CN104897838B - Method for separating and analyzing is extracted while a kind of highly polar organic acids and base mixture - Google Patents
Method for separating and analyzing is extracted while a kind of highly polar organic acids and base mixture Download PDFInfo
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Abstract
Extract and method for separating and analyzing while the invention provides a kind of highly polar organic acids and base mixture, surfactant dynamic modification ODS solid-phase extraction column is used to be used in combination with ion pair chromatography, the method specificity is higher, linear relationship is good, especially preferable to highly polar both sexes medicine and acidic drug concentration and separation effect, extract while may be used for highly polar ionic drug mixture and separate analysis, it is adaptable to pharmaceutical factory surrounding enviroment water sample has the monitoring analysis of related substance.
Description
Technical field
The present invention relates to a kind of water sample extracts method for separating and analyzing while highly polar organic acids and base mixture, specifically relate to
And dynamic modification Solid-Phase Extraction coupled ion is to high performance liquid chromatography multiple highly polar ionic drug in environmental water sample
Method for separating and analyzing is extracted while intermediate.
Background technology
In the monitoring of chemicals synthetic reaction, environmental pollution monitoring, wild animal resources and drug disposition are analyzed, multiple
Extracting and separate analysis while polar medicine the most highly polar ion-type mixture is the most important step.Environmental sample
Or the extraction of the nonpolar and low pole compound in biological sample and separation use common liquid-liquid extraction or Solid-Phase Extraction knot
Close reversed phase liquid chromatography, can efficiently separate and analyze.But, in real work, it is sometimes desirable to in sample some
The mixture of highly polar acidity, alkalescence and/or both sexes organic compound extracts simultaneously and analyzes, due to its medicine itself
Polarity and Acidity of Aikalinity, the polarity of solvent and the impact of the factors such as dissolubility, sample matrices interference, it is difficult to these chemical combination
Thing extracts simultaneously and analyzes.In some samples, organic acid or organic base can also take respectively corresponding anion or sun from
Sub-exchange resin extracts, and extracts while highly polar organic acid, organic base and/or amphoteric compound and be difficult to common liquid
Liquid extraction and Solid-Phase Extraction solve.Such as, cation exchange resin can extract alkalescent medicine, but can not extract acid simultaneously
Property medicine or other low pole, non-polar drug.
Beta-lactam antibiotic (β-lactam antibiotics) refers in chemical constitution containing beta-lactam nucleus
One class antibiotic, predominantly penicillins and cephalosporins, due to such, antibiotic effect is strong, toxicity is low, has a broad antifungal spectrum, because of
And apply quite varied.When synthesizing the beta-lactam antibiotics such as amoxicillin, reaction system successively to be used hydroxyl
The intermediate such as phenylglycine (p-HPG), 4-Hydroxyphenyl hydantoin (p-HPH) and 6-amino-penicillanic acid (6-APA), along with technique
Difference, there is also a small amount of phenylacetic acid (PAA) in some response systems.Therefore in antibiotic actual production process, plant chimney stalk
Discharge may exist one or several of these intermediate, in being monitored factory's surrounding enviroment water sample analyzing, need
Want a kind of method that can extract simultaneously and analyze these intermediate.Owing to p-HPH, p-HPG, 6-APA and PAA belong to highly polar
Ionic compound, and p-HPG and 6-APA is highly polar amphoteric compound, and p-HPH and PAA is highly polar acid compound,
Extract highly difficult while these medicines in environmental water sample.At present this kind of highly polar organic acids and base is typically used individually extraction
Method with analyzing, wastes time and energy.Additionally, in conventional reversed-phase liquid chromatography is analyzed, p-HPH, p-HPG and the reservation of 6-APA
Time is the shortest, is difficult to efficiently separate with other impurity.These situations show, need to develop effective sample-pretreating method and liquid
Phase chromatography separating method extracts and analyzes while these highly polar organic acids and base mixture.
The purpose of this research is to set up one to be suitable for the multiple highly polar intermediate of Beta-lactam medicine in environmental water sample
While extraction and analytical method.
Technology contents
In order to overcome defect present in prior art, while the present invention provides a kind of highly polar organic acids and base mixture
Extract and the method for analysis.
