CN106442769A - Method for simultaneously detecting clavulanic acid and tazobactam in milk with high performance liquid chromatography - Google Patents
Method for simultaneously detecting clavulanic acid and tazobactam in milk with high performance liquid chromatography Download PDFInfo
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- CN106442769A CN106442769A CN201610800938.0A CN201610800938A CN106442769A CN 106442769 A CN106442769 A CN 106442769A CN 201610800938 A CN201610800938 A CN 201610800938A CN 106442769 A CN106442769 A CN 106442769A
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- clavulanic acid
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Abstract
The invention discloses a method for simultaneously detecting clavulanic acid and tazobactam in milk with high performance liquid chromatography, and belongs to the technical field of food detection. The method comprises the steps of conducting determination according to the following chromatographic condition with high performance liquid chromatography after conducting pretreatment on the sample. According to the chromatographic condition, the chromatographic column is a C18 chromatographic column; the detecting wavelength is 210nm-220nm; the mobile phase is phosphoric acid solution-methyl alcohol; the flow velocity is 0.7mL/min-1.2mL/min; the sample amount is 10 microliter; the elution time is 10min. The result demonstrates that a good linear relation is shown when the content of the clavulanic acid and the content of the tazobactam are in a concentration range of 12.5-200 microgrammes per millimeter, and the correlation coefficient is 0.9990 and 0.9992. Standard calibration is conducted with a method that blank matrix matches a standard solution, it is showed that the average recovery rate of both the clavulanic acid and the average recovery rate of the tazobactam are 81%-92%, and the relative standard deviation is smaller than 1.650%. The method for simultaneously detecting the clavulanic acid and the tazobactam in the milk with the high performance liquid chromatography is applicable to simultaneous detection of a beta-lactamase inhibitor, namely clavulanic acid, and the tazobactam in the milk.
Description
Technical field
The present invention relates to a kind of method using high performance liquid chromatography simultaneously to detect Ruzhong clavulanic acid and Tazobactam Sodium,
Belong to technical field of food detection.
Background technology
Clavulanic acid and Tazobactam Sodium are all competitive beta-lactamase inhibitors, can make after being combined with beta-lactamase
Its inactivation, has the effect of suppression gram-positive bacteria and negative bacterium.
The extensive application of antibiotic, causes its residue problem in Ruzhong to receive significant attention.In order to hide antibiotic
Detection, some people add beta-lactamase, in order to antibiotic of degrading, thus cannot judge whether Ruzhong contains antibiosis in Ruzhong
Element.So, for this phenomenon, establish the detection method with regard to Ruzhong beta-lactamase.But, some people utilize again β-interior
The character that lactamase inhibitor can irreversibly be combined with beta-lactamase, Ruzhong add beta-lactamase inhibitor, make β-
Lactamase loses activity, and causes positive milk not to be detected.
Detection method with regard to Ruzhong beta-lactamase inhibitor does not at home and abroad also have existing examination criteria to issue, state
Outer due to the difference of its culture development degree, mainly target is placed on the service condition in clinical treatment, does not see carat
Dimension acid and the report in Ruzhong for the Tazobactam Sodium；And the domestic report for clavulanic acid and the detection method of Tazobactam Sodium is little,
The method of document report is concentrated mainly on again microbial method, enzyme process, capillary electrophoresis etc..
Owing to there is no good effective detection means at present, set up Ruzhong beta-lactamase inhibitor clavulanic acid and his azoles
The detection method of Batan is extremely important.
Content of the invention
For solving the deficiencies in the prior art, the invention provides a kind of employing high performance liquid chromatography and detect Ruzhong gram simultaneously
Clavulanic acid and the method for Tazobactam Sodium, the technical scheme of employing is as follows：
It is an object of the invention to provide a kind of employing high performance liquid chromatography and detect Ruzhong beta-lactam enzyme level simultaneously
Agent clavulanic acid and the method for Tazobactam Sodium, the method step is as follows：
1) sample is carried out pre-treatment；
2) high performance liquid chromatography is utilized to be measured according to following chromatographic condition；Described chromatographic condition is：Chromatographic column is C18
Chromatographic column；Detection wavelength is 210nm-220nm；Flowing is phosphoric acid solution-methyl alcohol mutually；Flow velocity is 0.7mL/min-1.2mL/min；
Sample size is 10 μ L；Elution time is 10min.
