CN116106435B

CN116106435B - Preparation method and application of standard solution containing 9 antibiotics

Info

Publication number: CN116106435B
Application number: CN202211105464.XA
Authority: CN
Inventors: 贾永娟; 刘春冉; 刘杏立; 张�杰; 倪君君
Original assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Current assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Priority date: 2022-07-25
Filing date: 2022-09-09
Publication date: 2023-10-24
Anticipated expiration: 2042-09-09
Also published as: CN116087350A; CN116183736B; CN116183736A; CN116087350B; CN116148372B; CN116148372A; CN116106435A

Abstract

The present disclosure relates to a preparation method and application of a standard solution containing 9 antibiotics, wherein the 9 antibiotics include cefoperazone, ceftazidime, meropenem, linezolid, voriconazole, fluconazole, piperacillin, posaconazole and itraconazole, and the preparation method comprises: (1) Dissolving respective standard substances of 9 antibiotics by using a solvent to obtain respective mother solutions, and diluting the mother solutions by using a diluent to obtain standard intermediate solution; (2) Mixing the standard intermediate solutions of the 9 antibiotics respectively, and diluting the mixed solution by using a diluent to obtain a standard working solution; (2) The internal standard working solution is sequentially mixed with the standard working solution and the protein precipitant, and the supernatant is taken and diluted by ultrapure water to obtain the standard solution. The preparation method provided by the disclosure can obtain the standard solution containing 9 antibiotics simultaneously for subsequent standard curve determination, and the obtained standard solution has excellent stability, and the accuracy of the standard solution is basically stable after being placed at-20 ℃ for 3 months.

Description

Preparation method and application of standard solution containing 9 antibiotics

Technical Field

The disclosure relates to the technical field of antibiotic concentration detection, in particular to a preparation method and application of a standard solution containing 9 antibiotics.

Background

Bacterial infections are an important source of morbidity and mortality, and the use of antibiotic therapy is an important aspect of hospitalized patient management. However, it has now been demonstrated that antibiotic-treated patients based on standard dosing regimens often fail to achieve effective therapeutic concentrations in their blood levels, which greatly affects the therapeutic efficacy and may cause patients to develop resistance to multiple bacteria; because of individual differences in pharmacokinetics of different patients, simply increasing the drug dose may cause dangerous toxic side effects. Thus, there is a need for strict in vivo drug concentration monitoring, particularly for drugs with a narrow therapeutic concentration.

Cefperazone, ceftazidime, meropenem, voriconazole, itraconazole, posaconazole, fluconazole, linezolid, piperacillin are the commonly used 9 antibiotics, respectively aiming at different bacterial infections; however, the method has the defects that the treatment window is narrow, the individual difference is large, and the blood concentration of most severe patients cannot reach the treatment target range by using an empirical administration scheme, so that the blood concentration monitoring is needed to optimize the treatment scheme of the severe patients.

In addition, in practical situations, there are often cases where 9 antibiotics are combined, and in order to save detection time and reduce detection cost, several antibiotics may be generally placed in the same method package, but the solubility, stability, and the like of the 9 antibiotics are different, and if the 9 antibiotics are included in the same method package, there may be problems that the solubility of a part of the standard substance is poor, or the stability after dissolution is poor, and the like.

Therefore, it is desirable to provide a method for preparing a standard solution containing 9 antibiotics for subsequent detection and analysis.

Disclosure of Invention

In order to solve the technical problems, the disclosure provides a preparation method and application of a standard solution containing 9 antibiotics.

