WO2000062052A1

WO2000062052A1 - Method and device for analyzing chemical substances

Info

Publication number: WO2000062052A1
Application number: PCT/JP2000/001514
Authority: WO
Inventors: Kazuyoshi Nozaki
Original assignee: Fujisawa Pharmaceutical Co., Ltd.
Priority date: 1999-04-07
Filing date: 2000-03-13
Publication date: 2000-10-19

Abstract

A method for analyzing a sample on a mass spectrometer, wherein the sample is electrochemically treated before being assigned to a mass spectrometer, whereby molecular structure-related information (structural information) can be constantly obtained from fragment ions measured by a mass spectrometer to thereby provide a high-accuracy analyzing method in terms of specificity and sensitivity.

Description

Specification

Chemical substance analysis method and analyzer

The present invention relates to a method and an apparatus for analyzing a sample, and more particularly, to a method and an apparatus for analyzing a sample obtained by combining a method of electrochemically performing Z reduction with mass spectrometry.

Background art

In general, a mass spectrometer (MS) converts particles such as atoms, molecules, clusters, and organic compounds into gaseous ions and moves them in a vacuum, and separates samples according to the mass-Z charge ratio (mZz). · A device that can be detected. By analyzing the mass spectrum obtained using this device, it is possible to determine the mass (molecular weight information) from the molecular weight-related ions, the molecular structure (structural information) from the fragment ions, and the height and pattern of the isotope ions. The type and number of elements (elemental information) can be obtained.

Therefore, mass spectrometers have traditionally been selected as LC-MS by connecting to a liquid chromatograph (LC), and MSZMS, MSZMS / MS, etc. by connecting multiple mass spectrometers. As a highly versatile analytical instrument, it is used for sample quantification and structural analysis in various fields.

On the other hand, one of the detectors of liquid chromatographs is an electrochemical detector (ECD), which has a higher specificity, a wider measurement concentration range and better sensitivity than an ultraviolet absorption detector. Used for many substances.

However, when an MS is connected to an LC, it is important in mass spectrometry to ionize compounds that are difficult to ionize, since the low ionization and stability of the target substance are directly related to the quantitative sensitivity. . In addition, there is a problem in that analysis by MS cannot provide as much structural information as NMR (nuclear magnetic resonance).

Furthermore, although LC-ECD is a commonly used analyzer for certain types of compounds, the compound to be analyzed is detected using a redox reaction. Only compounds that are easily oxidized or reduced can be analyzed, and their targets are limited. In addition, increasing the potential applied for the oxidation-reduction reaction increases the background, reduces the selectivity of the compound, and requires time to stabilize the current signal in order to perform highly sensitive measurements. There are issues. Disclosure of the invention

According to the present invention, there is provided a method of analyzing a sample by subjecting the sample to a mass spectrometer and subjecting the sample to electrochemical analysis and then subjecting the sample to a mass spectrometer.

Further, according to the present invention, in a method of subjecting a sample to a mass spectrometer for analysis, the sample is electrochemically treated and then subjected to a mass spectrometer to obtain a mass spectrum. And a mass spectrum obtained by directly subjecting the sample to a mass spectrometer.

Further, according to the present invention, there is provided a sample analyzer in which a device capable of electrochemically processing a sample is incorporated in a sample introduction portion of a mass spectrometer. That is, the present invention provides a method for stabilizing structural information in mass spectrometry to analyze or quantify a target compound with high sensitivity, and to obtain molecular structural information that cannot be obtained only by using a mass spectrometer alone. It is an object of the present invention to provide a highly useful analysis method and an analysis device that can perform the analysis. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a conceptual diagram of a main part showing a series of analyzers used in the analysis method of the present invention.

FIGS. 2 (a) to 2 (d) are mass spectra of compound (1) measured in MS in positive ion mode.

FIGS. 3 (a) to 3 (c) are mass spectra of compound (2) measured in MS in positive ion mode.

FIGS. 4 (a) to (c) are mass spectra of compound (2) measured in negative ion mode with MS.

Fig. 5 (a) to (d) show that compound (3) was measured in positive ion mode by MS. It is a specified mass spectrum.

Fig. 6 (a) to (c) are mass spectra of compound (3) measured by negative ion mode using MS.

FIGS. 7 (a) to 7 (d) are mass spectra of compound (4) measured in the positive ion mode by MS.

