CN110715992A - Method for detecting content of chlormezanone in blood - Google Patents
Method for detecting content of chlormezanone in blood Download PDFInfo
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Abstract
The invention provides a method for detecting the content of chlormezanone in blood, which comprises the following steps: preparing at least three standard working solutions containing a standard substance of chlormezanone with known concentration; mixing the standard working solution, the internal standard working solution and the blank blood sample, and performing sample pretreatment to obtain a standard solution; respectively detecting each standard solution by using a high performance liquid chromatograph to obtain a chromatogram of each standard solution; fitting to obtain a standard curve equation according to the chromatogram of each standard solution; mixing the internal standard working solution with a blood sample obtained by processing at least 2mL of blood to be detected, and carrying out the same sample pretreatment to obtain a sample to be detected; detecting the sample to be detected to obtain a chromatogram map; and calculating the content of the chlormezanone in the blood sample according to the chromatogram and the standard curve equation of the sample to be detected. The scheme is combined with an internal standard method to realize detection, so that the accuracy of a detection result is improved.
Description
Technical Field
The invention relates to the technical field of clinical chemistry, in particular to a method for detecting the content of chlormezanone in blood.
Background
Anxiety disorder (anxiety), also known as anxiety neurosis, is the most common one of the major group of neurological disorders, and is characterized by an anxious emotional experience, which can be divided into two forms, chronic anxiety, generalized anxiety, and acute anxiety, panic attack. Anxiety disorders are characterized by a lack of stress in the clear objective subject, restlessness, and vegetative nerve dysfunction symptoms such as palpitation, trembling hands, sweating, frequent urination, and motor restlessness. Attention is paid to distinguish normal anxiety states, such as where the severity of the anxiety is significantly inconsistent with objective facts or situations, or where the duration is too long, which may be pathological.
The clomezanone has effects of resisting anxiety, tranquilizing, hypnotizing and relieving muscular tension, and can be used for tranquilizing and improving sleep for people with emotional tension, anxiety, and dysphoria. However, the clomezanone can inhibit the central nervous system and the respiratory system, can cause vasodilatation, reduce the effective blood volume, cause deep coma due to severe drug poisoning and be accompanied by shock to endanger life. With the wide clinical application of the medicine, the accidents of taking by mistake, suicide or poison are happened. Therefore, the method for quantitatively detecting the chlormezanone component in the blood of the human body has great practical use value, for example, the monitoring of the blood concentration is necessary in the medication process.
At present, the content of the chlormezanone in blood is detected by adopting a gas chromatography-mass spectrometry combined method, but the detection is basically realized by combining an external standard method. And the external standard method is easy to cause lower accuracy of the detection result.
Disclosure of Invention
The invention provides a method for detecting the content of chlormezanone in blood, which combines an internal standard method to realize detection, thereby improving the accuracy of a detection result.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention provides a method for detecting the content of chlormezanone in blood, which comprises the following steps:
preparing at least three standard working solutions, wherein the standard working solutions contain chlormezanone standard substances with known concentrations, and the chlormezanone standard substances in different standard working solutions have different concentrations;
mixing a certain amount of standard working solution, internal standard working solution and a blank blood sample, and performing sample pretreatment to obtain standard solution, wherein the internal standard working solution contains an internal standard substance with known concentration;
respectively detecting at least three standard solutions by using a high performance liquid chromatograph under certain detection conditions to obtain chromatograms of the standard solutions;
fitting to obtain a standard curve equation of the chlormezanone according to the chromatogram of each standard solution;
mixing a certain amount of internal standard working solution with a blood sample, and performing the same sample pretreatment to obtain a sample to be detected, wherein the blood sample is obtained by treating at least 2mL of blood to be detected;
detecting a sample to be detected by using a high performance liquid chromatograph under the same detection condition to obtain a chromatogram of the sample to be detected;
and calculating the content of the chlormezanone in the blood sample according to the chromatogram of the sample to be detected and the standard curve equation of the chlormezanone.
Preferably, the performing of sample pretreatment comprises the following steps of A1-A4:
a1: the mixed solution is evenly mixed for 1-2min in a vortex mode at the rotating speed of 2500-3000 rpm;
a2: adding 800-;
a3: transferring 700-1000 mu L of supernatant into a centrifuge tube, and drying by using nitrogen at normal temperature;
a4: adding 80-120 μ L of the complex solution into the centrifuge tube blown dry by the upper clear air, uniformly mixing the complex solution in a vortex manner at the rotating speed of 2500 plus 3000rpm for 2-4min, and then centrifuging the complex solution at the rotating speed of 10000 plus 15000rpm for 4-6min to obtain supernatant.
Preferably, the detection conditions include: phenomenex kinetex EVO C18 chromatographic column, the length of chromatographic column is 150mm, internal diameter is 2.1mm, and packing particle size is 5 mu m.
Preferably, the detection conditions include: the column temperature is 35-45 deg.C, the mobile phase is water and acetonitrile, the flow rate is 0.2-0.4mL/min, the sample amount is 5-15 μ L, and the analysis time is 7-9 min.
