Lenalidomide metabolites in urine and its application

Abstract

The present invention establishes a rapid and sensitive analytical method for simultaneously determining 11 lenalidomide metabolites in human urine; the metabolites are identified by mass spectrometry; thereby providing the possibility of analyzing the metabolism of lenalidomide in the body and the pathological and physiological conditions of the body; the differences in metabolites between multiple myeloma patients in remission (EG), with no change in symptoms (SG), and with worsening symptoms (IG) are compared. From the perspective of metabolite analysis, the use of urinary metabolites to identify lenalidomide resistance is studied, and at the same time, the possibility of early and rapid screening of lenalidomide-resistant patients is provided, thereby avoiding worsening of patients' symptoms and unnecessary treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an analysis method for lenalidomide metabolites in human urine, mass spectrometry identification, and use of the metabolites as diagnostic markers in the preparation of products for identifying lenalidomide resistance in multiple myeloma patients. Background Art

[0002] Lenalidomide is approved for the treatment of various cancers including multiple myeloma (MM), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), etc.

[0003] Lenalidomide is mainly eliminated in the kidneys, with about 90% eliminated through urine. Metabolites in urine reflect the final result of the interaction between the drug and the body, and can directly and accurately reflect the drug's metabolism in the body, the body's pathological and physiological conditions. More importantly, urine is convenient, simple, non-invasive, fast and easy to obtain. Urine metabolites have become an ideal means of evaluating drug metabolism in the body and for clinical screening and early diagnosis.

[0004] Luo Liya et al. from Chengdu University of Traditional Chinese Medicine developed a UPLC-MS / MS method to study the tissue distribution of lenalidomide in mice[1]. Muzaffar Iqbal et al. developed a tandem mass spectrometry method to determine lenalidomide in rabbit and human plasma[2]. However, both methods used animal or human plasma and tissues to detect lenalidomide, but did not detect and identify lenalidomide metabolites in human urine.

[0005] Among patients treated with lenalidomide, a small number experience worsening of their disease, while others remain stable or improve. Therefore, if we can identify drug-resistant patients before their symptoms worsen and offer them alternative treatment options, we can avoid worsening of symptoms and costly, unnecessary treatment.

[0006] NgYLD et al. reported that bone marrow samples were collected from 5 patients before treatment and at the time of relapse. They performed proteomic and phosphorylation proteomic analysis and RNA sequencing based on tandem mass spectrometry (TMT) technology and confirmed that CDK6 is a major regulatory factor in the treatment of refractory relapsed multiple myeloma[3]. However, since bone marrow samples were used, sampling was invasive and difficult. The Department of Hematology of the Tenth People's Hospital Affiliated to Tongji University reported that CRBN gene mutations can lead to lenalidomide resistance[4]. However, identification requires genetic testing, which is expensive and complicated.

[0007] Yael C. Cohen et al. from the Sackler School of Medicine at Tel Aviv University reported using single-cell sequencing of patient blood or bone marrow to identify drug resistance pathways and therapeutic targets in patients with relapsed multiple myeloma [5]. However, the use of patient blood or bone marrow is invasive and complex to operate, and currently only a few laboratories can perform single-cell genomic analysis.

[0008] Prior art literature

[0009] [1] Luo Liya, Deng Xing, Gou Liping, et al. Study on the tissue distribution of lenalidomide in mice by UPLC-MS / MS[J]. Chinese Journal of Clinical Pharmacology, 2019, 35(14):5.

[0010] [2]Iqbal M, Wani TA, Nasr Y Khalil…Development and validation of ultra-performance liquid chromatographic method with tandem mass spectrometry for determination of lenalidomide in rabbit and human plasma[J]. ChemistryCentral Journal, 2013, 7(1):7.

[0011] [3] Li Xin, Guo Caihong, Huang Zhongxia. Application of lenalidomide in the treatment of multiple myeloma[J]. Journal of Clinical Drug Therapy, 2013.

[0012] [4] Hu Ke, Tao Yi, Shi Jumei. Precision medicine for multiple myeloma: exploration of drug resistance mechanisms and related biological markers[J]. Shanghai Medical Journal, 2021, 44(6):5.

[0013] [5]Zada, M., Wang, SY, Bornstein, C., David, E., Moshe, A., Li, B.,... & Amit, I. Identification of resistance pathways and therapeutic targets in relapsed multiple myeloma patients through single-cell sequencing. Nature Medicine. Summary of the Invention

[0014] Problems to be solved by the invention:

[0015] The present invention establishes a rapid and sensitive analysis method for 11 lenalidomide metabolites in human urine; the metabolites are identified by mass spectrometry; the differences in metabolites between multiple myeloma patients in remission (EG), with no change in symptoms (SG), and with worsening symptoms (IG) are compared, and from the perspective of metabolite analysis, the use of urinary metabolites to identify lenalidomide resistance is studied.