In a first aspect, the present disclosure provides a method of preparing a standard solution containing 9 antibiotics, the standard solution being detected using LC-MS/MS for determining a standard curve of 9 antibiotics, the 9 antibiotics including cefoperazone, ceftazidime, meropenem, linezolid, voriconazole, fluconazole, piperacillin, posaconazole, and itraconazole, the method of preparing comprising:

(1) Dissolving respective standard substances of 9 antibiotics by using a solvent to obtain respective mother solutions, and diluting the mother solutions by using a diluent to obtain standard intermediate solution;

(2) Mixing the standard intermediate solutions of the 9 antibiotics respectively, and diluting the mixed solution by using a diluent to obtain a standard working solution;

(2) The internal standard working solution is sequentially mixed with the standard working solution and the protein precipitant, and the supernatant is taken and diluted by ultrapure water to obtain the standard solution.

The standard solution provided by the disclosure contains 9 antibiotics simultaneously, and when the LC-MS/MS is utilized for detection, 9 antibiotics can be detected simultaneously by one needle, and a standard curve is established, so that the time and cost for preparing the standard solution can be greatly saved.

Because the preparation of the mother liquor is related to the stability of the subsequent standard solution, as a preferred embodiment of the present disclosure, the mother liquor is prepared by:

the solvents used for the meropenem standard and the ceftazidime standard are respectively and independently selected from 30-50% methanol aqueous solution or 30-50% acetonitrile aqueous solution.

The 30-50% aqueous methanol solution may be 32%, 35%, 38%, 40%, 42%, 45%, 48%, etc. The aqueous acetonitrile solution with the concentration of 30-50% can be 32%, 35%, 38%, 40%, 42%, 45%, 48% and the like.

In the determination of standard curves, the stability of the standard solution is extremely important, and the present disclosure finds that although meropenem and ceftazidime have excellent solubility in methanol, the stability is extremely poor, and although the stability still has higher recovery rate in the subsequent liquid chromatography-mass spectrometry detection, the detection accuracy cannot be practically ensured; when the method is used for preparing the mother solution of meropenem and ceftazidime, the stability and the solubility of meropenem and ceftazidime can be ensured by selecting a specific dissolving solvent and matching with a subsequent diluent for use, and further, the accuracy of subsequent detection is ensured.

The solvents used for the cefoperazone and itraconazole standard are independently selected from methanol solution of 50-80% dichloroethane or acetonitrile solution of 50-80% dichloroethane.

The methanol solution of 50-80% dichloroethane can be 55%, 60%, 65%, 70%, 75%, etc. The acetonitrile solution of 50-80% dichloroethane can be 55%, 60%, 65%, 70%, 75%, etc.

The specific discovery of the present disclosure, which uses methanol commonly used at present as a solvent to dissolve cefoperazone and itraconazole, adopts 50-80% dichloroethane methanol/acetonitrile solution to prepare mother liquor, not only ensures the solubility of cefoperazone and itraconazole, but also ensures that the standard working solution prepared later cannot be used due to the volatilization of the solvent in the placing process.

The solvent used by the posaconazole standard substance is selected from 60-100% methanol aqueous solution or 60-100% acetonitrile aqueous solution, and the methanol aqueous solution and the acetonitrile aqueous solution also contain 0.4-1% formic acid.

The preparation method of posaconazole mother liquor provided by the disclosure can ensure the solubility and storage stability of posaconazole.

The 60-100% may be 65%, 70%, 80%, 90%, etc. The 0.4-1% may be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, etc.

The solvents used for the linezolid, fluconazole, piperacillin and voriconazole standard are respectively and independently selected from 70-90% methanol aqueous solution or 70-90% acetonitrile aqueous solution.

The 70-90% may be 72%, 75%, 78%, 80%, 82%, 85%, 88%, etc.

In the present disclosure, the above concentrations refer to volume concentrations, and are exemplarily explained as follows: a methanol solution of 80% dichloroethane means that the volume ratio of dichloromethane to methanol is 8:2.

As a preferred embodiment of the present disclosure, the internal standard used for the 9 antibiotics includes cefoperazone-D5, ceftazidime-D5, meropenem-D6, linezolid-D3, voriconazole-D3, fluconazole-D4, piperacillin-D5, posaconazole-D4, and itraconazole-D5.

