CN109633022B - Method for detecting additive in midazolam medicament - Google Patents
Method for detecting additive in midazolam medicament Download PDFInfo
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Abstract
The invention provides an analysis method for the content of dibutyl phthalate in midazolam process liquid, which is characterized by comprising the following steps: s1: preparing a sample mother solution; s2: preparing a sample solution; and (3) dissolving the sample mother liquor obtained in the step (S1) into the mixed solution, shaking up, standing, separating to obtain an organic phase, adding anhydrous magnesium sulfate, dehydrating, and taking a supernatant to be tested.
Description
Technical Field
The invention relates to the field of detection and analysis, and particularly relates to a method for detecting an additive in a midazolam medicament.
Background
Phthalate esters are one of the most commonly used plasticizers at present, and are widely used in PVC container materials because of their ability to significantly improve the processability and strength of PVC plastics. Because these substances and PVC plastics are mainly bonded by intermolecular force, they are easily released in use, causing pollution. Researches show that the endocrine of organisms is disordered, and serious consequences such as reproductive lesions, canceration of tissues and the like can be caused by long-term contact of the phthalate plasticizer. Therefore, the use of phthalate plasticizers is strictly limited in various countries, and the amount of such plasticizers is limited. With the wide application of PVC materials, pollution caused by the PVC materials cannot be ignored, but at present, relevant analysis and standards of the PVC materials in the drug production process are not reported. In future research, the detection of related components and the establishment of content standards become one direction.
At present, the methods for detecting phthalic acid substances at home and abroad mainly comprise Solid Phase Extraction (SPE) purification, supercritical extraction (SFE), gel chromatography (GPC), Accelerated Solvent Extraction (ASE) and purification volume block chromatography (SEC); the testing techniques mainly employ Gas Chromatography (GC), gas chromatography-mass spectrometry (GC MS), liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC MS), and the like.
At present, DBP detection methods and related researches for food packaging bags are more, but DBP detection for medicinal materials, particularly midazolam, is not reported.
Disclosure of Invention
In order to solve the technical problems, the invention provides an analysis method for the content of dibutyl phthalate in midazolam process liquid, which comprises the following steps:
s1: preparation of sample mother liquor
S2: preparation of sample solution
Dissolving the sample mother liquor obtained in the step S1 into the mixed solution, shaking up, standing, separating to obtain an organic phase, adding anhydrous magnesium sulfate, dehydrating, and taking a supernatant to be tested;
s3: GC-MS testing
The mixed solution is a mixed solution of heptane, butyl acetate and acetic acid.
As a preferred technical scheme, the preparation of the sample mother liquor comprises the following steps:
dissolving midazolam and dilute hydrochloric acid in a sodium chloride solution, adjusting the pH value with sodium hydroxide, and adding water to dilute to a scale.
As a preferred technical scheme, the pH is 3-3.5.
As a preferred technical scheme, the preparation of the sample mother liquor comprises the following steps:
adding midazolam into a dilute hydrochloric acid solution, and stirring until the midazolam is completely dissolved to obtain a midazolam solution; preparing a sodium chloride solution with the concentration of 0.8-1.2g/mL as a blank solution, adding the midazolam solution into the blank solution, adjusting the pH to 3.1-3.3, and adding water to dilute to the scale.
As a preferable technical scheme, the weight ratio of the heptane to the butyl acetate to the acetic acid is (35-40): (5-10): 2.
as a preferred technical scheme, the weight ratio of heptane, butyl acetate and acetic acid is 38: 8: 2.
as a preferred technical solution, the parameters of the GC-MS are set as follows:
a chromatographic column: HP-5MS, injection port temperature: 280-320 ℃, sample injection mode: no diversion, sample introduction: 1 μ L, carrier gas: inert gas, column flow rate: 0.8-1.2 mL/min.
As a preferred technical solution, the temperature rise program of the GC-MS is set as follows:
raising the temperature to 250 ℃ at the column temperature of 140 ℃ and 160 ℃ at every 15-25 ℃/min, and keeping the temperature for 0.5-2 min; then raising the temperature to 300 ℃ at the speed of 20-30 ℃/min, and keeping the temperature for 2-6 min.
