CN110749666A

CN110749666A - Liquid chromatography tandem mass spectrometry method for detecting busulfan in plasma

Info

Publication number: CN110749666A
Application number: CN201910857269.4A
Authority: CN
Inventors: 朱月红; 王金文; 吴亦宵
Original assignee: Shanghai Yaoming Kangde Medical Laboratory Co Ltd
Current assignee: Shanghai Yaoming Kangde Medical Laboratory Co Ltd
Priority date: 2019-09-09
Filing date: 2019-09-09
Publication date: 2020-02-04

Abstract

The invention provides a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for detecting busulfan in blood plasma, which comprises the following steps: preparing a working solution; pre-treating; LC-MS/MS analysis; extracting a chromatogram and fitting a calibration curve; and establishing a medicine time curve chart. The LC-MS/MS method optimizes the method for detecting busulfan in human plasma, shortens the analysis time by 2 minutes, ensures the analysis accuracy and precision and the data stability, and has simple pretreatment process, short time and easy operation. The detection time is saved, the detection accuracy and reliability are guaranteed, and more accurate, stable, high-precision and high-sensitivity detection data can be provided in a shorter time.

Description

Liquid chromatography tandem mass spectrometry method for detecting busulfan in plasma

Technical Field

The invention belongs to the technical field of therapeutic drug monitoring and pharmacokinetic analysis, and particularly relates to a method for detecting busulfan in blood plasma by using a liquid chromatography-tandem mass spectrometry mode.

Background

Busulfan is an alkylating agent, belongs to a double-function alkylating agent of dimethyl sulfonate, is a cell cycle nonspecific drug, and can be used for ablating bone marrow cells before hematopoietic stem cell transplantation. After the busulfan enters a human body, the cyclic structure of the sulfonate group of the busulfan is opened, and the structure and the function of DNA are damaged through alkylation with guanine in cellular DNA.

Busulfan is typically administered intravenously at a dose of 0.8mg/kg every 6 hours for 4 days for a total of 16 doses. However, repeated administration of busulfan can gradually accumulate in vivo, and overdose can cause hepatic vein occlusive disease. Thus to ensure that the dose of busulfan is sufficient to completely ablate bone marrow cells while avoiding toxicity, intravenous administration is guided by Pharmacokinetic (PK) assessment of the area under the plasma curve (AUC) and clearance after the first dose; PK assessments should be performed at the end of the first dose and PK test results may be used to facilitate dose adjustments for subsequent doses.

At present, the method for monitoring busulfan is mainly a chromatographic analysis method, and comprises a gas chromatography-mass spectrometry (GC-MS), a liquid chromatography tandem mass spectrometry (LC-MS/MS), a high performance liquid chromatography (HPLC-UV) and the like. However, the existing busulfan detection methods have respective disadvantages, for example, some methods have insufficient analysis sensitivity, higher lower limit and lower limit of quantification, some methods have relatively complex treatment processes and more steps or the methods are relatively simple but have longer treatment time; and other instruments have long analysis time and large sample usage amount.

Disclosure of Invention

The invention mainly aims to provide a method for quickly and accurately detecting the concentration of busulfan in human plasma, and provide effective support for clinical follow-up administration and treatment.

In order to achieve the above object, the present invention provides a method for detecting busulfan in plasma by liquid chromatography tandem mass spectrometry, which is characterized by comprising the steps of:

1) preparing a standard substance working solution, a quality control substance working solution and an internal standard working solution: accurately weighing the Busulfan standard substance, diluting with methanol, and fixing volume to obtain multiple standard substance working solutions with certain concentration gradient; precisely weighing the busulfan quality control product, dissolving the busulfan quality control product with methanol, and fixing the volume to obtain a plurality of quality control product working solutions with a certain concentration gradient; accurately weighing the Busulfan-d 8 internal standard, dissolving with methanol and fixing the volume to obtain an internal standard working solution;

2) pretreatment: precisely measuring a standard substance working solution, a quality control substance working solution and a sample to be detected, respectively adding an internal standard working solution to perform protein precipitation, oscillating and mixing uniformly, then centrifuging, and taking supernatant to dilute;

