Method for detecting TCEP content in ADC by LC-MS/MS

Abstract

The present disclosure relates to a method for detecting TCEP content in ADC by LC-MS/MS. The ADC samples are derivatized by Ellman reaction, and then the TCEP content is measured by LC-MS/MS. This method can exclude the interference caused by the substances that may react with Ellman in complex ADC samples, which provides with no need for sample pretreatment, and with strong specificity.

Claims

1. A method for detecting TCEP content in ADC samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS), wherein the detected ADC samples are derivatized by Ellman reaction, and a C18 chromatographic column is used, without need for sample pretreatment.

2. The method according to claim 1, wherein a mobile phase A used in the C18 chromatographic column is an aqueous solution comprising formic acid; preferably an aqueous solution comprising 0.1% formic acid.

3. The method according to claim 2, wherein a mobile phase B used in the C18 chromatographic column is an acetonitrile/methanol solution comprising formic acid; preferably an acetonitrile/methanol solution (80/20, v/v) comprising 0.1% formic acid.

4. The method according to claim 3, wherein concentration of the added Ellman reagent is about 5-20 g/ml, more preferably 10.0 g/ml.

5. The method according to claim 4, wherein the method uses TCEP to prepare standard solution.

6. The method according to claim 5, wherein the standard solution is preferably a TCEP solution of 1.0 g/ml.

7. The method according to claim 6, wherein the preparation of the TCEP solution and the preparation of the series solutions for standard curve comprise the following steps: 1) preparation of TCEP working solution: weighing 510 mg of TCEP.Math.HCl reference substance into an EP tube and dissolving with an appropriate amount of sample dissolving solution, and the weighed mass is expressed as m, calculating the TCEP concentration by a molar ratio of TCEP.Math.HCl and TCEP of 1:1, and performing dilution stepwise to a TCEP concentration of 1.0 g/ml; 2) preparation of series solutions for standard curve: diluting 1.0 g/ml TCEP standard solution with diluent to the TCEP standard solutions with concentrations of 16 ng/ml, 8 ng/ml, 4 ng/ml, 2 ng/ml, 1 ng/ml, and 0.5 ng/ml, respectively.

8. The method according to claim 7, wherein the method further comprises using quality control solutions, which are prepared by diluting a 1.0 g/ml TCEP solution with diluent, and the concentrations of the quality control solutions are 12 ng/ml, 3 ng/ml, and 1 ng/ml of TCEP quality control solutions.

9. The method according to claim 7, wherein the diluent is a 50% aqueous methanol solution.

10. The method according to claim 1, wherein the liquid chromatograph includes but is not limited to Agilent 1200 liquid chromatograph from Agilent USA, Teledyne Isco high pressure preparative liquid chromatograph, and Agilent HPLC 1260 high performance liquid chromatograph.

11. The method according to claim 1, wherein the tandem mass spectrometer includes but is not limited to API 4000 tandem mass spectrometer from AB Sciex USA, Shimadzu triple quadrupole liquid chromatography mass spectrometer, and GBC inductively coupled plasma orthogonal acceleration time-of-flight mass spectrometer Optimass.

12. The method according to claim 1, wherein the C18 chromatographic column includes but is not limited to YMC-C18, Agilent Extard-C18, and Shiseido PAK CR-18.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Linear fitting standard curve

(2) FIG. 2: Determination of TCEP content by LC-MS/MS

DETAILED DESCRIPTION OF EMBODIMENTS

Example 1. Determination of TCEP Content by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method

(3) 1) Preparation of Working Solution

(4) Preparation of TCEP working solution: 510 mg of TCEP.Math.HCl reference substance was precisely weighed into an EP tube and dissolved with an appropriate amount of sample dissolving solution, and the weighed mass is expressed as m. After the TCEP concentration (TCEP concentration=250.19 m/286.65) was calculated by the molar ratio of TCEP.Math.HCl and TCEP (1:1), it was diluted with diluent stepwise to a TCEP concentration of 1.0 g/ml.

