CN114646719A - Liquid chromatography-mass spectrometry detection method for MMAE in plasma - Google Patents

Abstract

The invention discloses a liquid chromatography-mass spectrometry detection method of MMAE in blood plasma, which comprises the following steps: s1, pretreatment of a plasma sample: extracting a sample to be detected by adopting a one-step organic solvent precipitation method so as to obtain an extracting solution to be detected; and S2, carrying out LC-MS detection on the extracting solution to be detected so as to determine the concentration of MMAE in the sample to be detected. The method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of MMAE; the linear range of the plasma standard curve of the method is 0.010-10.0ng/mL, the precision RSD in batch and between batches is less than +/-15%, and the method has the advantages of high recovery rate, good reproducibility and no obvious matrix effect, and is suitable for measuring the MMAE concentration in plasma.

Description

Liquid chromatography-mass spectrometry detection method for MMAE in plasma

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 liquid chromatography-mass spectrometry detection method for MMAE in blood plasma.

Background

Monomethyl auristatin E MMAE is (microtubule-associated)Inhibitors) are synthetic antineoplastic agents. Its chemical name (S) -2- (((3R,4S,5S) -1- ((S) -2- ((1R,2R) -3- (((1S,2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidin-1-yl) -3-methoxy-5-methyl-1-oxoheptan-4-yl) (methyl) amino) -3-methyl-N- (methyl-L-valyl) butanamide, formula: c₃₉H₆₇N₅O₇Molecular weight: 717.98, having the chemical formula:

MMAE is a synthetic derivative of dolastatin 10, which itself is very toxic and cannot be used as a drug by inhibiting tubulin polymerization to effect effective mitotic inhibition, but it is linked to monoclonal antibodies which direct it to cancer cells. MMAE is therefore widely used as a cytotoxic component in the manufacture of Antibody Drug Conjugates (ADCs) to treat cancer. The antibody coupling drug is that the monoclonal antibody with high targeting property is coupled with the antitumor drug with cytotoxicity through a specific section of connecting fragment, so that the high selectivity of the antibody and the antitumor activity of the drug are combined into a whole. Currently, ADC drugs using MMAE as a toxic small molecule drug are mainly approved in 2011 as addetris (seattle) against CD 30; polivy (roche) for CD77 β, approved in 2019; padcev (Seattle) against Nectin-4, approved in 2019; weidesintemab to Her2 (glogchang organism) obtained in 2021; tivdak against TF (Genmab/Seagen) approved in 2021. Among 14 types of ADC drugs on the market worldwide, 5 types of drugs use MMAE as an anti-tumor toxic small molecule drug, and hundreds of types of ADC drugs are under development worldwide by 2022, and most of MMAE is selected as a toxic small molecule drug, which is enough to show the prominent position of MMAE in the field of tumor treatment. Because the ADC medicine has targeting property and is a key treatment means in tumor treatment, the global sales of the ADC medicine is about 50 billion dollars in 2021 by incomplete statistics, and the global sales of the ADC medicine using MMAE as a toxic small-molecule medicine exceeds 16 billion dollars, so that great economic and social effects are shown.

MMAE has strong toxicity, and also has strong killing effect on normal cells of a human body during antitumor treatment, and since ADC drugs also have strong technical limit in the research and development process, free MMAE drug detection has an extremely important effect in the drug clinical monitoring stage regardless of a new drug research and development stage or after drugs are on the market.

Because the ADC medicament is targeted and enters cells through endocytosis, the free concentration is extremely low, and the requirement on a method for detecting free MMAE is high, a method which is rapid, accurate and reliable, and has high sensitivity and high selectivity needs to be established.

Disclosure of Invention

It is an object of the present invention to provide a method for LC-MS detection of MMAE in plasma that solves one or more of the above-mentioned problems of the prior art.

The invention provides a liquid chromatography-mass spectrometry detection method of MMAE in blood plasma, which comprises the following steps:

s1, pretreatment of a plasma sample: extracting a sample to be detected by adopting a one-step protein precipitation method so as to obtain an extracting solution to be detected;

s2, carrying out LC-MS detection on the extracting solution to be detected so as to determine the concentration of MMAE in the sample to be detected;

wherein: determining the parameter conditions of the detection and analysis by adopting the liquid chromatography-mass spectrometry, wherein the chromatographic conditions comprise:

a chromatographic column: agilent hilc Plus, 3.5 μm, column format 2.1 × 100 mm;

temperature of the chromatographic column: 40 ℃;

mobile phase A: h₂O/FA/1M HCOONH ₄100/0.1/1 (V/V); mobile phase B: acetonitrile;

washing liquid: acetonitrile/water 80/20 (V/V);

the temperature of the autosampler is 4 ℃;

gradient elution with flow rate of 0.6mL/min, sample size of 10 μ L, and analysis time of 4.2 min;

mass spectrum conditions: an electrospray ion source is adopted, and a scanning mode of multiple reaction detection is adopted.

