Method for simultaneously detecting concentrations of riluzole and edaravone

Abstract

The invention belongs to the technical field of chromatography-mass spectrometry detection, and relates to a method for simultaneously detecting concentrations of riluzole and edaravone. The invention establishes an analysis method for simultaneously detecting riluzole and edaravone based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) for the first time. Mass spectrum response signals of the two drugs are remarkably improved by optimizing key conditions such as mass spectrum ion source parameters, collision energy and declustering voltage. Experimental results show that the quantitation limits of riluzole and edaravone in the method provided by the invention are as low as 1 ng / mL, the monitoring requirement under low blood concentration can be met, and the method is particularly suitable for pharmacokinetic research and trace concentration monitoring of patients taking medicine for a long time.

Description

Technical Field

[0001] This invention belongs to the field of chromatography-mass spectrometry detection technology, and relates to a method for simultaneously detecting the concentrations of riluzole and edaravone. Background Technology

[0002] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the death of motor neurons in the brain, brainstem, and spinal cord, leading to progressive, painless muscle weakness and often resulting in death from respiratory failure within 2 to 5 years after the onset of symptoms. [1, 2] Because the disease is rare, has low public awareness, symptoms are not fully recognized, and there is a lack of early and appropriate specialist intervention, the average time interval from the first symptom to diagnosis is as long as 10 to 16 months. [3, 4] The pathogenesis of ALS stems from a complex interaction between age, sex, and genetic factors, and its main clinical manifestations are dysfunction of the upper motor neuron (UMN) and lower motor neuron (LMN). [5] The incidence of ALS is approximately 2.16 cases per 100,000 people per year, with a male-to-female ratio of approximately 1.3:1. [2 , 6] .

[0003] Riluzole, the first drug approved for the treatment of ALS, is a glutamatergic neurotransmitter inhibitor. It was approved by the U.S. Food and Drug Administration (FDA) in 1995 for the treatment of ALS. [7] Riluzole exerts its neuroprotective effect by inhibiting the release of glutamate from central neurons, thus blocking excessive glutamatergic neurotransmission. [8] Although riluzole cannot cure ALS, it can extend the life expectancy of ALS patients. [9] However, the pharmacokinetic characteristics of riluzole exhibit significant individual variability, primarily attributed to first-pass metabolism, hepatic metabolic enzyme polymorphism, extrahepatic metabolic pathways, and other factors.

[10] This individualized difference limits the efficacy and clinical application of riluzole. Another drug approved for the treatment of ALS is edaravone, a free radical scavenger that received FDA approval in 2017.

[10] Riluzole and edaravone are currently the main drugs used clinically to treat amyotrophic lateral sclerosis (ALS), and monitoring their blood drug concentrations is a key prerequisite for achieving personalized precision medicine. Liquid chromatography-mass spectrometry (LC-MS / MS) technology has broad application prospects in therapeutic drug monitoring due to its advantages such as simultaneous quantitative analysis of multiple drugs, low limit of quantitation, short analysis time, and minimal interference from the blood matrix. However, there are currently no reports on LC-MS / MS methods for the simultaneous detection of riluzole and edaravone. Therefore, it is necessary to establish an LC-MS / MS method capable of simultaneously determining the blood concentrations of riluzole and edaravone to support precision medicine strategies for ALS patients, thereby effectively reducing the risk of adverse drug reactions and improving clinical treatment outcomes.

[0004] References:

[0005] [1] Feldman, EL; Goutman, SA; Petri, S.; Mazzini, L.;Savelieff, MG; Shaw, PJ; Sobue, G., Amyotrophic lateral sclerosis. TheLancet 2022, 400 (10360), 1363-1380.

[0006] [2] Riva, N.; Domi, T.; Pozzi, L.; Lunetta, C.; Schito, P.; Spinelli,EG; Cabras, S.; Matteoni, E.; Consonni, M.; Bella, ED; 2024, 271 (7), 4693-4723.

[0007] [3] Richards, D.; Morren, J. A.; Pioro, E. P., Time to diagnosis andfactors affecting diagnostic delay in amyotrophic lateral sclerosis. Journalof the Neurological Sciences 2020, 417, 117054.

[0008] [4] Galvin, M.; Ryan, P.; Maguire, S.; Heverin, M.; Madden, C.;Vajda, A.; Normand, C.; Hardiman, O., The path to specialistmultidisciplinary care in amyotrophic lateral sclerosis: A population- basedstudy of consultations, interventions and costs. PLoS One 2017, 12 (6),e0179796.

[0009] [5] Goutman, S. A.; Hardiman, O.; Al-Chalabi, A.; Chió, A.;Savelieff, M. G.; Kiernan, M. C.; Feldman, E. L., Recent advances in thediagnosis and prognosis of amyotrophic lateral sclerosis. The LancetNeurology 2022, 21 (5), 480-493.

[0010] [6] Xu, L.; Liu, T.; Liu, L.; Yao, X.; Chen, L.; Fan, D.; Zhan, S.;Wang, S., Global variation in prevalence and incidence of amyotrophic lateralsclerosis: a systematic review and meta-analysis. Journal of Neurology 2020,267 (4), 944-953.

[0011] [7] Miller, R. G.; Mitchell, J. D.; Moore, D. H., Riluzole foramyotrophic lateral sclerosis (ALS) / motor neuron disease (MND). CochraneDatabase of Systematic Reviews 2012, (3), Cd001447.

[0012] [8] Doble, A., The pharmacology and mechanism of action of riluzole.Neurology 1996, 47 (6_suppl_4), 233S-241S.

[0013] [9] M, H.; A, S., Riluzole: real-world evidence supports significantextension of median survival times in patients with amyotrophic lateralsclerosis. Degenerative Neurological and Neuromuscular Disease 2017, 7, 61-70.

