CN105181872A - UPLC/MS/MS (ultra-performance liquid chromatography/tandem mass spectrometry) detection method for concentration of 6 beta-hydroxytestosterone in liver microsome - Google Patents
UPLC/MS/MS (ultra-performance liquid chromatography/tandem mass spectrometry) detection method for concentration of 6 beta-hydroxytestosterone in liver microsome Download PDFInfo
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Abstract
The invention discloses a UPLC/MS/MS (ultra-performance liquid chromatography/tandem mass spectrometry) detection method for concentration of 6 beta-hydroxytestosterone in liver microsome. The method comprises steps as follows: (1), an incubation system for the liver microsome is established, an internal standard substance and a 6 beta-hydroxytestosterone reference substance with serial concentration are added, and a standard curve sample is prepared; (2), the standard curve sample is injected into a UPLC/MS/MS liquid chromatograph/mass spectrometer for detection, and a standard curve of 6 beta-hydroxytestosterone is obtained according to a detection result; (3), a to-be-detected sample is prepared with the same method in the step (1), injected to the UPLC/MS/MS liquid chromatograph/mass spectrometer and detected under the same conditions in the step (2), and the concentration of 6 beta-hydroxytestosterone in the to-be-detected sample is obtained according to the standard curve. According to the detection method, the analysis time is short, the sensitivity is high, the sample amount is small, and the method meets requirements of methodology validation, is suitable for measurement of the concentration of 6 beta-hydroxytestosterone in a liver microsome incubation sample and has a wide application prospect.
Description
Technical field
The present invention relates to the detection method of medicine, be specifically related to the UPLC/MS/MS detection method of 6 beta-hydroxy testosterone concentrations in hepatomicrosome.
Background technology
CYP450 enzyme is the Major Enzymes system participating in drug metabolism, comprises the isodynamic enzyme closely-related with drug metabolism such as CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4.Research shows, the generation of most of drug-drug interactions is to the induction of CYP450 enzymatic activity and suppress relevant, wherein causes primarily of enzyme level.Utilize in-vitro method to carry out drug metabolism study, can effectively shorten the new drug development cycle, directly observe enzyme to the selectivity metabolism of substrate and substrate to the induction of enzyme and suppression, determine drug-metabolic pathway, drug interaction potential in predictor.At present, conventional in-vitro method is probe medicament method, and the method incubates system by setting up liver particle body temperature, thus establishes drug metabolism study model.
6 beta-hydroxy testosterones (6 β-hydroxyl-testosterone) are the metabolic products of CYP3A4 probe substrate testosterone.At present, the detection method of 6 beta-hydroxy testosterone concentrations in a kind of simple and quick hepatomicrosome is needed badly.
Summary of the invention
For solving the problem, the invention provides the UPLC/MS/MS detection method of 6 beta-hydroxy testosterone concentrations in a kind of hepatomicrosome, comprising the following steps:
(1) set up liver particle body temperature and incubate system, preparation contains the typical curve sample of 6 beta-hydroxy testosterone reference substances and internal standard compound;
(2) typical curve sample is injected UPLC/MS/MS LC-MS instrument to detect;
Chromatographic condition is as follows:
Chromatographic column: C18 chromatographic column;
Mobile phase: mobile phase A is 0.1% aqueous formic acid, Mobile phase B is 0.1% formic acid acetonitrile solution;
Condition of gradient elution is:
Working time is 3 minutes;
Mass Spectrometry Conditions is as follows:
Ion gun: electric spray ion source;
Detection mode: positive ion multiple-reaction monitoring, the ion pair of monitoring is: the parent-daughter ion of 6 beta-hydroxy testosterones is to the parent-daughter ion pair with internal standard compound;
The typical curve of 6 beta-hydroxy testosterones is obtained according to testing result;
(3) according to the method that step (1) is identical, system of substrate testosterone and liver particle body temperature being incubated is total to temperature and incubates 20min, prepare the testing sample containing internal standard compound, testing sample is injected UPLC/MS/MS LC-MS instrument, the condition identical according to step (2) detects, and obtains 6 beta-hydroxy testosterone concentrations in testing sample according to typical curve.
Wherein, the time that temperature is incubated is 20min.
Preferably, described internal standard compound is brocasipal.
Preferably, in described chromatographic condition, the flow velocity of mobile phase is 0.3mL/min.
