CN110759948B - A kind of platinum-containing compound, its preparation method, separation method and application - Google Patents
A kind of platinum-containing compound, its preparation method, separation method and application Download PDFInfo
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- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 title claims abstract description 141
- 150000001875 compounds Chemical class 0.000 title claims abstract description 74
- 229910052697 platinum Inorganic materials 0.000 title claims abstract description 70
- 238000000926 separation method Methods 0.000 title claims abstract description 10
- 238000002360 preparation method Methods 0.000 title abstract description 18
- 229950007221 nedaplatin Drugs 0.000 claims abstract description 97
- 239000007857 degradation product Substances 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 25
- 239000000126 substance Substances 0.000 claims abstract description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 19
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 17
- 238000010525 oxidative degradation reaction Methods 0.000 claims abstract description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 23
- 238000001514 detection method Methods 0.000 claims description 16
- 238000010828 elution Methods 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 150000003058 platinum compounds Chemical class 0.000 claims description 6
- 238000001704 evaporation Methods 0.000 claims description 5
- 238000002013 hydrophilic interaction chromatography Methods 0.000 claims description 5
- 230000008020 evaporation Effects 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 3
- 238000002390 rotary evaporation Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- GRHLMSBCOPRFNA-UHFFFAOYSA-M azanide 2-oxidoacetate platinum(4+) Chemical compound N[Pt]1(N)OCC(=O)O1 GRHLMSBCOPRFNA-UHFFFAOYSA-M 0.000 claims 14
- 238000002347 injection Methods 0.000 claims 1
- 239000007924 injection Substances 0.000 claims 1
- KLNFSAOEKUDMFA-UHFFFAOYSA-N azanide;2-hydroxyacetic acid;platinum(2+) Chemical compound [NH2-].[NH2-].[Pt+2].OCC(O)=O KLNFSAOEKUDMFA-UHFFFAOYSA-N 0.000 abstract description 82
- 238000011160 research Methods 0.000 abstract description 7
- 150000003839 salts Chemical class 0.000 abstract description 6
- 238000003908 quality control method Methods 0.000 abstract description 4
- 239000013558 reference substance Substances 0.000 abstract description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 16
- 230000014759 maintenance of location Effects 0.000 description 16
- 239000012071 phase Substances 0.000 description 10
- 238000001819 mass spectrum Methods 0.000 description 9
- 238000006731 degradation reaction Methods 0.000 description 8
- 239000012535 impurity Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 238000001228 spectrum Methods 0.000 description 8
- 239000003814 drug Substances 0.000 description 7
- 239000003643 water by type Substances 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000012488 sample solution Substances 0.000 description 4
- BMVXCPBXGZKUPN-UHFFFAOYSA-N 1-hexanamine Chemical compound CCCCCCN BMVXCPBXGZKUPN-UHFFFAOYSA-N 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 2
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical group O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 2
- 125000000129 anionic group Chemical group 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000132 electrospray ionisation Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 230000000155 isotopic effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 2
- 239000007800 oxidant agent Substances 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000004543 DNA replication Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000012494 forced degradation Methods 0.000 description 1
- 238000004896 high resolution mass spectrometry Methods 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- -1 nedaplatin amine Chemical class 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0086—Platinum compounds
- C07F15/0093—Platinum compounds without a metal-carbon linkage
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
本发明公开了一种如式1所示的含铂化合物。所述含铂化合物的制备方法包括以下步骤：将奈达铂与过氧化氢在水中进行氧化降解反应，即可。本发明还提供了一种含有如式1所示的含铂化合物的奈达铂降解产物的分离方法，所述分离方法包括以下步骤：将含有如式1所示的含铂化合物的奈达铂降解产物在色谱柱中洗脱，即可。本发明的如式1所示的含铂化合物在奈达铂或其盐的质量控制中可作为有关物质对照品进行应用，从而为奈达铂有关物质的研究提供了有力的工具。 The invention discloses a platinum-containing compound represented by formula 1. The preparation method of the platinum-containing compound includes the following steps: performing an oxidative degradation reaction with nedaplatin and hydrogen peroxide in water. The present invention also provides a method for separating nedaplatin degradation products containing the platinum-containing compound shown in formula 1, the separation method comprising the steps of: separating the nedaplatin containing the platinum-containing compound shown in formula 1 Degradation products are eluted in the column and that is enough. The platinum-containing compound shown in formula 1 of the present invention can be used as a reference substance of related substances in the quality control of nedaplatin or its salt, thereby providing a powerful tool for the research of related substances of nedaplatin. 