[0014]

[10] Dash, RP; Babu, RJ; Srinivas, NR, Two Decades-LongJourney from Riluzole to Edaravone: Revisiting the Clinical Pharmacokinetics of the Only Two Amyotrophic Lateral Sclerosis Therapeutics. ClinicalPharmacokinetics 2018, 57 (11), 1385-1398. Summary of the Invention

[0015] The technical problem to be solved by the present invention is to provide a method for simultaneously detecting the concentrations of riluzole and edaravone, which is capable of simultaneously determining the drug concentrations of riluzole and edaravone in biological samples, in order to address the shortcomings of the prior art.

[0016] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0017] This invention discloses a kit for simultaneously detecting the concentrations of riluzole and edaravone, the kit comprising the following reagents:

[0018] (1) Series of standard curve working solutions

[0019] The series of standard curve working solutions are prepared by mixing riluzole standard stock solution and edaravone standard stock solution of known concentration and then diluting with a first solvent to form standard curve working solutions with 8 different concentration levels.

[0020] The eight different concentration levels of the standard curve working solution are as follows:

[0021] Riluzole and edaravone were administered at the same concentrations, with eight concentrations in total: 5000.00 ng / mL, 1000.00 ng / mL, 500.00 ng / mL, 250.00 ng / mL, 100.00 ng / mL, 50.00 ng / mL, 25.00 ng / mL, and 10.00 ng / mL.

[0022] (2) Series of quality control working solutions

[0023] The series of quality control working solutions are prepared by mixing riluzole standard stock solution and edaravone standard stock solution of known concentration and then diluting with a second solvent to form quality control working solutions with four different concentration levels.

[0024] The four different concentration levels of the quality control working solution are as follows:

[0025] Riluzole and edaravone were administered at the same concentrations, with four concentrations of 4000.00 ng / mL, 300.00 ng / mL, 30.00 ng / mL, and 10.00 ng / mL, respectively.

[0026] (3) Internal standard working solution

[0027] The internal standard working solution is riluzole of known concentration. 13 C, 15 N2 isotope internal standard stock solution and edaravone-d at known concentrations 5 After the isotope internal standard stock solution is mixed, it is diluted with a third solvent to prepare the internal standard working solution;

[0028] The internal standard working solution contains riluzole- 13 C, 15 N2 isotope internal standard and edaravone-d 5 The concentrations of the isotope internal standards were all the same, at 100.00 ng / mL;

[0029] (4) Sample stabilizer: The sample stabilizer is one or more of the following: sodium sulfite, sodium bisulfite, sodium dithionite, sodium metabisulfite, vitamin C, sodium thiosulfate, 25wt%~35wt% ammonia, sodium bicarbonate, sodium carbonate, sodium acetate, sodium phosphate, disodium hydrogen phosphate, calcium hydroxide, calcium carbonate, ethylenediamine, and triethylamine; wherein, when the sample stabilizer is not 25wt%~35wt% ammonia, the sample stabilizer exists in the form of a sample stabilizer solution, the solvent in the sample stabilizer solution is water, and the concentration of the solute sample stabilizer is 5.00 mg / mL~200.00 mg / mL;

[0030] (5) Protein precipitant: a methanol solution containing 0.1 wt% to 10.0 wt% formic acid;

[0031] (6) Sample enhancement solution: formic acid;

[0032] (7) Mobile phase A: An aqueous solution containing 0.1 wt% formic acid;

[0033] (8) Mobile phase B: Methanol solution containing 0.1 vt% formic acid.

[0034] In some embodiments, the first solvent is a 50% (v / v) aqueous methanol solution; the second solvent is a 50% (v / v) aqueous methanol solution; and the third solvent is methanol.

[0035] In some embodiments, the riluzole standard stock solution of known concentration is prepared by the following method: weighing riluzole standard, adding a fourth solvent, mixing well to obtain riluzole standard stock solution; wherein, the fourth solvent is methanol; the concentration of riluzole standard in the riluzole standard stock solution is 5.00 mg / mL.

[0036] In some embodiments, the edaravone standard stock solution of known concentration is prepared by the following method: Edaravone standard is weighed, a fifth solvent and stabilizer A solution are added, and the mixture is stirred to obtain the edaravone standard stock solution; wherein, the fifth solvent is methanol; the stabilizer A solution contains one or more of sodium sulfite, sodium bisulfite, sodium dithionite, sodium metabisulfite, vitamin C, sodium thiosulfate, 25wt%~35wt% ammonia, sodium bicarbonate, sodium carbonate, sodium acetate, sodium phosphate, disodium hydrogen phosphate, calcium hydroxide, calcium carbonate, ethylenediamine, and triethylamine, further optionally vitamin C; wherein, when the stabilizer A in the stabilizer A solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer A is 10.00 mg / mL~100.00 mg / mL. mg / mL; the volume ratio of the fifth solvent to the stabilizer A solution is 1:2 to 10:1; the concentration of edaravone standard in the edaravone standard stock solution is 5.00 mg / mL.

[0037] In some embodiments, when the stabilizer A in the stabilizer A solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer A is 10.00 mg / mL~50.00 mg / mL.

[0038] In some embodiments, when the stabilizer A in the stabilizer A solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer A is 20.00 mg / mL~40.00 mg / mL.

[0039] In some embodiments, when the stabilizer A in the stabilizer A solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer A is 30.00 mg / mL.

[0040] In some embodiments, the volume ratio of the fifth solvent to the stabilizer A solution is 7:3.

[0041] In some embodiments, the known concentration of riluzole- 13 C, 15 N2 isotope internal standard stock solution was prepared as follows: Weigh riluzole- 13 C, 15N2 isotope internal standard, added to the sixth solvent, mixed well to obtain riluzole- 13 C, 15 N2 isotope internal standard stock solution; wherein, the sixth solvent is methanol; the riluzole- 13 C, 15 Riluzole in N2 isotope internal standard stock solution 13 C, 15 The concentration of the N2 isotope internal standard was 1.00 mg / mL.

