CN111044659A

CN111044659A - Method for determining concentration of azithromycin in blood plasma by liquid chromatography-mass spectrometry

Info

Publication number: CN111044659A
Application number: CN201911409612.5A
Authority: CN
Inventors: 林明弘
Original assignee: Nanjing Lishun Kangda Pharmaceutical Technology Co Ltd
Current assignee: Nanjing Lishun Kangda Pharmaceutical Technology Co Ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2020-04-21

Abstract

The invention discloses a method for determining the concentration of azithromycin in blood plasma by liquid chromatography-mass spectrometry, which adopts a liquid chromatography-mass spectrometry system to determine, firstly takes a sample to be determined, adds a certain amount of mixed organic solvent to extract, pretreats, separates by a chromatographic column, and detects by a mass spectrometer. The method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for the determination of the blood concentration of the azithromycin; the plasma standard curve linear range of the method is 1-500 ng/mL, the precision RSD in batch and between batches is less than +/-15%, and the method is suitable for measuring the concentration of azithromycin in plasma.

Description

Method for determining concentration of azithromycin in blood plasma by liquid chromatography-mass spectrometry

Technical Field

The invention belongs to the technical field of medicines, particularly relates to a method for determining a medicine, and particularly relates to a method for determining the concentration of azithromycin in blood plasma by liquid chromatography-mass spectrometry.

Background

Azithromycin (Azithromycin) is a second generation macrolide antibiotic that was successfully developed by the company Nanslev Pliva in the last 80 s of the century. The azithromycin has good tolerance and low adverse reaction rate, and has wide clinical application.

Azithromycin is a semi-synthetic pentadecatomic ring lactone antibiotic, is a broad-spectrum antibiotic obtained by modifying the chemical structure of erythromycin, has an antibacterial effect on most bacteria, and also has good activity on chlamydia and mycoplasma. The mechanism of action is the same as that of erythromycin, and it binds mainly to the 50S subunit of bacterial ribosomes, inhibiting RNA-dependent protein synthesis. The medicament is widely used for treating acute pharyngitis and acute tonsillitis caused by streptococcus pyogenes clinically; sinusitis, otitis media, acute bronchitis and acute attack of chronic bronchitis caused by sensitive bacteria; pneumonia caused by streptococcus pneumoniae, haemophilus influenzae, and mycoplasma pneumoniae; urethritis and cervicitis caused by chlamydia trachomatis and non-multiple drug-resistant neisseria gonorrhoeae; infection of skin soft tissue by sensitive bacteria.

At present, the speed, the precision, the sensitivity and the selectivity of the existing azithromycin measuring method need to be improved.

Disclosure of Invention

The invention aims to provide a method for determining the concentration of azithromycin in blood plasma by liquid chromatography-mass spectrometry, which can improve the sensitivity, precision, selectivity and speed of detection.

In order to realize the aim, the invention provides a method for determining the concentration of azithromycin in blood plasma by liquid chromatography-mass spectrometry, wherein a blood plasma sample is pretreated and then the concentration of the blood plasma sample is detected by high performance liquid chromatography-tandem mass spectrometry, and the specific method comprises the following steps:

(1) plasma sample pretreatment:

plasma with K₂EDTA as anticoagulant, azithromycin-¹³C-d3 is an internal standard; precisely adding 100 μ L of plasma sample into a 96-deep-well plate, adding 5 μ L of plasma sample at a volume ratio of 1: 1 acetonitrile aqueous solution, and after mixing uniformly, 5 mu L of 1 ng/mu L of internal standard azithromycin-¹³Mixing the C-d3 solution uniformly, adding 1000 mu L of methanol, carrying out vortex mixing for 1min, centrifuging at 6 ℃ at 3000rpm for 10min, taking 50 mu L of supernatant liquid to another 96 deep-hole plate filled with 1000 mu L of mixed organic solvent, carrying out vortex mixing uniformly, centrifuging at 6 ℃ at 3000rpm for 5min, and taking the supernatant liquid as a test sample to be detected; wherein: the mixed organic solvent is water: acetonitrile: formic acid is mixed according to a volume ratio of 80: 20: 0.2 mixing the obtained mixture; in the processes, except for vortex and centrifugation, other processes are operated under the ice bath condition;