Preferably, described pre-treatment, is to weigh 1 portion of milk to mix with acetonitrile, and concussion is centrifugal after mixing, take supernatant with just
Hexane mixes, and concussion is centrifugal after mixing, and discards n-hexane layer, after nitrogen dries up, redissolves mutually with flowing, finally crosses miillpore filter.
Preferably, described phosphoric acid solution concentration is 0.01%-0.03%；PH is 3.5-4.5.
It is highly preferred that described phosphoric acid solution concentration is 0.02%；PH is 4.
Preferably, described phosphoric acid solution-methyl alcohol, volume ratio is 95:5-90:10.
It is highly preferred that described phosphoric acid solution-methyl alcohol, volume ratio is 90:10.
Preferably, described C18 chromatographic column, specification is 4.6mm × 250mm, 5 μm.
Preferably, comprise the following steps that：
1) sample is carried out pre-treatment：Weigh 1 portion of milk and 3 parts of-5 parts of acetonitriles are mixed in centrifuge tube, after concussion mixes
Centrifugal, take supernatant and 4 parts of-6 parts of n-hexanes are mixed in another centrifuge tube, concussion is centrifugal after mixing, and discards n-hexane layer,
After 35 DEG C of-45 DEG C of nitrogen dry up, redissolve mutually with flowing, finally cross miillpore filter；
2) high performance liquid chromatography is utilized to be measured according to following chromatographic condition；Described chromatographic condition is：Chromatographic column is
4.6mm × 250mm, the C18 chromatographic column of 5 μm；Detection wavelength is 210-220nm；Flowing is 95 for volume ratio mutually:5-90:10
Phosphoric acid solution-methyl alcohol；Flow velocity is 0.7mL/min-1.2mL/min；Sample size is 10 μ L；Elution time is 10min；Described phosphoric acid
Solution concentration is 0.01%-0.03%, and pH is 3.5-4.5.
It is highly preferred that comprise the following steps that：
1) sample is carried out pre-treatment：Weighing 1 portion of milk and 4 parts of acetonitriles being mixed in centrifuge tube, concussion is centrifugal after mixing,
Taking supernatant and 5 parts of n-hexanes being mixed in another centrifuge tube, concussion is centrifugal after mixing, and discards n-hexane layer, at 40 DEG C of nitrogen
After drying up, redissolve mutually with flowing, finally cross miillpore filter；
2) high performance liquid chromatography is utilized to be measured according to following chromatographic condition；Described chromatographic condition is：Chromatographic column is
4.6mm × 250mm, the C18 chromatographic column of 5 μm；Detection wavelength is 215nm；Flowing is 90 for volume ratio mutually:The phosphoric acid solution of 10-
Methyl alcohol；Flow velocity：1.0mL/min；Sample size is 10 μ L；Elution time is 10min；Described phosphoric acid solution concentration is 0.02%, pH
It is 4.
The above either method detects the application in clavulanic acid and Tazobactam Sodium at the same time.
The present invention is to the look using HPLC method to detect Ruzhong beta-lactamase inhibitor clavulanic acid and Tazobactam Sodium simultaneously
Spectral condition is optimized, and obtains optimum chromatogram condition, then carries out clavulanic acid and Tazobactam Sodium methodology for this condition
Checking.
Beneficial effect of the present invention：
The detection while present invention uses high performance liquid chromatography to establish Ruzhong beta-lactamase inhibitor clavulanic acid
Method, sample uses acetonitrile removing protein, and the method for n-hexane degreasing, optimum chromatogram condition is：Chromatographic column is Hypersil ODS-
2C18 (4.6mm × 250mm, 5 μm), flowing is 0.02% phosphoric acid solution (pH4)-methyl alcohol (90 mutually:10), detecting wavelength is
215nm, flow velocity is 1mL/min.The range of linearity of the method is 12.5～200 μ g/mL, and coefficient correlation is 0.9990 He
0.9992, average recovery rate is between 81%～92%, and relative standard deviation is less than 1.650%.The inventive method is easy and simple to handle,
Reliable results, for ensure product quality, protection consumer health have a very important role, have certain reality and
Directive significance.It is applicable to detection while Ruzhong beta-lactamase inhibitor clavulanic acid and Tazobactam Sodium.
