CN104133030A - Method for simultaneous determination of contents of 6 kinds of sedative-hypnotic drugs in serum by UHPLC-CAD technology - Google Patents
Method for simultaneous determination of contents of 6 kinds of sedative-hypnotic drugs in serum by UHPLC-CAD technology Download PDFInfo
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Abstract
The invention relates to a method for simultaneous determination of the contents of 6 kinds of sedative-hypnotic drugs in serum by a UHPLC-CAD combined technology, wherein the 6 kinds of sedative-hypnotic drugs are diazepam, lorazepam, alprazolam, estazolam, clonazepam and zolpidem tartrate. The method is characterized by including the following steps: 1) reference substance preparation, 2) internal standard solution preparation, 3) sample solution preparation, 4) mobile phase solution preparation, 5) chromatographic condition setting, 6) electrospray detector condition optimization, 7) sample determination, 8) gradient-concentration reference substance solution preparation, 9) standard curve preparation, and 10) data calculation and analysis. The method is advanced, has good clinical application prospects and effects, can rationally use drugs for clinic, increases drug curative effects, lowers toxic and side effects and drug costs, and plays a positive role.
Description
Technical field
The present invention relates to a kind of drug world that belongs to, relate in particular to a kind of employing high efficiency liquid phase-electron spray and detect coupling technique, measure the detection method of 6 kinds of tranquilizing soporific class medicines in serum simultaneously.
Background technology
Modern society's life is due to work dog-eat-dog, rhythm of life is nervous, and extraneous various stimuluss affect people's mood, produces insomnia, anxiety, and then develop into various psychology and the physiological problems such as anxiety, depression, all cause huge pressure to family and society.An external investigation shows that the adult of 50%-60% suffers from sleep-disorder in various degree, and the incidence of disease of China's insomnia is up to 40%.Barbiturate is as 1st generation hypnotic sedative agent, owing to there being bad reaction such as breathings of inhibition grade, substantially by other, replaced about medicine in recent years.At present conventional calmness, hypnosis and anxiolytic drugs is benzodiazepine (2nd generation sedative hypnotic drug) clinically, and the Non-benzodiazepine medicine (the 3rd generation sedative hypnotic drug) of a new generation, all can cause the inhibition of central nervous system different parts, along with the increasing of consumption, clinical manifestation can even be gone into a coma to hypnosis from slight calmness.In benzodiazepine, represent that medicine mainly contains diazepam, alprazolam, Lorazepam etc., its main mechanism be by with central nervous system in main inhibition acceptor GABA
aγ-subunit combination in (γ-aminobutyric acid A) acceptor complex, activation GABA
aacceptor, promotes GABA (γ-aminobutyric acid) and GABA
athe combination of acceptor, makes Cl
-channel opener frequency increases, and induces more Cl
-interior stream, strengthens GABA to neural effect.In the 3rd generation Non-benzodiazepine medicine, represent that medicine mainly contains Zolpidemtar Trate, Zaleplon, zopiclone etc., wherein the mechanism of action of Zolpidemtar Trate is for optionally acting on the BZ in Benzodiazepine set site
1(ω
1) hypotype, promote GABA to GABA
areceptor affinity, causes Cl
-channel opener.Said medicine is generally used for the treatment of sleep-disorder, but at major depressive disorder, the relevant phrenoblabia of culture, in the symptomatic treatment of the mental illness such as obsession and schizophrenia, be also widely used, with antidepressant drug, anti-schizophrenia medicine and other antipsychotics use in conjunction, the mental symptom determined curative effects such as, anxiety manic to alleviating, vain hope, significantly improve comprehensive therapeutic effect.
Yet, tranquilizing soporific class medicine is often pressed drug half-life successive administration or according to the administration of clinician's experience clinically, due to conditions of patients degree difference and individual difference, beginning onset time of medicine and to reach in vivo the time difference of stable state larger, often cause the excessive use of medicine.Moreover due to this type of patient's singularity, compliance is poor, in drug use, often occur timing taking excessive or subtracting voluntarily the phenomenons such as medicine, directly affect result for the treatment of, and easily cause abstinence syndrome, even may there is serious complication.Therefore the reasonable use of tranquilizing soporific class medicine is and clinical efficacy and the closely-related key factor of patient safety, if can accomplish the fast detecting of blood concentration, and carry out Real-Time Monitoring, during treatment, can assign regimen according to individual patients feature, may adopt minimum effective dose, reach the object of performance curative effect, avoid the excessive use of psychotropic agent.