3) injecting the diluted sample, and performing liquid phase secondary mass spectrometry:

wherein, the chromatographic conditions are as follows:

a detection instrument: a UPLC liquid chromatograph;

a chromatographic column: an ultra high pressure UPLC 18 chromatography column;

the mobile phase consists of a mobile phase A and a mobile phase B, wherein the mobile phase A is a mixed solution of 5 mM-15 mM ammonium acetate, 0.05% -2% formic acid, 0.01% -0.05% trifluoroacetic acid and water, and the mobile phase B is a mixed solution of 5 mM-15 mM ammonium acetate, 0.05% -2% formic acid, 0.01% -0.05% trifluoroacetic acid and methanol;

gradient elution was used, with the following elution gradient: mobile phase A + mobile phase B is 100%; keeping the volume percentage of the mobile phase B at 10 percent for 0-0.3 min; the volume percentage of the mobile phase B is increased from 10 percent to 90 percent in 0.3-1.1 min; 1.1-1.7 min, and keeping the volume percentage of the mobile phase B at 90%; 1.7-1.8min, the volume percentage of the mobile phase B is reduced from 90% to 10%; 1.8-2.0 min, keeping the volume percentage of the mobile phase B at 10%;

wherein, the mass spectrum conditions are as follows: ESI ion source, positive ion mode, MRM scanning mode;

4) extracting an MRM chromatogram, and fitting a calibration curve;

5) establishing a medicine time curve chart: and calculating the concentration of the sample to be detected according to the calibration curve, and establishing a medicine time curve graph by taking the concentration of the sample to be detected as an abscissa and taking the area under the curve of the concentration of the sample to be detected as an ordinate.

Wherein the mass spectrometry conditions further comprise: the spray voltage was 5500V, the ion source temperature was 350 ℃, the gas curtain pressure was 10psi, the collision gas pressure was 3psi, the atomization gas pressure was 50psi, and the auxiliary gas pressure was 20 psi.

Wherein the sample to be detected is a blood sample obtained by intravenous blood sampling at different time nodes after intravenous injection administration of a patient; in a specific example, the time nodes are 2h, 3h, 6h, 8h, 10h post injection, respectively.

Wherein, the calibration curve in the step 4) takes the concentration of the working solution of the calibrator as the abscissa, the peak area ratio of the working solution of the calibrator to the internal standard as the ordinate, and the weight is set to be 1/x²Neglecting the origin, a linear calibration curve is fitted.

The ammonium acetate in the mixture of mobile phase A is preferably 8 mM-12 mM, and the ammonium acetate in the mixture of mobile phase B is preferably 8 mM-12 mM.

In a specific example, mobile phase a is 10mM ammonium acetate + 0.1% formic acid + 0.01% trifluoroacetic acid + water; mobile phase B was 10mM ammonium acetate + 0.1% formic acid + 0.01% trifluoroacetic acid + methanol.

Wherein the flow rate of the gradient elution is 0.5mL/min to 1mL/min, and the column temperature is 35 ℃ to 40 ℃. In a specific example, the flow rate of the gradient elution is 0.6mL/min and the column temperature is 40 ℃.

Wherein the sample amount is 1-5 mul; in one specific example, the sample size is 3. mu.l.

In a specific example, the concentration gradient of the standard working solution is 25, 200, 1000, 3000 and 7500ng/mL in sequence.

In a specific example, the concentration gradient of the quality control product working solution is 300ng/mL, 2500ng/mL and 5000ng/mL in sequence.

In a specific example, the internal standard working solution has a concentration of 100 ng/mL.

Compared with the prior art, the invention has the beneficial effects that:

compared with the conventional pretreatment method of liquid-liquid extraction with complicated steps, the method for detecting busulfan in human plasma disclosed by the invention adopts a protein precipitation method for pretreatment, and is simple in flow, short in time consumption and easy to operate; the sample amount required by the method is small, the analysis time is shortened to 2 minutes, and the detection time is saved; the working solution preparation method of the method enables the working solution to have higher accuracy, enables the standard curve to be more accurate, and ensures the accuracy and reliability of detection, thereby ensuring that more accurate, stable, high-precision and high-sensitivity detection data can be provided in a shorter time.

