CN102565203B - Method for separating and determining methyhaaltrexone bromide and impurities thereof with liquid chromatography - Google Patents
Method for separating and determining methyhaaltrexone bromide and impurities thereof with liquid chromatography Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 43
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 title claims abstract description 13
- 238000004811 liquid chromatography Methods 0.000 title claims abstract description 10
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims abstract description 26
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000002360 preparation method Methods 0.000 claims abstract description 18
- 239000007853 buffer solution Substances 0.000 claims abstract description 16
- 238000000926 separation method Methods 0.000 claims abstract description 12
- 239000003960 organic solvent Substances 0.000 claims abstract description 11
- 238000010828 elution Methods 0.000 claims abstract description 9
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 5
- IFGIYSGOEZJNBE-LHJYHSJWSA-N (3s,4r,4as,7ar,12bs)-3-(cyclopropylmethyl)-4a,9-dihydroxy-3-methyl-2,4,5,6,7a,13-hexahydro-1h-4,12-methanobenzofuro[3,2-e]isoquinoline-3-ium-7-one;bromide Chemical compound [Br-].C([N@@+]1(C)[C@@H]2CC=3C4=C(C(=CC=3)O)O[C@@H]3[C@]4([C@@]2(O)CCC3=O)CC1)C1CC1 IFGIYSGOEZJNBE-LHJYHSJWSA-N 0.000 claims description 56
- 229960002834 methylnaltrexone bromide Drugs 0.000 claims description 56
- 239000000243 solution Substances 0.000 claims description 49
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
- -1 morphinan formic acid compound Chemical class 0.000 claims description 24
- 238000012360 testing method Methods 0.000 claims description 24
- 239000012749 thinning agent Substances 0.000 claims description 20
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 238000002347 injection Methods 0.000 claims description 12
- 239000007924 injection Substances 0.000 claims description 12
- 150000001343 alkyl silanes Chemical group 0.000 claims description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 8
- 239000000523 sample Substances 0.000 claims description 8
- 239000000741 silica gel Substances 0.000 claims description 8
- 229910002027 silica gel Inorganic materials 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 7
- 239000000945 filler Substances 0.000 claims description 5
- 235000019253 formic acid Nutrition 0.000 claims description 4
- INAXVFBXDYWQFN-XHSDSOJGSA-N morphinan Chemical compound C1C2=CC=CC=C2[C@]23CCCC[C@H]3[C@@H]1NCC2 INAXVFBXDYWQFN-XHSDSOJGSA-N 0.000 claims description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 2
- 239000000499 gel Substances 0.000 abstract 1
- 238000012856 packing Methods 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 69
- AZQWKYJCGOJGHM-UHFFFAOYSA-N para-benzoquinone Natural products O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 20
- ZXKXJHAOUFHNAS-FVGYRXGTSA-N (S)-fenfluramine hydrochloride Chemical compound [Cl-].CC[NH2+][C@@H](C)CC1=CC=CC(C(F)(F)F)=C1 ZXKXJHAOUFHNAS-FVGYRXGTSA-N 0.000 description 11
- 238000004587 chromatography analysis Methods 0.000 description 9
- 239000007791 liquid phase Substances 0.000 description 9
- 229940079593 drug Drugs 0.000 description 8
- 239000003814 drug Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 125000004433 nitrogen atom Chemical group N* 0.000 description 4
- 239000013558 reference substance Substances 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- KOWKYHVVFKUDLI-NKBVYPNQSA-N (1S,9R,10R)-12-methyl-17-azatetracyclo[7.5.3.01,10.02,7]heptadeca-2,4,6-trien-13-one Chemical compound C1C(=O)C(C)C[C@H]2[C@]3([H])NCC[C@@]21C1=CC=CC=C1C3 KOWKYHVVFKUDLI-NKBVYPNQSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000031709 bromination Effects 0.000 description 1
- 238000005893 bromination reaction Methods 0.000 description 1
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- 229940079358 peripheral opioid receptor antagonist Drugs 0.000 description 1
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- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
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- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention provides a method for separating and determining methyhaaltrexone bromide and impurities and contents thereof with liquid chromatography. The method comprises the following steps: (a) adopting a chromatographic column using alkyl-silicane bonded silica gels as packing to be a separation column; (b) using a trifluoroacetic acid buffer solution as a flowing phase A, and using an organic solvent as a flowing phase B; and (c) and adopting gradient elution for the flowing phases, and separating and determining the impurities and contents of methyhaaltrexone bromide and a preparation thereof. According to the method, methyhaaltrexone and other known intermediate impurities as well as unknown impurities can be effectively separated and determined. The method has strong specificity, high accuracy and convenience for operation.
