CN116297999A - Method for detecting content of gluconate radicals in calcium zinc gluconate oral solution - Google Patents
Method for detecting content of gluconate radicals in calcium zinc gluconate oral solution Download PDFInfo
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- CN116297999A CN116297999A CN202211664603.2A CN202211664603A CN116297999A CN 116297999 A CN116297999 A CN 116297999A CN 202211664603 A CN202211664603 A CN 202211664603A CN 116297999 A CN116297999 A CN 116297999A
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- gluconate
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- 238000000034 method Methods 0.000 title claims abstract description 58
- 229940050410 gluconate Drugs 0.000 title claims abstract description 34
- IHBCFWWEZXPPLG-UHFFFAOYSA-N [Ca].[Zn] Chemical compound [Ca].[Zn] IHBCFWWEZXPPLG-UHFFFAOYSA-N 0.000 title claims abstract description 18
- 239000011670 zinc gluconate Substances 0.000 title claims abstract description 18
- 235000011478 zinc gluconate Nutrition 0.000 title claims abstract description 18
- 229960000306 zinc gluconate Drugs 0.000 title claims abstract description 18
- 229940100688 oral solution Drugs 0.000 title claims abstract description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 42
- 239000000243 solution Substances 0.000 claims abstract description 29
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 28
- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 claims abstract description 23
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000001514 detection method Methods 0.000 claims abstract description 21
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 15
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000012085 test solution Substances 0.000 claims abstract description 13
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 11
- AEQDJSLRWYMAQI-UHFFFAOYSA-N 2,3,9,10-tetramethoxy-6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinoline Chemical compound C1CN2CC(C(=C(OC)C=C3)OC)=C3CC2C2=C1C=C(OC)C(OC)=C2 AEQDJSLRWYMAQI-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000000176 sodium gluconate Substances 0.000 claims abstract description 10
- 235000012207 sodium gluconate Nutrition 0.000 claims abstract description 10
- 229940005574 sodium gluconate Drugs 0.000 claims abstract description 10
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims abstract description 7
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims abstract description 7
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims abstract description 7
- 239000013558 reference substance Substances 0.000 claims abstract description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000000945 filler Substances 0.000 claims abstract description 5
- 239000000741 silica gel Substances 0.000 claims abstract description 5
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 5
- 238000010812 external standard method Methods 0.000 claims abstract description 4
- 238000003756 stirring Methods 0.000 claims description 54
- 238000002156 mixing Methods 0.000 claims description 52
- 239000000523 sample Substances 0.000 claims description 32
- 239000007788 liquid Substances 0.000 claims description 22
- 238000012360 testing method Methods 0.000 claims description 20
- 239000012488 sample solution Substances 0.000 claims description 14
- 238000002425 crystallisation Methods 0.000 claims description 11
- 230000008025 crystallization Effects 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 8
- 238000007865 diluting Methods 0.000 claims description 6
- 238000012545 processing Methods 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 3
- BDOYKFSQFYNPKF-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;sodium Chemical compound [Na].[Na].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O BDOYKFSQFYNPKF-UHFFFAOYSA-N 0.000 claims description 2
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 2
- 238000010790 dilution Methods 0.000 abstract 1
- 239000012895 dilution Substances 0.000 abstract 1
- 238000004090 dissolution Methods 0.000 abstract 1
- 239000007864 aqueous solution Substances 0.000 description 8
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 4
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 3
- 239000004227 calcium gluconate Substances 0.000 description 3
- 229960004494 calcium gluconate Drugs 0.000 description 3
- 235000013927 calcium gluconate Nutrition 0.000 description 3