Wherein: the plasma is the most common clinical sample in clinical tests, is easy to obtain and can accurately reflect the in-vivo condition of the medicine.

In certain embodiments, step S1 specifically includes the steps of:

with K₂EDTA is anticoagulant, 50 mu L of EDTA is taken out to be placed in a 96-deep-well plate after a sample is prepared, 450 mu L of acetonitrile is added to be mixed for 2min in a vortex mode, the mixture is centrifuged for 10min at the temperature of 4 ℃ at 2600g, 300 mu L of supernatant is taken to be placed in the 96-deep-well plate, 10 mu L of 1M ammonium formate is precisely added to be mixed for 2min in a vortex mode, and the mixture is used as a test sample to be detected.

In certain embodiments, the procedure for gradient elution in step S2 is:

total time (min)	Mobile phase A (%)	Mobile phase B (%)
			0.20	10	90
1.80	35	65
			2.20	35	65
2.21	10	90
			4.20	10	90

Wherein: gradient elution can provide the method with extremely high sensitivity and resolution.

In certain embodiments, the mass spectrometry conditions in step S2 are:

the ion source adopts an electrospray ion source, the spraying voltage is 5500V, the atomizing temperature is 500 ℃, the spraying air pressure is 50Psi, the auxiliary heating air pressure is 50Psi, the air curtain air pressure is 25Psi, the collision air pressure is 8Psi, and the declustering voltage is MMAE of 30eV respectively;

the collision cell inlet voltages were respectively 10eV MMAE;

MMAE with collision voltages of 40eV, respectively;

the collision cell outlet voltages are respectively 10eV MMAE;

detecting in a positive ion mode;

the scanning mode is a multiple reaction detection.

In certain embodiments, the mass spectrometry conditions in step S2 are:

the ion pairs used for the quantitative analysis for MMAE were: m/z718.5 → m/z 686.5;

qualitative ion pair m/z718.5 → m/z 506.4.

Wherein: the ion pair is selected for quantification, and has high specificity and high sensitivity.

In certain embodiments, the step of determining the sample in step S2 is:

and (3) injecting 10 mu L of test sample into a high performance liquid chromatography-tandem mass spectrometer, detecting the chromatographic peak of the MMAE in the sample, and calculating the MMAE concentration in the plasma sample according to the chromatographic peak.

In certain embodiments, the concentration of MMAE in the plasma sample is calculated in step 2 by substituting the peak area of MMAE into a standard curve equation.

In certain embodiments, the establishing of the standard curve equation comprises the steps of:

and respectively injecting 10 mu L of standard samples into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of MMAE in the samples, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of the MMAE in the plasma.

In certain embodiments, the method of preparing a standard sample comprises the steps of:

putting 8 parts of 190 mu L blank plasma into a 1.5mL centrifuge tube, adding 10 mu L MMAE solution with the concentration of 0.200ng/mL, 0.400ng/mL, 1.60ng/mL, 4.00ng/mL, 12.0ng/mL, 40.0ng/mL, 160ng/mL and 200ng/mL in the form of stock solution to 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 standard sample 7 and a standard sample 8, respectively taking the standard sample 1, the standard sample 2, the standard sample 3, the standard sample 4, the standard sample 5, the standard sample 6, the standard sample 7, the standard sample 8 and a zero-concentration sample 50 mu L into a 96 deep-well plate, adding 450 mu L vortex mixing for 2min, centrifuging at 2600g for 10min at 4 ℃, taking 300 mu L supernatant into the 96-well plate, adding 10 mu L1M ammonium formate mixing for 2min, and taking the supernatant as a precision acetonitrile sample to be detected.

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

(1) the pretreatment method is simple and convenient, and the one-step organic solvent precipitation method is suitable for conventional determination;

(2) the specificity is strong: under the chromatographic conditions adopted in the experiment, the MMAE retention time is about 1.999 min. The MMAE has good peak type, no interference of miscellaneous peaks in measurement and stable baseline;

(3) the sensitivity is high: the minimum limit of quantitation of the plasma is 0.010ng/mL, the concentration of MMAE 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 measuring the blood concentration of MMAE. The linear range of the plasma standard curve of the method is 0.010-10.0ng/mL, and the precision RSD in and among batches is less than +/-15%.