FIG. 8 is a mass spectrum of compound (5) measured in positive ion mode by MS.

FIGS. 9 (a) to 9 (d) are mass spectra of compound (5) directly measured by MS ZMS.

FIG. 10 is a mass spectrum measured by MSMS after passing compound (5) through an ECD cell without applying a voltage.

FIGS. 11 (a) to (c) are mass spectra measured by MS ZMS after passing compound (5) through an ECD cell without applying a voltage. FIG. 12 is a mass spectrum showing the average of 104 continuous mass spectra measured by MSZMS ^/ MS after passing compound (5) through an ECD cell without applying a voltage.

Figures 13 (a) and (b) are mass spectra measured with MS after applying voltage to compound (5) and passing it through the cells of an ECD.

FIG. 14 is a mass spectrum measured by MSZ.MS after compound (5) was passed through an ECD cell by applying a voltage.

FIGS. 15 (a) to 15 (c) are mass spectra measured by MSZMSZMS after applying a voltage to compound (5) through an ECD cell.

FIG. 16 is a mass spectrum showing the average of 35 continuous mass spectra measured by MSZMSZMS after applying a voltage to compound (5) and passing it through an ECD cell.

FIGS. 17 (a) and (b) are mass spectra of compound (6) measured in positive ion mode by MS.

FIGS. 18 (a) to 18 (c) are mass spectra of compound (7) measured in positive ion mode by MS.

FIGS. 19 (a) to (d) are mass spectra of compound (8) measured in positive ion mode by MS. BEST MODE FOR CARRYING OUT THE INVENTION

The present invention mainly comprises subjecting a sample to be analyzed by a mass spectrometer to an electrochemical treatment in advance.

The sample in the present invention means a test substance containing a chemical substance to be measured, and is particularly preferably a liquid substance dissolved in a suitable solvent such as water or an organic solvent. Further, in the present invention, it is preferable that the chemical substance to be measured is present almost purely in the sample, and it is preferable to use the one after the separation or purification treatment. The separation or purification treatment can be performed by a conventional method in the relevant field, for example, by subjecting to a liquid chromatography (LC) or the like.

Chemical substances include various substances such as substances extracted from natural animals and plants, synthesized organic substances, inorganic substances, and metabolites thereof. For example, by subjecting to electrochemical treatment, electrochemically oxidized or reduced such that is easily compound, or oxidized or reduced, etc. are susceptible functional groups (e.g., single CHO, -CO, -NO, One N0 _2, = C = C =, = C = S, = C = N—, 1 N = N—, — N = N_N =, 1〇1 O—, _S—S—, organometallic compound residue, 1 C3 N , One N = S, -SO, one S〇 ₂ , —SX, -CX, = N—X, = P—R, etc .; amino group, phenolic OH group, = P—O—, one SH, R ₂ H - C (= S) one S-, one NH - CO - NH -, -NH -C S -NH -, -NH (-R) 2, - NH- NH 2, - CS-NH- R , etc. (Where R represents an alkyl group and X represents a halogen atom). Among them, a compound which can be oxidized or reduced or a compound having such a functional group when a certain voltage is applied to a sample dissolved in an appropriate solvent such as water or an organic solvent is advantageous. The sample of the present invention may be applied to the case where the sample contains a chemical substance other than the compound which is susceptible to electrochemical oxidation or reduction as described above, or a compound having a functional group which is susceptible to oxidation or reduction. Can be.

A mass spectrometer used in the present invention; a device which can be generally used for performing mass spectrometry, mainly after ionizing a sample and separating the sample according to a mass Z charge number (m / z); Means a device for measuring a mass spectrum by electrically detecting a target or detecting a specific ion. General The mass spectrometer is composed of an ion source for ionizing a sample, an analyzer for separating ions according to m / z, and a Z recording unit for detecting separated ions. Specifically, ion trap mass spectrometer (ITMS), quadrupole mass spectrometer (QMS), double focusing mass spectrometer, time-of-flight mass spectrometer (T〇F-MS), ion cyclotron resonance mass There are various analyzers used in this field, such as analyzers (ICR-MS). The ion source of the mass spectrometer may be of any type, but is preferably of a type capable of ionizing a liquid sample. For example, ion sources that can be ionized by the electrospray ionization (ESI) method, the atmospheric pressure chemical ionization (APCI) method, the fast atom bombardment (FAB) method, and the like.