Drawings

FIG. 1 is a chromatogram interface example of a blank sample (100% bovine plasma heparin sodium anticoagulation) (upper: total ion; middle: Busulfan internal standard; lower: Busulfan)

FIG. 2 is a chromatogram interface example of a low concentration C1 busulfan matrix sample (upper: total ions; middle: busulfan internal standard; lower: busulfan)

FIG. 3 is a chromatogram interface example of a high concentration C5 busulfan matrix sample (upper: total ions; middle: busulfan internal standard, lower: busulfan)

FIG. 4 is an exemplary chromatogram interface of an actual patient sample (upper: total ions; middle: Busulfan internal standard; lower: Busulfan)

FIG. 5 is a linear model of example two (2)

FIG. 6 is the time curve of the three cases 1 in the example

FIG. 7 is the time curve of the three cases 2 in the example

FIG. 8 is the time curve of the three cases of example 3

FIG. 9 is the time curve of the three cases 4

FIG. 10 is the time curve of the three cases 5 in the example

FIG. 11 is the time chart of the three cases of example 6

FIG. 12 is a scattergram of the administered dose versus AUC according to cases 1-6

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

The first embodiment is as follows: detailed description of the invention

1. Preparation of primary stock solution of standard substance/quality control substance

1.1 standard primary Stock Solutions (SSC): the Busulfan standard methanol solution (Cerilliant) with the concentration of 1mg/mL is SSC, and is stored at-25 to-15 ℃ until the validity period of a manufacturer;

1.2 quality control Primary Stock Solution (SSQC): weighing 10mg of busulfan (TRC/Sigma) quality control product by a million-level analytical balance, transferring the product into a 10mL volumetric flask, fixing the volume to 10mL by pure methanol, preparing a first-level stock solution of the quality control product with the concentration of 1mg/mL, oscillating, uniformly mixing, performing ultrasonic treatment, transferring the product into a brown glass vial, wherein the number of the bottle is SSQC, and storing the product at-25 to-15 ℃ for one year in an effective period.

2. Preparation of standard/quality control secondary stock solution

2.1 standard secondary stock solution (sub-SSC): transferring a proper amount of the primary stock solution (1.0mg/mL) of the quality control product, dissolving the primary stock solution with pure methanol to prepare a secondary stock solution of the calibrator with the concentration of 50 mu g/mL, shaking and uniformly mixing the solution, transferring the solution to a brown glass vial with the number of sub-SSC, and storing the solution at the temperature of between 25 ℃ below zero and 15 ℃ below zero for one year;

2.2 quality control Secondary stock solution (sub-SSQC): transferring a proper amount of the primary stock solution (1.0mg/mL) of the quality control product, dissolving the primary stock solution with pure methanol to prepare a secondary stock solution of the quality control product with the concentration of 50 mu g/mL, oscillating and uniformly mixing the solution, transferring the solution to a brown glass vial with the number of sub-SSQC, and storing the solution for one year at the validity period of-25 to-15 ℃;

in order to ensure the accuracy and avoid weighing errors and human errors as much as possible, the primary stock solution of the calibrator and the primary stock solution of the quality control product cannot be weighed or prepared simultaneously, and are respectively weighed and prepared by two persons.

3. Internal standard stock/working solution preparation

3.1 internal standard stock (SSIS): weighing 1mg of busulfan-d 8(TRC) internal standard by a million-level analytical balance, transferring the internal standard into a 10mL volumetric flask, fixing the volume to 10mL by pure methanol, preparing an internal standard stock solution with the concentration of 100 mug/mL, shaking, uniformly mixing, transferring the internal standard stock solution into a brown glass vial with the number of SSIS, and storing the internal standard stock solution at the temperature of between 25 ℃ below zero and 15 ℃ below zero for one year.

3.2 internal standard working solution (WSIS): transferring 100 mu L of the internal standard stock solution into a 100mL volumetric flask, using pure methanol to fix the volume to 100mL, preparing the internal standard working solution with the concentration of 100ng/mL, shaking, uniformly mixing, transferring into a reagent bottle, numbering as WSIS, and storing in a refrigerator at 4 ℃.

