CN106290591B - A kind of detection method of Gentamicin C1a - Google Patents
A kind of detection method of Gentamicin C1a Download PDFInfo
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Abstract
The invention discloses a kind of detection methods of Gentamicin C1a, it includes the following steps: for the sample to be tested containing Gentamicin C1a and derivatization reagent to be uniformly mixed and carries out derivative reaction, then liquid chromatogram or liquid phase chromatogram-mass spectrometry combination method detection are carried out；Wherein, the derivatization reagent includes solvent, o-phthalaldehyde, thioacetic acid and buffer solution these types component；The molar ratio of o-phthalaldehyde and thioacetic acid is 1:3.8~1:0.5 in the derivatization reagent；PH=10~11 of the derivatization reagent；The ratio of the mole of Gentamicin C1a measured in the mole of o-phthalaldehyde and the sample to be tested in derivatization reagent used need to be more than or equal to 3.59 in single testing result.The detection method of Gentamicin C1a of the invention is easy to operate, and accuracy is higher when Gentamicin C1a concentration in detecting production sample, the range of linearity is wide.
Description
Technical field
The present invention relates to a kind of detection methods of Gentamicin C1a.
Background technique
Derivatization detection method before the HPLC column of existing Gentamicin C1a, in generally United States Pharmacopeia USP35-nf30 editions
To the detection method of sulmycin.For example, accurately measure 10mL sample into volumetric flask, be added 5mL isopropanol and
4mLOPA derivatization reagent mixes, then is settled to 25mL with isopropanol, mixes well rear closed, and 60 DEG C of water-bath 15min are placed on
Reaction is terminated in ice water, 12000rmp centrifugation can carry out HPLC detection after five minutes.Wherein, used OPA derivatization reagent
Formula: o-phthalaldehyde 1g (0.0074mol) is dissolved in 5ml methanol, be added thioacetic acid 2ml (0.0288mol), finally plus
0.4mol/L boric acid (45% sodium hydroxide tune pH value 10.4) 95ml is mixed, then with 45% sodium hydroxide tune pH 10.4, puts refrigerator
It is kept in dark place.This method restricted application in the industrial production, the range of linearity is narrow, leads to the inspection of Gentamicin C1a concentration
It is excessively poor to survey result accuracy.And in actual production process, concentration is higher and the lower Gentamicin C1a sample of purity is people
Therefore the object often to be detected develops a kind of derivatization for the Gentamicin C1a sample that accuracy is higher, the range of linearity is wide
Reagent is this field urgent problem to be solved.
Summary of the invention
The technical problem to be solved by the present invention is in order to overcome, Gentamicin C1a is in detectable concentration in the prior art, line
The defects of property narrow range, restricted application, and provide a kind of detection method of Gentamicin C1a.Celebrating of the invention is mould greatly
The detection method of plain C1a is easy to operate, and accuracy is higher, the range of linearity when Gentamicin C1a concentration in detecting production sample
Extensively.
The present invention provides a kind of detection methods of Gentamicin C1a comprising following steps: will contain gentamicin
The sample to be tested and derivatization reagent of C1a is uniformly mixed and carries out derivative reaction, then carries out liquid chromatogram or liquid chromatogram-
Mass spectrometry combination method detection；
Wherein, the derivatization reagent includes solvent, o-phthalaldehyde, thioacetic acid and buffer solution these types component；
The molar ratio of o-phthalaldehyde and thioacetic acid is 1:3.8~1:0.5 in the derivatization reagent；The pH of the derivatization reagent
=10~11；
If in single testing result in derivatization reagent used in the mole of o-phthalaldehyde and the sample to be tested
The ratio of the mole of measured Gentamicin C1a is more than or equal to 3.59, as testing result；If in single testing result
The mole of the mole of o-phthalaldehyde and Gentamicin C1a measured in the sample to be tested in derivatization reagent used
Ratio less than 3.59 when, Gentamicin C1a in o-phthalaldehyde and the sample to be tested described in single detection need to be improved
Practical molar ratio, is detected again, until " the mole of o-phthalaldehyde in derivatization reagent used in single testing result
With the ratio of the mole of Gentamicin C1a measured in the sample to be tested " be more than or equal to 3.59 until；
The sample to be tested containing Gentamicin C1a is that the fermentation liquid in tunning containing Gentamicin C1a passes through
Sample to be tested is formed by after purifying pretreatment.
