CN105136963A - UPLC/MS/MS detection method of 4'-hydroxymephenytoin concentration in liver microsome - Google Patents
UPLC/MS/MS detection method of 4'-hydroxymephenytoin concentration in liver microsome Download PDFInfo
- Publication number
- CN105136963A CN105136963A CN201510642504.8A CN201510642504A CN105136963A CN 105136963 A CN105136963 A CN 105136963A CN 201510642504 A CN201510642504 A CN 201510642504A CN 105136963 A CN105136963 A CN 105136963A
- Authority
- CN
- China
- Prior art keywords
- detection method
- mephenetoin
- sample
- hydroxyl
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a UPLC/MS/MS detection method of the 4'-hydroxymephenytoin concentration in a liver microsome. The UPLC/MS/MS detection method comprises the following steps that 1, a liver microsome incubation system is established, internal standard substance and serial concentrations of 4'-hydroxymephenytoin comparison products are added to prepare a standard curve sample; 2, the standard curve sample is injected into a UPLC/MS/MS liquid chromatograph/mass spectrometer for detection, and a standard 4'-hydroxymephenytoin curve is obtained according to a detection result; 3, a sample to be detected is prepared by adopting the same method as the step 1 and is injected into the UPLC/MS/MS liquid chromatograph/mass spectrometer, detection is performed under the same conditions as the step 2, and the 4'-hydroxymephenytoin concentration in the sample to be detected is obtained according to the standard curve. The UPLC/MS/MS detection method is short in analysis time, high in sensitivity and small in sample size, meets the methodological validation requirement, is suitable for determination of the 4'-hydroxymephenytoin concentration in the liver microsome incubation sample and has 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 4 '-hydroxyl Mephenetoin concentration 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.
4 '-hydroxyl Mephenetoin (S-4-Hydroxymephenytoin) is the metabolic product of CYP2C19 probe substrate S-Mephenetoin.At present, existing patent CN102080122A reports the HPLC/MS/MS detection method of 4 '-hydroxyl Mephenetoin concentration in a kind of hepatomicrosome.But the gradient elution program of the method continues 8 minutes, consuming time longer, sample size is comparatively large, is unsuitable for the concentration detecting separately 4 '-hydroxyl Mephenetoin.
Therefore, need a kind of specificity at present badly stronger, more fast the detection method of 4 '-hydroxyl Mephenetoin concentration in hepatomicrosome.
Summary of the invention
For solving the problem, the invention provides the UPLC/MS/MS detection method of 4 '-hydroxyl Mephenetoin concentration in a kind of hepatomicrosome, comprising the following steps:
(1) set up liver particle body temperature and incubate system, add 4 '-hydroxyl Mephenetoin reference substance 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 ion pair of monitoring is: the parent-daughter ion of 4 '-hydroxyl Mephenetoin is to the parent-daughter ion pair with internal standard compound;
The typical curve of 4 '-hydroxyl Mephenetoin is obtained according to testing result;
(3) according to the method that step (1) is identical, system of substrate S-Mephenetoin 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 the 4 '-hydroxyl Mephenetoin concentration in testing sample according to typical curve.
Wherein, the time that temperature is incubated is 40min.
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 2 μ L.
Preferably, in described positive ion multiple-reaction monitoring, the parent-daughter ion of 4 '-hydroxyl Mephenetoin is to being m/z235.05 → m/z150.01, and taper hole voltage is 35V, and collision voltage is 16V; 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: 4 '-hydroxyl Mephenetoin is 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 reference standards 4 '-hydroxyl Mephenetoin 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.
Be different from existing HPLC method, the present invention is UPLC method, and exclusively detect 4 ' in hepatomicrosome-hydroxyl Mephenetoin concentration, the stratographic analysis time only needs 3 minutes, and sample size only needs 2 μ L, and analysis time is short, highly sensitive, sample size is little.Simultaneously, detection method specificity is strong, matrix effect is little, linear and scope wide, batch in and batch between veracity and precision high, sample injection disc stability is high, meet Method validation requirement, be applicable to the mensuration that liver particle body temperature incubates 4 '-hydroxyl Mephenetoin concentration 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
S-4-OH-mephenytoin represents 4 '-hydroxyl Mephenetoin.
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/z235.05 → m/z150.01, taper hole voltage 35V, collision voltage 16V, is also Mass Spectrometry Conditions of the present invention.
The Mass Spectrometry Conditions of Fig. 3 is: ion pair: m/z235.05 → m/z133.09, taper hole voltage 35V, collision voltage 18V.
The Mass Spectrometry Conditions of Fig. 4 is: ion pair: m/z235.05 → m/z105.11, taper hole voltage 35V, collision voltage 34V.
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 4 '-hydroxyl Mephenetoin representative standard curve
Embodiment
Embodiment 1
Described reagent and instrument are all from commercially available commodity.