Description
Technical Field
The invention relates to a platinum-containing compound, a preparation method, a separation method and application thereof.
Background
Nedaplatin (Nedaplatin), a broad-spectrum anticancer drug, is a new generation of platinum anticancer drugs with good therapeutic effect and few toxic and side effects. The action mechanism of the compound is similar to that of cisplatin, and the compound is combined with DNA basic groups of tumor cells to block DNA replication so as to exert the anti-tumor effect of the compound. Nedaplatin with the molecular formula C2H8N2O3Pt, having a relative molecular weight of 303.17, and having the following structure:
however, since the development of nedaplatin in the eighties of the last century, the research on related substances is less, and the research on related substances is related to the stability, quality research and medication safety of nedaplatin bulk drugs; therefore, there is a need in the art to study materials related to nedaplatin.
Disclosure of Invention
The invention aims to overcome the defects that the research on related substances related to nedaplatin is less, the preparation and separation methods for related substances are lacked, and the structures of related substances cannot be identified and confirmed in the field, and further provides a platinum-containing compound, and a preparation method, a separation method and application thereof.
The present invention arose from the surprising discovery by researchers that exposure of nedaplatin raw material to air results in the appearance of an unknown substance in an amount that gradually increases with time. In the subsequent forced degradation investigation of the nedaplatin crude drug, researchers have conducted intensive studies on the unknown substance and determined the structure of the unknown substance.
The preparation and separation method can effectively prepare and separate the substances generated in the production and/or storage process of nedaplatin, and can further detect trace impurities with high sensitivity through mass spectrum detection, and identify and confirm the structure of the nedaplatin, thereby controlling the medicine quality of the nedaplatin and laying a good foundation for the research of unknown nedaplatin impurities. The platinum-containing compound can be used for researching the influence of degradation products and degradation ways possibly existing in the medicine on content determination and impurity determination, and investigating the specificity and sensitivity of the detection method. The platinum-containing compounds of the present invention are a necessity for quality control of nedaplatin.
The invention solves the technical problem through the following technical scheme.
The invention provides a platinum-containing compound shown as a formula 1,
the invention provides a preparation method of a platinum-containing compound shown as a formula 1, which comprises the following steps: carrying out oxidative degradation reaction on the nedaplatin and hydrogen peroxide in water.
The preparation method of the platinum-containing compound shown in the formula 1 is characterized in that the initial concentration of the hydrogen peroxide in the water is preferably 0.05-0.15%, more preferably 0.06-0.12%, and the percentage is mass percent.
The preparation method of the platinum-containing compound shown in the formula 1 is characterized in that the time of the oxidative degradation reaction is preferably 0.5h-5h, and more preferably 1-3 h.
The preparation method of the platinum-containing compound shown in formula 1, wherein the initial concentration of nedaplatin in water is preferably 1-5mg/ml, more preferably 2-3mg/ml, for example 2.5 mg/ml.
The preparation method of the platinum-containing compound shown in the formula 1, wherein the temperature of the oxidative degradation reaction is preferably room temperature.
The method for preparing the platinum-containing compound represented by formula 1, wherein the water may be water that is conventional in the art, and is not particularly limited herein, and may be deionized water, for example.
The invention also provides a method for separating the nedaplatin degradation product containing the platinum-containing compound shown in the formula 1, which comprises the following steps: eluting nedaplatin degradation products containing the platinum-containing compound shown as the formula 1 in the invention in a chromatographic column; the chromatographic column is a HILIC chromatographic column; the eluted mobile phase is a mixed solution of water and acetonitrile, and the volume ratio of the water to the acetonitrile is (30-40): (60-70).
The method for separating the nedaplatin degradation product comprises the step of separating the nedaplatin degradation product from the acetonitrile in a volume ratio of 30:70 or 40: 60.
The method for separating nedaplatin degradation products, wherein the nedaplatin degradation products containing the platinum-containing compound represented by formula 1 are preferably in the form of an aqueous solution.
The method for separating the nedaplatin degradation product comprises the step of separating the nedaplatin degradation product containing the platinum-containing compound shown in the formula 1 from the platinum-containing compound, wherein the nedaplatin degradation product can also contain nedaplatin.
The method for separating the nedaplatin degradation product comprises the step of carrying out oxidative degradation reaction on the nedaplatin and hydrogen peroxide in water to obtain the nedaplatin degradation product containing the platinum-containing compound shown in the formula 1.