4. Preparation of standard working solution/quality control working solution

4.1 standard working solution: diluting the primary/secondary stock solutions (SSC, sub-SSC) with 100% bovine plasma (heparin sodium) by using a pipette and a volumetric flask, preparing standard working solutions (shown in table one), wherein the standard working solutions are numbered as C1-C5, have corresponding concentrations of 25, 200, 1000, 3000 and 7500ng/mL respectively, and are stored at-80 ℃ for one year of validity.

4.2 quality control product working solution: diluting the primary/secondary stock solution (SSQC, sub-SSQC) of the quality control product with 100% bovine plasma (heparin sodium) by using a pipette and a volumetric flask to prepare a working solution (shown in the table I) of the quality control product, wherein the working solution is numbered as LQC, MQC and HQC, the corresponding concentrations are 300, 2500 and 5000ng/mL respectively, and the working solution is stored at-80 ℃.

Preparing standard working solution (C1-C5) and quality control working solution (LQC, MQC, HQC)

5. Quality control material assignment

5.1 using the working solution of the calibrator (C1-C5) as a standard curve, using the working solution of the quality control product (LQC, MQC and HQC) as analytes to detect the respective concentration of the analytes, performing detection of a plurality of batches in a batch by a plurality of days, and calculating the corresponding uncertainty by using the mean value of the obtained results, namely the assigned concentration of the working solution of the quality control product.

6. Sample pretreatment method

6.1 dissolving blank matrix (anticoagulation of 100% bovine plasma heparin sodium), working solution of calibrator, working solution of quality control product and sample to be tested at room temperature, and respectively shaking and mixing uniformly for 1min on a shaking and mixing instrument at the rotating speed of 1000 rpm; the samples to be detected are 5 samples obtained by blood sampling for 2h, 3h, 6h, 8h and 10h of intravenous infusion administration;

6.2 adding 50 mu L of the sample into a 96-hole deep-hole plate respectively, and adding 450 mu L of internal standard working solution respectively;

6.3 evenly mixing the mixture on an oscillation mixer at 1000rpm for 5min, and centrifuging the mixture for 10min at the room temperature of 4000 rpm;

6.4 adding 100 μ L of the supernatant solution into a new 96-well deep-well plate, respectively, adding 200 μ L of mobile phase A (10 mM ammonium acetate aqueous solution containing 0.1% formic acid and 0.01% trifluoroacetic acid) for dilution, and sealing with a heat sealing machine;

6.5 Each sample was analyzed by feeding 3. mu.L of sample in an LC-MS/MS system.

7. Liquid chromatography-tandem mass spectrometer (LC-MS/MS) quantitative analysis method

7.1 Instrument: waters Acquity UPLC + API 4000

7.2 chromatographic column: waters ACQUITY UPLC BEH C18, 2.1 mm. times.50 mm, 1.7 μm

7.3 mobile phase, needle wash:

(1) mobile phase A: 10mM ammonium acetate, 0.1% formic acid, 0.01% trifluoroacetic acid and water (1L of ultrapure water is measured by a measuring cylinder, the weighed 0.7708g of ammonium acetate is added into a reagent bottle, 1mL of formic acid and 100 mu L of trifluoroacetic acid are added inwards after the ammonium acetate is dissolved, and the mixture is uniformly mixed and subjected to ultrasonic treatment for 10 minutes);

(2) mobile phase B: 10mM ammonium acetate + 0.1% formic acid + 0.01% trifluoroacetic acid + methanol (1L of methanol is measured by a measuring cylinder, the weighed 0.7708g of ammonium acetate is added into a reagent bottle, 1mL of formic acid and 100 mu L of trifluoroacetic acid are added inwards after the ammonium acetate is dissolved, and the mixture is uniformly mixed and is subjected to ultrasonic treatment for 10 minutes);

(3) strong needle washing liquid: 10% water + 90% acetonitrile + 0.1% formic acid (900 mL acetonitrile and 100mL water are taken by a measuring cylinder, poured into a reagent bottle, and then 1mL formic acid is added);

(4) weak needle washing liquid: 90% water + 10% acetonitrile + 0.1% formic acid (100 mL acetonitrile and 900mL water taken with a graduated cylinder are poured into a reagent bottle and 1mL formic acid is added).

