CN105241992B - The UPLC/MS/MS detection methods of 4 ' hydroxyl diclofenac concentration in a kind of hepatomicrosome - Google Patents
The UPLC/MS/MS detection methods of 4 ' hydroxyl diclofenac concentration in a kind of hepatomicrosome Download PDFInfo
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Abstract
The invention discloses in a kind of hepatomicrosome 4 ' hydroxyl diclofenac concentration UPLC/MS/MS detection methods, comprise the following steps：(1) set up liver particle body temperature and incubate system, add 4 ' hydroxyl diclofenac reference substances of internal standard substance and series concentration, prepare standard curve sample；(2) standard curve sample injection UPLC/MS/MS LC-MS instrument is detected；The standard curve of 4 ' hydroxyl diclofenacs is obtained according to testing result；(3) testing sample is prepared according to step (1) identical method, testing sample is injected into UPLC/MS/MS LC-MS instrument, detected according to step (2) identical condition, 4 ' hydroxyl diclofenac concentration in testing sample are obtained according to standard curve.Detection method analysis time is short, sensitivity is high, sample size is little, meets Method validation requirement, it is adaptable to which liver particle body temperature incubates the measure of 4 ' hydroxyl diclofenac concentration in sample, is with a wide range of applications.
Description
Technical field
The present invention relates to the detection method of medicine, and in particular to 4 ' in hepatomicrosome-UPLC/ of hydroxyl diclofenac concentration
MS/MS detection methods.
Background technology
CYP450 enzymes be participate in drug metabolism main enzyme system, including CYP1A2, CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6 and CYP3A4 etc. closely related with drug metabolism isozyme.Research shows, most of drug-drug phase
The generation of interaction is related to the induction of CYP450 enzymatic activitys and suppression, wherein mainly being caused by enzyme level.Using in vitro method
Drug metabolism study is carried out, can effectively shorten the new drug development cycle, directly observe selectivity metabolism and substrate pair of the enzyme to substrate
The induction and suppression of enzyme, determines drug-metabolic pathway, the internal potential drug interaction of prediction.At present, conventional external side
Method is probe medicament method, and the method incubates system by setting up liver particle body temperature, so as to establish drug metabolism study model.
4 '-hydroxyl diclofenac is the metabolite of CYP2C9 probe substrate diclofenacs.Therefore, need a kind of liver at present badly
4 ' in microsome-detection method of hydroxyl diclofenac concentration.
The content of the invention
To solve the above problems, the invention provides in a kind of hepatomicrosome 4 '-hydroxyl diclofenac concentration UPLC/
MS/MS detection methods, comprise the following steps：
(1) set up liver particle body temperature and incubate system, add 4 '-hydroxyl diclofenac reference substance of internal standard substance and series concentration,
Prepare standard curve sample；
(2) standard curve sample injection UPLC/MS/MS LC-MS instrument is detected；
Chromatographic condition is as follows：
Chromatographic column：C18 chromatographic columns；
Mobile phase：Mobile phase A is 0.1% aqueous formic acid, and Mobile phase B is 0.1% formic acid acetonitrile solution；
Condition of gradient elution：
Run time is 3 minutes；
Mass Spectrometry Conditions are as follows：
Ion source：Electric spray ion source；
Detection mode：Cation multiple-reaction monitoring, the ion pair of monitoring is：Parent ion-the son of 4 '-hydroxyl diclofenac from
Son to and internal standard substance parent-daughter ion pair；
The standard curve of 4 '-hydroxyl diclofenac is obtained according to testing result；
(3) according to step (1) identical method, substrate diclofenac and liver particle body temperature are incubated system, and temperature is incubated altogether, according to
Step (1) identical method prepares the testing sample containing internal standard substance, by testing sample injection UPLC/MS/MS liquid matter connection
With instrument, detected according to step (2) identical condition, the double chlorine of 4 '-hydroxyl obtained according to standard curve in testing sample are fragrant
Acid concentration.
Wherein, the time that temperature is incubated is 20min.
Preferably, the internal standard substance is chlorzoxazone.
Preferably, in the chromatographic condition, the flow velocity of mobile phase is 0.3mL/min.
Preferably, in the chromatographic condition, column temperature is 40 DEG C.
