CN118243831A

CN118243831A - Determination of Z in posaconazole injection3gLC-MS/MS method of content

Info

Publication number: CN118243831A
Application number: CN202211657087.0A
Authority: CN
Inventors: 倪科; 颜波; 薛倩; 刘靖榆
Original assignee: Chongqing Huapont Pharm Co Ltd
Current assignee: Chongqing Huapont Pharm Co Ltd
Priority date: 2022-12-22
Filing date: 2022-12-22
Publication date: 2024-06-25

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to an LC-MS/MS method for determining Z _3g content in posaconazole injection. The invention adopts a mixed solution of acetonitrile, N-dimethylacetamide and ammonia water as an extractant to prepare a sample to be detected, the obtained sample to be detected adopts octadecylsilane chemically bonded silica filler as a stationary phase, a mixed solution of ammonium acetate aqueous solution and formic acid as a mobile phase A, methanol as a mobile phase B for gradient elution, and finally a serial quadrupole mass spectrum detector is adopted for detection. The LC-MS/MS method has high sensitivity, strong specificity and good repeatability, effectively solves the matrix effect of cyclodextrin in posaconazole injection on impurity Z _3g, and realizes the separation and determination of impurity Z _3g in posaconazole injection.

Description

LC-MS/MS method for determining Z 3g content in posaconazole injection

Technical Field

The invention belongs to the technical field of analytical chemistry, and particularly relates to an LC-MS/MS method for determining Z _3g content in posaconazole injection.

Background

Posaconazole is the latest generation of triazole antifungal drugs, and the structural formula is shown in formula I. Posaconazole is the most broad-spectrum antifungal agent so far, and the indication is prevention of invasive aspergillus and candida infections. Posaconazole currently marketed in batches includes oral suspension formulations, enteric coated tablet formulations and injectable formulations.

The impurity Z _3g is a high-risk impurity existing in the preparation process of posaconazole injection, and contains a warning structure, and the structural formula of the impurity is shown as formula II. The impurity limit must be controlled below 2.5ppm according to ICH control requirements, however conventional liquid chromatography cannot meet the sensitivity requirements of impurity Z _3g and cannot be controlled within specified limits. Meanwhile, posaconazole injection contains a large amount of cyclodextrin, the cyclodextrin has a strong matrix effect on impurity Z _3g, detection of Z _3g is seriously interfered, and the research shows that the man skilled in the art cannot eliminate the matrix effect of the cyclodextrin by adjusting the liquid phase condition.

Xue Qian et al disclose an LC-MS/MS method which can realize the separation and determination of Z _3g in posaconazole, however, the posaconazole main body related to the method is a bulk drug, rather than an injection, and cyclodextrin is not contained in the bulk drug, so that the problem of matrix effect is avoided. No method for detecting the impurity Z _3g in posaconazole injection by using LC-MS/MS (triple quadrupole mass spectrometry) is found in the existing data or patent, so a method with low detection limit and high sensitivity is needed to realize separation and determination of the impurity Z _3g in posaconazole injection.

Disclosure of Invention

One of the purposes of the invention is to provide a method for determining the content of impurity Z _3g by using an LC-MS/MS method, which can realize the determination of the content of impurity Z _3g in posaconazole injection within 30 minutes.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

A method for determining the content of impurity Z _3g by LC-MS/MS method, comprising the steps of:

(1) Sample pretreatment: treating the sample by using an extractant to obtain a sample to be detected;

(2) Separating: the sample to be detected obtained in the step (1) adopts octadecylsilane chemically bonded silica filler as a stationary phase, a mixed solution of ammonium acetate aqueous solution and formic acid as a mobile phase A, methanol as a mobile phase B for gradient elution, and the posaconazole and the impurity Z _3g are sequentially separated through gradient elution;

(3) Quantitative determination: detecting by using a tandem quadrupole mass spectrum detector to obtain a chromatogram;

The structural formula of the impurity Z _3g is shown in a formula II; the extractant is a mixed solution of acetonitrile, N-dimethylacetamide and ammonia water;

The patent discovers that the N, N-dimethylacetamide and ammonia water can effectively cause the precipitation and layering of cyclodextrin, and the acetonitrile can extract Z _3g to achieve the separation effect through multiple solvent screening; in addition, the invention discovers that the sample solution must be added in two times during the test process to optimize the recovery stability. And it is necessary to separate Z _3g from posaconazole by liquid phase gradient conditions, excluding the interference of posaconazole.