As a preferred technical scheme of the disclosure, the internal standard working solution is diluted by an internal standard intermediate solution by a diluent, and the internal standard intermediate solution is diluted by an internal standard mother solution by the diluent.

The stability of the internal standard in the standard solution is also important when using the internal standard method for LC-MS/MS detection, as a preferred technical solution of the present disclosure, when preparing the internal standard mother liquor:

the solvents used by meropenem-D6 and ceftazidime-D5 are respectively and independently selected from 30-50% methanol aqueous solution or 30-50% acetonitrile aqueous solution.

The solvent provided by the present disclosure also can ensure solubility and storage stability of the internal standard when preparing the internal standard mother liquor.

The solvents used for the cefoperazone-D5 and itraconazole-D5 standard are independently selected from methanol solution of 50-80% dichloroethane or acetonitrile solution of 50-80% dichloroethane.

The solvent used for the posaconazole-D4 standard substance is selected from 60-100% methanol aqueous solution or 60-100% acetonitrile aqueous solution, and the methanol aqueous solution and the acetonitrile aqueous solution also contain 0.4-1% formic acid.

The 50-100% may be 60%, 70%, 80%, 90%, etc. The 0.4-1% may be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, etc.

The solvents used for the linezolid-D3, voriconazole-D3, fluconazole-D4 and piperacillin-D5 standard are respectively and independently selected from 70-90% methanol aqueous solution or 70-90% acetonitrile aqueous solution.

The 70-90% may be 72%, 75%, 78%, 80%, 82%, 85%, 88%, etc.

In order to ensure stability of the standard solution and further ensure accuracy of the detection result, as a preferred technical scheme of the disclosure, the diluent is an aqueous methanol solution or an aqueous acetonitrile solution, and the volume ratio of the methanol or the acetonitrile to the water is 3:7-5:5, for example, 3.5:6.5, 4:6, 4.5:5.5, etc.

The diluent disclosed by the disclosure can ensure the storage stability of the standard working solution and the internal standard working solution, and under certain storage conditions, the content of 9 antibiotics contained in the diluent is kept stable, so that the time of current detection requiring on-site preparation is reduced, the detection time is shortened, and the detection efficiency is improved.

According to the method, the solubility and the storage stability of 9 antibiotics in the prepared standard solution can be guaranteed by selecting the specific dissolving reagent and the diluent, the accuracy of on-site detection can be realized, the concentration of the 9 antibiotics is basically unchanged after the standard working solution, the internal standard working solution or the standard solution is placed for a certain time, the accuracy of detection by using the stored solution can be further guaranteed, the requirement of on-site detection can be avoided, and the detection time is saved.

As a preferred embodiment of the present disclosure, the protein precipitant is selected from acetonitrile.

As a preferable technical scheme of the disclosure, the volume ratio of the internal standard working solution to the standard working solution is 1 (2-10), for example, 1:3, 1:5, 1:6, 1:8, 1:9 and the like.

As a preferred technical scheme of the disclosure, the volume ratio of the standard working solution to the protein precipitant is 1 (5-10), such as 1:6, 1:7, 1:8, 1:9, etc.

As a preferred embodiment of the present disclosure, the volume ratio of the supernatant to the ultrapure water is 1 (5-10), such as 1:6, 1:7, 1:8, 1:9, etc.

As a specific embodiment of the present disclosure, the preparation of the standard solution includes: and respectively transferring 20 mu L of standard working solution and 10 mu L of internal standard working solution by using a pipette, respectively placing the standard working solution and the 10 mu L of internal standard working solution into 1.5mL centrifuge tubes, mixing to prepare standard solutions, adding 100 mu L of acetonitrile into each centrifuge tube, respectively and uniformly vortex the standard solutions for 30s-1min at the rotating speed of 1000-2000rpm, taking 40 mu L of supernatant, and adding 200 mu L of water to obtain the standard solutions.

In a second aspect, the present disclosure provides a standard solution prepared by the preparation method of the first aspect.

