CN102175780A - Method for measuring concentration of volatile anesthetics in samples by chromatography - Google Patents
Method for measuring concentration of volatile anesthetics in samples by chromatography Download PDFInfo
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- CN102175780A CN102175780A CN2010106005655A CN201010600565A CN102175780A CN 102175780 A CN102175780 A CN 102175780A CN 2010106005655 A CN2010106005655 A CN 2010106005655A CN 201010600565 A CN201010600565 A CN 201010600565A CN 102175780 A CN102175780 A CN 102175780A
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Abstract
The invention discloses a method for measuring concentration of volatile anesthetics in samples by automatic headspace gas chromatography. The method is suitable for measuring the concentrations of volatile anesthetics like methoxyflurane, enflurane, isoflurane, sevoflurane and desflurane in samples. The samples can be solutions containing the substances to be measured, as well as can be in vitro biological samples containing the substances to be measured, such as blood, urine, tissue fluid. The method is simple, fast and exact, suitable for measuring and analyzing laboratory samples, and suitable for result measurement of a plurality of samples in clinical monitoring.
Description
Technical field
The invention belongs to field of biomedicine technology, be specifically related to the method for volatile anesthetic concentration in the automatic Headspace Gas Chromatography sample of a kind of usefulness.
Background technology
Volatile anesthetic is just the same with the general last purpose of intravenous anesthetics, but when the inhalation route administration, after at first must in the anesthesia cyclic system, reaching finite concentration, be absorbed in the blood with the concentration partial pressure difference through lung, be distributed to surrounding tissue through the circulation system again, arrive central nervous system and bring into play its anesthetic action, the blood concentration of measuring volatile anesthetic such as methoxyflurane, peace fluothane, isoflurane, sevoflurane and Desflurane etc. in the blood thus is very crucial index.
Liu advances, Liu Mingzheng etc. (the secondary balancing method is measured inhalation anesthetic liquid/gas partition factor. " China's anesthesia pharmaceutical journal ", 1996,16 (12): 604～607.) disclose a kind of method of measuring inhalation anesthetic concentration with syringe head space secondary balancing method.Arcotic standard gas such as isoflurane standard gas at first need be prepared and proofread and correct to this method, and process comprises 1, extracts blood 5～7ml with syringe; 2, made isoflurane in blood, gas two-phase, reach balance (about 2.5 hours altogether) for the first time in 1 hour in jolting water-bath under 37 ℃ of water bath with thermostatic control conditions; 3, make isoflurane in blood, gas two-phase, reach balance (1～1.5 hour) for the second time in jolting water-bath under 37 ℃ of water bath with thermostatic control conditions; 4,, write down before and after twice balance the volume of gas phase and liquid phase in the syringe by the isoflurane concentration in the gas phase of twice balance of gas chromatograph for determination; 5, calculate isoflurane concentration in the blood.
This method is measured accurately, can calculate simultaneously the liquid/gas partition factor of arcotic, but sample pretreatment process is consuming time longer, and twice equilibration time adds up 3.5 hours, measures a sample fully and takes 4 hours, determination efficiency is low, and in the practice process, blood sample preparation product can't store in syringe for a long time, can be influential to the result who measures behind the blood denaturation, so this method is not suitable for carrying out the mensuration processing of batch samples, so can't satisfy the needs of clinical monitoring fast measuring and batch processing.
Summary of the invention
The invention provides the method for the automatic Headspace Gas Chromatography volatile anesthetic of a kind of usefulness concentration.This method has overcome the shortcoming that standard items prepare and sample preparation is loaded down with trivial details, minute long, storage is inconvenient in the prior art, provide a kind of easy, fast, efficient, accurately, and can extensive treatments the method for volatile anesthetic concentration determination of gross sample and the application in clinical monitoring.
The method that the purpose of this invention is to provide volatile anesthetic concentration in the automatic Headspace Gas Chromatography sample of a kind of usefulness.
Specifically, the invention provides the method for volatile anesthetic concentration in a kind of working sample, it adopts automatic headspace gas chromatography that the sample that comprises volatile anesthetic is detected, here, described sample is the solution that comprises volatile anesthetic, optional from containing the organic solvent of volatile anesthetic and/or the solution of inorganic solvent, the perhaps biological sample of Li Tiing, for example blood, urine or tissue fluid.
