WO2005116629A1 - アミノ官能性化合物の分析方法及び装置 - Google Patents
アミノ官能性化合物の分析方法及び装置 Download PDFInfo
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- WO2005116629A1 WO2005116629A1 PCT/JP2005/009618 JP2005009618W WO2005116629A1 WO 2005116629 A1 WO2005116629 A1 WO 2005116629A1 JP 2005009618 W JP2005009618 W JP 2005009618W WO 2005116629 A1 WO2005116629 A1 WO 2005116629A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
- G01N33/6809—Determination of free amino acids involving fluorescent derivatizing reagents reacting non-specifically with all amino acids
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8818—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving amino acids
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
- Y10T436/145555—Hetero-N
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/24—Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry
Definitions
- the present invention provides a method for analyzing amino-functional compounds such as amino acids, peptides, and amino acid analogs by liquid chromatography Z mass spectrometry (LCZMS) quickly, simply, and with high sensitivity, and an apparatus therefor. Etc.
- LCZMS liquid chromatography Z mass spectrometry
- Amino acids in a living body are useful for identifying diseases such as amino acid metabolism disorders. For example, it is used for diagnosing congenital amino acid metabolism disorders, determining the severity of liver dysfunction and indices for treatment, and ascertaining the condition of patients with poor nutrition.
- diseases such as amino acid metabolism disorders.
- it is used for diagnosing congenital amino acid metabolism disorders, determining the severity of liver dysfunction and indices for treatment, and ascertaining the condition of patients with poor nutrition.
- palin, leucine, and isoleucine are called branched-chain amino acids (BCAAs), and their synthesis is reduced in severe liver diseases such as fulminant hepatitis and liver failure.
- phenylalanine which is an aromatic amino acid (AAA)
- AAA aromatic amino acid
- the ratio of BCAAZAAA which is the ratio of the two, is low, and the degree reflects the severity of liver disease.
- the BCAAZAAA ratio is generally called the Fischer ratio (Fischer ratio), and has been conventionally used for determining the severity of liver disease (for example, see Non-Patent Document 1). It is known that in diabetics, pheninolealanine, tyrosine, isoleucine, leucine, and parin increase and alanine decreases (for example, see Non-Patent Document 2).
- an amino acid analyzer based on post-column derivatization is often used.
- ninhydrin color development is usually performed after amino acids are separated by a cation exchange column, but the disadvantage is that the analysis time is long.
- the analysis time of the standard analysis method for about 20 types of amino acids of protein hydrolyzate has been reduced to about 20 minutes by devising the separation column and the flow rate of the buffer solution. 1).
- an amino acid analysis method based on a precolumn derivatization reaction is also being actively studied.
- amino acid analogs in living organisms that are not only protein constituent amino acids.
- Typical examples are taurine, O-phosphoethanolamine, hydroxyproline, methionine sulfoxide, sarcosine, a -aminoadipic acid, citrulline, a-amino-n-butyric acid, pipecolic acid, homocystin, homocitrulline, and aroisoleucine.
- amino-functional conjugates amino acids and amino acid analogs (collectively called amino-functional conjugates) by a cation exchange column and performs ninhydrin coloring
- the analysis time of the 41 components of these amino-functional compounds is at least 60 minutes, and that of the 53 components is 148 minutes.
- Such a long analysis time is one of the major factors hindering the study of the physiological function of amino acids. Even if new findings linking amino acids to health and disease are discovered, such information cannot be used effectively with such processing capabilities.
- Patent Document 1 JP-A-2002-243715
- Patent Document 2 International Publication No. 03Z069328 pamphlet
- Patent Document 3 JP 2002-71660 A
- Non-Patent Document 1 Fischer JE and five others, "Surgery" July 1976, Vol. 80, p. 77-91
- Non-Patent Document 2 Furigsch (Felig P) and three others, ⁇ Diabetes '', 1970, October, Vol. 19, p. 727-728
- the present invention provides various kinds of amino acids and their derivatives contained in living bodies, and amino acids
- An object of the present invention is to provide a method and an apparatus for analyzing an amino-functional conjugate containing a similar substance extremely quickly, conveniently and with high sensitivity as compared with the conventional method.
- the present inventors have made various improvements and optimizations of a method for analyzing an amino-functional compound by LCZMS. That is, a plurality of amino-functional conjugates, which are difficult to separate and detect due to the same mass in mass spectrometry, are separated and eluted in advance by liquid chromatography, and subsequently, analyzed by mass spectrometry.
