CN106290678A - A kind of detection method of Gentamicin C1a - Google Patents
A kind of detection method of Gentamicin C1a Download PDFInfo
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- CN106290678A CN106290678A CN201510245922.3A CN201510245922A CN106290678A CN 106290678 A CN106290678 A CN 106290678A CN 201510245922 A CN201510245922 A CN 201510245922A CN 106290678 A CN106290678 A CN 106290678A
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Abstract
The invention discloses the detection method of a kind of Gentamicin C1a, it comprises the steps: the testing sample containing Gentamicin C1a and derivatization reagent mix homogeneously and carries out derivative reaction, then liquid chromatograph or liquid phase chromatogram-mass spectrometry combination method detection are carried out,；Wherein, described derivatization reagent comprises these several components of solvent, o-phthalaldehyde(OPA), TGA and buffer solution；In described derivatization reagent, o-phthalaldehyde(OPA) is 1:8～1:3.89 with the mol ratio of TGA；The pH=10～11 of described derivatization reagent；In single testing result, in derivatization reagent used, the ratio of the mole of the Gentamicin C1a that the mole of o-phthalaldehyde(OPA) is measured with described testing sample need to be more than or equal to 1.6.The detection method of the Gentamicin C1a of the present invention is simple to operate, and in detecting production sample, during Gentamicin C1a concentration, accuracy is higher, the range of linearity is wide.
Description
Technical field
The present invention relates to the detection method of a kind of Gentamicin C1a.
Background technology
Detection method to sulmycin in derivatization detection method before the HPLC column of existing Gentamicin C1a, generally American Pharmacopeia USP35-nf30 version.Such as, accurately measure 10mL sample in volumetric flask, add 5mL isopropanol and the mixing of 4mLOPA derivatization reagent, it is settled to 25mL again with isopropanol, fully airtight after mixing, 60 DEG C of water-bath 15min are placed in frozen water termination reaction, and 12000rmp can carry out HPLC detection after being centrifuged 5 minutes.Wherein, the OPA derivatization reagent formula used: o-phthalaldehyde(OPA) 1g (0.0074mol), it is dissolved in 5ml methanol, add TGA 2ml (0.0288mol), finally add 0.4mol/L boric acid (45% sodium hydroxide adjusts pH value 10.4) 95ml mixing, adjust pH 10.4 with 45% sodium hydroxide again, put refrigerator and keep in Dark Place.This method restricted application in the industrial production, the range of linearity is narrow, and the testing result accuracy causing Gentamicin C1a concentration is excessively poor.And in actual production process, the Gentamicin C1a sample that concentration is higher and purity is relatively low is the object that people are to be detected, therefore, develop the derivatization reagent of the Gentamicin C1a sample that a kind of accuracy is higher, the range of linearity is wide, be this area problem demanding prompt solution.
Summary of the invention
The technical problem to be solved in the present invention be in order to overcome in prior art Gentamicin C1a when detectable concentration, the defect such as the range of linearity is narrow, restricted application, and provide the detection method of a kind of Gentamicin C1a.The detection method of the Gentamicin C1a of the present invention is simple to operate, and in detecting production sample, during Gentamicin C1a concentration, accuracy is higher, the range of linearity is wide.
