CN115480005A

CN115480005A - Detection method and application of 2-hexyldecanoic acid

Info

Publication number: CN115480005A
Application number: CN202211074923.2A
Authority: CN
Inventors: 赵鹿; 张雅慧; 何平; 赵宣
Original assignee: Liaoning Kai Kai Technology Co ltd
Current assignee: Liaoning Kai Kai Technology Co ltd
Priority date: 2022-09-02
Filing date: 2022-09-02
Publication date: 2022-12-16

Abstract

The invention discloses a detection method of 2-hexyldecanoic acid and application thereof. The method can rapidly and accurately separate the product and related substances through HPLC-CAD, can accurately measure the purity of the product in a linear range, is convenient and fast to operate and good in separation, and effectively controls the purity of the product through a high-low concentration method. The method meets the standard in the aspects of quantification, linear range and the like.

Description

Detection method and application of 2-hexyldecanoic acid

Technical Field

The invention relates to the technical field of chemical analysis, in particular to a detection method of ALC-0315 starting material 2-hexyldecanoic acid and application thereof.

Background

ALC-0315 is a cationic biodegradable lipid, which is a colorless oily substance with the chemical name ((4-hydroxybutyl) azedinyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), and is attracting attention as a component of a novel coronavirus (SARS) vaccine BNT162b2 produced by BioNTech and fevere.

Chinese patent document CN114249662A discloses a preparation method of ALC-0315, which comprises the following steps: s1, performing azeotropic dehydration on 6-halogenated hexanol, 2-hexyldecanoic acid and a water-carrying agent to perform esterification reaction to obtain 6-halogenated hexanol-2-hexyldecanoate; s2, in the presence of an acid binding agent, performing amination reaction on 6-halogenated hexanol-2-hexyldecanoate and 4-aminobutanol in a solvent to obtain ALC-0315.

2-hexyldecanoic acid is an important raw material for producing ALC-0315, and no report specific to detection of 2-hexyldecanoic acid is found.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a detection method of 2-hexyldecanoic acid and application thereof.

In a first aspect of the present invention, a method for detecting 2-hexyldecanoic acid is provided, which comprises the step of detecting a sample to be detected by a high performance liquid chromatography-electrospray detector (HPLC-CAD).

In particular, the stationary phase of HPLC-CAD is octadecyl bonded silica, such as the ACQUITY UPLC BEH series.

In particular, the stationary phase has a particle size of 1 to 10 μm (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μm), especially 1 to 5 μm, e.g. 1.7 μm.

In one embodiment of the invention, the stationary phase is Waters, acquisition UPLC BEH,2.1mm x 100mm;1.7 μm.

In some embodiments of the invention, the high performance liquid chromatograph used is a ThermoUltiMate 3000 high performance liquid chromatograph.

In some embodiments of the invention, the chromatographic station is Chromeleon.

In particular, the column temperature of the HPLC-CAD is in the range of 30-50 deg.C (e.g. 30, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50 deg.C), in particular 35-45 deg.C, e.g. 40 deg.C.

Specifically, the mobile phase of HPLC-CAD consists of a mobile phase aqueous phase (a) and a mobile phase organic phase (B).

In particular, the mobile phase a is an aqueous formic acid solution having a concentration of 0.01 to 1% (by volume) (for example 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.4%, 0.5%, 1%), in particular 0.05 to 0.2%, for example 0.1%.

In particular, mobile phase B is a formic acid solution in acetonitrile at a concentration of 0.01 to 1% (by volume) (for example 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.4%, 0.5%, 1%), in particular 0.05 to 0.2%, for example 0.1%.

In particular, the flow rate of the mobile phase is from 0.1 to 1mL/min (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mL/min), particularly from 0.1 to 0.5mL/min, e.g., 0.3mL/min.

