CN105548420B - The detection method of MOPS residual quantity - Google Patents
The detection method of MOPS residual quantity Download PDFInfo
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- CN105548420B CN105548420B CN201511022203.1A CN201511022203A CN105548420B CN 105548420 B CN105548420 B CN 105548420B CN 201511022203 A CN201511022203 A CN 201511022203A CN 105548420 B CN105548420 B CN 105548420B
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The present invention provides a kind of detection method of MOPS residual quantity, is detected with high performance liquid chromatography, is quantified using liner sum up method to MOPS.The high performance liquid chromatography uses Acclaim Trinity P1 chromatographic column and EFI fog detector, and mobile phase is acetonitrile and ammonium acetate solution mixed liquor, and the volume ratio of acetonitrile and ammonium acetate solution is 1:1~2:1, and flow rate of mobile phase is 0.4~0.6mL/min.The detection method of MOPS residual quantity of the invention, the micro MOPS ingredient being capable of detecting when in chemical titration and the HPV vaccine that the inspection of C18 column liquid chromatographic does not measure, with good detection precision, and its detection sensitivity and accuracy are high, can be widely applied to the MOPS residues detection of biological products.
Description
Technical field
It is related to the invention belongs to MOPS detection technique field more particularly to a kind of detection method of MOPS residual quantity.
Background technique
3- (N- morpholine) propane sulfonic acid, English name: 3- (N-Morpholino) propane-sulfonic acid, abbreviation
For MOPS, molecular formula: C7H15NO4S, molecular weight: 209.2633, pH value range: 6.5-7.9, is white crystalline powder, and fusing point is
Water solubility at 277-282 DEG C, 20 DEG C is 1000g/L, relative density 1.298g/cm3, structural formula is as follows:
MOPS can be used for the production purifying of more biological products as a kind of common biological buffer solutions.Therefore, biological
The residual quantity of MOPS and the purity of biological products are directly related in product, and detection MOPS residual quantity has great importance.However
In residual solvent measuring method and " ICH Q3c impurity: the guideline of middle residual solvent " in Chinese Pharmacopoeia, United States Pharmacopeia,
MOPS is not admitted to the bound requirements of first to fourth class solvent.In Chinese Academy of Sciences's chemical toxicity database, MOPS quail
The oral LD50 of quail is > 316mg/kg, and conversion adult human dose is > 52.67mg/kg.
Shanghai Bo Wei Biotechnology Co., Ltd makes in the purification process of production recombinant human papilla virus vaccine (HPV)
MOPS is used to be needed as buffer solution for the safety for guaranteeing vaccine to all possible toxic used in production process
Substance carry out residues detection.MOPS residual quantity conversion adult human dose is 0.0009mg/kg in HPV vaccine finished product after purification
(far smaller than 52.67mg/kg) is not measured with the inspection of conventional chemical titration.Meanwhile MOPS is amphoteric compound, is had larger
Polarity, with conventional C18 column carry out high performance liquid chromatography (HPLC) analysis when, also without reserve or without UV absorption.For micro
There is presently no relevant reports for the detection method of MOPS detection method or MOPS residual quantity.Therefore there is an urgent need to a kind of detection is micro
The method of MOPS residual quantity guarantees the peace of biological products to determine that MOPS residual quantity in HPV vaccine finished product meets the requirement of purity
Quan Xing.
Summary of the invention
The purpose of the present invention is to provide a kind of detection methods of MOPS residual quantity, micro- to overcome the prior art that cannot detect
The remaining defect of MOPS is measured, is capable of detecting when MOPS residual quantity in HPV vaccine finished product, it is ensured that the safety of HPV vaccine finished product,
Have the characteristics that detection precision, detection sensitivity and accuracy are high.
The first aspect of the invention provides a kind of detection method of MOPS residual quantity, and this method is with high-efficient liquid phase color
Spectrometry is detected, and is quantified using liner sum up method to MOPS.
The chromatographic column of the high performance liquid chromatography is Acclaim Trinity P1.
The high performance liquid chromatography uses EFI fog detector.
The mobile phase of the high performance liquid chromatography is acetonitrile and ammonium acetate solution mixed liquor, and volume ratio is 1:1~2:1.
The flow velocity of the mobile phase is 0.4~0.6ml/min.
Another aspect of the present invention, the detection method for providing the MOPS residual quantity are residual in the MOPS of biological products
Application in allowance detection.
Preferred embodiment in accordance with the present invention, the biological products are HPV vaccine.
Compared with prior art, the present invention having the following beneficial effects:
1) detection method of a kind of MOPS residual quantity of the invention, is detected, chromatographic column is using high performance liquid chromatography
It is micro- in the HPV vaccine not measured to be capable of detecting when that chemical titration and C18 column liquid chromatographic are examined by Acclaim Trinity P1
MOPS ingredient is measured, the safety for HPV vaccine or containing micro MOPS ingredients Biogenic product provides good guarantee.