Preferably, the detection conditions include: single column dual pump elution mode;
the double-pump switching mode is as follows: switching from the analysis pump to the cleaning pump at 5.5min, and switching from the cleaning pump to the analysis pump at 6.5 min;
the elution mode is as follows: the volume ratio of water to acetonitrile is 74-76% in other elution time ranges except 5.5-6.5 min: 26 to 24 percent.
Preferably, the ultraviolet detector in the high performance liquid chromatograph is a DAD-3000 detector, the detection wavelength is 225-235nm, the acquisition frequency is 5Hz, and the bandwidth is 4 nm.
Preferably, the on-line filter used by the high performance liquid chromatograph is SSI COL PRE-FILTER WATER 1/160.5M.
Preferably, the internal standard is pirfenidone.
Preferably, the concentration of the standard substance of the chlormezanone in the standard working solution is 10-640 mug/mL, and the diluent is 50-80% methanol water solution;
the concentration of the pirfenidone standard substance in the internal standard working solution is 20-30 mug/mL, and the diluent is 40-60% methanol water solution.
Preferably, the concentration of the chlormezanone standard substance in the at least three standard working solutions is at least three of 10 mu g/mL, 20 mu g/mL, 40 mu g/mL, 80 mu g/mL, 160 mu g/mL, 320 mu g/mL and 640 mu g/mL.
The invention provides a method for detecting the content of chlormezanone in blood, which comprises the following steps: preparing at least three standard working solutions containing a standard substance of chlormezanone with known concentration; mixing the standard working solution, the internal standard working solution and the blank blood sample, and performing sample pretreatment to obtain a standard solution; respectively detecting each standard solution by using a high performance liquid chromatograph to obtain a chromatogram of each standard solution; fitting to obtain a standard curve equation according to the chromatogram of each standard solution; mixing the internal standard working solution with a blood sample obtained by processing at least 2mL of blood to be detected, and carrying out the same sample pretreatment to obtain a sample to be detected; detecting the sample to be detected to obtain a chromatogram map; and calculating the content of the chlormezanone in the blood sample according to the chromatogram and the standard curve equation of the sample to be detected. The invention combines the internal standard method to realize detection, thereby improving the accuracy of the detection result.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a flow chart of a method for detecting the content of clomazanone in blood according to an embodiment of the present invention;
FIG. 2 is a chemical formula of chlormezanone provided by an embodiment of the present invention;
FIG. 3 is a chromatogram of a standard and an internal standard of chlormezanone in a standard solution according to an embodiment of the present invention;
fig. 4 is a chromatogram of chlormezanone and an internal standard in a sample to be tested according to an embodiment of the present invention;
FIG. 5 is a linear relationship diagram of chlormezanone provided by an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete, the technical solutions in the embodiments of the present invention will be described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention, and based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts belong to the scope of the present invention.
In order to avoid the fixed value deviation caused by misoperation in the pretreatment process, an internal standard method can be adopted to detect the content of the chlormezanone in the blood sample so as to ensure the detection accuracy.
The research shows that the extraction rates of the chlormezanone and the internal standard substance in the blood sample pretreatment process are different. For example, the results of the extraction rate test using pirfenidone as an internal standard are shown in table 1 below.
TABLE 1
| Internal standard substance | Chlormeptazinone | |
| Peak area of substance in standard solution without pretreatment | 2.3343 | 16.0023 |
| Peak area of substance in standard solution at pretreatment | 0.3876 | 0.7383 |
| Extraction rate | 16.6％ | 4.6％ |
In table 1, standard solutions, i.e., standard working solutions and internal standard working solutions, were directly prepared by dilution and analyzed by loading. And (4) performing pretreatment, namely adding blank blood into the standard working solution and the internal standard working solution, performing pretreatment operation which is the same as that of blood sample pretreatment, obtaining a standard solution, and loading and analyzing the standard solution.
Referring to table 1, the peak area of the internal standard substance without pretreatment is 2.3343, while the peak area of the internal standard substance with pretreatment is 0.3876, which shows that the pretreatment process has significant influence on the extraction of the internal standard substance, and the extraction rate is 16.6%.
Referring to table 1, the peak area of clomezanone without pretreatment is 16.0023, while the peak area of internal standard substance with pretreatment is 0.7383, which shows that the pretreatment process has a significant effect on the extraction of clomezanone, and the extraction rate is 4.6%.
It can be seen that the extraction rate of the internal standard substance is different from that of the chlormezanone and has a large difference in the pretreatment process. Therefore, in order to correct the fixed value error caused by inconsistent extraction rates of the chlormezanone and the internal standard substance, a blood adding pretreatment mode can be adopted to obtain the standard curve, so that the pretreatment process for preparing the standard solution according to the standard working solution is the same as the pretreatment process for preparing the sample to be detected according to the blood sample, and the accuracy of the detection result is ensured.