Brief description
The chromatogram of clavulanic acid and Tazobactam Sodium standard liquid under Fig. 1 optimum chromatogram condition.
Raw milk is added under Fig. 2 optimum chromatogram condition the chromatogram of clavulanic acid and Tazobactam Sodium.
Fig. 3 clavulanic acid and the calibration curve of Tazobactam Sodium.
Detailed description of the invention
Below in conjunction with the accompanying drawings technical scheme is further described, but is not limited thereto, every to this
Inventive technique scheme is modified or equivalent, without deviating from the spirit and scope of technical solution of the present invention, all should cover
In protection scope of the present invention.
Embodiment 1：
First, the preparation of standard reserving solution
Accurately weigh clavulanic acid and Tazobactam Sodium standard items, be made into the standard reserving solution that concentration is 1mg/mL, make
Used time becomes required concentration with flowing phase dilution, and all solution needed the miillpore filter of 0.45 μm before using.
2nd, liquid phase chromatogram condition
Chromatographic column：Hypersil ODS-2C18 (4.6mm × 250mm, 5 μm)；Detection wavelength：210-220nm is (preferably
215nm)；Flowing phase：0.02% phosphoric acid solution (pH4)-methyl alcohol (90:10)；Flow velocity：0.7mL/min-1.2mL/min is (preferably
1mL/min)；Sample size：10μl；The gradient elution time：10min.
3rd, sample-pretreating method
Weighing 1 portion of milk and 3 parts of-5 parts of acetonitriles being mixed in centrifuge tube, concussion is centrifugal after mixing, and takes supernatant and 4 part-6
Part n-hexane is mixed in another centrifuge tube, and concussion is centrifugal after mixing, and discards n-hexane layer, after 35 DEG C of-45 DEG C of nitrogen dry up,
Redissolve mutually with flowing, finally cross miillpore filter.
Optimum volume ratio through test discovery milk and acetonitrile is 1:4, milk is 1 with the optimum volume ratio of n-hexane:5
When, it is 40 DEG C that nitrogen dries up optimum temperature, i.e. weighs 1 portion of (such as 1mL) milk and 4 parts of (such as 4mL) acetonitriles are mixed in centrifuge tube,
Concussion mixes (such as 1min) and centrifuges (such as 4000r/min, 15min) afterwards, takes supernatant and 5 parts of (such as 5mL) n-hexanes are mixed in separately
In one centrifuge tube, concussion mixes (such as 1min) and centrifuges (such as 4000r/min, 8min) afterwards, discards n-hexane layer, repeats grease removal 2 times,
After 40 DEG C of nitrogen dry up, redissolve mutually with flowing, finally cross miillpore filter (such as 0.45 μm).
4th, the optimization of chromatographic condition
1st, the optimization of flowing selection and proportioning mutually
This test has primarily looked at methanol-water (10 under equal conditions：90) and acetonitrile-water (10：90) two kinds of flowings are relatively
The impact of material separating effect.Result shows, when flowing is mutually for acetonitrile-water, chromatographic peak trails；When flowing is methanol-water mutually
When, peak type is good, and separating degree is high, the wash-out effect that test substance can obtain.Therefore, this test determines methanol-water for flowing phase.
After determining that methanol-water is flowing phase, investigate methyl alcohol and water in different proportions in the presence of, effect is separated to test substance
The impact of fruit.Result shows, methanol ratio is from the change procedure of 5% to 35%, with the reduction of methanol concentration, retention time
Gradually extend (table-1).But when methanol concentration is less than 10%, separation condition is not reaching to optimum state, impurity and clavulanic acid
Not completely separable with Tazobactam Sodium.Therefore methanol-water optimum volume ratio=10:90.