2) detection method of MOPS residual quantity of the invention has good detection precision, and its detection sensitivity and standard
True property is high, can be widely applied to the detection of the MOPS residual quantity of biological products.
Detailed description of the invention
Fig. 1 is the MOPS standard solution separating spectrum of concentration 1.0mmol/L；
Fig. 2 is the control map of blank control and 1.0mmol/L MOPS standard solution (blank control dilution), in figure compared with
On curve indicate 1.0mmol/L MOPS standard solution (blank control dilution) separate curve, relatively under curve expression blank pair
According to separation curve.
Fig. 3 is the linear superposition map of the MOPS standard solution of concentration 0.1mmol/L~5.0mmol/L, in figure by down toward
Upper curve concentration is respectively as follows: 0.1mmol/L, 0.2mmol/L, 0.5mmol/L, 1.0mmol/L, 5.0mmol/L.
Fig. 4 is the separating spectrum of four batch samples.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described.It should be understood that following embodiment is merely to illustrate this
The range of invention and is not intended to limit the present invention.
Reagent, drug or the instrument that the present invention uses be all it is commercially available, wherein (the poly- biotechnology in Shanghai source has MOPS standard items
Limit company, purity: 99.6%), acetonitrile (chromatographically pure), high performance liquid chromatograph (model: Ultimate 3000).
Analytical solution preparation steps:
Mobile phase preparation:
1) it weighs 1.54g ammonium acetate to be dissolved in 1000ml deionized water, stirs evenly the acetic acid that 0.02mol/L is prepared
Vinegar acid for adjusting pH is added dropwise to 5.0 in ammonium salt solution.
2) mixed solution of acetonitrile and ammonium acetate solution is configured, volume ratio 1.5:1 shakes up to obtain mobile phase.Concentration
The preparation of 0.1mmol/L~5.0mmol/LMOPS standard solution:
Accurately weigh the standard that 1.045 grams of MOPS standard items add deionized water constant volume to be 5.0mmol/L to get concentration to 1L
Solution, then it is diluted to the MOPS standard solution of 1.0,0.5,0.2,0.1mmol/L respectively.
The preparation of blank control (sample blank buffer solution): weighing NaCl 28.4g, and poly- mountain is added in histidine 3.08g
After pear ester -800.2ml, adds appropriate ultrapure water to dissolve, with hydrochloric acid tune pH value to 6.2, ultrapure water is added to be settled to 1000ml.
1.0mmol/L MOPS standard solution (blank control dilution): 5.0mmol/L is diluted with sample blank buffer solution
Standard solution to 1.0mmol/LMOPS standard solution.
Specific chromatographic condition:Using the micro MOPS residual in high-efficient liquid phase chromatogram technology analysis HPV vaccine, design parameter
As shown in table 1.
1 chromatographic condition of table
Reference example 1:
High performance liquid chromatography detectable concentration is used as the standard solution of 1.0mmol/L, obtains the separation figure of standard solution
Spectrum, as shown in Figure 1.As seen from Figure 1, the MOPS standard solution that concentration is 1.0mmol/L is at 2.47min in retention time
There is MOPS characteristic peak.
Reference example 2:
It is molten to blank control (0mmol/L MOPS) and 1.0mmol/L MOPS standard respectively using high performance liquid chromatography
Liquid (blank control dilution) is detected, and it is as shown in Figure 2 to obtain control map.
As seen from Figure 2, it is not detected MOPS characteristic peak after blank control sample introduction, and 1.0mmol/L MOPS standard
Solution (blank control dilution) is that can detect that apparent MOPS characteristic peak at 2.49min in retention time.
Note: MOPS is using the normal retention time of liquid chromatographic detection between 2.40 ± 0.10min range.
Embodiment 1-3:
Quantitative detection, testing conditions: chromatographic column: Acclaim are carried out to the MOPS standard solution of 3 parts of 1.0mmol/L respectively
Trinity P1, column temperature are respectively 30 DEG C, and 35 DEG C, 40 DEG C, atomization temperature is respectively 25 DEG C, and 30 DEG C, 35 DEG C, mobile phase: acetonitrile
With the mixed liquor of ammonium acetate solution, volume ratio is respectively 1.1:1,1.5:1,2:1, and flow velocity is respectively as follows: 0.4ml/min, 0.5ml/
Min, 0.6ml/min, sample volume are respectively as follows: 3 μ L, 5 μ L, 10 μ L.Testing result is as shown in table 1.
Testing result of the table 11.0mmol/L MOPS standard items under different testing conditions
As can be seen from Table 1, MOPS can be detected under the testing conditions of embodiment 1-3, and retention time is in 2.44min
Left and right, peak area is close, illustrates that the chromatographic condition of embodiment 1-3 is able to achieve the detection of micro MOPS, and under the conditions of embodiment 2
The symmetry of gained chromatographic peak is best, therefore the chromatographic condition of embodiment 2 is more excellent.