For achieving the above object, the present invention is by the following technical solutions:
Extract and analysis method while a kind of highly polar organic acids and base mixture, it is characterised in that: use surface activity
Agent dynamic modification ODS solid-phase extraction column is used in combination with ion pair chromatography；Described surfactant dynamic modification ODS solid phase extracts
Take post to be prepared by following steps:
After activating ODS solid-phase extraction column with methanol, phosphoric acid solution successively, loading proper amount of surfactant SDS, solid to ODS
Phase extraction column carries out dynamic modification, makes the upper saturated SDS of extraction column absorption.
In order to increase the activation effect of ODS solid-phase extraction column, above-mentioned phosphoric acid solution preferred volume percentage concentration is 0.05%
Phosphoric acid solution.
In order to increase the adsorption effect of SDS on extraction column further, the concentration of preferred surfactant SDS is 20mmol/
L。
The consumption of surfactant SDS is determined according to practical situation by those skilled in the art.
Carry while the organic acids and base mixture that above-mentioned SDS modification solid phase extraction method is applicable to p-HPG, 6-APA and PAA
Take analysis.
Described ion pair chromatography includes herein below: with ODS silicagel column as Stationary Phase for HPLC, sodium heptanesulfonate
It is flowing phase with the mixed solution of phosphorus aqueous acid with methanol composition, uses 225nm wavelength detecting.In order to strengthen separation effect
Really, use gradient elution separation.
The concentration of sodium heptanesulfonate, the pH value of phosphoric acid solution, the initial proportion of methanol, and gradient elution time, by this
Skilled person determines according to concrete condition.
Above-mentioned ion pair chromatography, it is adaptable to while the organic acids and base mixture containing p-HPH, p-HPG, 6-APA and PAA
Separate and analyze.
Specifically,
Extract and analysis method while a kind of highly polar organic acids and base mixture, analyze p-HPH, p-HPG for simultaneously,
6-APA and PAA, or extraction and analysis p-HPG, 6-APA and PAA simultaneously, it is characterised in that: use surfactant dynamic modification
ODS solid-phase extraction column is used in combination with ion pair chromatography；Described surfactant dynamic modification ODS solid-phase extraction column is by following
Prepared by step:
Successively with after methanol, 0.05% phosphoric acid solution activation ODS solid-phase extraction column, live in the surface of the appropriate 20mmol/L of loading
Property agent SDS, ODS solid-phase extraction column is carried out dynamic modification, makes extraction column absorption the most saturated upper SDS.
Further,
Extraction and analytical method while a kind of highly polar organic acids and base mixture, analyzes p-HPH, p-HPG, 6-for simultaneously
APA and PAA, or extraction and analysis p-HPG, 6-APA and PAA simultaneously, it is characterised in that employing following steps:
(1) preparation of surfactant dynamic modification ODS solid-phase extraction column,
Successively with methanol, 0.05% phosphoric acid solution activation ODS solid-phase extraction column, the then surface activity of loading 20mmol/L
Agent SDS is appropriate, ODS solid-phase extraction column is carried out dynamic modification, makes extraction column fully adsorb SDS.
(2) preparation of test sample,
It is appropriate that precision weighs p-HPH standard substance, 6-APA standard substance, PAA standard substance and accurate absorption p-HPG standard substance, uses
Water dilution prepares the mixed solution of p-HPH, p-HPG, 6-APA and PAA of debita spissitudo, after regulation pH, by molten for prepared mixing
The dynamic modification Solid-Phase Extraction column extracting that liquid prepares through step (1), obtains the need testing solution for chromatography.
(3) ion pair chromatography separation detection is used,
With having the high performance liquid chromatograph of DAD or UV detector, use C18Chromatographic column, with the heptane sulphur containing debita spissitudo
Acid sodium solution is flowing phase with the mixed solution of methanol, and with phosphorus acid for adjusting pH, detection wavelength is 225nm；Accurate absorption test sample
Solution, injects in chromatograph of liquid, is analyzed measuring.
Simultaneously for expanding this method range of application, standard substance storing solution environmental water sample is diluted suitable multiple, obtains
Biased sample solution, extracts through method same as described above, obtains the need testing solution for chromatography.