The method for separating the nedaplatin degradation product comprises the step of separating the nedaplatin degradation product containing the platinum-containing compound shown in the formula 1 from the platinum-containing compound shown in the formula 1, wherein the nedaplatin degradation product containing the platinum-containing compound shown in the formula 1 can be composed of nedaplatin, the platinum-containing compound shown in the formula 1 and water.
The method for separating nedaplatin degradation products comprises the step of separating the nedaplatin degradation products containing the platinum-containing compound shown in the formula 1 to obtain the target product, namely the platinum-containing compound shown in the formula 1.
The separation method of nedaplatin degradation products, wherein the HILIC chromatographic column can be a HILIC chromatographic column conventional in the field, and is preferably TSK GEL AMIDE-HR80 chromatographic column.
The method for separating nedaplatin degradation products, wherein the flow rate of the eluted mobile phase may be a flow rate of a mobile phase conventional in the art, preferably 0.8-1.2ml/min, and more preferably 1 ml/min.
The method for separating nedaplatin degradation products, wherein the detection wavelength of the elution can be the conventional detection wavelength in the art, preferably 210-230nm, more preferably 220 nm.
The method for separating nedaplatin degradation products, wherein the sample amount of elution can be a sample amount conventional in the art, preferably 50-100ul, more preferably 80 ul.
The method for separating the nedaplatin degradation product, wherein the elution further comprises a step of collecting an effluent. The effluent is preferably 9.5-12 min; the effluent liquid is collected and then subjected to a evaporation drying step, preferably rotary evaporation drying, and the substance obtained after evaporation drying is the standard substance containing the platinum compound.
The invention also provides application of the platinum-containing compound shown in the formula 1 as a related substance reference substance in quality control of nedaplatin or salts thereof.
In the present invention, the related substances refer to starting materials, intermediates, polymers, side reaction products and degradation impurities which may be present during the preparation and storage of the drug. The degradation impurity is a substance obtained by degrading nedaplatin or salt thereof or naturally degrading the nedaplatin or the salt thereof; the degradation reaction may be a degradation reaction conventional in the art, such as an oxidative degradation reaction.
In the present invention, the definition of room temperature may be a definition of room temperature which is conventional in the art, and preferably means 10 to 30 ℃.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the preparation and separation method can effectively prepare and separate substances generated in the oxidative degradation process of nedaplatin, and can further detect trace impurities with high sensitivity through mass spectrum detection, identify and confirm the structure of the nedaplatin, so that the quality of the nedaplatin or the salt thereof is controlled, and a good foundation is laid for the research of unknown impurities of nedaplatin; the platinum-containing compound represented by formula 1 of the present invention is a necessity for quality control of nedaplatin or a salt thereof.
Drawings
In fig. 1, a is an HPLC analysis profile of an aqueous solution of nedaplatin raw material after being left at room temperature for 3 days; and B is an HPLC analysis spectrum of the aqueous solution of nedaplatin in A after being placed for another 15 days at room temperature.
FIG. 2 is the mass spectrum of the main component with a retention time of 11.17min in example 1B.
FIG. 3 is a first order mass spectrum of the platinum-containing compound of example 1.
FIG. 4 is a close-up view of the primary mass spectrum of the platinum-containing compound of example 1.
FIG. 5 is a secondary mass spectrum of the platinum-containing compound of example 1.
FIG. 6 shows the preparation of nedaplatin as in example 113And (C) a map.
FIG. 7 shows the platinum-containing compound of example 113And (C) a map.
FIG. 8 is an HPLC chromatogram of the oxidative degradation reaction solution of nedaplatin of example 2.
Fig. 9 is an HPLC profile of the nedaplatin oxidative degradation reaction solution of example 3.
FIG. 10 is an HPLC chromatogram of the oxidative degradation reaction solution of nedaplatin of example 4.
FIG. 11 is an HPLC plot of the platinum-containing compound of example 5.
FIG. 12 is a LC-MS (UV) chart of the platinum-containing compound solution of example 6.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1 production of platinum-containing Compounds as shown in formula 1
This example examined the production of a platinum-containing compound as shown in formula 1 after nedaplatin has been allowed to stand for a period of time.
The instrument used in this example was:
model 2695 high performance liquid chromatography-mass spectrometry system [ equipped with UV detector, ESI source and MassLynx 4.1 data processing system ] (Waters Co.).