In the present invention, the term “electrochemical treatment” generally means that an electrochemical oxidation or reduction reaction is performed by applying a predetermined voltage to a sample containing a chemical substance to be analyzed. I do. Such an electrochemical treatment is generally performed by applying to an electrochemical detector (ECD) which has been conventionally used as an LC detector. As the ECD, a coulometric detector having a reaction rate close to 100% is preferable. For example, various detectors used in this field, such as an amberometric detector, can be used. These ECDs are mainly composed of cells in which a pair of inert electrodes are placed in an electrolyte solution containing a sample and a predetermined voltage is applied between the electrodes. Here, the magnitude of the applied voltage can be appropriately adjusted according to the type of the electrode, the solvent used, the configuration of the cell used, and the properties of the chemical substance to be analyzed contained in the sample. Preferably, the voltage is set to a value suitable for the sample to be oxidized or reduced. Specifically, when a coulometric cell manufactured by ESA is used, a voltage of about ± 200 to 100 OmV is applied.

In the present invention, by subjecting a sample to electrochemical treatment and then subjecting the sample to a mass spectrometer, the molecular weight information and structural information normally obtained in the mass spectrometer can be obtained more stably and with higher sensitivity. be able to. In addition, it is possible to obtain information on the types of functional groups of a specific chemical substance in a sample and the bonding positions of the functional groups. In particular, in the present invention, a sample is electrochemically treated and then subjected to mass spectrometry to obtain a mass spectrum, and the obtained mass spectrum is analyzed by mass spectrometry without subjecting the sample to electrochemical treatment. By comparing with the mass spectrum obtained by the measurement, the mass number of the mass spectrum (mZz) and the ionic strength changed by the electrochemical treatment and the ionic strength Various information about the structure can be obtained.

Specifically, by subjecting a sample to electrochemical treatment, the mass of the chemical substance, which is easily or less likely to be oxidized or reduced, is compared with a mass spectrometer not subjected to the electrochemical treatment. The ionic strength of the vector increases or the peak is obtained stably, and stable fragmentation can be observed. In addition, when the compound which is liable to be oxidized or reduced electrochemically or the functional group which is liable to be oxidized or reduced is desorbed or changed by the electrochemical treatment, the sample is removed. Before and after the electrochemical treatment, the mass number increases and decreases, and thus can be used as an index when specifying a compound or a functional group. In addition, even for chemical substances of the same mass, by subjecting them to electrochemical treatment, the positions of functional groups that are easily oxidized or reduced electrochemically, the bonding state of atoms near those functional groups, and the presence of other functional groups Depending on the presence or the like, the degree of oxidization or reduction is changed, or the number of different masses is increased / decreased. Therefore, information on the position of the functional group can be obtained.

In the present invention, after the sample has been subjected to the electrochemical treatment, the sample may be applied only once to the mass spectrometer, or may be applied continuously two or more times. In particular, more stable fragmentation can be observed when the sample is continuously applied to the mass spectrometer two or more times.

The analysis method of the present invention can be easily implemented by, for example, an analyzer configured by incorporating a device capable of electrochemically processing a sample into a sample introduction portion of a mass spectrometer described later. Specifically, a sample containing a chemical substance to be analyzed can be electrochemically treated with a mobile phase having a composition that does not react with the chemical substance at a constant flow rate for a predetermined time, if desired. The sample is introduced into the instrument and a predetermined voltage is applied to the introduced sample. At this time, in order to stably apply a voltage to the sample, the specified phase was introduced in advance to a device that can electrochemically process only the mobile phase not containing the sample It is preferable to apply this voltage beforehand and then introduce the sample together with the mobile phase into an apparatus capable of electrochemically processing the sample. The sample may be introduced using a syringe infusion known in the art, a syringe infusion provided in a commercially available mass spectrometer, or may be electrochemically introduced. When using an analyzer that incorporates a liquid chromatograph, etc. in the introduction part of the equipment that can be processed, the sample discharged from the liquid chromatograph is kept while maintaining the mobile phase of the liquid chromatograph at a constant flow rate. It may be introduced into equipment that can be processed directly electrochemically.

The sample, which is attached to a device that can be processed electrochemically and discharged from this device, is directly introduced into the ion source, which is the interface of the mass spectrometer. At this time, the sample may be introduced by any means as long as the sample can be introduced at a constant flow rate for a predetermined time. For example, the introduction means usually provided in the mass spectrometer may be used, or the mobile phase discharge means from the liquid chromatograph may be used.