In embodiments of the invention, described volatile anesthetic is selected from methoxyflurane, peace fluothane, isoflurane, sevoflurane or Desflurane.
In embodiments of the invention, described automatic headspace gas chromatography comprises the automatic head-space sampler sample introduction of employing.
In a preferred embodiment of the invention, the invention provides volatile anesthetic concentration method in a kind of working sample, it adopts automatic headspace gas chromatography, comprises the steps:
(1) chromatographic condition and system suitability test
Experimental apparatus is the Agilent6890N gas chromatograph; Chromatographic column is the polarity capillary column, adopts ascending order to heat up; Injector temperature is 100～200 ℃, and detecting device is flame ionization ditector (FID), checks 200～300 ℃ of temperature; Carrier gas is nitrogen, hydrogen or helium; The chromatographic peak of volatile anesthetic to be measured and impurity peaks degree of separation are greater than 2.5; Automatic headspace sampling, 85 ℃ of insulation 30min, quantitatively ring and 90～150 ℃ of transmission line temperature, sample size 0.5～1ml;
(2) preparation of typical curve solution
It is an amount of to get volatile anesthetic, accurate claims surely, and storing solution is made in solubilizer dissolving also dilution, and stand-by storage liquid is diluted to 6～10 parts of variable concentrations solution as typical curve solution, gets 0.5～1ml top set empty bottle respectively, and the envelope bottle promptly;
(3) solution 0.5～1ml top set empty bottle to be measured is got in the preparation of need testing solution, and the envelope bottle promptly;
(4) mensuration is got typical curve solution and need testing solution respectively, measures, calculates, promptly with automatic head-space sampler inject gas chromatograph.
In embodiments of the invention, wherein, chromatographic column is the polarity capillary column in the step (1), and preferred DB-WAX etc. are the capillary column of packed column with the polyglycol.
In embodiments of the invention, wherein, preferred injector temperature is 100～200 ℃ in the step (1), and detecting device is flame ionization ditector (FID), 200～300 ℃ of detector temperatures; The employing ascending order heats up; Keep 2～10min for preferred initial 40～60 ℃, rise to 150～200 ℃, keep 2～10min with 5～10 ℃/min speed.
In embodiments of the invention, wherein, preferred carrier gas is a nitrogen in the step (1), and flow velocity is 1.0～3.0ml/min, and split ratio is 1: 1～1: 100.
In embodiments of the invention, wherein, to be preferably water or to contain volume ratio be 10～100% N to solvent in the step (2), the aqueous solution of dinethylformamide or dimethyl sulfoxide (DMSO).
In embodiments of the invention, wherein, preferred head space preheating condition is 70～90 ℃ of insulation 20～30min in the step (4).
Detection method provided by the invention, the standard value and the measured value (being typical curve and sample curve) that contain the volatile anesthetic sample with automatic Headspace Gas Chromatography, calculate volatile anesthetic concentration in the sample, and the application of this method in clinical monitoring and lab analysis
Useful technique effect of the present invention:
The present invention provides easy, quick for the concentration determination of volatile anesthetic, accurately universal method.
1, greatly simplified the sample preparation process, only need extract in test the gland packing to the head space bottle of 0.5～1ml blood or other fluid samples and get final product.
2, this becomes the titer than the easily dilutable preparation to typical curve by preparation volatile anesthetic standard gas, and the result is accurate equally.
3, greatly shortened the sample detection time, as making the minimum need of sample of the syringe secondary balancing 3 hours consuming time, and one day while working sample can not be carried out by the water bath restriction in batches, and can measure 30～40 samples in one day with automatic headspace sampling, and mensuration need not hand sampling fully automatically.
4, use the head space auto injection, the impurity that can avoid biological sample to bring into causes interference to analysis, thereby reduce the accurate of analysis result is guaranteed in the pollution of chromatographic column and injection port.
5, through study on the stability, volatile anesthetic such as isoflurane can be preserved in the head space bottle 7 days, and it is convenient to store, and the results are shown in Table 1.
Following 15 days study on the stability that keep sample of 25 ℃ of conditions of table 1 isoflurane sample normal temperature
Table 1 result shows the clinical whole blood sample that adds isoflurane, and the isoflurane sample stability is good under 25 ℃ of conditions of 7 days normal temperature, reduces slightly in 15 days, keeps stable more than 90% but still have.Only need in time sample is added in the head space bottle gland packing during therefore clinical sampling and preserve, can obviously prolong the sample time to be measured.
