CN107085064B - High performance liquid chromatography-tandem mass spectrometry simultaneous detection method for fusarium acetate C and bis (methylthio) gliotoxin in sputum - Google Patents
High performance liquid chromatography-tandem mass spectrometry simultaneous detection method for fusarium acetate C and bis (methylthio) gliotoxin in sputum Download PDFInfo
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- CN107085064B CN107085064B CN201710428772.9A CN201710428772A CN107085064B CN 107085064 B CN107085064 B CN 107085064B CN 201710428772 A CN201710428772 A CN 201710428772A CN 107085064 B CN107085064 B CN 107085064B
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- fusarinine
- acetic acid
- phenacetin
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- 238000001514 detection method Methods 0.000 title claims abstract description 33
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 title claims abstract description 14
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 title abstract 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 title abstract 2
- 241000223218 Fusarium Species 0.000 title abstract 2
- OVBAGMZLGLXSBN-UOVKNHIHSA-N (3r,5as,6s,10ar)-6-hydroxy-3-(hydroxymethyl)-2-methyl-3,10a-bis(methylsulfanyl)-6,10-dihydro-5ah-pyrazino[1,2-a]indole-1,4-dione Chemical compound C1C2=CC=C[C@H](O)[C@H]2N2[C@]1(SC)C(=O)N(C)[C@](CO)(SC)C2=O OVBAGMZLGLXSBN-UOVKNHIHSA-N 0.000 title description 7
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- CPJSUEIXXCENMM-UHFFFAOYSA-N phenacetin Chemical compound CCOC1=CC=C(NC(C)=O)C=C1 CPJSUEIXXCENMM-UHFFFAOYSA-N 0.000 claims abstract description 106
- 229960003893 phenacetin Drugs 0.000 claims abstract description 53
- 238000000034 method Methods 0.000 claims abstract description 28
- 238000012360 testing method Methods 0.000 claims abstract description 5
- 229940103893 gliotoxin Drugs 0.000 claims abstract 2
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 168
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 74
- 239000000243 solution Substances 0.000 claims description 57
- 239000007788 liquid Substances 0.000 claims description 53
- 239000012071 phase Substances 0.000 claims description 52
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical group ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 30
- -1 methyl mercapto Chemical class 0.000 claims description 26
- WRFIKQWBKYAFNH-UHFFFAOYSA-N Fusarinine Natural products CC(=C/C(=O)N(O)CCCC(N)C(=O)O)CCO WRFIKQWBKYAFNH-UHFFFAOYSA-N 0.000 claims description 25
- 229930190252 gliotoxin Natural products 0.000 claims description 25
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 24
- 239000000284 extract Substances 0.000 claims description 21
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- 239000003480 eluent Substances 0.000 claims description 15
- 238000013467 fragmentation Methods 0.000 claims description 15
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- 239000007787 solid Substances 0.000 claims description 14
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 10
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- 238000010828 elution Methods 0.000 claims description 9
- 238000012417 linear regression Methods 0.000 claims description 9
- 238000000605 extraction Methods 0.000 claims description 7
- 238000004949 mass spectrometry Methods 0.000 claims description 7
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- 238000005259 measurement Methods 0.000 claims description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 5
- 238000000132 electrospray ionisation Methods 0.000 claims description 5
- 235000019253 formic acid Nutrition 0.000 claims description 5
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 235000006508 Nelumbo nucifera Nutrition 0.000 claims description 4
- 240000002853 Nelumbo nucifera Species 0.000 claims description 4
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- 239000007791 liquid phase Substances 0.000 claims description 4
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- 150000001242 acetic acid derivatives Chemical class 0.000 claims 1
- FIVPIPIDMRVLAY-UHFFFAOYSA-N aspergillin Natural products C1C2=CC=CC(O)C2N2C1(SS1)C(=O)N(C)C1(CO)C2=O FIVPIPIDMRVLAY-UHFFFAOYSA-N 0.000 claims 1
- FIVPIPIDMRVLAY-RBJBARPLSA-N gliotoxin Chemical compound C1C2=CC=C[C@H](O)[C@H]2N2[C@]1(SS1)C(=O)N(C)[C@@]1(CO)C2=O FIVPIPIDMRVLAY-RBJBARPLSA-N 0.000 claims 1
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- UIVTVLVVAXJPKM-IJGXQDLJSA-N N',N'',N'''-triacetylfusarinine C Chemical compound O1N2CCC[C@H](NC(C)=O)C(=O)OCC\C(C)=C/C(N(CCC[C@H](NC(C)=O)C(=O)OCC\C(C)=C/C3=[O+]4)O5)=[O+][Fe-3]1451ON3CCC[C@H](NC(=O)C)C(=O)OCC\C(C)=C/C2=[O+]1 UIVTVLVVAXJPKM-IJGXQDLJSA-N 0.000 description 1
- 239000000589 Siderophore Substances 0.000 description 1
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 description 1
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- 238000004128 high performance liquid chromatography Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
The invention provides a high performance liquid chromatography-tandem mass spectrometry simultaneous detection method of fusarium c acetate (tafc) and bimethylthio-gliotoxin (bmGT) in sputum, which comprises the following steps: (1) preparing a standard sample and a test sample; (2) measuring the standard samples and the samples obtained in the step (1) by adopting a liquid chromatography-tandem mass spectrometry method to obtain spectrograms of the standard samples and the samples, and recording peak areas of TAFC, bmGT and phenacetin in the spectrograms of the standard samples and the samples; (3) and drawing working curves of the TAFC and the bmGT, and calculating the concentrations of the TAFC and the bmGT in the sample according to the peak area ratio of the TAFC to the phenacetin in the sample spectrogram, the peak area ratio of the bmGT to the phenacetin and the working curves. The invention realizes the simultaneous detection of the TAFC and the bmGT in the sputum, provides a new way for the detection of the TAFC and the bmGT, and is beneficial to improving the detection efficiency.
