CN107014944A - The method that derivatization HPLC DAD methods determine acyl chlorides in medicine or its synthetic intermediate - Google Patents
The method that derivatization HPLC DAD methods determine acyl chlorides in medicine or its synthetic intermediate Download PDFInfo
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Abstract
The invention discloses a kind of method that derivatization HPLC DAD methods determine acyl chlorides in medicine or its synthetic intermediate, including at room temperature, there is the product absorbed more by force in ultraviolet visible light region to being generated after acyl chlorides derivatization using nitrobenzene hydrazine derivatization reagent；Derivative reaction liquid is as sample introduction sample, and the principle based on RPLC separates in ultraviolet visible light region and determines the derivatization product of acyl chlorides, so as to realize the qualitative or quantitative detection to acyl chlorides.Fast reaction characteristic of the invention based on acyl chlorides and diazanyl, and ultraviolet-visible absorption characteristic of the acyl chlorides with generating product after nitrophenyl hydrazine derivative reaction, the interference that medicine or its synthetic intermediate matrix are determined to acyl chlorides is effectively prevented from, so as to establish a kind of method that simple, general derivatization HPLC DAD methods determine acyl chlorides in medicine or its synthetic intermediate.The result of Method validation shows that this method specificity and sensitivity are good.
Description
Technical field
The invention belongs to drugs analysis detection field, it is related to a kind of method that derivatization HPLC-DAD methods determine acyl chlorides, especially
It is related to the method that derivatization HPLC-DAD methods determine acyl chlorides in medicine or its synthetic intermediate.
Background technology
In recent years, in medicine genotoxicity impurity (Genotoxic impurities, GTIs) to the mankind due to depositing
It is in widespread attention, it is necessary to be controlled in a level of security in potential carcinogenicity[1].Gas chromatography (GC), efficiently
Liquid chromatography (HPLC) or Capillary Electrophoresis chromatography (CE) method are with mass spectrum (MS) GC-MS in genotoxicity impurity point
Played an important role in analysis[2-3].Some analysis strategies such as chemical derivatization[4]The detectability of analyte can be improved, or
The design (DoE) of person's experiment[5]The system optimization of carry out method condition can be assisted.Acyl chlorides is important acylation in pharmaceutical synthesis
Reagent[6-12].For example, chloroacetic chloride and 2- chlorpromazine chlorides are to can be used for synthesis brufen；Chloracetyl chloride is usually used in the system of Tadalafei
It is standby；5- chlorothiophene -2- formyl chlorides are to synthesize important intermediate of razaxaban etc..Querying acyl chlorides in spite of document causes gene to be dashed forward
The possibility of change[13], but regulator still attaches great importance to the monitoring that acyl chlorides is remained in medicine[14].Therefore need to set up one
The method that specificity is strong, sensitivity is high is planted to be monitored this kind of impurity in medicine.
Typically, since acyl chlorides is very active, the result of mistake is directly often produced with HPLC methods or the analysis of GC methods.
Therefore it is used to detect acyl chlorides frequently with derivatization method.Water is to detect one of derivatization reagent that acyl chlorides is commonly used, and acyl chlorides and water spread out
Product passes through CE after biochemistry[14]Or HPLC-MS[15]Analyzed, but Hydrolyze method cannot distinguish between acyl chlorides and medicine exists in itself
Corresponding carboxylic acid, cause determine result it is higher[16].Also once report used GC-MS to document[17]Or GC-FID[18]Method is to acyl chlorides
Detected after Derivatization of methanol, but this method can usually be competed by moisture, and reaction rate is slow, it is necessary to be catalyzed
Agent.And the rate of recovery of the both approaches in non-protonic solvent is all poor[16].The reaction speed of acyl chlorides and alkali compounds
Rate is higher than the reaction rate of acyl chlorides and water or alcohols, and amino or diazanyl are common basic group, and applicant attempts selection and contained
The compound for having this two kinds of groups performs the derivatization measure.
In various method for separating and analyzing, HPLC-DAD methods become the head in most of laboratories because its is simple and practical
Choosing method, but be due to that most drug and its synthetic intermediate have stronger UV absorption, therefore specificity deficiency is the method
Major defect.Applicant passes through the ultraviolet suction to Amino-substituted compounds or diazanyl substituted compound and acyl chlorides derivatization product
Spectrum is received to compare, find the product generated after nitrobenzene hydrazine derivatization reagent and acyl chlorides derivative reaction can avoid medicine and
The matrix interference of its synthetic intermediate, is further optimized by derivatising condition, has been invented a kind of derivatization HPLC-DAD methods and has been surveyed
Determine the method for acyl chlorides in medicine or its synthetic intermediate.
The content of the invention
The deficiency of the present invention is that there is provided the method that derivatization HPLC-DAD methods determine acyl chlorides in view of the shortcomings of the prior art.
Acyl chlorides in medicine or its synthetic intermediate is determined it is a further object to provide derivatization HPLC-DAD methods
Method.
The purpose of the present invention can be achieved through the following technical solutions：
The method that derivatization HPLC-DAD methods determine acyl chlorides, is comprised the steps of：
(1), at room temperature, acyl chlorides derivatization is generated in ultraviolet visible light region (320 using nitrophenyl hydrazine derivatization reagent
~450nm) in have the product absorbed more by force；
(2), with step (1) derivedization reaction product, using HPLC reversed phase partition chromatographies in ultraviolet visible light region
(320~450nm) determines the derivatization product of wherein acyl chlorides, so as to realize the qualitative or quantitative detection to acyl chlorides.