- the present inventors have found that various kinds of amino-functional conjugates can be analyzed very quickly, easily and with high sensitivity by separation and detection, thereby completing the present invention.
- the present invention provides a method for reacting an amino-functional conjugate in a sample containing the amino-functional conjugate with a derivatizing reagent to provide an amino-functional compound represented by the following general formula (I):
- a step of generating a compound derivative a step of eluting the amino-functional compound derivative by liquid chromatography using a stepwise concentration gradient elution means, and a step of eluted with the amino-functional compound derivative eluted by the liquid chromatography
- a method for analyzing amino-functional conjugates which comprises the step of detecting the amino-functional compounds by mass spectrometry.
- Ar represents a hydrocarbon which may have a substituent, or a substituent containing an aromatic carbon ring or a heterocyclic ring
- R represents a hydrogen atom or an alkyl which may have a substituent.
- a saturated or unsaturated alkyl group which may have a substituent or
- an amino-functional compound in a sample is reacted with a derivative to produce an amino-functional compound derivative represented by the above general formula (I).
- the method of the present invention about 40 types of typical biological amino-functional conjugates can be analyzed in a short time, for example, within 10 minutes. The analysis time can be significantly reduced. In the past, simultaneous analysis of amino acids with the same mass, such as leucine and isoleucine, was difficult by mass spectrometry alone. According to the method of the present invention, more than 100 amino functional groups containing these biological amino acids It is possible to analyze the conjugated product simultaneously and in a very short time.
- FIG. 1 shows a typical reaction formula for producing an amino-functional compound derivative in the method of the present invention.
- FIG. 2 shows a typical example of a stepwise concentration gradient elution pattern of liquid chromatography in the method of the present invention.
- FIG. 3 is a configuration diagram of an analyzer for an amino-functional conjugate according to one embodiment of the present invention.
- FIG. 4 (a) is a chromatogram showing the results of analyzing 40 kinds of amino-functional conjugates in Example 2 of Analysis: Analysis Example 1.
- FIG. 4 (b) is a chromatogram showing the results of analyzing 40 kinds of amino-functional conjugates in Example 2 of Analysis: Analysis Example 1.
- FIG. 4 (c) is a chromatogram showing the results of analyzing 40 kinds of amino-functional conjugates in Example 2: Analysis Example 1.
- FIG. 5 (a) is a chromatogram showing the results of analyzing 40 kinds of amino-functional conjugates in Example 3: Analysis Example 2.
- FIG. 5 (b) is a chromatogram showing the results of analyzing 40 kinds of amino-functional conjugates in Example 3: Analysis Example 2.
- Example 3 In Example 2, 40 kinds of amino-functional conjugates were analyzed. It is a chromatogram showing a result.
- Example 4 is a chromatogram showing the results of analyzing 39 kinds of amino-functional conjugates in Analysis Example 3.
- Example 4 is a chromatogram showing the results of analyzing 39 kinds of amino-functional conjugates in Analysis Example 3.
- Example 4 is a chromatogram showing the results of analyzing 39 kinds of amino-functional conjugates in Analysis Example 3.
- Example 5 A chromatogram showing the results of analyzing 39 kinds of amino-functional conjugates in Analysis Example 4.
- Example 5 A chromatogram showing the results of analyzing 39 kinds of amino-functional conjugates in Analysis Example 4.
- Example 5 A chromatogram showing the results of analyzing 39 kinds of amino-functional conjugates in Analysis Example 4.
- Example 6 A chromatogram showing the results of analyzing 39 kinds of amino-functional conjugates in Analysis Example 5.
- Example 6 A chromatogram showing the results of analyzing 39 kinds of amino-functional conjugates in Analysis Example 5.
- Example 6 A chromatogram showing the results of analyzing 39 kinds of amino-functional conjugates in Analysis Example 5.
- Example 7 of Example This is a chromatogram showing the result of analyzing 17 types of aminofunctional conjugates in Analysis Example 6.
- FIG. 10 (a) is a chromatogram showing the results of analyzing 106 types of amino-functional compounds in Example 9 of Analysis: Analysis example 7.
- FIG. 10 (b) is a chromatogram showing the results of analyzing 106 kinds of amino-functional compounds in Example 9 of Analysis: Analysis example 7.
- FIG. 10 (c) is a chromatogram showing the results of analyzing 106 types of amino-functional compounds in Example 9: Analysis example 7.
- Example 9 In analysis example 7, 106 kinds of amino-functional compounds were analyzed. 5 is a chromatogram showing the results.
- FIG. 10 (e) is a chromatogram showing the result of analyzing 106 kinds of amino-functional compounds in Example 9 of Analysis: Analysis example 7.