In particular,
Extraction and analytical method while a kind of highly polar organic acids and base mixture, analyzes p-HPH, p-HPG, 6-for simultaneously
APA and PAA, or extraction and analysis p-HPG, 6-APA and PAA simultaneously, it is characterised in that employing following steps:
(1) preparation of surfactant dynamic modification ODS solid-phase extraction column,
Successively with 90% methanol aqueous solution, 0.05% phosphoric acid solution activation ODS solid-phase extraction column, the table of loading 20mmol/L
Face activating agent SDS solution, carries out dynamic modification to solid-phase extraction column, makes extraction column fully adsorb SDS, more molten with 0.05% phosphoric acid
Liquid rinses extraction column；
(2) preparation of test sample,
It is appropriate that precision weighs p-HPH standard substance, 6-APA standard substance, PAA standard substance and accurate absorption p-HPG standard substance, adds
0.05% phosphoric acid solution dilution prepares concentration and is respectively p-HPH 0-0.40mg/mL, p-HPG 0-0.2mg/mL, 6-APA 0-
1.00mg/mL and the mixed solution of PAA 0-1.00mg/mL, the dynamic modification that prepared mixed solution is prepared through step (1)
Solid-Phase Extraction column extracting, first cleans with 0.05% phosphoric acid solution, then the ammonia hydroxide/methanol drip washing with 5%, discards eluent
First 0.1ml, collects eluent interval for the 0.2-0.3mL 0.2ml, N altogether of ammonia meoh eluate2With 0.05% after drying up
Phosphoric acid solution dissolves, and obtains the need testing solution for chromatography.
(3) ion pair chromatography separation detection is used,
With having the high performance liquid chromatograph of DAD detector, with XDB-C18 chromatographic column for fixing phase, with the heptan containing 5mmol/L
The mixed solvent that 0.05% phosphoric acid solution and the methanol of alkyl sulfonic acid sodium is formed in appropriate proportions is flowing phase, and its pH is about 2.5,
Accurate need testing solution of drawing, in injection chromatograph of liquid, flow velocity is 1mL/min, and column temperature is 20 DEG C, and sample size is 20uL, adopts
Separating with gradient elution, detection wavelength is 225nm.
Above-mentioned gradient elution mode is preferably from 0-8min, and flowing middle methanol ratio mutually is by 20% linear rise to 60%
, and maintain 4min (v/v).
Simultaneously for expanding this method range of application, by standard substance storing solution environmental water sample or certain sewage draining exit downstream, pharmaceutical factory about
At 1km, Yangtze River Water dilutes suitable multiple, with phosphorus acid for adjusting pH to about 2.5, obtains biased sample solution after filtration, through with upper
State same procedure extraction, obtain the need testing solution for chromatography.
During analysis, described percentage ratio is percent by volume, and the concentration of sample preparation, diluted sample situation are by ability
Territory those of ordinary skill according to test it needs to be determined that.
Beneficial effects of the present invention has:
1. the invention provides a kind of Ion-pair chromalography separating analysis being suitable for multiple highly polar organic acids and base mixture
Method, the method separates while can be used for p-HPH, p-HPG, 6-APA and PAA and analyzes, and the method specificity is higher, linear relationship
Well, separating degree is high, favorable reproducibility, is suitable for highly polar both sexes medicine and separates analysis while acidic drug mixture.Mesh
Before there is not yet and use this chromatography of ions based on sodium heptanesulfonate to concurrently separate the report of analysis for these mixture.
2. the invention provides a kind of dynamic modification solid phase extraction method adsorbing SDS on common ODS filler, have concurrently quiet
Electro ultrafiltration and hydrophobic interaction, can be used for highly polar organic acids and base medicine, both sexes medicine and low pole in water sample and pharmaceutical factory sewer
Extraction and analysis while medicine, respond well on extraction and analysis while p-HPG, 6-APA and PAA, coupled ion pair
Chromatography, it is adaptable to surrounding enviroment water sample monitoring in antibiotic pharmaceutical factory is analyzed, drug synthetic reaction monitors and drug disposition analysis.
Have not yet to see and use this solid phase extraction modified based on SDS for these organic acids and base mixture extraction and analysis simultaneously
Report.
3. beta-lactam antibiotic intermediate p-HPH, p-HPG, 6-APA and PAA are extracted point by the present invention simultaneously
Analysis, preferable to the separating degree of four kinds of intermediate, the most do not substantially interfere with, in wherein p-HPG, 6-APA and PAA tri-kinds
While mesosome, effect of extracting is preferable, has obvious enrichment.
4. the present invention environmental water sample detection for certain downstream, pharmaceutical factory, the detection limit of p-HPG, 6-APA, PAA is respectively
0.04,0.14,0.25 μ g/mL (S/N >=3), to p-HPG, the enrichment times of 6-APA, PAA is between 33-48 times.