Content method for tranquilizing soporific class medicine mainly contains the analytical approachs such as high performance liquid chromatography-UV-detector (HPLC-UV), High Performance Liquid Chromatography/Photodiode Array Detection (HPLC-DAD), gas chromatography (GC), mass spectrum (MS) at present.GC and GC-MS have advantage to detecting low boiling sample, not best to the detection of benzodiazepine, and instrument is expensive, and maintenance cost is high, and popularity is inadequate, and generalization is poor; HPLC-UV and HPLC-DAD are as the document of detection means, and the differences that adopt detect wavelength more, carry out respectively analyzing and testing, for the patient who takes two or more tranquilizing soporific class medicine, the figure spectrum information that cannot provide a survey to comment simultaneously more.
Electron spray detecting device (CAD) is current general detecting device, during with HPLC coupling, the effect of HPLC eluent nitrogen in atomizer and atomization, wherein larger drop flows out through sewer pipe under the effect of impinger, less solute (analyte) drop is at room temperature dry, forms particles of solute.Meanwhile, (containing High Voltage Pt spun gold electrode) forms positively charged nitrogen particle through corona-type device for the nitrogen of carrier gas, to shunt the second strand of nitrogen stream forming, and while oppositely meeting with particles of solute, through colliding, particles of solute become positively charged.It does not rely on the structure of analyte, do not need analyte to ionize yet, as long as this material belongs to half volatile or non-volatile compounds just can be detected, and sensitivity can reach pg rank, reappearance is good, is that the strong of the conventional detecting devices such as ultraviolet (UV), mass spectrum (MS), differential refraction (RI) and evaporative light-scattering (ELSD) supplements.This project adopts the detection technique of UHPLC-CAD, analyzes the more conventional liquid phase of pressure and increases to some extent, and obviously shortened analysis time, has improved analysis efficiency; The lower inferior position of response when simultaneously detecting device can overcome DAD detecting device and detects for trace constituent, can solve again the problem that the medicine of different uv absorption wavelength cannot disposable detection, obtain the assay result of low detectability, realize multiple such medicine disposable, measure fast, and this technology is compared to analytical technologies such as GC, HPLC-MS, easy and simple to handle, maintenance and use cost are lower.
Therefore, the detection for multiple tranquilizing soporific class medicine that the present invention sets up, analysis time is short, highly sensitive, favorable reproducibility, can realize the detection method that a survey is commented more, can provide detection method for clinical medicine concentration detects, reach and improve drug use efficiency, reduce the object of toxicity.By literature search, adopt at present UHPLC-CAD method measure in serum 6 kinds of tranquilizing soporific class medicines (comprise 2 generations and 3 generation medicine) method of content, and patients in psychiatric department blood medicine monitoring and measuring application, has no bibliographical information and patent open.
Summary of the invention
The object of the invention is to set up a kind of detection method of simultaneously measuring 6 kinds of benzodiazepine content in serum, for clinical medicine concentration monitor provides scheme.
To achieve these goals, the present invention has set up a kind of application high liquid chromatography-electron spray detecting device coupling technique (UHPLC-CAD), measures the method for 6 kinds of benzodiazepine content of serum simultaneously, and the method comprises the following steps:
1) preparation of reference substance
Precision takes diazepam, Lorazepam, alprazolam, estazolam, the western reference substance of chlorine nitre, is placed in respectively volumetric flask, adds acetonitrile to dissolve, be made into the reference substance solution of required mass concentration, standby, precision takes Zolpidemtar Trate reference substance, is placed in volumetric flask, add pure water to dissolve, be made into the reference substance solution of required mass concentration, standby, precision measures 6 kinds of reference substance solution, be made into the mixing reference substance solution of required mass concentration, standby;
2) preparation of inner mark solution
Precision takes colchicin reference substance, is placed in volumetric flask, adds acetonitrile to dissolve, and is made into the inner mark solution of required mass concentration, standby;
3) sample solution preparation
Precision measure sleep-disorder patient take medicine after serum, be placed in tool plug centrifuge tube, quantitatively add step 2) colchicin inner mark solution, vortex mixes, after high speed centrifugation, separation of supernatant; In supernatant, be added to the SPE post after the activation of first alcohol and water, after loading, use water wash, the methanol-eluted fractions of debita spissitudo, eluent volatilizes, and with acetonitrile, dissolves, and obtains sample solution, standby;