It is preferred that, the method that described derivatization HPLC-DAD methods determine acyl chlorides is comprised the steps of：
(1) it is anti-to acyl chlorides derivatization using nitrobenzene hydrazine derivatization reagent, at normal temperatures, using acetonitrile as reaction system
The product for having stronger absorption in ultraviolet visible light region (330~420nm) should be generated；
(2), with step (1) derivedization reaction product, using HPLC-DAD methods, existed based on reversed phase partition chromatography principle
Ultraviolet visible light region (330~420nm) determines the derivatization product of wherein acyl chlorides, so as to realize the qualitative or quantitative inspection to acyl chlorides
Survey.
Described acyl chlorides includes aliphatic acyl chlorides or aromatic series acyl chlorides, such asShown, R is selected from substitution or unsubstituted
C1~C6 alkyl, substituted or unsubstituted five to nine circle heterocycles containing S, N, O, substituted or unsubstituted phenyl ring, naphthalene nucleus, anthracene
Ring, phenanthrene ring；Wherein, the substituent of C1~C6 alkyl is halogen such as chlorine, bromine；Heterocycle or phenyl ring, naphthalene nucleus, anthracene nucleus, the substitution of phenanthrene ring
Base is chlorine, bromine, nitro, amino, substituted or unsubstituted C1~C6 alkyl, substituted or unsubstituted C1~C4 alkoxies, alkyl
Or the substituent of alkoxy is halogen.
R be preferably substituted or unsubstituted C1~C4 alkyl, substituted or unsubstituted five yuan containing S, N, O or hexa-atomic miscellaneous
Ring, substituted or unsubstituted chlorobenzoyl chloride；The substituent of C1~C4 alkyl is halogen such as chlorine, bromine；Five yuan or hexa-member heterocycle, benzene first
The substituent of acyl chlorides is chlorine, bromine, nitro, amino, substituted or unsubstituted C1~C6 alkyl, substituted or unsubstituted C1~C4 alkane
The substituent of epoxide, alkyl or alkoxy is halogen.
Described acyl chlorides further preferably from chloroacetic chloride, chloracetyl chloride, 2- chlorpromazine chlorides, isobutyryl chloride, chlorobenzoyl chloride and
Any one in 5- chlorothiophene -2- formyl chlorides.
Nitrobenzene hydrazine derivatization reagent preferably is selected from 2- nitrophenyl hydrazines, 3- nitrophenyl hydrazines, 4- nitrophenyl hydrazines, 2,3- dinitros
Any one in phenylhydrazine, DNPH, 3,4- dinitrophenylhydrazines, preferably 2- nitrophenyl hydrazines.
It is further preferred that the condition of derivative reaction：Using acetonitrile as reaction system, 2- nitrophenyl hydrazines rub with total acyl chlorides
You are than being 1.45:1~145:1, react 0.5~3h；It is preferred that using acetonitrile as reaction system, mole of 2- nitrophenyl hydrazines and total acyl chlorides
Than for 14.5:1, the reaction time is 0.5h.
It is preferred that, described HPLC-DAD methods：Using HPLC chromatogram instrument；Using reversed phase partition chromatography；With nonpolar bond
It is mutually stationary phase to close, and using polarity mobile phase, Detection wavelength is located between 320~450nm, further preferably positioned at 330~
Between 420nm, still more preferably between 390~400nm.
Still more preferably, the instrument that described HPLC-DAD methods are used is Shimadzu LC 20AT liquid chromatograies
Instrument, the chromatograph is configured with online vacuum degassing machine, binary gradient pump, automatic sampler, column oven, DAD detectors and LC-
Solution chromatographic work stations；Chromatographic column uses 250mm × 4.6mm, the Diamonsil of 5 μm of Di Ma scientific ＆ technical corporationTM C18
Post；Sample size：20μL；Flow rate of mobile phase：1.0mL/min；Eluent gradient：A phases are acetonitrile, and B phases are 0.1% phosphoric acid, 0min
30%A phases, 5min 40%A phases, 10min 40%A phases, 15min 60%A phases, 20min 60%A phases, 22min 70%A phases,
25min 70%A phases；Column temperature：30℃；Detection wavelength：395nm.
Method of the present invention be used for several samples in acyl chlorides qualitative and quantitative detection, preferably determine medicine and its
The application of acyl chlorides in synthetic intermediate.
The method that derivatization HPLC-DAD methods determine acyl chlorides in medicine and its synthetic intermediate, is comprised the steps of：
(1) 0.5~3h of reaction, at room temperature, is performed the derivatization using nitrobenzene hydrazine derivatization reagent, reaction solution is used as sample
Product sample introduction is determined；
(2) reaction solution after terminating, is reacted as sample introduction sample using step (1) derivedization, existed using HPLC-DAD methods
320-450nm determines the derivatization product of wherein acyl chlorides, so as to realize the qualitative or quantitative detection to acyl chlorides in medicine.