(2) and (3) sample measurement:

injecting 10 mu L of test sample into a high performance liquid chromatography-tandem mass spectrometer, and detecting azithromycin and internal standard azithromycin-¹³Chromatographic peaks of C-d3 and calculating therefrom the concentration of azithromycin in said plasma sample;

the liquid chromatography determination conditions were: the chromatographic column is Synergi^TM4um Polar-RP

The column specification is 50 x 2 mm; the temperature of the chromatographic column is 40 ℃; the mobile phase A is water: formic acid is mixed according to the volume percentage of 100: 0.2 mixing the obtained mixture; the mobile phase B is acetonitrile: formic acid is mixed according to the volume percentage of 100: 0.2 mixing the obtained mixture; the washing liquid is acetonitrile: water according to the volume percentage of 50: 50 mixing the obtained mixture; the temperature of the autosampler is 6 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10 μ L, and analysis time of 3.5 min;

the mass spectrometry conditions are as follows: the ion source is an electrospray ion source, the spraying voltage is 4500V, the atomizing temperature is 450 ℃, the spraying air pressure is 30Psi, the auxiliary heating air pressure is 30Psi, the air curtain air pressure is 30Psi, the collision air pressure is 8Psi, and the azithromycin-¹³The declustering voltage of C-d3 is 20 eV; azithromycin and azithromycin-¹³The collision cell inlet voltages of C-d3 are all 10 eV; azithromycin and azithromycin-¹³The collision voltage of C-d3 is 40 eV; azithromycin and azithromycin-¹³The outlet voltages of the collision chambers of C-d3 are all 10 eV; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z749.5 → m/z591.4, which is azithromycin; and m/z753.5 → m/z595.5, which is azithromycin-¹³C-d3。

Preferably, the gradient elution in step (2) is performed by the following procedure:

total time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	78	22
1.0	78	22
			1.1	15	85
2.0	15	85
			2.1	78	22
3.5	78	22

Preferably, in the step (2), an internal standard method is adopted, and azithromycin and internal standard azithromycin are used as the internal standard¹³The peak area ratio of C-d3 was substituted into a standard curve equation to calculate the concentration of azithromycin in the plasma sample.

Preferably, the establishment of the standard curve equation comprises the following steps:

ten 100 mu L blank blood plasma are placed in a 96-deep-well plate and are sequentially named as ten samples including a lowest quantitative lower limit sample, a standard sample 1, a standard sample 2, a standard sample 3, a standard sample 4, a standard sample 5, a standard sample 6, a highest quantitative upper limit sample, a zero concentration sample and a blank sample, wherein the zero concentration sample contains an internal standard azithromycin-¹³C-d3 solution without azithromycin solution for eliminating internal standard azithromycin-¹³Interference caused by the C-d3 solution on the detection result; the blank sample does not contain azithromycin solution and internal standard azithromycin-¹³C-d3 solution for eliminating the interference of blank plasma on the detection result;

adding 5 mul of azithromycin solution with the concentration of 0.02 ng/mul, 0.04 ng/mul, 0.1 ng/mul, 0.2 ng/mul, 1 ng/mul, 2 ng/mul, 6 ng/mul and 10 ng/mul into a minimum quantitative lower limit sample, a standard sample of 1-6 and a maximum quantitative upper limit sample in the form of stock solution, and adding 5 mul of azithromycin solution into a zero-concentration sample and a blank sample respectively in a volume ratio of 1: 1, respectively mixing the ten samples, and then respectively adding 5 mu L of 1 ng/mu L of internal standard azithromycin-¹³C-d3 solution, 5. mu.L of a 1: 1, respectively and uniformly mixing the ten samples, respectively adding 1000 mu L of methanol into the ten samples, carrying out vortex mixing for 1min, centrifuging at 6 ℃ for 10min at 3000rpm, taking 50 mu L of supernatant of the ten samples, transferring the supernatant into another 96 deep-hole plate filled with 1000 mu L of mixed organic solvent, carrying out vortex mixing, centrifuging at 6 ℃ for 5min at 3000rpm, and taking the supernatant as ten standard samples to be detected; the mixed organic solvent is water: acetonitrile: formic acid is mixed according to a volume ratio of 80: 20: 0.2 mixing the obtained mixture; in the processes, except for vortex and centrifugation, other processes are operated under the ice bath condition;