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- BVHLGVCQOALMSV-JEDNCBNOSA-N L-lysine hydrochloride Chemical compound Cl.NCCCC[C@H](N)C(O)=O BVHLGVCQOALMSV-JEDNCBNOSA-N 0.000 description 2
- WHMDKBIGKVEYHS-IYEMJOQQSA-L Zinc gluconate Chemical compound [Zn+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O WHMDKBIGKVEYHS-IYEMJOQQSA-L 0.000 description 2
- 239000000337 buffer salt Substances 0.000 description 2
- 229940042228 calcium gluconate oral solution Drugs 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- SHFJWMWCIHQNCP-UHFFFAOYSA-M hydron;tetrabutylazanium;sulfate Chemical compound OS([O-])(=O)=O.CCCC[N+](CCCC)(CCCC)CCCC SHFJWMWCIHQNCP-UHFFFAOYSA-M 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 229960005337 lysine hydrochloride Drugs 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical group [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses a method for detecting gluconate in calcium zinc gluconate oral solution, which comprises the following steps: 1ml of the product is precisely measured, the product is placed in a 100ml measuring flask, 3ml of disodium ethylenediamine tetraacetate solution (0.05 mol/L) and sodium hydroxide test solution are added, the product is diluted to scale by water and is uniformly shaken, the sample is required to be placed at room temperature for 1h, (2) a proper amount of sodium gluconate reference substance is additionally taken, precisely weighed, water is added for dissolution and quantitative dilution are carried out to prepare a solution containing 0.6+/-0.1 mg of sodium gluconate reference substance per 1ml, (3) the product is calculated according to an external standard method and calculated according to peak areas, (4) chromatographic conditions are as follows: amino-bonded silica gel as filler (Phenomnex)
5μmNH2
) 50mmol/L potassium dihydrogen phosphate solution (pH value is adjusted to 2.0 by phosphoric acid) -acetonitrile (35:65) is taken as a mobile phase; the flow rate is 0.6ml per minute; the detection wavelength is 210nm; the column temperature is 30 ℃; the relative standard deviation of the gluconate content measured by the method is less than 2.0 percent, and the precision is good.
Description
Technical Field
The invention relates to the field of general detection systems and methods, in particular to a method for detecting the content of gluconate in a calcium zinc gluconate oral solution.
Background
The calcium zinc gluconate oral solution is a calcium zinc compound preparation marketed in 2001 by Australian (China) pharmaceutical Co., ltd, and comprises the main components of calcium gluconate, zinc gluconate and lysine hydrochloride, the product name is zinc calcium, the specification is 10ml: 0.6g of calcium gluconate, 0.03g of zinc gluconate and 0.1g of lysine hydrochloride, wherein the medicine bag material directly contacted with the medicine is a PVC/PE composite hard sheet, the effective period of the medicine is 24 months,
the specific method comprises the following steps: the existing gluconate content determination method comprises an ion chromatograph and an HPLC-UV method, and the ion chromatograph has the following chromatographic conditions: chromatographic column: dionex IonPac AS18 (250 mm. Times.4 mm) Dionex IonPac AG18 (50 mm. Times.4 mm); eluent: potassium hydroxide solution, gradient elution: 0 to 20min,1 multiplied by 10 < -3 > mol/L < -1 >; 20-25 min, 40X 10-3mol/L-1; 25-35 min, 1X 10-3mol/L-1; flow rate: 1.0mL/min; a suppressor: dionex AERS500 (4 mm); suppression current: 99mA; sample injection volume: 20. Mu.L; column temperature: 30 ℃; detecting the temperature of the cell: 5 ℃; a detector: conductivity detector, HPLC-UV chromatography conditions were as follows: octadecylsilane chemically bonded silica gel is used as a filler, 10mmol/L dipotassium hydrogen phosphate solution-5 mmol/L tetrabutylammonium hydrogen sulfate solution (pH value 7.0) -methanol (98:2) is used as a mobile phase, the flow rate is 0.6ml per minute, the column temperature is 30 ℃, the detection wavelength is 210nm, the sample injection amount is 25 mu L, the reproducibility of ion chromatography is poor, the chromatographic column and a protection column are expensive and not durable, the detection cost is high, the HPLC-UV method is recommended to use ion pair reagents in the national drug standard revision draft of calcium gluconate oral solution, one chromatographic column is consumed basically in one test, the detection cost is extremely high, and obvious impurity peaks interfere with the detection of gluconate when the calcium zinc gluconate oral solution is detected.