Table 1 proportion of mobile phase and the relation of retention time
2nd, the optimization of the selection of cushioning liquid and concentration
In chromatography, adding cushioning liquid, reach to prevent sample from dissociating in aqueous phase, peak type trails, and increases method
The effect such as reliability.This experiment investigation TBAH and the shadow to separating effect for the two kinds of cushioning liquid of dipotassium hydrogen phosphate
Ring.Result shows, during with TBAH for cushioning liquid, bag peak phenomenon occurs in the chromatographic peak of clavulanic acid, separates effect
Really poor；And when selecting dipotassium hydrogen phosphate, clavulanic acid is preferably eluted, and separating degree is good.Therefore, this test select with
Phosphoric acid is as cushioning liquid.
Add phosphoric acid in aqueous phase, be configured to the phosphoric acid solution of variable concentrations, according to methyl alcohol determined above with water
Good volume ratio mixes, the impact on test substance separating effect for the concentration of investigation cushioning liquid.Result shows, at certain model
In enclosing, the ionic strength of flowing phase is higher, then retention time longer (table-2), but considers the restriction to concentration for the liquid chromatogram,
Ensure on the premise of efficiently separate, the relatively low buffering liquid of ionic strength should be selected as flowing phase.Therefore, this test is true
The optium concentration determining phosphoric acid is 0.02%.
Table 2 phosphoric acid concentration and the relation of retention time
3rd, the optimization of pH value of buffer solution
The selection of pH value of buffer solution is extremely important for the result of test, if the incorrect meeting of the selection of pH value is caused a split
Peak, asymmetrical peak dissymmetric peak, acromion or broad peak etc..The impact on retention time and separating effect for the different pH value of this experiment investigation.Knot
Fruit display, the change of pH value is little (table-3) on retention time impact, but can affect the peak type of chromatographic peak and the weight of experiment
Existing property.When pH value is 4, chromatogram peak-to-peak type is good, separating effect might as well, and with pH value during 0.02% phosphoric acid closest to.
Therefore, this test Optimal pH is 4.
Table 3 pH value and the relation of retention time
5th, methodological checking
1st, system suitability
By Fig. 1, Fig. 2 it can be seen that solvent does not disturb main peak and has the mensuration at related substance peak, clavulanic acid and Tazobactam Sodium
The retention time base of clavulanic acid and Tazobactam Sodium in retention time under optimum chromatogram condition for the standard solution and raw milk
This is consistent.To sum up show, detection application while clavulanic acid and Tazobactam Sodium be applicable to dairy products of this optimum chromatogram condition.
2nd, calibration curve and detection limits
Precision weighs clavulanic acid and Tazobactam Sodium standard items 25mg is placed in 25mL volumetric flask respectively, adds flowing phase dilution
To scale, shake up；Then precision measures above-mentioned solution respectively, is the 12.5th, the 25th, the 50th, the 100th, 200 by mobility progressively diluted concentration
The standard liquid of μ g/mL, is measured by above-mentioned optimum chromatogram condition sample introduction.Each concentration parallel determination 5 times, is put down by gained peak area
The concentration (μ g/mL) to clavulanic acid and Tazobactam Sodium for the average (y) does calibration curve, draws returning of clavulanic acid and Tazobactam Sodium
Return equation and coefficient correlation (Fig. 3).
3rd, the rate of recovery and precision
This test employing blank adds calibration method and carries out the rate of recovery and precision mensuration.By the 5th, the 50th, 100 μ g/mL 3 dense
The standard items of degree add in blank sample, process according to the method for 1.3.2 joint, and each concentration samples measures 5 times, record
Peak area, brings regression equation respectively into, and according to formula：The rate of recovery=sample measurements/Standard entertion amount × 100% is surveyed
Fixed, result such as table 4.By result in table it can be seen that the average recovery rate of the method is between 81%～92%, relative standard is inclined
Difference is less than 1.650%.