respectively injecting 10 mu L of standard sample into a high performance liquid chromatography-tandem mass spectrometer to detect the azithromycin and the internal standard azithromycin-¹³Chromatographic peaks of C-d3 and a standard curve was derived therefrom for calculating the concentration of azithromycin in said plasma.

Further, the liquid chromatography determination conditions in the step (3) further include: the volume of the syringe washing needle of the automatic sample injector is 500 mu L; the depth of a sample injection needle of the automatic sample injector is 45 mm; the cleaning speed of the automatic sample injector is 35 mu L/s; the sample injection speed of the automatic sample injector is 5 mu L/s; the soaking time is 5s when the sample injection needle of the automatic sample injector is cleaned; the automatic sample injector cleaning mode is before sample injection and after sample injection.

Compared with the prior art, the invention has the following advantages:

(1) the pretreatment method is simple and convenient, two-step organic solution extraction is adopted, and the method is suitable for conventional determination;

(2) the specificity is strong: under the chromatographic conditions adopted by the experiment, the retention time of the azithromycin is about 1.488min, and the internal standard azithromycin-¹³The retention time of the C-d3 is about 1.483min, and the azithromycin and the internal standard azithromycin-¹³The peak shape of C-d3 is good, no miscellaneous peak interference is caused to determine, and the baseline is stable;

(3) the sensitivity is high: the minimum limit of the plasma quantification is 1ng/mL, the concentration of the azithromycin in the plasma can be accurately determined, the sensitivity is high, and the specificity is strong;

(4) the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for determining the blood concentration of the azithromycin. The linear range of the plasma standard curve of the method is 1-500 ng/mL, and the precision RSD in batch and between batches is less than +/-15%.

Drawings

FIG. 1 is a standard graph of azithromycin in human plasma as measured by HPLC-MS/MS;

FIG. 2 is a HPLC-MS/MS graph of human blank plasma;

FIG. 3 shows the addition of azithromycin to human blank plasma¹³HPLC-MS/MS plot of C-d 3;

figure 4 shows that azithromycin and azithromycin are added into human blank plasma¹³HPLC-MS/MS plot of C-d 3;

figure 5 shows that the plasma sample is added with the internal standard azithromycin after the healthy subject takes the azithromycin drug orally¹³HPLC-MS/MS profile of C-d 3.

Detailed Description

The present invention will be described in further detail with reference to examples.

Example (b): human K₂Determination of Azithromycin concentration in EDTA plasma

First, experimental material and analytical equipment

Azithromycin (analyte): chinese food and drug testing research institute or standard substance with same or higher grade

Azithromycin-¹³C-d3 (internal standard): TLC Pharmaceutical Standards or equivalent, higher-grade Standards

The reagents used are shown in table 1 below:

TABLE 1 details of reagents

Note: the same or higher level of reagents may also be used

The analytical equipment used is shown in table 2 below:

TABLE 2 details of the devices used

Assembly	Model number	Manufacturer(s)
			Binary pump (Binary pump)	AC Pump	AB SCIEX
Degasser (deaerator)	Degasser	AB SCIEX
			Column oven (constant temperature Column box)	AC Column oven	AB SCIEX
Autosampler (automatic sampler)	AC Autosampler	AB SCIEX
			Sample rack	Rack Changer	AB SCIEX
Mass spectrometer	QTRAP^TM6500+	AB SCIEX
			Data processor	Analyst 1.6.3(software)	AB SCIEX

The same LC/MS system may also be used.

