CN105758984A - Method using derivatization HPLC-DAD method to determine small-molecule halogenated carboxylic acid in medicine - Google Patents
Method using derivatization HPLC-DAD method to determine small-molecule halogenated carboxylic acid in medicine Download PDFInfo
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Abstract
The invention discloses a method using a derivatization HPLC-DAD method to determine small-molecule halogenated carboxylic acid in medicine.The method includes: under normal temperature, using nitrobenzene hydrazine derivatization reagent to derivatize the small-molecule halogenated carboxylic acid so as to generate products with high absorption in the ultraviolet visible region; using the reaction liquid after the derivatization reaction as the feeding sample, and using the HPLC-DAD method to determine the derivatization products of halogenated carboxylic acid in the feeding sample in the ultraviolet visible region on the basis of the reversed phase partition chromatography principle so as to achieve the qualitative or quantitative detection of the small-molecule halogenated carboxylic acid.Due to the fact that the ultraviolet absorption band of the carboxylic acid derivatization products of the nitrobenzene hydrazine derivatization reagent has evident redshift effect due to the existence of nitro electron-withdrawing group on benzene ring and most medicine and impurities thereof are weak in absorption in the ultraviolet visible region (300-450 nanometers), the simple and universal method using pre-column derivatization HPLC-DAD to determine the small-molecule halogenated carboxylic acid is built.Methodology validation shows that the method is good in specificity.
Description
Technical field
The invention belongs to drugs analysis detection field, relate to derivatization HPLC-DAD method and measure the side of little molecule halogenated carboxylic acid
Method, particularly relates to the method that derivatization HPLC-DAD method measures medicine small molecular halogenated carboxylic acid.
Background technology
Little molecule halogenated carboxylic acid (Halogenated carboxylic acids, HCAs), as main in water environment
Disinfection byproduct (DBP), owing to having potential genotoxicity and carcinogenecity[1,2], receive many environment supervision departments very early and include
Environmental Protection Agency USA (USEPA)[3], World Health Organization (WHO) (WHO)[4]Deng common concern, and be listed in the normal of water quality research
One of rule monitoring index.In medical industry, this compounds is because having higher reactivity and special physiologically active
(being introduced by halogen element)[5], it is often used as acylating agent active in organic synthesis or alkylating agent, in pesticide, medicine and other fields
Extensive application[6-13], such as, monoxone can be used for synthetic vitamin B6, tadanafil, the intermediate of epinephrine etc.；2-
Chloropropionic acid is commonly used for the initiation material of ibuprofen green production process；Bromoacetic acid and bromo-propionic acid class are usually used in herbicide, insecticide
Preparation in pesticide.In addition to commercial production, affected by external condition during medicine storage and also can be produced halogenated carboxylic acid, such as
Chloromycetin degraded generates dichloroacetic acid[14].Therefore this compounds tends to remain in medicine, and Excess free enthalpy may be right
Human body works the mischief.The most commonly used toxicology pays close attention to threshold value (threshold of toxicological at present
Concern, TTC) genotoxicity impurity is carried out limit handling[15-17].The limit of genotoxicity impurity is the most relatively low, therefore
Need to set up high-sensitive analysis method this compounds in medicine is monitored.
Now, the method for most of documents report is both in the halogen acetic acid (Haloacetic measured in water environment
acids,HAAs).Gas chromatogram serial electronic acquisition detector (GC-ECD) is classified as detection water ring by EPA (USEPA)
The standard method of halogen acetic acid in border[18-21], the method needs to derive determinand the esters of chemical conversion volatile, then liquid liquid extraction
Take enrichment, can be analyzed.The chromatography of ions (IC)[22,23]With reversed phase high-performance liquid chromatography (RP-LC)[24,25]Etc. liquid phase
Chromatographic process, combined with electrochemical detector (ECD) or UV-detector (UV, 210nm) have been reported for the halogen in analysis water-like
Acetic acid.These methods concentrate before generally requiring loaded down with trivial details post and clean operation could obtain enough specificities and sensitivity.Matter
Spectrometry (MS), including hydrophilic Interaction Chromatography tandem mass spectrum (HILIC-MS)[26]、GC-MS[27,28]、LC-MS[29,30]And IC-MS[31 ,32]Content etc. the halogen acetic acid being also widely used in mensuration drinking water.Although mass detector is a kind of sensitive and general
Detector, but fancy price limits promoting the use of of it.