Preferably, the molar ratio of o-phthalaldehyde and thioacetic acid is 1:3.24~1:0.5 in the derivatization reagent, more
It goodly is 1:1.2~1:0.8.
Practical mole for improving Gentamicin C1a in o-phthalaldehyde described in single detection and the sample to be tested
The method of ratio, preferably, in the item that the derivatization reagent each component concentration, the sample to be tested each component concentration are constant
The mass ratio of derivatization reagent and the sample to be tested described in detection architecture is improved under part；It or is to be tried in the derivatization
Under conditions of the mass ratio of agent and the sample to be tested is constant, the sample to be tested is diluted and/or to the derivatization
Reagent is concentrated.
The fermentation liquid containing Gentamicin C1a can be the microorganism of the various general metabolic Gentamicin C1as in this field
Obtained by cultivation and fermentation.Preferably, the microorganism of the metabolism Gentamicin C1a is deep red micromonospora (Micromonmspora
purpurea).The deep red micromonospora is preferably purchased from Chinese industrial Microbiological Culture Collection administrative center (CICC) guarantor
The strain number of hiding is the deep red micromonospora of CICC11015.Above-mentioned deep red micromonospora can be bought in above-mentioned collection
It obtains.
Preferably, the fermentation liquid is to put tank fermentation liquid.
Preferably, the purifying pretreatment are as follows: release Gentamicin C1a fermentation liquid progress acidification
Come, and after separation of solid and liquid, filtrate is the sample to be tested containing Gentamicin C1a.
Preferably, the purifying pretreatment may also include the steps of: after the separation of solid and liquid, by the filtrate sun
Ion exchange resin is adsorbed, and is then eluted with buck, and eluent is the sample to be tested containing Gentamicin C1a.
Preferably, the cation exchange resin is HZ-732 cation exchange resin.
Preferably, the acidification be by the fermentation liquid aqueous sulfuric acid tune pH to 1.5~2.5 (more preferably for
1.5~2.0).Preferably, the process of the acidification is also stood.Preferably, the time of the standing is 1~4 small
When.Preferably, sulfuric acid concentration is 4~18M, such as 9M in the aqueous sulfuric acid.Preferably, described be separated by solid-liquid separation to use
Sheet frame is separated by solid-liquid separation or is centrifugated.Preferably, the buck is 1mol/L~5mol/LNaOH aqueous solution.Preferably,
The sour water is 0.2mol/L~2mol/L aqueous sulfuric acid.
Preferably, the derivatization reagent is that its each component is formed by solution after evenly mixing.
Preferably, the mass volume ratio of o-phthalaldehyde and the derivatization reagent is 0.01g/ in the derivatization reagent
ML~0.08g/mL；It is more preferably 0.012g/mL~0.04g/mL.
The solvent can be the various Conventional solvents in this field, preferably in alcohols solvent, esters solvent and ether solvent
It is one or more.Preferably, the alcohols solvent is one of methanol, ethyl alcohol and propyl alcohol or a variety of.
The dosage of the solvent can be this field conventional amount used, preferably, the body of the solvent and the o-phthalaldehyde
Product mass ratio is 1mL/g~10mL/g；It is more preferably 1.25mL/g~5mL/g.
The buffer solution can be the various conventional buffer solutions in this field, preferably boric acid aqueous solution, borax solution
With one of phosphate aqueous solution or a variety of.Preferably, the molar concentration of boric acid is 0.2mol/L in the boric acid aqueous solution
~0.5mol/L；It is more preferably 0.4mol/L~0.45mol/L.Preferably, the pH of the boric acid aqueous solution is 10~11；More preferably
Ground is 10.4~11.0.Preferably, the pH of the boric acid aqueous solution is is 40%~50% by mass fraction (more preferably for
45%) or saturation sodium hydroxide aqueous solution and/or mass fraction be 40%~50% or saturation potassium hydroxide (more preferably
It is adjusted for aqueous solution 45%).