Description
Technical field
The invention belongs to analytical chemistry field, be specifically related to the method with liquid phase chromatography (HPLC) separation determination methylnaltrexone bromide and impurity thereof, the method comprises a to adopt alkyl silane bonded silica gel to be the chromatographic column of filler be separating column: b take buffer solution as mobile phase A, and organic solvent is Mobile phase B; C mobile phase adopts gradient elution mode.The method can by methylnaltrexone bromide and the effective separation determination of its related substances.
Background technology
Methylnaltrexone bromide (Methylnaltrexone bromide) is a kind of peripheral opioid receptor antagonist, and its molecular formula is C
21h
26o
4nBr, under its structural formula is shown in (I).The chemistry of methylnaltrexone bromide is called: bromination 17-(ring third methyl)-4,5 α-epoxy-3,14-sodium catchol disulfonate 7-methyl-6-oxo-morphinan, has a nitrogen-atoms chiral center.Term used herein " R " and " S " represent the ad hoc structure of nitrogen-atoms chiral center.The nitrogen-atoms of methylnaltrexone bromide as herein described is (R) configuration, and " S-N isomeride " refers to that nitrogen-atoms is the epimer of the methylnaltrexone bromide of (S) configuration.
Report according to pertinent literature (WO2010039851A1), methylnaltrexone bromide under solution state to heat and illumination all unstable, may produce containing α, the catabolite of alpha, beta-unsaturated ketone carbonyl structure, its structure implies that this catabolite may have potential genetoxic.For methylnaltrexone bromide, the known impurities of priority control is needed to have two, single hydroxyl quinone: (17R)-17-(ring third methyl)-10,11-bis-dehydrogenation-3,11-dihydro-4,14-dihydroxy-17-methyl-3,6-dioxo morphinan formic acid compound, its structural formula is shown in following formula (II); Two hydroxyl quinone: (5 α, 17R)-17-(ring third methyl)-10,11-bis-dehydrogenation-3,11-dihydro-4,5,14-trihydroxy-17-methyl-3,6-dioxo morphinan bromide, its structural formula is shown in following formula (III).
CN101405031A, WO2010039851A1 disclose a kind of using the mixed liquor of 0.1% trifluoroacetic acid solution and methyl alcohol different proportion as mobile phase A (0.1% trifluoroacetic acid in the water/methyl alcohol of 95: 5 (V/V)) and Mobile phase B (0.1% trifluoroacetic acid in the water/methyl alcohol of 25: 75 (V/V) or 35: 65 (V/V)), and the HPLC then carrying out gradient elution detects the method for methylnaltrexone bromide.But " the S-N isomeride " of methylnaltrexone bromide can not effectively be separated with the catabolite list hydroxyl quinone of methylnaltrexone bromide by the method, its relative retention time basically identical (relative retention time is 0.89), thus effective priority control or detection can not be carried out to single hydroxyl quinone impurity.
The impurity that synthesis methylnaltrexone bromide is introduced and catabolite impurity thereof, no matter be all need to carry out quality control at methylnaltrexone bromide bulk drug or at the preparation containing methylnaltrexone bromide, the especially separated island form of impurity.Therefore, the effective separation determination realizing methylnaltrexone bromide and impurity thereof has great importance in the quality control of methylnaltrexone bromide bulk drug and preparation.
Because methylnaltrexone bromide can not be separated with its impurity such as " S-N isomeride ", single hydroxyl quinone by existing method effectively, in order to control the quality of methylnaltrexone bromide exactly, monitoring impurity, be necessary to find a kind of can simply, fast, be separated the method detecting methylnaltrexone bromide and relative substance thereof accurately and efficiently.
Summary of the invention
The object of the present invention is to provide the method for one liquid phase chromatography (HPLC) separation determination methylnaltrexone bromide and impurity thereof, thus realize the effective control to methylnaltrexone bromide and the quality of the pharmaceutical preparations thereof.