Preferably, in the chromatographic condition, chromatographic column is ACQUITY UPLC-BEH C18 chromatographic columns, and specification is 1.7 μm,
2.1×50mm。
Preferably, in the chromatographic condition, sample size is 1 μ L.
Preferably, in the anion multiple-reaction monitoring, the parent-daughter ion of 4 '-hydroxyl diclofenac is to for m/
Z309.95 → m/z 265.90, taper hole voltage are 20V, and collision voltage is 14V；The parent-daughter ion of chlorzoxazone is to for m/
167.88 → m/z of z 132.02, taper hole voltage are 36V, and collision voltage is 20V.
Preferably, in the Mass Spectrometry Conditions, capillary voltage is 3000V, and ion source temperature is 150 DEG C, desolvation temperature
Spend for 350 DEG C, desolvation gas flow rate is 650L/h.
Preferably, in the Mass Spectrometry Conditions, taper hole gas flow rate is 150L/h, and atomization gas pressure is 6.0bar.
Preferably, the standard curve sample preparation methods of the step (1) are：Add liver micro- 4 '-hydroxyl diclofenac
Plastochondria temperature incubates system, and the liver particle body temperature incubates system comprising hepatomicrosome, G-6-P, G-6-P dehydrogenation
Enzyme, MgCl2, PBS and NADP, add the acetonitrile solution of containing the internal standard, mix, centrifugation, take supernatant, obtain final product standard curve sample
Product.
Wherein, the preparation of standard curve sample can be carried out by the following method：Respectively by the reference standard of series concentration
Product diclofenac adds liver particle body temperature to incubate system (comprising hepatomicrosome, G-6-P, G-6-P dehydrogenation
Enzyme, MgCl2, PBS and NADP), add the acetonitrile solution of containing the internal standard, fully mix 3min, in 4 DEG C, 13000rpm centrifugations
10min, takes supernatant and obtains final product.Liver particle can adopt people, rat, mice, dog, Rhesus Macacus, the liver particle of machin.
The present invention exclusively detects that, to 4 ' in hepatomicrosome-hydroxyl diclofenac concentration, the chromatography time only needs 3 points
Clock, sample size only need 1 μ L, and analysis time is short, sensitivity is high, sample size is little.Meanwhile, detection method specificity is strong, base
Mass effect is little, the range of linearity is wide, batch in and batch between veracity and precision is high, sample injection disc stability is high, working solution stability is high,
Meet Method validation requirement, it is adaptable to which liver particle body temperature incubates the measure of 4 '-hydroxyl diclofenac concentration in sample, with extensive
Application prospect.
Obviously, the above of the invention, according to the ordinary technical knowledge and customary means of this area, without departing from
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification of other various ways can also be made, is replaced or is changed.
The specific embodiment of form, remakes further specifically to the above of the present invention by the following examples
It is bright.But this scope for being interpreted as above-mentioned theme of the invention should not be only limitted to Examples below.It is all based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Description of the drawings
Hepatomicrosome behaviour hepatomicrosome used.
Fig. 1 is the preferred result of parent ion and taper hole voltage.
The Mass Spectrometry Conditions of Fig. 2 are：Ion pair：309.95 → m/z of m/z 265.90, taper hole voltage 20V, collision voltage
14V, namely the Mass Spectrometry Conditions of the present invention.
The Mass Spectrometry Conditions of Fig. 3 are：Ion pair：309.95 → m/z of m/z 194.08, taper hole voltage 20V, collision voltage
22V。
The Mass Spectrometry Conditions of Fig. 4 are：Ion pair：309.95 → m/z of m/z 229.86, taper hole voltage 20V, collision voltage 6V.
Chromatograms of the Fig. 5 for dummy.
Fig. 6 is LLOQ (lower limit of quantitation) sample chromatogram.
Fig. 7 is sample to be tested chromatogram.
Fig. 8 is 4 '-hydroxyl diclofenac representative standard curve.
Specific embodiment
Embodiment 1
The reagent and instrument are all from commercially available commodity.
1st, reference substance and internal standard
4 '-hydroxyl diclofenac (OH-DCF)：Molecular weight 312.15, purity 99.5%, lot number SZBC286XV (sigma-
aldrich)；
Chlorzoxazone (anion internal standard, LZSZ)：Lot number 100364-200301 (National Institute for Food and Drugs Control).