Description
Technical field
The invention belongs to instrument analysis detection field, it is related to acetic acid fusarinine C (N, N', N "-in a kind of sputum
Triacetylfusarinine C, TAFC) and double methyl mercapto gliotoxins (bis (methylthio) gliotoxin, bmGT)
High performance liquid chromatography-tandem mass Simultaneous Detection.
Background technique
Aspergillus is a kind of fungi being widely present in nature, can more quickly in relative warmth and wet environment
Growth and breeding.The fungal spore of disperse can enter respiratory tract field planting by the breathing of human body in air.In order to understand in depth
Distribution situation of the Aspergillus in crowd, home and abroad researcher are constantly exploring the side for whether having Aspergillus to be colonized in respiratory tract
Method.The method for whether having Aspergillus to be colonized in identification human respiratory tract at present is mainly sputum fungal culture, however this method is quick
Perceptual very low and cultivation cycle is very long.Therefore, more and more researchs are exploring the inspection for passing through Aspergillus marker at present
It surveys to judge that respiratory tract is colonized with the presence or absence of Aspergillus.
TAFC is a kind of siderophore that Aspergillus is synthesized and discharged under iron deficiency environment.BmGT is synthesized and is divided by Aspergillus
Another compound secreted.Currently, there is document report using high performance liquid chromatography-tandem mass (UPLC-MS/MS) from serum
The method for detecting TAFC also has researcher to detect the side of bmGT from serum using high performance thin layer chromatography analyzer (HPTLC)
Method.However, the detection drawback lower there are sensibility of serum, the Samples detection based on local organs and tissue may can be more
Reflect the distribution situation of Aspergillus privileged site in human body well.Sputum is respiratory secretions, therefore Aspergillus in sputum
The detection of Research of predicting markers may more directly reflect the field planting of respiratory tract Aspergillus.In addition, existing method is not possible to realize
It is detected while both compounds of TAFC and bmGT.Therefore, if can develop from sputum while detect TAFC's and bmGT
Method, for further investigation Aspergillus the distribution of human respiratory tract and colonisation, abundant TAFC and bmGT detection approach,
Positive meaning will all be generated by improving detection efficiency.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide the efficient liquid phases of TAFC and bmGT in sputum a kind of
Chromatography-tandem mass spectrum Simultaneous Detection improves detection efficiency to enrich the detection approach of TAFC and bmGT.
The high performance liquid chromatography-tandem mass Simultaneous Detection of TAFC and bmGT, step in sputum provided by the invention
It is as follows:
(1) preparation of standard specimen and sample
1. TAFC and bmGT are dissolved with methanol, and a series of TAFC and bmGT concentration gradients are obtained with dilution in acetonitrile and are changed
Hybrid standard liquid；Phenacetin solution is prepared as inner mark solution；
2. taking isometric each hybrid standard liquid, isometric blank Sputum samples are separately added into each hybrid standard liquid
With isometric inner mark solution, be then uniformly mixed so as to obtain a series of mixed liquors, respectively into each mixed liquor be added extractant extraction 2~
3 time, the extract liquor that same mixed liquor obtains will be extracted and merged, extractant is removed under nitrogen protection, obtain a series of solid matters,
Each solid matter is dissolved with acetonitrile solution to respectively and is settled to same volume, is centrifuged, a series of supernatants of gained are to mark
Sample；
3. inner mark solution is added into Sputum samples to be measured and mixes, then extraction is added into the Sputum samples of gained containing the internal standard
Agent is taken to extract 2~3 times, combining extraction liquid simultaneously removes extractant under nitrogen protection, by gained solid matter acetonitrile solution
Dissolution, is then centrifuged for, gained supernatant is sample；
(2) measurement of standard specimen and sample
Using Liquid Chromatography-Tandem Mass Spectrometry to each standard specimen and sample obtained by step (1) according to following chromatographic condition and matter
Spectral condition is measured, and obtains standard specimen and sample spectrogram, records TAFC, bmGT and phenacetin in each standard specimen and sample spectrogram
Peak area；
Chromatographic condition: using Agilent Extend C18 chromatographic column, carries out gradient using mobile phase A and Mobile phase B and washes
De-, mobile phase A is the aqueous formic acid of 0.1wt%~0.15wt%, and Mobile phase B is acetonitrile, condition of gradient elution are as follows: in 0min
To 1min, the percentage by volume of Mobile phase B is 25% in eluent, the percentage by volume of mobile phase A is 75%, is being greater than 1min
To 4min, in eluent the percentage by volume of Mobile phase B by 25% be linearly increasing to the percentage by volume of 95%, mobile phase A by
75% linear reduction is being greater than 4min, the percentage by volume of Mobile phase B is the volume of 95%, mobile phase A in eluent to 5%
Percentage is 5%；
Mass Spectrometry Conditions: electro-spray ionization source, positive ion mode, multiple-reaction monitoring mode, capillary voltage 3500~
3550V, dry 350~360 DEG C of temperature degree, dry gas stream 5~6L/min of speed, 350~360 DEG C of sheath temperature degree, sheath gas 11
300~320V of~12L/min, Delta EMV；The mass-to-charge ratio m/z for measuring the parent ion of acetic acid fusarinine C is 928.3, son
The mass-to-charge ratio m/z of ion is 251, and fragmentation voltage is 240V, collision voltage 65V；Measure double methyl mercapto gliotoxins it is female from
The mass-to-charge ratio m/z of son is 357.1, and the mass-to-charge ratio m/z of daughter ion is 309.1, and fragmentation voltage is 76V, collision voltage 5V；Measurement
The mass-to-charge ratio m/z of phenacetin is 180.1, and the mass-to-charge ratio m/z of daughter ion is 138.3, and fragmentation voltage is 120V, and collision voltage is
15V；
(3) acquisition of testing result
Using the concentration of TAFC in each standard specimen as abscissa, with the peak area ratio of TAFC and phenacetin in each standard specimen spectrogram
For ordinate, the working curve of TAFC is drawn；Using the concentration of bmGT in each standard specimen as abscissa, with bmGT in each standard specimen spectrogram with
The peak area ratio of phenacetin is ordinate, draws the working curve of double methyl mercapto gliotoxins；
By the peak area ratio difference of the peak area ratio of TAFC and phenacetin, bmGT and phenacetin in sample spectrogram
It brings into the equation of linear regression of the working curve of TAFC and bmGT, calculates the concentration of TAFC and bmGT in sample.