Compared to water sample analysis, measure the little molecule halogenated carboxylic acid of residual in medicine and be faced with more challenge[14,16,33]。
First the complexity of medicine substrate is far above water sample, especially contain a large amount of the most similar to little molecule halogenated carboxylic acid character organic little
Molecule, therefore it is required that analysis method possesses the specificity of height[16,33], so could effectively reduce interference；Another problem is
Medicine itself is easily subject to extraneous factor impact degraded in operation and generates new little molecule halogenated carboxylic acid[14]Impact measures
Result, the accuracy to result the simplest for this Pretreatment, gentle is the most favourable.In view of the above-mentioned fact, it is intended that
These problems are improved by liquid phase Derivative.Liquid phase Derivative introduces specific by chemical reaction to determinand
Group, is possible not only to improve detection sensitivity, moreover it is possible to improve the separating effect of determinand, be particularly suitable for little molecular polarity material
Qualitative and quantitative analysis[34].Simone Jr etc.[35]After using post, the halogen acetic acid in drinking water is measured by fluorescence derivation,
It is its derivatization reagent with nicotiamide, by laboratory homemade self-reacting device device to realize derivatization.But it is specific
Device not only increases experimental cost, and is difficult to promote for conventional medicine analysis.Ghassempour et al.[36]Establish
Glycine betaine sample monoxone and dichloroacetic acid are measured by pre-column derivatization-HPLC method.The method uses naphthalidine as spreading out
Biochemical reagents, react 30min, detect subsequently under 220nm wavelength at 90 DEG C.The major defect of this method is: on the one hand, and high temperature holds
It is easily generated degradation impurity[37]；On the other hand, baseline noise is detected under low wavelength big, serious interference[38]。
Nitrophenyl hydrazine (Nitro-substituted phenylhydrazines, NPHs) is classical carboxylic acids derivatization
Reagent[38-45].Compared to phenyl amines, the carboxylic acid derivatization product of this kind of reagent is due to the depositing of nitro electron withdraw group on phenyl ring
, there is obvious redshift effect in ultraviolet absorption band[39-41].Most drug and impurity thereof are purple near visible district (400nm)
Outer absorption is the most weak, so being measured being greatly improved the specificity of method, summarized chromatogram separation condition at this wavelength.2-
Nitrophenyl hydrazine (2-NPH)[38-43], 3-nitrophenyl hydrazine (3-NPH)[44], 4-nitrophenyl hydrazine (4-NPH)[45]And 2,4 dinitrophenyl hydrazine
(2,4-DNPH)[43]It is the conventional commercialization Nitrobenzol hydrazine derivatization reagent of report at present, but about this kind of reagent
Systematic comparison does not also have been reported that.Noonan etc.[38]People once with 2-nitrophenyl hydrazine as derivatization reagent, pyridine and 1-ethyl-
Under the catalytic activation of (3-dimethylaminopropyl) carbodiimide hydrochloride, successfully the Fluoroethanoic acid in food is derived
Change.This article mainly stresses the application at LC-MS, does not has the ultraviolet absorption characteristic to its product to carry out deep excavation.Therefore,
Those skilled in the art cannot be speculated whether to use HPLC-DAD to derive Fluoroethanoic acid by information disclosed in the prior art
Compound qualitatively or quantitatively detects.
Research shows that carboxylic acid and amido reaction need through 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride
The activation of (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, EDC HCl)
Can carry out, and activation process is frequently added pyridine as catalyst[41].Therefore, inventor initially selects EDC HCl and pyrrole
Pyridine accelerates hydrazides synthesis as catalyst system and catalyzing, and is successfully realized chloroacetic derivatization；But, when this condition is extended
During to other detection objects, derivatization product occurs in that abnormal instability.We have found that conventional catalyst pyridine is easily made
Become the dehalogenation phenomenon of alpha-halogen derivant.By adjusting reaction temperature, system pH and changing the type of nitrophenyl hydrazine the most not
Can effectively solve the problems referred to above, this hampers the popularization and application of the method significantly.Also can through observing during discovery derived
There is chlorination, and present in chlorination and system, hydrochlorates are relevant in a large number, and hydrochlorate is mainly derived from system
EDC HCl and 2-NPH HCl.Although the probability that chlorination occurs can be reduced by reducing the consumption of hydrochlorate,
But but can not check dechlorination reaction to occur.Reagent plus current commercialization all contains hydrochlorate, without hydrochlorate
EDC and 2-NPH dissolubility is poor, considers, and the concentration only by reduction activator and derivatization reagent is effectively to change
Kind derivant stability.
Summary of the invention
It is an object of the invention to the above-mentioned deficiency for prior art, it is provided that derivatization HPLC-DAD method measures little molecule halogen
Method for carboxylic acid.
It is a further object of the present invention to provide the method that derivatization HPLC-DAD method measures medicine small molecular halogenated carboxylic acid.
The purpose of the present invention can be achieved through the following technical solutions:
Derivatization HPLC-DAD method measures the method for little molecule halogenated carboxylic acid, comprises the steps of
(1) at normal temperatures, Nitrobenzol hydrazine derivatization reagent is used to generate little molecule halogenated carboxylic acid derivatization in ultraviolet
There is the product of stronger absorption visible region (320-450nm)；
(2) reactant liquor after terminating using derivative reaction in step (1), as sample introduction sample, utilizes HPLC anti-phase distribution color
Spectrometry measures the derivatization product of wherein halogenated carboxylic acid at ultraviolet-visible district (320-450nm), thus realizes little molecule halogen
Qualitative or quantitative detection for carboxylic acid.
Described derivatization HPLC-DAD method measures the method for little molecule halogenated carboxylic acid, preferably comprises following steps:
(1) at normal temperatures, with acetonitrile-water as reaction system, with the hydrochlorate of carbodiimide class coupling agent as activator, make
With Nitrobenzol hydrazine derivatization reagent, the generation of little molecule halogenated carboxylic acid derivative reaction there is stronger absorption 330～420nm
Product；
(2) with derivative reaction product in step (1), utilize HPLC-DAD method, exist based on reversed phase partition chromatography principle
330～the 420nm derivatization products measuring wherein halogenated carboxylic acid, thus realize the qualitative or quantitative inspection to little molecule halogenated carboxylic acid
Survey.
Wherein, described little molecule halogenated carboxylic acid preferably is selected from the carboxylic acid of C1～C6 of halogen substiuted, further preferred halogen
The carboxylic acid of substituted C1～C4, further preferably monoxone, bromoacetic acid, dichloroacetic acid, 2-chloropropionic acid, 3-chloropropionic acid or 2-bromine
Any one in propanoic acid.
Described carbon carbodiimide class coupling agent preferably is selected from dicyclohexylcarbodiimide, N, N'-DIC,
Any one in 1-ethyl-(3-dimethylaminopropyl) carbodiimide；Further preferably 1-ethyl-(3-dimethylamino
Propyl group) carbodiimide.