Further, the chemical substance analyzer according to the present invention is configured by incorporating a device capable of electrochemically processing a sample into a sample introduction part of a mass spectrometer, preferably immediately before an ion source of the mass spectrometer. You. As described above, a mass spectrometer and an electrochemical detector known in the art can be used as the mass spectrometer and the device capable of performing the electrochemical treatment used here. Furthermore, in the analyzer of the present invention, a liquid chromatograph for separating and purifying a sample is further incorporated in the introduction portion of the device capable of electrochemical processing, preferably immediately before the device. Is preferred. With such a configuration, the sample to be applied to the analyzer of the present invention can be easily purified to a more pure state. In the analyzer of the present invention, a device which can be electrochemically processed and a mass spectrometer are connected to tubes generally used in the field, for example, PEEK tubes, stainless steel tubes, etc. (0.13 to 0.25 mm, outer diameter 1.6 mm) .If a liquid chromatograph is further incorporated, it is processed electrochemically with the liquid chromatograph. The resulting device can be connected using a tube as described above. Hereinafter, embodiments of the sample analysis method of the present invention will be described.

Embodiment 1

As shown in Fig. 1, a fixed amount of the mobile phase L was flowed from the high-performance liquid chromatograph (HPLC) 10, and the sample M was mixed with the mobile phase L via the T-tube 12 using the syringe 11 and then mixed. The measurement sample M was measured by introducing it into the cell 14 of an electrochemical processing device (electrochemical detector, ECD) 13 and then continuously introducing it into a mass spectrometer (MS) 15. Note that a control unit 16 for controlling an applied voltage and the like is connected to the cell 14 of the ECD 13.

Measurement sample

Compounds (1) represented by the following structural formulas, compounds (2) and (3) which are metabolites of compound (1), and compounds (4) in which the phenolic hydroxyl group of compound (2) is converted to a methoxy group ) Was used as a measurement sample. Compound (1) is a compound described in JP-A-2-111774, and compounds (2) and (3) are compounds described in J. Pharm. Biomed. Anal., 17 (1998) ρ1381392. Yes, the compound (4) was produced according to the method described in JP-A-2-111774.

measuring device

HP L C: Waters Model 2690 Alliance

ECD: Esa Coulochem II and Analytical Cell Model 5010

(Coolometric type, cell is used by applying voltage in DC mode to only the first of two stages)

MS: LCQ (ion trap type) manufactured by ThermoQuest, Inc. Electrospray method (ESI) was used as the interface.

In addition, the syringe infusion used was attached to MS.

Measurement method and results

The following two solutions A and B were used as mobile phases for HPLC, and B was set to 50%.

A: 2 OmM ammonium formate aqueous solution containing 5% acetonitrile B: only acetonitrile

Degassing was performed with the online degasser attached to HP LC. The column was not used and the mobile phase flow rate was set to 0.2 mL / min.

The measurement sample was prepared by dissolving with acetonitrile so that the concentration of each of the above compounds would be 5 O AiM, then diluting with about 40% aqueous solution of acetonitrile, and using a syringe at a rate of 5 L / min. Injected into the mobile phase before it was introduced.

The MS used an electrospray method (ESI) as the interface, set the heating capillary temperature to 250 ° C, the sheath gas volume to 75 psi, and the auxiliary gas volume to 1 Opsi. The spray voltage was 4.5 kV in the positive ion mode and 4 kV in the negative ion mode. Optimization of the tube lens voltage, etc. was performed using the autotune program attached to the equipment.

• Measurement of compound (1)

First, compound (1) was introduced into MS without applying the ECD voltage, and was measured by full scan in positive ion mode. The molecular weight-related ion [M + H] ⁺ was found to be m / z 439 (Fig. 2 (a)).

When the compound (1) was measured by MS with the ECD voltage set to an oxidation potential of 80 OmV or 90 OmV or a reduction potential of 180 OmV, there was no change in the peak of mZz439 (Fig. 2). (b) to (d)).

Furthermore, when compound (1) was similarly measured in negative ion mode, [M—H] —, a molecular weight-related ion, was observed, but the ionic strength was weak.