Preferably, in described chromatographic condition, column temperature is 40 DEG C.
Preferably, in described chromatographic condition, chromatographic column is ACQUITYUPLC-BEHC18 chromatographic column, and specification is 1.7 μm, 2.1 × 50mm.
Preferably, in described chromatographic condition, sample size is 1 μ L.
Preferably, in described positive ion multiple-reaction monitoring, the parent-daughter ion of 6 beta-hydroxy testosterones is to being m/z305.11 → m/z287.35, and taper hole voltage is 44V, and collision voltage is 14V; The parent-daughter ion of brocasipal is to being m/z270.10 → m/z181.08, and taper hole voltage is 15V, and collision voltage is 12V.
Preferably, in described Mass Spectrometry Conditions, capillary voltage is 3000V, and ion source temperature is 150 DEG C, and desolvation temperature is 350 DEG C, and desolvation gas flow rate is 650L/h.
Preferably, in described Mass Spectrometry Conditions, taper hole gas flow rate is 150L/h, and atomization gas pressure is 6.0bar.
Preferably, the typical curve sample preparation methods of described step (1) is: 6 beta-hydroxy testosterones are added liver particle body temperature and incubates system, and the described liver particle body temperature system of incubating comprises hepatomicrosome, G-6-P, glucose-6-phosphate dehydrogenase (G6PD), MgCl
2, PBS and NADP, then add containing interior target acetonitrile solution, mixing, centrifugal, get supernatant, obtain typical curve sample.
Wherein, the preparation of typical curve sample can be carried out by the following method: respectively the reference standards 6 beta-hydroxy testosterone of series concentration is added liver particle body temperature system of incubating and (comprise hepatomicrosome, G-6-P, glucose-6-phosphate dehydrogenase (G6PD), MgCl
2, PBS and NADP), then add containing interior target acetonitrile solution, fully mix 3min, in 4 DEG C, the centrifugal 10min of 13000rpm, gets supernatant and get final product.Liver particle can adopt the liver particle of people, rat, mouse, dog, rhesus macaque, machin.
The present invention exclusively detects 6 beta-hydroxy testosterone concentrations in hepatomicrosome, and the stratographic analysis time only needs 3 minutes, and sample size only needs 1 μ L, and analysis time is short, highly sensitive, sample size is little.Simultaneously, detection method specificity is strong, matrix effect is little, the range of linearity is wide, batch in and batch between veracity and precision high, sample injection disc stability is high, working fluid stability is high, meet Method validation requirement, be applicable to the mensuration that liver particle body temperature incubates 6 beta-hydroxy testosterone concentrations in sample, be with a wide range of applications.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
Hepatomicrosome behaviour hepatomicrosome used.
Fig. 1 is the preferred result of parent ion and taper hole voltage.
The Mass Spectrometry Conditions of Fig. 2 is: ion pair: m/z305.11 → m/z226.69, taper hole voltage 44V, collision voltage 12V.
The Mass Spectrometry Conditions of Fig. 3 is: ion pair: m/z305.11 → m/z287.35, taper hole voltage 44V, collision voltage 14V, is also Mass Spectrometry Conditions of the present invention.
The Mass Spectrometry Conditions of Fig. 4 is: ion pair: m/z305.11 → m/z105.31, taper hole voltage 44V, collision voltage 38V.
Fig. 5 is the chromatogram of dummy.
Fig. 6 is LLOQ (lower limit of quantitation) sample chromatogram.
Fig. 7 is sample to be tested chromatogram.
Fig. 8 is 6 beta-hydroxy testosterone representative standard curves.
Embodiment
Embodiment 1
Described reagent and instrument are all from commercially available commodity.
1, reference substance and interior mark
6 beta-hydroxy testosterones (OH-TS): molecular weight 304.42, purity 99%, lot number 051M1145V (sigma-aldrich);
Orphenadrine citrate (in positive ion mark): purity 99%, lot number 048F0510V (sigma-aldrich).
2, key instrument
3, main software used is tested
UNIFI1.6.1:UPLC/MS/MS data acquisition and concentration calculate.