Nitrobenzol hydrazine derivatization reagent preferably is selected from 2-nitrophenyl hydrazine hydrochloride, 3-nitrophenyl hydrazine hydrochloride, 4-Nitrobenzol
Any one in hydrazine hydrochloride and 2,4 dinitrophenyl hydrazine, further preferred 2-nitrophenyl hydrazine hydrochloride.
The condition of derivative reaction further preferably with acetonitrile-water=10:90～90:10 (V:V) as reaction system, 2-nitre
Base phenyl hydrazine hydrochloride salinity is 0.2～3mg/mL, and EDC HCl concentration is 1～4mg/mL, reacts 1～3h；The most preferred
Under room temperature, with acetonitrile-water=70:30 (V:V) as reaction system, 2-nitrophenyl hydrazine hydrochloride concentration is 1mg/mL, EDC HCl
2mg/mL, the response time is 2h.
Described HPLC-DAD method is preferred: use HPLC chromatogram instrument；Use reversed phase partition chromatography；With non-polar linkage phase
For fixing phase, use polarity flowing phase, detection wavelength between 320-450nm, be further preferably positioned at 300～420nm it
Between, further it is preferably placed between 390～400nm.
The instrument that the HPLC-DAD method stated further is preferably used is Shimadzu LC 20AT chromatograph of liquid, this color
Spectrometer is formulated with online vacuum degassing machine, binary gradient pump, automatic sampler, column oven, DAD detector and LC-solution
Chromatographic work station；Chromatographic column uses 250mm × 4.6mm, the Shimadzu Inertsil ODS-3 of 5 μm；Sample size: 20 μ L；Flowing phase
Flow velocity: 1.0mL/min；Eluent gradient: A phase is acetonitrile, B phase is 0.1% phosphoric acid, 0min 28%A phase, 10min 40%A
Phase, 14min 40%A phase, 20min 55%A phase；Column temperature: 40 DEG C；Detection wavelength: 392nm.
Method of the present invention can be used for the qualitative and quantitative detection of several samples small molecular halogenated carboxylic acid, preferably exists
Measure the application in medicine small molecular halogenated carboxylic acid.
Derivatization HPLC-DAD method measures the method for medicine small molecular halogenated carboxylic acid, comprises the steps of
(1) under room temperature, under the activation of the hydrochlorate of carbodiimide class coupling agent, 2-nitrophenyl hydrazine hydrochloride is used
Medicine to be measured performs the derivatization reaction 1～3h, and reactant liquor measures as sample feeding；Described carbodiimide class coupling agent choosing
From dicyclohexylcarbodiimide, N, N'-DIC, in 1-ethyl-(3-dimethylaminopropyl) carbodiimide
One, preferably 1-ethyl-(3-dimethylaminopropyl) carbodiimide；
(2) reactant liquor after terminating using derivative reaction in step (1), as sample introduction sample, utilizes HPLC-DAD method to exist
320-450nm measures the derivatization product of wherein halogenated carboxylic acid, thus realize the qualitative of medicine small molecular halogenated carboxylic acid or
Detection by quantitative.
Wherein, wherein, described little molecule halogenated carboxylic acid preferably is selected from the carboxylic acid of C1～C6 of halogen substiuted, further preferably
The carboxylic acid of C1～C4 of halogen substiuted, further preferably monoxone, bromoacetic acid, dichloroacetic acid, 2-chloropropionic acid, 3-chloropropionic acid or
Any one in 2 bromopropionic acid.
Under the preferred room temperature of condition of derivative reaction, with acetonitrile-water that acetonitrile volumetric concentration is 10%～90% for reaction
System, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride concentration is 1～4mg/mL, 2-nitrophenyl hydrazine hydrochloride
Concentration is 0.2～3mg/mL, and the response time is 1～3h；The condition of described derivative reaction further preferably with acetonitrile-water=
70:30 (V:V) is reaction system, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride 2mg/mL, 2-Nitrobenzol
Hydrazine hydrochloride concentration is 1mg/mL, and the response time is 2h.
HPLC-DAD method described in step (2) preferably employs HPLC chromatogram instrument；Use reversed phase partition chromatography；With non-pole
Property Bonded Phase be fixing phase, use polarity flowing phase, detection wavelength, between 320-450nm, is preferably placed at 330～420nm
Between, further preferably between 390～400nm；The instrument that described HPLC-DAD method is preferably used is Shimadzu LC
20AT chromatograph of liquid, this chromatograph is formulated with online vacuum degassing machine, binary gradient pump, automatic sampler, column oven, DAD
Detector and LC-solution chromatographic work station；Chromatographic column uses 250mm × 4.6mm, the Shimadzu Inertsil ODS-3 of 5 μm；
Sample size: 20 μ L；Flow rate of mobile phase: 1.0mL/min；Eluent gradient: A phase is acetonitrile, B phase is 0.1% phosphoric acid, 0min
28%A phase, 10min 40%A phase, 14min 40%A phase, 20min 55%A phase；Column temperature: 40 DEG C；Detection wavelength: 392nm.
What room temperature of the present invention referred to routine analyzes the temperature that carries out of experiment, usually between 10～30 DEG C.