Accompanying drawing explanation
Fig. 1 SDS dynamic modification solid-phase extraction column capacity indicator figure
Fig. 2 SDS dynamic modification solid-phase extraction column has investigated the pH impact on extraction
Fig. 3 SDS dynamic modification solid-phase extraction column has investigated the salinity impact on extraction in solution
Fig. 4 SDS dynamic modification solid-phase extraction column has investigated the ethane nitrile content impact on extraction in solution
Fig. 5 modification ODS-SPE post and ODS-SPE and MCX extraction column effect of extracting comparison diagram
Fig. 6 modification ODS-SPE post is for the extraction collection of illustrative plates of storing solution
Fig. 7 sample specificity investigates high-efficient liquid phase chromatogram, is followed successively by blank river, standard substance and river from top to bottom and adds
Standard specimen product HPLC spectrogram after extraction
Fig. 8 separates comparison diagram mixed with the river of medicine after the extraction of blank river
Detailed description of the invention
With detailed description of the invention, technical scheme is described in further detail below in conjunction with the accompanying drawings.
Key instrument:
Agilent 1200 high performance liquid chromatograph (joins DAD detector, Anjelen Sci. & Tech. Inc of the U.S.), electronics sky
Flat (Germany's Sartorius industry), pH meter (Switzerland's prunus mume (sieb.) sieb.et zucc. Teller-torr benefit Internaional, Inc), Milli-Q A10 is ultrapure
Water system (Millipore company of the U.S.).
Reagent is commercially available prod, and main agents is:
4-Hydroxyphenyl hydantoin standard substance (purity ＞ 99.5%, Zhuhai United Laboratories Ltd)；Para hydroxybenzene is sweet
Propylhomoserin standard substance (Beijing lark prestige Science and Technology Ltd.)；(Zhuhai federation pharmacy share is limited for 6-amino-penicillanic acid standard substance
Company)；Phenylacetic acid standard substance (Sen Beijia bio tech ltd, Nanjing)；1-sodium heptanesulfonate (traditional Chinese medicines group chemical reagent
Company limited)；Methanol (U.S. TEDIA world Reagent Company)；Acetonitrile (U.S. TEDIA world Reagent Company)；Water is deionization
Water；ODS extraction column (100mg/, Tianjin Tian Hao company)；MCX extraction column (60mg/, Tianjin Tian Hao company).
Embodiment 1
1 storing solution and the preparation of flowing phase
The preparation of 1.1 standard solutions
Precision weighs 4-Hydroxyphenyl hydantoin standard substance 0.2000g, uses 0.05%H3PO4Aqueous dissolution is also settled to
100mL, prepared concentration is the p-HPH standard substance storing solution of 2.00mg/mL.
Precision weighs D-pHPG standard substance 0.1000g, uses 0.05%H3PO4Aqueous dissolution is also settled to
100mL, prepared concentration is the D-HPG standard substance storing solution of 1.00mg/mL.
Precision weighs 6-APA standard substance 0.4000g, uses 0.05%H3PO4Aqueous dissolution is also settled to 100mL, prepares dense
Degree is the 6-APA standard substance storing solution of 4.00mg/mL.
Precision weighs phenylacetic acid standard substance 0.4000g, uses 0.05%H3PO4Aqueous dissolution is also settled to 100mL (ability
Territory those of ordinary skill can add a small amount of methanol hydrotropy according to actual needs), prepared concentration is the PAA standard substance storage of 4.00mg/mL
Standby liquid.
It addition, it is appropriate to take above-mentioned each solution, use 0.05%H3PO4Aqueous solution dilution prepares concentration and is respectively p-HPH, p-
HPG, 6-APA and PAA are respectively four kinds of standard substance mixing of 0.40mg/mL, 0.04mg/mL, 1.00mg/mL and 1.00mg/mL
Solution；Or, three kinds of standard substance mixing that p-HPG, 6-APA and PAA are respectively 0.2mg/mL, 1.00mg/mL and 1.00mg/mL are molten
Liquid, room temperature is placed, is newly joined, stand-by.
Above-mentioned each solution uses 0.05%H as required3PO4Aqueous solution is molten is diluted to debita spissitudo.