Preferably, the HPLC-CAD detection employs a gradient elution; more preferably, the elution procedure comprises:

0-0.01min, mobile phase B,0 → 60-70% (e.g., 60%, 62%, 64%, 65%, 66%, 68%, 70%), mobile phase a, the balance;

0.01-17min, mobile phase B,60-70% (e.g., 60%, 62%, 64%, 65%, 66%, 68%, 70%), mobile phase a, the balance;

17-17.1min, mobile phase B,60-70% (e.g., 60%, 62%, 64%, 65%, 66%, 68%, 70%) → 100%, mobile phase a, and the balance

17.1-27min, mobile phase B,100%;

27-27.1min, mobile phase B,100% → 60-70% (e.g., 60%, 62%, 64%, 65%, 66%, 68%, 70%), mobile phase a, the balance;

27.1-37min, mobile phase B,60-70% (e.g., 60%, 62%, 64%, 65%, 66%, 68%, 70%), mobile phase a, the balance;

37min，stop。

in a preferred embodiment of the invention, the elution procedure comprises:

0-0.01min, mobile phase B,0 → 65%, mobile phase A, and the balance;

0.01-17min, 65% of mobile phase B,65% of mobile phase A and the balance;

17-17.1min, mobile phase B,65% → 100%, mobile phase A, and the balance

17.1-27min, mobile phase B,100%;

27-27.1min, mobile phase B,100% → 65%, mobile phase A, and the balance;

27.1-37min, mobile phase B,65%, mobile phase A, and the balance;

37min，stop。

in some embodiments of the invention, the CAD parameters are: a Filter:1.0sec, range:100PA, CAD Temp:35 ℃ is carried out.

Specifically, the amount of sample is 1 to 10. Mu.L (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. Mu.L), particularly 5. Mu.L.

Specifically, the method may also suitably comprise the step of diluting the sample before the sample injection detection; more specifically, the solvent used for dilution is an organic solvent, such as one of acetonitrile, methanol and tetrahydrofuran, particularly acetonitrile.

Specifically, the method may further include a step of detecting a blank solution, which may be a diluting solvent, such as one of acetonitrile, methanol, and tetrahydrofuran, particularly acetonitrile.

In particular, the detection may be a qualitative detection or a quantitative detection, in particular a quantitative detection.

Specifically, the quantitative determination adopts a self-control method of 1% high and low concentration, and more specifically, the method further comprises the following steps: standard solutions of 100% and 1% concentration were prepared and subjected to HPLC-CAD detection.

In one embodiment of the invention, the sample is a 2-hexyldecanoic acid product, and the detection is carried out for detecting the purity of 2-hexyldecanoic acid in the product so as to provide a reference for subsequent application.

In a second aspect of the invention, there is provided the use of the method of the first aspect for the determination of the purity of 2-hexyldecanoic acid.

In a third aspect of the present invention, there is provided an application of the method of the first aspect in quality control and quality evaluation of ALC-0315.

Specifically, in the above application, ALC-0315 is prepared from 2-hexyldecanoic acid as a raw material, for example, as described in CN 114249662A:

the invention establishes an HPLC-CAD method for measuring the purity of the 2-hexyldecanoic acid, and provides information of Q-TOF high-resolution mass spectrum for larger impurities in the 2-hexyldecanoic acid, so that the information can be used as reference for subsequent quality control. The detection method provided by the invention can be used for quickly and accurately separating the product and related substances through HPLC-CAD, can be used for accurately measuring the purity of the product in a linear range, is convenient and fast to operate and good in separation, and can be used for effectively controlling the purity of the product through a high-low concentration method. The method meets the standard in the aspects of quantification, linear range and the like.

Drawings

FIG. 1 shows a liquid chromatogram for detecting a blank solution by the method of the present invention.

FIG. 2 is a liquid chromatogram of a sample solution detected by the method of the present invention.

Figure 3 shows an enlarged portion of the spectrum of figure 2.

FIG. 4 is a chromatogram showing the limit of quantitation (50 ug/mL, 0.5%) of a sample detected by the method of the present invention.