4, detection method
(1) preparation standard curve sample, adds internal standard compound;
(2) typical curve sample is injected UPLC/MS/MS LC-MS instrument to detect; Testing conditions is as follows:
Chromatographic column: ACQUITYUPLC-BEHC18,1.7 μm, 2.1 × 50mm (Waters company)
Mobile phase: A:0.1% aqueous formic acid
B:0.1% formic acid acetonitrile solution
Flow velocity: 0.3mL/min
Column temperature: 40 DEG C
Sample injection disc temperature: 4 DEG C
Ion gun: electro-spray ionization source (ESI);
Interior mark (IS): 25ng/mL brocasipal acetonitrile solution
Gradient elution:
Sample size: 1 μ L
Working time: 3min
Retention time: t
r (OH-TS)≈ 1.51min; t
r (IS)≈ 1.57min
Capillary voltage (Capillaryvoltage): 3.00kV
Ion source temperature (SourceTemperature): 150 DEG C
Desolvation temperature (DesolvationTemperature): 350 DEG C
Desolvation gas flow rate (Desolvationgasflow): 650L/h
Taper hole gas flow rate (Conegasflow): 150L/h
Atomization gas pressure (Nebulizergaspressure): 6.0bar
Detection mode: positive ion multiple-reaction monitoring (MRM)
Ion pair: m/z305.11 → m/z287.35 (OH-TS), m/z270.10 → m/z181.08 (IS)
Taper hole voltage (Conevoltage): 44V (OH-TS) and 15V (IS)
Collision voltage (Collisionenergy): 14V (OH-TS) and 12V (IS)
The typical curve of 6 beta-hydroxy testosterones is obtained according to testing result;
(3) according to the method that step (1) is identical, system of substrate testosterone and liver particle body temperature being incubated is total to temperature and incubates 20min, prepare the testing sample containing internal standard compound, testing sample is injected UPLC/MS/MS LC-MS instrument, the condition identical according to step (2) detects, and obtains 6 beta-hydroxy testosterone concentrations in testing sample according to typical curve.
Determining that in detection method various process parameters of the present invention, inventor screens the chromatographic condition of the inventive method and Mass Spectrometry Conditions, result as Figure 1-4.
Fig. 1 is the preferred result of parent ion and taper hole voltage.
The testing result of three kinds of ion pairs is as follows:
(1) Mass Spectrometry Conditions of Fig. 2 is:
Ion pair: m/z305.11 → m/z226.69, taper hole voltage 44V, collision voltage 12V.
Result display intensity is 479905.
(2) Mass Spectrometry Conditions of Fig. 3 is:
Ion pair: m/z305.11 → m/z287.35, taper hole voltage 44V, collision voltage 14V are also Mass Spectrometry Conditions of the present invention.
Result display intensity is 342908.
(3) Mass Spectrometry Conditions of Fig. 4 is:
Ion pair: m/z305.11 → m/z105.31, taper hole voltage 44V, collision voltage 38V.
Result display intensity is 318717.
Consider the result of above-mentioned collection of illustrative plates and intensity, detection method of the present invention is best.
5, methodological study
Investigated respectively the specificity of detection method, matrix effect, linear and scope, batch in and batch between veracity and precision, sample injection disc stability, working fluid stability etc.Specific as follows:
5.1 specificity
Get liver particle respectively, prepare blank temperature and incubate system sample, investigate endogenous material to determinand and the impact of interior target.In dummy, the interference of endogenous material should not exceed 20% of determinand lower limit of quantitation (LLOQ) and 5% of interior mark response.
The typical color spectrogram of dummy, 6 beta-hydroxy testosterones and interior mark brocasipal as illustrated in figs. 5-7.
Result shows, and liver particle temperature to incubate in system endogenous material to 6 beta-hydroxy testosterones and interior mark brocasipal without impact.
5.2 matrix effect
Prepare blank liver particle body temperature and incubate system sample, add the determinand reference substance working fluid of basic, normal, high three concentration and interior mark respectively, prepare not containing matrix sample simultaneously, investigate its matrix effect, should more than 85% ~ 115%.
Result is as shown in table 1.
Table 16 beta-hydroxy testosterone matrix effect is investigated
Result shows, and 6 beta-hydroxy testosterone matrix effects are 102.5% ~ 106.5%, prove that the inventive method matrix effect is little.
5.3 typical curve
Typical curve: the determinand typical curve sample of preparation finite concentration gradient, with the ratio of determinand and interior mark peak area for ordinate (y), concentration is horizontal ordinate (x), through weighted least-squares method (W=1/x
2) return calculating linear equation.
Result is as shown in table 2 and Fig. 8.