As a preferred technical solution, the temperature rise program of the GC-MS is set as follows:
raising the temperature to 250 ℃ at the column temperature of 150 ℃ at a rate of 20 ℃/min, and keeping the temperature for 1 min; then the temperature is increased to 300 ℃ at the speed of 25 ℃/min and kept for 4 min.
As a preferred technical solution, the steps further include preparation of a quantitative limiting solution and preparation of an accuracy solution.
Drawings
FIG. 1: a specific chromatogram (solvent blank);
FIG. 2: a specific chromatogram (sample solution-1);
FIG. 3: a specific chromatogram (sample solution-2);
FIG. 4: specificity chromatograms (accuracy solutions).
Detailed Description
For purposes of the following detailed description, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Moreover, other than in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
Moreover, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of "1 to 10" is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, i.e., having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10.
No mention is made in the present invention of the availability of the pharmaceutical product or the components for purchase or preparation.
In order to solve the technical problems, the invention provides an analysis method for the content of dibutyl phthalate in midazolam process liquid.
In a specific embodiment, the method for analyzing the content of dibutyl phthalate in the midazolam process liquid comprises the following steps:
s1: preparation of sample mother liquor
S2: preparation of sample solution
Dissolving the sample mother liquor obtained in the step S1 into the mixed solution, shaking up, standing, separating to obtain an organic phase, adding anhydrous magnesium sulfate, dehydrating, and taking a supernatant to be tested;
s3: GC-MS testing
The mixed solution is a mixed solution of heptane, butyl acetate and acetic acid.
In the manufacturing process of the medicine, the silicone tube contains different plasticizers, such as: the direct GC-MS test of fatty acid plasticizers has an effect on the DBP peak. The mixed solvent composed of heptane, butyl acetate and acetic acid has synergistic effect, carboxylic acid group and ester group interact with polar group of DBP, and alkyl group interacts with nonpolar group of DBP, so as to extract DBP from original solution.
In a specific embodiment, the preparation of the sample mother liquor comprises the following steps:
dissolving midazolam and dilute hydrochloric acid in a sodium chloride solution, adjusting the pH value with sodium hydroxide, and adding water to dilute to a scale.
In particular embodiments, the pH is 3-3.5; in a preferred embodiment, the pH is 3.1 to 3.3; in a further preferred embodiment, the pH is 3.204.
In a specific embodiment, the preparation of the sample mother liquor comprises the following steps:
adding midazolam into a dilute hydrochloric acid solution, and stirring until the midazolam is completely dissolved to obtain a midazolam solution; preparing a sodium chloride solution with the concentration of 0.8-1.2g/mL as a blank solution, adding the midazolam solution into the blank solution, adjusting the pH to 3.1-3.3, and adding water to dilute to the scale. In this application the water is deionized water.
In a preferred embodiment, the preparation of the sample mother liquor comprises the following steps:
adding midazolam into a dilute hydrochloric acid solution, and stirring until the midazolam is completely dissolved to obtain a midazolam solution; preparing a sodium chloride solution with the concentration of 1g/mL as a blank solution, adding the midazolam solution into the blank solution, adjusting the pH to 3.204, and adding water to dilute to the scale.
In a specific embodiment, the weight ratio of heptane, butyl acetate and acetic acid is (35-40): (5-10): 2.
in a preferred embodiment, the weight ratio of heptane, butyl acetate and acetic acid is 38: 8: 2.
in a specific embodiment, the parameter settings of the GC-MS are as follows:
a chromatographic column: HP-5MS, injection port temperature: 280-320 ℃, sample injection mode: no diversion, sample introduction: 1 μ L, carrier gas: inert gas, column flow rate: 0.8-1.2 mL/min. The GC-MS is a gas chromatography-mass spectrometer testing method.
In a preferred embodiment, the parameter settings of the GC-MS are as follows:
a chromatographic column: HP-5MS, injection port temperature: 300 ℃, sample injection mode: no diversion, sample introduction: 1 μ L, carrier gas: he, column flow rate: 1 mL/min. He refers to helium.