Drawings

FIG. 1 is a standard curve of MMAE in human plasma measured by HPLC-MS/MS method;

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

FIG. 3 is a HPLC-MS/MS plot of human blank plasma plus MMAE.

Detailed Description

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

EXAMPLE 1 Experimental materials and analytical Equipment

MMAE (analyte): ZZ STANDARDS or equivalent, higher-grade standard

The reagents used are shown in table 1 below:

TABLE 1 details of reagents

Name of reagent	Rank of	Manufacturer(s)
			Acetonitrile	HPLC	Fisher
Ammonium formate	HPLC	Fisher
			Formic acid	LC/MS	Aladdin
DMSO	Analytical purity	Fisher

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	Species of	Manufacturer(s)
			Binary pump (Binary pump)	AD Pump	SHIMADZU
Degasser (deaerator)	Degasser	SHIMADZU
			Column oven (constant temperature Column box)	AD Column oven	SHIMADZU
Autosampler (automatic sampler)	AC Autosampler	SHIMADZU
			Sample rack	Rack Changer	SHIMADZU
Mass spectrometer	TRIPLE QUAD 6500+	SCIEX
			Data processor	Analyst(software)	SCIEX

The same LC-MS/MS system may also be used.

Example 2 conditions for liquid quality detection

1. Conditions of liquid chromatography

A chromatographic column: agilent hilc Plus, 3.5 μm, column format 2.1 × 100 mm; temperature of the chromatographic column: 40 ℃; a mobile phase A: h₂O/FA/1M HCOONH ₄100/0.1/1 (volume ratio); mobile phase B: acetonitrile; washing liquid: the volume ratio of acetonitrile/water is 80/20; the temperature of the autosampler is 4 ℃; gradient elution with flow rate of 0.6mL/min, sample size of 10 μ L, and analysis time of 4.2 min;

TABLE 3 gradient elution procedure

2. Conditions of Mass Spectrometry

The ion source adopts an electrospray ion source, the spraying voltage is 5500V, the atomizing temperature is 500 ℃, the spraying air pressure is 50Psi, the auxiliary heating air pressure is 50Psi, the air curtain air pressure is 25Psi, the collision air pressure is 8Psi, and the declustering voltage is MMAE of 30eV respectively; the collision cell inlet voltages were respectively 10eV MMAE; MMAE with collision voltages of 40eV, respectively; the collision cell outlet voltages are respectively 10eV MMAE; detecting in a positive ion mode; the scanning mode is multiple reaction detection;

the ion pairs used for the quantitative analysis for MMAE were: m/z718.5 → m/z 686.5; qualitative ion pair m/z718.5 → m/z 506.4.

Example 3 test procedure

1. Preparation of MMAE standard solution

Preparation of MMAE standard solution: 2.000mg of MMAE (analyte) is precisely weighed, DMSO is added to be dissolved to 1.00mg/mL, 50% acetonitrile aqueous solution is dissolved to be sequentially diluted to prepare an MMAE standard solution, and the specific dilution concentration is shown in the following table 4:

TABLE 4MMAE Standard solution preparation concentrations

Source solution (ng/mL)	Volume of source solution (μ L)	Volume of solvent (μ L)	Final concentration (ng/mL)
				1000000	10	4990	2000
2000	200	1800	200
				2000	160	1840	160
2000	40	1960	40.0
				160	150	1850	12.0
160	50	1950	4.00
				40.0	80	1920	1.60
40.0	20	1980	0.400
				4.00	100	1900	0.200

The MMAE standard solution is stored in brown glass container and refrigerator (-20 deg.C) when not used, and the volume can be increased or decreased according to the proportion.

2. Linear test

Putting the blank plasma into a water bath at room temperature for unfreezing; transfer 190. mu.L of blank plasma 10 aliquots into 96 deep well plates (per standard curve)Sample, blank sample-00 and zero concentration sample-0), according to the list in the following table 5, respectively and precisely adding 10 μ L of MMAE standard solution or diluted solution with different concentrations to prepare each sample, mixing uniformly to prepare medicated plasma with different concentrations, and operating according to the 'pretreatment of plasma sample'. The peak area value Y was used for the regression calculation of the blood concentration X, and the results are shown in FIG. 1 and Table 6. Carrying out regression calculation on the blood concentration X by using Y to obtain a regression equation Y which is 691000X-770, wherein the correlation coefficient (R) is 0.9996, and the fitting degree (R) is²) Is 0.99920016. Weight coefficient W is 1/X²The minimum limit of quantitation of blood levels of MMAE measured by this method is: 0.010 ng/mL.