[0016] The present invention solves the problem that there is no rapid and sensitive analysis method for lenalidomide metabolites in urine and no mass spectrometry identification of lenalidomide metabolites in urine, thereby providing the possibility of analyzing the metabolism of lenalidomide in the body and the pathological and physiological conditions of the body; it solves the problem that there is no metabolite in urine as a drug resistance marker, thereby providing the possibility of early and rapid screening of lenalidomide-resistant patients, avoiding worsening of patients' symptoms and unnecessary treatment.

[0017] To achieve the above object, the present invention adopts the following technical solutions:

[0018] The first aspect of the present invention is to provide a method for analyzing lenalidomide metabolites in human urine, comprising the following steps:

[0019] (1) Sample

[0020] Urine samples from multiple myeloma patients not treated with lenalidomide served as blank samples (BS), while urine samples from multiple myeloma patients treated with lenalidomide served as analytical samples. The analytical samples were divided into three groups: those from patients in remission (EG), those with no change in symptoms (SG), and those with worsening symptoms (IG).

[0021] (2) Preparation of test solution and reference solution

[0022] Test solution: Each 500-1000 μL urine sample was dried in a freeze dryer and then dissolved in 1-5 mL of a mixture of methanol, acetonitrile, and water (1:3:5). The solution was stored at -20 to -40°C and filtered through a 0.22 μm filter membrane before analysis.

[0023] Reference solution: Accurately weigh lenalidomide, dissolve it in methanol, and dilute it to a solution containing 0.1-10 μg per 1 ml.

[0024] (3) UPLC-MS / MS analysis of the test solution and the reference solution

[0025] UPLC conditions: column temperature 40°C; mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile; injection volume 2 μl; flow rate 0.1-0.5 mL / min; gradient elution, the gradient elution conditions are as follows:

[0026] 0 min 5% B; 5 min 95% B

[0027] Mass spectrometry conditions: atmospheric pressure chemical ionization or electrospray ionization; mass analyzer: linear or three-dimensional ion hydrazine mass spectrometry, quadrupole time-of-flight mass spectrometry, electrostatic field orbital ion hydrazine mass spectrometry, or triple quadrupole mass spectrometry; primary and secondary mass spectra were collected in positive ion scanning mode with an acquisition range of m / z 50-2000, and a secondary mass spectrometry collision energy of 10-70V.

[0028] Furthermore, the analysis method comprises the following steps:

[0029] Test solution: Each 600 μL urine sample was dried in a freeze dryer and then dissolved in 2 ml of a mixture of methanol, acetonitrile, and water (1:3:5). The solution was stored at -20°C and filtered through a 0.22 μm filter membrane before analysis.

[0030] Reference solution: Lenalidomide was accurately weighed, dissolved in methanol, and diluted to a solution containing 0.5 μg per 1 ml.

[0031] UPLC conditions: column temperature 40°C; mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile; injection volume 2 μl; flow rate 0.35 mL / min; gradient elution, the gradient elution conditions are as follows:

[0032] 0 min 5% B; 5 min 95% B

[0033] Mass spectrometry conditions: electrospray ionization; quadrupole time-of-flight mass spectrometer; positive ion scan mode for collecting primary and secondary mass spectra, acquisition range m / z 100-1500, secondary mass spectrometry collision energy 20-60V.

[0034] Furthermore, the temperature of freeze-drying urine is -50 to -80°C.

[0035] Furthermore, the temperature of freeze-drying urine is -60 to -70°C.

[0036] The second aspect of the present invention is the analysis of 11 lenalidomide metabolites in urine.

[0037] We used ADME predictors to predict the class I and class II metabolites of lenalidomide. PeakView 1.2 software was used to process mass data based on MS and MS / MS fragment ions. Based on this extensive data set, we analyzed the metabolites of lenalidomide in each sample. The structures of each metabolite were analyzed within a reasonable error range (an error of less than 10 ppm between the measured and theoretical molecular weights is considered reasonable), and differences between samples were compared.

[0038] ADME prediction showed 14 metabolites, including class I and class II metabolites of lenalidomide. Metabolism assays detected a total of 192 binomial metabolites, including 79 metabolites in EG, 95 metabolites in SG, and 94 metabolites in IG. Combining the prediction results with metabolomics assay results, 11 metabolites of lenalidomide were characterized by UPLC-Q-TOF-MS.

[0039] The base peak chromatogram (BPC) of lenalidomide and BS, EG, SG, and IG is as follows: Figure 1 As shown in the results, the retention time of lenalidomide was 2.947 min, and there was no significant difference in the BPC values of BS, EG, SG, and IG. Therefore, we analyzed the metabolites for further study.

