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CN-122017089-A - Method for simultaneously detecting concentration of riluzole and edaravone

CN122017089ACN 122017089 ACN122017089 ACN 122017089ACN-122017089-A

Abstract

The invention belongs to the technical field of chromatographic mass spectrometry detection, and relates to a method for simultaneously detecting the concentration 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. By optimizing key conditions such as mass spectrum ion source parameters, collision energy, declustering voltage and the like, the mass spectrum response signals of the two medicaments are obviously improved. Experimental results show that the quantitative limit of riluzole and edaravone in the method provided by the invention is as low as 1 ng/mL, so that the monitoring requirement under low blood concentration can be met, and the method is especially suitable for pharmacokinetic research and micro concentration monitoring of patients taking medicines for a long time.

Inventors

  • FENG GUOBING
  • LUO YING
  • ZHANG DAN
  • WANG XIANG
  • SUN YAN

Assignees

  • 杭州市老年病医院

Dates

Publication Date
20260512
Application Date
20260415

Claims (10)

  1. 1. A kit for simultaneously detecting riluzole and edaravone concentrations, comprising the following reagents: (1) Working fluid with series standard curves The series of standard curve working solutions are standard curve working solutions with 8 different concentration levels, which are formed by diluting a first solvent after uniformly mixing a standard substance stock solution of riluzole with a known concentration and a standard substance stock solution of edaravone with a known concentration; Wherein, 8 different concentration levels of the standard curve working solution are as follows: the concentrations of riluzole and edaravone are the same, and 8 concentrations are in sequence 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、10.00 ng/mL; (2) Working liquid for series quality control products The series of quality control product working solutions are quality control product working solutions with 4 different concentration levels, which are formed by diluting a second solvent after uniformly mixing a standard riluzole stock solution with a known concentration and a standard edaravone stock solution with a known concentration; Wherein, 4 different concentration levels of the quality control product working solution are as follows: The concentrations of riluzole and edaravone are the same, and the 4 concentrations are 4000.00 ng/mL, 300.00 ng/mL, 30.00 ng/mL and 10.00 ng/mL in sequence; (3) Internal standard working fluid The internal standard working solution is obtained by uniformly mixing a standard stock solution in a riluzole- 13 C, 15 N 2 isotope with a known concentration and an edaravone-d 5 isotope with a known concentration, and then diluting the mixture with a third solvent to obtain an internal standard working solution; The concentration of the riluzole- 13 C, 15 N 2 isotope internal standard in the internal standard working solution is 100.00 ng/mL as that of the edaravone-d 5 isotope internal standard; (4) The sample stabilizer is one or more of sodium sulfite, sodium bisulfite, sodium dithionite, sodium metabisulfite, vitamin C, sodium thiosulfate, 25-35 wt% ammonia water, 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 25-35 wt% ammonia water, the sample stabilizer exists in the form of a sample stabilizer solution, a solvent in the sample stabilizer solution is water, and the concentration of solute sample stabilizer is 5.00-5.00 mg.00-200.00 mg/mL; (5) Protein precipitant is methanol solution containing 0.1vt% -10.0 vt% formic acid; (6) Sample enhancement solution, formic acid; (7) Mobile phase A is aqueous solution containing 0.1vt% formic acid; (8) Mobile phase B, methanol solution containing 0.1vt% formic acid.
  2. 2. The kit of claim 1, wherein the first solvent is 50% by volume aqueous methanol, the second solvent is 50% by volume aqueous methanol, and the third solvent is methanol.
  3. 3. The kit according to claim 1, wherein, The stock solution of the riluzole standard substance with the known concentration is prepared by weighing the riluzole standard substance, adding a fourth solvent, and uniformly mixing to obtain the stock solution of the riluzole standard substance, wherein the fourth solvent is methanol, and the concentration of the riluzole standard substance in the stock solution of the riluzole standard substance is 5.00 mg/mL; And/or the number of the groups of groups, The edaravone standard stock solution with the known concentration is prepared by weighing an edaravone standard product, adding a fifth solvent and a stabilizer A solution, uniformly mixing to obtain the edaravone standard product stock solution, wherein the fifth solvent is methanol, the stabilizer A in the stabilizer A solution is one or more of sodium sulfite, sodium bisulfite, sodium dithionite, sodium metabisulfite, vitamin C, sodium thiosulfate, 25-35 wt% of ammonia water, sodium bicarbonate, sodium carbonate, sodium acetate, sodium phosphate, disodium hydrogen phosphate, calcium hydroxide, calcium carbonate, ethylenediamine and triethylamine, and further optionally vitamin C, when the stabilizer A in the stabilizer A solution is not 25-35 wt% of ammonia water, the solvent in the solution is water, the concentration of the stabilizer A is 10.00-100.00 mg/mL, the volume ratio of the fifth solvent to the stabilizer A solution is 1:2-10:1, and the stabilizer A solution is 5300-5.00.