Beneficial effect:
Present invention carboxylic acid based on Nitrobenzol hydrazine derivatization reagent derivatization product is due to nitro electron-withdrawing group on phenyl ring
The existence of group, there is obvious redshift effect in ultraviolet absorption band.And most drug and impurity thereof are inhaled near visible district ultraviolet
Receive the most weak, establish a kind of method that simple, general pre-column derivatization HPLC-DAD measures little molecule halogenated carboxylic acid.Methodology
The result display medicine of checking common are machine acid and analysis all will not be interfered by other impurity, show that the method is exclusive
Property is good.Additionally, the detection of method is limited to 0.02-0.05 μ g/mL, being quantitatively limited to 0.05-0.12 μ g/mL, linear relationship is good
(r>0.999)；1.98% and 4.39% in a few days it is respectively smaller than with day to day precision；Average recovery rate is between 82.4-105.6%
(RSD < 4.30%), without obvious matrix interference；And derivatization product is good at 5h internal stability.
For the dechlorination reaction occurred during derivatization and chlorination, creatively find, by pyridine from derivatization
The generation of dechlorination reaction and chlorination can be avoided when the catalyst system and catalyzing of reaction is rejected.Find based on this, to derivatization body
System is optimized, and not only can ensure that halogenated carboxylic acid is by complete derivatization, moreover it is possible to be prevented effectively from the degraded of product.
Accompanying drawing explanation
Fig. 1: respectively with 2-nitrophenyl hydrazine (A), 3-nitrophenyl hydrazine (B), 4-nitrophenyl hydrazine (C), 2,4 dinitrophenyl hydrazine (D)
The uv absorption spectra of product is generated as derivatization reagent and chloroacetate reaction.
Fig. 2: when trace dichloroacetic acid in chloromycetin (15mg/mL) (2.5 μ g/mL) being performed the derivatization with 2-nitrophenyl hydrazine
The chromatogram (a-e represents different detection wavelength) obtained: a.224nm, b.275nm, c.337nm, d.356nm, e.392nm.
(Imp 1-5 represents other endogenous impurity contained in chloromycetin crude drug)
Fig. 3: blank solution (a), formic acid (b), acetic acid (c), trifluoroacetic acid (d), methanesulfonic acid (e), benzene first under the same terms
Acid (f), benzenesulfonic acid (g), p-methyl benzenesulfonic acid (h) and the chromatogram of 6 kinds little molecule halogenated carboxylic acid (i) derivatization products: (1)
Monoxone derivant；(2) bromoacetic acid derivative；(3) 3-chloropropionic acid derivant；(4) 2-chloropropionic acid derivant；(5) 2 bromopropionic acid
Derivant；(6) dichloroacetic acid derivant；(7) trifluoroacetic acid derivant；(8) benzoic acid derivative.The concentration of organic acid is 1 μ
g/mL.Fig. 4: the reaction condition impact on halogenated carboxylic acid derivatization: (A) pyridine impact on halogenated carboxylic acid derivant stability；
(B) the organic facies ratio impact on derivatization efficiency in system；(C) the 2-nitrophenyl hydrazine concentration impact on derivatization efficiency；(D)
The response time impact on derivatization efficiency.B, C, D are all the results investigated under conditions of non-pyridine participates in.Determinand dense
Degree is 1 μ g/mL.
Fig. 5: for verifying that pyridine is to the typical color spectrogram of derivatization stability influence and mass spectrum: (A) at pyridine and
Individually dichloroacetic acid (a) and monoxone (b) are performed the derivatization in the presence of EDC HCl；(B) exist at pyridine and EDC HCl
The most individually bromoacetic acid (d) and monoxone (b) are performed the derivatization；(C) individually to 2-bromine third in the presence of pyridine and EDC HCl
Acid (f) and 2-chloropropionic acid (g) perform the derivatization.C, e, h represent respectively under the conditions of not having pyridine to participate in individually to dichloroacetic acid,
Bromoacetic acid and 2 bromopropionic acid perform the derivatization.Testing concentration is 5 μ g/mL.
Detailed description of the invention
1.1. instrument
Shimadzu LC 20AT chromatograph of liquid (is contained in line vacuum degasser, binary gradient pump, automatic sampler, post
Incubator, DAD detector and LC-solution chromatographic work station)；Aglient 6520LC-TOF Liquid Chromatography-Tandem Mass Spectrometry instrument
(it is contained in line vacuum degasser, high pressure binary gradient pump, automatic sampler, column oven, DAD detector, electron spray (ESI) interface
With Agilent Mass Hunter Acquisition Software Ver.A.01.00 work station)；METTLER TOLEDO
AB135-S analytical balance (prunus mume (sieb.) sieb.et zucc. Teller swedish company)；Sartorious BS110 analytical balance (the limited public affairs of Beijing Sai Duolisi
Department).
1.2. reagent
Monoxone (98%), bromoacetic acid (98%), dichloroacetic acid (98%), 2-chloropropionic acid (97%), 2 bromopropionic acid
(98%), 3-chloropropionic acid (98%), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (97%), 2-Nitrobenzol
Hydrazine hydrochloride (97%), 3-nitrophenyl hydrazine hydrochloride (98%), 4-nitrophenyl hydrazine hydrochloride (98%) and 2,4 dinitrophenyl hydrazine
(99.0%), tadanafil (97%), chloromycetin (95%), vitamin B6(98%), ibuprofen (98%), 5-(piperazine-1-
Base) benzofuran-2-carboxamides (vilazodone intermediate, 99.2%), 6-hydroxyl-3,4-dihydro-2-quinolinone (Xi Luota
Azoles, 99.5%), metalaxyl (97%), Chloramphenicol Eye Drop (8mL:20mg).
Purified water, acetonitrile (TEDIA, chromatographically pure), phosphoric acid (analytical pure), formic acid (analytical pure).