Based on the above, as shown in fig. 1, an embodiment of the present invention provides a method for detecting the content of clomezanone in blood, which may include the following steps:
step 101: preparing at least three standard working solutions, wherein the standard working solutions contain the standard substance of the chlormezanone with known concentration, and the concentrations of the standard substances of the chlormezanone in different standard working solutions are different.
Step 102: and mixing a certain amount of standard working solution, internal standard working solution and a blank blood sample, and performing sample pretreatment to obtain the standard solution, wherein the internal standard working solution contains an internal standard substance with known concentration.
In detail, the blank blood sample may be serum or plasma without the chlormezanone and the internal standard substance, and the content detection result of the chlormezanone and the internal standard substance of the blank blood sample is not detected.
Step 103: and respectively detecting at least three standard solutions by using a high performance liquid chromatograph under certain detection conditions to obtain the chromatogram of each standard solution.
Step 104: and fitting to obtain a standard curve equation of the chlormezanone according to the chromatogram of each standard solution.
Step 105: and mixing a certain amount of internal standard working solution with the blood sample, and performing the same sample pretreatment to obtain a sample to be detected, wherein the blood sample is obtained by treating at least 2mL of blood to be detected.
Step 106: and detecting the sample to be detected by using a high performance liquid chromatograph under the same detection condition to obtain a chromatogram of the sample to be detected.
Step 107: and calculating the content of the chlormezanone in the blood sample according to the chromatogram of the sample to be detected and the standard curve equation of the chlormezanone.
Referring to fig. 2, fig. 2 shows the chemical structural formula of chlormezanone.
In general, at least three coordinate points are required for establishing the standard curve equation to ensure the accuracy of the established equation, so at least three standard solutions are required to be prepared, and the standard curve equation of the clomazazone can be fitted according to the chromatogram obtained by detecting each standard solution.
In detail, the standard curve equation fitted to the resulting clomezanone may be generally y ═ k × x + b. The two variables x and y can be the peak area ratio of the standard substance and the internal standard substance of the chlormezanone in the chromatogram of each standard solution, and the concentration ratio of the standard substance and the internal standard substance of the chlormezanone in each standard solution. Therefore, the concentration of the chlormezanone in the sample to be detected can be calculated by substituting the peak area ratio of the chlormezanone to the internal standard substance and the concentration of the internal standard substance in the sample to be detected into a standard curve equation in the chromatogram of the sample to be detected, so that the content of the chlormezanone in the blood sample can be obtained.
In the embodiment of the invention, the dosage of the blood to be detected is not high and can be as low as 2mL, and the blood sampling amount is less, so that the blood sampling experience of a detected person is good. After the blood to be detected is sampled, corresponding treatment can be carried out to obtain a blood sample. For example, at least 2mL of blood to be detected is taken, centrifuged at 3500rpm for 10min, and the supernatant is taken to obtain serum or plasma, thus obtaining the blood sample. Serum or plasma samples can be stored frozen at-20 ℃ until ready for analysis.
After the blood sample is obtained, the sample to be detected is obtained through the same pretreatment, and the sample to be detected is detected under the same detection condition to obtain a chromatogram map of the sample to be detected. Based on the chromatogram and the standard curve, the content of the chlormezanone in the blood sample can be obtained as described above.
In summary, the method for detecting the content of clomezanone in blood provided by the embodiment of the invention combines the internal standard method and the high performance liquid chromatography, so that the interference factors are greatly reduced, and the method has the advantages of accurate quantification, good reproducibility, strong specificity, high sensitivity, more accurate detection result, low cost and short analysis time. And the analysis time of the gas chromatography is long, the analysis time of the gas chromatography-mass spectrometry combined method is long, the analysis cost is high, the requirement on the field is high, and the operation is required by professional personnel, so the embodiment of the invention is favorable for detecting the large-flux blood sample.
In an embodiment of the present invention, the performing the sample pretreatment includes the following steps a1 to a 4:
a1: the mixed solution is evenly mixed for 1-2min in a vortex mode at the rotating speed of 2500-3000 rpm;
a2: adding 800-;
a3: transferring 700-1000 mu L of supernatant into a centrifuge tube, and drying by using nitrogen at normal temperature;
a4: adding 80-120 μ L of the complex solution into the centrifuge tube blown dry by the upper clear air, uniformly mixing the complex solution in a vortex manner at the rotating speed of 2500 plus 3000rpm for 2-4min, and then centrifuging the complex solution at the rotating speed of 10000 plus 15000rpm for 4-6min to obtain supernatant.
For example, the value of the swirl rate may be 2500, 2600, 2700, 2800, 2900, or 3000; the value of the vortex time in A1 can be 1, 1.2, 1.5, 1.7 or 2; the dosage of the extractant can be 800, 900, 1000, 1100 or 1200; the value of the vortex time in A2 can be 3, 4, 5, 6 or 7; the value of the centrifugal rotation speed can be 10000, 11000, 12000, 13000, 14000 or 15000; the value of the centrifugation time in A2 can be 3, 4, 5, 6 or 7; the dosage of the supernatant in A3 can be 700, 80, 900 or 1000; the dosage of the complex solution can be 80, 90, 100, 110 or 120; the value of the vortex time in A4 can be 2, 2.5, 3, 3.5 or 4; the value of the centrifugation time in A4 can be 4, 4.5, 5, 5.5 or 6.