The water used for preparing the experimental reagent is laboratory clinical water (the resistivity reaches 18.2M omega cm);

other reagents are HPLC grade, and the calibrator, the quality control product and the internal standard are proved compounds with quality inspection reports.

7.4 elution gradient: watch two

Time(min)	A(％)	B(％)
			Initial	90	10
0.3	90	10
			1.1	10	90
1.7	10	90
			1.8	90	10
2.0	90	10

Flow rate: column temperature 0.6 mL/min: 40 deg.C

7.5 MS parameter settings

7.6 ion pair information: watch III

8. Extracting MRM chromatogram, fitting calibration curve

Respectively extracting MRM chromatograms of a blank sample (anticoagulation of 100% bovine plasma heparin sodium), each concentration sample of a calibrator working solution (C1-C5) and a sample to be detected by using Analyst software.

As shown in fig. 1 to 4, which are interface examples of the analysis software for extracting MRM chromatogram of each sample, fig. 1 is chromatogram of blank sample (anticoagulated by 100% bovine plasma heparin sodium), fig. 2 is chromatogram of low-concentration (25ng/mL) busulfan matrix sample, fig. 3 is chromatogram of high-concentration C5(7500ng/mL) busulfan matrix sample, and fig. 4 is chromatogram of sample to be tested; the middle part and the lower part of the diagram correspond to the interior label of busulfan and busulfan respectively, and the upper part of the diagram corresponds to the total ions of the interior label of busulfan and busulfan.

Respectively obtaining the peak areas of the busulfan and the busulfan internal standard of each sample and the ratio of the peak areas (busulfan peak area/busulfan internal standard peak area) of the two; taking the concentration of the working solution of the calibrator as the abscissa and the peak area ratio (Busulfan peak area/Busulfan internal standard peak area) of Busulfan and Busulfan internal standard as the ordinate, and setting the weight as 1/x²Neglecting the originalAnd (4) point fitting a linear calibration curve.

9. Calculating the concentration of the sample to be measured according to the calibration curve, and calculating AUC

The AUC is the area under the curve obtained by using the blood concentration as the ordinate and the time as the abscissa after administration, and can be generally determined by the integration method or the trapezoidal method. And (3) substituting the value of the Busulfan peak area/Busulfan internal standard peak area of the sample to be detected in the MRM chromatogram into the calibration curve to obtain the corresponding concentration of the sample to be detected, namely the blood concentration. Therefore, a drug time curve graph can be established, and the AUC of the busulfan in the plasma can be calculated.

Taking the case one in the following example three as an example, in the present case, the intravenous administration dose of busulfan is 120mg, the intravenous blood collection is completed 2 hours after the intravenous injection of the drug, and the intravenous blood collection is performed 1, 4, 6 and 8 hours after the first blood collection, respectively. Carrying out sample pretreatment and sample feeding analysis on a machine, calculating the blood concentration, and recording the following table (table four); the drug time curve was established based on the blood concentration, and the results are shown in fig. 6.

Table four cases-blood concentration data table

Sample numbering	Time of infusion	Post-infusion sampling time point (min)	Blood concentration (mu M)	Blood concentration (ng/mL)
						9:45	0	0.00	0
1	11:45	120	8.65	2128
					2	12:45	180	7.33	1804
3	15:46	361	3.17	780
					4	17:48	483	1.98	487
5	19:49	604	1.26	309

Example two: methodology validation

First, precision

Adding a corresponding amount of busulfan standard substance into a blank matrix (100% bovine plasma heparin sodium anticoagulation), and preparing low, medium and high concentration levels of busulfan standard substance solutions in a linear range (25-7500ng/mL) of a calibration curve, wherein the concentrations are respectively as follows: 300ng/mL, 2500ng/mL, 5000ng/mL, each concentration level has 3 parallel samples, is a batch; a total of five batches, verified on day 5, one batch per day.