The invention provides the detection method of a kind of Gentamicin C1a, it comprises the steps: the testing sample containing Gentamicin C1a and derivatization reagent mix homogeneously and carries out derivative reaction, then liquid chromatograph or liquid phase chromatogram-mass spectrometry combination method detection are carried out,；
Wherein, described derivatization reagent comprises these several components of solvent, o-phthalaldehyde(OPA), TGA and buffer solution；In described derivatization reagent, o-phthalaldehyde(OPA) is 1:8～1:3.89 with the mol ratio of TGA；
If in derivatization reagent used, the ratio of the mole of the Gentamicin C1a that the mole of o-phthalaldehyde(OPA) is measured with described testing sample, more than or equal to 1.6, is testing result in single testing result；If in derivatization reagent used, the ratio of the mole of the Gentamicin C1a that the mole of o-phthalaldehyde(OPA) is measured with described testing sample is less than 1.6 in single testing result, o-phthalaldehyde(OPA) described in single detection and the actual mol ratio of Gentamicin C1a in described testing sample need to be improved, again detect, till in single testing result, " ratio of the mole of the Gentamicin C1a that the mole of o-phthalaldehyde(OPA) is measured with described testing sample in derivatization reagent used " is more than or equal to 1.6；
The testing sample that the described testing sample containing Gentamicin C1a is formed after purification pretreatment by the fermentation liquid containing Gentamicin C1a in tunning.
It is preferred that in the detection method of described Gentamicin C1a, in described derivatization reagent, o-phthalaldehyde(OPA) is 1:6～1:3.89 with the mol ratio of TGA；It is more preferably 1:5～1:3.89.
O-phthalaldehyde(OPA) described in the detection of described raising single and the method for the actual mol ratio of Gentamicin C1a in described testing sample, preferably, under conditions of each concentration of component of described derivatization reagent, each concentration of component of described testing sample are the most constant, improve the mass ratio of derivatization reagent described in detection system and described testing sample；Or it is, under conditions of the mass ratio of described derivatization reagent Yu described testing sample is constant, described testing sample to be diluted and/or described derivatization reagent is concentrated.
The described fermentation liquid containing Gentamicin C1a can be the microorganism culturing fermentation gained of this area various general metabolic Gentamicin C1a.It is preferred that the microorganism of described metabolism Gentamicin C1a is deep red micromonospora (Micromonmspora purpurea).Described deep red micromonospora is preferably purchased from the deep red micromonospora that strain number is CICC11015 of Chinese industrial Microbiological Culture Collection administrative center (CICC) preservation.Above-mentioned deep red micromonospora all can be commercially available at above-mentioned preservation center.
It is preferred that described fermentation liquid is for putting tank fermentation liquid.
It is preferred that described purification pretreatment is: described fermentation liquid is carried out acidification and makes Gentamicin C1a discharge, and after solid-liquid separation, filtrate is the described testing sample containing Gentamicin C1a.
It is preferred that described purification pretreatment may also include the steps of: after described solid-liquid separation, described filtrate being adsorbed with cation exchange resin, then use aqueous alkali eluting, eluent is the described testing sample containing Gentamicin C1a.It is preferred that described cation exchange resin is HZ-732 cation exchange resin.
It is preferred that described acidification is for adjusting pH to 1.5～2.5 (being more preferably 1.5～2.0) by described fermentation liquid aqueous sulfuric acid.It is preferred that the process of described acidification also stands.It is preferred that the time of described standing is 1～4 hour.It is preferred that sulfuric acid concentration is 4～18M in described aqueous sulfuric acid, such as 9M.It is preferred that described solid-liquid separation was for using sheet frame to carry out solid-liquid separation or centrifugation.It is preferred that described aqueous alkali is 1mol/L～5mol/LNaOH aqueous solution.
It is preferred that the solution that described derivatization reagent each component that is it is formed after uniformly mixing.
It is preferred that o-phthalaldehyde(OPA) is 0.01g/mL～0.08g/mL with the mass volume ratio of described derivatization reagent in described derivatization reagent；It is more preferably 0.04g/mL～0.06g/mL.
Described solvent can be one or more in the various Conventional solvents in this area, preferably alcohols solvent, esters solvent and ether solvent.It is preferred that described alcohols solvent is one or more in methanol, ethanol and propanol.
The consumption of described solvent can be this area conventional amount used, it is preferred that the volume mass of described solvent and described o-phthalaldehyde(OPA) is than for 1mL/g～10mL/g；It is more preferably 1.25mL/g～5mL/g.