Preferably, the extractant may be n-hexane, and the redissolution may be a 40-60% aqueous methanol solution. For example, the percentage of methanol in the reconstituted solution may be 40%, 45%, 50%, 55%, or 60%.
The embodiment of the invention adopts an internal standard method to detect the content of the chlormezanone in the blood sample, can use the pretreatment mode, has simple operation, can avoid errors caused by operation, and can quantify more accurately. In addition, the pretreatment method is simple, so that the method is beneficial to popularization and application of the embodiment of the invention.
In one embodiment of the invention, the dosage of the standard working solution is 5 muL, the dosage of the internal standard working solution is 5 muL, and the dosage of the blank blood sample is 90-100 muL, and the three are mixed for sample pretreatment. For example, a blank blood sample may be used in an amount of 90 μ L, 95 μ L, or 100 μ L.
In one embodiment of the invention, the dosage of the internal standard working solution is 5 muL, the dosage of the blood sample is 90-110 muL, and the internal standard working solution and the blood sample are mixed and then used for sample pretreatment. For example, the amount of blood sample may be 90 μ L, 95 μ L, 100 μ L, or 110 μ L.
In one embodiment of the present invention, the detection condition includes: phenomenex kinetex EVO C18 chromatographic column, the length of chromatographic column is 150mm, internal diameter is 2.1mm, and packing particle size is 5 mu m.
In one embodiment of the present invention, the detection condition includes: the column temperature is 35-45 deg.C, the mobile phase is water and acetonitrile, the flow rate is 0.2-0.4mL/min, the sample amount is 5-15 μ L, and the analysis time is 7-9 min.
For example, the column temperature can be 35, 37, 40, 42, or 45; the flow rate can take the value of 0.2, 0.25, 0.3, 0.35 or 0.4; the value of the sample volume can be 5, 7, 10, 12 or 15; the value of the analysis time may be 7, 7.5, 8, 8.5 or 9.
In the embodiment of the invention, based on the detection conditions, the analysis time can be about 8min, the analysis time is short, and the analysis efficiency is improved. In addition, the mobile phase in the embodiment of the invention is water and acetonitrile, and the liquid phase is simple and is beneficial to popularization and application.
In one embodiment of the invention, the volume ratio of water to acetonitrile is 74-76% during the target analysis phase of the total analysis time: 26 to 24 percent. For example, the volume ratio of water to acetonitrile may be 74:26, 74.5:25.5, 75:25, 75.5:24.5, or 76: 24.
In detail, the target to be analyzed includes chlormezanone and an internal standard.
Based on the above, in one embodiment of the present invention, the detection condition includes: single column dual pump elution mode;
the double-pump switching mode is as follows: switching from the analysis pump to the cleaning pump at 5.5min, and switching from the cleaning pump to the analysis pump at 6.5 min;
the elution mode is as follows: the volume ratio of water to acetonitrile is 74-76% in other elution time ranges except 5.5-6.5 min: 26 to 24 percent.
In the embodiment of the invention, the time period of 5.5-6.5min is the time period for cleaning the chromatographic column by cleaning the mobile phase of the pump, and the time period of 5.5min and 6.5min is the time period for analyzing the mobile phase of the pump to enter the chromatographic column for analyzing the target substance. Thus, for the analytical pump mobile phase, the volume ratio of water to acetonitrile is 74-76%: 26 to 24 percent. In one embodiment of the invention, the volume ratio of water to acetonitrile may be 30:70 for a clean pump mobile phase.
Compared with the single-pump gradient elution mode, the single-column double-pump elution mode can reduce the analysis time to the maximum extent under the condition of a single column. At the same time, the amount of organic reagent used can be reduced to some extent.
Therefore, the embodiment of the invention can shorten the analysis time, improve the detection flux and reduce the use amount of the organic solvent.
In one embodiment of the invention, the ultraviolet detector in the high performance liquid chromatograph is a DAD-3000 detector, the detection wavelength is 225-235nm, the acquisition frequency is 5Hz, and the bandwidth is 4 nm. For example, the detection wavelength may take on the value 225, 230, or 235.
In one embodiment of the present invention, the in-line filter used in the HPLC is SSI COLPRE-FILTER WATER 1/160.5M.
In one embodiment of the invention, the internal standard is pirfenidone.
In detail, pirfenidone is a drug for treating idiopathic pulmonary fibrosis. Compared with the use of psychotropic drugs with similar efficacies as internal standards, the pirfenidone used as the internal standard has great advantages, and the mutual interference brought to analysis by drug combination can be avoided. In addition, idiopathic pulmonary fibrosis is a rare disease with relatively low incidence, further avoiding the possibility of drug combination.