Table five precision data results

Busulfan medicine	Low concentration of	Middle concentration	High concentration
				Precision in batch (CV,%)	5.01	2.37	1.20
Precision between lots (CV,%)	3.43	3.79	3.04

The verification results are shown in the fifth table, and CV% of precision in batches and among batches are within 15% so as to meet the verification requirements; and the CV percent of the precision in the batch and between the batches is basically lower than 5 percent, and the precision is higher and far superior to the basic verification requirement.

Two, linear regression

Adding a corresponding amount of busulfan standard substance into a blank matrix (100% bovine plasma heparin sodium anticoagulation) to prepare 5 busulfan standard substance solutions with different concentration levels, wherein the concentrations are as follows: 25ng/mL, 200ng/mL, 1000ng/mL, 3000ng/mL, 7500ng/mL, with 3 replicates per concentration level, completed in the same batch.

Using least squares linear fit, the mean value of the measured concentration (y) of the sample_i) Theoretical concentration for the sample (x)_i) And performing regression calculation to obtain a linear regression equation y which is kx + b and calculating a correlation coefficient R.

Table six sample data

As shown in table six and fig. 5, the verification results were analyzed: the deviation of all concentration points is within + -15.0%, CV is within 20.0%, and R can be obtained by regression method²The slope is 0.9921, which meets the requirement of verification.

Third, sensitivity

LOB (limit of quantitation): the blank matrix (100% bovine plasma heparin sodium anticoagulation) and the C1 solution samples in table one, each with 4 replicates, were processed as one batch according to the sample pretreatment method described above 6; a total of five batches, verified on day 5, one batch per day.

TABLE VII: LOB result data

LOD (detection limit): preparing 4 samples with the concentration close to LOB, wherein the concentrations are 5ng/mL, 10ng/mL, 15ng/mL and 20ng/mL respectively, and each concentration point and the C1 solution sample respectively have 4 parallel samples which are one batch; a total of five batches, verified on day 5, one batch per day.

Table eight: LOD result data

LLOQ: and (3) analyzing the verification result of the performance LOD, wherein the lowest concentration sample (not less than LOD) meeting the standard that the CV is not more than 20% and the bias is not more than 15% is the LLOQ.

The verification results are shown in Table nine, the mean value of each concentration sample and the sample mean value +3SD are calculated, and the lowest concentration sample with the CV being less than or equal to 20% and the bias being less than or equal to 15% is obtained, namely the LLOQ is 20 ng/mL.

Table nine LLOQ data results

Substrate effect

Adding a corresponding amount of busulfan standard solution into a pure solvent to prepare 5 calibration curves, wherein the concentrations are as follows: 25ng/mL, 200ng/mL, 1000ng/mL, 3000ng/mL, 7500ng/mL, and the concentration of the standard working solution C1-C5.

Meanwhile, a corresponding amount of busulfan standard solution is respectively added into human plasma from 5 different sources to prepare 5 calibration curves, wherein the concentrations are as follows: 25ng/mL, 200ng/mL, 1000ng/mL, 3000ng/mL, 7500 ng/mL; the concentration of the standard working solution is equal to that of C1-C5;

5 calibration curves in pure solvent and human plasma matrix and signal mean values thereof at each concentration were obtained, and the two signal mean values were compared.

Results of the data in Table ten

The verification results are shown in table ten, the matrix deviation (bias) is within +/-25.0%, the CV is within 15.0%, no obvious ion inhibition exists, and the verification requirements are met; and CV% is within 10%, and the deviation is small and far superior to the basic verification requirement.

Fifth, dilution reliability

Adding a corresponding amount of busulfan standard substance into a blank matrix to prepare a busulfan standard substance solution with a specific concentration level, wherein the concentrations are 5000ng/mL and 15000ng/mL respectively, and then diluting the solution 2 or 10 times by using the blank matrix; 6 parallel samples were tested and completed in the same batch:

TABLE eleven results data

Busulfan medicine	DF2-5000ng/mL	DF10-5000ng/mL	DF10-15000ng/mL
				The recovery rate is high	16.67	18.70	5.56
CV％	3.0	1.3	1.6

The verification result is shown in the eleventh table, the average deviation of the recovery rate is within +/-20%, the CV is less than 15%, and the verification requirement is met; and CV% is not more than 3%, and the dilution precision is higher.