2nd, key instrument
3rd, main software used by testing
UNIFI 1.6.1：UPLC/MS/MS data acquisitions and concentration are calculated.
4th, detection method
(1) set up liver particle body temperature and incubate system, add 4 '-hydroxyl diclofenac reference substance of internal standard substance and series concentration,
Prepare standard curve sample；
(2) standard curve sample injection UPLC/MS/MS LC-MS instrument is detected；Testing conditions are as follows：
Chromatographic column：ACQUITY UPLC-BEH C18,1.7 μm, 2.1 × 50mm (Waters companies)
Mobile phase：A：0.1% aqueous formic acid
B：0.1% formic acid acetonitrile solution
Flow velocity：0.3mL/min
Column temperature：40℃
Sample injection disc temperature：4℃
Ion source：Electro-spray ionization source (ESI)；
Internal standard (IS)：40ng/mL chlorzoxazone acetonitrile solutions
Gradient elution：
Sample size：1μL
Run time：3min
Retention time：tR(OH-DCF)≈1.65min；tR(IS)≈1.59min
Capillary voltage (Capillary voltage)：3.00kV
Ion source temperature (Source Temperature)：150℃
Desolvation temperature (Desolvation Temperature)： 350℃
Desolvation gas flow rate (Desolvation gas flow)：650L/h
Taper hole gas flow rate (Cone gas flow)：150L/h
Atomization gas pressure (Nebulizer gas pressure)：6.0bar
Detection mode：Anion multiple-reaction monitoring (MRM)
Ion pair：309.95 → m/z of m/z 265.90 (OH-DCF), 167.88 → m/z of m/z 132.02 (IS)
Taper hole voltage (Cone voltage)：20V (OH-DCF) and 36V (IS)
Collision voltage (Collision energy)：14V (OH-DCF) and 20V (IS)
The standard curve of 4 '-hydroxyl diclofenac is obtained according to testing result；
(3) according to step (1) identical method, substrate diclofenac and liver particle body temperature are incubated system, and temperature incubates 20min altogether,
The testing sample containing internal standard substance is prepared, testing sample UPLC/MS/MS LC-MS instrument is injected into, according to step (2) phase
With condition detected, the 4 '-hydroxyl diclofenac concentration in testing sample is obtained according to standard curve.
It is determined that in the detection method various process parameters of the present invention, chromatographic condition and matter of the inventor to the inventive method
Spectral condition is screened, as a 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 are：
Ion pair：The matter of 309.95 → m/z of m/z 265.90, taper hole voltage 20V, collision voltage 14V, namely the present invention
Spectral condition.
As a result show that intensity is 1321370.
(2) Mass Spectrometry Conditions of Fig. 3 are：
Ion pair：309.95 → m/z of m/z 194.08, taper hole voltage 20V, collision voltage 22V.
As a result show that intensity is 362162.
(3) Mass Spectrometry Conditions of Fig. 4 are：
Ion pair：309.95 → m/z of m/z 229.86, taper hole voltage 20V, collision voltage 6V.
As a result show that intensity is 323917.
From the result of above-mentioned collection of illustrative plates and intensity, it is apparent that the detection method of the present invention is optimal.
5th, methodological study
Investigated respectively the specificity of detection method, matrix effect, linear and scope, batch in and batch between accuracy
With precision, sample injection disc stability, working solution stability etc..It is specific as follows：
5.1 specificity
Liver particle being taken respectively, blank temperature being prepared and is incubated system sample, investigating endogenous material affects on determinand and interior target.
The interference of endogenous material in dummy is not to be exceeded the 5% of the 20% and internal standard response of determinand lower limit of quantitation (LLOQ).
The typical chromatogram of dummy, 4 '-hydroxyl diclofenac and internal standard chlorzoxazone is as illustrated in figs. 5-7.
As a result show, liver particle temperature incubate in system endogenous material to 4 '-hydroxyl diclofenac and internal standard chlorzoxazone without
Affect.
5.2 matrix effect
Prepare blank liver particle body temperature and incubate system sample, be separately added into the determinand reference substance work of basic, normal, high three concentration
Make liquid and internal standard, while preparing without matrix sample, investigate its matrix effect, be not to be exceeded 85%～115%.
As a result it is as shown in table 1.
