Estrogen derivatization method, mass spectrometry method, derivatization reagent, and derivatization reagent kit

Abstract

To provide a novel technique for improving the detection sensitivity of estrogen in an analytical method in which estrogen is ionized.SOLUTION: The present invention provides an estrogen derivatization method using a quaternary cation-containing 5-fluoro-2,4-dinitrophenyl compound represented by Formula (1) [where X is a quaternary cation].SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for derivatizing an estrogen, a mass spectrometry method, a derivatization reagent, and a derivatization reagent kit. [Background technology]

[0002] Liquid chromatography tandem mass spectrometry (LC-MS / MS) and matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) are extremely powerful analytical techniques for quantifying trace amounts of metabolites and other substances. In these analytical techniques, the target compound is ionized. In analyses in which the target compound is ionized, some target compounds have low ionization efficiency, and there are many cases in which the target compound cannot be detected or the detection accuracy for quantification cannot be ensured. Therefore, the target compound may be derivatized to increase the detection sensitivity.

[0003] For example, a derivatization reagent for hydroxysteroids is disclosed in the following Non-Patent Document 1. Non-Patent Document 1 also discloses a technique that can improve the detection sensitivity of estrogen by derivatizing estrogen under specific conditions, compared to the case where estrogen is not derivatized. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Development and application of electrospray-active derivatization reagents for hydroxysteroids, Nishio, T.; Higashi, T.; Funaishi, A.; Tanaka, J.; Shimada, K., J. Pharm. Biomed. Anal., 2007, 44, 786-795. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a new method for increasing the detection sensitivity of estrogen in an analytical method in which estrogen is ionized. [Means for solving the problem]

[0006] The present inventors have found that 5-fluoro-2,4-dinitrophenyl compounds containing a quaternary cation (X-DNPF) are suitable for derivatization to enhance the detection sensitivity of estrogens.

[0007] That is, the present invention provides a method for derivatizing an estrogen using a 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation represented by the following formula (1). [ka] [In formula (1), X is a quaternary cation.] In the formula (1), the quaternary cation may be a quaternary ammonium cation. In this case, the quaternary ammonium cation may be a group represented by any one of the following formulae (2) to (4). [ka] [ka] [In formula (3), m is an integer of 1 to 8.] [ka] [In the formula (4), Y is a substituted or unsubstituted linear or branched alkyl group having 1 to 20 carbon atoms.] In addition, in the formula (1), the quaternary cation may be a quaternary phosphonium cation. In this case, the quaternary phosphonium cation may be a group represented by the following formula (5). [ka] [In formula (5), R 1 , R 2 , and R 3 are each independently a group represented by the following formula (6): [ka] In formula (6), m is an integer of 0 to 8. The method for derivatizing the estrogen may further include using an organic base. The organic base may be a tertiary amine. The present invention also provides a method for mass spectrometry, which comprises derivatizing an estrogen by the method for derivatizing an estrogen. The present invention also provides a derivatization reagent for derivatizing an estrogen, comprising a 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation represented by the following formula (1): [ka] [In formula (1), X is a quaternary cation.] The present invention also provides a derivatization reagent kit for derivatizing estrogen, comprising the above-mentioned derivatization reagent. Effect of the Invention

[0008] According to the present invention, it is possible to increase the detection sensitivity of estrogen in analytical methods in which estrogen is ionized, such as mass spectrometry methods. The effects of the present invention are not limited to those described herein, and may be any of the effects described in this specification. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a diagram showing an example of a reaction formula for estrogen derivatization. [Diagram 2] FIG. 1 is a diagram showing an example of a reaction formula for estrogen derivatization. [Diagram 3] This is an SRM chromatogram of MP-DNP-E2 obtained by the conventional two-step derivatization method. [Figure 4] 1 is an SRM chromatogram of MP-DNP-E2 obtained by the one-step derivatization method according to the present invention. [Diagram 5] 1 shows SRM chromatograms of MP-DNP-E2 in E2 standards and pooled serum. [Figure 6] 1 is a graph comparing peak areas in SRM chromatograms of MP-DNP-E2 and Dns-E2 in E2 standards and pooled serum. [Figure 7] FIG. 13 is a comparison of SRM chromatograms of MP-DNP-E2 and Dns-E2 in a low concentration E2 sample. [Figure 8] This is an SRM chromatogram when samples of E1, E2, and E3, each with a concentration of 62.5 pg / mL, were derivatized with MP-DNPF. [Figure 9A] 1 is a plot of the response ratio between the E1 component and the internal standard of E1, and the sample concentration. [Figure 9B] This is a plot of the response ratio between the E2 component and the E2 internal standard, and the sample concentration. [Figure 9C] This is a plot of the response ratio between the E3 component and the E3 internal standard, and the sample concentration. [Figure 10] This is an SRM chromatogram when pooled serum was derivatized with MP-DNPF. [Figure 11] This is an SRM chromatogram when serum containing a standard sample was derivatized with MP-DNPF. [Figure 12] This is an SRM chromatogram when pooled serum was derivatized with MP-DNPF. [Figure 13]NMR spectrum data. [Figure 14] NMR spectrum data. [Figure 15] NMR spectrum data. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The following describes preferred embodiments of the present invention. However, the present invention is not limited to the following preferred embodiments and can be freely modified within the scope of the present invention.