Preferably, it is 40%~50% (being more preferably 45%) or full that the pH of the derivatization reagent, which is by mass fraction,
The aqueous solution and/or mass fraction of the sodium hydroxide of sum are the potassium hydroxide (being more preferably 45%) of 40%~50% or saturation
Aqueous solution be adjusted.
The dosage of the buffer solution can be the various conventional amount useds in this field, preferably, the solvent and the buffering are molten
The volume ratio of liquid is 1:12~1:20；It is more preferably 1:19~1:13.
Preferably, reaction system before progress, is also uniformly mixed by the derivative reaction with isopropanol.Preferably, institute
The volume ratio for stating isopropanol and the reaction system is 1:1~1:2.Preferably, the derivative reaction is in confined conditions
It carries out.Preferably, the temperature of the derivative reaction is 40~60 DEG C；It is more preferably 60~65 DEG C.Preferably, the derivatization
The time of reaction is 10~20min；It is more preferably 15~17min.Preferably, the derivative reaction after, further include
The operation of post-processing.Preferably, the operation of the post-processing are as follows: reaction is terminated using reaction system is placed in -10~0 DEG C,
Centrifuging and taking supernatant.Preferably, the revolving speed of the centrifugation is 12000rmp~14000rmp.Preferably, the time of the centrifugation
For 5~10min.
Preferably, chromatographic column used is C18 chromatography in the liquid chromatogram or liquid phase chromatogram-mass spectrometry combination method detection
Column.The C18 chromatographic column be preferably receive micro- (Nano-Micro) company model Unisil 5-120C18 C18 chromatography
Column.Preferably, the packing material size of the C18 chromatographic column is 5 μm.Preferably, the length of the C18 chromatographic column is 250mm.Preferably
Ground, the diameter of the C18 chromatographic column are 4.6mm.Preferably, the liquid chromatogram or liquid phase chromatogram-mass spectrometry combination method detection
In, mobile phase used is the mixed liquor of heptanesulfonic acid sodium water solution, glacial acetic acid and methanol that volume ratio is 4:1:15；The heptane
The mass concentration of sodium heptanesulfonate is 5g/L~10g/L in sulfonic acid sodium water solution.Preferably, the liquid chromatogram or liquid phase color
In spectrum-mass spectrometry combination method detection, the dosage of mobile phase is 1L~1.5L.Preferably, the liquid chromatogram or liquid chromatogram-matter
It composes in method for combined use detection, the flow velocity of mobile phase is 1.0mL/min~1.2mL/min.Preferably, the liquid chromatogram or liquid phase
In the detection of chromatograph-mass spectrometer coupling method, sample volume is 5~20 μ L.Preferably, the liquid chromatogram or liquid phase chromatogram-mass spectrometry combination
In being detected with method, column temperature is 20~45 DEG C.Preferably, in the liquid chromatogram or liquid phase chromatogram-mass spectrometry combination method detection,
Liquid chromatographic detection wavelength is 330nm~335nm.
Preferably, in the liquid phase chromatogram-mass spectrometry combination method detection, using electro-spray ionization ionization source.Preferably,
The spray voltage of the electro-spray ionization ionization source is 4000~5000V.Preferably, the liquid chromatograph mass spectrography side
In method detection, dryer temperature is 300~350 DEG C.Preferably, being used in the liquid phase chromatogram-mass spectrometry combination method detection
Cation scan pattern.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that: the detection method of Gentamicin C1a of the invention is in detection gentamicin
Accuracy easy to operate is higher when C1a concentration, the range of linearity is wide.
Detailed description of the invention
Fig. 1 is Dependence Results obtained in reference implementation example 3
Fig. 2 is Dependence Results obtained in comparative example 1
Fig. 3 is Dependence Results obtained in embodiment 2
Fig. 4 is Dependence Results obtained in embodiment 3
Fig. 5 is Dependence Results obtained in embodiment 5
Fig. 6 is Dependence Results obtained in embodiment 6
Fig. 7 is Dependence Results obtained in embodiment 8
Fig. 8 is Dependence Results obtained in embodiment 9
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
Instrument in following embodiment: 1200 type liquid chromatograph of Agilent, including G1322A type on-line degassing machine,
G1311A type autosampler (U.S.'s Agilent)；DK-8D type electric heating constant temperature sink, Shanghai precision experimental facilities Co., Ltd.