  4. 4. The kit according to claim 1, wherein, The stock solution of the riluzole- 13 C, 15 N 2 isotope internal standard with known concentration is prepared by weighing the riluzole- 13 C, 15 N 2 isotope internal standard, adding a sixth solvent, and uniformly mixing to obtain the stock solution of the riluzole- 13 C, 15 N 2 isotope internal standard, wherein the sixth solvent is methanol, and the concentration of the riluzole- 13 C, 15 N 2 isotope internal standard in the stock solution of the riluzole- 13 C, 15 N 2 isotope internal standard is 1.00 mg/mL; And/or the number of the groups of groups, The edaravone-d 5 isotope internal standard stock solution with the known concentration is prepared by weighing one or more of edaravone-d 5 isotope internal standard stock solution, adding a seventh solvent and a stabilizer B solution, uniformly mixing to obtain the edaravone-d 5 isotope internal standard stock solution, wherein the seventh solvent is methanol, the stabilizer B in the stabilizer B solution is sodium sulfite, sodium bisulphite, sodium dithionite, sodium metabisulfite, vitamin C, sodium thiosulfate, 25-35 wt% ammonia water, sodium bicarbonate, sodium carbonate, sodium acetate, sodium phosphate, disodium hydrogen phosphate, calcium hydroxide, ethylenediamine and triethylamine, and further optionally vitamin C, when the stabilizer B in the stabilizer B solution is not 25-35 wt% ammonia water, the concentration of the stabilizer B is 20.00-60.00 mg/mL, the volume ratio of the seventh solvent to the stabilizer B in the stabilizer B solution is 531:5-57 isotope internal standard stock solution, and the volume ratio of the stabilizer B in the stabilizer B solution is 20.00-mg.00-60.00 isotope internal standard stock solution is 531:5-57 isotope.
  5. 5. The kit according to claim 1, wherein the sample stabilizer is 25wt% to 35wt% ammonia water, further optionally 25wt% to 28wt% ammonia water, the protein precipitant is a methanol solution containing 1.0vt% to 5.0vt% formic acid, further optionally a methanol solution containing 1.0vt% to 3.0vt% formic acid, further optionally a methanol solution containing 2.0vt% formic acid.
  6. 6. The use of a kit according to any one of claims 1 to 5 for the preparation of a diagnostic reagent for simultaneous detection of riluzole and edaravone in a biological sample of the human body, said biological sample being whole blood, plasma, serum, urine, cerebrospinal fluid, interstitial fluid or tissue homogenate.
  7. 7. The use of the kit according to any one of claims 1 to 5 for simultaneously detecting riluzole and edaravone in a human biological sample by using high performance liquid chromatography tandem mass spectrometry technology.
  8. 8. The use according to claim 7, wherein the human biological sample is human whole blood, human plasma, human serum, human urine, human cerebrospinal fluid, human tissue fluid or human tissue homogenate; the method for detecting the riluzole and the edaravone in the human biological sample comprises the following steps: ① Mixing a human body blank biological sample or a human body biological sample with a sample stabilizer to obtain a sample to be tested; ② Mixing the sample to be detected obtained in ① with the series of standard curve working solutions or the series of quality control product working solutions respectively, adding the internal standard product working solution and the protein precipitant, uniformly mixing, centrifuging, sucking the supernatant after centrifuging, adding a sample enhancement solution into the supernatant, uniformly mixing, and performing LC-MS/MS detection analysis; Or alternatively And secondly, directly adding an internal standard working solution and a protein precipitant into the sample to be detected obtained in ①, uniformly mixing, centrifuging, sucking the centrifuged supernatant, adding a sample enhancement solution into the supernatant, and uniformly mixing and then carrying out LC-MS/MS detection analysis.
  9. 9. The use according to claim 8, characterized in that in step ① the human blank biological sample is derived from a biological sample of a healthy human not taking riluzole and edaravone, and the human biological sample is derived from a biological sample of a patient taking riluzole and/or edaravone.
  10. 10. The use according to claim 8, wherein the chromatographic conditions of the LC-MS/MS detection are as follows: The conditions of liquid chromatography are Kinetex F, 2.6 μm,100×2.1: 2.1 mm, column temperature 40 ℃, mobile phase A, methanol solution containing 0.1vt% formic acid, mobile phase B, 2. Mu.L of sample, sample flow rate 0.25: 0.25 mL/min, gradient elution, and gradient elution procedure as shown in the following table: The mass spectrum chromatographic conditions comprise electrospray ion source, multi-reaction monitoring mode as acquisition mode, positive ion mode as acquisition mode, gas curtain gas of 30 psi, collision gas of 8 psi, electrospray voltage of 3000V, ion source temperature of 650 ℃, atomization gas of 45 psi, auxiliary gas of 60 psi, inlet potential of 10V and collision cell outlet potential of 13V.