In a specific embodiment, the temperature rise program of the GC-MS is set as follows:
raising the temperature to 250 ℃ at the column temperature of 150 ℃ at a rate of 20 ℃/min, and keeping the temperature for 1 min; then the temperature is increased to 300 ℃ at the speed of 25 ℃/min and kept for 4 min.
In the prior art, the temperature rising rate of DBP detected by GC-MS is not more than 20 ℃/min, and an overlapping peak appears when the temperature rising rate is too high; the invention overcomes the technical resistance and realizes the effect of obtaining a clear spectrogram under the condition of rapid temperature rise. The heating rates are respectively 20 ℃/min and 25 ℃/min, and dibutyl phthalate (DBP) and other substances in midazolam can be separated quickly by adopting quick heating in the invention, so that the detection sensitivity of the GCMS method can be effectively improved, the signal-to-noise ratio is improved, and the RSD can reach 1.5%.
In particular embodiments, the steps further comprise the preparation of a quantitative limiting solution and the preparation of an accuracy solution.
In a specific embodiment, the preparation of the quantitation limit solution comprises the steps of:
and (3) sampling the solution, adding the DBP reference substance mother liquor, adding the mixed solution, shaking up, standing, separating an organic phase, adding anhydrous magnesium carbonate for dehydration, and taking supernatant liquid for testing.
In a specific embodiment, the preparation of the accuracy solution comprises the steps of:
and (3) sampling the solution, adding the DBP reference substance mother liquor, adding the mixed solution, shaking up, standing, separating an organic phase, adding anhydrous magnesium carbonate for dehydration, and taking supernatant liquid for testing.
The invention selects heptane: butyl acetate: the acetic acid is 38: 8: 2, extracting the sample solution by the mixed solvent combined by the components, and controlling various parameters of GC-MS, wherein the effect is obvious, and the method comprises the following specific steps:
(1) heptane, butyl acetate and acetic acid are used as a first pretreatment procedure, so that the influence of other components in midazolam on DBP can be reduced; the clear GBP spectrogram is obtained in GC-MS;
(2) because midazolam can be processed by various devices in production, and the types and contents of plasticizers in different batches of medicines are different, if only a mixed solvent formed by compounding and combining heptane, butyl acetate and acetic acid is adopted for extraction, pure DBP cannot be obtained, and the DBP and other interference factors can be further separated under the parameter of GC-MS;
(3) the boiling points of heptane, butyl acetate and acetic acid are different, the column temperature is too low, the solvent can be independently evaporated in the temperature rising process, and the solvent evaporated at different temperatures can take away part of DBP, so that the peak emergence of DBP is influenced. When the column temperature is 140-160 ℃, the temperature is rapidly increased at the speed of 25 ℃/min, and dibutyl phthalate (DBP) and other substances in midazolam can be rapidly separated.
The following description will be given by way of specific examples.
Examples
Example 1
Example 1 provides a method for analyzing the content of dibutyl phthalate in midazolam process liquid, comprising the following steps:
s1: preparation of sample mother liquor
14mL of 1 wt% diluted hydrochloric acid is added with 1.000g of midazolam, and the mixture is stirred until the midazolam is completely dissolved; dissolving 8.001g of sodium chloride in 800mL of water to prepare a blank solution; adding the midazolam solution into the blank solution, uniformly stirring, and adjusting the pH value to 3.204 by using 0.02mol/L sodium hydroxide; water was added to 1000 mL.
S2: preparation of sample solution
Taking 25mL of sample mother solution, adding 5mL of mixed solution into an analysis bottle, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested.
S3: preparation of limiting of quantitation (LOQ) solution
Sampling 25mL of mother solution, putting the sample into an analysis bottle, adding 10 mu L of DBP reference mother solution, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 3 parts of LOQ solution were prepared in parallel in the same manner. The DBP loading concentration was 20. mu.g/L.
S4: preparation of accuracy solutions
Sampling 25mL of mother solution, adding 30 mu L of DBP reference mother solution into an analysis bottle, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 6 accurate solutions were prepared in parallel in the same way. The DBP loading concentration was 60. mu.g/L.