Second, liquid condition

1. Conditions of liquid chromatography

The chromatographic column is Synergi^TM4um Polar-RP

The column specification is 50 x 2 mm; the temperature of the chromatographic column is 40 ℃; the mobile phase A is water: formic acid is mixed according to the volume percentage of 100: 0.2 mixing the obtained mixture; the mobile phase B is acetonitrile: formic acid is mixed according to the volume percentage of 100: 0.2 mixing the obtained mixture; the washing liquid is acetonitrile: water according to the volume percentage of 50: 50 mixing the obtained mixture; the temperature of the autosampler is 6 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10 μ L, and analysis time of 3.5 min; the volume of the syringe washing needle of the automatic sample injector is 500 mu L; the depth of a sample injection needle of the automatic sample injector is 45 mm; the cleaning speed of the automatic sample injector is 35 mu L/s; the sample injection speed of the automatic sample injector is 5 mu L/s; the soaking time is 5s when the sample injection needle of the automatic sample injector is cleaned;the automatic sample injector cleaning mode is before sample injection and after sample injection.

TABLE 3 gradient elution procedure

2. Conditions of Mass Spectrometry

The ion source is an electrospray ion source, the spraying voltage is 4500V, the atomizing temperature is 450 ℃, the spraying air pressure is 30Psi, the auxiliary heating air pressure is 30Psi, the air curtain air pressure is 30Psi, the collision air pressure is 8Psi, and the azithromycin-¹³The declustering voltage of C-d3 is 20 eV; azithromycin and azithromycin-¹³The collision cell inlet voltages of C-d3 are all 10 eV; azithromycin and azithromycin-¹³The collision voltage of C-d3 is 40 eV; azithromycin and azithromycin-¹³The outlet voltages of the collision chambers of C-d3 are all 10 eV; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z749.5 → m/z591.4, which is azithromycin; and m/z753.5 → m/z595.5, which is azithromycin-¹³C-d3。

Third, the experimental process

1. Preparation of azithromycin standard solution

The weighing and preparation process of the standard solution (containing the stock solution and the working solution) for the azithromycin standard curve is as follows:

weighing weight (mg)	Dissolved volume (μ l)	Final concentration (ng/. mu.l)
			1.089	10283	100

Preparing 100 ng/mu L of azithromycin stock solution according to the preparation process, and then mixing the azithromycin stock solution with the volume ratio of 1: the acetonitrile aqueous solution of 1 is sequentially diluted to prepare an azithromycin standard solution, and the specific dilution concentration is shown in the following table 4:

TABLE 4 Azithromycin Standard solution preparation concentration

Source solution (ng/. mu.L)	Volume of source solution (μ L)	Final volume (mL)	Final concentration (ng/. mu.L)
				100^a	1000	10	10
100^a	600	10	6
				100^a	200	10	2
100^a	100	10	1
				100^a	20	10	0.2
10	100	10	0.1
				10	40	10	0.04
10	20	10	0.02
				10	60	10	0.06

a: prepared directly from azithromycin (analyte)

The azithromycin standard solution is stored in a plastic container and a refrigerator (20 ℃ below zero) when not used, and the volume can be increased or reduced according to the proportion as required.

2. Azithromycin-¹³Preparation of C-d3 internal standard solution

Azithromycin-¹³The weighing and preparation process of the standard solution of the C-d3 internal standard is as follows:

weighing weight (mg)	Dissolved volume (μ l)	Final concentration (ng/. mu.l)
			2.064	25232	80

Obtaining 80 ng/. mu.L of azithromycin-¹³C-d3 stock solution, and then mixing the stock solution with a volume ratio of 1: 1 acetonitrile aqueous solution is diluted to prepare azithromycin with the concentration of 1 ng/mu L¹³The specific dilution concentrations of the C-d3 internal standard solutions are shown in Table 5 below:

TABLE 5 Azithromycin-¹³C-d3 standard solution preparation concentration

Source solution (ng/. mu.L)	Volume of source solution (μ L)	Final volume (mL)	Final concentration (ng/. mu.L)
				80^a	1250	100	1^b

a: directly from azithromycin-¹³C-d3 (internal standard)

b: for sample preparation procedures

Azithromycin-¹³The standard solution of C-d3 internal standard is stored in plastic container and refrigerator (20 deg.C) when not in use, and the volume can be increased or decreased according to the need.