In addition, based on the detection conditions, the pirfenidone can peak before the chlormezanone under the analysis conditions, and the interference of impurities in the matrix is avoided, so that the analysis time can be effectively shortened.
Based on the above, in one embodiment of the present invention, the concentration of the chlormezanone standard substance in the standard working solution is 10-640 μ g/mL, and the diluent is 50-80% methanol aqueous solution;
the concentration of the pirfenidone standard substance in the internal standard working solution is 20-30 mug/mL, and the diluent is 40-60% methanol water solution.
In detail, a linear range can be set by combining the detected population, the dosage of blood to be detected and the approximate content range of the chlormezanone in the human body, so as to ensure that most of the detection results of the clinical samples fall within a reportable range.
Based on the above, in one embodiment of the present invention, the concentration of the clomazadone standard in the at least three standard working solutions is at least three of 10 μ g/mL, 20 μ g/mL, 40 μ g/mL, 80 μ g/mL, 160 μ g/mL, 320 μ g/mL, and 640 μ g/mL.
Preferably, the number of standard solutions is 7.
In summary, the method for detecting the content of clomezanone in blood provided by the embodiment of the invention combines the internal standard method and the high performance liquid chromatography, so that the interference factors are greatly reduced, and the method has the advantages of accurate quantification, good reproducibility, strong specificity, high sensitivity, more accurate detection result, low cost and short analysis time, and is beneficial to the detection of large-flux blood samples.
The embodiment of the invention can detect the content of the chlormezanone in blood, so that the detection method provided by the embodiment of the invention can be used for monitoring the content of the chlormezanone in the body of a patient in clinical treatment, and provides an experimental basis for personalized administration of the chlormezanone and reduction of toxic and side reactions, thereby being more beneficial to guiding the administration of the patient.
The present invention will be described in detail below by way of examples, but the present invention is not limited to the following examples.
Example 1
The embodiment of the invention is used for obtaining the standard curve equation.
1.1 preparation of Standard stock solutions
Standard stock solutions: 10mg of the clomazalone standard substance is accurately weighed and placed in a 10mL volumetric flask, dissolved by methanol and dissolved in 10mL to obtain a standard stock solution.
1.2 preparation of stock solutions for internal standards
Internal standard stock solution: accurately weighing 5mg of pirfenidone standard in a 5mL volumetric flask, dissolving with methanol, and fixing the volume to 5mL to obtain an internal standard stock solution.
1.3 the detection instrument Saimeishafi U3000 high performance liquid chromatograph.
1.4 detection conditions
1.4.1 chromatography columns
Phenomenex Kinetex EVO C18A chromatographic column, the length of the chromatographic column is 150mm, the inner diameter is 2.1mm, and the particle size of the filler is 5 μm.
1.4.2 mobile phase water and acetonitrile.
1.4.3 elution mode
Single column dual pump elution mode. The dual pump switching scheme is shown in table 2 below.
The volume ratio of water to acetonitrile is 75:25 within 0-5.5min and 6.5-8 min; the volume ratio of water to acetonitrile is 30:70 in 5.5-6.5 min.
TABLE 2
| Serial number | Time/min | ValveLeft |
| 1 | { initial time } | 10_1 |
| 2 | 5.500 | 1_2 |
| 3 | 6.500 | 10_1 |
In table 2, 10_1 identifies the analysis pump, and 1_2 identifies the switch from the analysis pump to the cleaning pump.
The control of the cleaning pump is shown in table 3 below.
TABLE 3
The control of the analysis pump is shown in table 4 below.
TABLE 4
1.4.4 other
The analysis time is 8 min; the column temperature was 40 ℃; the sample injection amount is 10 mu L; the flow rate was 0.3 mL/min.
1.4.5 Detector
The ultraviolet detector is a DAD-3000 detector, the detection wavelength is 230nm, the acquisition frequency is 5Hz, and the bandwidth is 4 nm.
1.4.6 in-line Filter
The in-line filter is SSI COL PRE-FILTER WATER 1/160.5M.
1.5 preparation of Standard working solution
Standard working solution: taking a proper amount of standard stock solution, diluting with 70% methanol aqueous solution to obtain a primary diluent of which the concentration of the chlormezanone is 640 mu g/mL, then continuously diluting with 70% methanol aqueous solution to prepare standard working solutions of which the concentrations of the chlormezanone standard substance are respectively 10 mu g/mL, 20 mu g/mL, 40 mu g/mL, 80 mu g/mL, 160 mu g/mL, 320 mu g/mL and 640 mu g/mL, and storing at-80 ℃.
It can be seen that the concentration of the chlormezanone standard substance in the seven standard working solutions is different and is increased sequentially.
1.6 preparation of internal standard working solution
Internal standard working solution: diluting a proper amount of internal standard stock solution by using 50% methanol aqueous solution to obtain internal standard working solution with the concentration of pirfenidone being 25 mug/mL, and storing the internal standard working solution at the temperature of-80 ℃.