1.3. the preparation of solution
Each halogenated carboxylic acid storing solution (hereinafter referred to as HCA): take appropriate HCA, accurately weighed, it is placed in 10mL measuring bottle, uses
70% dilution in acetonitrile, to graduation mark, shakes up.Under each storing solution room temperature, 8h internal stability is good.
2-nitrophenyl hydrazine hydrochloride, 3-nitrophenyl hydrazine hydrochloride, 4-nitrophenyl hydrazine hydrochloride and 2,4 dinitrophenyl hydrazine examination
Liquid (hereinafter referred to as 2-NPH HCl, 3-NPH HCl, 4-NPH HCl and DNPH): take each reagent about 100mg, accurately weighed,
It is placed in 10mL measuring bottle, by 80% dilution in acetonitrile to scale, shakes up.Need Fresh.
1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution (hereinafter referred to as EDC HCl): take 1-second
Base-(3-dimethylaminopropyl) carbodiimide hydrochloride 200mg, accurately weighed, it is placed in 10mL measuring bottle, dilute with 60% acetonitrile
Release to scale, shake up.Need Fresh.
1.4. derivatization experiment condition
Accurately weighed appropriate medicine, or precision pipettes 100 μ L halogenated carboxylic acid reference substance test solutions, is placed in 5mL measuring bottle, respectively
Add 500 μ L derivatization reagents and 500 μ L EDC HCl solution, add 70% dilution in acetonitrile to scale, vortex 10s, shake up.Keep away
Photoreaction 2h, shakes up, and takes 20 μ L direct injected after filtration.
Embodiment 1
With monoxone as representative, with the 4 of same concentrations kinds of Nitrobenzol hydrazine derivatization reagents, it is performed the derivatization.Derivative
Change experiment condition and see 1.4.Under identical chromatographic condition, scan each derivatization product, the spectrogram of record product and color with DAD
Spectrogram, evaluates the quality of derivatization reagent with the maximum absorption wavelength of product and absorption intensity.
Fig. 1 shows, in addition to 3-nitrophenyl hydrazine, and the product uv absorption that other three kinds of derivatization reagents generate with monoxone
Band all presents certain redshift effect, maximum absorption wavelength be respectively 392nm (2-nitrophenyl hydrazine) 356nm (4-nitrophenyl hydrazine) and
337nm (2,4 dinitrophenyl hydrazine).This experiment is ideally measured near visible district (400nm) place, can make
Ambient interferences is preferably minimized, summarized chromatogram separation condition, improves method specificity[40,41].From fig. 1, 2-nitrophenyl hydrazine produces
The redshift effect of thing is the most obvious, and other halogenated carboxylic acid derivants also show similar absorption characteristic.Therefore 2-nitrophenyl hydrazine is
For properly；And from the point of view of the uv absorption intensity of each product, the absorption intensity of 2-nitrophenyl hydrazine product is higher than other three kinds.Therefore
And, the present invention selects 2-nitrophenyl hydrazine to carry out follow-up study.
In actual test sample, due to the existence of high amount of drug substrate, shadow for the halogenated carboxylic acid derivatization product of trace
Ringing relatively big, if promoting the use of, each medicine will be set up a chromatographic condition and go to separate medicine and impurity, and interference is many, sensitivity
Low (baseline noise is big).And when using 2-nitrophenyl hydrazine as derivatization reagent, owing to most drug and impurity thereof closely may be used
Jian Guang district (400nm) uv absorption is the most weak, so being measured being greatly improved the specificity of method at this wavelength, simplifies
Chromatographic separation condition.Such as, when with 2-nitrophenyl hydrazine, the dichloroacetic acid in chloromycetin being performed the derivatization, and DAD record is used
When difference detects the chromatogram under wavelength, (Fig. 2) finds, compared to other shorter wavelengths, the chromatogram under 392nm wavelength detecting
The baseline more balanced and less Impurity Absorption peak can be provided, be conducive to improving baseline noise, reduce ambient interferences, improve
The specificity of method and sensitivity[39,40].The fact that also have with other drug as the advantage that 2-nitrophenyl hydrazine is important
Embody.
Embodiment 2
Prepared by need testing solution: accurately weighed appropriate medicine, is placed in 5mL measuring bottle, is separately added into 500 μ L derivatization reagents
With 500 μ L EDC HCl solution, add 70% dilution in acetonitrile to scale, vortex 10s, shake up, lucifuge reaction 2h, shake up, obtain confession
Test sample solution.20 μ L direct injected are taken after filtration.
Prepared by reference substance solution: precision pipettes 100 μ L variable concentrations halogenated carboxylic acid storing solutions, is placed in 5mL measuring bottle, respectively
Add 500 μ L derivatization reagents and 500 μ L EDC HCl solution, add 70% dilution in acetonitrile to scale, vortex 10s, shake up, keep away
Photoreaction 2h, shakes up, and obtains reference substance solution.20 μ L direct injected are taken after filtration.
Chromatographic condition: Shimadzu LC 20AT chromatograph of liquid (is contained in line vacuum degasser, binary gradient pump, automatically
Injector, column oven, DAD detector and LC-solution chromatographic work station)；Chromatographic column: Shimadzu Inertsil ODS-3
(250mm×4.6mm,5μm)；Flowing phase: acetonitrile (A phase)-0.1% phosphoric acid (B phase), gradient elution is shown in Table 1, flow velocity: 1.0mL/
min；Sample size: 20 μ L；Column temperature: 40 DEG C；Detection wavelength: 392nm.