3. Linear test

Putting the blank plasma into a water bath at room temperature for unfreezing; transferring 10 portions of 100 μ L of blank plasma to a 96-well plate (each standard curve sample, blank sample-00 and zero concentration sample-0), precisely adding 5 μ L of azithromycin standard solution or diluted solution with different concentrations respectively to prepare each sample according to the following table 6, mixing uniformly to prepare drug-containing plasma with different concentrations, and carrying out the operation according to 'plasma sample pretreatment'. Calculating the peak area As of the azithromycin and the internal standard azithromycin-¹³The peak area ratio Y of C-d3 to the peak area Ai (Y ═ As/Ai) was used for the regression calculation of the blood concentration X, and the results are shown in fig. 1 and table 7. The average ratio Y is used for carrying out regression calculation on the blood concentration X to obtain a regression equation Y which is 0.0181X-0.000433, r is 0.9994, and a weight coefficient W is 1/X²The lowest quantitative limit of the blood concentration of the azithromycin, which is measured by the method, is as follows: 1 ng/mL.

TABLE 6 Azithromycin Standard Curve formulation concentration

b: diluted solution of analyte: ACN/H₂O＝50/50

TABLE 7 Standard Curve of azithromycin in human plasma by HPLC-MS/MS method (n ═ 16)

4. Accuracy and precision

Putting the blank plasma into a water bath at room temperature for unfreezing; appropriate volumes of blank plasma were transferred to appropriate containers and azithromycin standard solution was added to prepare 5 drug-containing plasma quality control samples (LLOQ, QL, QLM, QM, QH) of different concentrations and a follow-up standard curve, which were processed as "plasma sample pretreatment" and prepared as shown in table 8 below. Making one batch and one following standard curve every day for 3 days, making 6 samples for each concentration of the first batch and the second batch, making 16 samples for each concentration of the third batch, calculating the peak area As of the azithromycin and the internal standard azithromycin-¹³And substituting the ratio Y of the peak areas Ai of the C-d3 into the standard curve on the day to obtain the measured concentration, calculating the precision between batches according to the measured concentration, and determining the ratio of the measured concentration to the added concentration as the accuracy, wherein the result is shown in the table 9. The result shows that the precision and the accuracy of the azithromycin plasma sample in batches and among batches are less than +/-15 percent and meet the requirements.

TABLE 8 quality control sample preparation concentration

a: final volume is source solution volume + plasma volume

Sufficient volume was dispensed into the labeled sample vials as required for each assay batch and stored at the theoretical temperature-80 ℃. The volume may be scaled up or down as desired.

TABLE 9 Intra-batch, inter-batch precision and accuracy of HPLC-MS/MS method for determining azithromycin in plasma

Note: the data of the evaluation results are from the data of 28 groups of quality control samples in 3 batches of table 9.

5. Interference

Nine different blank plasma samples are respectively from different healthy human bodies, and the nine different blank plasma samples are prepared and analyzed in the same analysis batch according to the sample preparation steps to evaluate the azithromycin analytes and the internal standard azithromycin-¹³Interference of C-d 3.

After nine blank healthy human plasma samples from different sources were prepared and analyzed, the interference peak responses at the time of compliance with azithromycin retention were all less than 20.0% of the azithromycin response of the quantitative lower limit sample in the standard curve of the analysis batch, with the results shown in table 10. The result shows that the analysis method has specificity to the analysis of the azithromycin.

After nine blank healthy human plasma samples with different sources are prepared and analyzed, the blood sample conforms to the internal standard azithromycin-¹³The interference peak responses at the retention time of C-d3 are all lower than the internal standard azithromycin of the quantitative lower limit sample in the standard curve of the analysis batch¹³5.0% of the C-d3 response, see Table 11 in the appendix. The result shows that the analysis method has no toxic or side effect on the internal standard azithromycin¹³The analysis of C-d3 was selective.