- FIG. 11 (a) is a chromatogram showing the result of analyzing 38 kinds of amino-functional compounds in Example 10 of Analysis: Analysis Example 8.
- FIG. 11 (b) is a chromatogram showing the result of analyzing 38 kinds of amino-functional compounds in Example 10 of Analysis: Analysis Example 8.
- FIG. 11 (c) is a chromatogram showing the result of analyzing 38 kinds of amino-functional compounds in Example 10 of Analysis: Analysis Example 8.
- FIG. 12 (a) is a chromatogram showing the result of analyzing 39 kinds of amino-functional compounds in Example 12 of Analysis: Analysis Example 9.
- FIG. 12 (b) is a chromatogram showing the result of analyzing 39 kinds of amino-functional compounds in Example 12 of Analysis: Analysis Example 9.
- FIG. 12 (c) is a chromatogram showing the result of analyzing 39 kinds of amino-functional compounds in Example 12 of Analysis: Analysis Example 9.
- amino-functional conjugate means a compound having a primary amine and Z or a secondary amine in a molecule (which may be in the form of a salt).
- the amine ⁇ secondary amine may be one or more.
- the amino-functional compound present in the sample may be one kind or a mixture of plural kinds.
- amines primary Amines, secondary amines, etc.
- amino acids amino acids, peptides, proteins, polyamines and the like, and specifically include those shown in Table 1 below.
- a plurality of such compounds may be contained. Examples thereof include a mixture of a plurality of amino acids, a mixture of one or more amino acids and one or more peptides, and a mixture of one or more amino acids and one or more amines.
- derivatizing reagent refers to a reagent capable of reacting with the amino-functional conjugate to produce an amino-functional conjugate derivative represented by the following general formula (I). [0018] [Formula 2]
- Ai ⁇ represents a hydrocarbon which may have a substituent, or a substituent containing an aromatic carbon ring or a heterocyclic ring, and R represents a hydrogen atom or a substituent.
- R represents a hydrogen atom or a substituent.
- the bond between Ar and the nitrogen atom is not only when a carbocyclic or heterocyclic ring showing aromaticity is directly bonded to the nitrogen atom, but also when the nitrogen is It may be bonded to an atom, or the nitrogen atom may constitute a part of a carbocyclic or heterocyclic ring.
- Examples of the structure of such an amino-functional derivative compound include, for example:
- R represents an alkyl group having at least a carboxyl group as a substituent.
- the non-functional compound contains various compounds other than amino acids, in which case R is
- alkyl group which may have a substituent other than a xyl group, for example, a hydroxyl group, a sulfonic acid group, a phenyl group and the like.
- the amino-functional conjugate derivative is a compound represented by the following general formula (II).
- Ar represents a carbocyclic compound residue or a heterocyclic compound residue showing aromaticity
- Y represents an oxygen atom, a sulfur atom, a secondary amine, a tertiary amine, or an optionally substituted methylene group
- R represents a hydrogen atom or a substituent
- the amino-functional conjugate is a phenylcarbamylamino-functional compound, a 3-pyridylcarbamylamino-functional conjugate, a phenylthiocarbamylamino-functional ⁇ ⁇ ⁇ , 3-pyridyl thiol rubamyl amino-functional ⁇ ⁇ , P-trimethylammo-dimethyl-rubamylamino-functional compound or p-dimethylammo-dimethyl-rubamylamino-functional compound.
- the derivatizing reagent is represented by the following general formula ( ⁇ ) or (IV). Examples include a sothiocyanate aromatic compound, a substituted succinate aromatic compound, a substituted succinimidyl carbamate aromatic compound, a substituted rubamoyl halide aromatic compound, or a substituted carbamoylalkoxy aromatic compound.
- Ar represents a carbocyclic compound residue or a heterocyclic compound residue exhibiting aromaticity
- X represents an oxygen atom or a sulfur atom
- Y represents an oxygen atom
- a sulfur atom Represents a teramine, a tertiary amine or a methylene group which may have a substituent.
- Ar bonded to the nitrogen atom of the olebamate group is a carbocyclic compound showing an aromatic attribute
- the atom and the nitrogen atom of the olebamate group are bonded, and the olebamate conjugate may be in the form of a salt.
- Ar has a substituent as a carbocyclic compound residue.
- a naphthyl group (1 and 2-naphthyl group), an anthryl group (11, 2 and 5 anthryl group), etc., which may be substituted, and a substituent as a heterocyclic compound residue.