1.7 preparation of Standard solution
Respectively transferring 5 mu L of standard working solution, 5 mu L of internal standard working solution and 95 mu L of blank serum or plasma by using a pipette, respectively placing the standard working solution, the internal standard working solution and the blank serum or plasma in 1.5mL of centrifuge tubes, uniformly mixing the standard working solution and the internal standard working solution in a vortex manner at the rotating speed of 2800rpm for 1min, adding 1000 mu L of N-hexane, uniformly mixing the N-hexane in a vortex manner at the rotating speed of 2800rpm for 5min, then centrifuging the N-hexane in a high speed at the rotating speed of 14000rpm for 5min, transferring 900 mu L of supernatant into another 1.5mL of centrifuge tube, slowly blowing the supernatant to dry at normal temperature by using N2, adding 100 mu L of 50% methanol aqueous solution (methanol: water is 50:50) into the blow-dried centrifuge tube, then, uniformly mixing the mixture in a vortex manner at the rotating speed of 2800.
Thus, seven standard solutions can be obtained for seven standard working solutions.
1.8 detecting the standard solution to generate a standard curve equation
After obtaining each standard solution, the high performance liquid chromatograph can be used for respectively detecting the seven standard solutions, and the chromatogram of each standard solution is correspondingly obtained.
Referring to fig. 3, fig. 3 shows chromatograms of a chlormezanone standard and an internal standard (i.e., pirfenidone standard) in a standard solution.
From the chromatogram of the standard solution, the chromatographic peak area of the chlormezanone standard substance and the chromatographic peak area of the pirfenidone standard substance can be obtained, and then the known concentrations of the chlormezanone standard substance and the pirfenidone standard substance in each standard solution are combined, so that the standard curve equation of the chlormezanone can be obtained.
Referring to fig. 5, fig. 5 shows the linear relationship diagram of the obtained chlormezanone, and according to the linear relationship diagram, the standard curve equation of the chlormezanone can be obtained: 27.9082 XX +0.1673, coefficient of correlation R20.99903, Y is the concentration ratio of the chlormezanone to the internal standard, and X is the area ratio of the chlormezanone to the internal standard.
It can be seen that the correlation coefficient R2 of the chlormezanone is greater than 0.9900 within the linear range of 0.5-32 mug/mL, which shows that the linear relation is good, and the accuracy is high and the error is small when the content of the chlormezanone in the blood sample is calculated based on the standard curve equation.
And after a standard curve equation is obtained, pretreating the blood sample to obtain a sample to be detected, detecting the sample to be detected under the same detection condition, and combining the obtained standard curve equation to obtain the content of the chlormezanone in the blood sample.
Example 2
The embodiment of the invention is used for detecting the content of the chlormezanone in venous blood.
2.1 obtaining blood samples
The blood sample is obtained by treating at least 2mL of blood to be tested. After the blood sample is obtained, pretreatment can be carried out to obtain a corresponding sample to be detected which can be directly loaded.
2.2 blood sample pretreatment
Transferring 5 mu L of internal standard working solution into a 1.5mL centrifuge tube by using a liquid transfer gun, then adding 100 mu L of blood sample, uniformly mixing in a vortex manner at the rotating speed of 2800rpm for 1min, adding 1000 mu L of n-hexane, uniformly mixing in a vortex manner at the rotating speed of 2800rpm for 5min, and then centrifuging at a high speed at the rotating speed of 14000rpm for 5 min; putting 900 mu L of supernatant into another 1.5mL centrifuge tube, and slowly drying by N2 at normal temperature; adding 100 mu L of 50% methanol water solution (methanol: water is 50:50) into a blow-dried centrifugal tube, uniformly mixing for 3min at the rotating speed of 2800rpm in a vortex manner, and then centrifuging for 5min at the rotating speed of 14000rpm at a high speed to obtain supernatant, namely the sample to be detected.
2.3 detection of samples to be tested
Under the detection conditions of example 1, the same high performance liquid chromatograph is used to detect the sample to be detected, and the chromatogram of the sample to be detected is obtained.
Referring to fig. 4, fig. 4 shows chromatograms of clomazadone and an internal standard (i.e., pirfenidone standard) in a sample to be tested.
Referring to fig. 3 and 4, the retention time of the chlormezanone in the sample to be detected is consistent with that of the chlormezanone standard in the standard solution, and pirfenidone is used as the internal standard substance, so that the identification of the target compound is more accurate, the analysis time is short, the interference is small, the internal standard is appropriate in quantification, the specificity is strong, and the accuracy and the sensitivity are high.
In addition, as mentioned above, in order to avoid the fixed value deviation caused by the inconsistent extraction rate of the chlormezanone and the internal standard, the standard working solution and the blood sample are subjected to the same pretreatment process, so that after the same pretreatment, chromatograms of the standard solution and the sample to be detected generally only have chromatographic peaks of the chlormezanone and the internal standard, solvent peaks at the elution stage of the chromatographic column are also basically consistent, and no other obvious impurities are found. Based on this, it is also demonstrated that the above pretreatment method is reasonable and that other substances are not introduced into the substrate.