Disclosure of Invention
The invention mainly aims to provide a method for detecting the content of gluconate with strong operability, high accuracy and good sensitivity, which adopts a novel mobile phase mixing strategy to avoid crystallization interference in the online mixing process and has good chromatographic column tolerance, so that the problems in the background technology can be effectively solved: ion chromatography reproducibility is poor, chromatographic columns and protection columns are high in price and not durable, detection cost is high, an HPLC-UV method is recommended to use ion pair reagents for a method recommended in national drug standards revised draft of calcium gluconate oral solution, one chromatographic column is consumed basically in one test, detection cost is extremely high, and obvious impurity peaks interfere with the detection of gluconate when calcium zinc gluconate oral solution is detected.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the method for detecting the gluconate content in the calcium zinc gluconate oral solution comprises the steps of taking 50mmol/L potassium dihydrogen phosphate solution (pH value is regulated to 2.0 by phosphoric acid) -acetonitrile (35:65) as a mobile phase, stirring and mixing the mobile phase by adopting a mobile phase stirring and mixing method in order to avoid the problem of crystallization in the online mixing process, wherein the mobile phase is firstly regulated to 2.0 and then mixed with acetonitrile, or 50mmol/L potassium dihydrogen phosphate solution-acetonitrile (35:65) is firstly prepared and uniformly mixed and then added with phosphoric acid to regulate the pH value to 3.0, the two modes are respectively provided with uniform base lines of the mobile phase, the peak-to-peak of the gluconate is normal, and the retention time can be kept at 15min;
wherein the flow isThe phase stirring and mixing method comprises the following specific steps: 101. placing a mobile phase mixing bottle to be stirred on a stirring table, dividing the surface of the stirring table to cover n multiplied by n coordinate units of a coordinate system, arranging a first light intensity sensor at the bottom of each coordinate unit, acquiring the light intensity Izs refracted after incident light enters the mobile phase mixing liquid, arranging an LED direct light source parallel to the n multiplied by n of the liquid level of the mixing bottle above the mixing bottle, corresponding to the position of the coordinate units, and arranging the light intensity of the light emitted by two pairs of LED direct light sources of the light intensity sensors I fs is collected, and the input end of the LED light source, the first light intensity sensor and the second light intensity sensor are respectively connected with processing equipment; 102. stirring and mixing the mobile phase mixed liquid in the mixing bottle by using a stirring rod, standing on a stirring table until the liquid is stable after mixing, measuring the turbidity of each coordinate unit, wherein a calculation formula of the turbidity is as follows:
wherein Izs is the intensity of the incident light after entering the mobile phase mixture, ifs is the emitted intensity of the two pairs of LED direct light sources of the light intensity sensor, kp is the proportionality coefficient, rpj is the distance from the LED direct light sources to the liquid surface, and a turbidity sequence (c) is obtained hzd1 ,c hzd2 ,c hzd3 ,c hzd4 ,…c hzdn ) And average turbidity->
Then calculating the variance of the turbidity, wherein the calculation formula of the turbidity variance is as follows:
comparing the variance value with a variance threshold value to observe whether stirring is uniform or not; 103. if the variance value of the turbidity is larger than the variance threshold, the stirring is not completed, the stirring is required to be continued, if the variance value of the turbidity is smaller than the variance threshold, the stirring is completed, and the next operation is carried out, so that the calculation of the turbidity is arranged, the situation of crystallization in the online mixing process is well known as soon as possible, and the situation of crystallization is well knownAnd (3) the time treatment is carried out, and the mixing efficiency of the mobile phase is intuitively known through calculating the turbidity variance, so that the stirring process is effectively mastered.