4) preparation of mobile phase solution
Get a certain amount of trifluoroacetic acid aqueous solution, as mobile phase A phase, standby; Get a certain amount of pure water, as Mobile phase B phase, standby;
5) setting of chromatographic condition
Selection be take octadecylsilane chemically bonded silica as filling agent, and specification is 4.6 * 250mm, and filler internal diameter is the chromatographic column of 5 μ m; Column temperature is 30 ℃, and flow rate of mobile phase is 0.8mL/min; Get reference substance solution and the step 2 of step 1)) inner mark solution, quantitative sample injection, carries out gradient elution; Adopt electron spray detecting device to carry out chromatography peak detection;
6) optimization of electron spray detecting device condition
Select electron spray detecting device, nitrogen pressure is 35psi, range 100pA, 30 ℃ of atomization temperatures;
Get step 1) reference substance solution, step 2) inner mark solution, quantitative sample injection, and obtain thus the chromatogram of reference substance and internal standard compound;
7) sample determination
Get the sample solution of step 3), difference quantitative sample injection, under the condition of step 5) and step 6), measure, obtain respectively diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, Zolpidemtar Trate reference substance, the chromatogram of colchicin internal standard compound and sample, in sample collection of illustrative plates, with diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, Zolpidemtar Trate reference substance and the identical retention time of colchicin internal standard compound place, for the diazepam to be measured in blood serum sample, Lorazepam, alprazolam, estazolam, Clonazepam, Zolpidemtar Trate, and the colchicin internal standard compound quantitatively adding,
8) preparation of gradient concentration reference substance solution
Accurate diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, the Zolpidemtar Trate reference substance solution of drawing step 1), is mixed with mixing reference substance solution, adds acetonitrile, is diluted to the mixing reference substance solution of gradient concentration, standby;
9) typical curve preparation
Many parts, the blank tissue fluid of patient getting sleep-disorder, quantitatively adds respectively the gradient concentration of step 8) to mix reference substance solution, according to the operation of step 3) method, obtains gradient mark-on sample solution, under the condition of step 7), measures, with diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, the ratio of Zolpidemtar Trate peak area and internal standard compound peak area is to diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, the concentration at Zolpidemtar Trate peak is carried out linear regression, obtain regression equation, the typical curve of diazepam: y=0.0635x-0.5842 (r=0.9940), the typical curve of Lorazepam: y=0.0758x-0.5796 (r=0.9968), the typical curve of alprazolam: y=0.0813x-0.6502 (r=0.9946), the typical curve of estazolam: y=0.0666x-0.5455 (r=0.9939), the typical curve of Clonazepam: y=0.0622x-0.4402 (r=0.9972), the typical curve of Zolpidemtar Trate: y=0.1428x-0.6019 (r=0.9963).Wherein y is the ratio of measured object peak area and internal standard compound peak area, and x is measured object quality concentration;
10) data analysis is calculated
Record the peak area of diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, Zolpidemtar Trate and colchicin, calculate the ratio of 6 kinds of medicines and interior mark colchicin peak area, distinguish substitution typical curve separately, calculate the mass concentration of 6 kinds of medicines; Be the concentration of serum Chinese traditional medicine.
As further improvement, in above-mentioned step 5), eluent gradient elution program is as follows: 0-7.00min, 45%A phase and 55%B phase; 7.01min-15.00min, 70%A phase and 30%B phase; 15.01min-20.00min, 45%A phase and 55%B phase.
As further improvement, it is 183.16 μ g/mL that above-mentioned step 8) is quantitatively diluted to concentration gradient, 91.58 μ g/mL, 45.79 μ g/mL, 18.32 μ g/mL, 9.16 μ g/mL, 1.83 the diazepam reference substance solution of μ g/mL, 177.44 μ g/mL, 88.72 μ g/mL, 44.36 μ g/mL, 17.74 μ g/mL, 8.72 μ g/mL, 1.77 the estazolam reference substance solution of μ g/mL, 173.56 μ g/mL, 86.78 μ g/mL, 43.39 μ g/mL, 17.36 μ g/mL, 8.68 μ g/mL, 1.74 the Lorazepam reference substance solution of μ g/mL, 190.04 μ g/mL, 95.02 μ g/mL, 47.51 μ g/mL, 19.00 μ g/mL, 9.52 μ g/mL, 1.81 the Clonazepam reference substance solution of μ g/mL, 174.40 μ g/mL, 87.20 μ g/mL, 43.60 μ g/mL, 17.44 μ g/mL, 8.72 μ g/mL, the alprazolam reference substance solution of 1.74 μ g/mL, and 100.00 μ g/mL, 50.00 μ g/mL, 25.00 μ g/mL, 10.00 μ g/mL, 5.00 μ g/mL, the Zolpidemtar Trate reference substance solution of 1.00 μ g/mL.