Described acyl chlorides includes aliphatic acyl chlorides or aromatic series acyl chlorides, such asShown, R is selected from substitution or unsubstituted
C1~C6 alkyl, substituted or unsubstituted five to nine circle heterocycles containing S, N, O, substituted or unsubstituted phenyl ring, naphthalene nucleus, anthracene
Ring, phenanthrene ring；Wherein, the substituent of C1~C6 alkyl is halogen such as chlorine, bromine；Heterocycle or phenyl ring, naphthalene nucleus, anthracene nucleus, the substitution of phenanthrene ring
Base is chlorine, bromine, nitro, amino, substituted or unsubstituted C1~C6 alkyl, substituted or unsubstituted C1~C4 alkoxies, alkyl
Or the substituent of alkoxy is halogen.
R be preferably substituted or unsubstituted C1~C4 alkyl, substituted or unsubstituted five yuan containing S, N, O or hexa-atomic miscellaneous
Ring, substituted or unsubstituted chlorobenzoyl chloride；The substituent of C1~C4 alkyl is halogen such as chlorine, bromine；Five yuan or hexa-member heterocycle, benzene first
The substituent of acyl chlorides is chlorine, bromine, nitro, amino, substituted or unsubstituted C1~C6 alkyl, substituted or unsubstituted C1~C4 alkane
The substituent of epoxide, alkyl or alkoxy is halogen.
Described acyl chlorides further preferably from chloroacetic chloride, chloracetyl chloride, 2- chlorpromazine chlorides, isobutyryl chloride, chlorobenzoyl chloride and
Any one in 5- chlorothiophene -2- formyl chlorides.
Nitrobenzene hydrazine derivatization reagent preferably is selected from 2- nitrophenyl hydrazines, 3- nitrophenyl hydrazines, 4- nitrophenyl hydrazines, 2,3- dinitros
Any one in phenylhydrazine, DNPH, 3,4- dinitrophenylhydrazines, preferably 2- nitrophenyl hydrazines.
It is further preferred that the condition of derivative reaction：It is characterized in that using acetonitrile as reaction system, 2- nitrophenyl hydrazines are dense
Spend for 0.01~1mg/mL, 0.5~3h of reaction；It is preferred that using acetonitrile as reaction system, 2- nitrophenyl hydrazines concentration is 100 μ g/mL, instead
It is 0.5h between seasonable.
It is preferred that, described HPLC-DAD methods：Using HPLC chromatogram instrument；Using reversed phase partition chromatography；With nonpolar bond
It is mutually stationary phase to close, and using polarity mobile phase, Detection wavelength is located between 320~450nm, further preferably positioned at 330~
Between 420nm, still more preferably between 390~400nm.
Still more preferably, the instrument that described HPLC-DAD methods are used is Shimadzu LC 20AT liquid chromatograies
Instrument, the chromatograph is configured with online vacuum degassing machine, binary gradient pump, automatic sampler, column oven, DAD detectors and LC-
Solution chromatographic work stations；Chromatographic column uses 250mm × 4.6mm, the Diamonsil of 5 μm of Di Ma scientific ＆ technical corporationTM C18
Post；Sample size：20μL；Flow rate of mobile phase：1.0mL/min；Eluent gradient：A phases are acetonitrile, and B phases are 0.1% phosphoric acid, 0min
30%A phases, 5min 40%A phases, 10min 40%A phases, 15min 60%A phases, 20min 60%A phases, 22min 70%A phases,
25min 70%A phases；Column temperature：30℃；Detection wavelength：395nm.
Described medicine and its synthetic intermediate is preferably brufen, Tadalafei, razaxaban, Ketoprofen, pungent cuts down him
Spit of fland and vilazodone, but it is not limited to above-claimed cpd.
Room temperature of the present invention refers to the temperature that conventional analysis experiment is carried out, usually between 20~30 DEG C.
Beneficial effects of the present invention：
Most drug and its impurity are very weak near visible area UV absorption, and the acyl chlorides of the invention based on nitrophenyl hydrazine spreads out
Biochemical products due to the presence of nitro electron withdraw group on phenyl ring, therefore UV absorption take back the obvious redshift effect of generation, can
To avoid the matrix interference of medicine and its synthetic intermediate, establish a kind of simple, general pre-column derivatization HPLC-DAD and survey
Determine the method for acyl chlorides.The result of Method validation shows that the carboxylic acid contained in itself and impurity in medicine will not cause to do to analysis
Disturb, show that this method specificity is good.In addition, the detection of method is limited to 0.01-0.03 μ g/mL, 0.03-0.08 μ are quantitatively limited to
G/mL, the good (r of linear relationship>0.999)；Sample introduction precision is less than 0.97%；Average recovery rate is between 87.8-114.1%
(RSD<3.36%), without obvious matrix interference；And derivatization product is good in 8h internal stabilities.
2- is worked as in the non-appearance of 2- chlorpromazine chlorides occurred during optimizing for derivatising condition, discovery creatively
During nitrophenyl hydrazine excessive concentration, the derivatization product of acyl chlorides may degrade.Based on this discovery, to derivatization reagent and total acyl
The mol ratio of chlorine is optimized, and not only can ensure that acyl chlorides by complete derivatization, moreover it is possible to be prevented effectively from the degraded of product.