- Pyridyl groups (2-, 3- and 4-pyridyl groups), virazyl groups, quinolyl groups (2-8-quinolyl groups), ataridyl groups (1-4- and 9-ataridyl groups), and cumaryl groups (5 to 8-cumaryl group) and the like.
- the group can have one or more substituents on the aromatic ring.
- substituents examples include an alkyl group having 1 to 5 carbon atoms, an aromatic group such as a naphthyl group and a phenyl group, a halogen atom such as a chlorine atom, a bromine atom, a fluorine atom and an iodine atom, a carboxyl group, a hydroxyl group, and a nitro group.
- a polar substituent particularly a substituent which is easily ionized in a solution.
- Ar include the following groups:
- the alkyl groups of the above-mentioned dialkylamino group and trialkylammonium group can each independently represent an alkyl group having 1 to 5 carbon atoms.
- the method for preparing these derivatization reagents and the method for labeling amino acids using them are described in detail in Patent Document 2 described above, and are incorporated herein by reference.
- the reaction conditions are the general conditions for labeling with such a reagent (Iwaki, K., Yoshida, S "Nimura, N” Kinoshita, T., Takeda, K., and Ogura, H. Chromatographia 23 899 (1987)), but it is preferable to use a solution obtained by dissolving the amino-functional conjugate in a suitable organic solvent except alcohols in an environment having a pH value of about 8 to 10. Conditions such as heating to about 60 ° C. after mixing with the derivatization reagent can be suitably employed.
- the amount of the amino-functional compound is considered, and 10 to: about L000-fold molar equivalent (equivalent), preferably about 100 to 1000-fold molar equivalent (equivalent) to enable labeling for all amino groups and imino groups.
- An amino-functional conjugate for example, an amino acid
- an amino-functional conjugate can be converted into an amino-functional conjugate by a reaction as shown in FIG.
- Fig. 1 (a) when amino acids are reacted with different derivatizing reagents, Fe-N-hydroxysuccinimidyl diluvate (PAHS) or phenyl isocyanate (PIC)
- PAHS Fe-N-hydroxysuccinimidyl diluvate
- PIC phenyl isocyanate
- the amino acids are combined with 3-aminopyridyl-N-hydroxysuccimidyl diluvate (APDS) or pyridyl isocyanate (PvIC).
- APDS 3-aminopyridyl-N-hydroxysuccimidyl diluvate
- PvIC pyridyl isocyanate
- the method of the present invention is characterized in that the structure of the amino-functional compound derivative resulting from the derivatization reaction is the structure represented by the above general formula (I) or ( ⁇ ), A method of performing a reaction using any derivatizing reagent as long as it has a structure is also included in the scope of the present invention.
- Liquid chromatography in the analytical method of the present invention is a separation technique based on chromatography using a liquid as a mobile phase, and is a high-speed liquid that can be operated under pressure by selecting a stationary phase column. It is used for various purposes as chromatography (HPLC). Separation mechanisms are classified into various mechanisms, such as partitioning, adsorption, ion exchange, and size exclusion, but the most frequently used columns are separated by partitioning on a reversed-phase stationary phase, and the sample is less polar. It is retained on the stationary phase and eluted by the mobile phase with polarity.
- the "stepwise concentration gradient elution means (stepwise gradient)" is to change the polarity or the salt strength of the mobile phase in a stepwise manner. This means changing the ratio as shown in FIG. In Fig. 2, the ratio of the organic solvent contained in the eluent changes stepwise with the elapse of the analysis time, but from a state without a concentration gradient or a state with a gentle concentration gradient, it has a sharper concentration gradient than that Non-linear change to state.
- the ratio of the organic solvent is changed at least two times, more preferably three times or more, so that efficient separation can be performed.
- the optimum stepwise gradient differs depending on the type of the amino-functional compound derivative to be analyzed and the type of the organic solvent used.
- the same effect as in the present invention can be obtained by increasing the elution time even when a linear concentration gradient elution means (linear gradient method) is used.
- various solvents can be appropriately selected in accordance with the type of the column such as the packing material and the size of the column.For example, in the case of HPLC using a reversed-phase column, formic acid or acid is used as the acid.
- the pH is adjusted to 2.5 to 7 with a 5 to 50 mM aqueous solution of formic acid, acetic acid, ammonium formate, or ammonium acetate. It is preferable to use a solution adjusted to 5, and as the organic solvent, acetonitrile, methanol, ethanol, and a mixed solution thereof with water.