Sixth, evaluation of residue

The corresponding amount of Busulfan standard substance is added into the blank matrix to prepare Busulfan standard substance solution with the concentration of 2 times of ULOQ (15000 ng/mL). During sample injection, one sample of 2 times of ULOQ busulfan standard substance is firstly injected, and then 5 blank matrix samples are continuously injected to form a batch. For a total of five batches, verification was carried out in five days, one batch per day.

TABLE twelve results data

The verification result is shown in fig. 12, the peak area ratio LLOQ peak area of all blank substrates is less than 25.0%, and the verification meets the requirements and has no residue.

Seventh, accuracy

Preparing 20 correct-check samples in the linear range of the calibration curve (25-7500ng/mL) in pooled human plasma by one technician using another brand of busulfan standard (different from the linear validation brand described above); the mixed human plasma is prepared by mixing human plasma from different sources and then using the mixed human plasma for preparing 20 accurate examination samples.

The sample was tested as a blind sample by another technician for concentration. The 20 samples configured were tested as one batch, 1 time per sample.

TABLE thirteen comparison of the test results with the theoretical concentrations

Accurate sample numbering	Theoretical concentration of sample (ng/mL)	Sample measured concentration (ng/mL)	Bias*(％)
				A1	375	410	109
A2	625	686	110
				A3	400	404	101
A4	1000	1160	116
				A5	3500	3310	94.5
A6	2125	1880	88.4
				A7	350	344	98.4
A8	500	498	99.7
				A9	1875	1660	88.4
A10	3125	2790	89.4
				A11	625	670	107
A12	375	367	97.9
				A13	4500	4070	90.5
A14	1250	1400	112
				A15	2250	1810	80.5
A16	5125	4230	82.5
				A17	2500	2080	83.1
A18	4000	3740	93.5
				A19	5750	5210	90.6
A20	5500	5320	96.8

The verification results are shown in table thirteen, the deviation (Bias) of the actually measured concentration of 100% of the samples is within the range of +/-20%, and the verification requirements are met.

Conclusion of method validation:

example 3: patient sample monitoring case

Case 1:

basic information: the male is 47 years old, the weight is 75kg, the intravenous administration dosage of the busulfan is 120mg, the intravenous blood collection is completed 2 hours after the intravenous injection of the medicine, the intravenous blood collection is respectively carried out 1, 4, 6 and 8 hours after the first blood collection, and the plasma is centrifugally separated as soon as possible and added with heparin sodium for anticoagulation. A time curve diagram of drug is established after sample pre-treatment and machine sample analysis is performed, as shown in fig. 6.

Case 2:

basic information: female, age 32 years, weight 67.2kg, intravenous administration dose of Busulfan 108mg, completing intravenous blood collection 2 hours after intravenous injection of medicine, performing intravenous blood collection 1, 4, 6 and 8 hours after the first blood collection respectively, and centrifuging plasma as soon as possible to add heparin sodium for anticoagulation. A time curve diagram of drug is established after sample pre-treatment, machine sample introduction and analysis is carried out, as shown in fig. 7.

Case 3:

basic information: the male is 28 years old, 72kg in weight, 108mg in intravenous injection dosage of Busulfan, completes intravenous blood collection 2 hours after intravenous injection of medicine, performs intravenous blood collection 1, 4, 6 and 8 hours after the first blood collection respectively, and performs centrifugal separation of blood plasma as soon as possible to add heparin sodium for anticoagulation. A time curve diagram of drug administration is established after pre-treatment of the sample and machine sample introduction analysis, as shown in fig. 8.

Case 4:

basic information: the male is aged 34 years old, has a body weight of 67kg, and has a busulfan intravenous administration dosage of 102mg, and completes intravenous blood collection 2 hours after intravenous injection of the medicine, and performs intravenous blood collection 1, 4, 6 and 8 hours after the first blood collection respectively, and centrifugally separates plasma as soon as possible to add heparin sodium for anticoagulation. A time curve diagram of drug is established after sample pre-treatment and machine sample analysis is performed, as shown in fig. 9.