[0040] ADME prediction shows that lenalidomide has 14 class I and class II metabolites. Among them, C1-C7 are class I metabolites of lenalidomide catalyzed by CYP3A4. C11 is a class II metabolite of lenalidomide, metabolized by UGT1A6, UGT1A9 and UGT2B7. C8, C9, C10, C12, C13 and C14 are metabolites of C5 catalyzed by CYP3A4 (e.g. Figure 2 As shown), Figure 2 As shown in A, the first-level metabolites of lenalidomide are all hydroxylated lenalidomide metabolized by CYP3A4, which has a narrow range and cannot fully predict other metabolites metabolized by CYP450S. Figure 2 As shown in Figure B, C8, C9, C13, and C14, hydroxylated C5s, are the first-level metabolites of C5 catalyzed by CYP3A4, and C10 is the dehydrogenated C8. In addition, it also includes other types of metabolites, such as C12. These results preliminarily indicate that the main metabolites of lenalidomide can still be further metabolized.

[0041] According to BPC data, the retention time of lenalidomide is 2.947 min. A secondary mass spectrometry analysis of lenalidomide was performed, and the mass-to-charge ratio (m / z) of its characteristic ion was 260.1040 [M+H] + , with a mass-to-charge ratio (m / z) of 215.1079 [M+H-CO-NH3] + , with a mass-to-charge ratio (m / z) of 187.0868 [M+H-2CO-NH3] + , with a mass-to-charge ratio (m / z) of 149.0712 [M+H-2CO-NH3-C3H2] + and [M+H-2CO-NH3-C3H2-CONH] with a mass-to-charge ratio (m / z) of 106.0651 + The difference between the theoretical molecular weight and the actual measured value is reasonable. The structural formula of lenalidomide is shown in Figure 3 The mass spectra / mass spectra of 11 metabolites are as follows. Figure 4 As shown, the structures of 11 metabolites were analyzed as follows:

[0042] Lenalidomide metabolite M1, by [M+H] + The mass-to-charge ratio (m / z) of the ion is 245.0926, and its molecular formula is deduced to be C 13 H 12 N2O3, whose characteristic ion is [M+H] with a mass-to-charge ratio (m / z) of 245.0926 + , with a mass-to-charge ratio (m / z) of 172.0746 [M+H-C2NH2O2] + , the structure is C4-desamino lenalidomide, named phthalimide aminoglutarimide / isomer, which is a polyketide compound with the structural formula shown in M1:

[0043]

[0044] Lenalidomide metabolite M2, by [M+H] + The mass-to-charge ratio (m / z) of the ion is 245.0926, and its molecular formula is deduced to be C 13 H 12 The fragment ion of N2O3, M2 is [M+H] with a mass-to-charge ratio (m / z) of 245.0926. + , with a mass-to-charge ratio (m / z) of 170.0605 [M+H-C2NO2H5] + , with a mass-to-charge ratio (m / z) of 142.0652 [M+H-C3NO3H5] + , the structure is C1′-deamino lenalidomide, which was imported into the SCI-Finder website and found to be a potential new compound named denitrosated-2-phthalimide aminoglutarimide, belonging to the polyketone class, with the structural formula shown in M2:

[0045]

[0046] Lenalidomide metabolite M3, by [M+H] + The mass-to-charge ratio (m / z) of the ion is 149.0702, and its molecular formula is deduced to be C8H8N2O. The characteristic ion of M3 is [M+H] with a mass-to-charge ratio (m / z) of 149.0698. + , with a mass-to-charge ratio (m / z) of 106.0649 [M+H-CNOH] + , the structure is C2-C3' hydrogenated, deglutarimide lenalidomide, named 4-aminoisoindolin-1-one, the structural formula is shown in M3:

[0047]

[0048]

[0049] Lenalidomide metabolite M4, by [M+H] + The mass-to-charge ratio (m / z) of the ion is 134.0595, and its molecular formula is deduced to be C8H7NO. The main MS ion produced by M4 is [M+H] with a mass-to-charge ratio (m / z) of 134.0585. + , mass-to-charge ratio (m / z) of 116.0503 [M+H-H2O] + , with a mass-to-charge ratio (m / z) of 106.0665 [M+H-CO] + , with a mass-to-charge ratio (m / z) of 79.0550 [M+H-CO-CNH2] + , the structure is C4-deamino M3, named 1-dihydroisopentanediol / isomer, the structural formula is shown in M4:

[0050]

[0051] Lenalidomide metabolite M5, whose molecular formula is C8H7NO, is deduced from the mass-to-charge ratio (m / z) of [M+H]+ ion of 134.0594. The characteristic ion of M5 is [M+H]+ with a mass-to-charge ratio (m / z) of 134.0585. + , mass-to-charge ratio (m / z) of 116.0503 [M+H-H2O] + , with a mass-to-charge ratio (m / z) of 106.0665 [M+H-CO] + , with a mass-to-charge ratio (m / z) of 79.0550 [M+H-CO-CNH2] + , which is C2-deaminated M3, is an isomer of M4. Like M4, it is an alkaloid compound, and its structural formula is shown in M5:

[0052]