In the above method, the extractant is chloroform or methylene chloride.
The step of above method method (1) 2. in, the additional amount of each extractant is the hybrid working liquid product of containing the internal standard
3~4 times, step (1) 3. in, the additional amount of each extractant is to obtain 3~4 times of Sputum samples volume of containing the internal standard.
In the above method, the percentage by volume of acetonitrile is 35%~50% in the acetonitrile solution.
In the above method, using phenacetin use acetonitrile be configured to phenacetin concentration be 13~14ng/mL solution as
Inner mark solution.
Compared with prior art, the invention has the following advantages:
1. the method that the present invention provides a kind of to detect TAFC and bmGT simultaneously from sputum is the detection of TAFC and bmGT
New approach is provided, solving existing method cannot achieve the deficiency of TAFC and bmGT while detection, be conducive to improve detection
Efficiency.
2. experiments have shown that the method for the invention in 1.56~100ng/mL concentration range to TAFC and bmGT have it is good
Good is linear, detection TAFC and bmGT in a few days, day to day precision relative standard deviation RSD be no more than 8%, meet less than 15%
Requirement, accuracy relative deviation RE is no more than ± 9%, meets the requirement no more than ± 15% range.
3. the detection of TAFC and bmGT, operation letter can be realized using routine test instrument and reagent for the method for the invention
It is single, be conducive to promote and apply.
Detailed description of the invention
Fig. 1 is the spectrogram of 1# standard specimen in embodiment 1；
Fig. 2 is the working curve for the TAFC that embodiment 1 is drawn；
Fig. 3 is the working curve for the bmGT that embodiment 1 is drawn.
Specific embodiment
By the following examples simultaneously to the high performance liquid chromatography-tandem mass of TAFC and bmGT in sputum of the present invention
Detection method is described further.Instrument used in the examples is Agilent 1260-6460 type high performance liquid chromatography mass spectrum
Combined instrument.
Embodiment 1
In the present embodiment, standard specimen is tested, to investigate the range of linearity of the method for the invention, steps are as follows:
1. the preparation of standard specimen
(1) precise TAFC and bmGT are placed in volumetric flask, with methanol dissolution and constant volume, obtain TAFC and bmGT concentration
It is the mixing stock solution of 10mg/mL.
Take mixing 7 parts of stock solution, obtain 1#~7# hybrid standard liquid with dilution in acetonitrile, in 1# hybrid standard liquid TAFC and
BmGT concentration is 15.63ng/mL, TAFC and bmGT concentration is 31.25ng/mL, 3# hybrid standard in 2# hybrid standard liquid
TAFC and bmGT concentration is 62.50ng/mL in liquid, TAFC and bmGT concentration is 125.00ng/mL in 4# hybrid standard liquid,
TAFC and bmGT concentration is 250.00ng/mL in 5# hybrid standard liquid, TAFC and bmGT concentration is in 6# hybrid standard liquid
TAFC and bmGT concentration is 1000.00ng/mL in 500.00ng/mL, 7# hybrid standard liquid.
(2) phenacetin 10mg is weighed in 10mL volumetric flask, is added acetonitrile dissolution and constant volume, is obtained the Fei Naxi of 1mg/mL
Fourth stock solution takes appropriate phenacetin stock solution to obtain inner mark solution with dilution in acetonitrile, and phenacetin is dense in the inner mark solution
Degree is 13.8ng/mL.
(3) each 10 μ L of 1#~7# hybrid standard liquid is taken, 100 μ L blank sputums are added into 1#~7# hybrid standard liquid respectively
(sputum without TAFC and bmGT) and 10 μ L inner mark solutions are vortexed and mix, obtain 7 portions of mixed liquors, be added into each mixed liquor
Chloroform extracts 2 times, and 400 μ L of chloroform is added every time, will extract the extract liquor that same mixed liquor obtains and merges, then blows under a nitrogen
The acetonitrile solution that gained solid matter acetonitrile percentage by volume is 50% is dissolved and is settled to 100 μ L by dry doubling, is then centrifuged for,
Gained supernatant is standard specimen, is successively denoted as 1# standard specimen~7# standard specimen.