2.4 calculation of the content of Chlormetazalone in the sample to be tested
Substituting the chromatographic peak areas of the chlormezanone and the internal standard substance in the chromatogram of the sample to be detected and the known concentration of the internal standard substance in the sample to be detected into the standard curve equation to calculate the content of the chlormezanone in the sample to be detected.
Example 3
The embodiment of the invention is used for determining the quantitative limit and the detection limit.
The blood sample dilutions were prepared by selecting standard solutions of appropriate concentrations and diluting the blank blood to different degrees, and were measured according to the blood sample pretreatment method and measurement conditions in example 2. The detection and quantification limits of the chlormezanone are shown as follows:
(1) limit of detection (LOD): 0.1. mu.g/mL.
(2) Limit of quantitation (LOQ): 0.15. mu.g/mL.
The embodiment shows that the detection limit of the chlormezanone can be as low as 0.1 mu g/mL, the quantification limit can be as low as 0.15 mu g/mL, the sensitivity is high, the parallelism of low-concentration samples can be improved, and the accuracy is enhanced.
Due to the high sensitivity, the requirement of the sample volume of the blood to be detected in the embodiment of the invention can be wider, so that the overall accuracy of sample detection is improved. In addition, the embodiment of the invention can accurately quantify the biological sample with low content of the chlormezanone, and ensures the high accuracy and wide applicability of the detection method.
Example 4
The embodiment of the invention is used for measuring the recovery rate and the precision.
Taking standard working solution, preparing into high, medium and low 3 concentrations, carrying out sample adding recovery rate and precision experiments, measuring according to the detection method in the embodiment 2, analyzing and measuring 3 batches, wherein the recovery rate and precision of the chlormezanone are shown in Table 5.
TABLE 5
| Adding quantity of scalar | 1μg/mL | 4μg/mL | 16μg/mL |
| Average recoveryRate of change | 98.72％ | 101.43％ | 103.72％ |
| Precision RSD | 1.56％ | 0.72％ | 0.93％ |
It can be seen that the mean recovery rate of the chlormezanone is 98.72-103.72% in the range of 3 addition levels of low, medium and high, the reproducibility is good, the sample-adding recovery rate is good, the precision is 0.72-1.56%, the accuracy of the detection result is high, and the system error can be eliminated.
Comparative example 1
The gas chromatography for measuring the chlormezanone in serum has the publication numbers as follows: the China labor health occupational disease journal, Vol.5, 5, 2012, p.391-392.
Comparative example 2
The clinical significance of gas chromatography-mass spectrometer detection of blood chlorin-mezanone of poisoned patients is as follows: journal of Chinese medicine, vol.46, No. 6, p.504-505, 6.2007.
Detection method
Comparative example 1 used gas chromatography in combination with an external standard method, comparative example 2 used gas chromatography tandem mass spectrometry in combination with an external standard method, and the present example used liquid chromatography in combination with an internal standard method.
Compared with the comparative example 1, the embodiment of the invention can avoid the fixed value deviation caused by misoperation in the pretreatment process, and has high detection accuracy.
Compared with the comparative example 2, the embodiment of the invention can avoid the fixed value deviation caused by misoperation in the pretreatment process, has high detection accuracy, low analysis cost and lower requirement on the site, basically does not need professional operation, and is beneficial to wide development.
Second, sample pretreatment
Comparative example 1 venous blood was taken at 3mL to prepare a sample to be tested. The blood dosage of the embodiment of the invention can be as low as 2 mL.
Compared with comparative example 1, the blood dosage required by the embodiment of the invention is lower, the blood sampling amount of a patient is reduced, and the compliance of the patient is improved.
Third, analysis time
In comparative example 1, the chlormezanone peaks at 8.269-8.478 min, and the analysis time is 15 min. In the embodiment of the invention, the retention time of the internal standard substance pirfenidone is about 3.1min, the retention time of the clomezanone is about 4.6min, and the analysis time is 8 min.
In the total particle flow chart of the blood test of the patient with the clomezanone poisoning in the comparative example 2, the retention time of the clomezanone is 9.704min, while in the present invention example, the retention time of the internal standard substance pirfenidone is about 3.1min, the retention time of the clomezanone is about 4.6min, and the analysis time is 8 min.
Therefore, compared with the comparative examples 1 and 2, the analysis time of the embodiment of the invention is obviously shortened, the analysis efficiency is greatly improved, and the detection of the large-flux blood sample is facilitated.
Fourthly, detection limit, quantitative limit, recovery rate and precision
In comparative example 1, the chlormezanone is linear in the linear range of 5.0-100 mug/mL, the lowest detection concentration is 2.0 mug/mL, the recovery rate of the method is 82.5-101.3%, and the Relative Standard Deviation (RSD) is 3.5-4.91%.
In comparative example 2, the process recoveries were 84.7% and 82.1%, corresponding to Relative Standard Deviations (RSD) of 6.1% and 6.6%.