The standard solution provided by the disclosure has excellent storage stability, and is convenient for accurate detection of an LC-MS/MS analysis method.

In a third aspect, the present disclosure provides an LC-MS/MS analysis method for simultaneously detecting 9 antibiotics in a sample, based on a preparation method of a standard solution provided by the present disclosure or a standard solution provided by the present disclosure, the analysis method comprising:

(1) Preparing a standard solution using the preparation method of the first aspect, the standard solution comprising at least 3 grade concentrations;

(2) Respectively establishing standard curves of 9 antibiotics by using standard solutions;

(3) Detecting a sample to be detected by utilizing LC-MS/MS, and respectively determining the content of 9 antibiotics in the sample to be detected by utilizing the standard curve determined in the step (2);

or alternatively, the first and second heat exchangers may be,

(1) Establishing standard curves of 9 antibiotics respectively by using standard solutions of the second aspect comprising at least 3 levels of concentration;

(2) And (3) detecting the sample to be detected by using LC-MS/MS, and respectively determining the content of 9 antibiotics in the sample to be detected by using the standard curve determined in the step (2).

In the present disclosure, the sample to be tested may be selected from the current conventional samples in the art, and the pretreatment method and the subsequent detection method are not limited in this disclosure, and any pretreatment method or detection method that can meet the requirements may be used.

Compared with the prior art, the technical scheme provided by the embodiment of the disclosure has the following advantages:

(1) The preparation method provided by the disclosure can obtain the standard solution containing 9 antibiotics simultaneously for subsequent standard curve determination, and the obtained standard solution has excellent stability, and the accuracy of the standard solution is basically kept stable after being placed for 3 months at the temperature of minus 20 ℃;

(2) The standard solution provided by the disclosure can obtain an accurate standard curve, and further can ensure the accuracy of a detection result during subsequent LC-MS/MS analysis of a sample to be detected.

Detailed Description

In order that the above objects, features and advantages of the present disclosure may be more clearly understood, a further description of aspects of the present disclosure will be provided below. It should be noted that, without conflict, the embodiments of the present disclosure and features in the embodiments may be combined with each other.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure, but the present disclosure may be practiced otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the disclosure.

Example 1

The embodiment provides a preparation method of a standard working solution.

(1) Preparing 9 antibiotic standard mother liquor

3.55mg of ceftazidime standard substance is precisely weighed by a balance, 2mL of methanol-water=1:1 solution is added for dissolution, the purity is 85.8%, and mother solution with the concentration of 1524 mug/mL is obtained;

2.54mg of meropenem standard substance is precisely weighed by a balance, 2mL of methanol-water=1:1 solution is added for dissolution, the purity is 98%, and mother solution with the concentration of 1243 mug/mL is obtained;

12.60mg of cefoperazone standard is precisely weighed by a balance, 1mL of methanol is added, 1mL of dichloromethane is added for dissolution, the purity is 93.8%, and a mother solution with the concentration of 5908 mug/mL is obtained;

accurately weighing 5.39mg of linezolid standard substance by a balance, adding 2mL of methanol/water=1:1 solution for dissolution, wherein the purity is 96.1%, and obtaining a mother solution with the concentration of 2685 mug/mL;

accurately weighing 10.83mg of piperacillin standard by a balance, adding 2mL of methanol/water=1:1 solution for dissolution, wherein the purity is 99.6%, and obtaining a mother solution with the concentration of 5203 mug/mL;

accurately weighing 10.63mg of fluconazole standard substance by a balance, adding 2mL of methanol and water=1:1 for dissolution, wherein the purity is 99.8%, and obtaining mother solution with the concentration of 5303 mug/mL;

accurately weighing 16.498mg of voriconazole standard by a balance, adding 2mL of methanol for dissolution, wherein the purity is 99.8%, and the concentration is 8233 mug/mL;

accurately weighing 4.354mg of posaconazole standard substance by a balance, adding 2mL of methanol, adding 10 mu L of formic acid for dissolution, and obtaining mother liquor with the concentration of 2155 mu g/mL, wherein the purity is 99%;

the itraconazole standard 5.35mg is precisely weighed by a balance, 1mL of methanol is added, 1mL of dichloromethane is added for dissolution, the purity is 99.2%, and the mother solution with the concentration of 2655 mug/mL is obtained.