FIG. 5 shows a linear plot (SM 2 concentration 0.50% -10%) using the method of the present invention.

FIG. 6 shows a linear plot (SM 2 concentration 0.50% -3%) using the method of the present invention.

Fig. 7 shows a mass spectrum of impurity peak 1 (peak with a retention time of 0.623min, relative retention time of 0.04).

Fig. 8 shows a mass spectrum of impurity peak 1 (peak with a retention time of 0.701min, relative retention time of 0.04).

FIG. 9 shows the mass spectrum of impurity peaks 4 to 8.

FIG. 10 shows the mass spectrum of impurity peaks 4 to 8 (EIC extracted ion flux was confirmed for m/z 412.3791, which confirmed the origin of impurity peaks 4 to 8 and a subsequent cluster of peaks).

Fig. 11 shows a mass spectrum of impurity peak 9.

Fig. 12 shows a mass spectrum of the impurity peak 10.

Fig. 13 shows a mass spectrum of impurity peak 12.

FIG. 14 shows the mass spectrum of the main peak.

FIG. 15 shows an isocratic elution chromatogram with a mobile phase of 100% B.

FIG. 16 shows an isocratic elution chromatogram of 90% B (the remainder is A) of a mobile phase.

FIG. 17 shows an isocratic elution chromatogram of 80% B (balance A) of a mobile phase.

FIG. 18 shows an isocratic elution chromatogram of 75% B (balance A) of a mobile phase.

FIG. 19 shows an isocratic elution chromatogram with a mobile phase of 70% B (balance A).

FIG. 20 is a liquid chromatogram obtained by the elution procedure of example 3.

Detailed Description

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

The structural formula of the 2-hexyldecanoic acid is shown in the specification

Its CAS number is 25354-97-6, sometimes referred to as "SM2" for short in the present invention. The 2-hexyl decanoic acid related substances comprise: 2-hexyldecanol; 2-hexyloctanoic acid and other unknown impurities.

The ALC-0315 is ((4-hydroxybutyl) azadiyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) and has a structural formula of

CAS number 2036272-55-4.

The disclosures of the various publications, patents, and published patent specifications cited herein are hereby incorporated by reference in their entirety.

The technical solutions of the present invention will be described clearly and completely below with reference to embodiments of the present invention, and it should be apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: detection of purity of 2-hexyldecanoic acid related substances

1. Test materials and methods

The instrument comprises the following steps: a ThermoUltiMate 3000 hplc;

a chromatographic column: c18 reverse phase chromatography columns (Waters, ACQUITY UPLC BEH, 2.1mm. About.100mm, 1.7 μm, S/N: 186002352);

mobile phase: MPA:0.1% fa water; MPB:0.1% of FA acetonitrile;

the parameters of the CAD instrument are as follows: a Filter:1.0sec, range:100PA, CAD Temp: at 35 deg.c.

The elution procedure is shown in table 1:

TABLE 1 elution procedure

Time(min)	0.01	17	17.1	27	27.1	37
							B％	65	65	100	100	65	65

Flow rate: 0.3mL/min;

column temperature: at 40 ℃;

sample introduction amount: 5 mu L of the solution;

a workstation: chromeleon;

blank solution: acetonitrile;

control stock solutions: precisely weighing about 10mg of a 2-hexyldecanoic acid (SM 2) standard substance, placing the standard substance in a 10mL volumetric flask, adding a proper amount of acetonitrile for dissolving, adding the acetonitrile for constant volume until the volume is scaled, and shaking up;

sample solution: precisely weighing about 10mg of a 2-hexyldecanoic acid (SM 2) sample (Merrill, batch number (RMS 237-N22010)), precisely weighing, placing in a centrifuge tube, adding 1mL of acetonitrile for dissolving, uniformly mixing in a vortex manner to prepare a solution (100%) of 10mg/mL, and then gradually diluting to a solution (1%) of 100 mu g/mL;

limit of quantitation (LOQ) solution: precisely weighing about 10mg of 2-hexyldecanoic acid, placing the 2-hexyldecanoic acid into a centrifuge tube, adding 1mL of acetonitrile to dissolve, uniformly mixing by vortex, gradually diluting to 50 mu g/mL, and uniformly mixing by vortex.

