JP4800048B2 - Method for measuring isotope content - Google Patents

Description

  The present invention relates to a method for accurately measuring a desired isotope content in a compound such as an isotope-labeled compound.

For example, in the field of environmental science research, pharmacokinetic research, and the like, an isotope-labeled compound is a method of preparing an isotope of the target compound to track the movement of the target compound and tracking the movement of the isotope Is widely used. Such an isotope is called an isotope-labeled compound. Usually, an isotope-labeled compound obtained by substituting 1-10 light hydrogens in one molecule of the target compound with deuterium, a carbon atom in one molecule of the target compound ( An isotope-labeled compound in which 1 to 10 carbon atoms are substituted with 13 carbon atoms, and 1 to 5 nitrogen atoms (mass 14) in one molecule of the target compound are substituted with 15 nitrogen atoms. Isotope-labeled compounds.
In isotopically-labeled compounds, for example, the isotope-labeled compound when, prepared using the isotopically labeled compounds of the subject compounds 1 molecule four deuterium substituted isotope of (d ₄ bodies) as a main component , D ₄ isomers, usually isotopes in which two molecules of deuterium are substituted in one molecule of the target compound (d ₂ isomers), isotopes in which 3 deuteriums are substituted (d ₃ isomers), or completely deuterium There is a possibility that an isotope in which hydrogen is not substituted (d ₀ form) is included. As described above, in general, when the isotope-labeled compound contains isotopes having different mass numbers in addition to the desired isotope, the content of the desired isotope in the isotope-labeled compound to be used must be accurately measured in advance. However, there is a problem that the movement of the isotope-labeled compound cannot be traced sufficiently accurately.
As a method for tracking the movement of a target compound using an isotope-labeled compound, for example, an isotope in which three light hydrogens are substituted with deuterium in one molecule of vitamin D ₃ derivative is used as an internal standard substance, and a gas chromatograph is used. The method of measuring from the molecular ion peak area ratio by mass spectrometry (GC-MS) is mentioned.

JP-A-6-341980 [Claim 1, Example]

In Patent Document 1, in an isotope-labeled compound of a vitamin D ₃ derivative, a desired isotope in which three light hydrogens are substituted with deuterium in the molecule, and a mass number in which two in the molecule are substituted is different. The content of isotopes and the like is not disclosed.
For the isotope-labeled compound in which four deuteriums are substituted in one molecule of a commercially available phthalate ester, the present inventors have prepared an isotope (d) in which four deuteriums in the isotope-labeled compound are substituted. We tried to measure the content of ₄ bodies. Specifically, first, the isotope-labeled compound was separated by liquid chromatography and then subjected to mass spectrometry (LC-MS), and the obtained total ion chromatogram was subjected to data processing to obtain a mass spectrum. However, from the peak indicating the mass number corresponding to the isotope (d ₃ body) substituted with 3 deuteriums from the peak indicating the mass number corresponding to the isotope (d ₀ body) in which no deuterium is substituted at all. poor respective S / N ratio, or the peak derived from phthalic acid ester (d ₀ bodies to d ₃ body) can not determination of whether a peak derived from the background, more than 90% of d ₄ body to for the isotope-labeled compounds believed to contain, could not be accurately measured content of d ₄ body to percent.
An object of the present invention is to provide a method for accurately measuring the content of a desired isotope in a compound containing an isotope having a mass number different from that of the desired isotope.

The present invention is a method for measuring the content of any isotope in a compound comprising the following steps (1) to (4).
(1) Step of performing mass analysis after separating a sample containing the compound by chromatography (2) Processing the data of the total ion chromatogram obtained by the mass analysis of (1) to obtain the mass of the component containing any isotope Steps (3) and (2) for collecting spectra Data processing is performed on the mass spectra obtained in steps (3) and (2), and the mass chromatograms obtained in steps (4) and (3) are collected. And calculating the content of any isotope in the compound based on the above.