As further improvement, in above-mentioned step 9), get 6 parts of the blank serum of patient of 1.0mL sleep-disorder, number 1～No. 6, add 183.16 μ g/mL diazepams for No. 1,177.44 μ g/mL estazolams, 173.56 μ g/mL Lorazepams, 190.04 μ g/mL Clonazepams, 190.04 μ g/mL alprazolams, each 10 μ L of 100.00 μ g/mL Zolpidemtar Trate reference substance solution; Add 91.58 μ g/mL for No. 2,88.72 μ g/mL estazolams, 86.78 μ g/mL Lorazepams, 95.02 μ g/mL Clonazepams, 87.20 μ g/mL alprazolams, each 10 μ L of 50.00 μ g/mL Zolpidemtar Trate reference substance solution; Add 45.79 μ g/mL diazepams for No. 3,44.36 μ g/mL estazolams, 43.39 μ g/mL Lorazepams, 47.51 μ g/mL Clonazepams, 43.60 μ g/mL alprazolams, each 10 μ L of 25.00 μ g/mL Zolpidemtar Trate reference substance solution; Add 18.32 μ g/mL diazepams for No. 4,17.74 μ g/mL estazolams, 17.74 μ g/mL Lorazepams, 19.00 μ g/mL Clonazepams, 17.44 μ g/mL alprazolams, each 10 μ L of 10.00 μ g/mL Zolpidemtar Trate reference substance solution; Add 9.16 μ g/mL diazepams for No. 5,8.72 μ g/mL estazolams, 8.68 μ g/mL Lorazepams, 9.52 μ g/mL Clonazepams, 8.72 μ g/mL alprazolams, each 10 μ L of 8.72 μ g/mL Zolpidemtar Trate reference substance solution; Add 1.83 μ g/mL diazepams for No. 6,1.77 μ g/mL estazolams, 1.74 μ g/mL Lorazepams, 1.81 μ g/mL Clonazepams, 1.74 μ g/mL alprazolams, each 10 μ L of 1.00 μ g/mL Zolpidemtar Trate reference substance solution.
The present invention, owing to having adopted above-mentioned technical scheme and step, has following feature:
1, method is advanced
UHPLC-CAD coupling technique is current domestic more advanced analyzing and testing means, with the traditional coupling of liquid chromatography-UV-detector, gas chromatography, spectroscopic analysis methods ratio, analysis time, more conventional liquid phase significantly shortened, simultaneously owing to adopting all-purpose detector, when having realized the medicine of variety classes, different wave length, measure, there is the superiority that a survey is commented more.The present invention adopts this technology to measure the content of diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, Zolpidemtar Trate in sleep-disorder blood samples of patients first simultaneously, method advanced person is feasible, can provide experimental basis for the Simultaneous Determination application that this coupling technique is carried out polycompound.
2, potential applicability in clinical practice and function well
Detection of drug concentration, as the important means of clinical safety, the rational use of medicines, can provide vivo medicine concentration data.The method that this method is set up can provide for the monitor drug concentration of diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, Zolpidemtar Trate practicable method, potential applicability in clinical practice is good, it can be clinical rational drug use, improve curative effect of medication, lower toxic and side effect and consumption cost, bring into play positive effect.
Accompanying drawing explanation
Fig. 1 is for mixing reference substance chromatogram.
Fig. 2 is internal standard compound colchicin chromatogram.
Fig. 3 is clinical sample chromatogram.
Fig. 4 is diazepam canonical plotting.
Fig. 5 is Lorazepam canonical plotting.
Fig. 6 is Clonazepam canonical plotting.
Fig. 7 is estazolam canonical plotting.
Fig. 8 is alprazolam canonical plotting.
Fig. 9 is Zolpidemtar Trate canonical plotting.
Embodiment
The following specific embodiments of the present invention is to make a detailed explanation.