[0011] The present invention will be described in the following order. 1. Description of the Invention 2. First embodiment (method of derivatizing estrogen) 3. Second embodiment (mass spectrometry method) 4. Third embodiment (derivatization reagent) 5. Fourth embodiment (derivatization reagent kit) 6. Working Example

[0012] 1. Description of the Invention

[0013] Mass spectrometers such as the LC-MS / MS and MALDI-MS mentioned above are extremely powerful analytical methods for quantifying trace amounts of metabolites and other substances. However, depending on the compound, the ionization efficiency is low, so in many cases it is impossible to detect them, or it is possible to detect them but not with enough accuracy to enable quantification.

[0014] For samples that are available in a certain amount, such as foods and supplements, it is possible to cover the lack of sensitivity by increasing the amount of sample. However, for such samples, it is desirable to be able to detect or quantify the target compound with a smaller amount of sample.

[0015] For target compounds such as human metabolites, it is often not easy to increase the amount of sample containing the target compound, and it is therefore very important to increase the ionization efficiency and thus the detection sensitivity for such target compounds.

[0016] In order to improve detection sensitivity, not only have equipment such as mass spectrometers been improved to be more sensitive, but also target compounds have been modified (derivatized) to increase ionization efficiency. Generally, biological metabolites are organic compounds mainly composed of carbon, hydrogen, oxygen, and nitrogen, and oxygen and nitrogen atoms with unshared electron pairs are easily added with hydrogen ions (protons), resulting in high ionization efficiency. Nitrogen atoms in particular have a higher proton addition ability than oxygen atoms, and compounds containing nitrogen atoms tend to be more easily ionized than compounds that do not contain them.

[0017] Estrogen, known as a female hormone, does not contain a nitrogen atom, and therefore, in order to improve the detection sensitivity of estrogen, a technique for introducing a nitrogen atom by derivatization using a derivatization reagent containing a nitrogen atom has been developed. For example, 5-fluoro-2,4-dinitrophenyl compounds having various substituents at the 1-position have high electrophilicity at the 5-position due to the very strong electron-attracting effect of the two nitro groups at the 2- and 4-positions on the benzene ring. Taking advantage of this property, a derivatization reagent has been developed in which a substituent having a nitrogen atom is placed at the 1-position (see Non-Patent Document 1 above).

[0018] In the technology described in Non-Patent Document 1, 1-(5-fluoro-2,4-dinitrophenyl)-4-methylpiperazine (P-DNPF), which has a tertiary amine methylpiperazine at the 1-position, is reacted with estrogen in the presence of sodium bicarbonate, and then derivatized with methyl iodide (MeI) (MP-DNP-estrogen). This achieves an approximately 2000-fold improvement in detection sensitivity compared to the case without derivatization.

[0019] In response to this, the present inventors have found that the use of a 5-fluoro-2,4-dinitrophenyl compound (X-DNPF) containing a quaternary cation can enhance the detection sensitivity of estrogen in an analytical method in which estrogen is ionized. That is, the present invention provides a method for derivatizing estrogen using a 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation.

[0020] The method for derivatizing estrogen according to the present invention can perform derivatization in one step, which has been conventionally performed in two steps. Specifically, in the derivatization method disclosed in the above-mentioned Non-Patent Document 1, derivatization with P-DNPF is followed by quaternization with methyl iodide, that is, derivatization of estrogen is performed by a two-step reaction. On the other hand, the method for derivatizing estrogen according to the present invention can perform derivatization of estrogen in a one-step reaction by using a 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation. In this way, the method for derivatizing estrogen according to the present invention can reduce the number of reactions required for derivatization compared to conventional methods, thereby simplifying the reaction procedure.

[0021] Fig. 1 is a diagram showing an example of a reaction formula for estrogen derivatization in Non-Patent Document 1 and the present invention. As shown in Fig. 1, in the derivatization method of Non-Patent Document 1, an estrogen derivative is obtained by a two-step reaction of a reaction using P-DNPF and an inorganic base, and a reaction using methyl iodide. On the other hand, in the present invention, the same estrogen derivative as in Non-Patent Document 1 can be obtained by a one-step reaction by using a 5-fluoro-2,4-dinitrophenyl compound (represented as X-DNPF in Fig. 1) containing a quaternary cation.

[0022] The method for derivatizing estrogen according to the present invention does not require an inorganic base (sodium bicarbonate) used in the derivatization with P-DNPF in the derivatization method of Non-Patent Document 1, which is believed to contribute to improved detection sensitivity and simplified reaction procedures. In the method for derivatizing estrogen according to the present invention, for example, an organic base can be used instead of an inorganic base to derivatize estrogen. By not using an inorganic base, contamination of the ion source in the analyzer where ionization is performed is reduced, so that detection sensitivity can be improved compared to when an inorganic base is used. In addition, the present inventors considered that the reason why derivatization of estrogen by a one-step reaction was not realized in the derivatization method of Non-Patent Document 1 was due to the use of an inorganic base. In the method for derivatizing estrogen according to the present invention, the fact that the use of an inorganic base is not essential is believed to contribute to the realization of a one-step reaction, that is, the realization of simplified reaction procedures.