According to the present invention, even if a compound such as an isotope-labeled compound contains an isotope having a mass number different from that of the desired isotope, the desired isotope content can be accurately measured. For example, the content ratio of ¹ H (light hydrogen) and ² H (deuterium), the content ratio of ¹² C and ¹³ C, the content ratio of ¹⁴ N and ¹⁵ N can be accurately quantified. In addition, even if a small amount of isotopes having different mass numbers are contained, it can be distinguished from the background, so that even a compound consisting of a high-purity isotope can be accurately quantified. Furthermore, even if a mass spectrometer with a low resolution such as an ion trap type or a quadrupole type is used, it can be quantified sufficiently accurately.

The present invention will be described in detail below.
(1) is a step of performing mass spectrometry after separating a sample containing a compound by chromatography, and examples of the chromatography include liquid chromatography, gas chromatography, gel permeation chromatography and the like. Of these, liquid chromatography and gas chromatography are preferable because they can be directly connected to a mass spectrometer for analysis.

The compound measured by the method of the present invention may be any compound that can be separated by the chromatography. Among these, compounds containing isotopes such as deuterium, ¹⁴ C, and ¹⁵ N as main components are preferable because the isotope content can be accurately quantified.

  As ionization methods in mass spectrometry, electron ionization (EI) method, chemical ionization (CI) method, field ionization (FI) method, fast atom collision (FAB) method, matrix-assisted laser desorption ionization (MALDI) method, electrospray Examples include ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI). Among them, the electron ionization (EI) method and the chemical ionization (CI) method are suitable as gas chromatography ionization methods, and the electrospray ionization (ESI) method and atmospheric pressure chemical ionization (APCI) method are suitable for liquid chromatography. Suitable for ionization method.

Examples of the analyzer in mass spectrometry include a magnetic field deflection type, a quadrupole type, an ion trap type, a time-of-flight type, a tandem type, and a Fourier transform ion cyclotron resonance type. Among these, a quadrupole type, an ion trap type, and a time-of-flight type are preferable.
In the examples, Di-2-ethylhexyl Phthalate-3,4,5,6-d ₄ was separated by liquid chromatography (retention time 2.59 minutes, see FIG. 1) and ionized by electrospray ionization (ESI). Then, it was analyzed with an ion trap type mass spectrometer.

(2) is a step of processing the total ion chromatogram obtained by the mass spectrometry of (1) and collecting a mass spectrum of a component containing an arbitrary isotope.
A total ion chromatogram is a chromatogram that detects and records all ions, and its data processing involves the detection and recording of ions that are detected and recorded in the time zone in which components containing any isotope are detected from the total ion chromatogram. This is the operation that appears in the spectrum. The total ion chromatogram of the example is shown in FIG. 2, and the mass chromatograph of the example is shown in FIG. In this example, the d ₄ isomer (mass number 395, [M + H] ⁺ ) in the total ion chromatogram is shown as a peak having a peak top of 395.21.

  The component containing an arbitrary isotope is a component containing the mass number of an arbitrary isotope (hereinafter, sometimes referred to as a target isotope) that is a measurement target of an isotope content rate in a compound such as an isotope-labeled compound. Specifically, the mass range of the target isotope is ± 10 u, preferably ± 5 u. In mass spectrometers with low resolution, such as ion trap type and quadrupole type, each of the target isotopes and isotopes with different mass numbers from the target isotope are analyzed by narrowing the scan range and analyzing in a narrow mass range. By increasing the resolution of the peak corresponding to the mass number, the resolution of the mass spectrum can be improved as compared with the full scan method, and quantification can be performed more accurately.

(3) is a step of collecting the mass chromatogram of each isotope by processing the mass spectrum obtained in (2).
Specifically, first, the peak in the range of m / z = ± 0.1u to ± 0.3u of the largest peak in the mass spectrum of (2), preferably the peak in the range of m / z = ± 0.2u. The mass chromatogram is obtained for. Subsequently, since the mass numbers of the isotopes differ by one, a mass chromatogram of about m / z = ± 10 ± 1 is collected from the mass number of the peak top. In the example, m / z = 395 which is the base peak. .21 ± 0.2 (d ₄ body, mass number ^{395, [M + H] +} ) mass chromatograms in Figure 4, m / z = 394.21 ± 0.2 (d 3 bodies, mass number 394 , [M + H] ⁺ ), and FIG. 5 shows a mass chromatogram of m / z = 393.21 ± 0.2 (d ₂ bodies, mass number 393, [M + H] ⁺ ). 6, the mass chromatogram of m / z = 392.21 ± 0.2 (d ₁ body, mass number 392, [M + H] ⁺ ) is shown in FIG. 7, m / z = 391.21 ± 0.2. A mass chromatogram of (d ₀ isomer, mass number 391, [M + H] ⁺ ) is shown in FIG.