TABLE 5MMAE Standard Curve formulation concentrations

a: diluted solution of analyte: 50% aqueous acetonitrile solution

TABLE 6 Standard Curve of MMAE in human plasma by HPLC-MS/MS method (n-5)

3. Accuracy and precision

Putting the blank plasma into a water bath at room temperature for unfreezing; the appropriate volume of blank plasma was transferred to appropriate containers and MMAE standard solution was added to prepare 5 drug-containing plasma quality control samples (LLOQ QC, LQC, GMQC, MQC, HQC) of different concentrations and a running standard curve, operating as "plasma sample pretreatment" and the quality control sample preparation is shown in table 7 below. Making one batch and one following standard curve every day, continuously making 5 batches for 5 days, respectively making 6 samples of each concentration of the first batch, the second batch, the third batch, the fourth batch and the fifth batch, calculating the MMAE peak area, substituting the MMAE peak area into the standard curve of the day to obtain the actually measured concentration, calculating the precision between batches according to the actually measured concentration, wherein the ratio of the actually measured concentration to the theoretical concentration is the accuracy, and the result is shown in Table 8. The precision and accuracy of the MMAE plasma sample between batches and in batches are within +/-15 percent.

TABLE 7 quality control sample preparation concentration

Sufficient volume was dispensed into the labeled sample vial and stored at the theoretical temperature-20 ℃ as required for each assay batch. The volume may be scaled up or down as desired.

TABLE 8HPLC-MS/MS method for determining the accuracy and precision of MMAE in plasma

4. Interference

The interference of different blank plasma on MMAE analytes was evaluated by using 6 blank plasma samples from different sources, 3 hyperlipidic plasma from different sources and 3 haemolytic plasma from different sources for the same assay batch prepared and analyzed according to the sample preparation procedure.

After 12 blank plasma samples from different sources were prepared and analyzed, the interference peak responses at the time consistent with MMAE retention were all less than 20.0% of the MMAE response of the lower limit sample of the quantitation in the standard curve of the assay batch, and the results are shown in table 9. The results show that the analysis method is specific to the analysis of MMAE.

TABLE 912 comparison of interference data on MMAE analytes from blank plasma of different origins

As can be seen from table 9, blank plasma from different human bodies did not interfere with the detection of MMAE. Thus, the method can be used to detect MMAE concentrations in different human plasma.

5. Recovery rate

Analyte recovery will be calculated using LQC, MQC and HQC formulated with pooled plasma. Extraction was performed for 6 parallel samples and 18 DBs each of LQC, MQC and HQC. The analytes were added after DB extraction, so that their concentration in the DB extract added after extraction was the same as the extracted LQC, MQC and HQC samples. The extraction recovery was calculated by comparing the peak areas of the analyte and internal standard from QC with the average peak areas of the analyte and internal standard added after DB extraction, and the results are shown in table 10, with% CV of the analyte peak area and overall% CV being ± 15.0% at each concentration level of the analyte, and a recovery of 101.1%, indicating a high extraction recovery using this pretreatment method MMAE.

TABLE 10 extraction recovery of MMAE analytes

6. Matrix effect

Blank matrix samples from at least 6 different sources were processed. The analytes were added after extraction of DB to ensure that their concentration in the processed DB (1 sample per source per concentration) was the same as the processed LQC, MQC and HQC samples; solutions containing the analytes were prepared to final concentrations identical to the treated LQC, MQC and HQC samples, 6 replicates per concentration, with results in table 11, analyte peak area ratios% CVs all between ± 15%, indicating the absence of matrix effect problems.

TABLE 11 matrix Effect of MMAE analytes

In summary, the following steps: the invention provides a simple and convenient method for measuring the concentration of MMAE in blood plasma by a pretreatment method, which adopts a one-step organic solvent precipitation method and is suitable for conventional measurement; meanwhile, under the chromatographic conditions adopted in the experiment, the MMAE retention time is about 1.999min, the peak shape is good, the measurement is free from the interference of the miscellaneous peak, and the base line is stable; the method has high specificity, can accurately determine the concentration of MMAE in plasma, and has high sensitivity, and the minimum limit of quantitation of the plasma is 0.0100 ng/mL; meanwhile, the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of MMAE. The linear range of the plasma standard curve of the method is 0.0100-10.0 ng/mL, the precision RSD in batches and among batches is +/-15%, the extraction recovery rate of the analytes is high, and the matrix effect does not exist.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, many variations and modifications can be made without departing from the inventive concept, and these should also be considered as within the scope of the invention.