[0053] Lenalidomide metabolite M6, by [M+H] + The mass-to-charge ratio (m / z) of the ion is 114.0563, and its molecular formula is deduced to be C5H7NO2. The characteristic ion of M6 is [M+H] with a mass-to-charge ratio (m / z) of 114.0563. + , mass-to-charge ratio (m / z) 72.9390 [M+H-CNO] + The structure is C2-C3' hydrogenated, glutarimide-retained lenalidomide, named glutarimide / isomer, and the structural formula is shown in M6:

[0054]

[0055] Lenalidomide metabolite M7, by [M+H]+ The mass-to-charge ratio (m / z) of the ion is 161.0956, and its molecular formula is deduced to be C 10 H 12 The characteristic ions of N2 and M7 are [M+H] with a mass-to-charge ratio (m / z) of 161.0956. + , mass-to-charge ratio (m / z) 146.0692 [M+H-NH] + The structure of M7, C2-C8 de-CO and C3'-C5'de-C2HO2N, was imported into the SCI-Finder website, and it was found to be a potential new compound, named cyclopropane-deoxy 4-aminoisoindoline-1-one-1. It is an amino-modified hydrocarbon compound with the structural formula shown in M7:

[0056]

[0057] Lenalidomide metabolite M8, by [M+H] + The mass-to-charge ratio (m / z) of the ion is 149.0916, and its molecular formula is deduced to be C 10 H 11 The characteristic ion of N, M8 is [M+H] with a mass-to-charge ratio (m / z) of 146.0916. + , mass-to-charge ratio (m / z) 104.0703 [M+H-C3H6] + , the structure of M8 C2-C8 de-CO, C4-deoxy and C3'-C5'de-C2HO2N lenalidomide was imported into the SCI-Finder website, and it was found to be a potential new compound, named cyclopropane-C4 deoxy-aminoisoindolin-1-one-2, with the structural formula shown in M8:

[0058]

[0059] Lenalidomide metabolite M9, by [M+H] + The mass-to-charge ratio (m / z) of the ion is 278.1131, so its molecular formula is C 13 The fragment ion of H5N3O4, M9 is [M+H] with a mass-to-charge ratio (m / z) of 278.1131. + , mass-to-charge ratio (m / z) 261.0855 [M+H-NH3] + , mass-to-charge ratio (m / z) 233.0933 [M+H-NH3-CO] + , mass-to-charge ratio (m / z) 187.0856 [M+H-NH3-CO-CO2H2] +The structure of lenalidomide is hydrogenated at N1' and hydroxylated at C6', and is named 4-amino-γ-(aminocarbonyl)-1,3-dihydro-1-oxo-2H-isoindole-2-butyric acid. It belongs to the alkaloid class, and its structural formula is shown in M9:

[0060]

[0061] Lenalidomide metabolite M10, by [M+H] + The mass-to-charge ratio (m / z) of the ion is 297.1076, and its molecular formula is deduced to be C 13 The fragment ion of H6N2O6, M10 is [M+H] with a mass-to-charge ratio (m / z) of 297.1076. + , mass-to-charge ratio (m / z) 281.0522 [M+H-NH2] + The structure of M10 (N1' deamination, C2' hydroxylation, C6' hydroxylation, N2 hydrogenation, C1 hydroxylation of lenalidomide) was imported into the SCI-Finder website, and it was found to be a potential new compound named denitro-hydroxy 4-amino-γ-(aminocarbonyl)-1,3-2-hydrogen-1-oxo-2H-isoindole-2-butyric acid, with the structural formula shown in M10:

[0062]

[0063]

[0064] Lenalidomide metabolite M11, by [M+H] + The mass-to-charge ratio (m / z) of the ion is 276.0972, and its molecular formula is deduced to be C 13 H 13 The fragment ion of N3O4, M11 is [M+H] with a mass-to-charge ratio (m / z) of 276.0972. + , mass-to-charge ratio (m / z) 203.0790 [M+H-C2H3NO2] + , mass-to-charge ratio (m / z) 165.0660 [M+H-C5H5NO2] + , mass-to-charge ratio (m / z) 148.0381 [M+H-C5H5NO2-NH3] + , mass-to-charge ratio (m / z) 122.0578 [M+H-C5H5NO2-CONH] + , the structure is 5-hydroxy-lenalidomide, which belongs to the alkaloid class, and the structural formula is shown in M11:

[0065]

[0066] The structures, molecular formulas, and molecular weights of the 11 metabolites of lenalidomide in human urine are shown in Figure 5; 11 metabolites are shown in Table 1.

[0067]

[0068]

[0069] The third aspect of the present invention is to analyze the correlation between 11 metabolites and lenalidomide resistance. Metabolites M1-M11 appear in the samples listed in Table 2 respectively.