In the embodiment of the invention, the chlormezanone is linear in the linear range of 0.5-32 mug/mL, the detection limit is 0.1 mug/mL, the quantification limit is 0.15 mug/mL, the average recovery rate is 98.72-103.72%, and the precision (RSD) is 0.72-1.56%.
Compared with the comparative example 1, the detection limit, the quantification limit, the recovery rate and the precision of the embodiment of the invention are all superior to those of the comparative example 1. The detection limit and the quantification limit of the embodiment of the invention are both lower, the sensitivity of the detection method is higher, and the recovery rate and the precision are better, so that the requirements on the sample amount can be wider, and the overall accuracy of the sample detection is higher.
In addition, the recovery rate and the precision of the embodiment of the invention are better than those of the comparative example 2.
In summary, compared with comparative example 1 and comparative example 2, the method for detecting the content of the chlormezanone in the blood provided by the embodiment of the invention has the advantages of short analysis time, high detection sensitivity, good recovery rate and precision, high detection accuracy, low detection cost and the like, so the method has more advantages in the field of clinical detection.
Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (10)
1. The method for detecting the content of the chlormezanone in the blood is characterized by comprising the following steps:
preparing at least three standard working solutions, wherein the standard working solutions contain chlormezanone standard substances with known concentrations, and the chlormezanone standard substances in different standard working solutions have different concentrations;
mixing a certain amount of standard working solution, internal standard working solution and a blank blood sample, and performing sample pretreatment to obtain standard solution, wherein the internal standard working solution contains an internal standard substance with known concentration;
respectively detecting at least three standard solutions by using a high performance liquid chromatograph under certain detection conditions to obtain chromatograms of the standard solutions;
fitting to obtain a standard curve equation of the chlormezanone according to the chromatogram of each standard solution;
mixing a certain amount of internal standard working solution with a blood sample, and performing the same sample pretreatment to obtain a sample to be detected, wherein the blood sample is obtained by treating at least 2mL of blood to be detected;
detecting a sample to be detected by using a high performance liquid chromatograph under the same detection condition to obtain a chromatogram of the sample to be detected;
and calculating the content of the chlormezanone in the blood sample according to the chromatogram of the sample to be detected and the standard curve equation of the chlormezanone.
2. The method of claim 1,
the sample pretreatment comprises the following steps A1-A4:
a1: the mixed solution is evenly mixed for 1-2min in a vortex mode at the rotating speed of 2500-3000 rpm;
a2: adding 800-;
a3: transferring 700-1000 mu L of supernatant into a centrifuge tube, and drying by using nitrogen at normal temperature;
a4: adding 80-120 μ L of the complex solution into the centrifuge tube blown dry by the upper clear air, uniformly mixing the complex solution in a vortex manner at the rotating speed of 2500 plus 3000rpm for 2-4min, and then centrifuging the complex solution at the rotating speed of 10000 plus 15000rpm for 4-6min to obtain supernatant.
3. The method of claim 1,
the detection conditions include: phenomenex kinetex EVO C18 chromatographic column, the length of chromatographic column is 150mm, internal diameter is 2.1mm, and packing particle size is 5 mu m.
4. The method of claim 1,
the detection conditions include: the column temperature is 35-45 deg.C, the mobile phase is water and acetonitrile, the flow rate is 0.2-0.4mL/min, the sample amount is 5-15 μ L, and the analysis time is 7-9 min.
5. The method of claim 4,
the detection conditions include: single column dual pump elution mode;
the double-pump switching mode is as follows: switching from the analysis pump to the cleaning pump at 5.5min, and switching from the cleaning pump to the analysis pump at 6.5 min;
the elution mode is as follows: the volume ratio of water to acetonitrile is 74-76% in other elution time ranges except 5.5-6.5 min: 26 to 24 percent.
6. The method of claim 1,
the ultraviolet detector in the high performance liquid chromatograph is a DAD-3000 detector, the detection wavelength is 225-235nm, the acquisition frequency is 5Hz, and the bandwidth is 4 nm.
7. The method of claim 1,
the on-line filter used by the high performance liquid chromatograph is SSI COL PRE-FILTER WATER 1/160.5M.
8. The method according to any one of claims 1 to 7,
the internal standard substance is pirfenidone.
9. The method of claim 8,
the concentration of the chlormezanone standard substance in the standard working solution is 10-640 mug/mL, and the diluent is 50-80% methanol water solution;
the concentration of the pirfenidone standard substance in the internal standard working solution is 20-30 mug/mL, and the diluent is 40-60% methanol water solution.
10. The method of claim 9,
the concentration of the standard substance of the chlormezanone in the at least three standard working solutions is at least three of 10 mu g/mL, 20 mu g/mL, 40 mu g/mL, 80 mu g/mL, 160 mu g/mL, 320 mu g/mL and 640 mu g/mL.
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| CN105158358A (en) * | 2015-08-18 | 2015-12-16 | 江苏出入境检验检疫局动植物与食品检测中心 | Method for detecting illegally-added 42 chemicals in Chinese patent medicines and health care products simultaneously |
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