(2) Preparing a standard intermediate solution by using a diluted solution of methanol and water=1:1, mixing the standard intermediate solution, and diluting the standard intermediate solution by using a diluted solution of methanol and water=1:1 to obtain a standard working solution with 8 levels of concentration, wherein the specific details are shown in table 1:

TABLE 1

Comparative examples 1 to 2

The comparative example provides a method for preparing a standard mother liquor.

The difference from example 1 is that in the case of preparing mother solutions of ceftazidime and meropenem in this comparative example, the solvents used were methanol (comparative example 1); methanol (methanol: water=1:1) =1:1 (comparative example 2).

If methanol is used as the solvent, (methanol: water=1:1) =1:1, this means that methanol: water=1:1 is used for dissolution, and then an equal amount of methanol is added for mixing.

Performance analysis 1

(1) Meropenem-based master solutions provided in example 1 and comparative examples 1-2 were left at-20℃for 3 months, and meropenem-based standard intermediate solutions (designed concentration of 20. Mu.g/mL) having a concentration level of L8 were prepared in the manner provided in example 1, and the concentrations of the standard intermediate solutions were examined, and the results are shown in Table 2:

TABLE 2

Sample of	Solvent(s)	L8 Point Peak area	L8 concentration (μg/mL)
				Example 1	Methanol: water=1:1	2184220	20.05
Comparative example 1	Methanol	280077	2.62
				Comparative example 2	Methanol (methanol: water=1:1) =1:1	1022248	9.56

As can be seen from table 2, if the mother liquor of meropenem and the subsequent intermediate liquor are prepared using the solvent defined in the present disclosure, stability of meropenem can be ensured, and after 3 months of standing at-20 ℃, accuracy thereof is reduced by only 0.0025 times, and stability is basically maintained, but the stability of meropenem is greatly reduced without using the solvent defined in the present disclosure.

(2) A mother liquor of ceftazidime having a concentration similar to that of example 1 was prepared while preparing a mother liquor of ceftazidime having a concentration higher than that of example 1, left at-20 ℃ for 3 months, and then a ceftazidime standard intermediate solution (designed concentration of 100 μg/mL) having a concentration level of L8 was prepared in accordance with the method provided in example 1, and the concentration of the standard intermediate solution was detected, and the results are shown in table 3:

TABLE 3 Table 3

As can be seen from table 3, the method provided by the present disclosure can ensure the stability of solutions such as mother solution and standard intermediate solution, and further ensure the accuracy of the detection result on the premise of saving time. The dissolution method provided in comparative example 2 can lead to ceftazidime being taken out for use after being placed at the temperature of-20 ℃ for 2 months, precipitation phenomenon occurs, the ceftazidime cannot be dissolved again, ultrasound cannot be dissolved again, the concentration of mother liquor is reduced by about 5 times, and under the condition that the solvent is methanol (methanol: water=1:1) =1:1, the higher the concentration of the mother liquor is, the worse the stability is.

Comparative examples 3 to 5

The difference from example 1 is that in the case of preparing the mother liquor of cefoperazone and itraconazole in this comparative example, the solvents used were methanol (comparative example 3), 40% methylene chloride in methanol (comparative example 4), 90% methylene chloride in methanol (comparative example 5).