[0070] Table 2

[0071] metabolites Existing samples M1 All samples (EG, SG, IG) M2 SG, IG M3 Almost all samples (EG, SG, IG) M4 All samples (EG, SG, IG) M5 All samples (EG, SG, IG) M6 All samples (EG, SG, IG) M7 All samples (EG, SG, IG) M8 All samples (EG, SG, IG) M9 All samples (EG, SG, IG) M10 All samples (EG, SG, IG) M11 EG, IG

[0072] Statistical analysis of 11 metabolites revealed that M2 and M11 were key compounds distinguishing each sample. M2 was only detected in SG and IG, and M11 was only detected in EG and IG. We then selected four additional samples and performed LC / MS analysis without knowing their clinical effects. The final results showed that M2 was detectable in all four samples, while M11 showed no pattern in distribution. Based on these test results, we inferred that 17-20 patients should be classified as IG or SG. After several days of observation, the clinical parameters of these four patients remained relatively stable, indicating that M2 can serve as a diagnostic marker for identifying patients with multiple myeloma who are resistant to lenalidomide treatment.

[0073] Based on the above technical solution, the present invention has the following beneficial effects:

[0074] The present invention established a rapid and sensitive UPLC-MS / MS qualitative analysis method for lenalidomide metabolites, and identified a total of 11 metabolites, including 4 potential new compounds, thus providing the possibility of analyzing the metabolism of lenalidomide in the body and the pathological and physiological conditions of the body. In addition, it was found that metabolite M2 can be used as a diagnostic marker for identifying lenalidomide resistance in patients with multiple myeloma. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 Base peak chromatograms of lenalidomide and BS, EG, SG, and IG;

[0076] Figure 2 Prediction of phase I and phase II metabolites of lenalidomide using ADME predictors;

[0077] Figure 3 Structural formula of lenalidomide;

[0078] Figure 4 Mass spectra / mass spectra of 11 metabolites M1-M11;

[0079] Figure 5 Structural formula, molecular formula and molecular weight of 11 metabolites. DETAILED DESCRIPTION

[0080] All materials, reagents, etc. in the following examples, unless otherwise specified, can be obtained from commercial sources. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0081] Instruments and reagents

[0082] UPLC-TOF-MS / MS (SCIEX triple TOF 6600+ mass spectrometry system equipped with SCIEX SCIEX LC AD system);

[0083] Chromatographic column: CSH (C18 1.7 μm, 2.1*50 mm; water);

[0084] Pre-column: AC quality BEH Shield RP-18 1.7 μm precolumn;

[0085] Lenalidomide with a purity of more than 98% was used as a reference substance for qualitative analysis;

[0086] LC-MS grade acetonitrile, methanol, and formic acid were (Thermo Fisher Scientific, TMO, USA); water was prepared by a Millipore Alpha-Q water purification system (Bedford, MA, USA).

[0087] Example 1

[0088] This example provides a method for analyzing lenalidomide metabolites in human urine, comprising the following steps:

[0089] 1. Analytical Samples

[0090] Urine samples from multiple myeloma patients not treated with lenalidomide served as blank samples (BS), while urine samples from multiple myeloma patients treated with lenalidomide served as analytical samples. The analytical samples were divided into three groups: those from patients in remission (EG), those with no change in symptoms (SG), and those with worsening symptoms (IG).

[0091] 2. Preparation of test solution and reference solution

[0092] Test solution: Each 600 μL urine sample was freeze-dried at -60 to -70°C and dissolved in 2 mL of a mixture of methanol, acetonitrile, and water (1:3:5). The solution was stored at -20°C and filtered through a 0.22 μm filter membrane before analysis.

[0093] Reference solution: Accurately weigh lenalidomide, dissolve it in methanol, and dilute it to a solution containing 0.5 μg per 1 ml.

[0094] 3. Analyze the test solution and reference solution by UPLC-MS / MS

[0095] UPLC conditions: column temperature 40°C; mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile; injection volume 2 μl; flow rate 0.1-0.5 mL / min; gradient elution, the gradient elution conditions are as follows:

[0096] 1.0min 5% B; 5min 95% B

[0097] Mass spectrometry conditions: electrospray ionization; quadrupole time-of-flight mass spectrometer; positive ion scan mode for collecting primary and secondary mass spectra, acquisition range m / z 100-1500, secondary mass spectrometry collision energy 20-60V.

[0098] IV. Results

[0099] The retention times are shown in Table 1; the base peak chromatograms (BPC) of lenalidomide and its metabolites BS, EG, SG, and IG are shown in Figure 1 As shown; the spectra of 11 lenalidomide metabolites in urine are as follows Figure 4 shown.

[0100] Example 2

[0101] The structures of M1-M11 were elucidated by UPLC-MS / MS.

[0102] Lenalidomide metabolite M1, [M+H] + The mass-to-charge ratio (m / z) of the ion is 245.0926, which is 15 less than the molecular weight of lenalidomide. It is speculated that M1 is the product after lenalidomide loses NH. The molecular formula of M1 is deduced to be C 13 H 12 N2O3, similarly, the mass-to-charge ratio (m / z) of the M1 characteristic ion is 245.0926, which is [M+H] + , mass-to-charge ratio (m / z) 172.0746, [M+H-C2NH2O2] + , the M1 structure was deduced to be C4-desamino lenalidomide.