The conditions for HPLC analysis in this example were:
the chromatographic column is TSK GEL AMIDE-HR80(5) hydrophilic chromatographic column (4.6X 250mm, 5 μm); mobile phase 0.1% n-hexylamine (formic acid adjusted to ph5.50) -acetonitrile (30: 70); the detection wavelength is 220 nm; the flow rate is 1 ml/min; the column temperature was 30 ℃.
Mass spectrometry conditions: electrospray ionization source, positive ion detection.
The method A comprises the following steps: about 2mg of nedaplatin was precisely weighed out after naturally standing in air at room temperature for 3 days, and prepared into an aqueous solution having a concentration of 1mg/ml for HPLC analysis. The HPLC analysis result is shown in A in FIG. 1.
The method B comprises the following steps: the sample solution in method A was left to stand at room temperature for 15 days and then subjected to LC-MS analysis. The HPLC profile is shown in B in FIG. 1.
As can be seen from a of fig. 1, the peak at retention time 10.75min has a height of 1895227, a peak area of 761851.63, and a peak area ratio of 99.92%, which is the nedaplatin peak. The retention time was 20.32min peak with a peak height of 1120, peak area 625.33, peak area ratio of 0.08%.
As can be seen from B of fig. 1, the peak with retention time of 11.17min has height of 1594726, peak area 619799.44, peak area ratio of 94.98%, which is nedaplatin peak, and the mass spectrum of the peak is shown in fig. 2. FIG. 2 shows [ M + H ]]+303 Peak and [2M + H]+Peak 605, confirming a molecular weight of 302, consistent with the molecular weight of nedaplatin, and the isotopic peaks indicate the presence of platinum in the structure.
In fig. 1B, the peak with retention time of 21.25min has a height of 47937, a peak area of 32777.75 and a peak area ratio of 5.02%, and the mass spectrum and the enlarged partial view of the peak are shown in fig. 3 and fig. 4, respectively. FIGS. 3-4 show [ M + Na ]]+359 Peak, [ M + n-hexylamine + H]+439 Peak and [2M + H]+673 peak, the molecular weight of the compound confirming this retention time is 336, and the isotopic peaks indicate the presence of platinum in the structure. The compound is subjected to MS/MS analysis, and a secondary mass spectrum of the compound is shown in figure 5, so that the cleavage route is shown as follows:
since it generates ions with mass to charge ratios of 251, 227, 209, it was demonstrated that no degradation of the nedaplatin amine ligand moiety occurred, and therefore, it was assumed that the degradation of nedaplatin occurred at the anionic leaving group moiety. The cleavage pathway was verified by high resolution mass spectrometry, and the high resolution data are listed in table 1.
TABLE 1
Further performing nuclear magnetic resonance on nedaplatin crude drug and unknown substance with retention time of 21.25min by nuclear magnetic resonance spectroscopy13C spectrum detection, and nuclear magnetic spectrum is shown in FIGS. 6-7. Of nedaplatin13In the C spectrum, signals with chemical shifts of 67.915ppm and 194.699ppm are respectively attributed to methylene and carbonyl carbons on nedaplatin. In addition, the signals on the carbon spectrum of the unknown substance are similar to nedaplatin, and the chemical shifts are 191.508ppm and 69.523ppm respectively, and are also assigned to methylene and carbonyl carbons. The partial carbon spectrum data of both are shown in Table 2. Accordingly, it was judged that under the oxidative degradation conditions, no oxidation of the anionic group portion occurred.
TABLE 2
Through the above analysis and detection, it can be finally determined that the structure of the unknown substance at the retention time of 21.25min in B of FIG. 1 is
I.e., a platinum-containing compound as described herein.
In addition, it can be inferred from the natural standing conditions and the results of the analysis test that the structure of the compound having a peak with a retention time of 20.32min in a of fig. 1 is also the same as the above structure, i.e., the platinum-containing compound described in the present application.
In fig. 1 a and B, the peaks before the retention time of 5min were both solvent peaks.
Examples 2-4 preparation and isolation of platinum-containing Compounds of formula 1
The reagents used in examples 2-4 were as follows:
acetonitrile was chromatographically pure (j.t.baker); the mobile phase water is commercially available Wahaha purified water (filtered through a 0.22 μm filter membrane), 30% hydrogen peroxide (Hu test). Nedaplatin was prepared according to patent CN 102417522 a.