For realizing object of the present invention, one provided by the invention adopts the method for liquid phase chromatography (HPLC) separation determination methylnaltrexone bromide and impurity thereof, comprising:
A employing alkyl silane bonded silica gel is the chromatographic column of filler is separating column;
B is mobile phase A with buffer solution, take organic solvent as Mobile phase B;
C mobile phase adopts Gradient methods.
In said method, said alkyl silane bonded silica gel is octadecylsilane chemically bonded silica, said buffer solution is trifluoroacetic acid buffer solution, and the concentration of said (trifluoroacetic acid) buffer solution is 0.01% to 0.3%, preferably 0.05% to 0.15%; Said organic solvent is methyl alcohol or acetonitrile or its mixed solution, particular methanol.
In said method, said Gradient methods is: 0 minute to 10 minutes, and mobile phase A is 75% ~ 95% (V/V), and Mobile phase B is 5% ~ 25% (V/V); 10 minutes to 35 minutes, mobile phase A was linearly reduced to 50% ~ 70% (V/V), and Mobile phase B is linearly increased to 30% ~ 50% (V/V); 35 minutes to 45 minutes, mobile phase A was 50% ~ 70% (V/V), and Mobile phase B is 30% ~ 50% (V/V); 45.1 minutes to 55 minutes, mobile phase A was 75% ~ 95% (V/V), and Mobile phase B is 5% ~ 25% (V/V), was namely balance chromatographic column after 45.1 minutes.
The method of the invention described above, also further comprising the steps:
1) preparation of need testing solution: get methylnaltrexone bromide or the preparation containing methylnaltrexone bromide is appropriate, adds thinning agent and makes dissolving, and to be mixed with every 1ml containing the sample solution of methylnaltrexone bromide 0.1mg to 10mg be need testing solution;
2) preparation of contrast solution: it is appropriate that precision measures need testing solution, add thinning agent and make the solution that concentration is need testing solution concentration 5% ~ 15%, solution in contrast, wherein, thinning agent is the mixed solution of mobile phase A and Mobile phase B, and its volume ratio is 95: 5 ~ 75: 25;
3) flow velocity arranging mobile phase is 0.5 ~ 1.5ml/mim, arranging determined wavelength is 280nm to 320nm, the column temperature arranging chromatographic column is 20 DEG C to 50 DEG C, arranging sample size is 5 μ l to 100 μ l, precision measures equal-volume need testing solution and contrast solution respectively, injection liquid chromatography, record chromatogram, completes the separation determination of need testing solution related substance.
The method of the invention described above, step 1) described in mobile phase A and the volume ratio of Mobile phase B be 85: 15; Step 2) described in flow velocity be 1.0ml/min, described determined wavelength is 310nm, and column temperature is 30 DEG C, and sample size is 20 μ l.
The said impurity of the present invention refers to the impurity that produces in the process of synthesis methylnaltrexone bromide, or the impurity of degrading and producing deposited in process by bulk drug and preparation thereof.Described impurity comprises known impurities and unknown impuritie, the one in following compound is at least comprised: (5 α in known impurities, 17R)-17-(ring third methyl)-10,11-bis-dehydrogenation-3,11-dihydro-4,5,14-trihydroxy-17-methyl-3,6-dioxo morphinan bromide (being called for short two hydroxyl quinone), (17R)-17-(ring third methyl)-10,11-bis-dehydrogenation-3,11-dihydro-4,14-dihydroxy-17-methyl-3,6-dioxo morphinan formic acid compound (being called for short single hydroxyl quinone), and S-N-methylnaltrexone bromide (S-N isomeride).
Described assay method, mainly for separating of mensuration methylnaltrexone bromide and impurity thereof, is preferred for separation determination and contains impurity in methylnaltrexone bromide preparation.
In one embodiment, one of the present invention adopts the method for liquid phase chromatography (HPLC) separation determination methylnaltrexone bromide and impurity thereof, comprising:
A employing alkyl silane bonded silica gel is the chromatographic column of filler is separating column, preferred octadecylsilane chemically bonded silica;
B mobile phase A is trifluoroacetic acid buffer solution, and Mobile phase B is the organic solvent being selected from methyl alcohol, acetonitrile or its mixed solution, particular methanol;
C mobile phase adopts Gradient methods.