[0103] Lenalidomide metabolite M2, [M+H] + The mass-to-charge ratio (m / z) of the ion is 245.0926, which is 15 less than the molecular weight of lenalidomide. It is speculated that M2 is the product after lenalidomide loses NH. The molecular formula of M2 is deduced to be C 13 H 12N2O3, similarly, the fragment ion mass-to-charge ratio (m / z) of M2 is 245.0926, which is [M+H] + , with a mass-to-charge ratio (m / z) of 170.0605, which is [M+H-C2NO2H5] + , with a mass-to-charge ratio (m / z) of 142.0652, which is [M+H-C3NO3H5] + , the M2 structure was deduced to be C1′-desamino lenalidomide.

[0104] Lenalidomide metabolite M3, [M+H] + The mass-to-charge ratio (m / z) of the ion is 149.0702, which is 111 less than the molecular weight of lenalidomide. It is speculated that M3 is the product of lenalidomide losing C5H5NO2. The molecular formula of M3 is deduced to be C8H8N2O. Similarly, the characteristic ion mass-to-charge ratio (m / z) of M3 is 149.0698, which is [M+H] + , with a mass-to-charge ratio (m / z) of 106.0649, [M+H-CNOH] + , the M3 structure was deduced to be C2-C3' hydrogenated, deglutarimidate lenalidomide, and named 4-aminoisoindolin-1-one.

[0105] Lenalidomide metabolite M4, [M+H] + The mass-to-charge ratio (m / z) of the ion is 134.0595, which is 126 less than the molecular weight of lenalidomide. It is speculated that M4 is the product of lenalidomide losing C5H6N2O2. The molecular formula of M4 is deduced to be C8H7NO. Similarly, the main MS ion produced by M4 has a mass-to-charge ratio (m / z) of 134.0585, which is [M+H] + , mass-to-charge ratio (m / z) 116.0503, [M+H-H2O] + , with a mass-to-charge ratio (m / z) of 106.0665, [M+H-CO] + , with a mass-to-charge ratio (m / z) of 79.0550, [M+H-CO-CNH2] + , the structure of M4 was deduced to be C4-deamino M3, named 1-dihydroisopentanediol / isomer.

[0106] Lenalidomide metabolite M5, [M+H] + The mass-to-charge ratio (m / z) of the ion is 134.0594, which is 126 less than the molecular weight of lenalidomide. It is speculated that M5 is the product of lenalidomide losing C5H6N2O2. M5 is an isomer of M4, and its molecular formula is deduced to be C8H7NO. Similarly, the characteristic ion mass-to-charge ratio (m / z) of M5 is 134.0585, which is [M+H] + , mass-to-charge ratio (m / z) 116.0503, [M+H-H2O] +, with a mass-to-charge ratio (m / z) of 106.0665, [M+H-CO] + , with a mass-to-charge ratio (m / z) of 79.0550, [M+H-CO-CNH2] + , deducing that the structure of M5 is C2-deaminated M3.

[0107] Lenalidomide metabolite M6, [M+H] + The mass-to-charge ratio (m / z) of the ion is 114.0563, which is 146 less than the molecular weight of lenalidomide. It is speculated that M6 is the product of lenalidomide losing C8H6N2O. The molecular formula of M6 is deduced to be C5H7NO2. Similarly, the characteristic ion mass-to-charge ratio (m / z) of M6 is 114.0563, which is [M+H] + , with a mass-to-charge ratio (m / z) of 72.9390, [M+H-CNO] + , the M6 structure was deduced to be lenalidomide with C2-C3' hydrogenation and glutarimide retention, and was named glutarimide / isomer.

[0108] Lenalidomide metabolite M7, [M+H] + The mass-to-charge ratio (m / z) of the ion is 161.0956, which is 99 less than the molecular weight of lenalidomide. It is speculated that M7 is the product of lenalidomide losing C3HNO3. The molecular formula of M7 is deduced to be C 10 H 12 N2, similarly, the characteristic ion mass-to-charge ratio (m / z) of M7 is 161.0956, which is [M+H] + , with a mass-to-charge ratio (m / z) of 146.0692, [M+H-NH] + , the structure of M7 was deduced to be C2-C8 de-CO and C3'-C5'de-C2HO2N lenalidomide.

[0109] Lenalidomide metabolite M8, [M+H] + The mass-to-charge ratio (m / z) of the ion is 149.0916, which is 114 less than the molecular weight of lenalidomide. It is speculated that M8 is the product of lenalidomide losing C3H2N2O3. The molecular formula of M8 is deduced to be C 10 H 11 N, similarly, the characteristic ion mass-to-charge ratio (m / z) of M8 is 146.0916, which is [M+H] + , with a mass-to-charge ratio (m / z) of 104.0703, [M+H-C3H6] + , the structure of M8 was deduced to be C2-C8 de-CO, C4-deoxy and C3'-C5'de-C2HO2N lenalidomide.