[0023] As described above, the present invention makes it possible to derivatize estrogen in a single-step reaction, and further makes it possible to increase the detection sensitivity of estrogen in an analytical device in which ionization is performed.

[0024] Furthermore, the present invention makes it possible to detect trace metabolites using mass spectrometry with high sensitivity, for example, in the field of clinical testing, and also to quantify them with high sensitivity. The present invention can be used regardless of the type of mass spectrometer. For example, in order to widely disseminate the quantification of trace metabolites by mass spectrometry in the field of clinical testing, it is desirable that the same values ​​can be obtained anytime, anywhere, and with any manufacturer's device. The improvement in detection sensitivity according to the present invention can fill the gap between models of each manufacturer, thereby supporting the dissemination of the quantification of trace metabolites by mass spectrometry.

[0025] The invention will now be described in more detail.

[0026] 2. First embodiment (method of derivatizing estrogen)

[0027] The present invention provides a method for derivatizing an estrogen using a 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation represented by the following formula (1). That is, the method for derivatizing an estrogen includes a step of derivatizing an estrogen using a 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation represented by the following formula (1). By the method for derivatizing an estrogen according to the present invention, an estrogen derivative that can be detected with high sensitivity can be obtained in a one-step reaction.

[0028] 2-1. 5-Fluoro-2,4-dinitrophenyl compounds containing a quaternary cation of formula (1)

[0029] The 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation used in the above-mentioned method for derivatizing estrogen is represented by the following formula (1).

[0030] [ka] [In formula (1), X is a quaternary cation.]

[0031] In one embodiment, the quaternary cation may be a quaternary ammonium cation. The quaternary ammonium cation may be a group represented by any one of the following formulae (2) to (4). From the viewpoint of further increasing the detection sensitivity of estrogen in an analysis method in which ionization is performed, the quaternary cation is preferably a group represented by the following formula (2).

[0032] [ka] [ka] [In formula (3), m is an integer of 1 to 8.] [ka] [In the formula (4), Y is a substituted or unsubstituted linear or branched alkyl group having 1 to 20 carbon atoms.]

[0033] In Y of formula (4), the number of carbon atoms in the linear or branched alkyl group may be, for example, 1 to 18, 1 to 16, 1 to 14, 1 to 12, or 1 to 10. This number of carbon atoms does not include the number of carbon atoms in the substituent.

[0034] As an example, the linear or branched alkyl group in Y of formula (4) may be an unsubstituted alkyl group, i.e., an alkyl group having only hydrogen attached to the carbon chain. As another example, the linear or branched alkyl group in Y of formula (4) may be a substituted alkyl group, i.e., one or more substituents may be attached to the carbon chain in addition to hydrogen. Examples of the substituents include, but are not limited to, alkoxy groups, aryl groups, hydroxyl groups, and halogen atoms. When Y has two or more substituents, they may be the same or different.

[0035] In another embodiment, the quaternary cation may be a quaternary phosphonium cation. The quaternary phosphonium cation may be a group represented by the following formula (5).

[0036] [ka] [In formula (5), R 1 , R 2 , and R 3 are each independently a group represented by the following formula (6): [ka] In formula (6), m is an integer of 0 to 8.

[0037] In formula (6), m may be, for example, 0 to 6, 0 to 4, 0 to 2, or 1.

[0038] 2-2. Organic bases

[0039] In the above-mentioned method for derivatizing estrogen, an organic base may be further used. That is, the above-mentioned method for derivatizing estrogen may include a step of derivatizing estrogen using a 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation of formula (1) and an organic base. The organic base functions as a catalyst in the derivatization reaction of estrogen using the 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation of formula (1).

[0040] The organic base may, for example, be a tertiary amine, such as 4-dimethylaminopyridine or quinuclidine.

[0041] Estrogen

[0042] The compound to be derivatized by the 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation of formula (1) is estrogen. Estrogen is a type of female hormone and is also called follicular hormone. Generally, estrogens are known to include estrone (E1), estradiol (E2), estriol (E3), and estetrol (E4).

[0043] The estrogen as the compound to be derivatized may be at least one, two, or three selected from estrone, estradiol, estriol, and estetrol, or may be four of these. Alternatively, the estrogen as the compound to be derivatized may be at least one or two selected from estrone, estradiol, and estriol, or may be three of these.

[0044] The estrogen may be, for example, that contained in a biological sample. The biological sample that may contain the estrogen to be derivatized according to the present invention may be, for example, a body fluid or a sample derived from a body fluid, more specifically, any of serum, plasma, blood, urine, cerebrospinal fluid, and saliva. Such a biological sample may be subjected to a predetermined pretreatment according to each analysis method as necessary, and then the estrogen may be derivatized according to the present invention.

[0045] 2-4. Derivatization of estrogen

[0046] In the above-mentioned method for derivatizing an estrogen, the derivatization of an estrogen can be carried out by reacting an estrogen with a 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation of formula (1), and, if necessary, with the above-mentioned organic base.