- the difference in elution time between the tyramine derivative, cysteamine derivative, putrescine derivative, cystamine derivative and 2-phenylethylamine derivative, which has the longest elution time, is 3 to 20 minutes , More preferably 3 to: It can be set between LO minutes.
- the mass spectrometry method used in the present invention is characterized in that the amino-functional conjugate eluted by the liquid chromatography is subjected to electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and sonication.
- Positive ions are present by methods such as spray ionization (SSI)! / Is a method of ionizing into negative ions and measuring in the gas phase.
- the generated ions are applied to a mass separator such as a quadrupole type, an ion trap type, a time-of-flight type, and a magnetic field type, and are separated based on a mass-to-charge ratio.
- MSZMS mass separation devices in series
- a triple quadrupole device after measuring the mass of the amino-functional compound derivative at the first quadrupole, only the amino-functional compound derivative of interest is measured at the second quadrupole. It is sent to the quadrupole where it is fragmented by collision-induced dissociation (CID), and the mass of the fragment can be measured with very high sensitivity at the third quadrupole.
- CID collision-induced dissociation
- n Monitoring (SRM) method the first mass analyzer detects ions of a reactant of the amino-functional conjugate and the labeling reagent, and the second mass analyzer detects fragment ions derived from the reagent. Highly sensitive and highly sensitive analysis can be achieved.
- the derivatization reagents used in such a method include p-dimethylamino-allyl-N-hydroxysuccinimidyl-caprate (DAHS) and 3-aminopyridyl-N-hydroxys-succinimidyl-caprate (APDS). ), P-Trimethylammo-dimethyl-N-hydroxysuccinimidyl-rubbamate iodide (TAHS), Aminovirazyl N-hydroxysuccinimidyl-rubbamate, 6-Aminoquinolyl-N-hydroxysuccinimide carnomate (AQC ), 9-aminoacridyl N-hydroxysuccinimide carbamate and the like.
- DAHS p-dimethylamino-allyl-N-hydroxysuccinimidyl-caprate
- APDS 3-aminopyridyl-N-hydroxys-succinimidyl-caprate
- TAHS P-Tri
- a highly selective and highly sensitive amino acid separation can be achieved by detecting the ion of the reactant with the second mass analyzer to detect the ion derived from the amino acid whose structure derived from the reagent skeleton is -Eutral loss.
- the derivative reagent used in such a method include 1-naphthylamino-N-hydroxysuccinimidyl olebamate (NAHS).
- the mass spectrometry used in the present invention is not particularly limited, but some preferred examples will be described.
- a compound having a target mass can be detected with high accuracy by monitoring only a specific mZz ratio that has been preliminarily selected.
- the first mass analyzer (such as Q1) is specified.
- the ions in this range are dissociated by, for example, collision-induced dissociation in Q2.
- a precursor ion (parent ion) that generates a specific fragment ion is recorded.
- the target ion is selected by a first mass analyzer (such as Ql), this ion is dissociated in a collision cell, and a specific fragment ion (such as Q3) is used by a second mass analyzer (such as Q3).
- a first mass analyzer such as Ql
- Q3 a specific fragment ion
- Q3 a specific fragment ion
- a first mass analyzer eg, Q1
- ions in this range are dissociated by, for example, collision-induced dissociation at Q2.
- the second mass analyzer Q3, etc.
- a scanning method that detects all precursor ions (parent ions) from which a specific neutral molecule is desorbed is adopted.
- Mass spectrometry is widely used as an extremely selective detection method because it can detect ions caused by the mass of the substance to be analyzed.
- a derivatization reagent that causes regular cleavage of an analyte, a more selective detection method for an amino-functional compound can be obtained.
- a highly ionic derivatization reagent is designed, highly sensitive analysis of an amino-functional conjugate can be achieved.
- the detection limit of amino acids differs depending on the type of amino acid.
- an SRM mode is selected using a force quadrupole mass spectrometer, for example, an API365 type detector (Applied Biosystems), ⁇ -trimethylammo-puma About 2 to 40 fmol with -ryl N-hydroxysuccinimidyl potash iodide (TAHS), about 3 to 2000 fmol with p-dimethylaminoaryl-N-hydroxysuccinimidyl potash ibatide (DAHS), 3 Aminoviridyl-N-hydroxysuccinimidyl sorbate (APDS): 3-180 fmol, 6-aminoquinolyl-N-hydroxysuccinimide carbamate (AQC): 2-200 fmol, surpassing general fluorescent labeling reagents It is confirmed that it can be done.