Agents useful for same in following embodiment: methanol, sulfuric acid, boric acid, isopropanol, sodium hydroxide, o-phthalaldehyde (OPA) are equal
Pure to analyze, thioacetic acid, sodium heptanesulfonate are chromatographically pure.
Reference implementation example 1
Standard items: precision weighs Gentamicin C1a standard items, and being dissolved in and concentration is made in distilled water is the molten of 18mg/ml
Liquid, 4 DEG C of refrigerators save, and face the used time and are diluted.
Sample: (the fermentation fermented to purple-red single-spore bacteria Micromonmspora purpurea of tank fermentation liquid is put
Tank fermentation) with 4mol/L~18mol/IL sulfuric acid be acidified 1 hour after cross sheet frame be separated by solid-liquid separation, filtrate is handed over through HZ-732 ion
After changing resin adsorption, the elution of 1mol/L~5mol/LNaOH aqueous solution, eluent is sample to be tested, gentamicin in the sample
The content of C1a is 2~15mg/mL, and HPLC purity is 40%~60% or so.
1, derivatization reagent preparation method is 1.:
O-phthalaldehyde 1g (0.0074mol), is dissolved in 5ml methanol, is added thioacetic acid 2ml (0.0288mol), finally
Add 0.4mol/L boric acid (45% sodium hydroxide tune pH value 10.4) 95ml to mix, then with 45% sodium hydroxide tune pH 10.4, puts ice
Case is kept in dark place.
Here 1. derivatization reagent preparation method can refer to United States Pharmacopeia USP35-nf30 the 3rd~5 section of left column of version page 3326
In to the detection method of sulmycin.
2, derivatization reagent preparation method is 2.:
O-phthalaldehyde 4g (0.0296mol), is dissolved in 5ml methanol, is added thioacetic acid 2ml (0.0288mol), finally
Add 0.4mol/L boric acid (45% sodium hydroxide tune pH value 10.4) 95ml to mix, then with 45% sodium hydroxide tune pH 10.4, puts ice
Case is kept in dark place.
3, derivatization reagent preparation method is 3.:
O-phthalaldehyde 1.2g (0.00888mol), is dissolved in 5ml methanol, is added thioacetic acid 2ml (0.0288mol),
Finally plus 0.4mol/L boric acid (45% sodium hydroxide tune pH value 10.4) 95ml is mixed, then with 45% sodium hydroxide tune pH 10.4,
Refrigerator is put to be kept in dark place.
4, derivatization reagent preparation method is 4.:
O-phthalaldehyde 4g (0.0296mol), is dissolved in 5ml methanol, is added thioacetic acid 1ml (0.0144mol), finally
Add 0.4mol/L boric acid (45% sodium hydroxide tune pH value 10.4) 95ml to mix, then with 45% sodium hydroxide tune pH 10.4, puts ice
Case is kept in dark place.
5, derivative reaction step:
The accurate 10mL sample that measures is added 5mL isopropanol and 4mL derivatization reagent mixes into volumetric flask, then uses isopropyl
Alcohol is settled to 25mL, mix well it is rear closed, 60 DEG C of water-bath 15min be placed in ice water terminate reaction, 12000rmp be centrifuged 5 points
HPLC detection can be carried out after clock.
6, HPLC detection method:
Chromatographic condition: the model Unisil 5-120C18 of chromatographic column Nano-Micro company chromatographic column (5 μm, 4.6
×250mm)；Mobile phase: heptanesulfonic acid sodium water solution (5g/L, 200ml)-glacial acetic acid (50ml)-methanol (750ml)；Flow velocity
1.2ml/min；40 DEG C of column temperature；20 μ l of sample volume；Detection wavelength 330nm.Gentamicin C1a appearance time is about 9~11min.
Here HPLC detection method can refer to big to celebrating in United States Pharmacopeia USP35-nf30 the 3rd~5 section of left column of version page 3326
The detection method of doxycycline sulfate.
Reference implementation example 2 (precision test)
It is 1.155mg/ that the standard solution that concentration is 18mg/ml in reference implementation example 1, which is diluted with distilled water into concentration,
The standard solution of ml；Then derivatization reagent is 1. prepared according to changing preparation of reagents method derived from reference implementation example 1, and according to
1 derivedization reaction step of reference implementation example, HPLC detection method are performed the derivatization and are detected.It repeats sample introduction 5 times, calculates RSD,
It the results are shown in Table 1.