Detection method provided by the invention provides new technological means for the clinical monitoring of volatile anesthetic.
Description of drawings
Fig. 1 typical curve, regression equation and related coefficient
To concentration (X-axis) linear regression, the drawing standard curve draws regression equation with volatile anesthetic isoflurane peak area (Y-axis), and related coefficient meets " chemicals CLINICAL PHARMACOKINETIS STUDY ON technological guidance principle " greater than 0.995.
Isoflurane gas chromatographic analysis collection of illustrative plates in Fig. 2 people's whole blood
Precision is measured the 1ml human whole blood, quantitatively adds the isoflurane of concentration known, carries out gas chromatographic analysis, and the result shows that the isoflurane retention time is 7.199 minutes, separates well with other impurity peaks in the blood, and response is sensitive.Go out isoflurane concentration in the sample with external standard method by regression equation calculation shown in Figure 1.
Embodiment
The present invention is further elaborated in conjunction with instantiation, but it is not had any restriction.The mensuration of volatile anesthetic concentration in embodiment 1 blood
(1) chromatographic condition Agilent6890N gas chromatograph, the DB-WAX capillary chromatographic column, 160 ℃ of injector temperatures, carrier gas is high purity nitrogen, the post flow is 2ml/min.Temperature programme is kept 2min for initial 60 ℃, rises to 200 ℃ with 10 ℃/min speed, keeps 2min, moves 18min altogether.Detecting device is flame ionization detector (FID), and detector temperature is 300 ℃.Automatic headspace sampling, 85 ℃ of insulation 30min quantitatively encircle 90 ℃ of temperature, 120 ℃ of transmission line temperature, sample size 1ml.(chromatographic condition is the same)
(2) to get the sevoflurane reference substance an amount of for standard reserving solution preparation, use N, and dinethylformamide (DMF) dissolving is also diluted, and makes the solution that every 1ml contains about 14mg, as standard reserving solution; Facing the time spent now joins.
(3) sample preparation and mensuration sevoflurane standard reserving solution doubling dilution 9 times are respectively got 100 μ l and are added to gland packing in the head space bottle that has added 1ml water respectively, are sevoflurane typical curve solution.To concentration linear regression (weighted regression), draw regression equation with the sevoflurane peak area.Precision is measured blood sample 1ml to be measured gland packing to the head space bottle, and parallel 6 parts, the sample sevoflurane peak area that records goes out the concentration of sevoflurane in the sample by regression equation calculation.
Experimental result sees Table 2
Table 2
Automatic headspace injection method of embodiment 2 usefulness and syringe head space secondary balancing method are measured the isoflurane concentration of same whole blood sample.
Experimental implementation
Get two parts of each 20ml of blank whole blood, add a certain amount of isoflurane reference substance respectively, treat that 25 ℃ of balances of normal temperature after one day, obtain containing the blood sample 1 and the blood sample 2 of isoflurane, 1, the 2 use syringe secondary balancing method of taking a blood sample is measured isoflurane concentration; Blood sampling 1,2 each 1ml joins gland packing in the head space bottle in addition, measures isoflurane concentration with this law.
Experimental result
Automatic headspace injection method of table 3 and syringe head space secondary balancing method are measured the isoflurane concentration of same whole blood sample
(wherein accuracy is automatic headspace injection method measured value/syringe head space secondary balancing method measured value)
Table 3 result shows that the isoflurane concentration that automatic headspace injection method and syringe head space secondary balancing method measure same whole blood sample is almost consistent, and accuracy is higher.
The mensuration of volatile anesthetic concentration in the embodiment 3DMF aqueous solution
It is an amount of to get volatile anesthetic, accurate claim fixed, add the dissolving of DMF aqueous solution also dilution make storing solution, and stand-by storage liquid doubling dilution becomes 6～10 parts of variable concentrations solution as solution to be measured, gets 0.5～1ml top set empty bottle respectively, the envelope bottle; The peak area of measuring the variable concentrations sample under embodiment 1 chromatographic condition compares.To concentration (X-axis) linear regression, the drawing standard curve draws regression equation with volatile anesthetic isoflurane peak area (Y-axis)
Y=1.5969X+4.1027 R2=0.9994 presentation of results is linear good, meets " chemicals CLINICAL PHARMACOKINETIS STUDY ON technological guidance principle ".See Fig. 1.