(4) is a step of calculating the content of any isotope in the compound based on the mass chromatogram obtained in (3). Specifically, output values such as the peak area of each isotope are obtained from the obtained mass chromatogram, and the target isotope for the sum of the output values of the target isotope and the isotope different from the target isotope The ratio of the output value can be determined as the content of any isotope in the compound.
As the output value of the mass chromatogram, a peak area of the mass chromatogram or an electric signal value corresponding to the peak area is usually used.

As is apparent from the mass chromatograms of FIGS. 6 to 8, when a certain baseline is not observed, it is determined that no peak exists, and d ₂ bodies (mass number 393, [M + H] ⁺ ), d ₁ bodies It can be seen that there is no isotope of (mass number 392, [M + H] ⁺ ), d ₀ form (mass number 391, [M + H] ⁺ ).
As described above, according to the present invention, it is clear that even if a small amount of isotopes cannot be determined whether a peak exists in the mass spectrum of (2), such a isotope does not exist because a certain baseline is not observed. I can judge.

  Furthermore, by carrying out the steps (1) to (4), compounds and impurities may remain in the column, line, autosampler, ionized portion, etc., so use a solvent-only blank for each measurement ( It is preferable to perform washing by carrying out steps 1) to (4).

<Preparation of sample>
Weigh accurately 0.5 mg of a sample (Kanto Chemical Co., Ltd., for environmental analysis) whose main component is Di-2-ethylhexyl Phthalate-3,4,5,6-d ₄ (hereinafter referred to as d ₄ bodies) Sample A (concentration: about 100 ppm) was dissolved in 5 mL of acetonitrile. Sample A 1.25 mL was accurately weighed and acetonitrile was added to make exactly 5 mL, which was designated as Sample B (concentration: about 25 ppm solution).

<Step (1)>
For sample A, (1) was carried out under the following liquid chromatography conditions. The liquid chromatogram of Sample A is shown in FIG. Mass spectrometry of step (2) was performed on the component having a retention time of 2.6 minutes.
System: Agilent 1100 Series (Agilent Technology, step (2) mass
System including analyzer)
Column: ZORBAX SB-C18 Rapid Resolution Cartridge (3.5μm, 2.1mmφ × 30mm)
Mobile phase: Water / acetonitrile (containing 0.1% acetic acid) = 1: 9 (volume ratio)
Flow rate: 0.2 ml / min
Measurement wavelength: 254 nm (UV)
Column temperature: 40 ° C
Injection volume: 5 μl
Data processing: LC-MS workstation (Qual Browser Ver.1.3)

<Step (2)>
About the said component of the sample A, mass spectrometry was performed on the following conditions.
Mass spectrometer: LCQ DECA XP plus (Thermoelectron, ion trap type)
Ionization method: Electrospray ionization method (ESI)
Ion polarity: Positive
Measurement mode: Zoom scan method with m / z = 388.5 to 398.5 Data processing: LC-MS workstation (Qual Browser Ver.1.3)

<Step (3)>
Wherein the data obtained in, d ₄ body (mass number ^{395, [M + H] +} ) mass chromatograms in Figure 4, d ₃ body (mass number ^{394, [M + H] +} ) mass chromatogram of 5 shows the mass chromatogram of d ₂ (mass number 393, [M + H] ⁺ ), and FIG. 6 shows the mass chromatogram of d ₁ body (mass 392, [M + H] ⁺ ). FIG. 7 shows a mass chromatogram of d ₀ isomer (mass number 391, [M + H] ⁺ ).
In FIGS. 6 to 8, since no positive peak with respect to the baseline is observed, d ₂ body (mass number 393, [M + H] ⁺ ), d ₁ body (mass number 392, [M + H] ⁺ ) , D ₀ form (mass number 391, [M + H] ⁺ ) was determined not to exist.