2. the measurement of standard specimen
Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to 1# standard specimen~7# standard specimen according to following chromatographic condition and matter
Spectral condition is measured, and is obtained 1# standard specimen~7# standard specimen spectrogram, is recorded the peak of TAFC, bmGT and phenacetin in each standard specimen spectrogram
Area, wherein 1# standard specimen spectrogram is as shown in Figure 1, in Fig. 1, and the peak in three spectrograms is followed successively by TAFC, bmGT and interior from top to bottom
Mark the peak of object phenacetin (IS is denoted as in figure)；
Chromatographic condition: Agilent Extend C18 chromatographic column (3.0 × 100mm of specification, 3.5 μ of column packing partial size are used
M), gradient elution, 35 DEG C of column temperature, flow velocity 0.4mL/min, 3 μ L of sample volume, mobile phase A are carried out using mobile phase A and Mobile phase B
For the aqueous formic acid of 0.1wt%, Mobile phase B is acetonitrile.
Condition of gradient elution are as follows: the percentage by volume of Mobile phase B is 25%, mobile phase A in 0min to 1min, eluent
Percentage by volume be 75%, be greater than 1min to 4min, the percentage by volume of Mobile phase B is linearly increasing by 25% in eluent
Percentage by volume to 95%, mobile phase A is linearly reduced by 75% to 5%, is being greater than 4min to 6.5min, is being flowed in eluent
The percentage by volume of phase B is 95%, the percentage by volume of mobile phase A is 5%；
Mass Spectrometry Conditions: electro-spray ionization source, positive ion mode, multiple-reaction monitoring mode, capillary voltage 3500V are done
350 DEG C of pathogenic dryness temperature, dry gas stream speed 5L/min, 350 DEG C of sheath temperature degree, sheath gas 11L/min, Delta EMV 300V；
The mass-to-charge ratio m/z for measuring the parent ion of acetic acid fusarinine C is 928.3, and the mass-to-charge ratio m/z of daughter ion is 251, and fragmentation voltage is
240V, collision voltage 65V；The mass-to-charge ratio m/z for measuring the parent ion of double methyl mercapto gliotoxins is 357.1, the matter of daughter ion
Lotus ratio m/z is 309.1, and fragmentation voltage is 76V, collision voltage 5V；Measure phenacetin mass-to-charge ratio m/z be 180.1, son from
The mass-to-charge ratio m/z of son is 138.3, and fragmentation voltage is 120V, collision voltage 15V.
3. drawing working curve
Using the concentration of TAFC in each standard specimen as abscissa, with the peak area ratio of TAFC and phenacetin in each standard specimen spectrogram
For ordinate, TAFC is drawn in the working curve of 1.56~100ng/mL concentration range, as shown in Fig. 2, with weighting (W=1/x2)
Least square method carries out regressing calculation, and the equation of linear regression for obtaining TAFC working curve is Y=0.03095X+6.82 × 10-3,
In the equation of linear regression, Y is the peak area ratio of TAFC and phenacetin, and X is the concentration of TAFC, the phase of equation of linear regression
Closing property coefficient r value is 0.9997.
Using the concentration of bmGT in each standard specimen as abscissa, with the peak area ratio of bmGT and phenacetin in each standard specimen spectrogram
For ordinate, bmGT is drawn in the working curve of 1.56~100ng/mL concentration range, as shown in Figure 3 with weighting (W=1/x2)
Least square method carries out regressing calculation, and the equation of linear regression for obtaining bmGT working curve is Y=0.05647X+4.62 × 10-4,
In the equation of linear regression, Y is the peak area ratio of bmGT and phenacetin, and X is the concentration of bmGT, the phase of equation of linear regression
Closing property coefficient r value is 0.9980.
Embodiment 2
In the present embodiment, standard specimen is tested, to investigate the precision and accuracy of the method for the invention, step is such as
Under:
1. the preparation of standard specimen
(1) precise TAFC and bmGT are placed in volumetric flask, with methanol dissolution and constant volume, obtain TAFC and bmGT concentration
It is the mixing stock solution of 10mg/mL.
Take mixing 3 parts of stock solution, obtain 1#~3# hybrid standard liquid with dilution in acetonitrile, in 1# hybrid standard liquid TAFC and
BmGT concentration is 31.25ng/mL, TAFC and bmGT concentration is 125.00ng/mL, 3# hybrid standard in 2# hybrid standard liquid
TAFC and bmGT concentration is 500.00ng/mL in liquid.
(2) phenacetin 10mg is weighed in 10mL volumetric flask, is added acetonitrile dissolution and constant volume, is obtained the Fei Naxi of 1mg/mL
Fourth stock solution takes appropriate phenacetin stock solution to obtain inner mark solution with dilution in acetonitrile, and phenacetin is dense in the inner mark solution
Degree is 13.8ng/mL.