Wherein, described Gradient methods is: 0 minute to 10 minutes, and mobile phase A is 75% ~ 95% (V/V), and Mobile phase B is 5% ~ 25% (V/V); 10 minutes to 35 minutes, mobile phase A was linearly reduced to 50% ~ 70% (V/V), and Mobile phase B is linearly increased to 30% ~ 50% (V/V); 35 minutes to 45 minutes, mobile phase A was 50% ~ 70% (V/V), and Mobile phase B is 30% ~ 50% (V/V); 45.1 minutes to 55 minutes, mobile phase A was 75% ~ 95% (V/V), and Mobile phase B is 5% ~ 25% (V/V), was namely balance chromatographic column after 45.1 minutes.
In another embodiment, a kind of method adopting liquid phase chromatography (HPLC) separation determination methylnaltrexone bromide and impurity thereof, comprise: a employing alkyl silane bonded silica gel is the chromatographic column of filler is separating column, b mobile phase A is buffer solution, preferred trifluoroacetic acid buffer solution, Mobile phase B is organic solvent, and preferred organic solvent is selected from the organic solvent into methyl alcohol, acetonitrile or its mixed solution, more preferably methyl alcohol, c mobile phase adopts Gradient methods; Comprise the following steps:
1) preparation of need testing solution: get methylnaltrexone bromide or the preparation containing methylnaltrexone bromide is appropriate, adds thinning agent and makes dissolving, and to be mixed with every 1ml containing the sample solution of methylnaltrexone bromide 0.1mg to 10mg be need testing solution;
2) preparation of contrast solution: it is appropriate that precision measures need testing solution, add thinning agent and make the solution that concentration is need testing solution concentration 5% ~ 15%, solution in contrast, wherein, thinning agent is the mixed solution of mobile phase A and Mobile phase B, and its volume ratio is 95: 5 ~ 75: 25;
3) flow velocity arranging mobile phase is 0.5 ~ 1.5ml/mim, arranging determined wavelength is 280nm to 320nm, the column temperature arranging chromatographic column is 20 DEG C to 50 DEG C, arranging sample size is 5 μ l to 100 μ l, precision measures equal-volume need testing solution and contrast solution respectively, injection liquid chromatography, record chromatogram, completes the separation determination of need testing solution related substance.
In above-mentioned specific embodiments, said alkyl silane bonded silica gel is octadecylsilane chemically bonded silica, and said buffer solution is the buffer solution containing trifluoroacetic acid, and the concentration of said buffer solution is 0.01% to 0.3%, preferably 0.05% to 0.15%; Said organic solvent is methyl alcohol or acetonitrile or its mixed solution, particular methanol.
In above-mentioned specific embodiments, said Gradient methods is: 0 minute to 10 minutes, and mobile phase A is 75% ~ 95% (V/V), and Mobile phase B is 5% ~ 25% (V/V); 10 minutes to 35 minutes, mobile phase A was linearly reduced to 50% ~ 70% (V/V), and Mobile phase B is linearly increased to 30% ~ 50% (V/V); 35 minutes to 45 minutes, mobile phase A was 50% ~ 70% (V/V), and Mobile phase B is 30% ~ 50% (V/V); 45.1 minutes to 55 minutes, mobile phase A was 75% ~ 95% (V/V), and Mobile phase B is 5% ~ 25% (V/V), was namely balance chromatographic column after 45.1 minutes.
In above-mentioned specific embodiments, step 1) described in mobile phase A and the volume ratio of Mobile phase B be 85: 15; Step 2) described in flow velocity be 1.0ml/min, described determined wavelength is 310nm, and column temperature is 30 DEG C, and sample size is 20 μ l.
Method of the present invention, in mobile phase A, said buffer solution is trifluoroacetic acid solution, and its concentration is 0.01% to 0.3%, preferably 0.05% to 0.15, optimum 0.1%
Known impurities of the present invention, at least comprise the one in following known compound: (17R)-17-(ring third methyl)-10,11-bis-dehydrogenation-3,11-dihydro-4,14-dihydroxy-17-methyl-3,6-dioxo morphinan formic acid compound (being called for short single hydroxyl quinone), (5 α, 17R)-17-(ring third methyl)-10,11-bis-dehydrogenation-3,11-dihydro-4,5,14-trihydroxy-17-methyl-3,6-dioxo morphinan bromide (being called for short two hydroxyl quinone), and S-N-methylnaltrexone bromide (S-N isomeride).