Brief description of the drawings
Fig. 1 is respectively with aniline (A), 2- nitroanilines (B), 4- nitroanilines (C), phenylhydrazine (D), 2- nitrophenyl hydrazines (E)
The uv absorption spectra for generating product is reacted as derivatization reagent and isobutyryl chloride with 4- nitrophenyl hydrazine hydrochlorides (F).Spread out
The concentration of biochemical reagents is 100 μ g/mL.
Fig. 2 is the chromatogram (a-e obtained with Ketoprofen (2mg/mL) and 2- nitrophenyl hydrazines (100 μ g/mL) derivative reaction
Represent different Detection wavelengths)：a.224nm,b.274nm,c.314nm,d.356nm,e.395nm.(Imp 1-7 represent Ketoprofen
Contained unknown endogenous impurity in bulk drug).
Fig. 3 is influence of the reaction condition to acyl chlorides derivatization：(A) mol ratio of 2- nitrophenyl hydrazines/total acyl chlorides is to derivatization
The influence of reaction efficiency；(B) influence of the reaction temperature to derivative reaction efficiency；(C) reaction time is to derivative reaction efficiency
Influence；(D) influence of the water, methanol and the ethanol of 1% (v/v) content to derivative reaction efficiency.Derivative reaction efficiency is led to
Cross normalized peak area to represent, normalization peak area is the maximum peak area under testing with each derivative reaction parameter optimization
For 100% control, the ratio of other peak areas and maximum peak area is referred to as normalizing peak area.The concentration of chloroacetic chloride is 0.5 μ g/
ML, the concentration of remaining acyl chlorides is 1 μ g/mL.
Fig. 4 is blank solution (2-NPH) (a), acetic acid (b), monoxone (c), isobutyric acid (d), 2- chlorine third under the same terms
Sour (e), benzoic acid (f) and 5- chlorothiophene -2- formic acid (g) and 6 kinds of aliphatic and aromatic series acyl chlorides (h) derivatization product
Chromatogram：(1) acetyl chlorine derivative；(2) chloracetyl chloride derivative；(3) isobutyryl chloride derivative；(4) 2- chlorpromazine chlorides derive
Thing；(5) benzoyl chloride derivative；(6) 5- chlorothiophenes -2- formyl chlorine derivatives.The concentration of carboxylic acid is 10 μ g/mL.
For mixing acyl chlorides (1 μ g/mL) in same medicine with 2- nitrophenyl hydrazines (100 μ g/mL) derivative reaction occurs for Fig. 5
Chromatogram afterwards (a-f represents different medicines)：Brufen (a), Tadalafei (b), Simvastatin (c), Ketoprofen (d), profit
Cut down husky class (e) and vilazodone (f)；The derivatization product of six kinds of aliphatic and aromatic series acyl chlorides：(1) acetyl chlorine derivative；(2)
Chloracetyl chloride derivative；(3) isobutyryl chloride derivative；(4) 2- chlorpromazine chlorides derivative；(5) benzoyl chloride derivative；(6)5-
Chlorothiophene -2- formyl chlorine derivatives.
Embodiment
1.1. instrument
Shimadzu LC 20AT liquid chromatographs (are contained in line vacuum degasser, binary gradient pump, automatic sampler, post
Incubator, DAD detectors and LC-solution chromatographic work stations)；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 detectors, METTLERTOLEDO
AB135-S assay balances (plum Teller swedish company)；Sartorious BS110 assay balances (the limited public affairs of Beijing Sai Duolisi
Department).
1.2. reagent
Chloroacetic chloride (98%), chloracetyl chloride (97%), 2- chlorpromazine chlorides (97%), chlorobenzoyl chloride (98%), isobutyryl chloride
And 5- chlorothiophene -2- formyl chlorides (97%) (98%)；Aniline (99.5%), phenylhydrazine (98.0%), 2- nitroanilines (98.5%),
4- nitroanilines (99.5%), 2- nitrophenyl hydrazines (97%) and 4- nitrophenyl hydrazine hydrochlorides (97%)；Brufen (98%), he reaches
Draw non-(97%), razaxaban (99%), Ketoprofen (98%), Simvastatin (98%) and vilazodone (99.5%).
Purified water, acetonitrile (TEDIA, chromatographically pure), phosphoric acid (analysis is pure).
1.3. the preparation of solution
The storing solution of various acyl chlorides：Each acyl chlorides about 10mg acyl chlorides is taken, it is accurately weighed, it is placed in 10mL measuring bottles, it is dilute with acetonitrile
Release to graduation mark, shake up.8h internal stabilities are good at room temperature for each storing solution.
Aniline, 2- nitroanilines (2-NA), 4- nitroanilines (4-NA), phenylhydrazine, 2- nitrophenyl hydrazines (2-NPH) and 4- nitros
Hydrazinobenzene hydrochloride salt (4-NPHHCl) test solution：Each reagent about 20mg is taken, it is accurately weighed, it is placed in 10mL measuring bottles, it is dilute with pure acetonitrile
Release to scale, shake up.
1.4. derivatization experiment condition
Precision pipettes 100 μ L acyl chlorides mother liquor, is placed in 10mL measuring bottles, is separately added into 500 μ L derivatization reagents plus acetonitrile
Scale is diluted to, is shaken up.At room temperature after reaction 1h, 20 μ L direct injecteds are taken.