(3) each 10 μ L of 1#~3# hybrid standard liquid is taken, 100 μ L blank sputums are added into 1#~3# hybrid standard liquid respectively
It with 10 μ L inner mark solutions, is vortexed and mixes, obtain 3 portions of mixed liquors, chloroform is added into each mixed liquor and extracts 2 times, chlorine is added every time
Imitative 400 μ L will extract the extract liquor that same mixed liquor obtains and merge, then drying and by gained solid matter acetonitrile under a nitrogen
The acetonitrile solution that percentage by volume is 50% dissolves and is settled to 100 μ L, is then centrifuged for, and gained supernatant is standard specimen, according to
It is secondary to be denoted as 1# standard specimen~3# standard specimen.
2. the measurement of standard specimen and the calculating of preci-sion and accuracy
1# standard specimen~3# standard specimen is measured according to following chromatographic condition and Mass Spectrometry Conditions using LC-MS/MS method, each
The standard specimen of concentration is 6 parts parallel, is measured in parallel 3 days, records each standard specimen spectrogram, calculate in a few days, day to day precision and accuracy, as a result
As shown in table 1.
Chromatographic condition: Agilent Extend C18 chromatographic column (3.0 × 100mm of specification, 3.5 μ of column packing partial size are used
M), gradient elution, 35 DEG C of column temperature, flow velocity 0.4mL/min, 3 μ L of sample volume, mobile phase A are carried out using mobile phase A and Mobile phase B
For the aqueous formic acid of 0.1wt%, Mobile phase B is acetonitrile.
Condition of gradient elution are as follows: the percentage by volume of Mobile phase B is 25%, mobile phase A in 0min to 1min, eluent
Percentage by volume be 75%, be greater than 1min to 4min, the percentage by volume of Mobile phase B is linearly increasing by 25% in eluent
Percentage by volume to 95%, mobile phase A is linearly reduced by 75% to 5%, is being greater than 4min to 6.5min, is being flowed in eluent
The percentage by volume of phase B is 95%, the percentage by volume of mobile phase A is 5%；
Mass Spectrometry Conditions: electro-spray ionization source, positive ion mode, multiple-reaction monitoring mode, capillary voltage 3500V are done
350 DEG C of pathogenic dryness temperature, dry gas stream speed 5L/min, 350 DEG C of sheath temperature degree, sheath gas 11L/min, Delta EMV 300V；
The mass-to-charge ratio m/z for measuring the parent ion of acetic acid fusarinine C is 928.3, and the mass-to-charge ratio m/z of daughter ion is 251, and fragmentation voltage is
240V, collision voltage 65V；The mass-to-charge ratio m/z for measuring the parent ion of double methyl mercapto gliotoxins is 357.1, the matter of daughter ion
Lotus ratio m/z is 309.1, and fragmentation voltage is 76V, collision voltage 5V；Measure phenacetin mass-to-charge ratio m/z be 180.1, son from
The mass-to-charge ratio m/z of son is 138.3, and fragmentation voltage is 120V, collision voltage 15V.
Table 1
As shown in Table 1, the method for the invention detection TAFC and bmGT in a few days, day to day precision relative standard deviation
RSD is no more than 8%, meets the requirement less than 15%, and accuracy relative deviation RE is no more than ± 9%, meets and is no more than ± 15%
The requirement of range.
Embodiment 3
In the present embodiment, matrix effect and extraction recovery are investigated, steps are as follows:
1. preparing hybrid standard liquid and inner mark solution
(1) precise TAFC and bmGT are placed in volumetric flask, with methanol dissolution and constant volume, obtain TAFC and bmGT concentration
It is the mixing stock solution of 10mg/mL.
Take mixing 3 parts of stock solution, obtain 1#~3# hybrid standard liquid with dilution in acetonitrile, in 1# hybrid standard liquid TAFC and
BmGT concentration is 31.25ng/mL, TAFC and bmGT concentration is 125.00ng/mL, 3# hybrid standard in 2# hybrid standard liquid
TAFC and bmGT concentration is 500.00ng/mL in liquid.
(2) phenacetin 10mg is weighed in 10mL volumetric flask, is added acetonitrile dissolution and constant volume, is obtained the Fei Naxi of 1mg/mL
Fourth stock solution takes appropriate phenacetin stock solution to obtain inner mark solution with dilution in acetonitrile, and phenacetin is dense in the inner mark solution
Degree is 13.8ng/mL.
2. taking each 100 μ L of 3 parts of blank Sputum samples, 10 μ L of acetonitrile is added into each blank Sputum samples respectively, is vortexed mixed
It is even, it is then respectively adding chloroform and extracts 2 times, 400 μ L of chloroform is added every time, the extract liquor that same sample obtains will be extracted and merged,
Then it dries up under a nitrogen, it is respectively that gained solid matter is molten for 37.5% acetonitrile solution with 80 μ L acetonitrile percentage by volumes
Solution, is added 10 μ L1#~3# hybrid standard liquids, then be separately added into 10 μ L inner mark solutions into acquired solution respectively, is vortexed after mixing
LC-MS/MS analysis is carried out according to the condition in embodiment 2, each concentration investigates the blank Sputum samples of 6 separate sources, note
Record spectrogram.
Each 10 μ L of 1#~3# hybrid standard liquid is taken, 10 μ L inner mark solutions and 80 are added into 1#~3# hybrid standard liquid respectively
The acetonitrile solution that μ L acetonitrile percentage by volume is 37.5% is vortexed after mixing and carries out LC-MS/ according to the condition in embodiment 2
MS analysis, the sample of each concentration is 6 parts parallel, records spectrogram.