TABLE 10 comparison of interference data of blank healthy human plasma from nine different sources on azithromycin analyte

a: analyte peak area (selective sample)/analyte peak area (LLOQ of standard curve). times.100.0% to 20.0%

b: the area peak area is considered zero when "no significant peak can be integrated (or no peak)" or "the retention time of the peak area does not match the retention time of the analyte in the sample".

TABLE 11 nine blank healthy human plasma from different sources versus internal standard azithromycin-¹³Interference data comparison of C-d3Watch (A)

a: the area of the peak of the analyte (selective sample)/the area of the peak of the internal standard (LLOQ of the standard curve) multiplied by 100.0 percent is less than or equal to 5.0 percent

As can be seen from tables 10 and 11, blank plasma from different human bodies did not interfere with the results of the azithromycin test. Therefore, the method can be used for detecting the concentration of the azithromycin in the plasma of different human bodies.

6. Detection of human plasma samples

(1) Human blank plasma without azithromycin administration is precisely added with 100 μ L of blank plasma sample in a 96-deep well plate, 10 μ L of blank plasma sample is added in a volume ratio of 1: 1, adding 1000 mu L of methanol into acetonitrile water solution, carrying out vortex mixing for 1min, centrifuging at 6 ℃ for 10min at 3000rpm, taking 50 mu L of supernatant, transferring the supernatant into another 96 deep-hole plate filled with 1000 mu L of mixed organic solvent, carrying out vortex mixing, centrifuging at 6 ℃ for 5min at 3000rpm, taking 10 mu L of sample, and carrying out LC-MS/MS analysis, wherein the representative map result is shown in figure 2; the mixed organic solvent is water: acetonitrile: formic acid is mixed according to a volume ratio of 80: 20: 0.2 mixing the obtained mixture; in the above process, except for the vortex and the centrifugation, other processes are all operated under ice bath conditions.

(2) Human blank plasma without azithromycin administration is precisely added with 100 μ L of blank plasma sample in a 96-deep well plate, and 5 μ L of blank plasma sample is added in a volume ratio of 1: adding 5 mu L of an internal standard azithromycin-13C-d 3 solution with the concentration of 1 ng/mu L into acetonitrile aqueous solution of 1, adding 1000 mu L of methanol, carrying out vortex mixing for 1min, centrifuging at 6 ℃ for 10min at 3000rpm, taking 50 mu L of supernatant, transferring the supernatant into another 96 deep-well plate filled with 1000 mu L of mixed organic solvent, carrying out vortex mixing, centrifuging at 6 ℃ for 5min at 3000rpm, taking 10 mu L of sample, and carrying out LC-MS/MS analysis, wherein the representative map result is shown in figure 3; the mixed organic solvent is water: acetonitrile: formic acid is mixed according to a volume ratio of 80: 20: 0.2 mixing the obtained mixture; in the above process, except for the vortex and the centrifugation, other processes are all operated under ice bath conditions.

(3) Taking human blank plasma without azithromycin, precisely adding 100 mu L of blank plasma sample into a 96-deep-well plate, adding 5 mu L of azithromycin standard solutions with the concentrations of 0.02 ng/mu L, 0.04 ng/mu L, 0.1 ng/mu L, 0.2 ng/mu L, 1 ng/mu L, 2 ng/mu L, 6 ng/mu L and 10 ng/mu L respectively, and adding 5 mu L of 1 ng/mu L of internal standard azithromycin-¹³Adding 1000 mu L of methanol into the C-d3 solution, carrying out vortex mixing for 1min, centrifuging at 6 ℃ for 10min at 3000rpm, taking 50 mu L of supernatant, transferring the supernatant into another 96 deep-hole plate filled with 1000 mu L of mixed organic solvent, carrying out vortex mixing, centrifuging at 6 ℃ at 3000rpm for 5min, taking 10 mu L of sample added with 0.02 ng/mu L of azithromycin standard solution, and carrying out LC-MS/MS analysis, wherein the result of a representative map is shown in figure 4; the mixed organic solvent is water: acetonitrile: formic acid is mixed according to a volume ratio of 80: 20: 0.2 mixing the obtained mixture; in the above process, except for the vortex and the centrifugation, other processes are all operated under ice bath conditions.