Performance analysis 2

The mother solutions of cefoperazone and itraconazole provided in example 1 and comparative examples 3-5 were stored at-20℃and used repeatedly 5 times, and then standard working solutions of cefoperazone and itraconazole having concentration levels of L8 (designed concentrations of 100. Mu.g/mL and 20. Mu.g/mL, respectively) were prepared in the same manner as provided in example 1, and the concentrations of the standard working solutions were examined, and the results are shown in Table 4:

TABLE 4 Table 4

As can be seen from table 4, the preparation of the mother liquor and the subsequent intermediate liquor of cefoperazone and itraconazole using the solvents defined in the present disclosure can ensure the stability of both, and are substantially stable after storage and repeated use at-20 ℃, whereas the solvents provided in comparative examples 3 to 5 can cause the phenomenon that they are not completely dissolved or become large in concentration during the configuration, placement and use, i.e., the solution stability is greatly reduced.

Comparative examples 6 to 9

The difference from example 1 is that in the preparation of the mother liquor of posaconazole in this comparative example, the solvents used were methanol (comparative example 6), acetonitrile (comparative example 7), 60% aqueous methanol (comparative example 8), 60% aqueous acetonitrile (comparative example 9), 0.5% formic acid, 50% aqueous methanol (comparative example 10).

Performance analysis 3

The posaconazole mother liquors provided in example 1 and comparative examples 6 to 10 were stored at-20 ℃ and used repeatedly 10 times, and then a posaconazole standard working liquor (design concentration of 20 μg/mL) having a concentration level of L8 was prepared in the same manner as provided in example 1, and the concentration of the standard working liquor was measured, and the results are shown in table 5:

TABLE 5

Sample of	Solvent(s)	Posaconazole L8 concentration (μg/mL)
			Example 1	0.5% methanoic acid solution	20.03
Example 1-1	0.5% formic acid, 60% aqueous methanol solution	19.96
			Comparative example 6	Methanol	12.13 (incomplete dissolution, turbidity)
Comparative example 7	Acetonitrile	13.65 (incomplete dissolution, turbidity)
			Comparative example 8	60% aqueous methanol solution	6.15 (incomplete dissolution, precipitation of crystals)
Comparative example 9	60% acetonitrile aqueous solution	8.96 (incomplete dissolution, precipitation of crystals)
			Comparative example 10	0.5% formic acid, 50% aqueous methanol solution	18.15 (not completely dissolved, there are crystals)Precipitation of

As can be seen from table 5, the preparation of posaconazole mother liquor using the solvent defined in the present disclosure can ensure concentration stability during storage and repeated use. If an appropriate amount of acid is not added to the solvent or the content of the organic solvent (methanol/acetonitrile) is not within the limit of the present disclosure, posaconazole cannot be completely dissolved.

Comparative examples 11 to 12

The difference from example 1 is that in this comparative example, the diluent used is methanol or acetonitrile: water < 3:7 (comparative example 11), methanol or acetonitrile: water > 1:1 (comparative example 12).

Experiments show that if the water content in the used diluent is too high, the concentration of 9 substances is reduced to different degrees after the standard yeast working solution is placed in a refrigerator at the temperature of minus 20 ℃ and repeatedly frozen and thawed for 5 times; if the water content is too low, the meropenem, the ceftazidime and the internal standards of the two substances are placed for 3 months at the temperature of 20 ℃ below zero, and the reduction degree is more than 30 percent.

Example 2

The embodiment provides a preparation method of an internal standard working solution.

(1) Preparing internal standard mother liquor

ceftazidime-D5 standard substance with the specification of 1mg is dissolved by adding 2mL of methanol with water=1:1, the purity is 90.1%, and mother liquor with the concentration of 451 mug/mL is obtained;

Meropenem-D6 standard with the specification of 1mg is added with 2mL of methanol and water=1:1 for dissolution, the purity is 100%, and the mother solution with the concentration of 500 mug/mL is obtained;

the cefoperazone-D5 standard has the specification of 1.047mg, 500 mu L of methanol is added firstly, then 500 mu L of dichloromethane is added for dissolution, the purity is 90.1%, and the mother solution with the concentration of 953 mu g/mL is obtained;

linezolid-D3 standard with the specification of 1mg is added with 2mL of methanol for dissolution, the purity is 98.9%, and the mother liquor with the concentration of 495 mu g/mL is obtained;

the piperacillin-D5 standard substance has the specification of 1mg, 2mL of methanol is added for dissolution, the purity is 92.1%, and the mother solution with the concentration of 461 mu g/mL is obtained;