Embodiment 1
Using isobutyryl chloride as representative, with the aniline of same concentrations, 2- nitroanilines, 4- nitroanilines, phenylhydrazine, 2- nitrobenzene
Hydrazine, 4- nitrophenyl hydrazine derivatization reagents are performed the derivatization to it.Derivatization experiment condition is shown in 1.4.Under identical chromatographic condition,
Each derivatization product is scanned with DAD, the spectrogram and chromatogram of product are recorded, with the maximum absorption wavelength and absorption intensity of product
To evaluate the quality of derivatization reagent.
Figure 1A show the derivatization product uv-absorption maximum wavelength of aniline and its isobutyryl chloride be respectively 234nm and
241nm.And the maximum absorption wavelength red shift of 2- nitroanilines and 4- nitroanilines is to 408nm (Figure 1B) and 374nm (Fig. 1 C).But
It is that their own isobutyryl chloride derivatization product peak makes absorbing wavelength there occurs blue shift and absorption because weakening conjugation
Intensity diminishes.
Fig. 1 D show that phenylhydrazine and its isobutyryl chloride derivatization product have maximal ultraviolet absorption at 224nm and 233nm respectively
Wavelength.And 2- nitrophenyl hydrazines and 4- nitrophenyl hydrazines have absorption well strong in 383nm (Fig. 1 E) and 337nm (Fig. 1 F) place respectively
Degree, and be increased slightly with the maximum absorption wavelength and absorption intensity of product after isobutyryl chloride generation derivatization.This experiment is managed
Situation about thinking be near visible area (>380nm) place is measured, the base that medicine and its endogenous impurity can so produced
Matter interference is preferably minimized, so as to improve the specificity of method.From the maximum absorption wavelength and absorption intensity of Fig. 1 derivatization product
From the point of view of, nitrophenyl hydrazine analog derivative meets above-mentioned requirements, especially the most suitable with 2- nitrophenyl hydrazines.
In actual test sample, due to the presence of high amount of drug substrate, influenceed for micro acyl chlorides derivatization product compared with
Greatly, each medicine will set up a chromatographic condition and remove separation medicine and impurity if promoting the use of, and disturb many, sensitivity is low
(baseline noise is big).And when using 2- nitrophenyl hydrazines as derivatization reagent, because most drug and its impurity are near visible
Light area (>380nm) UV absorption is very weak, so the specificity of method can be greatly improved by being measured at this wavelength.For example
When being performed the derivatization with 2- nitrophenyl hydrazines to a certain amount of Ketoprofen, and the chromatogram under different Detection wavelengths recorded using DAD
(Fig. 2) has found, compared to other shorter wavelengths, the chromatogram under 395nm wavelength detectings can provide the baseline that more balances and
Less Impurity Absorption peak, so as to improve the specificity of method.The fact that as the important advantage of 2- nitrophenyl hydrazines at it
Also there is embodiment on his medicine.
Embodiment 2
It is prepared by need testing solution：Precision weighs medicine in right amount, is placed in 10mL measuring bottles, adds 500 μ L derivatization reagents, plus
Dilution in acetonitrile is shaken up to scale, and 0.5h is reacted at room temperature, produces need testing solution.Take 20 μ L direct injecteds.
It is prepared by reference substance solution：Precision pipettes 100 μ L various concentrations acyl chlorides storing solutions, is placed in 10mL measuring bottles, adds 500
μ L derivatization reagents, plus dilution in acetonitrile is to scale, shakes up, and 0.5h is reacted at room temperature, produces reference substance solution.20 μ L are taken directly to enter
Sample.
Chromatographic condition：Shimadzu LC 20AT liquid chromatographs (are contained in line vacuum degasser, binary gradient pump, automatically
Injector, column oven, DAD detectors and LC-solution chromatographic work stations)；Chromatographic column：Di Ma scientific ＆ technical corporation
DiamonsilTMC18(250mm×4.6mm,5μm)；Mobile phase：Acetonitrile (A phases) -0.1% phosphoric acid (B phases), gradient elution is shown in Table
1, flow velocity：1.0mL/min；Sample size：20μL；Column temperature：30℃；Detection wavelength：395nm.
The gradient elution program of table 1.
Specification Curve of Increasing method：Precision pipettes each reference substance storing solution and is placed in right amount in same 10mL measuring bottle respectively,
Plus dilution in acetonitrile is to scale, hybrid standard strain row strength solution is made.Performed the derivatization by 1.4. lower methods, take 20 μ L to note
Enter liquid chromatograph, determine the peak area of each derivatization product.With acyl chlorides concentration C (μ g/mL) for abscissa, derivatization product peak
Area (A) is ordinate, and linear regression analysis is carried out using least square method, calculates equation of linear regression and coefficient correlation.
Quantitative approach：Quantified using external standard method.
Embodiment 3
In order to ensure derivatization fast reaction completely, while avoiding product degradation, it is necessary to the derivatization reagent in system
Concentration, reaction time and reaction temperature investigated.Meanwhile, because the presence of water, methanol or ethanol can be with 2- nitrobenzene
Hydrazine competes acyl chlorides, so needing to investigate the content of the reagent of these three in reaction system.With the derivatization product of generation
Normalization peak area is index, the single factor exploration different mol ratio of the total acyl chlorides of 2-NPH/ (1.45,2.90,7.24,14.5,
29.0th, 72.4,145.0), different reaction temperature (25,40,60,80 DEG C), the different reaction time (15,30,60,90,
120th, 180min) and 1% content the influence to derivative reaction efficiency of water, methanol or ethanol.Concrete outcome is shown in Fig. 3.