(4) Collecting plasma of healthy subjects after oral administration of azithromycin or pharmaceutical salt thereof, precisely adding 100 mu L of collected human plasma samples into a 96-deep-well plate, and adding 5 mu L of the human plasma samples with the volume ratio of 1: 1 methanol aqueous solution, 5 mu L of 1 ng/mu L of internal standard azithromycin-¹³Adding 1000 mu L of methanol into the C-d3 solution, carrying out vortex mixing for 1min, centrifuging at 6 ℃ for 10min at 3000rpm, taking 50 mu L of supernatant, transferring into another 96 deep-well plate filled with 1000 mu L of mixed organic solvent, carrying out vortex mixing, centrifuging at 6 ℃ for 5min at 3000rpm, taking 10 mu L of sample, and carrying out LC-MS/MS analysis, wherein the representative map result is shown in FIG. 5; the mixed organic solvent is water: acetonitrile: formic acid is mixed according to a volume ratio of 80: 20: 0.2 mixing the obtained mixture; in the above process, except for the vortex and the centrifugation, other processes are all operated under ice bath conditions.

In conclusion, the invention provides a method for determining the concentration of azithromycin in blood plasma, which is simple and convenient in pretreatment method, adopts a two-step organic solution extraction method and is suitable for conventional determination; meanwhile, under the chromatographic conditions adopted in the experiment, the azithromycinThe retention time is about 1.488min, and the internal standard azithromycin-¹³The retention time of the C-d3 is about 1.483min, and the azithromycin and the internal standard azithromycin-¹³The peak shape of C-d3 is good, no miscellaneous peak interference is caused to determine, and the baseline is stable; the method has high specificity, can accurately measure the concentration of the azithromycin in the blood plasma, has high sensitivity, and has the minimum limit of blood plasma quantification of 1 ng/mL; meanwhile, the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for determining the blood concentration of the azithromycin. The linear range of the plasma standard curve of the method is 1-500 ng/mL, and the precision RSD in batch and between batches is less than +/-15%.

Claims

1. A method for determining the concentration of azithromycin in blood plasma by liquid chromatography-mass spectrometry is characterized in that: the plasma sample is pretreated and then the concentration of the plasma sample is detected by high performance liquid chromatography-tandem mass spectrometry, and the specific method comprises the following steps:

(1) plasma sample pretreatment:

(2) and (3) sample measurement:

2. The method for determining the concentration of azithromycin in blood plasma by LC-MS according to claim 1, which is characterized in that: the gradient elution procedure in the step (2) is as follows:

3. the method for determining the concentration of azithromycin in blood plasma by LC-MS according to claim 1 or 2, wherein: in the step (2), an internal standard method is adopted, and azithromycin and internal standard azithromycin-¹³The peak area ratio of C-d3 was brought to the standard curveThe concentration of azithromycin in the plasma sample is calculated.

4. The method for determining the concentration of azithromycin in blood plasma by LC-MS according to claim 3, wherein: the establishment of the standard curve equation comprises the following steps:

5. The method for determining the concentration of azithromycin in blood plasma by LC-MS according to claim 1 or 2, wherein: the conditions for the liquid chromatography determination in the step (2) further include: the volume of the syringe washing needle of the automatic sample injector is 500 mu L; the depth of a sample injection needle of the automatic sample injector is 45 mm; the cleaning speed of the automatic sample injector is 35 mu L/s; the sample injection speed of the automatic sample injector is 5 mu L/s; the soaking time is 5s when the sample injection needle of the automatic sample injector is cleaned; the automatic sample injector cleaning mode is before sample injection and after sample injection.