[0047] An example of a reaction scheme for estrogen derivatization by a 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation of formula (1) is shown in FIG. 2. In the figure, formula (7) represents estrogen, specifically estrone (E1), estradiol (E2), and estriol (E3). Also, formula (8) represents an estrogen derivative. As shown in the figure, estrogens (estrone, estradiol, and estriol) are derivatized by the compound of formula (1).

[0048] 3. Second embodiment (mass spectrometry method)

[0049] The present invention also provides a mass spectrometry method comprising derivatizing estrogen by the above-described estrogen derivatization method. That is, the mass spectrometry method comprises a derivatization step of derivatizing estrogen by the estrogen derivatization method. Details of the estrogen derivatization method are as described in 2 above, and the description also applies to this embodiment. Derivatizing estrogen can increase the detection sensitivity of estrogen in mass spectrometry.

[0050] The mass spectrometry method includes a mass spectrometry step after the derivatization step, specifically, the mass spectrometry step includes a mass spectrometry step after the derivatization step, the mass spectrometry step including ionizing the derivatized estrogen.

[0051] The ionization may be, for example, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), or matrix-assisted laser desorption ionization (MALDI) for more efficient ionization, but may also be ionization by other ionization methods.

[0052] By ionizing derivatized estrogens using these ionization methods, it is possible to measure or observe the quantification of estrogen and the distribution (imaging) of estrogen in tissues or cells with greater accuracy and higher resolution.

[0053] The mass spectrometry step may be performed, for example, by a liquid chromatography tandem mass spectrometer (LC-MS / MS) or a matrix-assisted laser desorption / ionization mass spectrometer (MALDI-MS), but the apparatus for performing the mass spectrometry step is not limited to these. Specific procedures in the mass spectrometry step can be appropriately set by those skilled in the art depending on, for example, the ionization method employed and the apparatus used.

[0054] 4. Third embodiment (derivatization reagent)

[0055] The present invention also provides a derivatization reagent for derivatizing an estrogen, comprising the 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation represented by the above-described formula (1). Details of the 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation represented by the formula (1) and the estrogen are as described in 2. above, and the description also applies to this embodiment.

[0056] The above-mentioned derivatization reagent may contain a solvent in addition to the 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation represented by formula (1). That is, in the derivatization reagent, the 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation represented by formula (1) may be present in a solvent. The solvent may be an organic solvent, for example, acetonitrile, acetone, tetrahydrofuran, dichloromethane, or dimethoxyethane.

[0057] The above-mentioned derivatization reagent may be used, for example, in the estrogen derivatization method described in 2. above, or in the mass spectrometry method described in 3. above.

[0058] 5. Fourth embodiment (derivatization reagent kit)

[0059] The present invention also provides a derivatization reagent kit for derivatizing estrogen, comprising the above-described derivatization reagent. The details of the derivatization reagent are as described in 4. above, and the description also applies to this embodiment.

[0060] The above-mentioned derivatization reagent kit may further include an organic base. Examples of the organic base include tertiary amines. Examples of the tertiary amines include 4-dimethylaminopyridine (DMAP) and quinuclidine.

[0061] The above-mentioned derivatization reagent kit may be used, for example, in the estrogen derivatization method described in 2. above, or in the mass spectrometry method described in 3. above.

[0062] When the derivatization reagent kit is used in a mass spectrometry method, the derivatization reagent kit may further include other reagents that can be used in mass spectrometry of estrogen. The other reagents may include, for example, at least one of a calibration solution, an internal standard, and a positive control (e.g., a control serum).

[0063] In the above-mentioned derivatization reagent kit, the 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation represented by formula (1), the organic base, and other reagents may be in any form such as solid or liquid. For example, one or more reagents contained in the derivatization reagent kit may be in a state dissolved in any solvent. Alternatively, the derivatization reagent kit may contain a reagent and a solvent for dissolving the reagent. In this case, the reagent can be used after dissolving when needed. EXAMPLES

[0064] 6. Working Example The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0065] 6-1. Synthesis examples 1-(5-fluoro-2,4-dinitrophenyl)-4-dimethylpiperazinium iodide (hereinafter referred to as "MP-DNPF") used in the following examples was synthesized. The synthesis was carried out according to a known document (Development and application of electrospray-active derivatization reagents for hydroxysteroids, T. Nishio et al., Journal of Pharmaceutical and Biomedical Analysis, 44 (2007) 786-795). The synthesized MP-DNPF was a compound represented by the above formula (1), and X in formula (1) was a group represented by the above formula (2).

[0066] 6-2. Example 1 As described above with reference to Figure 1, the conventional technique derivatizes estrogen through a two-step reaction, whereas the method for derivatizing estrogen according to the present invention derivatizes estrogen through a one-step reaction. Therefore, a test was conducted to compare the sensitivity of the conventional two-step derivatization with that of the one-step derivatization according to the present invention.