[0042] In some embodiments, the known concentration of edaravone-d 5 Isotope internal standard stock solution was prepared as follows: Weigh edaravone-d 5 An isotopic internal standard was added to a seventh solvent and stabilizer B solution, and after mixing, edaravone-d was obtained. 5 An isotope internal standard stock solution; wherein the seventh solvent is methanol; the stabilizer B in the stabilizer B solution is one or more of sodium sulfite, sodium bisulfite, sodium dithionite, sodium metabisulfite, vitamin C, sodium thiosulfate, 25wt%~35wt% ammonia, sodium bicarbonate, sodium carbonate, sodium acetate, sodium phosphate, disodium hydrogen phosphate, calcium hydroxide, calcium carbonate, ethylenediamine, and triethylamine, further optionally vitamin C; wherein, when the stabilizer B in the stabilizer B solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer B is 20.00 mg / mL~60.00 mg / mL; the volume ratio of the seventh solvent to the stabilizer B solution is 1:1~5:1; the edaravone-d 5 Idaravone-d in internal standard stock solution 5 The concentration of the isotope internal standard was 1.00 mg / mL.

[0043] In some embodiments, when the stabilizer B in the stabilizer B solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer B is 20.00 mg / mL~50.00 mg / mL.

[0044] In some embodiments, when the stabilizer B in the stabilizer B solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer B is 20.00 mg / mL~40.00 mg / mL.

[0045] In some embodiments, when the stabilizer B in the stabilizer B solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer B is 30.00 mg / mL.

[0046] In some embodiments, the volume ratio of the seventh solvent to the stabilizer B solution is 7:3.

[0047] In some embodiments, the sample stabilizer is 25wt%~35wt% ammonia water, more preferably 25wt%~28wt% ammonia water; the protein precipitant is a methanol solution containing 1.0vt%~5.0vt% formic acid, more preferably a methanol solution containing 1.0vt%~3.0vt% formic acid, and even more preferably a methanol solution containing 2.0vt% formic acid.

[0048] The application of the above-mentioned kit in the preparation of diagnostic reagents for the simultaneous detection of riluzole and edaravone in human biological samples is also within the scope of protection of this invention. The biological samples are whole blood, plasma, serum, urine, cerebrospinal fluid, tissue fluid, or tissue homogenate.

[0049] The application of the above-mentioned kit in the simultaneous detection of riluzole and edaravone in human biological samples using high performance liquid chromatography-tandem mass spectrometry is also within the scope of protection of this invention.

[0050] In some embodiments, the human biological sample is human whole blood, human plasma, human serum, human urine, human cerebrospinal fluid, human tissue fluid, or human tissue homogenate.

[0051] The steps for detecting riluzole and edaravone in human biological samples include:

[0052] ①The sample to be tested is obtained by mixing a blank human biological sample or a human biological sample with a sample stabilizer;

[0053] ② Method 1: Mix the sample to be tested obtained in ① with the series of standard curve working solutions or the series of quality control working solutions respectively, then add the internal standard working solution and protein precipitant, mix well, and centrifuge; aspirate the supernatant after centrifugation, add the sample enhancement solution to the supernatant, mix well, and then perform LC-MS / MS detection and analysis.

[0054] or,

[0055] Method 2: Add the internal standard working solution and protein precipitant directly to the sample to be tested obtained in step ①, mix well, and centrifuge; aspirate the supernatant after centrifugation, add the sample enhancement solution to the supernatant, mix well, and then perform LC-MS / MS detection and analysis.

[0056] In some embodiments, in step ①: the human blank biological sample is derived from biological samples of healthy individuals who have not taken riluzole and edaravone; the human biological sample is derived from biological samples of patients who have taken riluzole and / or edaravone.

[0057] In some embodiments, in step ①: the volume percentage of the sample stabilizer in the sample to be tested is 20% to 40%, optionally 20%.

[0058] In some embodiments, in step ②, the sample to be tested is obtained by mixing a blank human biological sample with a sample stabilizer.

[0059] In some embodiments, in step ②, the volume ratio of the sample to be tested to the working solution of the series of standard curves is 5:1 to 10:1, optionally 7:1 to 10:1, and further optionally 9:1.

[0060] In some embodiments, in step ②, the ratio of the total volume of the sample to be tested and the working solution of the series of standard curves to the volume of the internal standard working solution and the protein precipitant is 2:1:10 to 10:1:30, optionally 3:1:10 to 6:1:20, and further optionally 5:1:15.

[0061] In some embodiments, in step ②, the volume ratio of the sample to be tested to the working solution of the series of quality control products is 5:1 to 10:1, optionally 7:1 to 10:1, and further optionally 9:1.

[0062] In some embodiments, in step ②, the ratio of the total volume of the test sample and the series of quality control working solutions to the volume of the internal standard working solution and the protein precipitant is 2:1:10 to 10:1:30, optionally 3:1:10 to 6:1:20, and further optionally 5:1:15.

[0063] In some embodiments, in step ②, the sample to be tested is obtained by mixing a human biological sample with a sample stabilizer.

[0064] In some embodiments, in step ②, the volume ratio of the sample to be tested to the internal standard working solution and the protein precipitant is 2:1:10 to 10:1:30, optionally 3:1:10 to 6:1:20, and further optionally 5:1:15.

[0065] In some embodiments, in step ②: the centrifugation is performed at 2~8 ℃ and 6000~15000 rpm for 5~15 min.

[0066] In some embodiments, in step ②: the centrifugation is performed at 2~6 ℃ and 10000~15000 rpm for 8~12 min.

[0067] In some embodiments, in step ②: the centrifugation is performed at 4 ℃ and 13000 rpm for 10 min.