The preparation of 1.2 flowing phases
Precision weighing 0.5075g1-sodium heptanesulfonate, is dissolved in 500mL 0.05%H3PO4In aqueous solution, prepare 5mmol/L
Heptane sulfonic acid sodium salt, record pH=2.5.
2 Ion-pair Liquid Chromatography conditions
Agilent 1200 high performance liquid chromatograph (is furnished with DAD detector)
Chromatographic column: Agilent Eclipse XDB-C18 (250*4.6mm, 5 μm)
Flowing phase: A (0.05% phosphoric acid solution sodium heptanesulfonate Han 5mmol/L, pH=2.5) and B (MeOH) initial than
Example is 80/20 (v/v), gradient elution
Flow velocity: 1mL/min
Wavelength: 225nm
Column temperature: 20 DEG C
Sample size: 20uL
Table 2.1 eluent gradient elution program
3 sample extraction
The preparation of 3.1 extraction samples
Accurate draw prepare hybrid standard product solution (p-HPH, p-HPG, 6-APA and PAA be respectively 0.40mg/mL,
0.04g/mL, 1.00mg/mL and 1.00mg/mL), use 0.05%H3PO4Aqueous solution dilutes 50 times, draws the extraction of 5ml loading；Or
50 times are diluted with regulating to certain sewage draining exit downstream, pharmaceutical factory about 1km of pH2.5 river with phosphoric acid, vortex mixing 2min, continues
Continuous vortex mixing 2min, 7500r/min are centrifuged 5min and take supernatant, then draw the extraction of 5ml loading.
3.2 ODS extraction columns are modified
Successively with 5mL methanol, 2mL 0.05% phosphoric acid solution activation ODS solid phase extraction column, loading 5mL20mmol/L
Surfactant SDS, carries out dynamic modification to ODS solid-phase extraction column so that it is ensure that on extraction column, fully absorption is upper saturated
SDS, then rinse the SDS on unadsorbed with 0.3mL 0.05% phosphoric acid solution.
3.3 sample pre-treatments
Take 5mL through the dilution storing solution of 50 times or river through dynamic modification Solid-Phase Extraction column extracting, then use 0.5mL successively
0.05% phosphoric acid solution, 1mL 5% ammonia hydroxide/methanol drip washing, collect interval the washing of ammonia meoh eluate 0.2-0.3mL
De-liquid 0.2ml, N altogether2Dissolve with 0.1mL 0.05% phosphoric acid solution after drying up, carry out HPLC detection.
The investigation of some important factor in order in embodiment
(1) capacity indicator
The principle of ODS-SPE column extracting is mainly based upon hydrophobic interaction.To have strong ionic groups and chain alkyl will be dredged
The immobilized surface of ODS stationary phase material in SPE post of surfactant SDS of water base group, so can be obtained by SPE post and has concurrently
Ion exchanges the mixed type with hydrophobic interaction and fixes the characteristic of phase.In this project, surfactant SDS is that dynamic adsorption is filled out in ODS
Material surface, this adsorbance can have certain loss along with the increase being extracted aqueous sample volume.In this experiment, adopt
Extraction column be the solid-phase extraction column containing 100mg ODS filler, inventor uses 20mmol/L SDS solution, applied sample amount
For 20mL, so it is able to ensure that on extraction column that the SDS amount of absorption reaches saturated.
Inventor has investigated four kinds of medicines saturated extent of adsorption on modified extraction column.Data are shown in Fig. 1.Can send out from figure
Existing, D-pHPG and 6-amino-penicillanic acid adsorption capacity on modified extraction column are very big, and this explanation is at pH2.5
Left and right, owing to both both sexes medicines show as with clean positive charge, in extraction, electrostatic attraction accounts for leading, and adsorbance is the biggest.
Due to loading capacity test in, sample concentration is the highest, applied sample amount when 2-8mL, D-pHPG, 6-amino
Penicillanic acid and phenylacetic acid just reach saturated, and in follow-up extraction, sample concentration is the lowest, therefore sample applied sample amount is preferably 5mL.