UHPLC-CAD coupling technique is measured the method for 6 kinds of tranquilizing soporific class medicament contgs in serum simultaneously, and the method comprises the following steps:
1) preparation of reference substance
Precision takes diazepam reference substance 45.79mg, Lorazepam reference substance 43.39mg, Clonazepam reference substance 47.51mg, estazolam reference substance 44.36mg, alprazolam reference substance 43.60mg, Zolpidemtar Trate reference substance 25.00 mg are placed in respectively 100mL volumetric flask, add acetonitrile to dissolve, quantitatively being made into respectively mass concentration is the diazepam reference substance solution of 0.4579mg/mL, the Lorazepam reference substance solution of 0.4339mg/mL, the Clonazepam reference substance solution of 0.4751mg/mL, the estazolam reference substance solution of 0.4436mg/mL, the alprazolam reference substance solution of 0.4360mg/mL and the Zolpidemtar Trate reference substance solution of 0.2500mg/mL, standby.
2) preparation of inner mark solution
Precision takes colchicin reference substance 13.37mg, is placed in 10mL volumetric flask, adds acetonitrile to dissolve, the above-mentioned solution 2ml of accurate absorption, is placed in 10mL volumetric flask, adds acetonitrile to dissolve, be made into mass concentration and be the colchicin inner mark solution of 26.74 μ g/mL, standby.
3) sample solution preparation
Through the ethics committee member of hospital, approve after examination, after patient knows the inside story and to inform, the relevant phrenoblabia of culture (phonism illusion, persecutory delusion) patient takes after Lorazepam, gets blood 3mL, and separation obtains serum, precision measures serum 1.0mL, be placed in tool plug centrifuge tube, quantitatively add step 2) colchicin inner mark solution 25uL, vortex mixes, after 13000r/min high speed centrifugation 15min, get supernatant 60 uL and cross SPE post; SPE post is first with methyl alcohol 3mL and water 3mL activation SPE post, coutroi velocity 3-5min/mL during activation, after loading, use the water wash of 1mL, use again 80% methyl alcohol 1mL wash-out, coutroi velocity 1-2mL/min during wash-out, the solution eluting, water-bath volatilizes solvent, with 50 μ L acetonitriles, dissolve, obtain sample solution, standby.
4) preparation of mobile phase solution
Precision measures trifluoroacetic acid aqueous solution, as mobile phase A phase, standby; Precision measures pure water, as Mobile phase B phase, standby.
5) setting of chromatographic condition
Selection be take octadecylsilane chemically bonded silica as filling agent, and specification is 4.6 * 250mm, and filler internal diameter is the chromatographic column of 5.0 μ m; Column temperature is 30 ℃, and flow rate of mobile phase is 0.8mL/min; Get reference substance solution and the step 2 of step 1)) inner mark solution, sample introduction 10 μ L, the eluent gradient elution program of optimization: 0-7.00min, 45%A phase and 55%B phase; 7.01min-15.00min, 70%A phase and 30%B phase; 15.01min-20.00min 45%A phase and 55%B phase, carry out gradient elution; Adopt electron spray detecting device to carry out chromatography peak detection.
6) optimization of electron spray detecting device condition
Select electron spray detecting device, nitrogen pressure is 35psi, range 100pA, 30 ℃ of atomization temperatures;
Get step 1) reference substance solution, step 2) inner mark solution, sample introduction 10 μ L respectively, obtain the standard colors spectrogram of reference substance and internal standard compound thus.
7) sample determination
Get the sample solution of step 3), difference sample introduction 20 μ L, under the chromatographic condition of step 5) and the testing conditions of step 6), measure, obtain respectively diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, Zolpidemtar Trate mixing reference substance, the chromatogram of colchicin internal standard compound and sample, in sample chromatogram, with diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, Zolpidemtar Trate reference substance and the identical retention time of colchicin internal standard compound place, for the medicine to be measured in blood serum sample, and the colchicin internal standard compound quantitatively adding, and each material chromatogram is consistent with reference substance chromatogram.
8) preparation of gradient concentration reference substance solution
Each accurate mixing reference substance solution of drawing step 1), is placed in volumetric flask, adds acetonitrile, and it is 183.16 μ g/mL that quantitative dilution becomes concentration gradient, 91.58 μ g/mL, 45.79 μ g/mL, 18.32 μ g/mL, 9.16 μ g/mL, the diazepam reference substance solution of 1.83 μ g/mL, 177.44 μ g/mL, 88.72 μ g/mL, 44.36 μ g/mL, 17.74 μ g/mL, 8.72 μ g/mL, the estazolam reference substance solution of 1.77 μ g/mL, 173.56 μ g/mL, 86.78 μ g/mL, 43.39 μ g/mL, 17.36 μ g/mL, 8.68 μ g/mL, the Lorazepam reference substance solution of 1.74 μ g/mL, 190.04 μ g/mL, 95.02 μ g/mL, 47.51 μ g/mL, 19.00 μ g/mL, 9.52 μ g/mL, the Clonazepam reference substance solution of 1.81 μ g/mL, 174.40 μ g/mL, 87.20 μ g/mL, 43.60 μ g/mL, 17.44 μ g/mL, 8.72 μ g/mL, the alprazolam reference substance solution of 1.74 μ g/mL, and 100.00 μ g/mL, 50.00 μ g/mL, 25.00 μ g/mL, 10.00 μ g/mL, 5.00 μ g/mL, the Zolpidemtar Trate reference substance solution of 1.00 μ g/mL, standby.