[0068] In some embodiments, in step ②, the volume ratio of the supernatant to the sample enhancement solution is 2:1 to 10:1, optionally 3:1 to 6:1, and further optionally 5:1.

[0069] In some embodiments, the series of standard curve working solutions are standard curve working solutions with eight different concentration levels prepared by mixing a known concentration of riluzole standard stock solution and a known concentration of edaravone standard stock solution and diluting them with a first solvent; wherein, the eight different concentration levels of the standard curve working solution are: the concentrations of riluzole and edaravone are the same, and the eight concentrations are 5000.00 ng / mL, 1000.00 ng / mL, 500.00 ng / mL, 250.00 ng / mL, 100.00 ng / mL, 50.00 ng / mL, 25.00 ng / mL, and 10.00 ng / mL, respectively.

[0070] In some embodiments, the first solvent is a 50% (v / v) aqueous methanol solution.

[0071] In some embodiments, the series of quality control working solutions are quality control working solutions with four different concentration levels prepared by mixing a known concentration of riluzole standard stock solution and a known concentration of edaravone standard stock solution, and then diluting them with a second solvent; wherein the four different concentration levels of the quality control working solution are: the concentrations of riluzole and edaravone are the same, and the four concentrations are 4000.00 ng / mL, 300.00 ng / mL, 30.00 ng / mL, and 10.00 ng / mL, respectively.

[0072] In some embodiments, the second solvent is a 50% (v / v) aqueous methanol solution.

[0073] In some embodiments, the internal standard working solution is riluzole of a known concentration. 13 C, 15 N2 isotope internal standard stock solution and edaravone-d at known concentrations 5 After mixing the isotope internal standard stock solution, it was diluted with a third solvent to prepare the internal standard working solution; the internal standard working solution contained riluzole- 13 C, 15 N2 isotope internal standard and edaravone-d 5 The concentrations of the isotope internal standards were the same, all being 100.00 ng / mL.

[0074] In some embodiments, the third solvent is methanol.

[0075] In some embodiments, the sample stabilizer is one or more of sodium sulfite, sodium bisulfite, sodium dithionite, sodium metabisulfite, vitamin C, sodium thiosulfate, 25wt%~35wt% ammonia, sodium bicarbonate, sodium carbonate, sodium acetate, sodium phosphate, disodium hydrogen phosphate, calcium hydroxide, calcium carbonate, ethylenediamine, and triethylamine; wherein, when the sample stabilizer is not 25wt%~35wt% ammonia, the sample stabilizer exists in the form of a sample stabilizer solution, the solvent in the sample stabilizer solution is water, and the concentration of the solute sample stabilizer is 5.00 mg / mL~200.00 mg / mL.

[0076] In some embodiments, the sample stabilizer is 25wt% to 35wt% ammonia, and more preferably 25wt% to 28wt% ammonia.

[0077] In some embodiments, the protein precipitant is a methanol solution containing 0.1 vt% to 10.0 vt% formic acid.

[0078] In some embodiments, the protein precipitant is a methanol solution containing 1.0 vt% to 5.0 vt% formic acid, more preferably a methanol solution containing 1.0 vt% to 3.0 vt% formic acid, and even more preferably a methanol solution containing 2.0 vt% formic acid.

[0079] In some embodiments, the sample enhancement solution is formic acid.

[0080] In some embodiments, the riluzole standard stock solution of known concentration is prepared by the following method: weighing riluzole standard, adding a fourth solvent, mixing well to obtain riluzole standard stock solution; wherein, the fourth solvent is methanol; the concentration of riluzole standard in the riluzole standard stock solution is 5.00 mg / mL.

[0081] In some embodiments, the edaravone standard stock solution of known concentration is prepared by the following method: Edaravone standard is weighed, a fifth solvent and stabilizer A solution are added, and the mixture is stirred to obtain the edaravone standard stock solution; wherein, the fifth solvent is methanol; the stabilizer A solution contains one or more of sodium sulfite, sodium bisulfite, sodium dithionite, sodium metabisulfite, vitamin C, sodium thiosulfate, 25wt%~35wt% ammonia, sodium bicarbonate, sodium carbonate, sodium acetate, sodium phosphate, disodium hydrogen phosphate, calcium hydroxide, calcium carbonate, ethylenediamine, and triethylamine, further optionally vitamin C; wherein, when the stabilizer A in the stabilizer A solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer A is 10.00 mg / mL~100.00 mg / mL. mg / mL; the volume ratio of the fifth solvent to the stabilizer A solution is 1:2 to 10:1; the concentration of edaravone standard in the edaravone standard stock solution is 5.00 mg / mL.

[0082] In some embodiments, when the stabilizer A in the stabilizer A solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer A is 10.00 mg / mL~50.00 mg / mL.

[0083] In some embodiments, when the stabilizer A in the stabilizer A solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer A is 20.00 mg / mL~40.00 mg / mL.

[0084] In some embodiments, when the stabilizer A in the stabilizer A solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer A is 30.00 mg / mL.

[0085] In some embodiments, the volume ratio of the fifth solvent to the stabilizer A solution is 7:3.

[0086] In some embodiments, the known concentration of riluzole- 13 C, 15 N2 isotope internal standard stock solution was prepared as follows: Weigh riluzole- 13 C, 15 N2 isotope internal standard, added to the sixth solvent, mixed well to obtain riluzole- 13 C, 15 N2 isotope internal standard stock solution; wherein, the sixth solvent is methanol; the riluzole- 13 C, 15 Riluzole in N2 isotope internal standard stock solution 13 C, 15The concentration of the N2 isotope internal standard was 1.00 mg / mL.