[0067] [I. Conventional two-step derivatization (derivatization with P-DNPF + quaternization)] 1. Derivatization with P-DNPF 1) A solution of 1-(5-fluoro-2,4-dinitrophenyl)-4-methylpiperazine (P-DNPF) in acetonitrile (2 mg / mL, 20 μL) was added to a solution of estradiol (E2) (400 fg) in acetonitrile (20 μL). 2) Further, an aqueous solution of sodium hydrogen carbonate (1 M, 20 μL) was added. 3) Allow to stand at 60°C (60 minutes). 2. Desalting using solid-phase extraction cartridges 1) Strata TM A -X cartridge (60 mg) was conditioned sequentially with ethyl acetate (2 mL), methanol (2 mL), and water (2 mL). 2) The above derivatization reaction solution was diluted with methanol / water (50:50, v / v, 500 μL) and loaded onto the cartridge. 3) The cartridge was washed with water (2 mL). 4) P-DNP-E2 was eluted with ethyl acetate (1 mL). 5) The solvent was distilled off under a nitrogen stream to obtain P-DNP-E2. 3. Quaternization with methyl iodide 1) Iodomethane (100 μL) was added to P-DNP-E2. 2) Allow to stand at 60°C (30 minutes). 3) Excess iodomethane was distilled off under a nitrogen stream to obtain MP-DNP-E2. 4) The mixture was dissolved in mobile phase (40 μL) and used as a sample for LC-MS / MS analysis. 5) 10 μL of the sample was injected into the LC-MS / MS.

[0068] [II. One-step derivatization according to the present invention] 1) An acetonitrile solution of MP-DNPF (2 mg / mL, 20 μL) was added to an acetonitrile solution (20 μL) of E2 (200 fg). 2) Further, an acetonitrile solution of dimethylaminopyridine (DMAP) (1 mg / mL, 20 μL) was added. 3) It was allowed to stand at 60 °C (for 15 minutes). 4) The solvent was distilled off under a nitrogen stream to obtain MP-DNP-E2. 5) It was dissolved in the mobile phase (40 μL) to prepare a sample for LC-MS / MS analysis. 6) Of the sample, 10 μL was injected into LC-MS / MS.

[0069] [III. LC-MS / MS Analysis Conditions] <LC Analysis Conditions> Apparatus: Shimadzu LC-30AD Column: YMC-UltraHT Pro C18 (2.0 μm, 100 × 2.0 mm i.d.) Mobile phase: Methanol - 10 mM ammonium formate - formic acid (60:40:0.1, v / v / v) Flow rate: 0.3 mL / min <MS / MS Analysis Conditions> Apparatus: Shimadzu LCMS-8030 plus Ionization method: Positive ion detection ESI Collision activation energy: -43 eV SRM: m / z 551.2 → 504.3

[0070] The SRM chromatogram of MP-DNP-E2 obtained by the conventional two-step method described in I. above (injecting 100 fg (detection limit, S / N = 3) as E2) is shown in Figure 3. The SRM chromatogram of MP-DNP-E2 obtained by the one-step method described in II. above (injecting 50 fg (detection limit, S / N = 3) as E2) is shown in Figure 4.

[0071] As shown in Figures 3 and 4, when E2 was derivatized according to the present invention, the noise was about half that of the case derivatized by the conventional two-step method. From this result, it can be seen that the detection sensitivity of E2 can be improved by about 2 times according to the present invention.

[0072] 6-3. Example 2 A test was conducted to detect E2 in the E2 standard product and pooled serum using MP-DNPF.

[0073] [I. Derivatization with MP-DNPF] <Sample pretreatment> Sample pretreatment was performed according to the following procedure. 1. SLE (solid-liquid) extraction 1) 100 μL of serum and 100 μL of water were mixed. 2) 200 μL of the serum dilution was loaded onto an SLE column. 3) 600 μL of hexane / ethyl acetate (50 / 50, v / v) was added and eluted. This was repeated 3 times. 2. Drying: Nitrogen blowing or centrifugal concentration for about 20 minutes 3. Derivatization 1) MP-DNPF was dissolved in acetonitrile to a solution of 2 mg / mL. 2) 20 μL of DMAP (1 mg / mL, acetonitrile solution), 10 μL of the MP-DNPF solution, and 20 μL of acetonitrile were added to the dried sample. 3) It was left standing at 60 °C (for 15 minutes). 4. Drying: Nitrogen blowing or centrifugal concentration for about 10 minutes 5) 100 μL of 50% (v / v) aqueous acetonitrile solution was added to prepare a sample for LC-MS / MS analysis.

[0074] <LC analysis conditions> The LC analysis conditions were as follows. Apparatus: Waters ACQUITY UPLC I-Class Analysis column: Waters ACQUITY UPLC BEH C18 1.7 mm I.D × 50 mm Elution conditions: Flow rate 0.3 mL / min Solvent A: 0.1% formic acid - water B: 0.1% formic acid - acetonitrile Details of the elution conditions were as shown in the following table.

[0075]

Table 1

[0076] <MS / MS Analysis Conditions> The MS / MS analysis conditions were as follows. Instrument: Waters Xevo TQ-XS triple quadrupole mass spectrometer Ionization condition: ESI positive ion mode The SRM parameters were as shown in the following table.