Table 1
As seen from Table 1, according to the preparation method of the derivatization reagent in reference implementation example 1 1., derivative reaction step,
HPLC detection method is performed the derivatization and is detected, and precision is higher.
Reference implementation example 3 (standard items Drawing of Curve)
Precision draws Gentamicin C1a standard solution, the solution of various concentration is diluted to respectively, according to reference implementation example
The preparation method of derivatization reagent in 1 is 1., derivative reaction step, HPLC detection method perform the derivatization and detect.With knot
Gentamicin C1a peak area is ordinate (Y) in fruit, and Gentamicin C1a concentration is abscissa (X), carries out linear regression, must return
Return equation: y=0.6672x+13.146R2=0.9997.Should the result shows that, using this method to Gentamicin C1a carry out HPLC
Detection, being in Gentamicin C1a concentration is in good linear relationship, linear regression result such as Fig. 1 within the scope of 0.1~18mg/mL
It is shown.
Comparative example 1
Precision draws testing sample solution in reference implementation example 1, is diluted to the solution of various concentration respectively, according to reference reality
Apply the preparation method of the derivatization reagent in example 1 1., derivative reaction step, HPLC detection method perform the derivatization and detect.
Using Gentamicin C1a peak area in result as ordinate (Y), Gentamicin C1a concentration is abscissa (X), carries out linear regression,
Linear regression result is as shown in Figure 2.As it is clear from fig. 2 that carrying out HPLC detection to Gentamicin C1a sample 2 using this method, celebrating
When big mycin C1a concentration is greater than 2mg/mL, linear relationship is poor.
Wherein, when concentration value is less than or equal to 2mg/mL in X-axis, concentration values are according to 3 Plays product of reference implementation example
Concentration values corresponding to its peak area on curve；And when concentration values are greater than 2mg/mL in X-axis, it is relative to concentration with it
The cycles of concentration of the sample introduction liquid of 2mg/mL calculates its concentration value.
Embodiment 1 (precision test)
It is 1.155mg/ that the standard solution that concentration is 18mg/ml in reference implementation example 1, which is diluted with distilled water into concentration,
The standard solution of ml；Then derivatization reagent is 2. prepared according to changing preparation of reagents method derived from reference implementation example 1, and according to
1 derivedization reaction step of reference implementation example, HPLC detection method are performed the derivatization and are detected.It repeats sample introduction 5 times, calculates RSD,
It the results are shown in Table 2.
Table 2
As seen from Table 2, according to the preparation method of the derivatization reagent in reference implementation example 1 2., derivative reaction step,
HPLC detection method is performed the derivatization and is detected, and precision is higher.
Embodiment 2 (standard items Drawing of Curve)
Precision draws Gentamicin C1a standard solution, the solution of various concentration is diluted to respectively, according to reference implementation
The preparation method of derivatization reagent in example 1 is 2., derivative reaction step, HPLC detection method perform the derivatization and detect.With
As a result middle Gentamicin C1a peak area is ordinate (Y), and Gentamicin C1a concentration is abscissa (X), carries out linear regression, obtains
Regression equation: y=0.6669x+23.493, R2=0.9997.Should the result shows that, using this method to Gentamicin C1a carry out
It is in good linear relationship that HPLC detection, which is within the scope of 0.1~18mg/mL in Gentamicin C1a concentration, and linear regression result is such as
Shown in Fig. 3.
Embodiment 3
By sample to be tested according to the derivatization reagent in reference implementation example 1 preparation method 2., derivative reaction step,
HPLC detection method is performed the derivatization and is detected, and peak area is Y-axis, and concentration is X-axis, is carried out linear regression, is as a result seen Fig. 4.
Wherein, when concentration value is less than or equal to 14.6mg/mL in X-axis, concentration values are bent according to 2 Plays product of embodiment
Concentration values corresponding to its peak area on line；And when concentration values are greater than 14.6mg/mL in X-axis, it is relative to concentration with it
The cycles of concentration of the sample introduction liquid of 14.6mg/mL calculates its concentration value.