S5: GC-MS testing
A chromatographic column: HP-5MS, injection port temperature: 300 ℃, sample injection mode: no diversion, sample introduction: 1 μ L, carrier gas: he, column flow rate: 1mL/min, raising the temperature to 250 ℃ at the column temperature of 150 ℃ at every 20 ℃/min, and keeping the temperature for 1 min; then the temperature is increased to 300 ℃ at the speed of 25 ℃/min and kept for 4 min.
The mixed solution is prepared from heptane, butyl acetate and acetic acid according to the weight ratio of 38: 8: 2, mixing the solution.
Example 2
Example 2 provides a method for analyzing the content of dibutyl phthalate in midazolam process liquid, comprising the following steps:
s1: preparation of sample mother liquor
14mL of 1 wt% diluted hydrochloric acid is added with 1.000g of midazolam, and the mixture is stirred until the midazolam is completely dissolved; dissolving 8.001g of sodium chloride in 800mL of water to prepare a blank solution; adding the midazolam solution into the blank solution, uniformly stirring, and adjusting the pH value to 3 by using 0.02mol/L sodium hydroxide; water was added to 1000 mL.
S2: preparation of sample solution
Taking 25mL of sample mother solution, adding 5mL of mixed solution into an analysis bottle, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested.
S3: preparation of limiting of quantitation (LOQ) solution
Sampling 25mL of mother solution, putting the sample into an analysis bottle, adding 10 mu L of DBP reference mother solution, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 3 parts of LOQ solution were prepared in parallel in the same manner. The DBP loading concentration was 20. mu.g/L.
S4: preparation of accuracy solutions
Sampling 25mL of mother solution, adding 30 mu L of DBP reference mother solution into an analysis bottle, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 6 accurate solutions were prepared in parallel in the same way. The DBP loading concentration was 60. mu.g/L.
S5: GC-MS testing
A chromatographic column: HP-5MS, injection port temperature: 280 ℃, sample injection mode: no diversion, sample introduction: 1 μ L, carrier gas: he, column flow rate: 0.8mL/min, rising to 250 ℃ at every 15 ℃/min when the column temperature is 150 ℃, and keeping for 0.5 min; then the temperature is increased to 300 ℃ at the speed of 20 ℃/min and kept for 2 min.
The mixed solution is prepared from heptane, butyl acetate and acetic acid according to the weight ratio of 35: 5: 2, mixing the solution.
Example 3
Example 3 provides a method for analyzing the content of dibutyl phthalate in midazolam process liquid, comprising the following steps:
s1: preparation of sample mother liquor
14mL of 1 wt% diluted hydrochloric acid is added with 1.000g of midazolam, and the mixture is stirred until the midazolam is completely dissolved; dissolving 8.001g of sodium chloride in 800mL of water to prepare a blank solution; adding the midazolam solution into the blank solution, uniformly stirring, and adjusting the pH value to 3.5 by using 0.02mol/L sodium hydroxide; water was added to 1000 mL.
S2: preparation of sample solution
Taking 25mL of sample mother solution, adding 5mL of mixed solution into an analysis bottle, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested.
S3: preparation of limiting of quantitation (LOQ) solution
Sampling 25mL of mother solution, putting the sample into an analysis bottle, adding 10 mu L of DBP reference mother solution, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 3 parts of LOQ solution were prepared in parallel in the same manner. The DBP loading concentration was 20. mu.g/L.
S4: preparation of accuracy solutions
Sampling 25mL of mother solution, adding 30 mu L of DBP reference mother solution into an analysis bottle, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 6 accurate solutions were prepared in parallel in the same way. The DBP loading concentration was 60. mu.g/L.
S5: GC-MS testing
A chromatographic column: HP-5MS, injection port temperature: 300 ℃, sample injection mode: no diversion, sample introduction: 1 μ L, carrier gas: he, column flow rate: 1mL/min, raising the temperature to 250 ℃ at the column temperature of 150 ℃ at every 25 ℃/min, and keeping the temperature for 2 min; then the temperature is increased to 300 ℃ at the speed of 30 ℃/min and kept for 6 min.
The mixed solution is prepared from heptane, butyl acetate and acetic acid according to the weight ratio of 40: 10: 2, mixing the solution.