From Fig. 3 A, the peak area of derivatization product increases with the increase of derivatization reagent concentration, until 2- nitre
Base phenylhydrazine concentration is that 100 μ g/mL (are 14.5 equivalent to mol ratio：1) maximum is reached when, when 2- nitrophenyl hydrazine excessive concentrations
When, the derivatization product of acyl chlorides may degrade, so we have finally chosen behind the continuation of 100 μ g/mL 2- nitrophenyl hydrazines
Research.From Fig. 3 B, increase reaction temperature does not have much affect to derivatization peak areas, illustrates the derivative at room temperature
Changing reaction just can quickly be carried out.The reaction time for understanding 30min by 3C is just enough to make derivative reaction complete.From Fig. 3 D,
1% (v/v) water, methanol or ethanol is not interfered with to derivative reaction.
Embodiment 4
As described in Example 2, to chloroacetic chloride, chloracetyl chloride, isobutyryl chloride, 2- chlorpromazine chlorides, chlorobenzoyl chloride and 5- chlorine
Thiophene -2- formyl chlorides are investigated.As shown in Figure 4, under above-mentioned chromatographic condition, 6 kinds of derivatization reagent products and derivatization examination
Between agent separating degree preferably, in view of in medicine it is that may be present correspondence acyl chlorides endogenous carboxylic acid (acetic acid, monoxone, isobutyl
Acid, 2- chloropropionic acids, benzoic acid and 5- chlorothiophene -2- formic acid) it may be reacted with 2- nitrophenyl hydrazines, so this experiment is in phase
This six kinds of carboxylic acids are investigated with conditions of, studying it can or can not produce with derivative reaction under identical reaction conditions
Identical derivatization product.As a result as shown in figure 4, due to the reactionlessness of carboxylic acid and 2- nitrophenyl hydrazines under same reaction conditions,
It is the derivatization product peak that carboxylic acid is not observed at 395nm in absorbing wavelength.Illustrate that this method specificity is good.
Embodiment 5
Investigate the ability that this method distinguishes different acyl chlorides in identical medicine.Need the dissolving in acetonitrile according to medicine
Degree, precision weighs appropriate medicine, is placed in 10mL measuring bottles, adds 500 μ L derivatization reagents, adds the mixing of same concentrations
Solution of acid chloride (1 μ g/mL), plus dilution in acetonitrile is to scale, shakes up, and 0.5h is reacted at room temperature.Take 20 μ L direct injecteds.As a result as schemed
Shown in 5, illustrate that this method distinguishes that the ability of different acyl chlorides is good in identical drug matrices.
The Method validation of embodiment 6 and application
6.1. specificity
Specificity mainly investigates the corresponding carboxylic acid of acyl chlorides carboxylic (acetic acid, monoxone, isobutyric acid, 2- chloropropionic acids, benzoic acid and 5-
Chlorothiophene -2- formic acid) can or can not with 2- nitrophenyl hydrazines generate identical target derivative.As a result such as Fig. 4, due to carboxylic acid and 2-
NPH reactionlessness, is the derivatization product peak that carboxylic acid is not observed at 395nm in absorbing wavelength.Illustrate that this method is exclusive
Property is good.
6.2. linearity and range
Precision pipettes each acyl chlorides storing solution and is placed in right amount in same 10mL measuring bottle respectively, plus dilution in acetonitrile is to scale, makes
Into hybrid standard strain row strength solution.Performed the derivatization by 1.4. lower methods, take 20 μ L to inject liquid chromatograph, determine each
The peak area of derivatization product.With acyl chlorides concentration C (μ g/mL) for abscissa, derivatization peak areas (A) is ordinate, is adopted
Linear regression analysis is carried out with least square method, equation of linear regression and coefficient correlation is calculated.As a result show, chloroacetic chloride it is linear
Scope is 0.03-0.3 μ g/mL, and the range of linearity of chloracetyl chloride and isobutyryl chloride is 0.05-0.5 μ g/mL, 2- chlorpromazine chlorides
The range of linearity with chlorobenzoyl chloride is that the range of linearity of 0.06-0.6 μ g/mL, 5- chlorothiophene -2- formyl chlorides is 0.08-0.8 μ g/
mL.And good (the r of each determinand linear relationship>0.999).It the results are shown in Table 2.
6.3. test limit and quantitative limit
With signal to noise ratio 3:1 is the test limit of method, with signal to noise ratio 10:1 is the quantitative limit of method.As a result show, 6 kinds of halos
The test limit of carboxylic acid is between 0.01-.03 μ g/mL, and quantitative limit is between 0.03-0.08 μ g/mL.Specifically it is shown in Table 2.
6.3. sample introduction precision test
100% limit level (0.18-0.48 μ g/mL) mixed standard solution is prepared by 6.2. lower methods, is taken on 100 μ L
Solution is stated, is placed in 10mL volumetric flasks, adding derivatization reagent according to 1.4 lower methods is carried out after pre-treatment, repeats sample introduction 6
It is secondary, with derivatization product calculated by peak area RSD values.As shown in Table 2, the sample introduction precision of the method is good, and RSD is in 0.38-
Between 0.90%.