Described buffer solution can be one or more in this area various conventional buffer solution, preferably boric acid aqueous solution, borax solution and aqueous phosphatic.It is preferred that the molar concentration of described boric acid aqueous solution mesoboric acid is 0.2mol/L～0.5mol/L；It is more preferably 0.4mol/L～0.45mol/L.It is preferred that the pH of described boric acid aqueous solution is 10～11；It is more preferably 10.4～11.0.It is preferred that the pH of described boric acid aqueous solution is to be adjusted by the aqueous solution that mass fraction is 40%～50% (being more preferably 45%) or the aqueous solution of saturated sodium hydroxide and/or potassium hydroxide (being more preferably 45%) that mass fraction is 40%～50% or saturated.
It is preferred that the pH of described derivatization reagent is to be adjusted by the aqueous solution that mass fraction is 40%～50% (being more preferably 45%) or the aqueous solution of saturated sodium hydroxide and/or potassium hydroxide (being more preferably 45%) that mass fraction is 40%～50% or saturated.
The consumption of described buffer solution can be the various conventional amount used in this area, it is preferred that the volume ratio of described solvent and described buffer solution is 1:12～1:20；It is more preferably 1:19～1:13.
It is preferred that described derivative reaction is before carrying out, also reaction system is mixed homogeneously with isopropanol.It is preferred that the volume ratio of described isopropanol and described reaction system is 1:1～1:2.It is preferred that described derivative reaction is to carry out in confined conditions.It is preferred that the temperature of described derivative reaction is 40～60 DEG C；It is more preferably 60～65 DEG C.It is preferred that the time of described derivative reaction is 10～20min；It is more preferably 15～17min.It is preferred that described derivative reaction is after end, also include the operation of post processing.It is preferred that the operation of described post processing is: reaction system is placed in-10～0 DEG C and terminates reaction by employing, and/or, centrifuging and taking supernatant.It is preferred that described centrifugal rotating speed is 12000rmp～14000rmp.It is preferred that the described centrifugal time is 5～10min.
It is preferred that during described liquid chromatograph or liquid phase chromatogram-mass spectrometry combination method detect, chromatographic column used is C18 chromatographic column.Described C18 chromatographic column preferably receives the C18 chromatographic column that model is Unisil 5-120C18 of micro-(Nano-Micro) company.It is preferred that the packing material size of described C18 chromatographic column is 5 μm.It is preferred that a length of 250mm of described C18 chromatographic column.It is preferred that a diameter of 4.6mm of described C18 chromatographic column.It is preferred that in described liquid chromatograph or liquid phase chromatogram-mass spectrometry combination method detection, flowing used is mutually for heptanesulfonic acid sodium water solution, glacial acetic acid and the mixed liquor of methanol that volume ratio is 4:1:15；In described heptanesulfonic acid sodium water solution, the mass concentration of sodium heptanesulfonate is 5g/L～10g/L.It is preferred that during described liquid chromatograph or liquid phase chromatogram-mass spectrometry combination method detect, the consumption of flowing phase is 1L～1.5L.It is preferred that during described liquid chromatograph or liquid phase chromatogram-mass spectrometry combination method detect, the flow velocity of flowing phase is 1.0mL/min～1.2mL/min.It is preferred that during described liquid chromatograph or liquid phase chromatogram-mass spectrometry combination method detect, sample size is 5～20 μ L.It is preferred that during described liquid chromatograph or liquid phase chromatogram-mass spectrometry combination method detect, column temperature is 20～45 DEG C.It is preferred that during described liquid chromatograph or liquid phase chromatogram-mass spectrometry combination method detect, liquid chromatographic detection wavelength is 330nm～335nm.