Table 1. gradient elution program
The bent method for drafting of mark: precision pipettes each reference substance storing solution and is placed in right amount in same 5mL measuring bottle respectively, adds 70%
Dilution in acetonitrile, to scale, makes hybrid standard strain row strength solution.Perform the derivatization by method under 1.4. item, take 20 μ L and inject
Chromatograph of liquid, measures the peak area of each derivatization product.With halogenated carboxylic acid concentration C (μ g/mL) as abscissa, derivatization product
Peak area (A) is vertical coordinate, uses method of least square to carry out linear regression analysis, calculates equation of linear regression and correlation coefficient.
Quantitative approach: use external standard method to carry out quantitatively.
Embodiment 3
According to the method for embodiment 2, to monoxone, bromoacetic acid, 3-chloropropionic acid, 2-chloropropionic acid, 2 bromopropionic acid, dichloroacetic acid
Investigate.From the i of Fig. 3, under above-mentioned chromatographic condition, 6 kinds of halogenated carboxylic acid derivatization products and derivatization reagent, urge
Between agent, separating degree is preferable.In view of organic acid (formic acid, acetic acid, trifluoroacetic acid, methanesulfonic acid, benzene conventional in pharmaceutical synthesis
Formic acid, benzenesulfonic acid and p-methyl benzenesulfonic acid) may react with 2-nitrophenyl hydrazine, so this experiment is the most right
These seven kinds of organic acid are investigated, and study the mensuration of its material generated with 2-nitrophenyl hydrazine whether jamming target thing.Result is such as
Fig. 3, the product after organic acid derivatization conventional in these seven kinds of pharmaceutical synthesis does not interferes with at the peak of determinand, and the party is described
The specificity of method is good.
Embodiment 4
Research shows that carboxylic acid and amido reaction need through 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride
The activation of (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, EDC HCl)
Can carry out, and activation process is frequently added pyridine as catalyst[41].Therefore, this experiment initially selects EDC HCl and pyrrole
Pyridine accelerates hydrazides synthesis as catalyst system and catalyzing, and is successfully realized chloroacetic derivatization；But, when this condition is extended
During to other detection objects, derivatization product occurs in that abnormal instability.
Precision pipettes 100 μ L halogenated carboxylic acid reference substance test solution (50 μ g/mL), is placed in 5mL measuring bottle, adds 500 μ L and derives
Change reagent (10mg/mL), 500 μ L EDC HCl solution (20mg/mL) and 20 μ L pyridines, add 70% dilution in acetonitrile to scale, whirlpool
Rotation 10s, shakes up.Lucifuge reaction 2h, shakes up, takes 20 μ L direct injected after filtration.
The A of Fig. 4 shows, when performing the derivatization 6 kinds of halogenated carboxylic acids simultaneously, along with the increase in response time, two chloroethenes
The derivative peak response of acid, bromoacetic acid and 2 bromopropionic acid presents decline trend, correspondingly, and monoxone and the derivant of 2-chloropropionic acid
Peak response continues to increase.In order to illustrate this phenomenon, we have individually carried out derivatization to each halogenated carboxylic acid, find to add simultaneously
When adding EDC HCl and pyridine catalytic reaction, dichloroacetic acid derivant easily quick dealuminated USY cataluyst generates monoxone derivant；And
Bromoacetic acid and 2 bromopropionic acid derivant easy debrominate generation chlorination form monoxone and 2-chloropropionic acid derivant.This phenomenon can
Proved further from chromatographic retention and the mass spectral results of Fig. 5.By adjusting reaction temperature, system pH and change
The type of nitrophenyl hydrazine all can not effectively solve the problems referred to above, and this hampers the popularization and application of the method significantly.
Embodiment 5
Owing to lacking the catalysis of pyridine, therefore when using the activator EDC HCl of high concentration (2mg/mL), in order to really
Protect derivative reaction and avoid product degradation the most simultaneously, need the use of the ratio of organic facies, derivatization reagent in system
Amount and response time are investigated.With the peak area of derivatization product that generates as index, single factor exploration variable concentrations
2-nitrophenyl hydrazine hydrochloride (0.2,0.5,0.8,1.0,1.25mg/mL), the ratio (10%, 30%, 50%, 70% of organic facies
With 90%) and the response time (1,2,3,5,7h) impact on derivative reaction efficiency.Concrete outcome is shown in the B-D of Fig. 4.
Embodiment 6 Method validation and application
6.1. specificity
7 kinds of organic acid (formic acid, acetic acid, trifluoroacetic acid, first sulphur conventional in pharmaceutical synthesis has mainly been investigated in specificity test
Acid, benzoic acid, benzenesulfonic acid and p-methyl benzenesulfonic acid) material interference to target derivant that generates with 2-nitrophenyl hydrazine.Result is such as
Fig. 3, the product after these seven kinds of carboxylic acid derivatizations does not interferes with at the peak of determinand, illustrates that the specificity of the method is good.
6.2. linearity and range
Precision pipettes each reference substance storing solution and is placed in right amount in same 5mL measuring bottle respectively, adds 70% dilution in acetonitrile to carving
Degree, makes hybrid standard strain row strength solution.Perform the derivatization by method under 1.4. item, take 20 μ L and inject chromatograph of liquid,
Measure the peak area of each derivatization product.With halogenated carboxylic acid concentration C (μ g/mL) as abscissa, derivatization peak areas (A) is
Vertical coordinate, uses method of least square to carry out linear regression analysis, calculates equation of linear regression and correlation coefficient.Result shows, chlorine
Acetic acid and the 2-chloropropionic acid range of linearity are 0.05～0.5 μ g/mL, and bromoacetic acid, dichloroacetic acid and the 3-chloropropionic acid range of linearity are
0.08～0.8 μ g/mL, the 2 bromopropionic acid range of linearity is 0.12～1.2 μ g/mL.And each determinand linear relationship good (r >
0.999).The results are shown in Table 2.