(5) Preparation of DTNB working solution: 5-10 mg of DTNB was precisely weighed into an EP tube and dissolved with an appropriate amount of sample dissolving solution to a concentration of 10 mg/ml, and stepwise diluted with a diluent to 10 g/ml.

(6) Preparation of series solutions for standard curve: 1.0 g/ml TCEP (recorded as A1) was diluted with diluent according to Table 1 to TCEP reference solutions with the concentrations of 16 ng/ml, 8 ng/ml, 4 ng/ml, 2 ng/ml, 1 ng/ml, and 0.5 ng/ml, respectively. 200 l of the above solutions with different concentrations was taken, and each was added with 20 l DTNB working solution with a concentration of 10 g/ml. Then, it was directly tested on the machine after mixed by vortex and reacted at room temperature for 2 h in the dark.

(7) TABLE-US-00001 TABLE 1 working final working solution diluent total concen- sample solution volume volume volume tration name name (l) (l) (l) (ng/ml) A2 A1 50 450 500 100 STD6 A2 80 420 500 16 STD5 STD6 250 250 500 8 STD4 STD5 250 250 500 4 STD3 STD4 250 250 500 2 STD2 STD3 250 250 500 1 STD1 STD2 250 250 500 0.5

(8) Preparation of quality control solutions: 1.0 g/ml TCEP (recorded as A1) was diluted with diluent according to Table 2 to the TCEP quality control solutions with the concentrations of 400 ng/ml (HQC), 200 ng/ml (MQC) and 100 ng/ml (LQC), respectively. 200 l of the above TCEP quality control solutions with different concentrations was taken, and each was added with 20 l of DTNB working solution with a concentration of 10 g/ml. Then it was directly tested on the machine after mixed by vortex and reacted at room temperature for 10 min in the dark.

(9) TABLE-US-00002 TABLE 2 working final working solution diluent total TCEP con- sample solution volume volume volume centration name name (l) (l) (l) (ng/ml) HQC A1 400 600 1000 400 MQC HQC 500 500 1000 200 LQC MQC 500 500 1000 100
2) Determination by Liquid Chromatography-Tandem Mass Spectrometry:

(10) Treatment of the samples to be tested: The mobile phase (an acetonitrile/methanol (80/20, v/v) solution comprising 0.1% formic acid) was used as the diluent, and the ADC samples were diluted to an appropriate concentrations (the TCEP content of samples is within the linear range, if it exceeds the range then the dilution factor needs to be increased/decreased for the test again). 200 l of sample diluent was taken and added with 20 l of DTNB solution with a concentration of 10 g/ml. Then, it was directly tested on the machine after mixed by vortex and reacted at room temperature for 2 h in the dark.

(11) Preparation of mobile phase A: 800 ml of water was measured and added with 800 l of formic acid, and mixed well by ultrasonic.

(12) Preparation of mobile phase B: 800 ml of acetonitrile and 200 ml of methanol were measured and added with 1 ml of formic acid, and mixed well by ultrasonic.

(13) Mobile phase A and mobile phase B were used in elution according to the concentration gradient shown in Table 3.

(14) TABLE-US-00003 TABLE 3 elution time (min) module event parameter elution 0.2 Pumps Pump B ConC 5 gradient 4 Pumps Pump B ConC 60 5 Pumps Pump B ConC 95 6 Pumps Pump B ConC 95 6.1 Controller Stop /
3) Data Processing and Analysis

(15) Evaluation standard: r0.9800, the concentration range between the lower limit of quantification and the upper limit of quantification is the quantification range of the standard curve, and the recovery rate of the standard curve concentration points is between 85% and 115%, and the recovery rate of the concentration points of the upper limit of quantification and the lower limit of quantification is 80%-120%.

(16) The recovery rate of the quality control is 85%-115%. At least 50% of the quality control samples of each concentration meet this range, and at least 4 quality control concentration points of the same analytical batch meet the requirements.