The spectrogram peak area for comparing two kinds of samples in the step obtains TAFC, bmGT and phenacetin in 3 concentration
Matrix effect, the results are shown in Table 2.
3. taking each 10 μ L of 1#~3# hybrid standard liquid, 100 μ L blank sputums are added into 1#~3# hybrid standard liquid respectively
It with 10 μ L inner mark solutions, is vortexed and mixes, obtain 3 portions of mixed liquors, chloroform is added into each mixed liquor and extracts 2 times, chlorine is added every time
Imitative 400 μ L will extract the extract liquor that same mixed liquor obtains and merge, then drying and by gained solid matter acetonitrile under a nitrogen
The acetonitrile solution that percentage by volume is 50% dissolves and is settled to 100 μ L, is then centrifuged for, to gained supernatant according to embodiment
Condition in 2 carries out LC-MS/MS analysis, and each concentration investigates the blank Sputum samples of 6 separate sources, records spectrogram.
Each 100 μ L of 3 parts of blank Sputum samples is taken, 10 μ L of acetonitrile is added into each blank Sputum samples respectively, is vortexed and mixes,
It is then respectively adding chloroform to extract 2 times, 400 μ L of chloroform is added every time, the extract liquor that same sample obtains will be extracted and merged, then
It dries up, respectively dissolves the gained solid matter acetonitrile solution that 80 μ L acetonitrile percentage by volumes are 37.5% under a nitrogen,
10 μ L1#~3# hybrid standard liquids are added into acquired solution respectively, then are separately added into 10 μ L inner mark solutions, is vortexed after mixing and presses
LC-MS/MS analysis is carried out according to the condition in embodiment 2, each concentration investigates the blank Sputum samples of 6 separate sources, record
Spectrogram.
The spectrogram peak area for comparing two kinds of samples in the step obtains TAFC, bmGT and phenacetin in 3 concentration
The results are shown in Table 2 for absolute extraction recovery.
Table 2
Embodiment 4
The present embodiment investigates the stability of Sputum samples, and steps are as follows:
1. preparing hybrid standard liquid and inner mark solution
(1) precise TAFC and bmGT are placed in volumetric flask, with methanol dissolution and constant volume, obtain TAFC and bmGT concentration
It is the mixing stock solution of 10mg/mL.
Take mixing 3 parts of stock solution, obtain 1#~3# hybrid standard liquid with dilution in acetonitrile, in 1# hybrid standard liquid TAFC and
BmGT concentration is 31.25ng/mL, TAFC and bmGT concentration is 125.00ng/mL, 3# hybrid standard in 2# hybrid standard liquid
TAFC and bmGT concentration is 500.00ng/mL in liquid.
(2) phenacetin 10mg is weighed in 10mL volumetric flask, is added acetonitrile dissolution and constant volume, is obtained the Fei Naxi of 1mg/mL
Fourth stock solution takes appropriate phenacetin stock solution to obtain inner mark solution with dilution in acetonitrile, and phenacetin is dense in the inner mark solution
Degree is 13.8ng/mL.
2. untreated samples: taking 3 parts of each 100 μ L of the blank Sputum samples just acquired, respectively into each Sputum samples immediately
10 μ L of 1#~3# hybrid standard liquid, (25 DEG C) standing 4h of room temperature are added, are separately added into 10 μ L of inner mark solution, is vortexed and mixes, obtain 3
Part mixed liquor is added chloroform into each mixed liquor and extracts 2 times, 400 μ L of chloroform is added every time, will extract what same mixed liquor obtained
Extract liquor merges, and then dries up under a nitrogen and gained solid matter acetonitrile percentage by volume is molten for 50% acetonitrile solution
100 μ L are solved and be settled to, are then centrifuged for, LC-MS/MS analysis are carried out according to the condition in embodiment 2 to gained supernatant, each
Concentration is 3 parts parallel, records spectrogram.
Treated sample: taking 3 parts of each 100 μ L of the blank Sputum samples just acquired, respectively into each Sputum samples immediately
10 μ L of 1#~3# hybrid standard liquid and 10 μ L of inner mark solution is added, is vortexed and mixes, 3 portions of mixed liquors are obtained, into each mixed liquor
Chloroform is added to extract 2 times, 400 μ L of chloroform is added every time, the extract liquor that same mixed liquor obtains will be extracted and merged, then in nitrogen
The acetonitrile solution that gained solid matter acetonitrile percentage by volume is 50% is simultaneously dissolved and is settled to 100 μ L by lower drying, then
Gained supernatant is placed for 24 hours at (25 DEG C) of room temperature, then carries out LC-MS/MS analysis according to the condition in embodiment 2 by centrifugation,
Each concentration is 3 parts parallel, records spectrogram.
Precision relative standard deviation RSD and accuracy relative deviation RE in above-mentioned two situations is calculated, as a result such as table 3
Shown, as shown in Table 3, for untreated samples and treated sample, precision relative standard deviation RSD is no more than
10%, accuracy relative deviation RE are no more than ± 8%, this illustrates that the Sputum samples of acquisition are unprocessed and is no more than being placed at room temperature for
4h, acquisition Sputum samples after treatment be placed at room temperature for be no more than for 24 hours be it is stable, it is accurate to will not influence testing result
Property.
