédé, le diamètre de particule spécifique approprié pour la séparation de l'isomère de F2-isoprostane est de 4 μm ou moins, et la solution aqueuse de méthanol/acétonitrile a un rapport de concentration de 10:90 à 100:0.
PCT/JP2007/072146 2006-11-08 2007-11-08 Procédé de séparation/mesure d'un isomère de f2-isoprostane WO2008065895A1 (fr)

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EP2492679A1 (fr) 2011-02-28 2012-08-29 Tanita Corporation Procédé pour déterminer quantitativement le 8-isoprostane
EP2492680A1 (fr) 2011-02-28 2012-08-29 Tanita Corporation Procédé de purification du 8-isoprostane
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EP2492679A1 (fr) 2011-02-28 2012-08-29 Tanita Corporation Procédé pour déterminer quantitativement le 8-isoprostane
EP2492680A1 (fr) 2011-02-28 2012-08-29 Tanita Corporation Procédé de purification du 8-isoprostane
JP2012194177A (ja) * 2011-02-28 2012-10-11 Tanita Corp ８−イソプラスタンを精製する方法
JP2012194178A (ja) * 2011-02-28 2012-10-11 Tanita Corp ８−イソプラスタンの定量方法
US8652850B2 (en) 2011-02-28 2014-02-18 Tanita Corporation Method of quantitatively determining 8-isoprostane
US8692047B2 (en) 2011-02-28 2014-04-08 Tanita Corporation Method of purifying 8-isoprostane
JP2014228315A (ja) * 2013-05-20 2014-12-08 花王株式会社 多成分試料の質量分析方法
CN114216951A (zh) * 2021-12-08 2022-03-22 中国石油大学(北京) 污水中磺酸盐类化合物的分离方法和溶解性有机质分子组成分析方法

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