Table 14 '-hydroxyl diclofenac matrix effect is investigated
As a result show, 4 '-hydroxyl diclofenac matrix effect is 104.2%～112.8%, it was demonstrated that the inventive method substrate
Effect is little.
5.3 standard curve
Standard curve：The determinand standard curve sample of finite concentration gradient is prepared, with determinand and internal standard peak area
Ratio be vertical coordinate (y), concentration be abscissa (x), weighted method of least square (W=1/x2) regression Calculation linear equation.
As a result as shown in table 2 and Fig. 8.
Table 24 '-hydroxyl diclofenac standard curve
As a result show, in the range of 0.05～10 μM, linear relationship is good for detection method.
5.4 lower limit of quantitation
5 parts of lower limit of quantitation samples (standard curve least concentration) of parallel preparation, the mean deviation for determining concentration should be resonable
Within concentration ± 20%.
As a result it is as shown in table 3.
Table 34 '-hydroxyl diclofenac lower limit of quantitation
As a result show, the lower limit of quantitation of detection method is 0.05 μM, and sensitivity is high.
In 5.5 batches and batch between accuracy and precision
Veracity and precision：Prepare the Quality Control sample of basic, normal, high three concentration.Each concentration is parallel to prepare 5 parts, even
It is continuous to determine 3 batches, to calculate its concentration when batch standard curve, in batch and batch between deviations in accuracy Dev% ((determine concentration-theory dense
Degree)/theoretical concentration × 100) less than ± 15%, precision RSD % is less than 15%.
As a result it is as shown in table 4.
In 44 '-hydroxyl of table diclofenac batch and batch between accuracy and precision
As a result show, in batch and batch between accuracy (Dev%) between -14.40%～5.20%, precision (RSD%)
Between 2.38%～10.45%.
5.6 sample injection disc shelf-stabilities
Sample injection disc stability：Quality Control sample places after certain hour sample detection again in the sample injection disc.
As a result it is as shown in table 5.
54 '-hydroxyl of table diclofenac, 4 DEG C of sample injection disc shelf-stabilities
As a result show, place 2 days in 4 DEG C of sample injection discs and stablize.
5.7 working solution stability
As a result it is as shown in table 6.
Table 64 '-hydroxyl diclofenac working solution stability
As a result show, working solution is placed in 2～8 DEG C 89 days and stablized.
The application of 2 the inventive method of embodiment
1st, enzyme kineticss test
Determine enzyme kinetic parameter when, the concentration of probe substrate is generally selected between 1/3Km～3Km, metabolic rate should with it is anti-
It is linear between seasonable, and the substrate being metabolized should be less than 10%.Can generate can be quantitative metabolite in the case of should be using to the greatest extent
Protein concentration that may be low is reducing non-specific binding.With reference to the Km and trial test result of document report, hepatomicrosome is chosen
Protein concentration is 0.2mg/mL, and probe substrate diclofenac concentration is respectively 1,1.5,2,2.5,3,5,20 and 50 μM.
It is 0.1M phosphate buffers (PBS) that temperature incubates system medium, comprising probe substrate, hepatomicrosome (final concentration 0.2mg/
) and NADP regenerative systems (final concentration of 10mM G-6-P, 1U/mL G-6-PDH, 10mM MgCl mL2, 0.5mM NADP), eventually
200 μ L of volume, organic solvent content are less than 1%.First respectively by the probe substrate of variable concentrations and hepatomicrosome, G-6-P, G-
6-PDH、MgCl2, PBS mix after 37 DEG C of pre-temperatures incubate 5min, be subsequently adding NADP (37 DEG C of pre-temperatures are incubated) start reaction, reaction
The 200 μ L terminating reactions of acetonitrile solution (ice bath) of containing the internal standard are added after 20min, 3min is fully mixed, in 4 DEG C, 13000rpm from
Heart 10min, takes metabolite 4 '-hydroxyl diclofenac growing amount that supernatant determines diclofenac with the inventive method, respectively
Each concentration of substrate enzyme reaction speed is calculated, enzyme kineticss parameter Km, Vmax is calculated by nonlinear regression enzyme kineticss model.