Table 26 beta-hydroxy testosterone typical curve
Result shows, and detection method is within the scope of 0.1 ~ 10 μM, and linear relationship is good.
5.4 lower limit of quantitation
Parallel preparation 5 parts of lower limit of quantitation samples (typical curve least concentration), the mean deviation measuring concentration should within theoretical concentration ± 20%.
Result is as shown in table 3.
Table 36 beta-hydroxy testosterone lower limit of quantitation
Result shows, and the lower limit of quantitation of detection method is 0.1 μM, highly sensitive.
In 5.5 batches and batch between accuracy and precision
Veracity and precision: the Quality Control sample preparing basic, normal, high three concentration.The parallel preparation of each concentration 5 parts, METHOD FOR CONTINUOUS DETERMINATION 3 batches, to calculate its concentration when batch typical curve, in batch and batch between deviations in accuracy Dev% ((measuring concentration-theoretical concentration)/theoretical concentration × 100) be no more than ± 15%, precision RSD % is less than 15%.
Result is as shown in table 4.
Table 46 beta-hydroxy testosterone criticize interior and batch between accuracy and precision
Result shows, and in batch and between criticizing, accuracy (Dev%) is between-8.31% ~ 6.25%, and precision (RSD%) is between 1.45% ~ 8.14%.
5.6 sample injection disc shelf-stabilities
Sample injection disc stability: sample detection again to be placed after certain hour by Quality Control sample in sample injection disc.
Result is as shown in table 5.
Table 56 beta-hydroxy testosterone 4 DEG C of sample injection disc shelf-stabilities (48h)
Result shows, and places stablize for 2 days at 4 DEG C of sample injection discs.
5.7 working fluid stability
Result is as shown in table 6.
Table 66 beta-hydroxy testosterone working fluid stability
Result shows, and working fluid is placed in 2 ~ 8 DEG C and stablized for 86 days.
The application of embodiment 2 the inventive method
1, enzyme kinetics test
When measuring enzyme kinetic parameter, the concentration of probe substrate is selected between 1/3Km ~ 3Km usually, and metabolic rate should be linear with the reaction time, and should be less than 10% by the substrate of metabolism.When can generate can be quantitative metabolin alap protein concentration should be adopted to reduce non-specific binding.In conjunction with Km and the trial test result of bibliographical information, choosing hepatomicrosome protein concentration is 0.2mg/mL, and probe substrate testosterone concentration is respectively 5,6,8,10,20,50,100 and 300 μMs.
It is 0.1M phosphate buffer (PBS) that temperature incubates system medium, (final concentration is 10mMG-6-P to comprise probe substrate, hepatomicrosome (final concentration 0.2mg/mL) and NADP regenerative system, 1U/mLG-6-PDH, 10mMMgCl2,0.5mMNADP), final volume 200 μ L, organic solvent content is no more than 1%.First respectively by the probe substrate of variable concentrations and hepatomicrosome, G-6-P, G-6-PDH, MgCl
2, PBS mix rear 37 DEG C of pre-temperature incubate 5min, then add NADP (37 DEG C of pre-temperature are incubated) and start reaction, add containing interior target acetonitrile solution (ice bath) 200 μ L cessation reaction after reaction 20min, abundant mixing 3min, in 4 DEG C, the centrifugal 10min of 13000rpm, get the metabolin 6 beta-hydroxy testosterone growing amount that supernatant measures testosterone with the inventive method, calculate each concentration of substrate enzyme reaction speed respectively, calculate enzyme kinetics parameter Km, Vmax by non-linear regression enzyme kinetics model.
2, depression effect test
It is 0.1MPBS that temperature incubates system medium, and (final concentration is 10mMG-6-P, 1U/mLG-6-PDH, 10mMMgCl to comprise probe substrate, inhibitor, hepatomicrosome (final concentration 0.2mg/mL) and NADP regenerative system
2, 0.5mMNADP), final volume 200 μ L, organic solvent content is no more than 1%.The substrate testosterone concentration chosen is respectively 5,6,8,10,20,50,100 and 300 μMs, specific inhibitor ketoconazole concentration 0.02,0.1,0.5 and 1 μM.First by variable concentrations probe substrate and variable concentrations inhibitor and hepatomicrosome, G-6-P, G-6-PDH, MgCl
2, PBS mix rear 37 DEG C of pre-temperature incubate 5min, then add NADP (37 DEG C of pre-temperature are incubated) and start reaction, add containing interior target acetonitrile solution (ice bath) 200 μ L cessation reaction after 20min, abundant mixing 3min, in 4 DEG C, the centrifugal 10min of 13000rpm, gets the growing amount that supernatant measures 6 beta-hydroxy testosterones in the process of the present invention.Calculate each concentration level enzyme reaction speed, by the matching of non-linear regression enzyme kinetics inhibition, judge to suppress type and calculate to suppress constant Ki.