The invention further improves that the detection method comprises the following specific steps: (1) 1ml of a test sample is precisely measured, placed in a 100ml measuring flask, 3ml of disodium ethylenediamine tetraacetate solution (0.05 mol/L) and sodium hydroxide test solution are added, the mixture is diluted to the maximum scale with a test sample solvent, and the mixture is stirred and mixed by a mobile phase stirring and mixing method, and the sample is placed at room temperature for 1h. (2) And taking a proper amount of sodium gluconate reference substance, precisely weighing, adding water for dissolving, and quantitatively diluting to prepare a solution containing 0.6+/-0.1 mg of sodium gluconate per 1 ml. (3) calculating by an external standard method according to the peak area: the method comprises the following steps:
chromatographic conditions: amino-bonded silica gel is used as a filler, and 50mmol/L potassium dihydrogen phosphate solution (pH value is adjusted to 2.0 by phosphoric acid) -acetonitrile (35:65) is used as a mobile phase; the flow rate is 0.4ml/min-0.8ml/min; the detection wavelength is 210nm; the column temperature is 25-35 ℃; sample volume 20 μl, chromatographic conditions and system applicability: the theoretical plate number is not less than 3000 calculated according to gluconate radical peak.
The mobile phase is phosphate buffer salt with the pH value of 1.0-3.0, the selected buffer salt is monopotassium phosphate, 50mmol/L potassium dihydrogen phosphate solution-acetonitrile (35:65) is taken as the mobile phase, in order to avoid the problem of crystallization in the online mixing process, the mobile phase is stirred and mixed by adopting a mobile phase stirring and mixing method, the pH value of the mobile phase can be firstly adjusted to 2.0 and then mixed with acetonitrile, and the pH value can be adjusted to 3.0-4.0 after 50mmol/L potassium dihydrogen phosphate solution-acetonitrile (35:65) is uniformly mixed, the base line of the mobile phase is uniformly configured in the two modes, and the peak of gluconate is normal and the retention time is longer than 15min.
The invention is further improved in that the solvent of the test sample is aqueous solution of hydrochloric acid with pH of 1.0, aqueous solution of sulfuric acid with pH of 2.0, aqueous solution of phosphoric acid with pH of 2.0, aqueous solution of trifluoroacetic acid with pH of 2.0 or pure water, the solvent is preferably pure water, the water is used as the normal and unique peak of the solvent, and the gluconate can be completely dissociated.
The invention is further improved in that the disodium ethylenediamine tetraacetate solution (0.05 mol/L) and the sodium hydroxide test solution are added in the preparation process of the test solution, wherein the disodium ethylenediamine tetraacetate solution (0.05 mol/L) is between 2ml and 4ml, and the sodium hydroxide test solution is between 2ml and 3 ml.
The invention is further improved in that the sample solution is required to be placed for 1h at room temperature after the preparation.
The invention further improves that the high temperature is avoided in the process of preparing the sample solution.
Compared with the prior art, the invention has the following beneficial effects: the method has strong operability, high accuracy and good sensitivity, adopts a novel mobile phase mixing strategy to sense the crystallization condition in the online mixing process, intuitively knows the mixing efficiency of the mobile phase, is favorable for effectively grasping the stirring process, and has good chromatographic column tolerance.
Drawings
FIG. 1 is a chromatogram of pH2.0 hydrochloric acid using an assay according to an embodiment of the invention.
FIG. 2 is a chromatogram of sulfuric acid pH2.0 using a detection method according to an embodiment of the present invention.
FIG. 3 is a chromatogram of pH2.0 phosphoric acid using a detection method according to an embodiment of the present invention.
FIG. 4 is a chromatogram of trifluoroacetic acid at pH2.0 using a detection method according to an embodiment of the invention.
Fig. 5 is a chromatogram of empty white water using a detection method according to an embodiment of the present invention.
FIG. 6 is a chromatogram of a sodium gluconate control solution using a detection method according to an embodiment of the invention.