The apparatus used in examples 2-4 was as follows:
2695 type high performance liquid chromatography-mass spectrometry system [ equipped with UV detector, ESI source and MassLynx 4.1 data processing system ] (Waters corporation);
waters Xevo G2-XS QT of high resolution mass spectrometer (Waters Corp.);
mettler AE-163 electronic balance.
In examples 2-4, the oxidative degradation method of nedaplatin was: about 2.5mg of nedaplatin is precisely weighed, aqueous hydrogen peroxide solution with different concentrations is taken as an oxidant, 1ml of the oxidant is added, and the reaction is carried out at room temperature.
In examples 2-4, the elution conditions of the column after oxidative degradation of nedaplatin were: the chromatographic column is TSK GEL AMIDE-HR80(5) hydrophilic chromatographic column (4.6X 250mm, 5 μm); the mobile phase is water-acetonitrile (volume ratio is 40: 60); the detection wavelength is 220 nm; the flow rate is 1 ml/min; the amount of the sample was 80. mu.l.
In example 4, the effluent with a retention time of 9.5 to 12min was collected and the solvent was removed by rotary evaporation to obtain a standard platinum-containing compound.
In examples 2 to 4, the concentration of the aqueous hydrogen peroxide solution was defined as a mass percentage concentration.
In examples 2-4, the oxidative degradation conditions of nedaplatin and the resulting HPLC profile of the corresponding platinum-containing compound are shown in table 3.
TABLE 3
As can be seen from table 3 and fig. 8-10, the peak at 11.47min in fig. 8 is nedaplatin, and the peak at 22.07min is the platinum-containing compound; in FIG. 9, the peak at 6.67min is nedaplatin and the peak at 9.64min is the platinum-containing compound; whereas nedaplatin degraded almost completely due to the prolonged time of oxidative degradation in fig. 10, thus producing an impurity peak at 3.54min, while the peak-off time of the platinum-containing compound was 9.92 min.
Therefore, the concentration of the aqueous hydrogen peroxide solution, the time of the oxidative degradation reaction and the composition of the mobile phase have a certain influence on the preparation of the platinum-containing compound. The increase in the hydrogen peroxide concentration and the increase in the degradation reaction time contribute to the increase in the content of the platinum-containing compound, and the elution time of the platinum-containing compound is proportional to the proportion of the organic phase.
Example 5 purity analysis and Mass Spectrometry analysis of platinum-containing Compounds
The reagents used in this example were as follows:
acetonitrile was chromatographically pure (j.t.baker); the deuterated methanol is a product of Cambridge Isotrope Laboratories, Inc.; the mobile phase water is commercial Wahaha purified water (filtered by a 0.22 μm filter membrane).
The apparatus used in this example is as follows:
2695 type high performance liquid chromatography-mass spectrometry system [ equipped with UV detector, ESI source and MassLynx 4.1 data processing system ] (Waters corporation);
waters Xevo G2-XS QT of high resolution mass spectrometer (Waters Corp.);
a Varian 500M NMR spectrometer;
mettler AE-163 electronic balance.
The standard platinum-containing compound used in this example was prepared for example 4 of the present invention. The standards were formulated as solutions at a concentration of about 0.5 mg/ml.
The conditions for HPLC analysis in this example were:
the chromatographic column is TSK GEL AMIDE-HR80(5) hydrophilic chromatographic column (4.6X 250mm, 5 μm); the mobile phase is water-acetonitrile (30: 70); the detection wavelength is 220 nm; the flow rate is 1 ml/min; the amount of sample was 20. mu.l.
The standard substance containing platinum compound in this example was analyzed by HPLC, and its spectrum is shown in FIG. 11.
In FIG. 11, the peak height at retention time of 11.57min is 5031, the peak area is 2963.61, and the peak area ratio is 0.27%; the peak height at retention time 23.40min was 444979, peak area 1093513.63, peak area ratio was 99.73%, and in addition, the peak before retention time 5min was the solvent peak. Accordingly, the HPLC purity of the standard containing the platinum compound in this example was 99.73%.
The mass spectrometry adopts electrospray ionization source positive ion detection, and the mass spectrometry spectrum and the result of the platinum-containing compound are the same as those of the embodiment 1.
Example 6 comparative testing of Nedaplatin and platinum-containing Compounds
The instrument used in this example was:
2695 type high performance liquid chromatography-mass spectrometry system [ equipped with UV detector, ESI source and MassLynx 4.1 data processing system ] (Waters corporation);
the concentration of the sample solution of nedaplatin prepared in this example was 1 mg/ml.