The recovery of standard addition of table 4 Ruzhong clavulanic acid and Tazobactam Sodium and precision
4th, the mensuration of actual sample
15 kinds of liquid milks to market sale for the method for the high performance liquid chromatography set up according to this test are measured, temporarily
Do not find positive.
Although the present invention is open as above with preferred embodiment, but it is not limited to the present invention, any is familiar with this
The people of technology, without departing from the spirit and scope of the present invention, can do various change and modification, the therefore protection of the present invention
Scope should be with being as the criterion that claims are defined.
Claims (6)
1. one kind uses the method that high performance liquid chromatography detects Ruzhong clavulanic acid and Tazobactam Sodium simultaneously, it is characterised in that step
Rapid as follows：Step one：Sample is carried out pre-treatment；Step 2：High performance liquid chromatography is utilized to survey according to following chromatographic condition
Fixed；Described chromatographic condition is：Chromatographic column is C18 chromatographic column；Detection wavelength is 210nm-220nm；Flowing is phosphoric acid solution-first mutually
Alcohol；Flow velocity is 0.7mL/min-1.2mL/min；Sample size is 10 μ L；Elution time is 10min.
2. method according to claim 1, it is characterised in that pre-treatment described in step one is to weigh 1 portion of milk to mix with acetonitrile
Closing, concussion is centrifugal after mixing, and takes supernatant and mixes with n-hexane, and concussion is centrifugal after mixing, and discards n-hexane layer, and nitrogen dries up
After, redissolve mutually with flowing, finally cross miillpore filter.
3. method according to claim 1, it is characterised in that C18 chromatographic column described in step 2, specification is 4.6mm × 250mm,
5μm.
4. method according to claim 1, it is characterised in that described in step 2, phosphoric acid solution concentration is 0.02%；PH is 4.
5. method according to claim 1, it is characterised in that phosphoric acid solution-methyl alcohol described in step 2, volume ratio is 90:10.
6. method according to claim 1, it is characterised in that comprise the following steps that：Sample is carried out pre-treatment：Weigh 1 part
Milk and 4 parts of acetonitriles are mixed in centrifuge tube, and concussion is centrifugal after mixing, and take supernatant and 5 parts of n-hexanes are mixed in another and centrifuge
Guan Zhong, concussion is centrifugal after mixing, and discards n-hexane layer, after 40 DEG C of nitrogen dry up, redissolves mutually with flowing, finally crosses micropore filter
Film；High performance liquid chromatography is utilized to be measured according to following chromatographic condition；Described chromatographic condition is：Chromatographic column be 4.6mm ×
250mm, the C18 chromatographic column of 5 μm；Detection wavelength is 215nm；Flowing is 90 for volume ratio mutually:Phosphoric acid solution-the methyl alcohol of 10；Stream
Speed：1.0mL/min；Sample size is 10 μ L；Elution time is 10min；Described phosphoric acid solution concentration is 0.02%, and pH is 4.
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| CN201610800938.0A CN106442769A (en) | 2016-09-05 | 2016-09-05 | Method for simultaneously detecting clavulanic acid and tazobactam in milk with high performance liquid chromatography |
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| CN201610800938.0A CN106442769A (en) | 2016-09-05 | 2016-09-05 | Method for simultaneously detecting clavulanic acid and tazobactam in milk with high performance liquid chromatography |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110878332A (en) * | 2019-12-03 | 2020-03-13 | 国家食品安全风险评估中心 | Method for indirectly and quantitatively detecting illegally added sulbactam by enzyme method and kit thereof |
| CN111198266A (en) * | 2020-01-09 | 2020-05-26 | 深圳市博奥通科生物制品有限公司 | Rapid detection kit for sulbactam |
-
2016
- 2016-09-05 CN CN201610800938.0A patent/CN106442769A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110878332A (en) * | 2019-12-03 | 2020-03-13 | 国家食品安全风险评估中心 | Method for indirectly and quantitatively detecting illegally added sulbactam by enzyme method and kit thereof |
| CN111198266A (en) * | 2020-01-09 | 2020-05-26 | 深圳市博奥通科生物制品有限公司 | Rapid detection kit for sulbactam |
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Application publication date: 20170222 |