[0077]

Table 2

[0078] [Derivatization with II.DnsCl] <Sample Pretreatment> Sample pretreatment was performed according to the following procedure. 1. SLE (solid-liquid) extraction 1) 100 μL of serum and 100 μL of water were mixed. 2) 200 μL of the serum dilution solution was loaded onto an SLE column. 3) 600 μL of hexane / ethyl acetate (50 / 50, v / v) was added and eluted. This was repeated 3 times. 2. Drying: Nitrogen blowing or centrifugal concentration for about 20 minutes 3. Derivatization 1) The derivatization reagent Dansyl chloride (DnsCl) was dissolved in acetonitrile to a solution of 1 mg / mL. 2) 50 μL of an aqueous sodium bicarbonate solution (0.1 M) and 50 μL of the DnsCl solution were added to the dried sample. 3) It was allowed to stand at 60 °C for 10 minutes. 4) It was used as a sample for LC-MS / MS analysis.

[0079] <LC Analysis Conditions> The LC analysis conditions were as follows. Equipment: Waters ACQUITY UPLC I-Class Analytical column: Waters ACQUITY UPLC BEH C18 1.7 mm I.D × 50 mm Elution conditions: Flow rate 0.3 mL / min, Solvent A: 0.1% formic acid - water, Solvent B: 0.1% formic acid - acetonitrile The detailed elution conditions were as shown in the following table.

[0080]

Table 3

[0081] <MS / MS analysis conditions> The MS / MS analysis conditions were as follows. Equipment: Waters Xevo TQ-XS triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode The SRM parameters were as shown in the following table.

[0082]

Table 4

[0083] <Analysis results> Figure 5 is the SRM chromatogram of MP-DNP-E2 after derivatization of E2 standard (100 pg / mL) and pooled serum with MP-DNPF respectively. Figure 6 is a graph comparing the peak areas in the SRM chromatograms of MP-DNP-E2 and Dns-E2 after derivatization of E2 standard (100 pg / mL) and pooled serum with MP-DNPF or DnsCl respectively. Figure 7 is a comparison of the SRM chromatograms of MP-DNP-E2 and Dns-E2 after derivatization of a sample prepared by diluting pooled serum 10-fold with E2-free serum (MSG3000, Golden West Diagnostic) to a low concentration of E2 with MP-DNPF or DnsCl.

[0084] In the results shown in Figure 6, the peak area is smaller when derivatized with MP-DNPF than when derivatized with the conventional reagent DnsCl. However, the background noise is lower when derivatized with MP-DNPF. By derivatizing E2 according to the present invention, the background noise can be reduced compared to when DnsCl is used.

[0085] 7, in the case of a sample prepared with a low concentration of E2, the S / N ratio in the SRM chromatogram was about 10 times higher for the MP-DNP derivatization than for the Dns derivatization. This result shows that the present invention has good detection sensitivity when the E2 concentration is low. Compared with Dns derivatization, MP-DNP derivatization of pooled serum can significantly reduce the background noise level in SRM chromatograms, enabling highly sensitive analysis in the low E2 concentration range.

[0086] Example 3 Using MP-DNPF, the detection and quantification of estrone (E1), estradiol (E2), and estriol (E3) standards were confirmed.

[0087] [Derivatization with MP-DNPF] <Sample pretreatment> The sample was pretreated according to the following procedure. 1. Sample preparation 1) Measurement samples were prepared in 50% (v / v) acetonitrile aqueous solution so that the concentrations of E1, E2, and E3 were each 1000 pg / mL. 2) The measurement samples were further diluted with 50% (v / v) acetonitrile aqueous solution to prepare samples of 6.25 pg / mL, 12.5 pg / mL, 25 pg / mL, 62.5 pg / mL, 125 pg / mL, 250 pg / mL, and 500 pg / mL. 3) After mixing 100 μL of the sample prepared in 1) and 2) above with 100 μL of water, E1- 13 C 3 , E2- 13 C 3 , E3- 13 C 3 was added to the mixed solution at a concentration of 1 ng / mL each in a 50% (v / v) aqueous acetonitrile solution as an internal standard, and the mixture was stirred well. 2. Drying: Nitrogen blowing for about 20 minutes 3. Derivatization 1) MP-DNPF was dissolved in acetonitrile to form a 2 mg / mL solution. 2) 20 μL of DMAP (1 mg / mL, acetonitrile solution), 10 μL of MP-DNPF solution, and 20 μL of acetonitrile were added to the dried sample. 3) It was allowed to stand at 60 °C for 15 minutes. 4) Drying: Nitrogen blowing for about 10 minutes 5) 100 μL of 50% (v / v) aqueous acetonitrile solution was added to prepare a sample for LC-MS / MS analysis.

[0088] <LC analysis conditions> The LC analysis conditions were as follows. Apparatus: Waters ACQUITY UPLC I-Class Analysis column: Waters ACQUITY UPLC BEH C18 1.7 mm I.D × 50 mm Elution conditions: Flow rate 0.3 mL / min Solvent A: 0.1% formic acid - water B: 0.1% formic acid - acetonitrile The detailed elution conditions were as shown in the following table.

[0089]

Table 5

[0090] <MS / MS analysis conditions> The MS / MS analysis conditions were as follows. Equipment: Waters Xevo TQ-XS triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode The SRM parameters were as shown in the table below.