(2) the different surfaces activating agent impact on extraction
Inventor selects the ODS-SPE post through SDS dynamic adsorption is modified to be used for 4-Hydroxyphenyl hydantoin, the sweet ammonia of para hydroxybenzene
The extraction of acid, 6-amino-penicillanic acid, phenylacetic acid so some polar medicines.Although, CTAB (cetyltrimethylammonium base
Ammonium) it is more likely to be appropriate for the extraction of acidic drug such as 4-Hydroxyphenyl hydantoin, phenylacetic acid, but holding concurrently in view of extraction and piece-rate system
The medicine having in capacitive, and analyte, such as 6-amino-penicillanic acid, stability in the basic conditions, inventor is final
Choose SDS rather than CTAB, for the extraction of these medicines.
(3) pH impact on extraction
Inventor has investigated the pH impact on extraction, and data are shown in Fig. 2.Data from figure, pH is from 2.5-8.8, to hydroxyl
The recovery of extraction of base benzene glycolylurea is the most relatively low, although under pH6.86, unfired accounts for leading, and the repulsion between SDS is not
Very big, but owing to its hydrophilic is extremely strong, cause adsorbance on SPE post little.Blue or green for D-pHPG and 6-amino
Mould alkanoic acid, along with the reduction of pH value, the response rate dramatically increases, for both both sexes medicines, the reduction of pH value, on the one hand
Reduce the dissociation of carboxyl, on the other hand, add the protonation of amino, this contribute to increasing both sexes medicine and SDS it
Between electrostatic attraction, improve the response rate.For phenylacetic acid, its response rate raises with pH value and reduces, owing to its pKa value about exists
4.28 left and right, although there being certain electrostatic repulsion between it and SDS, but the hydrophobicity of phenyl is relatively strong, remains to obtain higher
The response rate.The result tested according to this, in dynamic modification below extracts, the pH value of loading extraction solution selects about 2.5
It is preferred.
(4) salinity impact on extraction in solution
In solution, the impact of extraction has been also carried out investigating by salinity, and data are shown in Fig. 3.It can be seen that the increase of salt amount,
Affecting complex, the extraction quantity of phenylacetic acid increased, but for D-pHPG and 6-amino-penicillanic acid, extraction
Taken amount has significantly reduction.This may be relevant to the Different Effects of three with ion exchange with hydrophobic interaction.For reducing sample
The salinity impact on extraction efficiency in product matrix, is used for extracting after the sample suggestion dilution certain multiple higher containing salinity.
(5) ethane nitrile content impact on extraction in solution
In solution, the impact of extraction has been also carried out investigating by ethane nitrile content, and data are shown in Fig. 4.It can be seen that acetonitrile is from 0%
Increasing to 20%, the extraction on D-pHPG, 6-amino-penicillanic acid, 4-Hydroxyphenyl hydantoin affects less, but, benzene
The extraction quantity of acetic acid has significantly minimizing.This demonstrates, in the extraction of phenylacetic acid, hydrophobic interaction accounts for leading；And to additionally
Both sexes medicine, electrostatic attraction accounts for leading, and hydrophobic interaction accounts for secondary role, therefore extraction influence is less.
(6) loading and elution requirement are investigated
After any of the above factor affecting effect of extracting is investigated, selection sample load solution condition is: pH2.5.
If this method to be used for the extraction of biological fluid, and use acetonitrile removing protein, it is proposed that before loading extraction, to sample according to experiment need
Suitably diluting, after controlling dilution, in sample, acetonitrile concentration is less than 10% (v/v).
Take in this experiment is the small-sized extraction column of 100mg specification, after upper complete sample, first selects 0.3mL 0.05% phosphoric acid
After aqueous cleaning, then with elution, contrast 1.0mL 5% ammonia methanol solution and 1.0mL 5% ammonia aqueous strip solution,
Realize that both of which can be by complete for object eluting in 0.3ml.In the extraction eluting of sample below, for the ease of the denseest
Contracting, the 0.2-0.3mL interval that collection ammonia methanol goes out as elution collects, and is concentrated into sample introduction after 0.1ml.
(7) modified capillary compares with ODS-SPE and MCX extraction column
Inventor has carried out this extracting process to contrast (extraction conditions is identical) with other several traditional extraction posts.From Fig. 5
It can be seen that tradition ODS-SPE post is to 4-Hydroxyphenyl hydantoin, D-pHPG, the effect of extracting of 6-amino-penicillanic acid
Very poor, but fine to the effect of extracting of phenylacetic acid.And MCX hybrid ionic exchange column is to D-pHPG, 6-amino penicillium sp
The effect of extracting of alkanoic acid and phenylacetic acid is the most undesirable.This experiment set up SDS modification ODS-SPE post to D-pHPG,
The effect of extracting of 6-amino-penicillanic acid and phenylacetic acid is superior to ODS-SPE post and MCX post.