Gained regression equation: y=0.5732x+187.06, R2=0.9994.Should the result shows that, using derivative of the invention
When changing reagent to detecting after the lower sample derivatization of purity, remained within the scope of 0.1~15mg/mL of sample concentration in good
Good linear relationship (see Fig. 4).
Embodiment 4 (precision test)
It is 1.155mg/ that the standard solution that concentration is 18mg/ml in reference implementation example 1, which is diluted with distilled water into concentration,
The standard solution of ml；Then derivatization reagent is 3. prepared according to changing preparation of reagents method derived from reference implementation example 1, and according to
1 derivedization reaction step of reference implementation example, HPLC detection method are performed the derivatization and are detected.It repeats sample introduction 5 times, calculates RSD,
It the results are shown in Table 3.
Table 3
As seen from Table 3, according to the preparation method of the derivatization reagent in reference implementation example 1 3., derivative reaction step,
HPLC detection method is performed the derivatization and is detected, and precision is higher.
Embodiment 5 (standard items Drawing of Curve)
Precision draws Gentamicin C1a standard solution, the solution of various concentration is diluted to respectively, according to reference implementation example
The preparation method of derivatization reagent in 1 is 3., derivative reaction step, HPLC detection method perform the derivatization and detect.With knot
Gentamicin C1a peak area is ordinate (Y) in fruit, and Gentamicin C1a concentration is abscissa (X), carries out linear regression, must return
Return equation: y=0.666x+20.017, R2=0.9998.Should the result shows that, using this method to Gentamicin C1a carry out HPLC
Detection, being in Gentamicin C1a concentration is in good linear relationship, linear regression result such as Fig. 5 within the scope of 0.1~18mg/mL
It is shown.
Embodiment 6
By sample to be tested according to the derivatization reagent in reference implementation example 1 preparation method 3., derivative reaction step,
HPLC detection method is performed the derivatization and is detected, and peak area is Y-axis, and concentration is X-axis, is carried out linear regression, is as a result seen Fig. 6.
Wherein, when concentration value is less than or equal to 14.6mg/mL in X-axis, concentration values are according to 5 Plays of reference implementation example
Concentration values corresponding to its peak area on product curve；And when concentration values are greater than 14.6mg/mL in X-axis, with it relative to dense
Degree is the cycles of concentration of the sample introduction liquid of 14.6mg/mL to calculate its concentration value.
Gained regression equation: y=0.5779x+190.39, R2=0.9991.Should the result shows that, using derivative of the invention
When changing reagent to detecting after the lower sample derivatization of purity, remained within the scope of 0.1~15mg/mL of sample concentration in good
Good linear relationship (see Fig. 6).
Embodiment 7 (precision test)
It is 1.155mg/ that the standard solution that concentration is 18mg/ml in reference implementation example 1, which is diluted with distilled water into concentration,
The standard solution of ml；Then derivatization reagent is 4. prepared according to changing preparation of reagents method derived from reference implementation example 1, and according to
1 derivedization reaction step of reference implementation example, HPLC detection method are performed the derivatization and are detected.It repeats sample introduction 5 times, calculates RSD,
It the results are shown in Table 4.
Table 4
As seen from Table 4, according to the preparation method of the derivatization reagent in reference implementation example 1 4., derivative reaction step,
HPLC detection method is performed the derivatization and is detected, and precision is higher.
Embodiment 8 (standard items Drawing of Curve)
Precision draws Gentamicin C1a standard solution, the solution of various concentration is diluted to respectively, according to reference implementation example
The preparation method of derivatization reagent in 1 is 4., derivative reaction step, HPLC detection method perform the derivatization and detect.With knot
Gentamicin C1a peak area is ordinate (Y) in fruit, and Gentamicin C1a concentration is abscissa (X), carries out linear regression, must return
Return equation: y=0.6639x+28.235, R2=0.9997.Should the result shows that, using this method to Gentamicin C1a carry out
It is in good linear relationship that HPLC detection, which is within the scope of 0.1~18mg/mL in Gentamicin C1a concentration, and linear regression result is such as
Shown in Fig. 7.
Embodiment 9
By sample to be tested according to the derivatization reagent in reference implementation example 1 preparation method 4., derivative reaction step,
HPLC detection method is performed the derivatization and is detected, and peak area is Y-axis, and concentration is X-axis, is carried out linear regression, is as a result seen Fig. 8.