Description

Method for simultaneously detecting concentration of riluzole and edaravone Technical Field The invention belongs to the technical field of chromatographic mass spectrometry detection, and relates to a method for simultaneously detecting the concentration of riluzole and edaravone. Background Amyotrophic lateral sclerosis (Amyotrophic Lateral Sclerosis, ALS) is a neurodegenerative disease [1, 2] that results from motor neuron death in the brain, brain stem and spinal cord, resulting in progressive, painless muscle weakness, and often death from respiratory failure within 2 to 5 years after symptoms appear. The average time interval from first symptoms to diagnosis is as long as 10 to 16 months [3, 4], due to the rarity of the disease, low public awareness, inadequate identification of symptoms, and lack of early and appropriate specialist intervention. The pathogenesis of ALS arises from complex interactions between age, sex and genetic factors, and is manifested clinically by upper motor neuron (Upper motor neuron, UMN) and lower motor neuron (Lower motor neuron, LMN) dysfunction [5]. The incidence of ALS is about 2.16 cases per 10 tens of thousands of people per year, with a male to female ratio of about 1.3:1 [2, 6]. Riluzole (Riluzole), the first drug for ALS treatment, is a glutamatergic neurotransmission inhibitor, approved by the U.S. food and drug administration (Food and Drug Administration, FDA) in 1995 for the treatment of ALS [7]. Riluzole exerts its neuroprotective effects [8] by inhibiting the release of glutamate from central neurons, blocking excessive glutamatergic neurotransmission. Although riluzole does not cure ALS, it can extend the life expectancy [9] of ALS patients. However, there are significant individual differences in the pharmacokinetic profile of riluzole, which are primarily due to first pass metabolism, liver metabolic enzyme polymorphisms, extrahepatic metabolic pathways, and other various factors [10]. Such individuation differences may limit the efficacy and clinical application of riluzole. Another drug approved for the treatment of ALS is Edaravone (Edaravone), which was approved by the FDA in the united states as a free radical scavenger [10] in 2017. Riluzole and edaravone are the main drugs for treating Amyotrophic Lateral Sclerosis (ALS) clinically at present, and monitoring the blood concentration of the drugs is a key precondition for realizing individual accurate medication. The liquid chromatography-mass spectrometry (LC-MS/MS) technology has the advantages of capability of simultaneously carrying out quantitative analysis on various medicines, low quantitative limit, short analysis time, small interference by blood matrixes and the like, and has wide application prospect in the monitoring of therapeutic medicines. However, no report on a liquid chromatography-mass spectrometry combined method for simultaneously detecting riluzole and edaravone is currently seen. Therefore, it is necessary to establish an LC-MS/MS method capable of synchronously determining the blood concentration of riluzole and edaravone, so as to support the accurate medication strategy of ALS patients, thereby effectively reducing the risk of adverse drug reaction and improving the clinical treatment effect. Reference is made to: [1] Feldman, E. L.; Goutman, S. A.; Petri, S.; Mazzini, L.; Savelieff, M. G.; Shaw, P. J.; Sobue, G., Amyotrophic lateral sclerosis. The Lancet 2022, 400 (10360), 1363-1380. [2] Riva, N.; Domi, T.; Pozzi, L.; Lunetta, C.; Schito, P.; Spinelli, E. G.; Cabras, S.; Matteoni, E.; Consonni, M.; Bella, E. D.; Agosta, F.; Filippi, M.; Calvo, A.; Quattrini, A., Update on recent advances in amyotrophic lateral sclerosis. Journal of Neurology 2024, 271 (7), 4693-4723. [3] Richards, D.; Morren, J. A.; Pioro, E. P., Time to diagnosis and factors affecting diagnostic delay in amyotrophic lateral sclerosis. Journal of the Neurological Sciences 2020, 417, 117054. [4] Galvin, M.; Ryan, P.; Maguire, S.; Heverin, M.; Madden, C.; Vajda, A.; Normand, C.; Hardiman, O., The path to specialist multidisciplinary care in amyotrophic lateral sclerosis: A population- based study of consultations, interventions and costs. PLoS One 2017, 12 (6), e0179796. [5] Goutman, S. A.; Hardiman, O.; Al-Chalabi, A.; Chió, A.; Savelieff, M. G.; Kiernan, M. C.; Feldman, E. L., Recent advances in the diagnosis and prognosis of amyotrophic lateral sclerosis. The Lancet Neurology 2022, 21 (5), 480-493. [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 lateral sclerosis: a systematic review and meta-analysis. Journal of Neurology 2020, 267 (4), 944-953. [7] Miller, R. G.; Mitchell, J. D.; Moore, D. H., Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND). Cochrane Database of Systematic Reviews 2012, (3), Cd001447. [8] Doble, A., The pharmacology and mechanism of action of riluzole. Neurology 1996, 47 (