9) typical curve preparation
Through the ethics committee member of hospital, approve after examination, after patient knows the inside story and to inform, get 1.0mL sleep-disorder patient's blank serum, be divided into 6 parts, number 1～No. 6, add 183.16 μ g/mL diazepams for No. 1,177.44 μ g/mL estazolams, 173.56 μ g/mL Lorazepams, 190.04 μ g/mL Clonazepams, 190.04 μ g/mL alprazolams, each 10 μ L of 100.00 μ g/mL Zolpidemtar Trate reference substance solution; Add 91.58 μ g/mL for No. 2,88.72 μ g/mL estazolams, 86.78 μ g/mL Lorazepams, 95.02 μ g/mL Clonazepams, 87.20 μ g/mL alprazolams, each 10 μ L of 50.00 μ g/mL Zolpidemtar Trate reference substance solution; Add 45.79 μ g/mL diazepams for No. 3,44.36 μ g/mL estazolams, 43.39 μ g/mL Lorazepams, 47.51 μ g/mL Clonazepams, 43.60 μ g/mL alprazolams, each 10 μ L of 25.00 μ g/mL Zolpidemtar Trate reference substance solution; Add 18.32 μ g/mL diazepams for No. 4,17.74 μ g/mL estazolams, 17.74 μ g/mL Lorazepams, 19.00 μ g/mL Clonazepams, 17.44 μ g/mL alprazolams, each 10 μ L of 10.00 μ g/mL Zolpidemtar Trate reference substance solution; Add 9.16 μ g/mL diazepams for No. 5,8.72 μ g/mL estazolams, 8.68 μ g/mL Lorazepams, 9.52 μ g/mL Clonazepams, 8.72 μ g/mL alprazolams, each 10 μ L of 8.72 μ g/mL Zolpidemtar Trate reference substance solution; Add 1.83 μ g/mL diazepams for No. 6,1.77 μ g/mL estazolams, 1.74 μ g/mL Lorazepams, 1.81 μ g/mL Clonazepams, 1.74 μ g/mL alprazolams, each 10 μ L of 1.00 μ g/mL Zolpidemtar Trate reference substance solution.According to the operation of step 3) method, obtain gradient mark-on sample solution; Under the condition of step 7), measure.With 6 kinds of medicines separately the ratio of peak area and internal standard compound peak area vancomycin concentration is carried out to linear regression, obtain regression equation, the typical curve of diazepam: y=0.0635x-0.5842 (r=0.9940), the typical curve of Lorazepam: y=0.0758x-0.5796 (r=0.9968), the typical curve of alprazolam: y=0.0813x-0.6502 (r=0.9946), the typical curve of estazolam: y=0.0666x-0.5455 (r=0.9939), the typical curve of Clonazepam: y=0.0622x-0.4402 (r=0.9972), the typical curve of Zolpidemtar Trate: y=0.1428x-0.6019 (r=0.9963).Wherein y is the ratio of measured object peak area and internal standard compound peak area, and x is measured object quality concentration.
10) data analysis is calculated
The peak area of Lorazepam and colchicin in record patient serum, calculates the ratio of medicine and interior mark colchicin peak area, and the typical curve of substitution Lorazepam calculates the mass concentration of medicine, is the drug concentration in serum.The results are shown in Table 1.