It is preferred that in the detection of described liquid phase chromatogram-mass spectrometry combination method, use electro-spray ionization ionization source.It is preferred that the spray voltage of described electro-spray ionization ionization source is 4000～5000V.It is preferred that in the detection of described liquid phase chromatogram-mass spectrometry combination method, dryer temperature is 300～350 DEG C.It is preferred that in the detection of described liquid phase chromatogram-mass spectrometry combination method, use cation scan pattern.
On the basis of meeting common sense in the field, above-mentioned each optimum condition, can combination in any, obtain the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are the most commercially.
The most progressive effect of the present invention is: the detection method of the Gentamicin C1a of the present invention is simple to operate when detecting Gentamicin C1a concentration, accuracy is higher, the range of linearity is wide.
Accompanying drawing explanation
Fig. 1 is the Dependence Results of gained in reference example 3
Fig. 2 is the Dependence Results of gained in comparative example 1
Fig. 3 is the Dependence Results of gained in embodiment 1
Fig. 4 is the Dependence Results of gained in embodiment 2
Detailed description of the invention
Further illustrate the present invention below by the mode of embodiment, but the most therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Instrument in following example: Agilent 1200 type chromatograph of liquid, including the online degasser of G1322A type, G1311A type automatic sampler (U.S.'s Agilent)；DK-8D type electric heating constant temperature tank, Shanghai precision experimental facilities company limited；Mass spectrum: LCQ Fleet Ion Trap MSn Mass Spectrometer, Thermo Fisher Scientific.
Agents useful for same in following example: methanol, sulphuric acid, boric acid, isopropanol, sodium hydroxide, o-phthalaldehyde(OPA) (OPA) are analytical pure, and TGA, sodium heptanesulfonate are chromatographically pure.
Reference example 1
Standard substance: precision weighs Gentamicin C1a standard substance, are dissolved in distilled water and make the solution that concentration is 18mg/ml, 4 DEG C of Refrigerator stores, face the used time and are diluted.
Sample: cross sheet frame after putting the acidifying of tank fermentation liquid (ferment tank that purple-red single-spore bacteria Micromonmspora purpurea is fermented) 4mol/L～18mol/IL sulphuric acid 1 hour and carry out solid-liquid separation, filtrate is after HZ-732 ion exchange resin adsorbs, 1mol/L～5mol/LNaOH aqueous solution eluting, eluent is testing sample, in this sample, the content of Gentamicin C1a is 2～15mg/mL, and HPLC purity is 40%～about 60%.
1, derivatization reagent compound method:
O-phthalaldehyde(OPA) 1g (0.0074mol), it is dissolved in 5ml methanol, add TGA 2ml (0.0288mol), finally add 0.4mol/L boric acid (45% sodium hydroxide adjusts pH value 10.4) 95ml mixing, adjust pH 10.4 with 45% sodium hydroxide again, put refrigerator and keep in Dark Place.
3, derivative reaction step:
Accurately measure 10mL sample in volumetric flask, add 5mL isopropanol and the mixing of 4mL derivatization reagent, then be settled to 25mL with isopropanol, fully airtight after mixing, 60 DEG C of water-bath 15min are placed in frozen water termination reaction, and 12000rmp can carry out HPLC-MS detection after being centrifuged 5 minutes.
4, HPLC detection method:
Chromatographic condition: the model of chromatographic column Nano-Micro company is the chromatographic column (5 μm, 4.6 × 250mm) of Unisil 5-120C18；Flowing phase heptanesulfonic acid sodium water solution (5g/L, 200ml)-glacial acetic acid (50ml)-methanol (750ml)；Flow velocity 1.2ml/min；Column temperature 40 DEG C；Sample size 20 μ l；Detection wavelength 330nm.Gentamicin C1a appearance time is about 9～11min.
Here detection method to sulmycin during derivatization reagent compound method and derivative reaction step refer to the 3rd～5 section of the left hurdle of American Pharmacopeia USP35-nf30 version page 3326.