[0110] Lenalidomide metabolite M9, [M+H] +The mass-to-charge ratio (m / z) of the ion is 278.1131, which is 18 more than the molecular weight of lenalidomide. It is speculated that M9 is the product of lenalidomide after adding one H2O. The molecular formula of M9 is deduced to be C 13 H5N3O4, similarly, the fragment ion mass-to-charge ratio (m / z) of M9 is 278.1131, which is [M+H] + , mass-to-charge ratio (m / z) 261.0855, [M+H-NH3] + , with a mass-to-charge ratio (m / z) of 233.0933, [M+H-NH3-CO] + , with a mass-to-charge ratio (m / z) of 187.0856, which is [M+H-NH3-CO-CO2H2] + , the structure of M9 was deduced to be N1' hydrogenated and C6' hydroxylated lenalidomide, and was named 4-amino-γ-(aminocarbonyl)-1,3-2hydro-1-oxo-2H-isoindole-2-butyric acid.

[0111] Lenalidomide metabolite M10, [M+H] + The mass-to-charge ratio (m / z) of the ion is 297.1076, which is 37 more than the molecular weight of lenalidomide. It is speculated that M10 is the product of lenalidomide adding 3 0H and losing 1 N. The molecular formula of M10 is deduced to be C 13 H6N2O6, similarly, the fragment ion mass-to-charge ratio (m / z) of M10 is 297.1076, which is [M+H] + , mass-to-charge ratio (m / z) 281.0522, [M+H-NH2] + , the structure of M10 was deduced as N1' deamination, C2' hydroxylation, C6' hydroxylation, N2 hydrogenation, and C1 hydroxylation of lenalidomide.

[0112] Lenalidomide metabolite M11, [M+H] + The mass-to-charge ratio (m / z) of the ion is 276.0972, which is 16 more than the molecular weight of lenalidomide. It is speculated that M11 is the product of one H of lenalidomide replaced by 0H, and its molecular formula is deduced to be C 13 H 13 N3O4, similarly, the fragment ion mass-to-charge ratio (m / z) of M11 is 276.0972, which is [M+H] + , with a mass-to-charge ratio (m / z) of 203.0790, which is [M+H-C2H3NO2] + , with a mass-to-charge ratio (m / z) of 165.0660, which is [M+H-C5H5NO2] + , with a mass-to-charge ratio (m / z) of 148.0381, which is [M+H-C5H5NO2-NH3] + , with a mass-to-charge ratio (m / z) of 122.0578, which is [M+H-C5H5NO2-CONH]+ , the structure of M11 was deduced to be 5-hydroxy-lenalidomide.

[0113] Example 3

[0114] Two differentially expressed metabolites in urine were screened and subjected to targeted bipolar analysis. Based on p-values less than 0.05, fold-changes greater than 15, and VIP greater than 1, the two metabolite structures with the highest AUCs were identified: M2 and M11 (see Table 3). M2 demonstrated good predictive effectiveness for lenalidomide resistance prediction, with sensitivity and specificity shown in Table 4.

[0115] Table 3. Results of identifying lenalidomide resistance using two metabolites, M2 and M11

[0116]

[0117] Table 4. Sensitivity and specificity of M2 for identifying lenalidomide resistance

[0118]

[0119] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for analyzing lenalidomide metabolites in human urine, characterized in that: The method comprises the following steps: (1) Preparation of test solution and reference solution: The test solution was prepared as follows: 500-1000 μL of urine sample was dried in a freeze dryer and dissolved in 1-5 mL of a solvent, wherein the solvent was a mixture of methanol, acetonitrile, and water in a volume ratio of 1:3:

5. The solution was stored at -20 to -40°C and filtered through a 0.22 μm filter membrane before analysis. The reference solution is prepared by accurately weighing lenalidomide, dissolving it in methanol, and diluting it into a solution containing 0.1 to 10 μg per 1 ml; (2) Analyze the test solution and reference solution by UPLC-MS / MS: The UPLC conditions were as follows: column temperature of 40° C.; mobile phase A of 0.1% formic acid in water; mobile phase B of 0.1% formic acid in acetonitrile; injection volume of 2 μl; flow rate of 0.1 to 0.5 mL / min; gradient elution, wherein the gradient elution conditions were as follows: 0 min 5% B; 5 min 95% B; Mass spectrometry conditions include atmospheric pressure chemical ionization or electrospray ionization; the mass analyzer can be a linear or three-dimensional ion hydrazine mass spectrometer, a quadrupole time-of-flight mass spectrometer, an electrostatic field orbital ion hydrazine mass spectrometer, or a triple quadrupole mass spectrometer; primary and secondary mass spectra are acquired in positive ion scan mode over the m / z range of 50-2000, and the secondary mass spectrometry collision energy is 10-70 V; The lenalidomide metabolites in human urine include M2 and M11; The structural formula of the lenalidomide metabolite M2 in human urine is shown as M2: M2's [M+H] + The mass-to-charge ratio of the ion is 245.0926, and the molecular formula is C 13 H 12 N2O3, for C1′-desamino lenalidomide; The structural formula of the lenalidomide metabolite M11 in human urine is shown as M11: M11's [M+H] + The mass-to-charge ratio of the ion is 276.0972, and the molecular formula is C 13 H 13 N3O4, is 5-hydroxy-lenalidomide.