6.5. derivative stability
100% limit level (0.18~0.48 μ g/mL) mixed standard solution is prepared by 6.2. lower methods, 100 μ L are taken
Above-mentioned solution, is placed in 10mL volumetric flasks, and adding derivatization reagent according to 1.4 lower methods is carried out after pre-treatment, respectively in room
Temperature decentralization set to 0,1,2,4,6,8h when be measured, with determinand peak area change investigate derivative stability.Can by table 2
Know, when derivatization product room temperature is placed, the RSD of peak area is less than 0.97% in 0-8h, that is, has good stability.
The methodology result of the test of table 2.
The concentration (μ g/mL) of x- determinands, y- peak areas, the coefficient correlation of r-regression equation
6.6. average recovery
In order to evaluate the accuracy of this method, we calculate the average recovery of each acyl chlorides respectively.The medicine being related to is included
Brufen (4mg/mL), Tadalafei (6mg/mL), vilazodone (1mg/mL), Simvastatin (1mg/mL), Ketoprofen (2mg/
ML), profit cuts down his class (2mg/mL).
Rate of recovery contrast solution：LOQs, 6LOQs and 10LOQs mixed standard solution not dosings are prepared by 6.2. lower methods
Thing background, is performed the derivatization by 1.4. lower methods, 20 μ L sample introductions is taken after filtering.Each 3 parts of concentration level.
Rate of recovery sample solution：Precision weighs appropriate each medicine background, is placed in 10mL measuring bottles, is separately added into phase in synthesis
Three limit level (LOQ, 6LOQ and 10LOQ) the single standard product solution that should be used, derivatization, is filtered after the same method
After take 20 μ L sample introductions.Each 3 parts of concentration level.
Average recovery=(sample peak area-background peak area)/reference substance peak area × 100%.
And calculate the average value and RSD of each concentration level average recovery.
As shown in Table 3, the average recovery of each acyl chlorides is between 87.8-114.1%, and RSD is respectively less than 3.36%.
The average recovery of this method of table 3.
Embodiment 7
The method set up is applied to the detection that acyl chlorides is remained in medicine.Precision weighs each medicine in right amount, is respectively placed in
In 10mL measuring bottles, 500 μ L 2- nitrophenyl hydrazines are added, acetonitrile constant volume is used, reacted at room temperature after 0.5h, take 20 μ L sample introductions, observation is
It is no to have acyl chlorides derivatization product peak.
As a result show, corresponding acyl chlorides is not detected in the medicine of six kinds of purifying, but the method is still in medical industry
In to remain acyl chlorides detection have important application.
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Claims (10)
1. the method that derivatization HPLC-DAD methods determine acyl chlorides, it is characterised in that comprise the steps of：
(1), at room temperature, acyl chlorides derivatization is generated in ultraviolet visible light region 320- using nitrobenzene hydrazine derivatization reagent
There is the product absorbed more by force in 450nm；
(2), with step (1) derivedization reaction product, using HPLC-DAD methods, based on reversed phase partition chromatography principle, in purple
Outer visible region 320-450nm determines the derivatization product of wherein acyl chlorides, so as to realize the qualitative or quantitative detection to acyl chlorides.
2. according to the method described in claim 1, it is characterised in that comprise the steps of：
(1), at room temperature, using acetonitrile as reaction system, acyl chlorides derivative reaction is given birth to using nitrobenzene hydrazine derivatization reagent
Into there is the product that absorbs more by force in ultraviolet visible light region 330-420nm；
(2), with step (1) derivedization reaction product, using HPLC-DAD methods, based on reversed phase partition chromatography principle, in purple
Outer visible region 330-420nm determines the derivatization product of wherein acyl chlorides, so as to realize the qualitative or quantitative detection to acyl chlorides.
3. method according to claim 2, it is characterised in that described acyl chlorides is selected fromR is selected from substitution or not taken
C1~C6 alkyl in generation, substituted or unsubstituted five to nine circle heterocycles containing S, N, O, substituted or unsubstituted phenyl ring, naphthalene nucleus,
Anthracene nucleus, phenanthrene ring, wherein, the substituent of C1~C6 alkyl is halogen, and bromine heterocycle or phenyl ring, naphthalene nucleus, anthracene nucleus, the substituent of phenanthrene ring are
Chlorine, bromine, nitro, amino, substituted or unsubstituted C1~C6 alkyl, substituted or unsubstituted C1~C4 alkoxies；Alkyl or alkane
The substituent of epoxide is halogen；R preferably is selected from substituted or unsubstituted C1~C4 alkyl, containing the substituted or unsubstituted of S, N, O
Five yuan or hexa-member heterocycle, substituted or unsubstituted chlorobenzoyl chloride, wherein, the substituent of C1~C4 alkyl is halogen；Five yuan or six
Circle heterocycles, the substituent of chlorobenzoyl chloride are chlorine, bromine, nitro, amino, substituted or unsubstituted C1~C6 alkyl, substitution or not taken
C1~C4 alkoxies in generation；
Described acyl chlorides preferably is selected from chloroacetic chloride, chloracetyl chloride, 2- chlorpromazine chlorides, isobutyryl chloride, chlorobenzoyl chloride and 5- chlorothiophenes -2-
Any one in formyl chloride；
Described nitrobenzene hydrazine derivatization reagent is selected from 2- nitrophenyl hydrazines, 3- nitrophenyl hydrazines, 4- nitrophenyl hydrazines, 2,3- dinitros
Any one in phenylhydrazine, DNPH, 3,4- dinitrophenylhydrazines, preferably 2- nitrophenyl hydrazines.