1, reference substance and interior mark
(S)-4 '-hydroxyl Mephenetoin (OH-MTP): molecular weight 234.25, purity 98%, lot number 6-DHL-127-1 (TCR);
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) set up liver particle body temperature and incubate system, add 4 '-hydroxyl Mephenetoin reference substance 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; 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): 5ng/mL brocasipal acetonitrile solution
Gradient elution:
The typical curve of 4 '-hydroxyl Mephenetoin is obtained according to testing result;
(3) according to the method that step (1) is identical, system of substrate S-Mephenetoin and liver particle body temperature being incubated is total to temperature and incubates 40min, 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 the 4 '-hydroxyl Mephenetoin concentration 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/z235.05 → m/z150.01, taper hole voltage 35V, collision voltage 16V are also Mass Spectrometry Conditions of the present invention.
Result display intensity is 7638454.
(2) Mass Spectrometry Conditions of Fig. 3 is:
Ion pair: m/z235.05 → m/z133.09, taper hole voltage 35V, collision voltage 18V.
Result display intensity is 2131694.
(3) Mass Spectrometry Conditions of Fig. 4 is:
Ion pair: m/z235.05 → m/z105.11, taper hole voltage 35V, collision voltage 34V.
Result display intensity is 1390469.
Passable as apparent from 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, 4 '-hydroxyl Mephenetoin and interior mark brocasipal as illustrated in figs. 5-7.
Result shows, and liver particle temperature to incubate in system endogenous material to 4 '-hydroxyl Mephenetoin 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 14 ' investigation of-hydroxyl Mephenetoin matrix effect
Result shows, and 4 '-hydroxyl Mephenetoin matrix effect is 91.6% ~ 101.9%, proves 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 24 '-hydroxyl Mephenetoin typical curve
Result shows, and detection method is within the scope of 5 ~ 500nM, 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 34 '-hydroxyl Mephenetoin lower limit of quantitation
Result shows, and the lower limit of quantitation of detection method is 5nM, highly sensitive.
5.5 batch in 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 44 '-hydroxyl Mephenetoin criticize interior and batch between accuracy and precision
Result shows, and in batch and between criticizing, accuracy (Dev%) is between-11.44% ~ 7.6%, and precision (RSD%) is between 1.52% ~ 8.64%.
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 54 '-hydroxyl Mephenetoin 4 DEG C of sample injection disc shelf-stabilities (72h)
Result shows, and places stablize for 3 days at 4 DEG C of sample injection discs.
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 that 0.2mg/mL, probe substrate S-Mephenetoin concentration is respectively 5,7.5,10,15,20,25,50 and 100 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 after reaction 40min, abundant mixing 3min, in 4 DEG C, the centrifugal 10min of 13000rpm, gets metabolin 4 '-hydroxyl Mephenetoin growing amount that supernatant measures S-Mephenetoin 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 S-Mephenetoin concentration chosen is respectively 5,7.5,10,15,20,25,50 and 100 μMs, specific inhibitor ticlopidine concentration 0.5,1,2 and 4 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) start reaction, fully mix 3min, in 4 DEG C, the centrifugal 10min of 13000rpm, gets the growing amount that supernatant measures 4 '-hydroxyl Mephenetoin in the process of the present invention.
In sum, detection method specificity is strong, matrix effect is little, linear and scope wide, batch in and batch between veracity and precision high, sample injection disc stability is high, meet Method validation requirement, be applicable to the mensuration that liver particle body temperature incubates 4 '-hydroxyl Mephenetoin concentration in sample, be with a wide range of applications.
Claims (10)
1. the UPLC/MS/MS detection method of 4 '-hydroxyl Mephenetoin concentration in hepatomicrosome, comprises the following steps:
(1) set up liver particle body temperature and incubate system, add 4 '-hydroxyl Mephenetoin reference substance 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:
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 4 '-hydroxyl Mephenetoin is to the parent-daughter ion pair with internal standard compound;
The typical curve of 4 '-hydroxyl Mephenetoin is obtained according to testing result;
(3) according to the method that step (1) is identical, system of substrate S-Mephenetoin and liver particle body temperature being incubated is total to temperature and incubates, the testing sample containing internal standard compound is prepared according to the method that step (1) is identical, testing sample is injected UPLC/MS/MS LC-MS instrument, the condition identical according to step (2) detects, and obtains the 4 '-hydroxyl Mephenetoin concentration 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 2 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 4 '-hydroxyl Mephenetoin is to being m/z235.05 → m/z150.01, and taper hole voltage is 35V, and collision voltage is 16V; 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: 4 '-hydroxyl Mephenetoin is 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510642504.8A CN105136963A (en) | 2015-09-30 | 2015-09-30 | UPLC/MS/MS detection method of 4'-hydroxymephenytoin concentration in liver microsome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510642504.8A CN105136963A (en) | 2015-09-30 | 2015-09-30 | UPLC/MS/MS detection method of 4'-hydroxymephenytoin concentration in liver microsome |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105136963A true CN105136963A (en) | 2015-12-09 |