Reference example 2 (precision test)
The standard solution that concentration in reference example 1 is 18mg/ml is diluted with distilled water into the standard solution that concentration is 1.155mg/ml；Then according to derivatization reagent compound method preparation derivatization reagent in reference example 1, and perform the derivatization according to derivative reaction step, HPLC detection method in reference example 1 and detect.Repeat sample introduction 5 times, calculate RSD, the results are shown in Table 1.
Table 1
As seen from Table 1, performing the derivatization according to the compound method of the derivatization reagent in reference example 1, derivative reaction step, HPLC detection method and detect, precision is higher.
Reference example 3 (standard substance curve plotting)
Accurate draw Gentamicin C1a standard solution, be diluted to the solution of variable concentrations respectively, perform the derivatization according to the compound method of the derivatization reagent in reference example 1, derivative reaction step, HPLC detection method and detect.In result, Gentamicin C1a peak area is as vertical coordinate (Y), and Gentamicin C1a concentration is abscissa (X), carries out linear regression, obtains regression equation: y=0.6672x+13.146R2=0.9997.This result shows, uses the method that Gentamicin C1a is carried out HPLC detection, and in good linear relationship in the range of Gentamicin C1a concentration is 0.1～18mg/mL, linear regression result is as shown in Figure 1.
Comparative example 1 (sample curves drafting)
Accurate draw testing sample solution in reference example 1, be diluted to the solution of variable concentrations respectively, perform the derivatization according to the compound method of the derivatization reagent in reference example 1, derivative reaction step, HPLC detection method and detect.In result, Gentamicin C1a peak area is as vertical coordinate (Y), and Gentamicin C1a concentration is abscissa (X), carries out linear regression, and linear regression result is as shown in Figure 2.As it is clear from fig. 2 that use the method that Gentamicin C1a sample 2 is carried out HPLC detection, when Gentamicin C1a concentration is more than 2mg/mL, linear relationship is poor.
Wherein, when in X-axis, concentration value is less than or equal to 2mg/mL, its concentration values is according to the concentration values corresponding to its peak area on reference example 3 Plays product curve；And when concentration values is more than 2mg/mL in X-axis, calculate its concentration value with it relative to the cycles of concentration of the concentration sample introduction liquid as 2mg/mL.
Embodiment 1 (surveying Gentamicin C1a concentration after testing sample dilution)
Accurate draw testing sample solution in reference example 1, be diluted to the solution of different multiples respectively, multiple such as: 1,1.2,1.4,1.6,1.8,2,4,6,8,10 times.Perform the derivatization according to the compound method of the derivatization reagent in reference example 1, derivative reaction step, HPLC detection method and detect, detection gained peak area finds corresponding concentration values according to reference example 3 Plays product curve, this concentration values is multiplied by corresponding to this sample introduction liquid in the extension rate of testing sample, the concentration of testing sample.
With extension rate as X-axis, detection gained testing sample concentration is Y-axis, does curve, and result is shown in Fig. 3.It can be seen from figure 3 that when testing sample carries out 1.8～10 times of dilutions, testing result is more accurate, and the R of the curve corresponding to this partial dot2=0.1767.
As can be known from Fig. 3, testing sample concentration is 14.6mg/mL, will carry out 1.8～10 times of dilutions, i.e. enter sample liquid concentration for less than 8.1mg/mL time, testing result is the most accurate.
Embodiment 2 (surveying Gentamicin C1a concentration after increasing derivatization reagent consumption)
Testing sample is performed the derivatization according to the compound method of the derivatization reagent in reference example 1, derivative reaction step, HPLC detection method and detects, and the consumption of the derivatization reagent in derivative reaction step therein increase to original 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, keep reaction system cumulative volume constant.Detection gained peak area finds corresponding concentration values according to reference example 3 Plays product curve, is the concentration of testing sample.