6.3. detection limit and quantitative limit
Detection limit with signal to noise ratio 3:1 as method, the quantitative limit with signal to noise ratio 10:1 as method.Result shows, 6 kinds of halos
The detection of carboxylic acid limits between 0.02～0.05 μ g/mL, and quantitative limit is between 0.05～1.2 μ g/mL.Specifically it is shown in Table 2.
6.4. precision test (withinday precision and day to day precision)
Prepare 100% limit level (0.2～0.6 μ g/mL) hybrid standard product solution by method under 6.2. item, take 100 μ L
Above-mentioned solution, is placed in 5ml measuring bottle, after under 1.4. item, method addition derivatization reagent and activator carry out pre-treatment, and day
Interior precision repeats sample introduction 6 times, and day to day precision repeats sample introduction 6 times, with derivatization product calculated by peak area every day for three days on end
RSD value.As shown in Table 2, the withinday precision of the method and day to day precision are good, and RSD is respectively smaller than 1.98% and 4.39%.
6.5. derivant stability
Prepare 100% limit level (0.2～0.6 μ g/mL) hybrid standard product solution by method under 6.2. item, take 100 μ L
Above-mentioned solution, is placed in 5ml measuring bottle, after method performs the derivatization under 1.4. item, sets to 0 respectively at ambient temperatare, 1,2,3,
It is measured during 5h, investigates the stability of derivant with the change of determinand peak area.As shown in Table 2, derivatization product room temperature
During placement, in 0～5h, the RSD of peak area is less than 4.62%, i.e. has good stability.
Table 2 methodology result of the test
The concentration (μ g/mL) of x-determinand, y-peak area, the correlation coefficient of r regression equation.
6.6. average recovery
In order to evaluate the accuracy of the method, we calculate the average recovery of each halogenated carboxylic acid respectively.The medicine related to
Thing background includes tadanafil (5mg/mL), chloromycetin (15mg/mL), vitamin B6(8mg/mL), ibuprofen (8mg/mL), first
Frost spirit (10mg/mL), vilazodone intermediate (1mg/mL), cilostazol intermediate (8mg/mL) and Chloramphenicol Eye Drop
(1mg/mL)。
Response rate contrast solution: prepare 100% limit level (0.2～0.6 μ g/mL) single standard by 6.2 lower methods
Product solution, is not added with medicine background, performs the derivatization by method under 1.4. item, takes 20 μ L sample introductions after filtration.Each concentration level 6
Part.
Response rate sample solution: precision weighs appropriate each medicine background, is placed in 5mL measuring bottle, is separately added into 100 μ L
100% limit horizontal single standard product solution, derivatization after the same method, take 20 μ L sample introductions after filtration.Each concentration water
Flat 6 parts.
Average recovery=(sample peak area background peak area)/reference substance peak area × 100%.
And calculate meansigma methods and the RSD of each concentration level average recovery.
As shown in Table 3, the average recovery of each halogenated carboxylic acid is all between 82.4～105.6%, and RSD is respectively less than
4.30%.
Embodiment 7
Maximum taking dose according to medicine and each impurity TTC value, prepare the need testing solution of finite concentration medicine.Press
1.4. item method performs the derivatization, and takes 20 μ L sample introductions according to embodiment 2 method after filtration, records the peak area of derivatization product,
Calculate determinand content (μ g/g) in medicine.
Result shows, except vitamin B6, outside vilazodone intermediate and cilostazol intermediate, remaining 5 kinds of medicine is equal
Corresponding halogenated carboxylic acid can be detected.Wherein, the monoxone in tadanafil sample, the 2-chloropropionic acid in ibuprofen sample and
2 bromopropionic acid content in metalaxyl sample is all not less than quantitative limit level (< 12 μ g/g).But, chloromycetin crude drug and chlorine are mould
Dichloroacetic acid content in element eye drop respectively reaches 167 μ g/g and 454 μ g/g, and be all above standard limit.
Table 3 the method application result in 8 kinds of medicine samples
a: detection limit (μ g/g) is expressed as [detection limit (μ g/mL) of determinand]/[concentration (mg/mL) of medicine].
b: quantitative limit (μ g/g) is expressed as [quantitative limit (μ g/mL) of determinand]/[concentration (mg/mL) of medicine].
ND: be not detected by.
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Claims (11)
1. the method that derivatization HPLC-DAD method measures little molecule halogenated carboxylic acid, it is characterised in that comprise the steps of
(1) at normal temperatures, use Nitrobenzol hydrazine derivatization reagent to little molecule halogenated carboxylic acid derivative reaction, generate at 320-
450nm has the relatively strong product absorbed；
(2) with derivative reaction product in step (1), HPLC-DAD method is utilized, based on reversed phase partition chromatography principle, at 320-
450nm measures the derivatization product of wherein halogenated carboxylic acid, thus realizes the qualitative or quantitative detection to little molecule halogenated carboxylic acid.
Method the most according to claim 1, it is characterised in that comprise the steps of
(1) at normal temperatures, with acetonitrile-water as reaction system, Nitrobenzol hydrazine derivatization reagent is used, with carbodiimide class coupling
The hydrochlorate of agent is activator, generates little molecule halogenated carboxylic acid derivative reaction and has the relatively strong product absorbed at 330～420 nm
Thing；
(2) with derivative reaction product in step (1), utilize HPLC-DAD method, based on reversed phase partition chromatography principle 330～
420 nm measure the derivatization product of wherein halogenated carboxylic acid, thus realize the qualitative or quantitative detection to little molecule halogenated carboxylic acid.