2mL of methanol is added to the standard product of fluconazole-D4 with the specification of 1mg for dissolution, the purity is 98.5%, and the mother solution with the concentration of 493 mug/mL is obtained;

the standard of voriconazole-D3 is 5mg, 10mL of methanol is added to dissolve with water=1:1, the purity is 100%, and the mother solution with the concentration of 500 mug/mL is obtained;

the posaconazole-D4 standard substance has the specification of 0.5mg, 2mL of methanol is added firstly, then 10 mu L of formic acid is added for dissolution, the purity is 97.6%, and the mother solution with the concentration of 243 mu g/mL is obtained;

itraconazole-D5 with the specification of 1mg is added with 1mL of methanol and then 1mL of dichloromethane for dissolution, the purity is 99.6%, and the mother liquor with the concentration of 498 mug/mL is obtained.

(2) Preparing an internal standard intermediate solution by using a diluted solution of methanol and water=1:1, and diluting the internal standard intermediate solution to obtain an internal standard working solution, wherein the specific details are shown in table 6:

TABLE 6

Antibiotics	Internal standard intermediate solution concentration (μg/mL)	Concentration of internal standard working solution (μg/mL)
			ceftazidime-D5	200	20
meropenem-D6	20	4
			cefoperazone-D5	500	10
linezolid-D3	20	0.5
			fluconazole-D4	20	1
piperacillin-D5	100	10
			voriconazole-D3	20	4
posaconazole-D4	20	1
			itraconazole-D5	20	2

Comparative example 13

The comparative example provides a preparation method of an internal standard working solution.

The only difference from example 2 is that in this comparative example, the solvents used for the internal standard mother liquors of meropenem-D6 and ceftazidime-D5 are both methanol.

Performance analysis 4

The internal standard mother liquor provided for example 2 and comparative example 13 was left at-20 ℃ for a period of time with the following results:

the concentration of ceftazidime-D5 was reduced by a factor of 10 after 6 months at-20℃and meropenem-D6 was not substantially detectable after 6 months at-20 ℃.

Therefore, the method for preparing the internal standard mother solution can ensure the stability of the internal standard mother solution, and further ensure the accuracy of subsequent detection.

As can be seen from the tests of examples 1-2 and comparative examples 1-13, the method for preparing the standard working solution and the internal standard working solution provided by the present disclosure can ensure the stability of 9 antibiotics and the internal standard thereof as much as possible, thereby ensuring the determination of standard curves and the accuracy of the detection results of the subsequent samples to be tested.

Example 3

The embodiment provides a preparation method of a standard solution.

Firstly, 20 mu L of standard working solution and 10 mu L of internal standard working solution are removed by a pipette and placed into a 1.5mL centrifuge tube to be mixed, then 100 mu L of acetonitrile is added, after vortex mixing is carried out for 0.5-1min at the rotating speed of 1000-2000rpm, 40 mu L of supernatant is taken, and then 200 mu L of water is added to be mixed, thus obtaining standard solution, and according to the method, a plurality of standard solutions with different concentrations are prepared in sequence.

Example 4

The present embodiment provides a method for determining a standard curve using LC-MS/MS analysis.

Detecting the standard solution by using a high performance liquid chromatography mass spectrometer, and respectively establishing standard curves of 9 antibiotics;

when the standard curve is established, the ratio of the peak area of the target object to the corresponding internal standard peak area is taken as Y, and the ratio of the concentration of the target object to the corresponding internal standard concentration is taken as X.

Example 5

The embodiment provides a sample pretreatment method and a detection method.