4. method according to claim 3, it is characterised in that using acetonitrile as reaction system, 2- nitrophenyl hydrazines and total acyl chlorides
Mol ratio is 1.45:1~145:1, react 0.5~3h；It is preferred that using acetonitrile as reaction system, 2- nitrophenyl hydrazines rub with total acyl chlorides
You are than being 14.5:1, the reaction time is 0.5h.
5. according to the method described in claim 1, it is characterised in that in step (2), described HPLC-DAD methods use HPLC colors
Spectrometer；Using reversed phase partition chromatography；Using non-polar linkage as stationary phase, using polarity mobile phase, Detection wavelength is located at 320
Between~450nm, it is preferably placed between 330~420nm, further preferably between 390~400nm.
6. the application of method according to any one of claims 1 to 5 acyl chlorides in medicine or its synthetic intermediate is determined.
7. the method that derivatization HPLC-DAD methods determine acyl chlorides in medicine or its synthetic intermediate, its feature mainly includes following step
Suddenly：
(1) at room temperature, 0.5~3h of reaction is performed the derivatization using nitrobenzene hydrazine derivatization reagent, reaction solution enters as sample
Sample is determined；
(2) reaction solution after terminating is reacted as sample introduction sample using step (1) derivedization, using HPLC-DAD methods in 320-
450nm determines the derivatization product of wherein acyl chlorides, thus realize in medicine or its synthetic intermediate acyl chlorides it is qualitative or quantitative
Detection.
8. method according to claim 7, it is characterised in that described acyl chlorides is selected fromR is selected from substitution or not taken
C1~C6 alkyl in generation, substituted or unsubstituted five to nine circle heterocycles containing S, N, O, substituted or unsubstituted phenyl ring, naphthalene nucleus,
Anthracene nucleus, phenanthrene ring, wherein, the substituent of C1~C6 alkyl is halogen, and bromine heterocycle or phenyl ring, naphthalene nucleus, anthracene nucleus, the substituent of phenanthrene ring are
Chlorine, bromine, nitro, amino, substituted or unsubstituted C1~C6 alkyl, substituted or unsubstituted C1~C4 alkoxies；Alkyl or alkane
The substituent of epoxide is halogen；R preferably is selected from substituted or unsubstituted C1~C4 alkyl, containing the substituted or unsubstituted of S, N, O
Five yuan or hexa-member heterocycle, substituted or unsubstituted chlorobenzoyl chloride, wherein, the substituent of C1~C4 alkyl is halogen；Five yuan or six
Circle heterocycles, the substituent of chlorobenzoyl chloride are chlorine, bromine, nitro, amino, substituted or unsubstituted C1~C6 alkyl, substitution or not taken
C1~C4 alkoxies in generation；
Described acyl chlorides preferably is selected from chloroacetic chloride, chloracetyl chloride, 2- chlorpromazine chlorides, isobutyryl chloride, chlorobenzoyl chloride and 5- chlorothiophenes -2-
Any one in formyl chloride；
Described nitrobenzene hydrazine derivatization reagent is selected from 2- nitrophenyl hydrazines, 3- nitrophenyl hydrazines, 4- nitrophenyl hydrazines, 2,3- dinitros
Any one in phenylhydrazine, DNPH, 3,4- dinitrophenylhydrazines, preferably 2- nitrophenyl hydrazines.
9. method according to claim 7, it is characterised in that using acetonitrile as reaction system, 2- nitrophenyl hydrazines concentration is 0.01
~1mg/mL, reacts 0.5~3h；It is preferred that using acetonitrile as reaction system, 2- nitrophenyl hydrazines concentration is 100 μ g/mL, and the reaction time is
0.5h。
10. method according to claim 7, it is characterised in that the HPLC-DAD methods described in step (2) use HPLC colors
Spectrometer；Using reversed phase partition chromatography；Using non-polar linkage as stationary phase, using polarity mobile phase, Detection wavelength is located at 320
Between~450nm, it is preferably placed between 330~420nm, further preferably between 390~400nm；Described HPLC-
The instrument that DAD methods are preferably used be Shimadzu LC 20AT liquid chromatographs, the chromatograph be configured with online vacuum degassing machine,
Binary gradient pump, automatic sampler, column oven, DAD detectors and LC-solution chromatographic work stations；Chromatographic column uses 250mm
× 4.6mm, the Diamonsil of 5 μm of Di Ma scientific ＆ technical corporationTMC18 posts；Sample size：20μL；Flow rate of mobile phase：1.0mL/min；
Eluent gradient：A phases be acetonitrile, B phases be 0.1% phosphoric acid, 0min 30%A phases, 5min 40%A phases, 10min 40%A phases,
15min 60%A phases, 20min 60%A phases, 22min 70%A phases, 25min 70%A phases；Column temperature：30℃；Detection wavelength：
395nm。
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