Fig. 7 is a chromatogram of an oral solution of calcium gluconate using an exemplary method of detection according to the present invention.
FIG. 8 is a schematic flow chart of the mobile phase stirring and mixing method of the invention.
Detailed Description
In order that the technical means, the creation characteristics, the achievement of the objects and the effects of the present invention may be easily understood, it should be noted that in the description of the present invention, the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings are merely for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the apparatus or elements to be referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as limiting the present invention. Furthermore, the terms "a", "an", "the" and "the" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. The invention is further described below in conjunction with the detailed description.
Examples
The invention discloses a method for detecting the content of gluconate in a calcium zinc gluconate oral solution, which adopts a high performance liquid chromatography method for detection, and comprises the following specific steps: (1) Mobile phase configuration, taking 50mmol/L potassium dihydrogen phosphate solution-acetonitrile (35:65) as mobile phase; (2) Precisely measuring 1ml of a test sample, placing the test sample into a 100ml measuring flask, adding an ethylenediamine tetraacetic acid disodium solution and a sodium hydroxide test solution, diluting the test sample solution to the maximum scale, and stirring and mixing the test sample solution by adopting a mobile phase stirring and mixing method to generate a test sample solution; (3) Taking a proper amount of sodium gluconate reference substance, precisely weighing, adding water for dissolving and quantitatively diluting to prepare a solution containing 0.6+/-0.1 mg of sodium gluconate in each 1 ml; (4) calculating by an external standard method according to the peak area: the method comprises the following steps:
chromatographic conditions: amino-bonded silica gel as filler (column of Phenomnex)
5μmNH2/>
4.6X1250 mm) with 50mmol/L potassium dihydrogen phosphate solution (pH adjusted to 2.0 with phosphoric acid) -acetonitrile (35:65) as mobile phase; the flow rate is 0.4ml/min-0.8ml/min; the detection wavelength is 210nm; the column temperature is 25-35 ℃; sample volume 20 μl, chromatographic conditions and system applicability: the theoretical plate number is not less than 3000 calculated according to gluconate radical peak.
High performance liquid chromatographyDetecting, wherein the mobile phase is phosphate buffer salt with the pH value of 1.0-3.0, the selected buffer salt is monopotassium phosphate, 50mmol/L potassium dihydrogen phosphate solution-acetonitrile (35:65) is taken as the mobile phase, the mobile phase is stirred and mixed by adopting a mobile phase stirring and mixing method to avoid crystallization in the online mixing process, the mobile phase can be firstly adjusted to be 2.0 in pH value and then mixed with acetonitrile, 50mmol/L potassium dihydrogen phosphate solution-acetonitrile (35:65) can be firstly prepared and uniformly mixed and then adjusted to be 3.0-4.0, the two modes are provided with uniform base line of the mobile phase, the peak-to-peak of gluconate is normal, the retention time is more than 15min, and the chromatographic column is Phenomenex
5μm NH2/>
4.6X1250 mm, the gluconate radical of the chromatographic column with the model has good peak shape, high sensitivity and good separation degree from front and back peaks.
In some embodiments, the sample solvent is aqueous solution of hydrochloric acid of pH1.0, aqueous solution of sulfuric acid of pH2.0, aqueous solution of phosphoric acid of pH2.0, aqueous solution of trifluoroacetic acid of pH2.0 or pure water, preferably pure water, and pure water is used as the normal and unique solvent peak, so that the gluconate can be completely dissociated.
In some embodiments, the test solution is allowed to stand for 1 hour after being prepared.
In some embodiments, the column temperature is 25 ℃ to 35 ℃, preferably 30 ℃.
In some embodiments, the flow rate of the mobile phase is from 0.4ml/min to 0.8ml/min, preferably 0.6/min.
In some embodiments, disodium ethylenediamine tetraacetate solution (0.05 mol/L) and sodium hydroxide solution are added during the preparation of the sample solution.