Example 4
Example 4 provides a method for analyzing the content of dibutyl phthalate in midazolam process liquid, comprising the following steps:
s1: preparation of sample mother liquor
14mL of 1 wt% diluted hydrochloric acid is added with 1.000g of midazolam, and the mixture is stirred until the midazolam is completely dissolved; dissolving 8.001g of sodium chloride in 800mL of water to prepare a blank solution; adding the midazolam solution into the blank solution, uniformly stirring, and adjusting the pH value to 3.204 by using 0.02mol/L sodium hydroxide; water was added to 1000 mL.
S2: preparation of sample solution
Taking 25mL of sample mother liquor, adding 5mL of n-hexane into an analysis bottle, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested.
S3: preparation of limiting of quantitation (LOQ) solution
Sampling 25mL of mother solution, putting the sample into an analysis bottle, adding 10 mu L of DBP reference mother solution, adding 5mL of n-hexane, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 3 parts of LOQ solution were prepared in parallel in the same manner. The DBP loading concentration was 20. mu.g/L.
S4: preparation of accuracy solutions
Sampling 25mL of mother solution, putting the sample into an analysis bottle, adding 30 mu L of DBP reference mother solution, adding 5mL of n-hexane, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 6 accurate solutions were prepared in parallel in the same way. The DBP loading concentration was 60. mu.g/L.
S5: GC-MS testing
A chromatographic column: HP-5MS, injection port temperature: 300 ℃, sample injection mode: no diversion, sample introduction: 1 μ L, carrier gas: he, column flow rate: 1mL/min, raising the temperature to 250 ℃ at the column temperature of 150 ℃ at every 20 ℃/min, and keeping the temperature for 1 min; then the temperature is increased to 300 ℃ at the speed of 25 ℃/min and kept for 4 min.
Example 5
Example 5 provides a method for analyzing the content of dibutyl phthalate in midazolam process liquid, comprising the following steps:
s1: preparation of sample mother liquor
14mL of 1 wt% diluted hydrochloric acid is added with 1.000g of midazolam, and the mixture is stirred until the midazolam is completely dissolved; dissolving 8.001g of sodium chloride in 800mL of water to prepare a blank solution; adding the midazolam solution into the blank solution, uniformly stirring, and adjusting the pH value to 3.204 by using 0.02mol/L sodium hydroxide; water was added to 1000 mL.
S2: preparation of sample solution
Taking 25mL of sample mother solution, adding 5mL of mixed solution into an analysis bottle, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested.
S3: preparation of limiting of quantitation (LOQ) solution
Sampling 25mL of mother solution, putting the sample into an analysis bottle, adding 10 mu L of DBP reference mother solution, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 3 parts of LOQ solution were prepared in parallel in the same manner. The DBP loading concentration was 20. mu.g/L.
S4: preparation of accuracy solutions
Sampling 25mL of mother solution, adding 30 mu L of DBP reference mother solution into an analysis bottle, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 6 accurate solutions were prepared in parallel in the same way. The DBP loading concentration was 60. mu.g/L.
S5: GC-MS testing
A chromatographic column: HP-5MS, injection port temperature: 300 ℃, sample injection mode: no diversion, sample introduction: 1 μ L, carrier gas: he, column flow rate: 1mL/min, raising the temperature to 250 ℃ at the column temperature of 150 ℃ at every 20 ℃/min, and keeping the temperature for 1 min; then the temperature is increased to 300 ℃ at the speed of 25 ℃/min and kept for 4 min.
The mixed solution is prepared from heptane, butyl acetate and acetic acid according to a weight ratio of 8: 38: 2, mixing the solution.
Example 6
Example 6 provides a method for analyzing the content of dibutyl phthalate in midazolam process liquid, comprising the steps of:
s1: preparation of sample mother liquor
14mL of 1 wt% diluted hydrochloric acid is added with 1.000g of midazolam, and the mixture is stirred until the midazolam is completely dissolved; dissolving 8.001g of sodium chloride in 800mL of water to prepare a blank solution; adding the midazolam solution into the blank solution, uniformly stirring, and adjusting the pH value to 3.204 by using 0.02mol/L sodium hydroxide; water was added to 1000 mL.