With the consumption of derivatization reagent as X-axis, with the testing sample concentration that obtains of detection as Y-axis, do curve, result is shown in Fig. 4.As seen from Figure 4, when improving the consumption of the derivatization reagent in the derivative reaction step in reference example 1 to 1.5～3 times, testing result is more accurate, and the R of the curve corresponding to this partial dot2=0.4219.
Claims (10)
1. the detection method of a Gentamicin C1a, it is characterised in that its comprise the steps: by
Testing sample containing Gentamicin C1a and derivatization reagent mix homogeneously also carry out derivative reaction, so
After carry out liquid chromatograph or liquid phase chromatogram-mass spectrometry combination method detection,；
Wherein, described derivatization reagent comprise solvent, o-phthalaldehyde(OPA), TGA and buffer solution this
Several components；In described derivatization reagent, o-phthalaldehyde(OPA) is 1:8～1:3.89 with the mol ratio of TGA；
The pH=10.0～11.0 of described derivatization reagent；
If in derivatization reagent used, the mole of o-phthalaldehyde(OPA) is treated with described in single testing result
The ratio of the mole of Gentamicin C1a measured in test sample product, more than or equal to 1.6, is detection knot
Really；If in derivatization reagent used, the mole of o-phthalaldehyde(OPA) is to be measured with described in single testing result
When the ratio of the mole of Gentamicin C1a measured in sample is less than 1.6, single detection need to be improved
Described in the actual mol ratio of Gentamicin C1a in o-phthalaldehyde(OPA) and described testing sample, carry out again
Detection, until " in derivatization reagent used, the mole of o-phthalaldehyde(OPA) is with described in single testing result
The ratio of the mole of Gentamicin C1a measured in testing sample " more than or equal to 1.6 till；
The described testing sample containing Gentamicin C1a is sending out containing Gentamicin C1a in tunning
The testing sample that ferment liquid is formed after purification pretreatment.
2. the detection method of Gentamicin C1a as claimed in claim 1, it is characterised in that described
In derivatization reagent, o-phthalaldehyde(OPA) is 1:6～1:3.89 with the mol ratio of TGA.
3. the detection method of Gentamicin C1a as claimed in claim 1, it is characterised in that described
In derivatization reagent, o-phthalaldehyde(OPA) is 1:5～1:3.89 with the mol ratio of TGA.
4. the detection method of the Gentamicin C1a as according to any one of claims 1 to 3, its feature
It is:
O-phthalaldehyde(OPA) described in the detection of described raising single and Gentamicin C1a in described testing sample
The method of actual mol ratio be, each group of each concentration of component of described derivatization reagent, described testing sample
Derivatization reagent described in detection system and described testing sample is improved under conditions of dividing concentration the most constant
Mass ratio；Or it is, under conditions of the mass ratio of described derivatization reagent Yu described testing sample is constant,
Described testing sample is diluted and/or described derivatization reagent is concentrated；
And/or, the described fermentation liquid containing Gentamicin C1a is the microorganism of metabolism Gentamicin C1a
Cultivation and fermentation gained；The microorganism of described metabolism Gentamicin C1a is deep red micromonospora；
And/or, described fermentation liquid is for putting tank fermentation liquid；
And/or, described purification pretreatment is: described fermentation liquid is carried out acidification and makes Gentamicin C1a
Discharge, and after solid-liquid separation, filtrate is the described testing sample containing Gentamicin C1a.
5. the detection method of Gentamicin C1a as claimed in claim 4, it is characterised in that:
Described acidification is that described fermentation liquid aqueous sulfuric acid is adjusted pH to 1.5～2.5；
And/or, the process of described acidification also stands；
And/or, described solid-liquid separation carried out solid-liquid separation or centrifugation for using sheet frame；
And/or, described purification pretreatment also comprises the steps: after described solid-liquid separation, by described filter
Liquid cation exchange resin adsorbs, and then uses aqueous alkali eluting, and eluent is described big containing celebrating
The testing sample of mycin C1a.