[0087] In some embodiments, the known concentration of edaravone-d 5 Isotope internal standard stock solution was prepared as follows: Weigh edaravone-d 5 An isotopic internal standard was added to a seventh solvent and stabilizer B solution, and after mixing, edaravone-d was obtained. 5 An isotope internal standard stock solution; wherein the seventh solvent is methanol; the stabilizer B in the stabilizer B solution is one or more of sodium sulfite, sodium bisulfite, sodium dithionite, sodium metabisulfite, vitamin C, sodium thiosulfate, 25wt%~35wt% ammonia, sodium bicarbonate, sodium carbonate, sodium acetate, sodium phosphate, disodium hydrogen phosphate, calcium hydroxide, calcium carbonate, ethylenediamine, and triethylamine, further optionally vitamin C; wherein, when the stabilizer B in the stabilizer B solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer B is 20.00 mg / mL~60.00 mg / mL; the volume ratio of the seventh solvent to the stabilizer B solution is 1:1~5:1; the edaravone-d 5 Idaravone-d in internal standard stock solution 5 The concentration of the isotope internal standard was 1.00 mg / mL.

[0088] In some embodiments, when the stabilizer B in the stabilizer B solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer B is 20.00 mg / mL~50.00 mg / mL.

[0089] In some embodiments, when the stabilizer B in the stabilizer B solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer B is 20.00 mg / mL~40.00 mg / mL.

[0090] In some embodiments, when the stabilizer B in the stabilizer B solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer B is 30.00 mg / mL.

[0091] In some embodiments, the volume ratio of the seventh solvent to the stabilizer B solution is 7:3.

[0092] In some embodiments, the chromatographic conditions for LC-MS / MS detection are as follows:

[0093] Liquid chromatography conditions: Column: Kinetex F5, 2.6 μm, 100 × 2.1 mm; Column temperature: 40 ℃; Mobile phase A: aqueous solution containing 0.1 vt% formic acid; Mobile phase B: methanol solution containing 0.1 vt% formic acid; Injection volume: 2 μL; Injection flow rate: 0.25 mL / min; Gradient elution; The gradient elution program is shown in Table 1 below:

[0094] Table 1

[0095]

[0096] Mass spectrometry chromatographic conditions: Electrospray ionization source was used; multiple reaction monitoring mode was used; positive ion mode was used; curtain gas: 30 psi; collision gas: 8 psi; electrospray voltage: 3000 V; ion source temperature: 650 ℃; nebulizer gas: 45 psi; auxiliary gas: 60 psi; inlet potential: 10 V; collision cell outlet potential: 13 V.

[0097] Beneficial effects:

[0098] Compared with existing technologies, the method for simultaneous detection of riluzole and edaravone concentrations provided by this invention has the following significant advantages:

[0099] (1) Extremely high sensitivity, with a detection limit at the ng / mL level.

[0100] This invention establishes for the first time an analytical method for the simultaneous detection of riluzole and edaravone based on liquid chromatography-tandem mass spectrometry (LC-MS / MS). By optimizing key conditions such as mass spectrometry ion source parameters, collision energy, and declustering voltage, the mass spectrometry response signals of both drugs were significantly improved. Experimental results show that the limits of quantitation for both riluzole and edaravone provided by this invention are as low as 1 ng / mL, which can meet the monitoring needs at low blood drug concentrations, and is particularly suitable for pharmacokinetic studies and trace concentration monitoring in long-term patients.

[0101] (2) It achieves efficient and rapid separation of two drugs using a single method.

[0102] This invention enables the simultaneous extraction and detection of riluzole and edaravone within the same analytical cycle. The total analysis time for a single sample is controlled within 6 minutes, and the retention times of the two drugs differ significantly (riluzole retention time: 3.12 min; edaravone retention time: 2.43 min), with no cross-interference. This setup significantly shortens sample analysis time, reduces the amount of biological samples used and detection costs, and improves the efficiency of high-throughput screening in clinical or laboratory settings.

[0103] (3) Excellent methodological performance, meeting the requirements of the guidelines for biological sample analysis.

[0104] After complete methodological validation, the detection method of this invention exhibits good linearity in the range of 1-500 ng / mL (correlation coefficient R0). 2 The method precision (RSD) was less than 15%, the accuracy (RE) was within ±15%, the extraction recovery was stable, and no obvious matrix effect was observed. These data indicate that the method of this invention has high specificity, high reproducibility, and good reliability, and fully complies with the relevant requirements of the "Guideline for Validation of Quantitative Analysis Methods for Biological Samples". Attached Figure Description

[0105] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0106] Figure 1 This is a typical chromatogram for riluzole with a limit of quantification of 1.00 ng / mL in an embodiment of the present invention.

[0107] Figure 2 This is a typical chromatogram for an edaravone quantification limit of 1.00 ng / mL in an embodiment of the present invention. Detailed Implementation

[0108] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0109] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0110] Riluzole: Purchased from the National Institutes for Food and Drug Control, 100 mg / vial, batch number 100684-202002.

[0111] Edaravone: Purchased from the National Institutes for Food and Drug Control, 100 mg / bottle, batch number 100620-202204.

[0112] Riluzole - 13 C, 15 N2: Purchased from MedChemExpress (MCE), 1 mg / bottle, catalog number HY-B0211S.

[0113] Edaravon-d 5 Purchased from MedChemExpress (MCE), 1 mg / bottle, catalog number HY-B0099S.

[0114] The blank human plasma used in the embodiments of the present invention is derived from the plasma of healthy individuals who have not taken riluzole and edaravone.

[0115] The plasma used in the embodiments of the present invention is derived from the plasma of patients who have taken riluzole or edaravone.

[0116] Example 1:

[0117] (1) Preparation of standard stock solution

[0118] Weigh 5.00 mg of riluzole standard, add 1.00 mL of methanol, and sonicate to obtain a 5.00 mg / mL riluzole standard stock solution.

[0119] Weigh 5.00 mg of edaravone standard, add 0.70 mL of methanol and 0.30 mL of vitamin C aqueous solution (30.00 mg / mL), and sonicate to obtain a 5.00 mg / mL edaravone standard stock solution.