Method the most according to claim 2, it is characterised in that described little molecule halogenated carboxylic acid is the C1 ~ C6 of halogen substiuted
Carboxylic acid；Described carbon carbodiimide class coupling agent is selected from dicyclohexylcarbodiimide, N, N'-DIC, 1-
Any one in ethyl-(3-dimethylaminopropyl) carbodiimide；Nitrobenzol hydrazine derivatization reagent is selected from 2-Nitrobenzol
Any one in hydrazine hydrochloride, 3-nitrophenyl hydrazine hydrochloride, 4-nitrophenyl hydrazine hydrochloride and 2,4 dinitrophenyl hydrazine.
Method the most according to claim 3, it is characterised in that described little molecule halogenated carboxylic acid is selected from monoxone, bromine second
Any one in acid, dichloroacetic acid, 2-chloropropionic acid, 3-chloropropionic acid or 2 bromopropionic acid；Described carbon carbodiimide class coupling agent
For 1-ethyl-(3-dimethylaminopropyl) carbodiimide, described Nitrobenzol hydrazine derivatization reagent is 2-Nitrobenzol hydrazonium salt
Hydrochlorate.
Method the most according to claim 4, it is characterised in that in acetonitrile-water reaction system the volumetric concentration of acetonitrile be 10% ~
90%；1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride concentration is 1 ~ 4 mg/mL, 2-nitrophenyl hydrazine hydrochloride
Concentration in derivative reaction system is 0.2～3 mg/mL；The derivative reaction time is 1 ~ 3h.
Method the most according to claim 1, it is characterised in that the HPLC-DAD method described in step (2) uses HPLC chromatogram
Instrument；Use reversed phase partition chromatography；With non-polar linkage for fixing phase, using polarity flowing phase, detection wavelength is positioned at 320-
Between 450nm, it is preferably placed between 330～420 nm, is further preferably positioned between 390 ~ 400nm.
7. the application in measuring medicine small molecular halogenated carboxylic acid of the method according to any one of claim 1 ~ 6.
8. the method that derivatization HPLC-DAD method measures medicine small molecular halogenated carboxylic acid, it is characterised in that comprise the steps of
(1) under room temperature, under the activation of the hydrochlorate of carbodiimide class coupling agent, Nitrobenzol hydrazine derivatization reagent is used
Medicine to be measured is performed the derivatization reaction, and reactant liquor measures as sample feeding；
(2) reactant liquor after terminating using derivative reaction in step (1), as sample introduction sample, utilizes HPLC-DAD method at 320-
450nm measures the derivatization product of wherein halogenated carboxylic acid, thus realize medicine small molecular halogenated carboxylic acid qualitative or quantitative
Detection.
Method the most according to claim 8, it is characterised in that described little molecule halogenated carboxylic acid selected from halogen substiuted C1 ~
Any one in the carboxylic acid of C6, preferably monoxone, bromoacetic acid, dichloroacetic acid, 2-chloropropionic acid, 3-chloropropionic acid or 2 bromopropionic acid；
Described carbodiimide class coupling agent salt is selected from dicyclohexylcarbodiimide, N, N'-DIC, 1-ethyl-(3-
Dimethylaminopropyl) one in carbodiimide, preferably 1-ethyl-(3-dimethylaminopropyl) carbodiimide；Described
Nitrobenzol hydrazine derivatization reagent selected from 2-nitrophenyl hydrazine hydrochloride, 3-nitrophenyl hydrazine hydrochloride, 4-nitrophenyl hydrazine hydrochloride
With any one in 2,4 dinitrophenyl hydrazine, preferably 2-nitrophenyl hydrazine hydrochloride.
Method the most according to claim 8, it is characterised in that the condition of derivative reaction is under room temperature, with acetonitrile volume
Concentration be the acetonitrile-water of 10% ~ 90% be reaction system, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride concentration
Being 1 ~ 4 mg/mL, 2-nitrophenyl hydrazine hydrochloride concentration is 0.2 ~ 3mg/mL, and the response time is 1 ~ 3h；Described derivative reaction
Condition preferably with acetonitrile-water=70:30(V:V) as reaction system, 1-ethyl-(3-dimethylaminopropyl) carbodiimide salt
Hydrochlorate 2 mg/mL, 2-nitrophenyl hydrazine hydrochloride concentration is 1mg/mL, and the response time is 2h.
11. methods according to claim 9, it is characterised in that the HPLC-DAD method described in step (2) uses HPLC color
Spectrometer；Use reversed phase partition chromatography；With non-polar linkage for fixing phase, using polarity flowing phase, detection wavelength is positioned at
Between 320-450nm, it is preferably placed between 330 ~ 420nm, is further preferably positioned between 390 ~ 400nm；Described HPLC-
The instrument that DAD method is preferably used is Shimadzu LC 20AT chromatograph of liquid, this chromatograph be formulated with online vacuum degassing machine,
Binary gradient pump, automatic sampler, column oven, DAD detector and LC-solution chromatographic work station；Chromatographic column uses 250
Mm × 4.6 mm, the Shimadzu Inertsil ODS-3 of 5 μm；Sample size: 20 μ L；Flow rate of mobile phase: 1.0 mL/min；Flowing phase
Gradient: A phase is acetonitrile, B phase is 0.1% phosphoric acid, 0min 28%A phase, 10min 40%A phase, 14min 40%A phase, 20min 55%
A phase；Column temperature: 40 DEG C；Detection wavelength: 392 nm.
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| CN115266986A (en) * | 2022-07-29 | 2022-11-01 | 江苏慧聚药业股份有限公司 | Method for measuring EDCI residue in Toxolitinib |
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