The present invention adopts Breeze AQ-C18 (4.6 × 250mm, 5 μm) chromatographic column, effectively can be separated the impurity detecting methylnaltrexone bromide and preparation thereof.Select gradient 0 minute to 10 minutes mobile phase such as ratio such as section such as degree such as grade as thinning agent sample dissolution, eliminate the interference of solvent peak; Column temperature is 30 DEG C, adopts gradient elution, effectively can be separated single hydroxyl quinone and " S-N isomeride ", guarantees good symmetry and the higher post effect of chromatographic peak simultaneously.
Methylnaltrexone bromide can effectively be separated with impurity peaks by method of the present invention, simultaneously can being effectively separated " S-N isomeride " and single hydroxyl quinone, impurity can accurately be measured, and peak shape symmetry is better, post effect is higher, thus solve the problem that methylnaltrexone bromide and preparation impurity thereof is difficult to separated island form, especially solve the problem that " S-N isomeride " and single hydroxyl quinone are difficult to be separated, thus ensure that the quality controllable of methylnaltrexone bromide and preparation thereof.Meanwhile, method of the present invention, compared with the conventional method, mobile phase A and B all do not adopt trifluoroacetic acid solution and organic potpourri, and mobile phase is simple, and easily prepare, simple to operate, cost is low.
Accompanying drawing explanation
The liquid chromatogram of the thinning agent of Fig. 1 embodiment 1
The liquid chromatogram of the methylnaltrexone bromide of Fig. 2 embodiment 1, " S-N isomeride ", single hydroxyl quinone, two hydroxyl quinone biased sample
The liquid chromatogram of the methyhaaltrexone bromide injection blank auxiliary need testing solution of Fig. 3 embodiment 1
The liquid chromatography baseline of the gradient elution of Fig. 4 embodiment 1
The liquid chromatogram of the methyhaaltrexone bromide injection determination of related substances need testing solution of Fig. 5 embodiment 2
The liquid chromatogram of methyhaaltrexone bromide injection related substance 10% contrast solution of Fig. 6 embodiment 2
Embodiment
Embodiment 1
Instrument and condition
Wear peace Ulitimate 3000 high performance liquid chromatograph and chem workstation; Auto injection; Be chromatographic column with Breeze AQ-C18 (4.6 × 250mm, 5 μm); Arranging UV-detector wavelength is 310nm; Mobile phase: with 0.1% trifluoroacetic acid solution for mobile phase A, take methyl alcohol as Mobile phase B, carries out gradient elution in the following manner:: 0 minute to 10 minutes, mobile phase A was 85% (V/V), and Mobile phase B is 15% (V/V); 10 minutes to 35 minutes, mobile phase A was linearly reduced to 60% (V/V), and Mobile phase B is linearly increased to 40% (V/V); 35 minutes to 45 minutes, mobile phase A was 60% (V/V), and Mobile phase B is 40% (V/V); 45.1 minutes to 55 minutes, mobile phase A was 85% (V/V), and Mobile phase B is 15% (V/V), was namely balance chromatographic column after 45.1 minutes.Column temperature is 30 DEG C, and flow velocity is 1.0ml/min, and sampling volume is 20 μ l.
Experimental procedure
Accurately take methylnaltrexone bromide respectively, S-N isomeride, single hydroxyl quinone, two hydroxyl quinone reference substance are appropriate, split in 10ml measuring bottle, dissolve with thinning agent (mobile phase A-Mobile phase B (85: 15)) and be diluted to scale, shaking up, as respective reference substance solution; Divide and get each reference substance in right amount, put in 10ml measuring bottle, be diluted to scale with thinning agent, shake up, as mixing reference substance solution.