Further, the sample is posaconazole injection, and the posaconazole injection contains the impurity Z _3g.

Further, the volume ratio of the acetonitrile, the N, N-dimethylacetamide and the ammonia water is as follows: 600:400:0.5.

Further, the sample to be measured is sampled for 2 times.

Further, the gradient elution procedure was:

Setting the volume fraction of the mobile phase A to be 65-75% and the volume fraction of the mobile phase B to be 35-25% in 0.01 min;

Setting the volume fraction of the mobile phase A to be 65-75% and the volume fraction of the mobile phase B to be 35-25% in 1 minute;

Setting the volume fraction of the mobile phase A to be 5-15% and the volume fraction of the mobile phase B to be 95-85% in 13 minutes;

setting the volume fraction of the mobile phase A to be 5-15% and the volume fraction of the mobile phase B to be 95-85% in 20 minutes;

Setting the volume fraction of the mobile phase A to be 65% -75% and the volume fraction of the mobile phase B to be 35% -25% in 20.1 minutes;

At 30 minutes, the volume fraction of the mobile phase A is set to be 65% -75%, and the volume fraction of the mobile phase B is set to be 35% -25%.

Further, the sample to be detected comprises a sample solution, a reference substance solution, a sensitivity solution and/or a labeling solution.

Further, in the mobile phase A, the concentration of the ammonium acetate aqueous solution is 10mM, and the mass percentage of the formic acid is 0.1%.

Further, the mobile phase flow rate is 1.0ml/min; the column temperature of the chromatographic column is 30 ℃; the temperature of the sample injector is 25+/-3 ℃; the sample volume was 5. Mu.l.

Further, the mass spectrometry conditions were: selecting ESI ion source and MRM positive ion mode; the atomization gas is 55psi, the heating auxiliary gas is 55psi, the door curtain gas is 35psi, the collision gas is 9psi, the spraying voltage is 5000V, and the ion source temperature is 550 ℃; the residence time was 100msce; the mass spectrum Resolution is Resolution Q1:Unit, resolution Q3:Unit.

Further, a mass-to-charge ratio 547.3.fwdarw.135.1 was used as the detection ion pair of the impurity Z _3g.

Further, preparing a sample solution, and detecting to obtain a sample solution chromatogram; preparing a reference substance solution, and detecting to obtain a reference substance solution chromatogram; the content of the impurity Z _3g is calculated according to the external standard method and the peak area.

Preferably, the mobile phase A is a mixed solution of an ammonium acetate aqueous solution and formic acid, wherein the mass percentage of the formic acid is 0.1%; the mobile phase B is methanol; the chromatographic column has the specification that: 4.6 mm. Times.250 mm. Times.5. Mu.m; the flow rate of the mobile phase is 1.0ml/min; column temperature of the chromatographic column is 30 ℃; the temperature of the sample injector is 25+/-3 ℃; the sample injection volume is 5 μl; linear gradient elution was performed and chromatograms were obtained according to the following procedure:

at 0.01 min, the volume fraction of mobile phase A was set to 70% and the volume fraction of mobile phase B was set to 30%;

setting the volume fraction of the mobile phase A as 70% and the volume fraction of the mobile phase B as 30% in 1 minute;

Setting the volume fraction of the mobile phase A as 10% and the volume fraction of the mobile phase B as 90% at 13 minutes;

Setting the volume fraction of the mobile phase A as 10% and the volume fraction of the mobile phase B as 90% in 20 minutes;

At 20.1 minutes, the volume fraction of mobile phase A was set to 70% and the volume fraction of mobile phase B was set to 30%;

at 30 minutes, the volume fraction of mobile phase A was set to 70% and the volume fraction of mobile phase B was set to 30%.