Embodiment 4:
Quantitative detection, testing conditions: chromatographic column Acclaim are carried out to the MOPS standard solution of 8 parts of 1mmol/L
Trinity P1, column temperature are 35 DEG C；Atomization temperature is 30 DEG C, and mobile phase: the mixed liquor of acetonitrile and ammonium acetate solution, volume ratio are
1.5:1, flow velocity: 0.5ml/min, sample volume: 5 μ L.
The relative standard deviation RSD (%) of gained MOPS are as follows: 1.89%, it is good to illustrate that the detection method of the application has
Precision.
Embodiment 5-9:
Respectively detectable concentration be 0.1mmol/L (quantitative limit), 0.5mmol/L, 1.0mmol/L, 2.0mmol/L,
The MOPS standard solution of 5.0mmol/L, testing conditions: chromatographic column: Acclaim Trinity P1, column temperature are 35 DEG C；Atomization temperature
Degree is 30 DEG C, mobile phase: the mixed liquor of acetonitrile and ammonium acetate solution, volume ratio 1.5:1, flow velocity: 0.5ml/min, sample volume:
5 μ L obtain linear superposition map as shown in figure 3, corresponding concentration-peak area corresponding relationship is as shown in table 2:
Table 2
As seen from Figure 3, the different MOPS standard solution of concentration is to occur MOPS spy at 2.49min in retention time
Levy peak.By table 2 it can be concluded that, coefficient R2It is 0.9990, so measuring MOPS residual quantity with this method, concentration is in quantitative limit
It is good linear to being presented between 5mmol/L.
The detection limit DL and quantitative limit QL of analysis method is calculated by signal-to-noise ratio (S/N), with method provided by the invention progress
Detection, detection are limited to 0.05mmol/L, are quantitatively limited to 0.1mmol/L.Illustrate detection method high sensitivity of the invention.
Embodiment 10:
By the sample of 4 batches, by high performance liquid chromatograph, sample introduction is analyzed, detects micro in the sample of four batches
MOPS residual quantity.Testing conditions: chromatographic column is Acclaim Trinity P1, and column temperature is 35 DEG C；Atomization temperature is 30 DEG C, flowing
Phase: the mixed liquor of acetonitrile and ammonium acetate solution, volume ratio 1.5:1, flow velocity are as follows: 0.5ml/min, sample volume are as follows: 5 μ L, four
The folded figure of the analysis of batch sample as shown in figure 4, corresponding MOPS residue analysis the results are shown in Table 3.
3 sample analysis result of table
As seen from Figure 4, there is MOPS characteristic peak, the spectrum of four samples in the sample of 4 different batches of detection
Figure curve is substantially overlapping, and retention time is in 2.40min or so, and MOPS characteristic peak area is essentially identical, illustrates of the invention
Technical solution is capable of detecting when MOPS micro in sample, and accuracy is high.
MOPS residual quantity finally is measured in 0.10mmoL/L or so it can be seen from the result of table 3, illustrates inspection of the invention
Survey method is capable of detecting when the micro MOPS residual in HPV vaccine, and detection performance is stablized, and accuracy is high.
Although above embodiments only detect the micro MOPS residual in HPV vaccine, the technology of this field
Personnel are readily appreciated that for method of the invention for using the other biological product of MOPS equally applicable in production process, this is aobvious
And it is clear to.To those skilled in the art, any equivalent modifications and substitution to the practical progress are also all in the present invention
Scope among.Therefore, made equal transformation and modification without departing from the spirit and scope of the invention, should all cover at this
In the range of invention.
Claims (4)
1. a kind of detection method of MOPS residual quantity, which is characterized in that detected with high performance liquid chromatography, using linearly returning
One method quantifies MOPS；
The chromatographic column of the high performance liquid chromatography is Acclaim Trinity P1, column temperature: 30~40 DEG C；
The high performance liquid chromatography uses EFI fog detector, atomization temperature: 25~35 DEG C；
The mobile phase of the high performance liquid chromatography is acetonitrile and ammonium acetate solution mixed liquor, the acetonitrile and ammonium acetate solution body
Product is than being 1:1~2:1.
2. a kind of detection method of MOPS residual quantity as described in claim 1, which is characterized in that the flow velocity of the mobile phase is
0.4~0.6ml/min.
3. the detection method such as MOPS residual quantity of any of claims 1-2 is examined in the MOPS residual quantity of biological products
Application in survey.
4. application as claimed in claim 3, which is characterized in that the biological products are HPV vaccine.
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Effective date of registration: 20190703 Address after: 201203 No. 333 Edison Road, China (Shanghai) Free Trade Pilot Area, Pudong New Area, Shanghai Applicant after: Shanghai Wison-Bio Co., Ltd. Address before: No. 12-13, Lane 908, Ziping Road, Pudong New Area, Shanghai, 201321 Applicant before: Shanghai Bovax Biological Science & Technology Co., Ltd. |
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