Table 3
Embodiment 5
In the present embodiment, 3 Sputum samples are measured using the method for the invention, by 3 parts of sputum sample numbers
For A#, B#, C# Sputum samples, steps are as follows:
1. the preparation of sample
(1) phenacetin 10mg is weighed in 10mL volumetric flask, is added acetonitrile dissolution and constant volume, is obtained the Fei Naxi of 1mg/mL
Fourth stock solution takes appropriate phenacetin stock solution to obtain inner mark solution with dilution in acetonitrile, and phenacetin is dense in the inner mark solution
Degree is 13.8ng/mL.
(2) each 100 μ L of A#, B#, C# Sputum samples is taken, 10 μ L of inner mark solution is wherein added respectively, is vortexed and mixes, obtain 3
Part mixed liquor is added chloroform into each mixed liquor and extracts 2 times, 400 μ L of chloroform is added every time, will extract what same mixed liquor obtained
Extract liquor merges, and then dries up under a nitrogen and gained solid matter acetonitrile percentage by volume is molten for 50% acetonitrile solution
100 μ L are solved and be settled to, are then centrifuged for, gained supernatant is sample, is successively denoted as A#, B#, C# sample.
2. the measurement of sample
A#, B#, C# sample are measured according to following chromatographic condition and Mass Spectrometry Conditions using LC-MS/MS method, obtained
A#, B#, C# sample spectrogram record the peak area of TAFC, bmGT and phenacetin in each sample spectrogram；
Chromatographic condition: Agilent Extend C18 chromatographic column (3.0 × 100mm of specification, 3.5 μ of column packing partial size are used
M), gradient elution, 35 DEG C of column temperature, flow velocity 0.4mL/min, 3 μ L of sample volume, mobile phase A are carried out using mobile phase A and Mobile phase B
For the aqueous formic acid of 0.1wt%, Mobile phase B is acetonitrile.
Condition of gradient elution are as follows: the percentage by volume of Mobile phase B is 25%, mobile phase A in 0min to 1min, eluent
Percentage by volume be 75%, be greater than 1min to 4min, the percentage by volume of Mobile phase B is linearly increasing by 25% in eluent
Percentage by volume to 95%, mobile phase A is linearly reduced by 75% to 5%, is being greater than 4min to 6.5min, is being flowed in eluent
The percentage by volume of phase B is 95%, the percentage by volume of mobile phase A is 5%；
Mass Spectrometry Conditions: electro-spray ionization source, positive ion mode, multiple-reaction monitoring mode, capillary voltage 3500V are done
350 DEG C of pathogenic dryness temperature, dry gas stream speed 5L/min, 350 DEG C of sheath temperature degree, sheath gas 11L/min, Delta EMV 300V；
The mass-to-charge ratio m/z for measuring the parent ion of acetic acid fusarinine C is 928.3, and the mass-to-charge ratio m/z of daughter ion is 251, and fragmentation voltage is
240V, collision voltage 65V；The mass-to-charge ratio m/z for measuring the parent ion of double methyl mercapto gliotoxins is 357.1, the matter of daughter ion
Lotus ratio m/z is 309.1, and fragmentation voltage is 76V, collision voltage 5V；Measure phenacetin mass-to-charge ratio m/z be 180.1, son from
The mass-to-charge ratio m/z of son is 138.3, and fragmentation voltage is 120V, collision voltage 15V.
3. the acquisition of testing result
By the peak area of the peak area ratio of TAFC and phenacetin, bmGT and phenacetin in A#, B#, C# sample spectrogram
Ratio is brought into respectively in the equation of linear regression of the working curve of the TAFC that embodiment 1 determines and bmGT, is calculated in each sample
The concentration of TAFC and bmGT, the results are shown in Table 4.
Table 4
Claims (9)
1. the high performance liquid chromatography-tandem mass of acetic acid fusarinine C and double methyl mercapto gliotoxins while the side of detection in sputum
Method, it is characterised in that steps are as follows:
(1) preparation of standard specimen and sample
1. acetic acid fusarinine C and double methyl mercapto gliotoxins are dissolved with methanol, and a series of acetic acid are obtained with dilution in acetonitrile
The hybrid standard liquid of fusarinine C and double methyl mercapto gliotoxin concentration gradient variations；Phenacetin solution is prepared as internal standard
Solution；
2. taking isometric each hybrid standard liquid, isometric blank Sputum samples are separately added into each hybrid standard liquid and waiting
Then the inner mark solution of volume is uniformly mixed so as to obtain a series of mixed liquors, extractant is added into each mixed liquor respectively and extracts 2~3 times,
The extract liquor that same mixed liquor obtains will be extracted to merge, extractant is removed under nitrogen protection, obtain a series of solid matters, it will be each
Solid matter dissolves with acetonitrile solution respectively and is settled to same volume, and centrifugation, a series of supernatants of gained are standard specimen；
3. inner mark solution is added into Sputum samples to be measured and mixes, then extractant is added into the Sputum samples of gained containing the internal standard
Extraction 2~3 times, combining extraction liquid simultaneously removes extractant under nitrogen protection, and gained solid matter is dissolved with acetonitrile solution,
It is then centrifuged for, gained supernatant is sample；
(2) measurement of standard specimen and sample
Using Liquid Chromatography-Tandem Mass Spectrometry to each standard specimen and sample obtained by step (1) according to following chromatographic condition and mass spectrum item