Further, the retention time was 13.77.+ -. 0.5min as impurity Z _3g.

Impurity Z _3g can be characterized in terms of retention time.

Further, the impurity Z _3g is in the range of 0.4465ng/ml to 4.465ng/ml, y=88700x+220, r=0.9994, where Y is the Y-axis, representing the peak area, X is the X-axis, representing the concentration, and r is the correlation coefficient.

Further, the detection limit concentration of the impurity Z _3g is 0.2233ng/ml, and the quantitative limit concentration is 0.4465ng/ml.

The invention has the beneficial effects that:

1. The invention adopts the mixed solution of acetonitrile, N, N-dimethylacetamide and ammonia water as the solvent, effectively solves the matrix effect of cyclodextrin on impurity Z _3g in posaconazole injection, and the N, N-dimethylacetamide and the ammonia water can effectively lead the precipitation of the cyclodextrin to be layered, and the acetonitrile can extract Z _3g, thereby achieving the separation effect. Meanwhile, impurities Z _3g and posaconazole are separated by optimizing a liquid phase gradient condition, so that interference of posaconazole is eliminated, and separation and determination of posaconazole injection impurities Z _3g are realized; in order to optimize the recovery rate and stability of the sample, the sample solution is injected in a mode of adding the sample solution twice, and the LC-MS/MS method has high sensitivity, strong specificity and good repeatability.

Drawings

FIG. 1 is a Z _3g full-scan mass spectrum;

FIG. 2 is a Z _3g secondary mass spectrum;

FIG. 3 is a blank solvent chromatogram;

FIG. 4 is a chromatogram of a control solution;

FIG. 5 is a chromatogram of a test solution;

FIG. 6 is a Z _3g -LOQ chromatogram;

FIG. 7 is a Z _3g LOD chromatogram;

FIG. 8 is a graph of the concentration of Z _3g plotted against peak area.

Detailed Description

The technical scheme of the present invention will be further clearly and completely described in connection with specific embodiments. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. Therefore, all other embodiments obtained by those skilled in the art without undue burden are within the scope of the invention based on the embodiments of the present invention.

Example 1

The measurement is strictly carried out according to high performance liquid chromatography-mass spectrometry (Chinese pharmacopoeia 2020 edition four general rules 0512 and 0431).

1. Solution preparation

The extractant comprises: 600ml of acetonitrile and 400ml of N, N-dimethylacetamide are taken, and then 0.5ml of ammonia water is added for mixing.

Test solution: accurately transferring 9.5ml of extractant into a 15ml centrifuge tube, adding 0.25ml of sample into the centrifuge tube, swirling for 5min, standing for 5min, taking 1ml of supernatant solution into a 2ml centrifuge tube, centrifuging at 12000rpm for 10min at 4 ℃, and taking supernatant for sample introduction if layering.

Control solution: taking a proper amount of Z _3g reference substance, precisely weighing, adding acetonitrile for dissolving, and diluting with an extractant to prepare a solution containing about 2.25ng of impurities in each 1ml of the solution serving as the reference substance solution.

Sensitivity solution: a proper amount of the reference solution was precisely measured, and diluted with an extractant to prepare a solution containing about 0.45ng of impurities per 1ml as a sensitivity solution.

2. Chromatographic/mass spectral conditions

The chromatographic/mass spectrometry conditions are shown in Table 1, and in this example, octadecylsilane chemically bonded silica is used as a filler (Shim-pack VP ODS 4.6mm. Times.250 mm,5 μm or a column with equivalent performance); gradient elution was performed according to table 2 using 10mM aqueous ammonium acetate (0.1% formic acid) as mobile phase a and methanol as mobile phase B; adopting a tandem quadrupole mass spectrum detector, performing multi-reaction detection (MRM) under an electrospray ion source (ESI) positive ion mode, and adopting mass-to-charge ratio (m/Z) 547.3-135.1 as Z _3g ion pair detection, wherein the details are shown in Table 3; the flow rate is 1.0ml per minute; the column temperature is 30 ℃; the temperature of the sample injector is room temperature; the sample volume was 5. Mu.l.