• Measurement of compound (2)

Next, the compound (2) having a phenolic hydroxyl group was measured by full scan in the positive ion mode without applying the ECD voltage, and the molecular weight-related ion [M + H] + was found to be mZz455. (Fig. 3 (a)).

When the compound (2) was measured at an oxidation potential of 45 OmV with the ECD voltage, the peak of mZz 455 did not change, but started to decrease from around 50 OmV. At 55 OmV, the peak of mZz 455 completely disappeared, and instead mZz 469 and other peaks having a mass number of 14 increased (FIG. 3 ()). At 80 OmV, the peak at m / z 469 was dominant (Fig. 3 (c)). At a reduction potential of one 80 OmV, as in the case of compound (1), no change was observed in the peak of mZz455 for the molecular weight-related ion [M + H] ⁺ .

Furthermore, when compound (2) was measured in the negative ion mode in the same manner, [M—H] —, a molecular weight-related ion, was observed in mZz 453 (Fig. 4

(a)).

When compound (2) was measured with the ECD voltage set to 55 OmV, the peak of mZz 453 disappeared completely as in the positive ion mode, and instead, mZz 467 and 14 A peak appears (Figure 4

(b)). At 70 OmV, the peak at mZz 467 was dominant and the ionic strength also increased (Fig. 4 (c)).

-Measurement of compound (3)

When the compound (3) having a fuminolic hydroxyl group at a binding position different from that of the compound (2) was measured by a full scan in the positive ion mode without applying an ECD voltage, the molecular weight-related ion was measured. M + H] ⁺ was found at m / z 455 (FIG. 5 (a)).

When compound (3) was measured with an ECD voltage of 50 OmV oxidation potential, mZz469 with a large mass number of 14, m / z 951 which is a dimer-related peak, and other peaks appeared (Fig. 5). (b)). When the voltage is set to 55 OmV, the peak of mZz 455 further decreases. Compound (2) Unlike the case of (1), it did not completely disappear (Fig. 5 (c)), and was the same even when the voltage was applied up to 700mV. When measured at a reduction potential of one 80 OmV, as in the case of compound (1), no change was observed in the peak of mZz 455 for the molecular weight-related ion [M + H] ⁺ (Fig. 5 (cl)). .

Furthermore, when compound (3) was similarly measured in negative ion mode, [M—H] —, a molecular weight-related ion, was found in mZz 453 (FIG. 6 (a)).

When compound (3) was measured at an ECD voltage of 50 OmV, unlike the positive ion mode, the peak of mZz 453 almost disappeared, and instead, the peak of mZz 467 and the dimer-related peak with a mass number of 14 increased. Certain mZz 92.1 and other peaks appeared (Fig. 6 (b)). At 70 OmV, the dimer-related peak became slightly larger (Fig. 6 (c-)).

'' Measurement of compound (4)

The compound (4) having a methoxy group instead of the phenolic hydroxyl group of the compound (2) was measured.

First, when compound (4) was measured by full-scan in positive ion mode without applying ECD voltage, [M + H] ⁺ , a molecular weight-related ion, was observed at m / z 469 (Fig. 7 (a)).

When the compound (4) was measured with an ECD voltage of 80 OmV or 90 OmV oxidation potential or -80 OmV reduction potential, there was no change in the peak of mZz 469 (Fig. 7 (b) ~ (D)).

Furthermore, when compound (4) was similarly measured in negative ion mode, [M_H] —, a molecular weight-related ion, was observed, but the ionic strength was weak.

As is clear from the above measurement results, the compounds (2) and (3) having a phenolic hydroxyl group, which is a functional group that is easily oxidized electrochemically, have a mass number of 14 by applying an oxidation potential. The change to a compound provided structural information that could not be obtained with MS alone. That is, structural information as to whether or not the compound is easily oxidized or reduced electrochemically was obtained by simple online measurement.

In addition, for compound (2), the voltage of the ECD especially in the negative ion mode By applying 70 OmV, the peak force of mZz 467 was about 15 times that of mZz 453 when OmV was applied, and the effect of enhancing ionic strength was recognized.

Furthermore, as shown in the measurement results of compounds (2) and (3), even if the compound has a phenolic hydroxyl group, a difference was observed in the peak if the bonding position was different. It was suggested that the location information could be obtained by the accumulation.

Embodiment 2

The sample was measured in the same manner as in the first embodiment.

Measurement sample

Zotepine (compound (5)) represented by the following structural formula was used as a measurement sample.