(8) modified ODS-SPE post is for the extraction of storing solution
Extracting after storing solution is diluted 50 times, as can be seen from Figure 6, several drugs is extracted by the condition after optimizing before employing
Taking, in addition to 4-Hydroxyphenyl hydantoin extraction is undesirable, remaining several drugs all has obvious enrichment.
4 methodological studies
The specificity of 4.1 methods
Under the conditions of this Ion-pair chromalography tested, tetra-kinds of intermediate of p-HPH, p-HPG, 6-APA, PAA have bigger chromatograph
Peak, without severe jamming (Fig. 7) in bare substrate, and separating degree and peak type are well, and the method specificity is higher.Institute with this understanding
The result recorded can represent constituent concentration to be measured.
The preparation of 4.2 standard curves
The preparation series of samples containing variable concentrations p-HPH, p-HPG, 6-APA and PAA, uses ion pair chromatography to separate,
Record chromatogram, with concentration C (μ g/mL) as abscissa (x), peak area A is that vertical coordinate (y) carries out linear regression analysis and obtains respectively
The standard curve of medicine.
Owing to the effect of extracting of p-HPH is undesirable, after only determine tri-kinds of materials of p-HPG, 6-APA and PAA, to p-
The standard curve of HPG, 6-APA and PAA and the range of linearity, be shown in Table 2.
The equation of linear regression of table 23 kinds of intermediate of beta-lactam antibiotic, correlation coefficient, the range of linearity
4.3 stability analyzing sample
Label taking quasi-mixing storing solution, by the ion pair chromatography analysis after optimizing, took respectively at 0,1,2,4,12,24 hours
Sample carries out HPLC analysis.Statistics p-HPG, the peak area of 6-APA, PAA, calculate to obtain the equal ＜ of relative standard deviation RSD 8.1%, and with
The RSD that retention time calculates is respectively less than 2.3%.Show that the method records result stability to standard substance mixed liquor in 24h good
Good.
4.4 response rate analyzing method and precision
Respectively preparation three concentration levels hybrid standard product solution (p-HPG:0.2,0.8,4.0 μ g/mL, 6-APA:
1.0,4.0,20.0 μ g/mL, PAA:1.0,4.0,20.0 μ g/mL), each concentration parallel analysis 5 times, warp after modified capillary extraction
HPLC analyzes, and is calculated the response rate and relative standard deviation (RSD).The average recovery rate of 3 kinds of intermediate is all at 69.0%-
Between 97.8%, relative standard deviation is less than 11.5%, meets Method validation requirement, the results are shown in Table 3.
The average recovery rate of 33 kinds of intermediate materials of table and relative standard deviation
The application in Yangtze River Water extraction at the 1km of certain blow-off line downstream, pharmaceutical factory of 5 modified ODS-SPE posts
Above-mentioned sample is mixed Yangtze River Water at the 1km of certain blow-off line downstream, pharmaceutical factory, vortex mixing 2min by inventor, adds phosphoric acid
Regulation pH to 2.5, takes supernatant extraction after being centrifuged.
Through the collection of illustrative plates that ion pair chromatography sample introduction analysis obtains, such as Fig. 8.In the method, the inspection of p-HPG, 6-APA, PAA
Rising limit is respectively 0.04,0.14,0.25 μ g/mL (S/N >=3).It is respectively 0.8 μ g/mL (p-with incorporation river drug concentration
HPG), 4.0 μ g/mL (6-APA), the sample of 4.0 μ g/mL (PAA) is for investigating object, and the response rate of calculating is respectively p-HPG,
96.7%；6-APA, 87.3%；PAA, 67.4%.Continuous extraction is analyzed, and is computed, RSD (peak area, the n=of each medicine
3) it is respectively p-HPG, 7.9%；6-APA, 6.1%；PAA, 5.8%.Compare directly dividing about medicine in the water liquid not extracted
Analysis, the present invention is to p-HPG, and the concentration effect of 6-APA, PAA is it will be apparent that enrichment times respectively reaches between 33-48 times, this
Show that the present invention is effective to extracting while highly polar both sexes medicine and acidic drug.
