CN110308217A - Based on before LC-MS/MS in Derivatization Determination human plasma Risedronic Acid method and its application - Google Patents
Based on before LC-MS/MS in Derivatization Determination human plasma Risedronic Acid method and its application Download PDFInfo
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Abstract
The present invention relates to based on before LC-MS/MS in Derivatization Determination human plasma Risedronic Acid method.The preceding derivatization step of this method are as follows: take 150 μ L of plasma sample, 5.00 μ L internal standard working solutions are added, it mixes, 150 μ L10% trichloroacetic acids are added, after vibrating 3min, 13000rpm is centrifuged 5min, and 100 μ L of supernatant is taken to add -25% ammonium hydroxide (50: 7 of 57.0 μ L methanol, v/v), 3min is vibrated；Then 400 μ L trimethylsilyldiazomwhiche whiches are added, 1600rpm vibrates derivative 4h, stands 2.00 μ L sample introduction of Hou Qu lower layer solution.Compared with prior art, the present invention dramatically simplifies the Sample pretreatment process of Risedronic Acid by directly performing the derivatization reaction after albumen precipitation, has the advantages that runing time is short, selective good, high sensitivity.
Description
Technical field
The invention belongs to Pharmaceutical Analysis technical field, it is related to based on benefit plug in Derivatization Determination human plasma before LC-MS/MS
The method and its application of phosphonic acids.
Background technique
Risedronic Acid (Risedronate, Fig. 1) is a kind of bis-phosphonic acids compounds, can inhibit osteoclast cell activation, is prevented
Valuation dissolution is clinically mainly used for treating and preventing the osteoporosis of postmenopausal women.Biphosphonates are by chemistry knot
Structure can be divided into three generations, and the representative drug of first generation diphosphonate is Etidronic Acid and clodronic acid pamidronic acid；Second generation diphosphonate is at it
Side chain introduces nitrogen-atoms, and representing drug has pamidronic acid, Allan phosphoric acid and Neridronic Acid；The third generation then has cyclic pendant, generation
Medicine administered to bring out the cold object has Risedronic Acid and zoledronic acid.Etidronic Acid (A), clodronic acid pamidronic acid (B), pamidronic acid (C), Allan phosphoric acid (D), how
The structural formula of vertical phosphonic acids (E), Risedronic Acid (F) and zoledronic acid (G) are as follows.
Containing there are two phosphonyl group in bisphosphonate compound structure, special structure has analysis test very
Big challenge.Bis-phosphonic acids compounds polarity is big, so that such compound is difficult to retain on common HPLC chromatogram column, and
It is easy to produce matrix effect.Most of diphosphonates are non-volatile compounds, and lack ultraviolet or fluorescent chromophore in structure,
It further limits gas-chromatography and liquid phase-is ultraviolet or the direct application of fluorescence detection.
The method that document report mainly uses Solid Phase Extraction combination derivatization at present measures bisphosphonate class of drugs in biological sample
Concentration in this.Such as Zhu establishes alendronic acid and Li Sai phosphine in diazomethane derivatization LC-MS/MS measurement human serum and urine
Acid performs the derivatization on solid-phase extraction column, but derivatization reagent diazomethane is unstable, and operation difficulty is big；Chen etc. is herein
On the basis of using safer trimethyl silicane diazomethane replace diazomethane as derivatization reagent, establish solid-phase extraction column
Upper Derivatization Determination alendronic acid is in the intracorporal blood concentration of people；Yang etc. is according to identical processing to the pharmacokinetics of minodronic acid
It is studied.
These methods are required to the method using Solid Phase Extraction, and spread out on column or to bis-phosphonic acids compounds after column
Biochemical reaction, but this method is limited by solid-phase extraction column, needs to use corresponding anion and cation exchange column, not only valence
Lattice are expensive, and in the actual operation process because on column pH influence, derivatization efficiency can be influenced to some extent, led
Poor reproducibility of the cause method between different experiments room affects the further exploitation of two banks drug.