Compound (5)

measuring device

The same device as that used in the first embodiment was used.

Measurement method and results

The mobile phase of HPLC and the flow rate of the mobile phase were set in the same manner as in the first embodiment. Degassing was performed with an online degasser attached to HP LC.

The measurement sample was prepared by dissolving the above compound (5) in acetonitrile so that the concentration of the compound (5) became 50 / xM, diluting it with about 40% acetonitrile aqueous solution, and using a syringe to prepare 5 L / min. The mobile phase was injected before it was introduced into the ECD at a rate.

The MS used the electrospray method (ESI) as an interface under the same conditions as in the first embodiment. • Measurement of compound (5)

First, compound (5) was introduced into MS without passing through ECD, and was measured by full scan in positive ion mode. As a result, [M + H] +, a molecular weight-related ion, was found in mZz332 (Fig. 8).

When compound (5) was measured in the negative ion mode in the same manner, [M—H] —, which is a molecular weight-related ion, was not observed.

Next, without passing through the ECD, MS / MS by collision for obtaining structural information was performed, and the dominant ion was measured. Even when the collision power was changed to about 15%, a constant fragment peak was observed. Was not obtained (FIGS. 9 (a) to 9 (d)).

Compound (5) was passed through an ECD cell without applying voltage, and MSZMS by collision was performed to measure the daughter ions. When the collision power was changed to about 15%, the fragment of mZz 317 was detected. Peaks have been observed (Fig. 10).

When this fragment was further introduced into MSZMSZMS (collision path: 18%), predetermined fragmentation was observed (FIGS. 11 (a;) to (c) and FIG. 12). 11 (a) to 11 (c) show mass spectra measured individually, and FIG. 12 shows an average of 104 continuous mass spectra on a computer.

Compound (5) was applied to the MS at an ECD voltage of 70 OmV, measured in the positive ion mode, and a new peak was found at mZz 330, which has a smaller mass number by 2 as a molecular weight-related ion. This peak becomes the main peak when the voltage is increased to 800 mV or more (Figs. 13 (a) and (b)), and the ion intensity is about 10 times that of when not passing through the ECD cell (see Fig. 8). It's big.

When the ECD voltage was applied at 80 OmV and MS / MS by collision was performed to measure the dormancy, a fragment peak of mZz 315 was observed when the collision power was changed to about 15%. (Fig. 14).

When this fragment was further introduced into MSZMSZMS (collision power 18%), the ion intensity of the fragment peak was large and stable. (Figs. 15 (a) to (c) and Fig. 16). FIGS. 15 (a) to 15 (c) show mass spectra measured individually, and FIG. 16 shows an average of 35 continuous mass spectra on a computer. As is evident from the above measurement results, by passing the voltage through the ECD cell and further applying a voltage, the intensity is increased, that is, the sensitivity is improved, as compared to the MS peak measured without applying the ECD. I was able to plan.

In addition, even for compounds where MSZMS fragmentation is difficult to obtain stably, the fragmentation as structural information can be stabilized and the peak intensity can be increased by applying a voltage to the cell through the ECD cell and further applying a voltage. Was completed.

Furthermore, a voltage was applied to the cell through the ECD cell, and a new peak with a small mass number of 2 appeared as a molecular weight-related ion, and a peak with a high ionic strength was obtained. The singularity and sensitivity of the device were dramatically improved.

Embodiment 3

The sample was measured in the same manner as in the first embodiment.

Measurement sample

Hydroxyacetamides having different hydroxyl positions (compounds (6), (7) and (8)) represented by the following structural formulas were used as measurement samples.

0, NH

Compound (8) The same device as that used in the first embodiment was used.

Measurement method and results

The mobile phase of HPLC and the flow rate of the mobile phase were set in the same manner as in Embodiment 1. Degassing was performed using an online degasser attached to HPLC.

The measurement sample is prepared by dissolving with acetonitrile so that the concentration of each of the above compounds becomes 1 mM, then diluting with about 40% aqueous acetonitrile solution, and introducing it into the CCD at a rate of 5 LZ using a syringe. Injected into the previous mobile phase.

The MS used an electrospray method (ESI) as an in-face, and was used under the same conditions as in the first embodiment.