S2: preparation of sample solution
Taking 25mL of sample mother solution, adding 5mL of mixed solution into an analysis bottle, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested.
S3: preparation of limiting of quantitation (LOQ) solution
Sampling 25mL of mother solution, putting the sample into an analysis bottle, adding 10 mu L of DBP reference mother solution, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 3 parts of LOQ solution were prepared in parallel in the same manner. The DBP loading concentration was 20. mu.g/L.
S4: preparation of accuracy solutions
Sampling 25mL of mother solution, adding 30 mu L of DBP reference mother solution into an analysis bottle, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 6 accurate solutions were prepared in parallel in the same way. The DBP loading concentration was 60. mu.g/L.
S5: GC-MS testing
A chromatographic column: HP-5MS, injection port temperature: 300 ℃, sample injection mode: no diversion, sample introduction: 1 μ L, carrier gas: he, column flow rate: 1mL/min, raising the temperature to 250 ℃ at the column temperature of 150 ℃ at every 40 ℃/min, and keeping the temperature for 3 min; then the temperature is increased to 300 ℃ at the speed of 10 ℃/min and kept for 1 min.
The mixed solution is prepared from heptane, butyl acetate and acetic acid according to the weight ratio of 38: 8: 2, mixing the solution.
Example 7
Example 7 provides a method for analyzing the content of dibutyl phthalate in midazolam process liquid, comprising the steps of:
s1: preparation of sample mother liquor
14mL of 1 wt% diluted hydrochloric acid is added with 1.000g of midazolam, and the mixture is stirred until the midazolam is completely dissolved; dissolving 8.001g of sodium chloride in 800mL of water to prepare a blank solution; adding the midazolam solution into the blank solution, uniformly stirring, and adjusting the pH value to 5 by using 0.02mol/L sodium hydroxide; water was added to 1000 mL.
S2: preparation of sample solution
Taking 25mL of sample mother solution, adding 5mL of mixed solution into an analysis bottle, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested.
S3: preparation of limiting of quantitation (LOQ) solution
Sampling 25mL of mother solution, putting the sample into an analysis bottle, adding 10 mu L of DBP reference mother solution, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 3 parts of LOQ solution were prepared in parallel in the same manner. The DBP loading concentration was 20. mu.g/L.
S4: preparation of accuracy solutions
Sampling 25mL of mother solution, adding 30 mu L of DBP reference mother solution into an analysis bottle, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 6 accurate solutions were prepared in parallel in the same way. The DBP loading concentration was 60. mu.g/L.
S5: GC-MS testing
A chromatographic column: HP-5MS, injection port temperature: 300 ℃, sample injection mode: no diversion, sample introduction: 1 μ L, carrier gas: he, column flow rate: 1mL/min, raising the temperature to 250 ℃ at the column temperature of 150 ℃ at every 20 ℃/min, and keeping the temperature for 1 min; then the temperature is increased to 300 ℃ at the speed of 25 ℃/min and kept for 4 min.
The mixed solution is prepared from heptane, butyl acetate and acetic acid according to the weight ratio of 38: 8: 2, mixing the solution.
Example 8
Example 8 provides a method for analyzing the content of dibutyl phthalate in midazolam process liquid, comprising the steps of:
s1: preparation of sample mother liquor
14mL of 1 wt% diluted hydrochloric acid is added with 1.000g of midazolam, and the mixture is stirred until the midazolam is completely dissolved; dissolving 8.001g of sodium chloride in 800mL of water to prepare a blank solution; adding the midazolam solution into the blank solution, uniformly stirring, and adjusting the pH value to 3.204 by using 0.02mol/L sodium hydroxide; water was added to 1000 mL.
S2: preparation of sample solution
Taking 25mL of sample mother solution, adding 5mL of mixed solution into an analysis bottle, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested.
S3: preparation of limiting of quantitation (LOQ) solution
Sampling 25mL of mother solution, putting the sample into an analysis bottle, adding 10 mu L of DBP reference mother solution, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 3 parts of LOQ solution were prepared in parallel in the same manner. The DBP loading concentration was 20. mu.g/L.
S4: preparation of accuracy solutions
Sampling 25mL of mother solution, adding 30 mu L of DBP reference mother solution into an analysis bottle, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 6 accurate solutions were prepared in parallel in the same way. The DBP loading concentration was 60. mu.g/L.