Get thinning agent and mixing contrast solution respectively, carry out liquid-phase chromatographic analysis by above-mentioned chromatographic condition, record chromatogram, the results are shown in Figure 1, Fig. 2;
Another precision measures the blank auxiliary 0.5ml of methyhaaltrexone bromide injection prescription ratio, puts in 10ml measuring bottle, is diluted to scale with thinning agent, shake up, as blank auxiliary need testing solution, carry out liquid-phase chromatographic analysis by above-mentioned chromatographic condition, record chromatogram, the results are shown in Figure 3.
In addition, not sample introduction, gathers gradient elution baseline by above-mentioned chromatographic condition, sees Fig. 4.
In Fig. 2, retention time is the chromatographic peak of 8.925min is two hydroxyl quinone, and the chromatographic peak of 15.075min is single hydroxyl quinone, and the chromatographic peak of 15.900min is " S-N isomeride ", and the chromatographic peak of 18.567min is methylnaltrexone bromide.
Fig. 1 proves, thinning agent is interference measurement not; Fig. 3 proves, blank auxiliary is interference measurement not; Fig. 4 proves, chromatographic system does not disturb; Fig. 2 proves, this method can effectively be separated the known impurities that may exist in methylnaltrexone bromide, and namely this method may be used for the impurity determination of methylnaltrexone bromide and preparation thereof.
Embodiment 2
The impurity of liquid chromatography for measuring methyhaaltrexone bromide injection.
Instrument and condition
Agilent 1200 high performance liquid chromatograph and chem workstation; Auto injection; Chromatographic condition is identical with embodiment 1.
Get methyhaaltrexone bromide injection appropriate (being about equivalent to methylnaltrexone bromide 10mg), put in 10ml measuring bottle, (mobile phase A: Mobile phase B=85: 15) make dissolving and be diluted to scale, shakes up, as need testing solution to add thinning agent; Precision measures 1.0ml, puts in 10ml measuring bottle, adds thinning agent and is diluted to scale, shake up, in contrast solution; Chromatographic condition according to embodiment 2 carries out liquid-phase chromatographic analysis, the results are shown in Figure 5, Fig. 6.Fig. 5, Fig. 6 show almost not detect in methylnaltrexone bromide bulk drug have impurity, and illustrate that methylnaltrexone bromide bulk drug purity is high, impurity content is extremely low.
Embodiment 3
The impurity of liquid chromatography for measuring methylnaltrexone bromide bulk drug
Instrument and condition
Agilent 1200 high performance liquid chromatograph and chem workstation; Auto injection; Chromatographic condition is identical with embodiment 1.
Experimental procedure
Get methylnaltrexone bromide and be about 10mg, accurately weighed, put in 10ml measuring bottle, (mobile phase A: Mobile phase B=85: 15) make dissolving and be diluted to scale, shakes up, as need testing solution to add thinning agent; Precision measures 1.0ml, puts in 10ml measuring bottle, adds thinning agent and is diluted to scale, shake up, in contrast solution; Carry out liquid-phase chromatographic analysis by above-mentioned chromatographic condition, whether the position observing single hydroxyl quinone and two hydroxyl quinone in the chromatogram of need testing solution has impurity peaks, if any, the area of impurity peaks must not be greater than 0.001 times (0.01%) of contrast solution main peak area.Result is basically identical with the result implementing 2, and also almost do not detect in methylnaltrexone bromide bulk drug have impurity, illustrate that methylnaltrexone bromide bulk drug purity is high, impurity content is extremely low, reaches the limitation almost examined and do not measure.
Embodiment 2 and 3 also proves that method of the present invention can the amount of effectively detection and control methylnaltrexone bromide and impurity thereof.
Claims (7)
1., by a method for liquid chromatography for separating and determining methylnaltrexone bromide and impurity thereof, comprising:
A employing alkyl silane bonded silica gel is the chromatographic column of filler is separating column;
B is mobile phase A with buffer solution, take organic solvent as Mobile phase B;
C mobile phase adopts gradient elution mode,
Wherein, said gradient elution mode is: 0 minute to 10 minutes, and mobile phase A is 75% ~ 95% (V/V), and Mobile phase B is 5% ~ 25% (V/V); 10 minutes to 35 minutes, mobile phase A was linearly reduced to 50% ~ 70% (V/V), and Mobile phase B is linearly increased to 30% ~ 50% (V/V); 35 minutes to 45 minutes, mobile phase A was 50% ~ 70% (V/V), and Mobile phase B is 30% ~ 50% (V/V); 45.1 minutes to 55 minutes, mobile phase A is 75% ~ 95% (V/V), Mobile phase B is 5% ~ 25% (V/V), namely be balance chromatographic column after 45.1 minutes, described mobile phase A is 0.1% trifluoroacetic acid solution, and described Mobile phase B is methyl alcohol, determined wavelength is 310nm, column temperature is 30 DEG C, and flow velocity is 1.0ml/min, and sampling volume is 20 μ l.