(1) Pretreatment method of sample to be tested

The pipette is used for taking 20 mu L of serum sample to be measured, 10 mu L of internal standard working solution and 100 mu L of acetonitrile are added, the mixture is uniformly mixed for 5min at 2000r/min, the mixture is centrifuged for 10min at 14000r/min, 40 mu L of supernatant is taken, 200 mu L of water is added, the mixture is uniformly mixed for 1min at 2000r/min, and 150 mu L of supernatant is taken as the sample to be measured.

(2) Detection of a sample to be tested

And detecting the sample to be detected by using a high performance liquid chromatography mass spectrometer, and determining the concentration of 9 antibiotics in the sample to be detected by using the established standard curve.

It should be noted that in this document, relational terms such as "first" and "second" and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

The foregoing is merely a specific embodiment of the disclosure to enable one skilled in the art to understand or practice the disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the disclosure. Thus, the present disclosure is not intended to be limited to the embodiments shown and described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a standard solution containing 9 antibiotics, wherein the standard solution is detected by LC-MS/MS for determining a standard curve of 9 antibiotics, and the 9 antibiotics include cefoperazone, ceftazidime, meropenem, linezolid, voriconazole, fluconazole, piperacillin, posaconazole, and itraconazole, the method comprising:

(3) Mixing the internal standard working solution with the standard working solution and the protein precipitant in sequence, taking supernatant and diluting with ultrapure water to obtain a standard solution;

in preparing the mother liquor:

the solvents used by the meropenem standard and the ceftazidime standard are respectively and independently selected from 50% methanol aqueous solution;

the solvents used for the cefoperazone and itraconazole standard are independently selected from 50% methylene chloride in methanol;

the solvent used by the posaconazole standard substance is selected from 60-100% methanol aqueous solution, and the methanol aqueous solution also contains 0.5% formic acid;

the solvents used by the linezolid, fluconazole, piperacillin and voriconazole standard are respectively and independently selected from 70-90% methanol aqueous solution;

the internal standard working solution is diluted by an internal standard intermediate solution by a diluent, and the internal standard intermediate solution is diluted by an internal standard mother solution by the diluent;

the diluent is methanol aqueous solution or acetonitrile aqueous solution, and the volume ratio of the methanol or acetonitrile to the water is 3:7-5:5;

when preparing the internal standard mother liquor:

the solvents used for the meropenem-D6 and ceftazidime-D5 standards are each independently selected from 50% strength aqueous methanol solutions;

the solvents used for the cefoperazone-D5 and itraconazole-D5 standard are independently selected from 50% strength methylene chloride in methanol;

the solvent used for posaconazole-D4 standard substance is selected from 60-100% methanol aqueous solution, wherein the methanol aqueous solution also contains 0.5% formic acid;

the solvents used for linezolid-D3, voriconazole-D3, fluconazole-D4, piperacillin-D5 standard are each independently selected from 70-90% aqueous methanol solutions.

2. The method of claim 1, wherein the protein precipitant is selected from acetonitrile.

3. The preparation method according to claim 1, wherein the volume ratio of the internal standard working solution to the standard working solution is 1 (2-10);

the volume ratio of the standard working solution to the protein precipitant is 1 (5-10);

the volume ratio of the supernatant to the ultrapure water is 1 (5-10).

4. A standard solution prepared by the preparation method of any one of claims 1 to 3.

5. A LC-MS/MS assay for simultaneous detection of 9 antibiotics in a sample, the assay comprising:

(1) Preparing a standard solution comprising at least 3 grade concentrations using the preparation method of any one of claims 1-3;

or alternatively, the first and second heat exchangers may be,

(1) Establishing standard curves of 9 antibiotics respectively using the standard solutions of claim 4 comprising at least 3 levels of concentration;

(2) And (3) detecting the sample to be detected by using LC-MS/MS, and respectively determining the content of 9 antibiotics in the sample to be detected by using the standard curve determined in the step (1).