In some embodiments, the mobile phase agitation mixing method comprises the specific steps of: 101. placing a mobile phase mixing bottle to be stirred on a stirring table, dividing the surface of the stirring table to cover n multiplied by n coordinate units of a coordinate system, arranging a first light intensity sensor at the bottom of each coordinate unit, and using the first light intensity sensorCollecting the light intensity Izs refracted after the incident light enters the mobile phase mixed liquid, arranging an LED direct light source parallel to the n multiplied by n of the liquid level of the mixed bottle above the mixed bottle, wherein the LED light source corresponds to the position of the coordinate unit, collecting the emitted light intensity Ifs of the LED direct light source by arranging a light intensity sensor II, and connecting the input ends of the LED light source, the light intensity sensor I and the input ends of the light intensity sensor II with processing equipment respectively; 102. stirring and mixing the mobile phase mixed liquid in the mixing bottle by using a stirring rod, standing on a stirring table until the liquid is stable after mixing, measuring the turbidity of each coordinate unit, wherein a calculation formula of the turbidity is as follows:
wherein Izs is the intensity of the incident light after entering the mobile phase mixture, ifs is the emitted intensity of the two pairs of LED direct light sources of the light intensity sensor, kp is the proportionality coefficient, rpj is the distance from the LED direct light sources to the liquid surface, and a turbidity sequence (c) is obtained hzd1 ,c hzd2 ,c hzd3 ,c hzd4 ,…c hzdn ) And average turbidity->
Then calculating the variance of the turbidity, wherein the calculation formula of the turbidity variance is as follows: />
Comparing the variance value with a variance threshold value to observe whether stirring is uniform or not; 103. if the variance value of the turbidity is larger than the variance threshold, the stirring is not completed, the stirring is required to be continued, if the variance value of the turbidity is smaller than the variance threshold, the stirring is completed, and the next operation is performed, so that the calculation of the turbidity is performed, the crystallization condition in the online mixing process can be known as soon as possible, the crystallization condition can be processed in time, the mixing efficiency of the mobile phase can be known intuitively through the calculation of the turbidity variance, and the effective grasping of the stirring process can be facilitated.
The invention is further illustrated by a specific example.
The sample preparation and detection operation specifically comprises the following steps:
1. mobile phase preparation: taking 6.8g of potassium dihydrogen phosphate, adding water to dissolve and dilute to 1000ml, regulating the pH value to 2.0 by phosphoric acid, taking 700ml of the solution, adding 1300ml of acetonitrile, placing a mobile phase mixing bottle to be stirred on a stirring table, dividing the surface of the stirring table to cover n multiplied by n coordinate units of a coordinate system, arranging a first light intensity sensor at the bottom of each coordinate unit, acquiring the light intensity Izs refracted after the incident light enters the mobile phase mixing liquid, arranging an LED direct light source parallel to the n multiplied by n of the liquid surface of the mixing bottle above the mixing bottle, acquiring the emitted light intensity Ifs of the LED light source corresponding to the position of the coordinate unit by arranging a second light intensity sensor, and connecting the input end of the LED light source, the first light intensity sensor and the input end of the second light intensity sensor with processing equipment respectively; stirring and mixing the mobile phase mixed liquid in the mixing bottle by using a stirring rod, standing on a stirring table until the liquid is stable after mixing, measuring the turbidity of each coordinate unit, wherein a calculation formula of the turbidity is as follows:
wherein Izs is the intensity of light refracted after incident light enters the mobile phase mixture, I fs The emitted light intensity k of the two pairs of LED direct light sources of the light intensity sensor p Is a proportionality coefficient, r pj Obtaining a turbidity sequence (c) for the distance from the LED direct light source to the liquid level hzdl ，c hzd2 ，c hzd3 ，c hzd4 ，…c hzdn ) And average turbidity->
Then calculating the variance of the turbidity, wherein the calculation formula of the turbidity variance is as follows: />
Comparing the variance value with a variance threshold value to observe whether stirring is uniform or not; if the variance value of the turbidity is larger than the variance threshold, the stirring is incomplete, stirring needs to be continued, if the turbidity isAnd if the variance value is smaller than the variance threshold, the stirring is completed, the next operation is carried out, and the filtering is carried out after stirring and mixing.