2. The analysis method according to claim 1, characterized in that The steps include: Test solution: Each 600 μL urine sample was dried in a freeze dryer and then dissolved in 2 mL of a solvent consisting of a mixture of methanol, acetonitrile, and water in a volume ratio of 1:3:

5. The solution was stored at -20°C and filtered through a 0.22 μm filter membrane before analysis. Reference solution: Accurately weigh lenalidomide, dissolve in methanol, and dilute to a solution containing 0.5 μg per 1 ml; The UPLC conditions were as follows: column temperature of 40° C.; mobile phase A of 0.1% formic acid in water; mobile phase B of 0.1% formic acid in acetonitrile; injection volume of 2 μl; flow rate of 0.35 mL / min; gradient elution, wherein the gradient elution conditions were as follows: 0 min 5% B; 5 min 95% B; The mass spectrometry conditions were electrospray ionization; the mass analyzer was a quadrupole time-of-flight mass spectrometer; the primary and secondary mass spectra were acquired in positive ion scanning mode with an acquisition range of m / z 100-1500, and the secondary mass spectrometry collision energy was 20-60V.

3. The analysis method according to claim 2, characterized in that The freeze-drying temperature is -50 to -80°C.

4. The analysis method according to claim 2, characterized in that The freeze-drying temperature is -60 to -70°C.

5. Lenalidomide metabolites in human urine include M1 to M11, characterized in that: Identified by the analytical method according to any one of claims 1 to 4, The structural formula of the lenalidomide metabolite M1 in human urine is shown as M1: M1's [M+H] + The mass-to-charge ratio of the ion is 245.0926, and the molecular formula is C 13 H 12 N2O3, for C4-desamino lenalidomide, The structural formula of the lenalidomide metabolite M2 in human urine is shown as M2: M2's [M+H] + The mass-to-charge ratio of the ion is 245.0926, and the molecular formula is C 13 H 12 N2O3, for C1′-desamino lenalidomide, The structural formula of the lenalidomide metabolite M3 in human urine is shown as M3: M3's [M+H] + The mass-to-charge ratio of the ion is 149.0702, the molecular formula is C8H8N2O, which is C2-C3' hydrogenated, deglutarimidate lenalidomide, The structural formula of the lenalidomide metabolite M4 in human urine is shown as M4: M4's [M+H] + The mass-to-charge ratio of the ion is 134.0595, the molecular formula is C8H7NO, and it is C4-deaminated M3. The structural formula of the lenalidomide metabolite M5 in human urine is shown as M5: M5's [M+H] + The mass-to-charge ratio of the ion is 134.0594, the molecular formula is C8H7NO, it is C2-deamino M3, an isomer of M4, The structural formula of the lenalidomide metabolite M6 in human urine is shown as M6: M6's [M+H] + The mass-to-charge ratio of the ion is 114.0563, and the molecular formula is C5H7NO2, which is lenalidomide with C2-C3' hydrogenation and glutarimide retention. The structural formula of the lenalidomide metabolite M7 in human urine is shown as M7: M's [M+H] + The mass-to-charge ratio of the ion is 161.0956 and the molecular formula is C 10 H 12 N2, C2-C8de-CO and C3'-C5'de-C2HO2N lenalidomide, The structural formula of the lenalidomide metabolite M8 in human urine is shown as M8: M8's [M+H] + The mass-to-charge ratio of the ion is 149.0916, and the molecular formula is C 10 H 11 N, is C2-C8de-CO, C4 deoxy and C3'-C5'de-C2HO2N lenalidomide, The structural formula of the lenalidomide metabolite M9 in human urine is shown as M9: M9's [M+H] + The mass-to-charge ratio of the ion is 278.1131 and the molecular formula is C 13 H5N3O4, for N1' hydrogenation, C6' hydroxylation of lenalidomide, The structural formula of the lenalidomide metabolite M10 in human urine is shown as M10: M10's [M+H] + The mass-to-charge ratio of the ion is 297.1076, and the molecular formula is C 13 H6N2O6, for N1' deamination, C2' hydroxylation, C6' hydroxylation, N2 hydrogenation, C1 hydroxylation lenalidomide, The structural formula of the lenalidomide metabolite M11 in human urine is shown as M11: M11's [M+H] + The mass-to-charge ratio of the ion is 276.0972, and the molecular formula is C 13 H 13 N3O4, is 5-hydroxy-lenalidomide.

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