Claims (9)

1. A liquid chromatography-mass spectrometry detection method for MMAE in plasma is characterized by comprising the following steps:

s1, pretreatment of a plasma sample: extracting a sample to be detected by adopting a one-step organic solvent precipitation method so as to obtain an extracting solution to be detected;

s2, carrying out LC-MS detection on the extracting solution to be detected so as to determine the concentration of MMAE in the sample to be detected;

wherein: determining the parameter conditions of the detection and analysis by adopting the liquid chromatography-mass spectrometry, wherein the chromatographic conditions comprise:

a chromatographic column: agilent hilc Plus, 3.5 μm, column format 2.1 × 100 mm;

temperature of the chromatographic column: 40 ℃;

mobile phase A: h₂O/FA/1M HCOONH₄100/0.1/1 (V/V); mobile phase B: acetonitrile;

washing liquid: acetonitrile/water 80/20 (V/V);

the temperature of the autosampler is 4 ℃;

gradient elution with flow rate of 0.6mL/min, sample size of 10 μ L, and analysis time of 4.2 min;

mass spectrum conditions: an electrospray ion source is adopted, and a scanning mode of multiple reaction detection is adopted.

2. The method according to claim 1, wherein the step S1 comprises the following steps:

with K₂EDTA is anticoagulant, 50 mu L of EDTA is taken out to be placed in a 96-deep-well plate after a sample is prepared, 450 mu L of acetonitrile is added to be mixed for 2min in a vortex mode, the mixture is centrifuged for 10min at the temperature of 4 ℃ at 2600g, 300 mu L of supernatant is taken to be placed in the 96-deep-well plate, 10 mu L of 1M ammonium formate is precisely added to be mixed for 2min in a vortex mode, and the mixture is used as a test sample to be detected.

3. The method according to claim 1, wherein the gradient elution in step S2 is performed by the following steps:

4. the method according to claim 1, wherein the mass spectrometry conditions in step S2 are as follows:

the ion source adopts an electrospray ion source, the spraying voltage is 5500V, the atomizing temperature is 500 ℃, the spraying air pressure is 50Psi, the auxiliary heating air pressure is 50Psi, the air curtain air pressure is 25Psi, the collision air pressure is 8Psi, and the declustering voltage is MMAE of 30eV respectively;

the collision cell inlet voltages were respectively 10eV MMAE;

MMAE with collision voltages of 40eV, respectively;

the outlet voltages of the collision chambers are respectively MMAE of 10 eV;

detecting in a positive ion mode;

the scanning mode is a multiple reaction detection.

5. The method according to claim 4, wherein the mass spectrometry conditions in step S2 are as follows:

the ion pairs used for the quantitative analysis for MMAE were: m/z718.5 → m/z 686.5;

qualitative ion pair m/z718.5 → m/z 506.4.

6. The method according to claim 1, wherein the step of determining the sample in step S2 comprises:

and (3) injecting 10 mu L of test sample into a high performance liquid chromatography-tandem mass spectrometer, detecting the chromatographic peak of the MMAE in the sample, and calculating the MMAE concentration in the plasma sample according to the chromatographic peak.

7. The method of claim 6, wherein the concentration of MMAE in the plasma sample is calculated in step 2 by substituting the MMAE peak area into a standard curve equation.

8. The method according to claim 7, wherein the standard curve equation is established by the following steps:

and respectively injecting 10 mu L of standard samples into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of MMAE in the samples, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of the MMAE in the plasma.

9. The method according to claim 8, wherein the method for preparing the standard sample comprises the following steps:

putting 8 parts of 190 mu L blank plasma into a 1.5mL centrifuge tube, adding 10 mu L MMAE solution with the concentration of 0.200ng/mL, 0.400ng/mL, 1.60ng/mL, 4.00ng/mL, 12.0ng/mL, 40.0ng/mL, 160ng/mL and 200ng/mL in the form of stock solution to 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 standard sample 7 and a standard sample 8, respectively taking the standard sample 1, the standard sample 2, the standard sample 3, the standard sample 4, the standard sample 5, the standard sample 6, the standard sample 7, the standard sample 8 and a zero-concentration sample 50 mu L into a 96 deep-well plate, adding 450 mu L vortex mixing for 2min, centrifuging at 2600g for 10min at 4 ℃, taking 300 mu L supernatant into the 96-well plate, adding 10 mu L1M ammonium formate mixing for 2min, and taking the supernatant as a precision acetonitrile sample to be detected.

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