- TAHS N-hydroxysuccinimidyl potash iodide
- DAHS p-dimethylaminoary
- 6-aminoquinolyl-N-hydroxysuccinimide carbamate is commercially available as a fluorescent labeling reagent. Its detection limit is reported to be several hundred fmol. In the SRM measurement, a sensitivity improvement of up to 100 times is seen, which is equivalent to the fluorescence method.
- the detection by the method of the present invention is performed with ions caused by the mass of the derivatized and labeled amino-functional compound
- other compounds such as a labeling reagent, a hydrolyzate of the labeling reagent, and the like may be used.
- the accuracy of measurement can be improved by using an amino-functional conjugate containing a stable isotope.
- a stable isotope having a low natural abundance such as 13 C, (D), 15 N, and 180 , or an amino-functional compound containing a radioisotope is used as an internal standard.
- the concentration in the sample, and the analysis conditions By selecting the optimal internal standard substance according to the type of the amino-functional compound to be analyzed, the concentration in the sample, and the analysis conditions, the quantification accuracy is dramatically improved.
- the detection step by the mass spectrometry comprises the following steps:
- an amino-functional conjugate derivative having the same mass is shown, and according to the method of the present invention, these derivatives are included in these groups.
- the conditions of the elution step can be set so that each of the amino-functional compound derivatives can be separated and detected. Therefore, these are simultaneously analyzed using a sample containing various kinds of amino-functional conjugates. It becomes possible. Therefore, the method of the present invention may use a sample containing all of these amino-functional conjugates, but it includes at least two or more amino-functional conjugates in which the power of leucine, isoleucine and norleucine is also selected.
- the method includes a method of simultaneously analyzing a plurality of these amino-functional derivatives using a sample.
- the analysis device of the present invention includes, for example, as shown in FIG. 3, components (parts) necessary for performing the above-described analysis method of the present invention.
- these components include, at least, a reaction part 10 for reacting the amino-functional compound in the sample with the derivatization reagent, and elution by liquid chromatography of the amino-functional conjugate compound derivative generated in the reaction part 10.
- the elution section 20 includes a separation column 21 And an elution system for supplying an eluate having a stepwise concentration gradient to the column, for example, a pump 23a (23b) for sending the eluate 24a (24b) to the column. Multiple pumps (23a, 23b) can be used to create a concentration gradient in the eluate. Further, the chromatography section 20 includes an injection valve 22 for introducing the amino-functional conjugate derivative generated in the reaction section 10.
- the amino-functional conjugate derivative eluted from the chromatography unit 20 is first ionized in the mass spectrometry unit 30.
- the most commonly used techniques for ionizing proteins and peptides are electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption ionization (MALDI).
- ESI electrospray ionization
- APCI atmospheric pressure chemical ionization
- MALDI matrix-assisted laser desorption ionization
- the force of the device of the present invention such as sonic spray ionization method (SSI), etc.
- SSI sonic spray ionization method
- a method for ionizing an amino-functional conjugate compound derivative contained in an eluate from liquid chromatography is as follows. It is preferable to use the ESI method. Many systems with efficient heat transfer and gasification methods for ionization are used.
- the mass spectrometry unit 30 separates the ions within the mass-to-charge ratio (m / z) selected from the ionized sample.
- the mass spectrometer plays an important role in the sensitivity and resolution of analytical data, the accuracy of mass, and the abundance of information obtained from mass spectral data.
- ion separation methods magnetic, electric, ion trap, time of flight (TOF), quadrupole, and Fourier. It is a conversion cyclotron type. These have advantages and disadvantages, respectively.
- a force that can be used alone or in combination with each other is usually used in a quadrupole mass spectrometry unit in ionization by ESI.
- the analyzer of the present invention can automatically connect the above-mentioned respective constituent elements (units), and can automate from the reaction section 10 to the mass analysis section 30.
- a unit for storing a sample such as plasma in a 96-well plate is added. From this, a predetermined amount of sample is collected by an automatic sampling device, transferred to the reaction section 10, and further derivatized amino functional groups.
- the sex conjugate can be automatically injected into the chromatographic section 20 using an autosampler or the like of liquid chromatography. This enables extremely high-throughput prayer.
- the separation column in reversed-phase HPLC uses a CAPCELL PAK AQ inner diameter of 2. Omm, a length of 5 Omm, a particle diameter of 3 m (Shiseido), a flow rate of 0.3 mLZmin, and gradient conditions as follows.