[0091] [Table 6]

[0092] <Analysis results> Figure 8 shows the SRM chromatograms of samples of E1, E2, and E3, each with a concentration of 62.5 pg / mL, derivatized with MP-DNPF. 9A to 9C are diagrams in which the response ratios of each of the components E1, E2, and E3 to their internal standards, and the sample concentrations are plotted.

[0093] The analysis results show that the derivatization of E1, E2, and E3 with MP-DNPF is possible, and that these components can be detected simultaneously in a concentration-dependent manner. This shows that the present invention allows the derivatization of E1, E2, and E3, and that the present invention allows the simultaneous detection of E1, E2, and E3 in a concentration-dependent manner.

[0094] 6-5. Example 4 Tests were performed to detect E1, E2, and E3 in pooled serum and serum spiked with standard samples.

[0095] [Derivatization with MP-DNPF] <Sample pretreatment> The sample was pretreated according to the following procedure. 1. SLE (solid-liquid) extraction 1) 100 μL of serum was mixed with 100 μL of water and 50% (v / v) acetonitrile solution (each concentration 1 ng / mL) of E1- as an internal standard. 13 C 3 , E2- 13 C 3 , E3- 13 C 3 30 μL of the mixed solution was added and thoroughly stirred. 2) 10 μL of the standard solution with each concentration of E1, E2, and E3 at 10 ng / mL was added to 90 μL of serum and stirred, and then the same operations as in 1) were performed. 3) 230 μL of the serum diluent was loaded onto the SLE column. 4) 600 μL of hexane / ethyl acetate (50 / 50, v / v) was added and eluted. This was repeated three times. 2. Drying: Nitrogen blowing for about 20 minutes 3. Derivatization 1) MP-DNPF was dissolved in acetonitrile to form a 2 mg / mL solution. 2) 20 μL of DMAP (1 mg / mL, acetonitrile solution), 10 μL of the solution with MP-DNPF, and 20 μL of acetonitrile were added to the dried sample. 3) It was allowed to stand at 60 °C (for 15 minutes). 4) Drying: Nitrogen blowing for about 10 minutes 5) 100 μL of 50% (v / v) aqueous acetonitrile solution was added to prepare a sample for LC-MS / MS analysis.

[0096] <LC analysis conditions> The LC analysis conditions were as follows. Apparatus: Waters ACQUITY UPLC I-Class Analysis column: Osaka Soda CAPCELL CORE C18 2.1 mm I.D × 75 mm Elution conditions: Flow rate 0.3 mL / min Solvent A: 0.1% formic acid - water B: 0.1% formic acid - acetonitrile Details of the elution conditions were as shown in the following table.

[0097]

Table 7

[0098] <MS / MS analysis conditions> The MS / MS analysis conditions were as follows. Apparatus: Waters Xevo TQ-XS triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode The SRM parameters were as shown in the table below.

[0099] [Table 8]

[0100] <Analysis results> FIG. 10 shows an SRM chromatogram of pooled serum derivatized with MP-DNPF. FIG. 11 shows an SRM chromatogram when serum containing a standard sample was derivatized with MP-DNPF.

[0101] As shown in Figure 10, E3 was not detected in the pooled serum (see "MP-DNP-E3" in Figure 10). This is because the concentration of E3 in the pooled serum was very low. On the other hand, as shown in Figure 11, E1, E2, and E3 were simultaneously detected in the serum to which E1, E2, and E3 standard samples were added to the pooled serum. It is believed that E1, E2, and E3 can be analyzed with high sensitivity by derivatizing E1, E2, and E3 in serum according to the present invention.

[0102] Example 5 A study was carried out to quantify E1 and E2 in pooled serum.

[0103] [Derivatization with MP-DNPF] <Sample pretreatment> The sample was pretreated according to the following procedure. 1. SLE (solid-liquid) extraction 1) 100 μL of serum was mixed with 100 μL of water and 50% (v / v) acetonitrile solution (each concentration 10 ng / mL) of E1- as an internal standard. 13 C 3 , E2- 13 C 3 20 μL of the mixed solution was added and thoroughly stirred. 2) E1 and E2 mixed standard samples were prepared to have concentrations of 7.9 pg / mL, 15.9 pg / mL, 31.8 pg / mL, 62.5 pg / mL, 125 pg / mL, and 500 pg / mL respectively in a 50% (v / v) aqueous acetonitrile solution. Then, 100 μL of water and 20 μL of a mixed solution prepared in a 50% (v / v) aqueous acetonitrile solution (10 ng / mL for each concentration) of E1- 13 C 3 , E2- 13 C 3 were added to 100 μL of the mixed standard sample and stirred well. 3) 220 μL of the serum diluent was loaded onto the SLE column. 4) 600 μL of hexane / ethyl acetate (75 / 25, v / v) was added and eluted. This was repeated twice. 2. Drying: Nitrogen blowing for about 20 minutes 3. Derivatization 1) The derivatization reagent MP-DNPF was dissolved in acetonitrile to form a 2 mg / mL solution. 2) 20 μL of DMAP (1 mg / mL, acetonitrile solution), 10 μL of the MP-DNPF solution, and 20 μL of acetonitrile were added to the dried sample. 3) It was allowed to stand at 60 °C for 15 minutes. 4) Drying: Nitrogen blowing for about 10 minutes 5) 50 μL of a 30% (v / v) aqueous acetonitrile solution was added to prepare a sample for LC-MS / MS analysis.