In sum, detection method specificity is strong, matrix effect is little, the range of linearity is wide, batch in and batch between veracity and precision high, sample injection disc stability is high, working fluid stability is high, dilution effect is little, meet Method validation requirement, be applicable to the mensuration that liver particle body temperature incubates 6 beta-hydroxy testosterone concentrations in sample, be with a wide range of applications.
Claims (10)
1. the UPLC/MS/MS detection method of 6 beta-hydroxy testosterone concentrations in hepatomicrosome, comprises the following steps:
(1) set up liver particle body temperature and incubate system, add 6 beta-hydroxy testosterone reference substances of internal standard compound and series concentration, preparation standard curve sample; ;
(2) typical curve sample is injected UPLC/MS/MS LC-MS instrument to detect;
Chromatographic condition is as follows:
Chromatographic column: C18 chromatographic column;
Mobile phase: mobile phase A is 0.1% aqueous formic acid, Mobile phase B is 0.1% formic acid acetonitrile solution;
Condition of gradient elution is:
Working time is 3 minutes;
Mass Spectrometry Conditions is as follows:
Ion gun: electric spray ion source;
Detection mode: positive ion multiple-reaction monitoring;
The typical curve of 6 beta-hydroxy testosterones is obtained according to testing result;
(3) according to the method that step (1) is identical, system of substrate testosterone and liver particle body temperature being incubated is total to temperature and incubates, prepare the testing sample containing internal standard compound, testing sample is injected UPLC/MS/MS LC-MS instrument, the condition identical according to step (2) detects, and obtains 6 beta-hydroxy testosterone concentrations in testing sample according to typical curve.
2. detection method according to claim 1, is characterized in that: described internal standard compound is brocasipal.
3. detection method according to claim 1, is characterized in that: in described chromatographic condition, and the flow velocity of mobile phase is 0.3mL/min.
4. detection method according to claim 1, is characterized in that: in described chromatographic condition, and column temperature is 40 DEG C.
5. detection method according to claim 1, is characterized in that: in described chromatographic condition, and chromatographic column is ACQUITYUPLC-BEHC18 chromatographic column, and specification is 1.7 μm, 2.1 × 50mm.
6. detection method according to claim 1, is characterized in that: in described chromatographic condition, and sample size is 1 μ L.
7. detection method according to claim 2, is characterized in that: in described positive ion multiple-reaction monitoring, and the parent-daughter ion of 6 beta-hydroxy testosterones is to being m/z305.11 → m/z287.35, and taper hole voltage is 44V, and collision voltage is 14V; The parent-daughter ion of brocasipal is to being m/z270.10 → m/z181.08, and taper hole voltage is 15V, and collision voltage is 12V.
8. the detection method according to any one of claim 1-7, is characterized in that: in described Mass Spectrometry Conditions, and capillary voltage is 3000V, and ion source temperature is 150 DEG C, and desolvation temperature is 350 DEG C, and desolvation gas flow rate is 650L/h.
9. the detection method according to any one of claim 1-7, is characterized in that: in described Mass Spectrometry Conditions, and taper hole gas flow rate is 150L/h, and atomization gas pressure is 6.0bar.
10. the detection method according to any one of claim 1-7, it is characterized in that: the typical curve sample preparation methods of described step (1) is: 6 beta-hydroxy testosterones are added liver particle body temperature and incubates system, the described liver particle body temperature system of incubating comprises hepatomicrosome, G-6-P, glucose-6-phosphate dehydrogenase (G6PD), MgCl
2, PBS and NADP, then add containing interior target acetonitrile solution, mixing, centrifugal, get supernatant, obtain typical curve sample.
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| CN112649535A (en) * | 2020-12-30 | 2021-04-13 | 安领生物医药(苏州)有限公司 | Establishment method and use method of testosterone and 6 beta-hydroxytestosterone detection platform in liver microsome incubation system |
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