- Time (ratio of mobile phase B) 0 min to 0.25 min (3%), 0.25 min to 1 min (3% Et 15 0/0), 1 component force et 1.75 minutes (15 0/0), 1.75 component force et 2.35 minutes (15 0/0 Power et 34 0/0), 2.35 min 5 Minutes (34% to 35%), 5.01 minutes to 5.1 minutes (50% to 70%), 5.1 minutes to 5.7 minutes (70%), 5.71 minutes to 12 minutes (3%).
- the gradient pattern representing the methanol concentration is shown in FIG.
- FIG. 4 shows the results of the analysis.
- the TIC Total Ion Chromatogram
- the extracted ion chromatogram shows a pattern in which these 40 compounds were separated and detected.
- the patterns in which the respective amino-functional conjugates were detected alone or as a mixture of several types are shown below them. As is evident from FIG. 4, it is important to be able to separate and detect each of the 40 amino-functional conjugates in about 9 minutes.
- a reverse column HPLC uses a CAPCELL PAK AQ separation column with an inner diameter of 2. Omm, a length of 5 Omm, a particle diameter of 3 m (Shiseido), a flow rate of 0.3 mLZmin, and gradient conditions as follows.
- Time (ratio of mobile phase B) 0 min 0.25 min (3%), 0.25 min 1 min (3% to 15 0/0), 1 component force et 1.75 minutes (15 0/0) , 1.75 component force et 2. 35 min (15 0/0 power et 34 0/0), (35% to 34%) 35 minutes to 5 minutes 2. from 5.01 minutes to 5.1 minutes (50% 70%), 5.1 minutes to 5.7 minutes (70%), 5.71 minutes 12 minutes (3%).
- FIG. 5 shows the result of the analysis. Also in this analysis example, it can be seen that each of the 40 kinds of amino-functional conjugates can be separated and detected in about 9 minutes similarly to the above-mentioned analysis example 1.
- FIG. 6 shows the result of the analysis. Also in this analysis example, it can be seen that each of the 39 kinds of amino-functional conjugates can be separated and detected in about 8 minutes similarly to the above analysis examples 1 and 2.
- FIG. 7 shows the result of the analysis.
- each of the 39 kinds of amino-functional conjugates can be separated and detected in about 8 minutes in the same manner as in the above analysis examples 1 to 3.
- the separation column in the reverse phase HPLC uses a SuperODS inner diameter of 2. Omm, a length of 50 mm, a particle diameter of 2 / ⁇ ( ⁇ 030 ⁇ ), a flow rate of 0.4 mLZmin, and the following gradient conditions.
- Time (ratio of mobile phase B) 0 min 0.01 min (13%), 0.01 min 2 min (60%), 2.5 min force et 4.5 minutes (100 0/0), 4.51 Bunkara 10 minutes (13 0/0).
- FIG. 8 shows the result of the analysis.
- this analysis example about 6 minutes as in the above analysis examples 1-4 It can be seen that each of the 39 kinds of amino-functional conjugates can be detected separately.
- 17 kinds of amino acid derivatives (leucine derivative, isoleucine derivative, norleucine derivative, sarcosine derivative, ⁇ -alanine derivative, prepared by the reaction conditions described in Alanine derivative, ⁇ ⁇ ⁇ amino- ⁇ -butyric acid derivative, ⁇ -aminoisobutyric acid derivative, ⁇ -amino ⁇ -butyric acid derivative, ⁇ -aminoisobutyric acid derivative, j8-amino- ⁇ -butyric acid derivative, 1-methylhistidine derivative, 3-methylhistidine derivative, homoserine Derivatives, threonine derivatives, palin derivatives, and norparin derivatives) were separated by reverse phase HPLC and detected in Selected Reaction Monitoring (positive mode).
- the column for reverse phase HPLC uses a CAPCELL PAK AQ with an inner diameter of 2. Omm, a length of 50 mm, and a particle size of 3 m (Shiseido).
- the flow rate is 0.3 mLZmin and the gradient conditions are as follows.
- Time (ratio of mobile phase B) 0 minute force is also 0.5 minute (13%), 0.51 minute to 4 minutes (50%), 4.01 minute to 6 minutes (80%), 6.01 minute to 12 minutes (13% %).
- FIG. 9 shows the results. As shown in the respective charts of FIG. 9, the fact that a plurality of amino-functional conjugates having the same mass number, for example, a sarcosine derivative, a ⁇ -alanine derivative, and an alanine derivative can be separated and detected.
- a 0.2-borate buffer ( ⁇ 8.8) was added to 20 ⁇ L of a standard mixed solution of the amino-functional conjugate.
- a solution of 3 aminoviridyl 1-hydroxysuccinimidyl olebamate reagent (10 mg of the derivatizing reagent dissolved in 1 mL of acetonitrile for LCZMS) with 20 / z L added.