<Step (4)>
From the peak area value of the mass chromatogram corresponding to [M + H] ⁺ of d ₀ to d ₄ bodies in which the peak area was observed using the data processing apparatus of step (2), d ₄ bodies were contained according to the following formula: The rate (%) was quantified.

The d ₀ to d ₄ body of [M + H] peak area of mass chromatogram value corresponding to ⁺ and d ₄ body content shown in Table 1.

<Confirmation of detection>
After repeating steps (1) to (4) using acetonitrile as a solvent and washing (hereinafter referred to as step (5)), Sample B was also quantified in steps (1) to (5).
D ₄ body sample B (mass number ^{395, [M + H] +} ) peak area values in the mass chromatogram of, d ₄ body obtained from the quantitative results of Sample A (mass number 395, [M ⁺ H] + ) Was found to be 28.2% of the peak area value. The results are shown in Table 2.

<Reproducibility of the system>
Determination of Sample A (step (1) to repeated six times (5), d ₄ body (mass number 395, to be a relative standard deviation of 1.7% of the peak area value of [M + H] ⁺⁾ The results are shown in Table 3.

  If the measurement method of the present invention is used, it should be used for the measurement of purity of isotope-labeled compounds, quantitative measurement of isotope-labeled compounds used in fields such as environmental science research and pharmacokinetic research, and measurement of the half-life of radioisotopes. Can do.

It is a chromatogram by liquid chromatography of Di-2-ethylhexyl Phthalate-3,4,5,6 -d 4. Is a total ion chromatogram obtained by mass spectrometry of Di-2-ethylhexyl Phthalate-3,4,5,6 -d 4. Said total ion chromatogram and data processing, is a mass spectrum of a component containing Di-2-ethylhexyl Phthalate-3,4,5,6 -d 4. It is a mass chromatogram of m / z = 395.21 ± 0.2 (equivalent to ₄ d-forms). It is a mass chromatogram of m / z = 394.21 ± 0.2 (equivalent to ₃ d bodies). It is a mass chromatogram of m / z = 393.21 ± 0.2 (corresponding to d ₂ isomers). It is a mass chromatogram of m / z = 392.21 ± 0.2 (equivalent to ₁ d body). It is a mass chromatogram of m / z = 391.21 ± 0.2 (equivalent to d ₀ isomer).

Claims (7)

A method for measuring the content of any isotope in an isotope-labeled compound, comprising the following steps (1) to (4):
(1) Step of performing mass analysis after separating sample containing isotope-labeled compound by chromatography (2) Processing the total ion chromatogram obtained by mass analysis of (1) to include any isotope Steps (3) and (2) for collecting mass spectra of the components are processed , and the peak top m / z = ± 0.1u to ± 0.3u for each isotope is obtained. the range of peak, steps on the basis of the mass chromatogram obtained in step (4) (3) collecting a mass chromatogram, and calculates the content of any isotopes in the isotope-labeled compound The method according to claim 1, wherein the chromatography of (1) is liquid chromatography, gas chromatography, or gel permeation chromatography. The ionization method of mass spectrometry (2) includes electron ionization (EI) method, chemical ionization (CI) method, field ionization (FI) method, fast atom collision (FAB) method, matrix-assisted laser desorption ionization (MALDI) method. The measurement method according to claim 1, which is an electrospray ionization (ESI) method, an atmospheric pressure chemical ionization (APCI) method, or an atmospheric pressure photoionization (APPI) method. The mass spectrometry of (2) is analyzed by a magnetic field deflection type, a quadrupole type, an ion trap type, a time of flight type, a tandem type, or a Fourier transform ion cyclotron resonance type. The measuring method as described in. The isotope-labeled compound is an isotope-labeled compound containing at least one selected from the group consisting of deuterium, a carbon atom having a mass number of 14 and a nitrogen atom having a mass number of 15. The measuring method in any one. The measurement method according to claim 1, further comprising the step of determining that the content rate of the isotope of the unstable mass chromatogram of the baseline is not present in the step of (4). The measurement method according to claim 1, further comprising the following step (5).
(5): A step of performing steps (1) to (4) with only the solvent used for dissolving the sample.

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