The relevant psychiatric patient of the clinical culture of table 1. is taken blood concentration table after Lorazepam
| Numbering | Get the blood time | Serum drug level (ng/mL) |
| 1 | 2014.07.01 22:00 | 14.33 |
| 2 | 2014.07.02 06:00 | 72.43 |
| 3 | 2014.07.02 22:00 | 93.79 |
| 4 | 2014.07.11 06:00 | 53.38 |
Claims (5)
1.UHPLC-CAD coupling technique is measured the method for 6 kinds of tranquilizing soporific class medicament contgs in serum simultaneously, and 6 kinds of described tranquilizing soporific class medicines are diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, get Zolpidemtar Trate is characterized in that the method comprises the following steps:
1) preparation of reference substance
Precision takes diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, gets Zolpidemtar Trate reference substance, be placed in respectively volumetric flask, add acetonitrile to dissolve, be made into the reference substance solution of required mass concentration, standby, precision measures 6 kinds of reference substance solution, is made into the mixing reference substance solution of required mass concentration, standby;
2) preparation of inner mark solution
Precision takes colchicin reference substance, is placed in volumetric flask, adds acetonitrile to dissolve, and is made into the inner mark solution of required mass concentration, standby;
3) sample solution preparation
According to sleep-disorder diagnostic criteria, selected patient, clinician gives a kind or several sedative hypnotic drugs according to symptom and treats, after patient takes medicine, regularly get blood, separation of serum, is placed in tool plug centrifuge tube, quantitatively add step 2) colchicin inner mark solution, vortex mixes, after high speed centrifugation, and separation of supernatant; In supernatant, be added to the SPE post after the activation of first alcohol and water, after loading, use water wash, the methanol-eluted fractions of debita spissitudo, eluent volatilizes, and with acetonitrile, dissolves, and obtains sample solution, standby;
4) preparation of mobile phase solution
Get trifluoroacetic acid aqueous solution, as mobile phase A phase, standby; Get pure water, as Mobile phase B phase, standby;
5) setting of chromatographic condition
Selection be take octadecylsilane chemically bonded silica as filling agent, and specification is 4.6 * 250mm, and filler internal diameter is the chromatographic column of 5.0 μ m; Column temperature is 30 ℃, and flow rate of mobile phase is 0.8mL/min; Get reference substance solution and the step 2 of step 1)) inner mark solution, quantitative sample injection, carries out gradient elution; Adopt electron spray detecting device to carry out chromatography peak detection;
6) electron spray detector strip piece optimization
Select electron spray detecting device, nitrogen pressure is 35psi, range 100pA, 30 ℃ of atomization temperatures;
Get step 1) reference substance solution, step 2) inner mark solution, quantitative sample injection, obtains the standard colors spectrogram of reference substance and internal standard compound thus;
7) sample determination
Get the sample solution of step 3), difference quantitative sample injection, under the condition of step 5) and step 6), measure, obtain respectively diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, Zolpidemtar Trate reference substance, the chromatogram of colchicin internal standard compound and sample, in sample collection of illustrative plates, with diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, Zolpidemtar Trate reference substance and the identical retention time of colchicin internal standard compound place, for the diazepam to be measured in blood serum sample, Lorazepam, alprazolam, estazolam, Clonazepam, Zolpidemtar Trate, and the colchicin internal standard compound quantitatively adding,
8) preparation of gradient concentration reference substance solution
Accurate diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, the Zolpidemtar Trate reference substance solution of drawing step 1), is mixed with mixing reference substance solution, adds acetonitrile, is diluted to the mixing reference substance solution of gradient concentration, standby;
9) typical curve preparation
Many parts of blank serum of patient getting sleep-disorder, quantitatively add respectively the gradient concentration of step 8) to mix reference substance solution, according to the operation of step 3) method, obtain gradient mark-on sample solution, under the condition of step 7), measure, with diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, the ratio of Zolpidemtar Trate peak area and internal standard compound peak area is to diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, the concentration at Zolpidemtar Trate peak is carried out linear regression, obtain regression equation, the typical curve of diazepam: y=0.0635x-0.5842 (r=0.9940), the typical curve of Lorazepam: y=0.0758x-0.5796 (r=0.9968), the typical curve of alprazolam: y=0.0813x-0.6502 (r=0.9946), the typical curve of estazolam: y=0.0666x-0.5455 (r=0.9939), the typical curve of Clonazepam: y=0.0622x-0.4402 (r=0.9972), the typical curve of Zolpidemtar Trate: y=0.1428x-0.6019 (r=0.9963), wherein y is the ratio of measured object peak area and internal standard compound peak area, and x is measured object quality concentration,
10) data analysis is calculated
Record the peak area of diazepam, Lorazepam, alprazolam, estazolam, Clonazepam, Zolpidemtar Trate and colchicin, calculate the ratio of 6 kinds of medicines and interior mark colchicin peak area, distinguish substitution typical curve separately, calculate the mass concentration of 6 kinds of medicines; Be the concentration of serum Chinese traditional medicine.