2nd, depression effect test
Temperature incubates system medium for 0.1M PBS, comprising probe substrate, inhibitor, hepatomicrosome (final concentration 0.2mg/mL) and
NADP regenerative systems (final concentration of 10mM G-6-P, 1U/mL G-6-PDH, 10mM MgCl2, 0.5mM NADP), final volume
200 μ L, organic solvent content are less than 1%.The substrate diclofenac concentration of selection is respectively 1,1.5,2,2.5,3,5,20 and
50 μM, 0.05,0.2,0.5 and 1 μM of specific inhibitor sulfaphenazole concentration.First by variable concentrations probe substrate with it is difference dense
Degree inhibitor and hepatomicrosome, G-6-P, G-6-PDH, MgCl2, PBS mix after 37 DEG C of pre-temperatures incubate 5min, be subsequently adding NADP
(37 DEG C of pre-temperatures are incubated) starts reaction, adds the 200 μ L terminating reactions of acetonitrile solution (ice bath) of containing the internal standard, fully mix after 20min
3min, in 4 DEG C, 13000rpm centrifugation 10min take the growing amount that supernatant determines 4 '-hydroxyl diclofenac in the process of the present invention.
Each concentration level enzyme reaction speed is calculated, is fitted by nonlinear regression enzyme kineticss inhibition, judge to suppress type and count
Calculate inhibition constant Ki.
In sum, detection method specificity is strong, matrix effect is little, the range of linearity is wide, batch in and batch between accurately
Degree is high with precision, sample injection disc stability is high, working solution stability is high, meets Method validation requirement, it is adaptable to hepatomicrosome
Temperature incubates the measure of 4 '-hydroxyl diclofenac concentration in sample, is with a wide range of applications.
Claims (4)
1. in a kind of hepatomicrosome 4 '-hydroxyl diclofenac concentration UPLC/MS/MS detection methods, comprise the following steps：
(1) set up liver particle body temperature and incubate system, add 4 '-hydroxyl diclofenac reference substance of internal standard substance and series concentration, prepare
Standard curve sample；
(2) standard curve sample injection UPLC/MS/MS LC-MS instrument is detected；
Chromatographic condition is as follows：
Chromatographic column：Chromatographic column is ACQUITY UPLC-BEH C18 chromatographic columns, and specification is 1.7 μm, 2.1 × 50mm；
Mobile phase：Mobile phase A is 0.1% aqueous formic acid, and Mobile phase B is 0.1% formic acid acetonitrile solution；The flow velocity of mobile phase is
0.3mL/min；
Condition of gradient elution is：
Run time is 3 minutes；
Mass Spectrometry Conditions are as follows：
Capillary voltage is 3000V, and ion source temperature is 150 DEG C, and desolvation temperature is 350 DEG C, desolvation gas flow rate
For 650L/h, taper hole gas flow rate is 150L/h, and atomization gas pressure is 6.0bar；
Ion source：Electric spray ion source；
Detection mode：Anion multiple-reaction monitoring；
The standard curve of 4 '-hydroxyl diclofenac is obtained according to testing result；
(3) according to step (1) identical method, substrate diclofenac and liver particle body temperature are incubated system, and temperature is incubated altogether, is prepared
Testing sample is injected UPLC/MS/MS LC-MS instrument, according to step (2) identical condition by the testing sample containing internal standard substance
Detected, the 4 '-hydroxyl diclofenac concentration in testing sample is obtained according to standard curve；
The internal standard substance is chlorzoxazone；
In the anion multiple-reaction monitoring, the parent-daughter ion of 4 '-hydroxyl diclofenac is to for 309.95 → m/ of m/z
Z265.90, taper hole voltage are 20V, and collision voltage is 14V；The parent-daughter ion of chlorzoxazone is to for 167.88 → m/ of m/z
Z 132.02, taper hole voltage are 36V, and collision voltage is 20V.
2. detection method according to claim 1, it is characterised in that：In the chromatographic condition, column temperature is 40 DEG C.
3. detection method according to claim 1, it is characterised in that：In the chromatographic condition, sample size is 1 μ L.
4. the detection method according to any one of claim 1-3, it is characterised in that：The standard curve sample of the step (1)
Product preparation method is：4 '-hydroxyl diclofenac addition liver particle body temperature is incubated into system, the liver particle body temperature incubates system comprising liver
Microsome, G-6-P, glucose-6-phosphate dehydrogenase (G6PD), MgCl2, PBS and NADP, the acetonitrile for adding containing the internal standard is molten
Liquid, mixes, and centrifugation takes supernatant, obtains final product standard curve sample.
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