TABLE 1 chromatographic/Mass Spectrometry conditions

TABLE 2 gradient elution program table

Time (min)	Mobile phase a	Mobile phase B
			0.01	70	30
1	70	30
			13	10	90
20	10	90
			20.1	70	30
30	70	30

TABLE 3 Mass Spectrometry detection parameter table

Parent ion	Ion	DP	CE	EP	CXP
						547.3	135.1	140	45	10	6

3. Measurement

Precisely measuring the sample solution and the reference substance solution, injecting into a high performance liquid chromatography-mass spectrometer, and recording the chromatograms. The system applicability is to select proper mass spectrum conditions, and the signal-to-noise ratio of a Z _3g peak in a sensitivity solution chromatogram should not be less than 10. The limit is that Z _3g in the test sample solution is less than 0.00025% calculated by the external standard method and calculated by peak area.

The full-scan mass spectrum of the impurity Z _3g is shown in figure 1, and the secondary mass spectrum of the impurity Z _3g is shown in figure 2.

Example 2 method verification

1. Blank diluent validation

Analysis was performed using a blank solvent and Z _3g in liquid chromatography, and the results are shown in FIG. 3, where the blank diluent had no chromatographic peak at the retention time of the Z _3g peak, and no interference with impurity detection.

2. Repeatability verification of reference solution

Preparing a reference substance solution of the impurity Z _3g, and detecting by using a high performance liquid chromatography-mass spectrometer, wherein the results are shown in table 4 and fig. 4 (fig. 4 is a chromatogram of the 1 st sample injection of the impurity Z _3g), and the repeatability of the sample injection of the reference substance solution is good.

TABLE 4 comparative sample solution repeatability verification results Table

Number of sample injections	Retention time (minutes)	Peak area
			1	13.71	170000
2	13.79	175000
			3	13.77	172000
4	13.77	172000
			5	13.78	178000
6	13.78	180000
			Average value of	13.77	174500
RSD(％)	0.21	2.23

3. Quantitative limit verification

The quantitative limit detection of the impurity Z _3g is carried out, the results are shown in table 5 and fig. 6 (fig. 6 is a chromatogram of the 1 st sample injection of the impurity Z _3g), the quantitative limit concentration of the impurity Z _3g is about 0.4465ng/ml respectively, and the signal to noise ratio is greater than 10.

TABLE 5 quantitative limit verification results Table

4. Detection limit verification

The detection limit of the impurity Z _3g is shown in Table 6 and FIG. 7 (FIG. 7 is a chromatogram of the 1 st sample of the impurity Z _3g), the detection limit concentration of Z _3g is about 0.2233ng/ml, and the signal to noise ratio is greater than 10.

TABLE 6 detection limit verification results Table

5. Linear relationship verification

In this example, the concentration and peak area of impurity Z _3g were linearly analyzed, and as shown in table 7 and fig. 8, impurity Z _3g was linearly related in the concentration range of 0.4465 to 4.465ng/ml, and the correlation coefficient r=0.9994.

TABLE 7 Linear relationship verification results Table

6. Precision verification

Test solution: accurately transferring 9.5ml of extractant into a 15ml centrifuge tube, adding 0.25ml of sample into the centrifuge tube, swirling for 5min, standing for 5min, taking about 1ml of upper solution into a 2ml centrifuge tube, centrifuging at 12000rpm for 10min at 4 ℃, and taking the supernatant as a sample solution. 6 parts in parallel.

Control solution: and precisely transferring 0.5ml of the impurity reference substance solution III, placing into a10 ml measuring flask, adding an extractant to dilute to a scale, and shaking uniformly to obtain the reference substance solution.