Claims (10)
1. the assay method of volatile anesthetic concentration in the sample, it adopts automatic headspace gas chromatography that the sample that comprises volatile anesthetic is detected, here, described sample is the solution that comprises volatile anesthetic, be selected from the organic solvent and/or the solution of inorganic solvent, the perhaps biological sample of Li Tiing that contain volatile anesthetic.
2. assay method according to claim 1, wherein, volatile anesthetic is selected from methoxyflurane, peace fluothane, isoflurane, sevoflurane or Desflurane.
3. assay method according to claim 1, wherein, described stripped biological sample, for example blood, urine or tissue fluid.
4. assay method according to claim 1, wherein, described automatic headspace gas chromatography comprises the automatic head-space sampler sample introduction of employing.
5. according to the described assay method of arbitrary claim in the claim 1 to 4, may further comprise the steps:
(1) chromatographic condition and system suitability test
Detector is a gas chromatograph; Chromatographic column is the polarity capillary column, adopts temperature programme; Injector temperature is 100～200 ℃, and detecting device is a flame ionization ditector, checks 200～300 ℃ of temperature, and carrier gas is nitrogen, hydrogen or helium; The chromatographic peak of volatile anesthetic to be measured and impurity peaks degree of separation should be greater than 2.5; Automatic headspace sampling;
(2) preparation of typical curve solution
It is an amount of to get volatile anesthetic, accurate claims surely, and storing solution is made in solubilizer dissolving also dilution, and stand-by storage liquid is diluted to 6～10 parts of variable concentrations solution as typical curve solution, gets 0.5～1ml top set empty bottle respectively, and the envelope bottle promptly;
(3) preparation of need testing solution
Get solution top set empty bottle to be measured, the envelope bottle promptly;
(4) measure
Get typical curve solution and need testing solution respectively, measure, calculate, promptly with automatic head-space sampler inject gas chromatograph.
6. assay method according to claim 5, wherein, chromatographic column is the polarity capillary column in the step (1), preferred DB-WAX etc. are the capillary column of packed column with the polyglycol.
7. detection method according to claim 5, wherein, preferred injector temperature is 100～200 ℃ in the step (1), detecting device is a flame ionization ditector, 200～300 ℃ of detector temperatures; The employing ascending order heats up; Keep 2～10min for preferred initial 40～60 ℃, rise to 150～200 ℃, keep 2～10min with 5～10 ℃/min speed.
8. detection method according to claim 5, wherein, carrier gas is a nitrogen in the step (1), and flow velocity is 1.0～3.0ml/min, and split ratio is 1: 1～1: 100.
9. detection method according to claim 5, wherein, the middle solvent of step (2) is selected from water or contains volume ratio is 10～100%N, the aqueous solution of dinethylformamide or dimethyl sulfoxide (DMSO).
10. detection method according to claim 5, wherein, the head space preheating method is 70～90 ℃ of insulation 20～30min in the step (4).
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| CN109470807A (en) * | 2018-11-07 | 2019-03-15 | 中国科学院合肥物质科学研究院 | A kind of detection method of the waste gas of anesthesia sevoflurane absorption of human body factor |
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| CN1342634A (en) * | 1994-04-08 | 2002-04-03 | 森陶硝子株式会社 | Manufacturing method of fluoromethyl-1,1,1,3,3,3-hexafluoroisopropylether |
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| FR814927A (en) * | 1936-03-17 | 1937-07-02 | Improvements to dolls or two-legged automatons | |
| DE4317139A1 (en) * | 1993-05-21 | 1994-11-24 | Volker Prof Dr Schurig | Process for the preparative gas-chromatographic enantiomer separation of inhalation anaesthetics (enflurane) on cyclodextrin derivatives in polysiloxane solution |
| CN101393196B (en) * | 2008-10-17 | 2013-06-05 | 复旦大学附属华山医院 | Method for simultaneously determining of concentration multi anesthesia medicament in blood plasma |
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| CN1342634A (en) * | 1994-04-08 | 2002-04-03 | 森陶硝子株式会社 | Manufacturing method of fluoromethyl-1,1,1,3,3,3-hexafluoroisopropylether |
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| CN109470807A (en) * | 2018-11-07 | 2019-03-15 | 中国科学院合肥物质科学研究院 | A kind of detection method of the waste gas of anesthesia sevoflurane absorption of human body factor |
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