2. Sample solution preparation: accurately measuring the lml of the calcium zinc gluconate oral solution, placing in a 100ml measuring flask, adding 3ml of EDTA (0.05 mol/l) and 3ml of sodium hydroxide test solution, diluting to the maximum scale value by using a solvent, and standing at room temperature for 1h.
3. Preparing a reference substance: taking sodium gluconate reference substance 15m g Put in a 25ml measuring flask, dissolve with solvent and dilute to the maximum scale.
4. System usability test: the theoretical plate number of gluconate radical peak in the reference solution chromatogram should be greater than 5000.
5. EDTA (0.05 mol/1) and sodium hydroxide solution addition amount investigation result
| Sample number | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | |
Sample 7 | Sample 8 |
| EDTA， |
0 | 1 | 2 | 3 | 4 | 3 | 3 | 3 |
| Sodium hydroxide solution, ml | 3 | 3 | 3 | 3 | 3 | 2 | 3 | 4 |
| Gluconate radical content% | 88.5 | 90.2 | 96.8 | 96.3 | 97.5 | 97.2 | 96.5 | 95.8 |
The result shows that the dosage of EDTA (0.05 mol/l) is between 2ml and 4ml, and the content measurement result of the gluconic acid root between 2ml and 3ml of sodium hydroxide test solution is relatively close to the theoretical value.
6. Examination of test solution standing time
| Time of placement | 0h | 10min | 30min | 60min | 120min | 240min | 480min |
| Gluconate radical content% | 90.3 | 93.5 | 95.8 | 96.8 | 96.7 | 97.2 | 96.9 |
The results show that after adding 3ml of EDTA (0.05 mol/l) and 3ml of sodium hydroxide, the measurement results of the gluconate content tend to be stable after the sample solution is placed for 1 hour.
7. Recovery test results
The result shows that the recovery rate of the gluconate content measured by the method is between 98.0% and 102.0%, the relative standard deviation is less than 2.0%, and the recovery rate is good.
8. Results of the repeatability test
The repeatability test is carried out by adopting the method, 6 samples are prepared by the same method, the content of the gluconate is measured, and the results are shown in the following table:
| sample number | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | |
RSD,％ |
| Gluconate radical content% | 96.5 | 95.8 | 96.3 | 95.7 | 96.2 | 96.5 | 0.4 |
The result shows that the relative standard deviation of the gluconate radical content measured by the method is less than 2.0 percent, and the precision is good.
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. A method for detecting the content of gluconate in a calcium zinc gluconate oral solution is characterized by comprising the following steps of: the method adopts high performance liquid chromatography to detect, and comprises the following specific steps: (1) Mobile phase configuration, wherein a mobile phase is prepared by taking a ratio of 50mmol/L potassium dihydrogen phosphate solution to acetonitrile as 35:65; (2) Precisely measuring 1ml of a test sample, placing the test sample into a 100ml measuring flask, adding an ethylenediamine tetraacetic acid disodium solution and a sodium hydroxide test solution, diluting the test sample solution to the maximum scale, and stirring and mixing the test sample solution by adopting a mobile phase stirring and mixing method to generate a test sample solution; (3) Taking a proper amount of sodium gluconate reference substance, precisely weighing, adding water for dissolving and quantitatively diluting to prepare a solution containing 0.6+/-0.1 mg of sodium gluconate in each 1 ml; (4) calculating by an external standard method according to the peak area: the method comprises the following steps:
wherein, chromatographic conditions: amino-bonded silica gel is used as a filler, and a mobile phase is prepared by adjusting the pH value to 2.0 by phosphoric acid before, wherein the ratio of 50mmol/L potassium dihydrogen phosphate solution to acetonitrile is 35:65; the flow rate is 0.4ml/min-0.8ml/min; the detection wavelength is 210nm; the column temperature is 25-35 ℃; sample volume 20 μl, chromatographic conditions and system applicability: the theoretical plate number is not less than 3000 calculated according to gluconate radical peak.