The preparation method of the platinum-containing compound solution in this example is as follows: 2ml of the nedaplatin sample solution was dissolved by adding 0.5mg of the standard platinum compound-containing solution prepared in example 4 to the sample solution to obtain a platinum compound-containing solution.
The conditions for HPLC analysis in this example were:
the chromatographic column is TSK GEL AMIDE-HR80(5) hydrophilic chromatographic column (4.6X 250mm, 5 μm); mobile phase 0.1% n-hexylamine (formic acid adjusted to ph5.50) -acetonitrile (30: 70); the detection wavelength is 220 nm; the flow rate is 1 ml/min; the column temperature was 30 ℃.
The HPLC chart of this example is shown in FIG. 12. In fig. 12, the chromatographic peak with retention time of 10.33min is nedaplatin, and the chromatographic peak with retention time of 18.71min is the platinum-containing compound according to the present invention. LC-MS detection results show that nedaplatin can be well separated from the platinum-containing compound.
Claims (6)
1. A method for separating nedaplatin degradation products containing a platinum-containing compound represented by formula 1, comprising the steps of: eluting nedaplatin degradation products containing the platinum-containing compound shown as the formula 1 in a chromatographic column; the chromatographic column is a HILIC chromatographic column; the HILIC chromatographic column is TSK GEL AMIDE-HR80 chromatographic column; the eluted mobile phase is a mixed solution of water and acetonitrile, wherein the volume ratio of the water to the acetonitrile is 30:70 or 40: 60; the nedaplatin degradation product containing the platinum-containing compound shown as the formula 1 is in the form of aqueous solution; the target product obtained by the method for separating the nedaplatin degradation product containing the platinum-containing compound shown in the formula 1 is the platinum-containing compound shown in the formula 1
2. The separation method of claim 1, wherein the nedaplatin degradation product comprising the platinum-containing compound of formula 1 further comprises nedaplatin.
3. The method of isolating nedaplatin degradation products of claim 1,
the nedaplatin degradation product containing the platinum-containing compound shown in the formula 1 is an aqueous solution system obtained by carrying out oxidative degradation reaction on nedaplatin and hydrogen peroxide in water.
4. The method for separating nedaplatin degradation product according to claim 2, wherein the nedaplatin degradation product containing the platinum-containing compound represented by formula 1 comprises nedaplatin, the platinum-containing compound represented by formula 1, and water.
5. The method of isolating nedaplatin degradation products of claim 1, wherein the flow rate of the eluting mobile phase is 0.8 to 1.2 ml/min;
and/or the detection wavelength of the elution is 210-230 nm;
and/or the sample injection amount of the elution is 50-100 ul;
and/or, the elution further comprises the step of collecting the effluent; the effluent is 9.5-12 min; the effluent liquid is collected and then subjected to a step of evaporating to dryness, wherein the evaporation to dryness is rotary evaporation to dryness; and the substance obtained after evaporation to dryness is the standard substance containing the platinum compound shown in the formula 1.
6. The method for separating nedaplatin degradation products of claim 5, wherein the flow rate of the eluting mobile phase is 1 ml/min;
and/or the detection wavelength of the elution is 220 nm;
and/or the sample size of the elution is 80 ul.
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| US4658048A (en) * | 1984-06-20 | 1987-04-14 | Shionogi & Co., Ltd. | Platinum complexes |
| CN101865894A (en) * | 2010-06-28 | 2010-10-20 | 江苏奥赛康药业有限公司 | Method for detecting impurities in freeze-dried powder injection of nedaplatin |
| CN108241032A (en) * | 2016-12-23 | 2018-07-03 | 江苏奥赛康药业股份有限公司 | A kind of analysis method of Nedaplatin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4658048A (en) * | 1984-06-20 | 1987-04-14 | Shionogi & Co., Ltd. | Platinum complexes |
| CN101865894A (en) * | 2010-06-28 | 2010-10-20 | 江苏奥赛康药业有限公司 | Method for detecting impurities in freeze-dried powder injection of nedaplatin |
| CN108241032A (en) * | 2016-12-23 | 2018-07-03 | 江苏奥赛康药业股份有限公司 | A kind of analysis method of Nedaplatin |
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| 奈达铂的HPLC测定法;毕同香等;《中国医药工业杂志》;20001020(第10期);457-458页 * |
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