[0120] (2) Preparation of internal standard stock solution

[0121] Weigh 1.00 mg riluzole - 13 C, 15 N2 isotope internal standard was added to 1.00 mL of methanol and sonicated to obtain 1.00 mg / mL riluzole. 13 C, 15 N2 isotope internal standard stock solution.

[0122] Weigh 1.00 mg edaravone-day 5 An isotope internal standard was added to 0.70 mL of methanol and 0.30 mL of vitamin C aqueous solution (30.00 mg / mL), and after ultrasonic mixing, 1.00 mg / mL edaravone-d was obtained. 5 Isotope internal standard stock solution.

[0123] (3) Preparation of a series of standard curve working solutions

[0124] Equal volumes of riluzole and edaravone standard stock solutions were mixed and continuously diluted with 50% (v / v) methanol aqueous solution to prepare a series of standard curve working solutions with riluzole / edaravone concentrations of 5000.00 ng / mL, 1000.00 ng / mL, 500.00 ng / mL, 250.00 ng / mL, 100.00 ng / mL, 50.00 ng / mL, 25.00 ng / mL, and 10.00 ng / mL, respectively; the concentrations of riluzole and edaravone were the same in all the standard curve working solutions.

[0125] (4) Preparation of a series of quality control working solutions

[0126] Take equal volumes of riluzole and edaravone standard stock solutions, mix them thoroughly, and continuously dilute them with 50% methanol aqueous solution to prepare a series of quality control working solutions with standard (riluzole / edaravone) concentrations of 4000.00 ng / mL, 300.00 ng / mL, 30.00 ng / mL, and 10.00 ng / mL, respectively; the concentrations of riluzole and edaravone in the series of quality control working solutions are all the same.

[0127] (5) Preparation of internal standard working solution

[0128] Measure an equal volume of riluzole- 13 C, 15 N2 isotope internal standard stock solution and edaravone-d 5 The isotope internal standard stock solution was mixed and diluted with methanol to prepare the internal standard (riluzole- 13 C, 15 N2 / Edaravone-d 5 Internal standard working solution with a concentration of 100.00 ng / mL; riluzole in the internal standard working solution 13 C, 15 N2 isotope internal standard and edaravone-d 5 The concentrations of the isotope internal standards were the same, all being 100.00 ng / mL.

[0129] (6) Prepare standard curve samples for detection

[0130] Preparation of standard curve sample for detection: Measure 45 μL of blank human plasma (containing 20 vt% of 25 wt% ammonia water; the human plasma used was derived from the plasma of healthy individuals who had not taken riluzole and edaravone), add 5 μL of standard curve working solution, 10 μL of internal standard working solution, and 150 μL of methanol solution containing 2 vt% formic acid in sequence, mix well, and centrifuge at 4 ℃ and 13000 rpm for 10 min; take 100 μL of supernatant, add 20 μL of formic acid, mix well, and obtain the standard curve sample for detection. High performance liquid chromatography-tandem mass spectrometry is used for drug detection and analysis.

[0131] (7) Prepare quality control samples for testing

[0132] Preparation of quality control samples for testing: Measure 45 μL of blank human plasma (containing 20 vt% of 25 wt% ammonia water; the human plasma used was derived from the plasma of healthy individuals who had not taken riluzole and edaravone), add 5 μL of quality control working solution, 10 μL of internal standard working solution, and 150 μL of methanol solution containing 2 vt% formic acid in sequence, mix well, and centrifuge at 4 ℃ and 13000 rpm for 10 min; take 100 μL of the supernatant, add 20 μL of formic acid, mix well, and obtain the quality control sample for testing. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) is used for drug detection and analysis.

[0133] (8) Preparation of biological samples to be tested

[0134] Take 50 μL of the plasma to be tested (containing 20 vt% of 25 wt% ammonia water; the human plasma used was derived from the plasma of patients who took riluzole or edaravone), add 10 μL of internal standard working solution and 150 μL of methanol solution containing 2 vt% formic acid, mix well, and centrifuge at 4 ℃ and 13000 rpm for 10 min; take 100 μL of the supernatant, add 20 μL of formic acid, mix well, and obtain the biological sample to be tested. The drug was detected and analyzed by high performance liquid chromatography-tandem mass spectrometry.

[0135] (9) Chromatographic conditions for detection using high performance liquid chromatography-tandem mass spectrometry in this embodiment

[0136] Liquid chromatography conditions: Column: Kinetex F5, 2.6 μm, 100 × 2.1 mm; Column temperature: 40 ℃; Mobile phase A: aqueous solution containing 0.1 vt% formic acid; Mobile phase B: methanol solution containing 0.1 vt% formic acid; Injection volume: 2 μL; Injection flow rate: 0.25 mL / min; Gradient elution; The gradient elution program is shown in Table 2 below.

[0137] Table 2 Gradient elution program for liquid chromatography

[0138]

[0139] Mass spectrometry conditions: Electrospray ionization (ESI) source; Multiple reaction monitoring (MRM) acquisition mode; Positive ion acquisition mode; Curtain gas (CUR): 30 psi; Collision gas (CAD): 8 psi; Electrospray voltage (IS): 3000 V; Ion source temperature (TEM): 650 ℃; Nebulizer gas (Gas 1): 45 psi; Auxiliary gas (Gas 2): 60 psi; Inlet potential (EP): 10 V; Collision cell outlet potential (CXP): 13 V.

[0140] (10) The standard curve working solutions and internal standard working solutions prepared under “(3) Preparation of a series of standard curve working solutions” above were injected into a liquid chromatography-tandem mass spectrometry (LC-MS / MS) instrument and detected by high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). For specific chromatographic conditions, please refer to “(9) Chromatographic conditions for detection by high performance liquid chromatography-tandem mass spectrometry in this embodiment” above. Detailed ion pair parameters are shown in Table 3. Note: In the table, Q1 is the parent ion, Q3 is the daughter ion, Dwell (msec) is the residence time, DP (V) is the declustering voltage, and CE (V) is the cleavage voltage.