And precisely measuring 5 mu l of each of the sample solution and the reference solution, and recording a chromatogram. To examine the effect of random variation on precision, tests were performed by different analysts at different times, the results are shown in table 8 and fig. 5.

TABLE 8 precision verification results Table

7. Accuracy verification

Test solution: accurately transferring 9.5ml of extractant into a 15ml centrifuge tube, adding 0.25ml of sample into the centrifuge tube, swirling for 5min, standing for 5min, taking about 1ml of upper solution into a 2ml centrifuge tube, centrifuging at 12000rpm for 10min at 4 ℃, and taking the supernatant as a sample solution.

Adding a marking solution: respectively precisely transferring 9.25mL, 9mL and 8.75mL of extractant into a 15mL centrifuge tube, correspondingly precisely adding 0.25mL, 0.5mL and 0.75mL of reference substance solution III, mixing, adding 0.25mL of sample, swirling for 5min, standing for 5min, taking about 1mL of upper solution into a 2mL centrifuge tube, centrifuging at 12000rpm for 10min at 4 ℃, and taking supernatant to obtain 50%, 100% and 150% standard solution (3 parts are prepared in parallel).

Control solution: and precisely transferring 0.5ml of the impurity reference substance solution III, placing into a 10ml measuring flask, adding an extractant to dilute to a scale, and shaking uniformly to obtain the reference substance solution. The chromatogram was recorded and the results are shown in table 9.

TABLE 9 accuracy verification results Table

8. Solution stability verification

The control solution, the test solution and the labeled test solution were sampled at the sample injector (room temperature) for about 0, 3, 6, 10, 17 hours, and the RSD of the peak area of the sample at each time point was calculated, and the results are shown in table 10. The results showed that the product was stable at room temperature for 17 hours.

TABLE 10 results of solution stability verification

Claims

1. A method for determining the content of impurity Z _3g by LC-MS/MS method, characterized by comprising the steps of:

2. The method of claim 1, wherein the sample is a posaconazole injection, the posaconazole injection containing the impurity Z _3g.

3. The method according to claim 1, wherein the volume ratio of the acetonitrile, the N, N-dimethylacetamide and the ammonia water is: 600:400:0.5.

4. The method of claim 1, wherein the gradient elution procedure is:

5. The method of claim 1, wherein the mass spectrometry conditions are: selecting ESI ion source and MRM positive ion mode; the atomization gas is 55psi, the heating auxiliary gas is 55psi, the door curtain gas is 35psi, the collision gas is 9psi, the spraying voltage is 5000V, and the ion source temperature is 550 ℃; the residence time was 100msce; the mass spectrum Resolution is Resolution Q1:Unit, resolution Q3:Unit.

6. The method according to claim 1, wherein a mass to charge ratio 547.3→135.1 is used as the detection ion pair of the impurity Z _3g.

7. The method of claim 1, wherein a sample solution is prepared and detected to obtain a sample solution chromatogram; preparing a reference substance solution, and detecting to obtain a reference substance solution chromatogram; the content of the impurity Z _3g is calculated according to the external standard method and the peak area.

8. The method according to claim 4, wherein the mobile phase A is a mixed solution of an ammonium acetate aqueous solution and formic acid, and the mass percentage of the formic acid is 0.1%; the mobile phase B is methanol; the chromatographic column has the specification that: 4.6 mm. Times.250 mm. Times.5. Mu.m; the flow rate of the mobile phase is 1.0ml/min; column temperature of the chromatographic column is 30 ℃; the temperature of the sample injector is 25+/-3 ℃; the sample injection volume is 5 μl; linear gradient elution was performed and chromatograms were obtained according to the following procedure:

9. The method according to claim 8, wherein the retention time is 13.77±0.5min as impurity Z _3g.

10. The method of claim 8, wherein the impurity Z _3g is in the range of 0.4465ng/ml to 4.465ng/ml, Y = 88700x+220, r = 0.9994, where Y is the Y-axis, represents peak area, X is the X-axis, represents concentration, and r is the correlation coefficient.