2. UHPLC-CAD technology according to claim 1 is measured the method for 6 kinds of tranquilizing soporific class medicament contgs in serum simultaneously, it is characterized in that the gradient elution program of step 5) is as follows: 0-7.00min, 45%A phase and 55%B phase; 7.01min-15.00min, 70%A phase and 30%B phase; 15.01min-20.00min, 45%A phase and 55%B phase.
3. UHPLC-CAD technology according to claim 1 is measured the method for 6 kinds of tranquilizing soporific class medicament contgs in serum simultaneously, it is characterized in that the electron spray detecting device condition of step 6) is as follows: nitrogen pressure is 35psi, range 100pA, 30 ℃ of atomization temperatures.
4. UHPLC-CAD technology according to claim 1 is measured the method for 6 kinds of tranquilizing soporific class medicament contgs in serum simultaneously, it is characterized in that it is 183.16 μ g/mL that step 8) is quantitatively diluted to concentration gradient, 91.58 μ g/mL, 45.79 μ g/mL, 18.32 μ g/mL, 9.16 μ g/mL, 1.83 the diazepam reference substance solution of μ g/mL, 177.44 μ g/mL, 88.72 μ g/mL, 44.36 μ g/mL, 17.74 μ g/mL, 8.72 μ g/mL, 1.77 the estazolam reference substance solution of μ g/mL, 173.56 μ g/mL, 86.78 μ g/mL, 43.39 μ g/mL, 17.36 μ g/mL, 8.68 μ g/mL, 1.74 the Lorazepam reference substance solution of μ g/mL, 190.04 μ g/mL, 95.02 μ g/mL, 47.51 μ g/mL, 19.00 μ g/mL, 9.52 μ g/mL, 1.81 the Clonazepam reference substance solution of μ g/mL, 174.40 μ g/mL, 87.20 μ g/mL, 43.60 μ g/mL, 17.44 μ g/mL, 8.72 μ g/mL, the alprazolam reference substance solution of 1.74 μ g/mL, and 100.00 μ g/mL, 50.00 μ g/mL, 25.00 μ g/mL, 10.00 μ g/mL, 5.00 μ g/mL, the Zolpidemtar Trate reference substance solution of 1.00 μ g/mL.
5. UHPLC-CAD technology according to claim 1 is measured the method for 6 kinds of tranquilizing soporific class medicament contgs in serum simultaneously, it is characterized in that getting in step 9) 6 parts of the blank serum of patient of 1.0mL sleep-disorder, number 1～No. 6, add 183.16 μ g/mL diazepams for No. 1,177.44 μ g/mL estazolams, 173.56 μ g/mL Lorazepams, 190.04 μ g/mL Clonazepams, 190.04 μ g/mL alprazolams, each 10 μ L of 100.00 μ g/mL Zolpidemtar Trate reference substance solution; Add 91.58 μ g/mL for No. 2,88.72 μ g/mL estazolams, 86.78 μ g/mL Lorazepams, 95.02 μ g/mL Clonazepams, 87.20 μ g/mL alprazolams, each 10 μ L of 50.00 μ g/mL Zolpidemtar Trate reference substance solution; Add 45.79 μ g/mL diazepams for No. 3,44.36 μ g/mL estazolams, 43.39 μ g/mL Lorazepams, 47.51 μ g/mL Clonazepams, 43.60 μ g/mL alprazolams, each 10 μ L of 25.00 μ g/mL Zolpidemtar Trate reference substance solution; Add 18.32 μ g/mL diazepams for No. 4,17.74 μ g/mL estazolams, 17.74 μ g/mL Lorazepams, 19.00 μ g/mL Clonazepams, 17.44 μ g/mL alprazolams, each 10 μ L of 10.00 μ g/mL Zolpidemtar Trate reference substance solution; Add 9.16 μ g/mL diazepams for No. 5,8.72 μ g/mL estazolams, 8.68 μ g/mL Lorazepams, 9.52 μ g/mL Clonazepams, 8.72 μ g/mL alprazolams, each 10 μ L of 8.72 μ g/mL Zolpidemtar Trate reference substance solution; Add 1.83 μ g/mL diazepams for No. 6,1.77 μ g/mL estazolams, 1.74 μ g/mL Lorazepams, 1.81 μ g/mL Clonazepams, 1.74 μ g/mL alprazolams, each 10 μ L of 1.00 μ g/mL Zolpidemtar Trate reference substance solution.
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