[0141] Table 3 Ion-pair parameters of riluzole and edaravone

[0142]

[0143] (11) The standard curve sample prepared under “(6) Preparation of standard curve sample for detection” and the quality control sample prepared under “(7) Preparation of quality control sample for detection” were used to detect and analyze the drug by high performance liquid chromatography-tandem mass spectrometry. For the specific chromatographic conditions used, please refer to the above “(9) Chromatographic conditions for detection by high performance liquid chromatography-tandem mass spectrometry in this example”.

[0144] (11-1) Specific standard curve data for riluzole and edaravone are shown in Tables 4 and 6, respectively. Specifically, the linear regression equation for riluzole is: y = 0.01965x - 5.5×10 -4 (weighting: 1 / x 2 The linear regression equation for edaravone is: y = 0.03772 x – 0.00517 (weighting: 1 / x) 2 ).

[0145] (11-2) The quality control samples were tested according to the standard curve. Each sample was tested 6 times. The test results are shown in Table 5 and Table 7 respectively.

[0146] Figure 1 The chromatogram shown in this embodiment of the invention is a typical chromatogram for riluzole with a limit of quantification of 1.00 ng / mL, with a peak elution time of 3.12 min and a peak area of ​​1.7 × 10⁻⁶. 4 Peak height 6.0×10 3 .

[0147] Figure 2 This is a typical chromatogram for edaravone with a limit of quantification of 1.00 ng / mL in an embodiment of the present invention, wherein the peak elution time is 2.43 min and the peak area is 1.8 × 10⁻⁶. 4 Peak height 4.2×10 3 .

[0148] The data results show that the assay method of this invention can simultaneously detect the concentrations of riluzole and edaravone. In biological matrices, the method achieves a limit of quantification of 1.00 ng / mL for both riluzole and edaravone, and exhibits high sensitivity and good accuracy.

[0149] Table 4. Standard Curve of Riluzole

[0150]

[0151] Table 5. Quality control samples for riluzole

[0152]

[0153] Table 6 Edaravone Standard Curve

[0154]

[0155] Table 7 Edaravone Quality Control Samples

[0156]

[0157] (11-3) A total of 12 ALS patients who took riluzole or edaravone were collected clinically and tested according to the method of “(8) Preparation of biological samples for testing”. The test results are shown in Table 8.

[0158] Table 8. Detection results of biological samples to be tested

[0159]

[0160] Note: * in the table indicates that the detection result is below the limit of quantitation (1 ng / mL).

[0161] This invention provides a method for simultaneously detecting the concentrations of riluzole and edaravone. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A kit for simultaneously detecting riluzole and edaravone concentrations, characterized in that, The kit contains the following reagents: (1) Series of standard curve working solutions The series of standard curve working solutions are prepared by mixing riluzole standard stock solution and edaravone standard stock solution of known concentration and then diluting with a first solvent to form standard curve working solutions with 8 different concentration levels. The eight different concentration levels of the standard curve working solution are as follows: Riluzole and edaravone were administered at the same concentrations, with eight concentrations in total: 5000.00 ng / mL, 1000.00 ng / mL, 500.00 ng / mL, 250.00 ng / mL, 100.00 ng / mL, 50.00 ng / mL, 25.00 ng / mL, and 10.00 ng / mL. (2) Series of quality control working solutions The series of quality control working solutions are prepared by mixing riluzole standard stock solution and edaravone standard stock solution of known concentration and then diluting with a second solvent to form quality control working solutions with four different concentration levels. The four different concentration levels of the quality control working solution are as follows: Riluzole and edaravone were administered at the same concentrations, with four concentrations of 4000.00 ng / mL, 300.00 ng / mL, 30.00 ng / mL, and 10.00 ng / mL, respectively. (3) Internal standard working solution The internal standard working solution is riluzole of known concentration. 13 C, 15 N2 isotope internal standard stock solution and edaravone-d at known concentrations 5 After the isotope internal standard stock solution is mixed, it is diluted with a third solvent to prepare the internal standard working solution; The internal standard working solution contains riluzole- 13 C, 15 N2 isotope internal standard and edaravone-d 5 The concentrations of the isotope internal standards were all the same, at 100.00 ng / mL; (4) Sample stabilizer: The sample stabilizer is one or more of the following: sodium sulfite, sodium bisulfite, sodium dithionite, sodium metabisulfite, vitamin C, sodium thiosulfate, 25wt%~35wt% ammonia, sodium bicarbonate, sodium carbonate, sodium acetate, sodium phosphate, disodium hydrogen phosphate, calcium hydroxide, calcium carbonate, ethylenediamine, and triethylamine; wherein, when the sample stabilizer is not 25wt%~35wt% ammonia, the sample stabilizer exists in the form of a sample stabilizer solution, the solvent in the sample stabilizer solution is water, and the concentration of the solute sample stabilizer is 5.00 mg / mL~200.00 mg / mL; (5) Protein precipitant: a methanol solution containing 0.1 wt% to 10.0 wt% formic acid; (6) Sample enhancement solution: formic acid; (7) Mobile phase A: An aqueous solution containing 0.1 wt% formic acid; (8) Mobile phase B: Methanol solution containing 0.1 vt% formic acid.

2. The reagent kit according to claim 1, characterized in that, The first solvent is a 50% (v / v) aqueous methanol solution; the second solvent is a 50% (v / v) aqueous methanol solution; and the third solvent is methanol.