[0104] <LC analysis conditions> The LC analysis conditions were as follows. Instrument: Agilent 1290 Infinity liquid chromatography system Analytical column: Osaka Soda CAPCELL CORE C18 2.1 mm I.D × 75 mm Elution conditions: Flow rate 0.4 mL / min Solvent A: 0.1% formic acid - water B: 0.1% formic acid - acetonitrile The detailed elution conditions were as shown in the following table.

[0105]

Table 9

[0106] <MS / MS Analysis Conditions> The MS / MS analysis conditions were as follows. Apparatus: SCIEX QTRAP4500 Ionization condition: ESI positive ion mode The SRM parameters were as shown in the following table.

[0107]

Table 10

[0108] <Analysis Results> Figure 12 is the SRM chromatogram when pooled serum was derivatized with MP-DNPF.

[0109] From the analysis results, it can be seen that E1 and E2 in the pooled serum can be quantified by the present invention.

[0110] 6 - 7. Example 6 According to the following procedure, (5-fluoro-2,4-dinitrophenyl)triethylphosphonium fluoride (TEP-DNPF) was synthesized.

[0111] 1,5-Difluoro-2,4-dinitrobenzene (100 mg, 0.49 mmol) was dissolved in 1 mL of acetonitrile, stirred in an ice bath, and triethylphosphine-tetrahydrofuran solution (1 M 0.49 mL, 0.49 mmol) was added. A white precipitate immediately formed. After stirring at room temperature for 30 minutes, the mixture was concentrated using a rotary evaporator. The residue was purified by silica gel column chromatography (column size: diameter 2 cm × length 5 cm, 50% ethyl acetate-hexane, 10% methanol-chloroform) to obtain (5-fluoro-2,4-dinitrophenyl)triethylphosphonium fluoride (65 mg, 0.2 mmol). The structure of (5-fluoro-2,4-dinitrophenyl)triethylphosphonium fluoride is represented by the above formula (1), where X in formula (1) is a group represented by the above formula (5), and R in formula (5) is a group represented by the above formula (6). 1 , R 2 , and R 3 are all groups represented by the above formula (6) (m=1). Regarding the obtained (5-fluoro-2,4-dinitrophenyl)triethylphosphonium fluoride, 1 H-NMR measurement, 13 C-NMR measurement, and 31 P-NMR measurements were performed, and the results were as follows: 1 H-NMR (CDCl 3 ) δ 9.07 (d, 4 J FH = 7.2 Hz, 1H), 7.0 (d, 3 J FH = 17.2 Hz, 1H), 2.53 (dq, 2 J PH = 11.2 Hz, 3 J HH = 8 Hz, 6H), 1.27 (dt, 3 J PH = 12 Hz, 3 J HH = 7.6 Hz, 9H) (Fig. 13); 13 C-NMR (CDCl 3) δ 145.3, 137.9, 129.5, 129.4, 127.0, 14.9, 6.8 (Fig. 14); 31 P-NMR (CDCl 3 ) δ 41.0 (Fig. 15).

Claims

1. A method for derivatizing an estrogen using a 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation represented by the following formula (1) and an organic base: 【Chemistry 1】 [In formula (1), X is a quaternary cation.]

2. 2. The method for derivatizing an estrogen according to claim 1, wherein in formula (1), the quaternary cation is a quaternary ammonium cation.

3. 3. The method for derivatizing an estrogen according to claim 2, wherein the quaternary ammonium cation is a group represented by any one of the following formulas (2) to (4): 【Chemistry 2】 【Chemistry 3】 [In formula (3), m is an integer of 1 to 8.] 【Chemistry 4】 [In formula (4), Y is a substituted or unsubstituted linear or branched alkyl group having 1 to 20 carbon atoms.]

4. 2. The method for derivatizing an estrogen according to claim 1, wherein in formula (1), the quaternary cation is a quaternary phosphonium cation.

5. The method for derivatizing an estrogen according to claim 4, wherein the quaternary phosphonium cation is a group represented by the following formula (5): 【Chemistry 5】 In formula (5), R1, R2, and R3 are each independently a group represented by formula (6): 【Chemistry 6】 In formula (6), m is an integer of 0 to 8.

6. 2. The method for derivatizing an estrogen according to claim 1, wherein the organic base is a tertiary amine.

7. A mass spectrometry method, comprising derivatizing an estrogen by the method for derivatizing an estrogen according to any one of claims 1 to 6.

8. A derivatization reagent kit for derivatizing estrogen, comprising a derivatization reagent, The derivatization reagent kit includes a 5-fluoro-2,4-dinitrophenyl compound containing a quaternary cation represented by the following formula (1), and the derivatization reagent kit includes an organic base: 【Chemistry 7】 [In formula (1), X is a quaternary cation.]

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