2. method according to claim 1, said alkyl silane bonded silica gel is octadecylsilane chemically bonded silica.
3. method according to claim 1, said organic solvent is the mixed solution of acetonitrile or acetonitrile and methyl alcohol.
4. method according to claim 1, the concentration of said buffer solution is 0.01% to 0.3%.
5. method according to claim 4, the concentration of said buffer solution is 0.05% to 0.15%.
6. method according to claim 1, is characterized in that: said method also comprises following steps:
1) preparation of need testing solution: get methylnaltrexone bromide or the preparation containing methylnaltrexone bromide is appropriate, adds thinning agent and makes dissolving, and to be mixed with every 1ml containing the sample solution of methylnaltrexone bromide 0.1mg to 10mg be need testing solution;
2) preparation of contrast solution: it is appropriate that precision measures need testing solution, add thinning agent and make the solution that concentration is need testing solution concentration 5% ~ 15%, solution in contrast, wherein, thinning agent is the mixed solution of mobile phase A and Mobile phase B, and its volume ratio is 95: 5 ~ 75: 25;
3) flow velocity arranging mobile phase is 1.0ml/mim, arranging determined wavelength is 310nm, the column temperature arranging chromatographic column is 30 DEG C, arranging sample size is 20 μ l, precision measures equal-volume need testing solution and contrast solution respectively, injection liquid chromatography, record chromatogram, completes the separation determination of need testing solution related substance.
7. method according to claim 1, described impurity comprises the one at least following two kinds of compounds:
(5 α, 17R)-17-(ring third methyl)-10,11-bis-dehydrogenation-3,11-dihydro-4,5,14-trihydroxy-17-methyl-3,6-dioxo morphinan bromide, (17R)-17-(ring third methyl)-10,11-bis-dehydrogenation-3,11-dihydro-4,14-dihydroxy-17-methyl-3,6-dioxo morphinan formic acid compound.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010577754.5A CN102565203B (en) | 2010-12-07 | 2010-12-07 | Method for separating and determining methyhaaltrexone bromide and impurities thereof with liquid chromatography |
Applications Claiming Priority (1)
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| CA2609662A1 (en) * | 2005-05-25 | 2006-11-30 | Progenics Pharmaceuticals, Inc. | (r)-n-methylnaltrexone, process for its synthesis and its pharmaceutical use |
| JP2008542286A (en) * | 2005-05-25 | 2008-11-27 | プロジェニックス ファーマシューティカルズ，インコーポレーテッド | (S) -N-methylnaltrexone, synthesis method thereof and pharmaceutical use thereof |
| CN101685084A (en) * | 2008-09-22 | 2010-03-31 | 重庆医药工业研究院有限责任公司 | Method for detecting methylnaltrexone bromide and impurity thereof by chromatography |
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| CA2609662A1 (en) * | 2005-05-25 | 2006-11-30 | Progenics Pharmaceuticals, Inc. | (r)-n-methylnaltrexone, process for its synthesis and its pharmaceutical use |
| JP2008542286A (en) * | 2005-05-25 | 2008-11-27 | プロジェニックス ファーマシューティカルズ，インコーポレーテッド | (S) -N-methylnaltrexone, synthesis method thereof and pharmaceutical use thereof |
| CN101685084A (en) * | 2008-09-22 | 2010-03-31 | 重庆医药工业研究院有限责任公司 | Method for detecting methylnaltrexone bromide and impurity thereof by chromatography |
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| Title |
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| Metabolism of Intravenous Methylnaltrexone in Mice, Rats, Dogs,and Humans;Appavu Chandrasekaran et al;《DRUG METABOLISM AND DISPOSITION》;20100106;第38卷(第4期);第606-616页 * |
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