Claims (4)
1. extract and an analysis method while highly polar organic acids and base mixture, analyze p-HPH, p-HPG, 6-for simultaneously
APA and PAA, or extraction and analysis p-HPG, 6-APA and PAA simultaneously, it is characterised in that: use surfactant dynamic modification ODS
Solid-phase extraction column is used in combination with ion pair chromatography；Described surfactant dynamic modification ODS solid-phase extraction column is by following step
Rapid preparation:
After activating ODS solid-phase extraction column with methanol, 0.05% phosphoric acid solution successively, the surfactant of the appropriate 20mmol/L of loading
SDS, carries out dynamic modification to ODS solid-phase extraction column, makes the upper saturated SDS of extraction column absorption.
2. the method for claim 1, analyzes p-HPH, p-HPG, 6-APA and PAA, or extraction and analysis simultaneously for simultaneously
P-HPG, 6-APA and PAA, it is characterised in that employing following steps:
(1) preparation of surfactant dynamic modification ODS solid-phase extraction column,
Successively with methanol, 0.05% phosphoric acid solution activation ODS solid-phase extraction column, the then surfactant SDS of loading 20mmol/L
In right amount, ODS solid-phase extraction column is carried out dynamic modification, make extraction column fully adsorb SDS；
(2) preparation of test sample,
It is appropriate that precision weighs p-HPH standard substance, 6-APA standard substance, PAA standard substance and accurate absorption p-HPG standard substance, uses water
Dilution prepares the mixed solution of p-HPH, p-HPG, 6-APA and PAA of debita spissitudo, after regulation pH, by prepared mixed solution
Through the dynamic modification Solid-Phase Extraction column extracting that step (1) prepares, obtain the need testing solution for chromatography；
(3) ion pair chromatography separation detection is used,
With having the high performance liquid chromatograph of DAD or UV detector, use C18 chromatographic column, with the heptanesulfonic acid containing debita spissitudo
Sodium solution is flowing phase with the mixed solution of methanol, and with phosphorus acid for adjusting pH, detection wavelength is 225nm；Accurate absorption test sample is molten
Liquid, injects in chromatograph of liquid, is analyzed measuring.
3. method as claimed in claim 2, analyzes p-HPH, p-HPG, 6-APA and PAA, or extraction and analysis simultaneously for simultaneously
P-HPG, 6-APA and PAA, it is characterised in that employing following steps:
(1) preparation of surfactant dynamic modification ODS solid-phase extraction column,
Successively with 90% methanol aqueous solution, 0.05% phosphoric acid solution activation ODS solid-phase extraction column, the surface activity of loading 20mmol/L
Agent SDS solution, carries out dynamic modification to ODS solid-phase extraction column, makes extraction column fully adsorb SDS, then uses 0.05% phosphoric acid solution
Rinse extraction column；
(2) preparation of test sample,
It is appropriate that precision weighs p-HPH standard substance, 6-APA standard substance, PAA standard substance and accurate absorption p-HPG standard substance, adds
0.05% phosphoric acid solution dilution prepares concentration and is respectively p-HPH 0.40mg/mL, p-HPG 0.2mg/mL, 6-APA 1.00mg/
The mixed solution of mL and PAA 1.00mg/mL, the dynamic modification solid-phase extraction column that prepared mixed solution is prepared through step (1)
Extraction, first cleans with 0.05% phosphoric acid solution, then the ammonia hydroxide/methanol drip washing with 5%, discards the 0.1ml that eluent is initial,
Collect eluent interval for the 0.2-0.3mL 0.2ml, N altogether of ammonia meoh eluate2After drying up molten with 0.05% phosphoric acid solution
Solve, obtain the need testing solution for chromatography；
(3) ion pair chromatography separation detection is used,
With having the high performance liquid chromatograph of DAD detector, with XDB-C18 as chromatographic column, with containing 5mmol/L sodium heptanesulfonate
The mixed solvent that 0.05% phosphoric acid solution and methanol are formed in appropriate proportions is flowing phase, and its pH is about 2.5, and accurate absorption supplies
Test sample solution, injects in chromatograph of liquid, and flow velocity is 1mL/min, and column temperature is 20 DEG C, and sample size is 20uL, uses gradient elution
Separating, detection wavelength is 225nm.
4. method as claimed in claim 3, it is characterised in that: described gradient elution mode is first from 0-8min, flowing mutually
Alcohol ratio is by 20% linear rise to 60%(v/v), and maintain 4min.
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