S5: GC-MS testing
A chromatographic column: HP-5MS, injection port temperature: 300 ℃, sample injection mode: no diversion, sample introduction: 1 μ L, carrier gas: he, column flow rate: 1mL/min, raising the temperature to 250 ℃ at the column temperature of 190 ℃ at every 20 ℃/min, and keeping the temperature for 1 min; then the temperature is increased to 300 ℃ at the speed of 25 ℃/min and kept for 4 min.
The mixed solution is prepared from heptane, butyl acetate and acetic acid according to the weight ratio of 38: 8: 2, mixing the solution.
Example 9
Example 9 provides a method for analyzing the content of dibutyl phthalate in midazolam process liquid, comprising the steps of:
s1: preparation of sample mother liquor
14mL of 1 wt% diluted hydrochloric acid is added with 1.000g of midazolam, and the mixture is stirred until the midazolam is completely dissolved; dissolving 8.001g of sodium chloride in 800mL of water to prepare a blank solution; adding the midazolam solution into the blank solution, uniformly stirring, and adjusting the pH value to 3.204 by using 0.02mol/L sodium hydroxide; water was added to 1000 mL.
S2: preparation of sample solution
Taking 25mL of sample mother solution, adding 5mL of mixed solution into an analysis bottle, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested.
S3: preparation of limiting of quantitation (LOQ) solution
Sampling 25mL of mother solution, putting the sample into an analysis bottle, adding 10 mu L of DBP reference mother solution, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 3 parts of LOQ solution were prepared in parallel in the same manner. The DBP loading concentration was 20. mu.g/L.
S4: preparation of accuracy solutions
Sampling 25mL of mother solution, adding 30 mu L of DBP reference mother solution into an analysis bottle, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 6 accurate solutions were prepared in parallel in the same way. The DBP loading concentration was 60. mu.g/L.
S5: GC-MS testing
A chromatographic column: HP-5MS, injection port temperature: 350 ℃, sample injection mode: no diversion, sample introduction: 2 μ L, carrier gas: he, column flow rate: 0.5mL/min, rising to 250 ℃ at the column temperature of 200 ℃ per 30 ℃/min, and keeping for 2 min; then the temperature is increased to 300 ℃ at the speed of 30 ℃/min and is kept for 3 min.
The mixed solution is prepared from heptane, butyl acetate and acetic acid according to the weight ratio of 38: 8: 2, mixing the solution.
Example 10
Example 10 provides a method for analyzing the content of dibutyl phthalate in midazolam process liquid, comprising the steps of:
s1: preparation of sample mother liquor
14mL of 1 wt% diluted hydrochloric acid is added with 1.000g of midazolam, and the mixture is stirred until the midazolam is completely dissolved; dissolving 8.001g of sodium chloride in 800mL of water to prepare a blank solution; adding the midazolam solution into the blank solution, uniformly stirring, and adjusting the pH value to 5 by using 0.01mol/L sodium hydroxide; water was added to 1000 mL.
S2: preparation of sample solution
Taking 25mL of sample mother solution, adding 5mL of mixed solution into an analysis bottle, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested.
S3: preparation of limiting of quantitation (LOQ) solution
Sampling 25mL of mother solution, putting the sample into an analysis bottle, adding 10 mu L of DBP reference mother solution, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 3 parts of LOQ solution were prepared in parallel in the same manner. The DBP loading concentration was 20. mu.g/L.
S4: preparation of accuracy solutions
Sampling 25mL of mother solution, adding 30 mu L of DBP reference mother solution into an analysis bottle, adding 5mL of mixed solution, shaking up, and standing; separating the organic phase, adding anhydrous magnesium sulfate for dehydration, and taking the supernatant to be tested. A total of 6 accurate solutions were prepared in parallel in the same way. The DBP loading concentration was 60. mu.g/L.
S5: GC-MS testing
A chromatographic column: HP-5MS, injection port temperature: 350 ℃, sample injection mode: no diversion, sample introduction: 2 μ L, carrier gas: he, column flow rate: 0.5mL/min, rising to 250 ℃ at the column temperature of 200 ℃ per 30 ℃/min, and keeping for 2 min; then the temperature is increased to 300 ℃ at the speed of 30 ℃/min and is kept for 3 min.