Case 5:

basic information: female, age 35 years, weight 51.5kg, intravenous administration dose of Busulfan 78mg, completing intravenous blood collection 2 hours after intravenous injection of medicine, performing intravenous blood collection 1, 4, 6, 8 hours after the first blood collection, centrifuging blood plasma as soon as possible, and adding heparin sodium for anticoagulation. A time curve diagram of drug administration is established after the pre-treatment of the sample and the machine-sample analysis, as shown in fig. 10.

Case 6:

basic information: female, age 40 years old, weight 57kg, busulfan intravenous injection dosage 90mg, completing intravenous blood collection 2 hours after intravenous injection of drug, performing intravenous blood collection 1, 4, 6, 8 hours after the first blood collection respectively, and adding heparin sodium for anticoagulation after centrifuging plasma as soon as possible. A time curve diagram of drug formulation was established after pre-treatment of the sample and machine sample analysis, as shown in fig. 11.

From the time chart (fig. 6-11) of the above case, it can be seen that after intravenous busulfan, the concentration of busulfan in human body shows a downward trend with time, the peak value of the concentration is 0 hour after the completion of the administration and injection, the decrease is obvious after one hour, and the concentration change is small after 4 hours. The results are in line with theoretical expectations, and it is known that the method can ensure the accuracy of analysis and the stability of data.

As shown in fig. 12, a scattergram of the administered dose and AUC in the above case shows the overall trend of the AUC in direct proportion to the administered dose, and can accurately reflect the difference of AUC of each patient due to age, sex, etc. under the condition of the same administered dose; the method can ensure the precision of analysis, is suitable for individual case analysis, has higher applicability, and can provide reference for medical staff in the aspects of subsequent administration modes, dosage and the like according to the self condition of a patient.

In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A liquid chromatography tandem mass spectrometry method for detecting busulfan in plasma is characterized by comprising the following steps:

wherein, the chromatographic conditions are as follows:

a detection instrument: a UPLC liquid chromatograph;

a chromatographic column: an ultra high pressure UPLC 18 chromatography column;

wherein, the mass spectrum conditions are as follows:

ESI ion source, positive ion mode, MRM scanning mode;

4) extracting an MRM chromatogram, and fitting a calibration curve;

2. The method of claim 1, wherein the concentration gradient of the standard working solution is 25, 200, 1000, 3000, 7500ng/mL in sequence.

3. The method of claim 1, wherein the concentration gradient of the quality control working solution is 300ng/mL, 2500ng/mL and 5000ng/mL in sequence.

4. The method of claim 1, wherein the internal standard working solution has a concentration of 100 ng/mL.

5. The method of claim 1, wherein the sample to be tested is a blood sample obtained by intravenous administration of a drug to a patient at different time nodes.

6. The method of claim 1, wherein the mass spectrometry conditions further comprise: the spray voltage was 5500V, the ion source temperature was 350 ℃, the gas curtain pressure was 10psi, the collision gas pressure was 3psi, the atomization gas pressure was 50psi, and the auxiliary gas pressure was 20 psi.

7. The method according to claim 1, wherein the calibration curve in step 4) is set to have a weight of 1/x by taking the concentration of the working solution of the calibrator as the abscissa and the peak area ratio of the working solution of the calibrator to the internal standard as the ordinate²Neglecting the origin, a linear calibration curve is fitted.

8. The method according to claim 1, wherein the flow rate of the gradient elution is 0.5mL/min to 1mL/min, and the column temperature is 35 ℃ to 40 ℃.

9. The method of claim 1, wherein the sample size is 1 to 5 μ l.

10. The method according to claim 1, wherein the mobile phase a is 10mM ammonium acetate + 0.1% formic acid + 0.01% trifluoroacetic acid + water; mobile phase B was 10mM ammonium acetate + 0.1% formic acid + 0.01% trifluoroacetic acid + methanol.