3. The reagent kit according to claim 1, characterized in that, The known concentration of riluzole standard stock solution is prepared as follows: riluzole standard is weighed, a fourth solvent is added, and the mixture is stirred to obtain the riluzole standard stock solution; wherein, the fourth solvent is methanol; the concentration of riluzole standard in the riluzole standard stock solution is 5.00 mg / mL; And / or, The known concentration of edaravone standard stock solution is prepared as follows: Edaravone standard is weighed, a fifth solvent and stabilizer A solution are added, and the mixture is stirred to obtain the edaravone standard stock solution; wherein, the fifth solvent is methanol; stabilizer A in the stabilizer A solution is one or more of sodium sulfite, sodium bisulfite, sodium dithionite, sodium metabisulfite, vitamin C, sodium thiosulfate, 25wt%~35wt% ammonia, sodium bicarbonate, sodium carbonate, sodium acetate, sodium phosphate, disodium hydrogen phosphate, calcium hydroxide, calcium carbonate, ethylenediamine, and triethylamine, further optionally selected as vitamin C; wherein, when stabilizer A in the stabilizer A solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer A is 10.00 mg / mL~100.00 mg / mL. mg / mL; the volume ratio of the fifth solvent to the stabilizer A solution is 1:2 to 10:1; the concentration of edaravone standard in the edaravone standard stock solution is 5.00 mg / mL.

4. The reagent kit according to claim 1, characterized in that, The known concentration of riluzole- 13 C, 15 N2 isotope internal standard stock solution was prepared as follows: Weigh riluzole- 13 C, 15 N2 isotope internal standard, added to the sixth solvent, mixed well to obtain riluzole- 13 C, 15 N2 isotope internal standard stock solution; wherein, the sixth solvent is methanol; the riluzole- 13 C, 15 Riluzole in N2 isotope internal standard stock solution 13 C, 15 The concentration of the N2 isotope internal standard was 1.00 mg / mL; And / or, The known concentration of edaravone-d 5 Isotope internal standard stock solution was prepared as follows: Weigh edaravone-d 5 An isotopic internal standard was added to a seventh solvent and stabilizer B solution, and after mixing, edaravone-d was obtained. 5 An isotope internal standard stock solution; wherein the seventh solvent is methanol; the stabilizer B in the stabilizer B solution is one or more of sodium sulfite, sodium bisulfite, sodium dithionite, sodium metabisulfite, vitamin C, sodium thiosulfate, 25wt%~35wt% ammonia, sodium bicarbonate, sodium carbonate, sodium acetate, sodium phosphate, disodium hydrogen phosphate, calcium hydroxide, calcium carbonate, ethylenediamine, and triethylamine, further optionally vitamin C; wherein, when the stabilizer B in the stabilizer B solution is not 25wt%~35wt% ammonia, the solvent in the solution is water, and the concentration of stabilizer B is 20.00 mg / mL~60.00 mg / mL; the volume ratio of the seventh solvent to the stabilizer B solution is 1:1~5:1; the edaravone-d 5 Idaravone-d in internal standard stock solution 5 The concentration of the isotope internal standard was 1.00 mg / mL.

5. The reagent kit according to claim 1, characterized in that, The sample stabilizer is 25wt%~35wt% ammonia water, more preferably 25wt%~28wt% ammonia water; the protein precipitant is a methanol solution containing 1.0vt%~5.0vt% formic acid, more preferably a methanol solution containing 1.0vt%~3.0vt% formic acid, and even more preferably a methanol solution containing 2.0vt% formic acid.

6. The use of the kit according to any one of claims 1 to 5 in the preparation of a diagnostic reagent for the simultaneous detection of riluzole and edaravone in human biological samples, wherein the biological sample is whole blood, plasma, serum, urine, cerebrospinal fluid, tissue fluid, or tissue homogenate.

7. The application of the kit according to any one of claims 1 to 5 in the simultaneous detection of riluzole and edaravone in human biological samples using high performance liquid chromatography-tandem mass spectrometry.

8. The application according to claim 7, characterized in that, The human biological samples are human whole blood, human plasma, human serum, human urine, human cerebrospinal fluid, human tissue fluid, or human tissue homogenate. The steps for detecting riluzole and edaravone in human biological samples include: ①The sample to be tested is obtained by mixing a blank human biological sample or a human biological sample with a sample stabilizer; ② Method 1: Mix the sample to be tested obtained in ① with the series of standard curve working solutions or the series of quality control working solutions respectively, then add the internal standard working solution and protein precipitant, mix well, and centrifuge; aspirate the supernatant after centrifugation, add the sample enhancement solution to the supernatant, mix well, and then perform LC-MS / MS detection and analysis. or, Method 2: Add the internal standard working solution and protein precipitant directly to the sample to be tested obtained in step ①, mix well, and centrifuge; aspirate the supernatant after centrifugation, add the sample enhancement solution to the supernatant, mix well, and then perform LC-MS / MS detection and analysis.

9. The application according to claim 8, characterized in that, In step ①: the blank human biological sample is derived from the biological sample of a healthy person who has not taken riluzole and edaravone; the human biological sample is derived from the biological sample of a patient who has taken riluzole and / or edaravone.

10. The application according to claim 8, characterized in that, The chromatographic conditions for LC-MS / MS detection are as follows: Liquid chromatography conditions: Column: Kinetex F5, 2.6 μm, 100 × 2.1 mm; Column temperature: 40 ℃; Mobile phase A: aqueous solution containing 0.1 vt% formic acid; Mobile phase B: methanol solution containing 0.1 vt% formic acid; Injection volume: 2 μL; Injection flow rate: 0.25 mL / min; Gradient elution; The gradient elution program is shown in the table below: Mass spectrometry chromatographic conditions: Electrospray ionization source used; Multiple reaction monitoring mode used; Positive ion mode used; Curtain gas: 30 psi; Collision gas: 8 psi; Electrospray voltage: 3000 V; Ion source temperature: 650 ℃; Nebulizer gas: 45 psi; Auxiliary gas: 60 psi; Inlet potential: 10 V; Collision pool outlet Potential: 13 V.

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