Family
ID=54722388
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510642504.8A Pending CN105136963A (en) | 2015-09-30 | 2015-09-30 | UPLC/MS/MS detection method of 4'-hydroxymephenytoin concentration in liver microsome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105136963A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010036440A1 (en) * | 2000-03-30 | 2001-11-01 | Friedman Hylar L. | Methods of rapid screening of cytochrome CYP2C19 status using mephenytoin |
| CN102080122A (en) * | 2010-11-26 | 2011-06-01 | 吉林大学 | Detecting method for simultaneously measuring metabolic products of probe medicines of five CYP450 enzymes |
| CN102816830A (en) * | 2012-07-23 | 2012-12-12 | 上海市第六人民医院 | Method for detecting inhibitory effect of drug on human liver cytochrome P450 |
| CN104928350A (en) * | 2015-03-24 | 2015-09-23 | 中国药科大学 | Method for rapidly screening in-vitro inhibitory effect of nine human liver CYP450 enzymes |
-
2015
- 2015-09-30 CN CN201510642504.8A patent/CN105136963A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010036440A1 (en) * | 2000-03-30 | 2001-11-01 | Friedman Hylar L. | Methods of rapid screening of cytochrome CYP2C19 status using mephenytoin |
| CN102080122A (en) * | 2010-11-26 | 2011-06-01 | 吉林大学 | Detecting method for simultaneously measuring metabolic products of probe medicines of five CYP450 enzymes |
| CN102816830A (en) * | 2012-07-23 | 2012-12-12 | 上海市第六人民医院 | Method for detecting inhibitory effect of drug on human liver cytochrome P450 |
| CN104928350A (en) * | 2015-03-24 | 2015-09-23 | 中国药科大学 | Method for rapidly screening in-vitro inhibitory effect of nine human liver CYP450 enzymes |
Non-Patent Citations (1)
| Title |
|---|
| LIES DE BOCK等: "Development and validation of a fast and sensitive UPLC–MS/MS method for the quantification of six probe metabolites for the in vitro determination of cytochrome P450 activity", 《TALANTA》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xue et al. | Chemical methods for the simultaneous quantitation of metabolites and proteins from single cells | |
| Qin et al. | A high‐throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography–tandem mass spectrometry | |
| Jain et al. | Adsorptive stripping voltammetric determination of pyridostigmine bromide in bulk, pharmaceutical formulations and biological fluid | |
| Hamuro et al. | High-resolution HDX-MS of Cytochrome c using pepsin/fungal protease type XIII mixed bed column | |
| Monton et al. | A Sol− Gel-Derived Acetylcholinesterase Microarray for Nanovolume Small-Molecule Screening | |
| Farcaş et al. | Capillary electrophoresis in the context of drug discovery | |
| Spaggiari et al. | Phenotyping of CYP450 in human liver microsomes using the cocktail approach | |
| Liuni et al. | Measuring kinetic isotope effects in enzyme reactions using time-resolved electrospray mass spectrometry | |
| Vela et al. | Simultaneous quantitation of the nucleotide analog adefovir, its phosphorylated anabolites and 2′-deoxyadenosine triphosphate by ion-pairing LC/MS/MS | |
| CN105136961A (en) | UPLC/MS/MS detection method of 6 alpha-hydroxypaclitaxel concentration in liver microsome | |
| Řemínek et al. | New capillary electrophoretic method for on‐line screenings of drug metabolism mediated by cytochrome P 450 enzymes | |
| Ye et al. | Study of the voltammetric behavior of jatrorrhizine and its sensitive determination at electrochemical pretreatment glassy carbon electrode | |
| CN103630598B (en) | Electrochemical detection method of cocaine based on rolling circle amplification and supramolecular aptamer | |
| Trashin et al. | Label‐free impedance aptasensor for major peanut allergen Ara h 1 | |
| Nouws et al. | Electroanalytical determination of paroxetine in pharmaceuticals | |
| CN105319296A (en) | Measuring method for methyl alcohol content | |
| Canoura et al. | Suite of aptamer-based sensors for the detection of fentanyl and its analogues | |
| CN105136962A (en) | UPLC/MS/MS detection method of 1-hydroxymidazolam concentration in liver microsome | |
| CN105277635A (en) | UPLC/MS/MS detection method of dextrorphan concentration of liver microsomes | |
| CN105158375A (en) | UPLC/MS/MS detection method for acetaminophen concentration in liver microsome | |
| CN105181872A (en) | UPLC/MS/MS (ultra-performance liquid chromatography/tandem mass spectrometry) detection method for concentration of 6 beta-hydroxytestosterone in liver microsome | |
| Bi et al. | Studies on metabolites and metabolic pathways of bulleyaconitine A in rat liver microsomes using LC‐MSn combined with specific inhibitors | |
| CN105136963A (en) | UPLC/MS/MS detection method of 4'-hydroxymephenytoin concentration in liver microsome | |
| CN105241992B (en) | The UPLC/MS/MS detection methods of 4 ' hydroxyl diclofenac concentration in a kind of hepatomicrosome | |
| CN105241974A (en) | UPLC/MS/MS detection method of concentration of hydroxybupropion in liver microsome |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151209 |
|
| RJ01 | Rejection of invention patent application after publication |