6. the detection method of Gentamicin C1a as claimed in claim 5, it is characterised in that:
The time of described standing is 1～4 hour；
And/or, in described aqueous sulfuric acid, sulfuric acid concentration is 4～18M；
And/or, described aqueous alkali is 1mol/L～5mol/LNaOH aqueous solution；
And/or, described cation exchange resin is HZ-732 cation exchange resin.
7. the detection method of the Gentamicin C1a as according to any one of claims 1 to 3, its feature
It is:
Described derivatization reagent is the solution formed after its each component uniformly mixes；
And/or, in described derivatization reagent, o-phthalaldehyde(OPA) with the mass volume ratio of described derivatization reagent is
0.01g/mL～0.08g/mL；
And/or, described solvent is one or more in alcohols solvent, esters solvent and ether solvent；
And/or, the volume mass of described solvent and described o-phthalaldehyde(OPA) is than for 1mL/g～10mL/g；
And/or, described buffer solution is in boric acid aqueous solution, borax solution and aqueous phosphatic
Plant or multiple；
And/or, the pH of described derivatization reagent is to be 40%～the hydrogen of 50% or saturated by mass fraction
The aqueous solution of sodium oxide and/or mass fraction are that the aqueous solution of the potassium hydroxide of 40%～50% or saturated is carried out
Regulation；
And/or, described solvent is 1:12～1:20 with the volume ratio of described buffer solution.
8. the detection method of Gentamicin C1a as claimed in claim 7, it is characterised in that: described
The molar concentration of boric acid aqueous solution mesoboric acid is 0.2mol/L～0.5mol/L；And/or, described boric acid is water-soluble
The pH of liquid is 10～11；And/or, the pH of described boric acid aqueous solution is to be 40%～50% by mass fraction
Or the potassium hydroxide that the aqueous solution of saturated sodium hydroxide and/or mass fraction are 40%～50% or saturated
Aqueous solution is adjusted；
And/or, described alcohols solvent is one or more in methanol, ethanol and propanol；
And/or, in described derivatization reagent, o-phthalaldehyde(OPA) with the mass volume ratio of described derivatization reagent is
0.04g/mL～0.06g/mL；
And/or, the volume mass of described solvent and described o-phthalaldehyde(OPA) is than for 1.25mL/g～5mL/g；
And/or, described solvent is 1:19～1:13 with the volume ratio of described buffer solution.
9. the detection method of the Gentamicin C1a as according to any one of claims 1 to 3, its feature
It is: reaction system, before carrying out, is also mixed homogeneously by described derivative reaction with isopropanol；Described different
Propanol is 1:1～1:2 with the volume ratio of described reaction system；And/or, described derivative reaction is close
Carry out under the conditions of closing；And/or, the temperature of described derivative reaction is 40～60 DEG C；And/or, described in spread out
The time of biochemical reaction is 10～20min；And/or, described derivative reaction is after end, after also including
The operation processed；The operation of described post processing is: reaction system is placed in-10～0 DEG C and terminates anti-by employing
Should, centrifuging and taking supernatant.
10. the detection method of the Gentamicin C1a as according to any one of claims 1 to 3, its feature
It is: in described liquid chromatograph or liquid phase chromatogram-mass spectrometry combination method detection, chromatographic column used is C18
Chromatographic column；And/or, flowing used is mutually for heptanesulfonic acid sodium water solution that volume ratio is 4:1:15, ice vinegar
Acid and the mixed liquor of methanol；In described heptanesulfonic acid sodium water solution, the mass concentration of sodium heptanesulfonate is
5g/L～10g/L；And/or, the consumption of flowing phase is 1L～1.5L；And/or, the flow velocity of flowing phase is
1.0mL/min～1.2mL/min；And/or, sample size is 5～20 μ L；And/or, column temperature is 20～45 DEG C；
And/or, liquid chromatographic detection wavelength is 330nm～335nm.
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