Part is measured, and obtains standard specimen and sample spectrogram, records acetic acid fusarinine C in each standard specimen and sample spectrogram, double methyl mercapto glue
The peak area of mould toxin and phenacetin；
Chromatographic condition: using Agilent Extend C18 chromatographic column, carries out gradient elution, stream using mobile phase A and Mobile phase B
Dynamic phase A is the aqueous formic acid of 0.1wt%~0.15wt%, and Mobile phase B is acetonitrile, condition of gradient elution are as follows: 0min extremely
1min, the percentage by volume of Mobile phase B is 25% in eluent, the percentage by volume of mobile phase A is 75%, be greater than 1min,
Less than or equal to 4min, the percentage by volume of Mobile phase B is linearly increasing to 95%, mobile phase A by 25% in eluent volume hundred
Score is being greater than 4min, the percentage by volume of Mobile phase B is 95%, mobile phase A in eluent by 75% linear reduction to 5%
Percentage by volume be 5%；
Mass Spectrometry Conditions: electro-spray ionization source, positive ion mode, multiple-reaction monitoring mode, 3500~3550V of capillary voltage,
Dry 350~360 DEG C of temperature degree, dry gas stream 5~6L/min of speed, 350~360 DEG C of sheath temperature degree, 11~12L/ of sheath gas
300~320V of min, Delta EMV；The mass-to-charge ratio m/z for measuring the parent ion of acetic acid fusarinine C is 928.3, daughter ion
Mass-to-charge ratio m/z is 251, and fragmentation voltage is 240V, collision voltage 65V；Measure the matter of the parent ion of double methyl mercapto gliotoxins
Lotus ratio m/z is 357.1, and the mass-to-charge ratio m/z of daughter ion is 309.1, and fragmentation voltage is 76V, collision voltage 5V；Measure Fei Naxi
The mass-to-charge ratio m/z of fourth is 180.1, and the mass-to-charge ratio m/z of daughter ion is 138.3, and fragmentation voltage is 120V, collision voltage 15V；
(3) acquisition of testing result
Using the concentration of acetic acid fusarinine C in each standard specimen as abscissa, with acetic acid fusarinine C and Fei Naxi in each standard specimen spectrogram
The peak area ratio of fourth is ordinate, draws the working curve of acetic acid fusarinine C；With methyl mercapto gliotoxins double in each standard specimen
Concentration be abscissa, using the peak area ratio of methyl mercapto gliotoxins double in each standard specimen spectrogram and phenacetin as ordinate,
Draw the working curve of double methyl mercapto gliotoxins；
By the peak area ratio of acetic acid fusarinine C and phenacetin, double methyl mercapto gliotoxins and Fei Naxi in sample spectrogram
The peak area ratio of fourth brings the equation of linear regression of the working curve of acetic acid fusarinine C and double methyl mercapto gliotoxins into respectively
In, calculate the concentration of acetic acid fusarinine C and double methyl mercapto gliotoxins in sample.
2. acetic acid fusarinine C and pair high performance liquid chromatography-string of methyl mercapto gliotoxins in sputum according to claim 1
Join mass spectrum Simultaneous Detection, it is characterised in that the extractant is chloroform or methylene chloride.
3. the high-efficient liquid phase color of acetic acid fusarinine C and double methyl mercapto gliotoxins in sputum according to claim 1 or claim 2
Spectrum-tandem mass spectrum Simultaneous Detection, which is characterized in that step (1) 2. in the additional amount of extractant every time be the mixed of containing the internal standard
Close 3~4 times of working solution volume, step (1) 3. in, the additional amount of each extractant is to obtain the Sputum samples volume of containing the internal standard
3~4 times.
4. the high-efficient liquid phase color of acetic acid fusarinine C and double methyl mercapto gliotoxins in sputum according to claim 1 or claim 2
Spectrum-tandem mass spectrum Simultaneous Detection, it is characterised in that in the acetonitrile solution percentage by volume of acetonitrile be 35%~
50%.
5. acetic acid fusarinine C and pair high performance liquid chromatography-string of methyl mercapto gliotoxins in sputum according to claim 3
Join mass spectrum Simultaneous Detection, it is characterised in that the percentage by volume of acetonitrile is 35%~50% in the acetonitrile solution.
6. the high-efficient liquid phase color of acetic acid fusarinine C and double methyl mercapto gliotoxins in sputum according to claim 1 or claim 2
Spectrum-tandem mass spectrum Simultaneous Detection, it is characterised in that by phenacetin with acetonitrile be configured to phenacetin concentration be 13~
The solution of 14ng/mL is as inner mark solution.
7. acetic acid fusarinine C and pair high performance liquid chromatography-string of methyl mercapto gliotoxins in sputum according to claim 3
Join mass spectrum Simultaneous Detection, it is characterised in that it is 13~14ng/mL that phenacetin, which is configured to phenacetin concentration with acetonitrile,
Solution as inner mark solution.
8. acetic acid fusarinine C and pair high performance liquid chromatography-string of methyl mercapto gliotoxins in sputum according to claim 4
Join mass spectrum Simultaneous Detection, it is characterised in that it is 13~14ng/mL that phenacetin, which is configured to phenacetin concentration with acetonitrile,
Solution as inner mark solution.
9. acetic acid fusarinine C and pair high performance liquid chromatography-string of methyl mercapto gliotoxins in sputum according to claim 5
Join mass spectrum Simultaneous Detection, it is characterised in that it is 13~14ng/mL that phenacetin, which is configured to phenacetin concentration with acetonitrile,
Solution as inner mark solution.
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