(17) The specific results are as follows:

(18) TABLE-US-00004 TABLE 4 Standard curve (see FIG. 1 for the specific fitting curve) actual calculated concen- reten- concen- sample sample tration tion tration name type (ng/ml) peak area time (ng/ml) accuracy STD1 Standard 2 2.85E+04 2.99 1.99 99.5 STD2 Standard 4 6.23E+04 2.98 4.127 103.16 STD3 Standard 8 1.18E+05 2.98 7.661 95.76 STD4 Standard 16 2.45E+05 2.98 15.684 98.02 STD5 Standard 32 5.21E+05 2.97 33.17 103.65 STD6 Standard 64 1.01E+06 2.98 63.932 99.89

(19) The standard curve obtained after linear fitting is as follows:

(20) y=16058x416744, R.sup.2=0.9936, according to the results shown in FIG. 1, R.sup.2>0.99, the fitting is excellent.

Example 2. Detection Method Results and Evaluation

(21) 1) Repeatability

(22) 1000 l of the test samples was taken and added with 8 l of TCEP standard (concentration of 1.0 g/ml). 200 l of the above solution was taken and added with 20 l DTNB solution of 100 g/ml, and mixed by vortex, and reacted at room temperature in the dark for 2 h. 6 copies were prepared in parallel, and the concentration results of the 6 copies were measured for evaluation. The specific results are shown in Table 5.

(23) TABLE-US-00005 TABLE 5 measured concentration of measured concentration TCEP in sample concentration of sample (ng/ml) (ng/ml) standard (ng/ml) 1 12.565 3.636 8.925 2 13.019 3.636 9.379 3 13.448 3.636 9.808 4 12.706 3.636 9.066 5 12.456 3.636 8.816 6 13.116 3.636 9.476 RSD(%) 4.1

(24) In the field of quantitative analysis, repeatability refers to the precision of the results obtained by the same analyst within a short interval under the same operating conditions. The acceptable standard for repeatability is: the main peak concentration RSDS 15.0%, this test RSD=4.1%, so the repeatability of this method is very good.

(25) 2) Accuracy

(26) An appropriate amount of the test product was taken, and the addition amount of TCEP of the high, medium and low concentrations and the final concentration of the standard were the LQC solution (4 ng/ml), the MQC solution (16 ng/ml) and the HQC solution (50 ng/ml) of the quality control solutions, respectively. The addition amounts of the test product and standard are shown in Table 6, and the accuracy results are shown in Table 7.

(27) TABLE-US-00006 TABLE 6 addition amount concentration of addition amount concentration of test product TCEP standard of standard (ng/ml) (l) (ng/ml) (l) 4 960 100 40 960 100 40 960 100 40 16 984 1000 16 984 1000 16 984 1000 16 50 950 1000 50 950 1000 50 950 1000 50

(28) TABLE-US-00007 TABLE 7 concentra- concentra- tion of measured concen- tion of standard concen- tration standard recovery added tration in sample added RSD rate (ng/ml) (ng/ml) (ng/ml) (ng/ml) (%) (%) 4 9.183 5.866 3.318 3.1 82.9 9.391 5.866 3.526 88.1 9.311 5.866 3.446 86.1 16 7.164 3.636 16.677 2.5 104.2 7.365 3.636 15.918 99.5 7.415 3.636 16.559 103.5 50 21.629 3.636 51.235 0.8 102.5 22.239 3.636 51.245 102.5 22.888 3.636 51.987 104.0

(29) Acceptable criteria for accuracy: The recovery rate is 70.0%-130.0%, RSD15.0%; the recovery rate in this experiment is 82.9%104.2%, and the RSD is 3.1%, 2.5%, and 0.8%, respectively. Therefore, according to the detection results, this method is with good accuracy, strong selectivity and high sensitivity, and is suitable for detecting the TCEP concentration in the antibody drug conjugate samples.

(30) The spirit of the present disclosure has been described in detail above through the preferred examples of the present disclosure. Any changes, equivalent changes and modifications made to the above examples according to the essence of the present disclosure all fall within the protection scope of the present disclosure.