- the resulting mixture was heated at 55 ° C for 10 minutes. After the heating, the resulting mixture of the amino-functional compound derivatives was separated by reversed-phase liquid chromatography and introduced into a mass spectrometer. At this time, the obtained mixture of the amino-functional compound derivatives is neutralized with 100 L of a 0.1% aqueous formic acid solution, and diluted with 300 ⁇ L of mobile phase A of liquid chromatography.
- the derivatized product of a mixture of 106 amino-functional conjugates prepared by the reaction conditions described in Example 8 above with 3-aminopyridyl-N-hydroxysuccinimidyl sorbate (APDS) was separated by reverse phase HPLC. And detected in Selected Reaction Monitoring (positive mode).
- the separation column used in the reverse phase HPLC was InertsilC8-3, having an inner diameter of 2.1 mm, a length of 50 mm, and a particle system of 3 m (GL Science).
- the flow rate was 0.3 mL / min, and the gradient conditions were as follows.
- Time (ratio of mobile phase B) 0 to 1.25 minutes (4%), 1.25 to 1.26 minutes (4% to 15%), 1.26 to 5 minutes (15% to 20%), 5 min 5.5 min (20 0/0 power et 50 0/0), 5.5 component force et 6.5 minutes (50 0/0 power et 95 0/0), 6.5 component force et 6.75 Minutes (95%), 6.76 minutes to 12 minutes (4%).
- FIG. 10 shows the result of the analysis. As shown in FIG. 10, it is important to be able to separate and detect each of the 106 amino-functional compounds in about 9 minutes.
- Time (Ratio of mobile phase B) 0 to 1.25 minutes (4%), 1.25 to 1.25 minutes (4% to 15%), 1.26 to 5 minutes (15% to 20%), 5 min 5.5 min (20 0/0 power et 50 0/0), 5.5 component force et 5.51 minutes (50 0/0 power et 95 0/0), 5.51 component force et 6.5 min (95 0/0), 6.51 to 12 minutes (4%).
- FIG. 11 shows the result of the analysis. As shown in FIG. 11, it is important to be able to separate and detect each of the 38 kinds of amino-functional conjugates in about 8 minutes.
- Example 11 Specific Procedure 3 for Derivatization of Amino Functional Compound>
- a 0.2-borate buffer ⁇ 8.8 was added to 20 ⁇ L of a standard mixed solution of the amino-functional conjugate.
- 3-aminopyridyl 1-hydroxysuccinimidyl olebamate reagent solution 10 mg of the derivatization reagent dissolved in 1 mL of acetonitrile for LCZMS
- the resulting mixture was heated at 55 ° C for 10 minutes.
- the resulting mixture of the amino-functional compound derivatives was separated by reversed-phase liquid chromatography and introduced into a mass spectrometer. At that time, the obtained amino-functional compound derivative mixture is neutralized with 100 L of a 0.1% aqueous formic acid solution, and then diluted by adding 300 ⁇ L of mobile phase A of liquid chromatography.
- Derivatized product of a mixture of 39 amino-functional conjugates prepared by the reaction conditions described in Example 11 above using 3-aminopyridyl-N-hydroxysuccinimidyl sorbate (APDS) was separated by reverse phase HPLC. And detected in Selected Reaction Monitoring (positive mode).
- the separation column used in the reverse phase HPLC was an Atlantis dC18 with an inner diameter of 2.1 mm, a length of 100 mm, and a particle system of 3 m (Waters).
- the flow rate was 0.3 mL / min, and the gradient conditions were as follows.
- Time (ratio of mobile phase B) 0 min 0.5 min (0%), 0.5 mowed 2.25 (0 0/0 Power et 12 0/0), 2.25 mowed 2.26 (12 0/0 power et 18 0/0), 2.26 mosquito et 5.5 minutes (18 0/0 power et 22 0/0), 5.5 component force et 6 minutes (22 0/0 power et al 50 0 / 0 ), 6 minutes power 6.25 minutes (50% to 90%), 6.25 minutes to 6.5 minutes (90%), 6.51 minutes to 12 minutes (0%).
- FIG. 12 shows the result of the analysis. As shown in FIG. 12, it is important to be able to separate and detect each of the 39 amino-functional compounds in about 10 minutes.
- 100 or more amino-functional conjugates containing biological amino acids can be analyzed in a very short time, for example, within 10 minutes. It is useful in the fields of food, medicine, medical and analytical instruments related to.
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JP5030586B2 (ja) | 2012-09-19 |
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