2. The method for detecting the gluconate content in the calcium zinc gluconate oral solution according to claim 1, which is characterized in that: the mobile phase stirring and mixing method comprises the following specific steps: 101. placing a mobile phase mixing bottle needing stirring in the containerOn the stirring table, dividing the surface of the stirring table to cover n multiplied by n coordinate units of a coordinate system, arranging a first light intensity sensor at the bottom of each coordinate unit, wherein the first light intensity sensor is used for collecting the light intensity I refracted after the incident light enters the mobile phase mixed liquid zs Simultaneously, an LED direct light source parallel to the liquid level of the mixing bottle and the same as n multiplied by n is arranged above the mixing bottle, the LED light source corresponds to the position of the coordinate unit, and the light intensity I emitted by two pairs of LED direct light sources is measured by arranging a light intensity sensor fs Collecting, namely connecting the input end of the LED light source, the input end of the first light intensity sensor and the input end of the second light intensity sensor with processing equipment respectively; 102. stirring and mixing the mobile phase mixed liquid in the mixing bottle by using a stirring rod, standing on a stirring table until the liquid is stable after mixing, measuring the turbidity of each coordinate unit, wherein a calculation formula of the turbidity is as follows:
wherein I is zs I is the intensity of the incident light after entering the mobile phase mixed liquid fs The emitted light intensity k of the two pairs of LED direct light sources of the light intensity sensor p Is a proportionality coefficient, r pj Obtaining a turbidity sequence (c) for the distance from the LED direct light source to the liquid level hzd1 ,c hzd2 ,c hzd3 ,c hzd4 ,...c hzdn ) And average turbidity->
Then calculating the variance of the turbidity, wherein the calculation formula of the turbidity variance is as follows:
comparing the variance value with a variance threshold value to observe whether stirring is uniform or not; 103. if the variance value of the turbidity is larger than the variance threshold, the stirring is not completed, the stirring is required to be continued, if the variance value of the turbidity is smaller than the variance threshold, the stirring is completed, and the next operation is carried out, so that the calculation of the turbidity is arranged, the crystallization condition in the online mixing process can be known as soon as possible, the crystallization condition can be processed in time, and by calculating the turbidity variance,the mixing efficiency of the mobile phase is intuitively known, and the stirring process is effectively mastered.
3. The method for detecting the gluconate content in the calcium zinc gluconate oral solution according to claim 2, which is characterized in that: the chromatographic column is Phenomenex
5μmNH2/>
4.6×250mm。
4. The method for detecting the gluconate content in the calcium zinc gluconate oral solution according to claim 3, wherein the method comprises the following steps of: the test sample solvent is aqueous hydrochloric acid solution with pH of 1.0, aqueous sulfuric acid solution with pH of 2.0, aqueous phosphoric acid solution with pH of 2.0, aqueous trifluoroacetic acid solution with pH of 2.0 or pure water.
5. The method for detecting the gluconate content in the calcium zinc gluconate oral solution according to claim 4, wherein the method comprises the following steps of: in the preparation process of the sample solution, 0.05mol/L disodium ethylenediamine tetraacetate solution and sodium hydroxide test solution are added, wherein the 0.05mol/L disodium ethylenediamine tetraacetate solution is between 2ml and 4ml, and the sodium hydroxide test solution is between 2ml and 3 ml.
6. The method for detecting the gluconate content in the calcium zinc gluconate oral solution according to claim 5, wherein the method comprises the following steps of: and the sample solution is required to be placed for 1h at room temperature after the preparation of the sample solution is completed.
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