Linear control solution: the control stock solutions (1000. Mu.g/mL) were accurately pipetted into 50. Mu.L, 80. Mu.L, 100. Mu.L, 200. Mu.L, 300. Mu.L, 400. Mu.L, 500. Mu.L, 800. Mu.L, and 1000. Mu.L volumetric flasks, and acetonitrile was added to the flasks to thereby obtain solutions of 50. Mu.g/mL, 80. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL, 300. Mu.g/mL, 400. Mu.g/mL, 500. Mu.g/mL, 800. Mu.g/mL, and 1000. Mu.g/mL.

TABLE 2 Linear control solution preparation

2. Results of the experiment

The liquid chromatogram of the blank solution, sample solution, and limit of quantitation (LOQ) solution are shown in FIGS. 1-4, respectively, the results of the concentrations of the linear control and the main peak areas are shown in Table 3, and the linear relationship is shown in FIGS. 5-6.

TABLE 3 Linear results for control

Concentration, μ g/mL (actual measurement value)	Main peak area, PA x min
		50.5	0.5496
80.8	0.9363
		101	1.2190
202	2.7555
		303	4.4203
404	6.0719
		505	8.4958
808	14.5521
		1010	19.1095

And (3) calculating the purity of the sample:

the blank of the diluent does not interfere at the peak position of the target substance, the purity of each peak except for a blank solvent in the sample is calculated by adopting an HPLC-CAD 1% high-low concentration self-contrast method, and each peak with the peak area percentage of more than 0.1% participates in the calculation of the purity of the 2-hexyldecanoic acid.

And (3) calculating:

C＝(As/Ats)*100％

c: purity of each substance

Namely: c2-hexyldecanoic acid: purity of 2-hexyldecanoic acid; c unknown impurities: purity of each unknown impurity

As: peak area of each substance

Namely: a2-hexyldecanoic acid: peak area of 2-hexyldecanoic acid (peak area in 100% concentration sample = peak area in 1% concentration sample 100%); unknown impurities of A: peak area of each unknown impurity

At: total peak area, i.e.: unknown impurity of A2-hexyldecanoic acid + Sigma A

The concentration calculation of substances corresponding to each peak in the detection spectrum of the sample solution shown in fig. 3 is shown in the following table:

table 4 shows the peak and the concentration calculation results of the corresponding substances

* Remarking: the main peak-off time is 14.762min, and the relative retention time is calculated according to the peak-off time of the main peak.

The mass spectrum is adopted to further detect and analyze substances corresponding to each peak, and the instrument parameters are shown in the following table:

TABLE 5 Instrument parameters

The results are shown in the following table:

TABLE 6 Mass spectrometric results

* Remarking: the peak emergence time of the 2-hexyldecanoate is 14.762min, and the relative retention time is calculated according to the peak emergence time of the main peak; the table lists TOF mass spectrum information of impurity peak area >0.1% peak in 2-hexyldecanoic acid; the remaining peak areas, peak TOF greater than 0.1%, did not yield ESI + ion response.

Example 2

The test was carried out with reference to the test materials and methods of example 1, wherein the elution procedure was isocratic elution, employing respectively 100% B, 90% B, 80% B, 75% B, 70% B (balance A) the mobile phase, the resulting spectra are respectively shown in FIGS. 15-19. From 60 to 70%, in particular 65%, of B is expected to be a suitable mobile phase proportion, depending on the main peak-out position.