The mixed solution is prepared from heptane, butyl acetate and acetic acid according to a weight ratio of 8: 38: 2, mixing the solution.
Evaluation of Performance test
1) Various performance tests
The assays were performed according to the protocol described in example 1 and the results were tested for specificity, linearity, accuracy, precision and quantitation limits.
1-1 Linear test
The test results are shown in Table 1.
TABLE 1
The correlation coefficient is not lower than 0.99, and the correlation coefficient of the test result is 0.9992, which meets the requirement. 1-2 accuracy test
The test results are shown in Table 2.
TABLE 2
The recovery rate is less than 15%, and the RSD of the test result is 1.5%, which completely meets the requirement. 1-3LOQ
The test results are shown in Table 3.
TABLE 3
The recovery rate of the LOQ solution is between 70 and 125 percent, and the RSD is less than 15 percent.
1-4 degree of precision
The specific test results are shown in table 4.
TABLE 4
1-5 specialization
The blank solution, sample solution and accuracy solution should not interfere with the control at the position where the peak appears, and if there is a peak nearby, the separation degree between the blank solution, sample solution and accuracy solution should not be less than 1.5.
The test results are shown in FIGS. 1-4. There was no significant interference at the control peak position.
2) Accuracy testing
The same products were measured by the methods of examples 1 to 10, and the results of RSD test are shown in Table 5.
TABLE 5
| Examples | Example 1 | Example 2 | Example 3 | Example 4 | Example 5 |
| RSD/％ | 1.5 | 1.6 | 1.6 | 17.5 | 12.8 |
| Examples | Example 6 | Example 7 | Example 8 | Example 9 | Example 10 |
| RSD/％ | 18.3 | 12.1 | 12.9 | 16 | 18 |
The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims.
Claims (4)
1. A method for analyzing the content of dibutyl phthalate in midazolam process liquid is characterized by comprising the following steps:
s1: preparation of sample mother liquor
The preparation of the sample mother liquor comprises the following steps:
adding midazolam into a dilute hydrochloric acid solution, and stirring until the midazolam is completely dissolved to obtain a midazolam solution; preparing a sodium chloride solution with the concentration of 0.8-1.2g/mL as a blank solution, adding the midazolam solution into the blank solution, adjusting the pH to 3.1-3.3, and adding water to dilute to a scale;
s2: preparation of sample solution
Dissolving the sample mother liquor obtained in the step S1 into the mixed solution, shaking up, standing, separating to obtain an organic phase, adding anhydrous magnesium sulfate, dehydrating, and taking a supernatant to be tested;
s3: GC-MS testing
The mixed solution is a mixed solution of heptane, butyl acetate and acetic acid;
the weight ratio of the heptane to the butyl acetate to the acetic acid is (35-40): (5-10): 2;
the parameter settings of the GC-MS are as follows:
a chromatographic column: HP-5MS, injection port temperature: 280-320 ℃, sample injection mode: no diversion, sample introduction: 1 μ L, carrier gas: inert gas, column flow rate: 0.8-1.2 mL/min;
the temperature program of the GC-MS is set as follows:
raising the temperature to 250 ℃ at the column temperature of 140 ℃ and 160 ℃ at every 15-25 ℃/min, and keeping the temperature for 0.5-2 min; then raising the temperature to 300 ℃ at the speed of 20-30 ℃/min, and keeping the temperature for 2-6 min.
2. The analytical method according to claim 1, wherein the weight ratio of heptane, butyl acetate and acetic acid is 38: 8: 2.
3. the analytical method of claim 1, wherein the GC-MS temperature program is set as follows:
raising the temperature to 250 ℃ at the column temperature of 150 ℃ at a rate of 20 ℃/min, and keeping the temperature for 1 min; then the temperature is increased to 300 ℃ at the speed of 25 ℃/min and kept for 4 min.
4. The assay of claim 1, wherein the steps further comprise preparation of a quantitative limiting solution and preparation of an accuracy solution.
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