Wherein, when concentration value is less than or equal to 14.6mg/mL in X-axis, concentration values are according to 8 Plays of reference implementation example
Concentration values corresponding to its peak area on product curve；And when concentration values are greater than 14.6mg/mL in X-axis, with it relative to dense
Degree is the cycles of concentration of the sample introduction liquid of 14.6mg/mL to calculate its concentration value.
Gained regression equation: y=0.5758x+181.81, R2=0.9993.Should the result shows that, using derivative of the invention
When changing reagent to detecting after the lower sample derivatization of purity, remained within the scope of 0.1~15mg/mL of sample concentration in good
Good linear relationship (see Fig. 8).
Claims (10)
1. a kind of detection method of Gentamicin C1a, which is characterized in that it includes the following steps: will be containing Gentamicin C1a
Sample to be tested and derivatization reagent are uniformly mixed and carry out derivative reaction, then carry out liquid-phase chromatography method detection；
Wherein, the derivatization reagent includes solvent, o-phthalaldehyde, thioacetic acid and buffer solution these types component；It is described
The molar ratio of o-phthalaldehyde and thioacetic acid is 1:3.24 ~ 1:0.5 in derivatization reagent；PH=10 of the derivatization reagent ~
11；
If surveyed in the mole of o-phthalaldehyde and the sample to be tested in derivatization reagent used in single testing result
The ratio of the mole of the Gentamicin C1a obtained is more than or equal to 3.59, as testing result；If used in single testing result
The ratio of the mole of o-phthalaldehyde and the mole of Gentamicin C1a measured in the sample to be tested in derivatization reagent
When value is less than 3.59, the reality of Gentamicin C1a in o-phthalaldehyde described in single detection and the sample to be tested need to be improved
Molar ratio is detected again, until " mole of o-phthalaldehyde and institute in derivatization reagent used in single testing result
Until the ratio of the mole of Gentamicin C1a measured by stating in sample to be tested " is more than or equal to 3.59；
The sample to be tested containing Gentamicin C1a is that the fermentation liquid in tunning containing Gentamicin C1a passes through purifying
Sample to be tested is formed by after pretreatment.
2. the detection method of Gentamicin C1a as described in claim 1, it is characterised in that: the liquid chromatogram is liquid phase
Chromatograph-mass spectrometer coupling.
3. the detection method of Gentamicin C1a as described in claim 1, it is characterised in that: adjacent benzene in the derivatization reagent
The molar ratio of dicarbaldehyde and thioacetic acid is 1:1.2 ~ 1:0.8.
4. the detection method of Gentamicin C1a as claimed in any one of claims 1-3, it is characterised in that:
It is described to improve the practical molar ratio of Gentamicin C1a in o-phthalaldehyde described in single detection and the sample to be tested
Method is that inspection is improved under conditions of the derivatization reagent each component concentration, the sample to be tested each component concentration are constant
The mass ratio of derivatization reagent described in survey system and the sample to be tested；Or be, the derivatization reagent and it is described to
Under conditions of the mass ratio of sample is constant, the sample to be tested is diluted and/or the derivatization reagent is carried out dense
Contracting；
And/or the fermentation liquid containing Gentamicin C1a is the microculture fermentation gained for being metabolized Gentamicin C1a；Institute
The microorganism for stating metabolism Gentamicin C1a is deep red micromonospora；
And/or the fermentation liquid is to put tank fermentation liquid；
And/or the purifying pretreatment are as follows: release Gentamicin C1a fermentation liquid progress acidification, and solid
After liquid separation, filtrate is the sample to be tested containing Gentamicin C1a.
5. the detection method of Gentamicin C1a as claimed in claim 4, it is characterised in that:
The acidification is by the fermentation liquid aqueous sulfuric acid tune pH to 1.5~2.5；
And/or the process of the acidification is also stood；
And/or described be separated by solid-liquid separation is that sheet frame was used to be separated by solid-liquid separation or be centrifugated；
And/or the purifying pretreatment further includes following steps: after the separation of solid and liquid, the filtrate being handed over cation
It changes resin to be adsorbed, then be eluted with buck, eluent is the sample to be tested containing Gentamicin C1a.