Example 3

The assays were performed with reference to the experimental materials and methods of example 1, with the elution procedure replaced as shown in the following table:

TABLE 7 elution procedure

Time(min)	0	6	16	16.1	26
						B％	10	100	100	10	10

Under the elution gradient, the retention time of the main peak in the detection spectrogram (figure 20) of the sample is 10.087min, and compared with the retention time of the main peak, the positions of other impurities in the figure 20 are close to the retention time of the main peak, so that the separation is poor. The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.

The foregoing embodiments and methods described in this disclosure may vary based on the abilities, experiences, and preferences of one of ordinary skill in the art.

The mere order in which the steps of a method are listed in the present invention does not constitute any limitation on the order of the steps of the method.

Claims

1. A detection method of 2-hexyldecanoic acid comprises the steps of detecting a sample to be detected by a high performance liquid chromatography-electrospray detector (HPLC-CAD);

preferably, the stationary phase of the HPLC-CAD is octadecyl bonded silica gel;

preferably, the mobile phase of the HPLC-CAD consists of a mobile phase A and a mobile phase B, wherein the mobile phase A is aqueous formic acid and has a concentration of 0.01-1% by volume; the mobile phase B is an acetonitrile solution of formic acid, and the volume percentage concentration of the mobile phase B is 0.01-1%;

preferably, the HPLC-CAD detection employs a gradient elution.

2. The method according to claim 1, wherein the stationary phase of the HPLC-CAD is of the acquire UPLC BEH series;

preferably, the stationary phase has a particle size of 1-10 μm, preferably 1-5 μm.

3. The method of claim 1, wherein the column temperature of the HPLC-CAD is 30-50 ℃, preferably 35-45 ℃, more preferably 40 ℃.

4. The method according to claim 1, characterized in that the mobile phase a is an aqueous solution of formic acid with a concentration of 0.05 to 0.2% by volume, preferably 0.1%;

the mobile phase B is acetonitrile solution of formic acid, and the volume percentage concentration of the acetonitrile solution of formic acid is 0.05-0.2%, and the volume percentage concentration of the acetonitrile solution of formic acid is preferably 0.1%.

5. The method according to any one of claims 1 to 4, wherein the flow rate of the mobile phase is 0.1 to 1mL/min, preferably 0.1 to 0.5mL/min.

6. The method of claim 5, wherein the elution procedure of the gradient elution comprises:

0-0.01min, mobile phase B,0 → 60-70%, mobile phase A, the rest;

0.01-17min, 60-70% of mobile phase B,60-70% of mobile phase A and the balance;

17-17.1min, mobile phase B,60-70% → 100%, mobile phase A, and the balance

17.1-27min, mobile phase B,100%;

27-27.1min, mobile phase B,100% → 60-70%, mobile phase A, and the balance;

27.1-37min, 60-70% of mobile phase B,60-70% of mobile phase A and the balance;

37min，stop；

preferably, the elution procedure of the gradient elution comprises:

0-0.01min, mobile phase B,0 → 65%, mobile phase A, and the balance;

0.01-17min, 65% of mobile phase B,65% of mobile phase A and the balance;

17-17.1min, mobile phase B,65% → 100%, mobile phase A, and the balance

17.1-27min, mobile phase B,100%;

27-27.1min, mobile phase B,100% → 65%, mobile phase A, the balance;

27.1-37min, mobile phase B,65%, mobile phase A, and the balance;

37min，stop。

7. the method of claim 1, further comprising the step of diluting the sample prior to sample injection detection, wherein the dilution is performed using a solvent selected from the group consisting of: one of acetonitrile, methanol and tetrahydrofuran, preferably acetonitrile.

8. The method of claim 1, wherein the CAD parameters are: a Filter:1.0sec, range:100PA, CAD Temp:35 ℃ is carried out.

9. The method of claim 1, wherein the detection is a qualitative or quantitative detection;

preferably, the quantitative detection is performed by using a self-control method with high and low concentration of 1%.

10. Use of the method of any one of claims 1-9 in quality control and quality assessment of ALC-0315.