• Measurement of compound (6)

First, when a compound (6) having a hydroxyl group at the 2-position (o-position) was measured by full scan in the positive ion mode without applying an ECD voltage, it was found that the molecular weight-related ion [M + H] + Was found in m-z 152 (FIG. 17 (a)).

Next, when the ECD voltage was set to the oxidation potential of 30 OmV, the peak of mZz 152 did not change, but when the ECD voltage was set to 40 OmV, mZz 299, 257, and 2 1 were used instead of the peak of mZz 152. 5 peaks began to be observed, and the ionic strength increased about 10 times (Fig. 17b).

• Measurement of compound (7)

First, when a compound (7) having a hydroxyl group at the 3-position (m-position) was measured by full scan in the positive ion mode without applying an ECD voltage, it was found that the molecular weight-related ion [M + H] + Was found in mZz 152 (FIG. 18 (a)).

Next, when the compound (7) was measured by applying an ECD voltage up to an oxidation potential of 55 OmV, unlike the compound (6), the mass spectrum was hardly changed (Fig. 18 ( c)).

When the ECD voltage was increased to 70 OmV, mZz 299 and other peaks were observed instead of the peak at m / z 152, and the ion intensity increased about 4 times (Fig. 18b).

-Measurement of compound (8)

First, a compound (8) having a hydroxyl group at the 4-position (p-position) was measured by full scan in positive ion mode without applying an ECD voltage. A linked ion, [M + H] +, was observed at m / z 152 (FIG. 19 (a)). Next, when the compound (8) was measured by applying an ECD voltage of 20 OmV, there was no change in the peak of mZz 152 (FIG. 19 (b)). MZz 2 observed in (6) and (7)

The peak of mZz 301 having a mass number 2 larger than 99 was observed (FIG. 19 (c)). The ionic strength at 40 OmV was about 6 times (Fig. 19 (d)).

As is clear from the above measurement results, the compounds (6), (7), and (8), which have different phenolic hydroxyl groups, which are functional groups that are easily oxidized electrochemically, have oxidation potentials whose peaks change. In addition, differences were found in the mass numbers of the changed compounds, and it was possible to obtain structural information on binding positions that cannot be obtained by MS alone.

In addition, it was found that the application of the oxidation potential of the ECD increased the ion intensity of the obtained peak, thereby improving the sensitivity in quantification by LC_MS.

Industrial applicability

According to the present invention, in a method of subjecting a sample to a mass spectrometer for analysis, the sample is electrochemically treated and then subjected to a mass spectrometer, whereby a molecule is converted from a fragment ion measured by the mass spectrometer. The structure (structural information) can be stably obtained, and an analysis method with higher accuracy can be provided in terms of specificity and sensitivity.

Further, according to the present invention, in a method of subjecting a sample to a mass spectrometer for analysis, the sample is electrochemically treated and then subjected to a mass spectrometer to obtain a mass spectrum. By comparing the sample with a mass spectrum obtained by subjecting the sample to a mass spectrometer without subjecting the sample to electrochemical treatment, the sample is susceptible to the electrochemical treatment, that is, a function that is easily oxidized or reduced. The presence of a group is expressed as a change in mass number, so that it is possible to obtain structural information on molecules that cannot be obtained by simply attaching it to a mass spectrometer, and to provide a more reliable and useful analytical method in terms of sensitivity. Can be provided.

Claims

The scope of the claims

1. A method for analyzing a sample by subjecting the sample to a mass spectrometer, comprising subjecting the sample to electrochemical treatment and then subjecting the sample to a mass spectrometer.

2. In a method of subjecting a sample to mass spectrometry for analysis, the sample is electrochemically treated and then subjected to a mass spectrometer to obtain a mass spectrum, and the mass spectrum is directly transferred to the sample. A method for analyzing a sample consisting of comparing it with a mass spectrum obtained by applying it to a mass spectrometer.

3. The sample analysis method according to claim 1 or 2, wherein the sample is electrochemically treated using an electrochemical detector.

4. The method for analyzing a sample according to any one of claims 1 to 3, wherein the sample is obtained by liquid chromatography.

5. The method according to any one of claims 1 to 4, wherein the sample is applied to a mass spectrometer in a liquid state.

6. A sample analyzer that incorporates a device capable of electrochemically processing a sample into the sample inlet of the mass spectrometer.

7. The sample analyzer according to claim 6, wherein a liquid chromatograph is further incorporated into an introduction portion of an apparatus capable of electrochemically processing the sample.