6. the detection method of Gentamicin C1a as claimed in claim 5, it is characterised in that:
The time of the standing is 1 ~ 4 hour；
And/or sulfuric acid concentration is 4 ~ 18M in the aqueous sulfuric acid；
And/or the buck is the mol/LNaOH aqueous solution of 1 mol/L ~ 5；
And/or the cation exchange resin is HZ-732 cation exchange resin.
7. the detection method of Gentamicin C1a as claimed in any one of claims 1-3, it is characterised in that:
The derivatization reagent is that its each component is formed by solution after evenly mixing；
And/or in the derivatization reagent mass volume ratio of o-phthalaldehyde and the derivatization reagent be 0.01g/mL ~
0.08 g/mL；
And/or the solvent is one of alcohols solvent, esters solvent and ether solvent or a variety of；
And/or the solvent and the volume mass ratio of the o-phthalaldehyde are 1mL/g ~ 10mL/g；
And/or the buffer solution is one of boric acid aqueous solution, borax solution and phosphate aqueous solution or a variety of；
And/or it is 40% ~ 50% or the aqueous solution of the sodium hydroxide of saturation that the pH of the derivatization reagent, which is by mass fraction,
And/or mass fraction is 40% ~ 50% or the aqueous solution of the potassium hydroxide of saturation is adjusted；
And/or the volume ratio of the solvent and the buffer solution is 1:12 ~ 1:20.
8. the detection method of Gentamicin C1a as claimed in claim 7, it is characterised in that: boric acid in the boric acid aqueous solution
Molar concentration be 0.2mol/L ~ 0.5mol/L；And/or the pH of the boric acid aqueous solution is 10 ~ 11；And/or the boric acid
The pH of aqueous solution is by mass fraction be 40% ~ 50% or the aqueous solution of the sodium hydroxide of saturation and/or mass fraction be 40% ~
50% or the aqueous solution of potassium hydroxide of saturation be adjusted；
And/or the alcohols solvent is one of methanol, ethyl alcohol and propyl alcohol or a variety of；
And/or in the derivatization reagent mass volume ratio of o-phthalaldehyde and the derivatization reagent be 0.012g/mL ~
0.04 g/mL；
And/or the solvent and the volume mass ratio of the o-phthalaldehyde are 1.25mL/g ~ 5mL/g；
And/or the volume ratio of the solvent and the buffer solution is 1:19 ~ 1:13.
9. the detection method of Gentamicin C1a as claimed in any one of claims 1-3, it is characterised in that: the derivatization
Reaction system is also uniformly mixed by reaction before progress with isopropanol；The isopropanol and the volume ratio of the reaction system are
1:1 ~ 1:2；And/or the derivative reaction is to carry out in confined conditions；And/or the temperature of the derivative reaction is 40
~60℃；And/or the time of the derivative reaction is 10 ~ 20min；And/or the derivative reaction after, also wrap
Include the operation of post-processing；The operation of the post-processing are as follows: terminate reaction, centrifuging and taking using reaction system is placed in -10 ~ 0 DEG C
Supernatant.
10. the detection method of Gentamicin C1a as claimed in any one of claims 1-3, it is characterised in that: the liquid phase color
In spectrum or liquid phase chromatogram-mass spectrometry combination method detection, chromatographic column used is C18 chromatographic column；And/or mobile phase used is volume
Than the mixed liquor of heptanesulfonic acid sodium water solution, glacial acetic acid and methanol for 4:1:15；Heptane in the heptanesulfonic acid sodium water solution
The mass concentration of sodium sulfonate is the g/L of 5g/L ~ 10；And/or the dosage of mobile phase is 1L ~ 1.5L；And/or the flow velocity of mobile phase
For 1.0mL/min ~ 1.2mL/min；And/or sample volume is 5 ~ 20 μ L；And/or column temperature is 20 ~ 45 DEG C；And/or liquid chromatogram
Detection wavelength is the nm of 330nm ~ 335.
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| 庆大霉素发酵液的分离提取;于洪静;《黑龙江科技信息》;20140118(第35期);第75页第2-3节 |
| 庆大霉素发酵液的分离提取;范一文等;《实验室研究与探索》;20110831;第30卷(第8期);第28-31页 |
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