Summary of the invention
The present invention surveys Risedronic Acid as derivatization reagent using trimethyl silicane diazomethane according to document report
It is fixed, on the basis of carrying out system optimization to derivatising condition, establish durable derivatization LC-MS/MS method measurement people's blood
Risedronic Acid in slurry, and it is applied to the pharmacokinetic studies of bis-phosphonic acids compounds.
It is of the present invention based on before LC-MS/MS in Derivatization Determination human plasma Risedronic Acid method, specific steps
Are as follows:
(1) derivatization
150 μ L of plasma sample is taken, internal standard working solution is added, is mixed, the 8-12% trichloroacetic acid of 100-200 μ L, vibration is added
After swinging 1-10min, 8000-20000rpm is centrifuged 1-15min, and 100 μ L of supernatant is taken to add 0.1-0.7 times of methanol measured: ammonium hydroxide=50:
7, vibrate 1-10min；Then 200-600 μ L trimethylsilyldiazomwhiche whiche is added, 1000-5000rpm vibrates derivative 1-
10h stands Hou Qu lower layer solution sample introduction LC-MS/MS analysis；
(2) Mass Spectrometry Conditions
Ion source is electrospray ionisation source；Source injection electric is 5000-6000V；Temperature is 500-600 DEG C；With N2For from
Source gas 1, pressure 50-70psi；With N2For ion source gas 2, pressure 60-80psi；With N2For curtain gas, pressure
For 20-30psi；Positive ion mode detection；Removing cluster voltage is 60-80V；Scanning mode is multiple-reaction monitoring, and collision energy is
25-40eV；For Risedronic Acid derivatization product and the ionic reaction of internal standard Risedronic Acid-D4 derivatization product quantitative analysis point
It Wei not 354.2 → m/z of m/z 228.1 and 358.2 → m/z of m/z 232.1；Collisional activation is dissociated into 4-8；Sweep time is
40-60ms。
It is of the present invention based on before LC-MS/MS in Derivatization Determination human plasma Risedronic Acid method, specific steps
Are as follows:
(1) derivatization
150 μ L of plasma sample is taken, 5.00 μ L internal standard working solutions are added, is mixed, 150 μ L, 10% trichloroacetic acid, oscillation is added
After 3min, 13000rpm is centrifuged 5min, and 100 μ L of supernatant is taken to add -25% ammonium hydroxide (50: 7, v/v) of 57.0 μ L methanol, vibrates 3min；
Then be added 400 μ L trimethylsilyldiazomwhiche whiches, 1600rpm vibrates derivative 4h, stand 2.00 μ L of Hou Qu lower layer solution into
Sample LC-MS/MS analysis；
(2) Mass Spectrometry Conditions
Ion source is electrospray ionisation source；Source injection electric is 5500V；Temperature is 550 DEG C；With N2For ion source gas 1,
Pressure is 60psi；With N2For ion source gas 2, pressure 70psi；With N2For curtain gas, pressure 25psi；Cation side
Formula detection；Removing cluster voltage is 70V；Scanning mode is multiple-reaction monitoring, and collision energy is 32eV；For Risedronic Acid derivatization
Product and the ionic reaction of internal standard Risedronic Acid-D4 derivatization product quantitative analysis are respectively 354.2 → m/z of m/z, 228.1 He
m/z 358.2→m/z232.1；Collisional activation is dissociated into 6；Sweep time is 50ms.
It is of the present invention based on before LC-MS/MS in Derivatization Determination human plasma Risedronic Acid method, liquid-phase condition
Are as follows: chromatographic column is Shim-packGIST-HPC18 column (50 × 2.1mm, 3 μm of partial sizes)；Mobile phase A is mutually that 10mM ammonium acetate is water-soluble
Liquid (contains 0.5% formic acid), and B phase is methanol, flow velocity 0.4mL/min, using gradient elution program, mobile phase A change procedure are as follows:
0-0.4min, 95-95%；0.4-1.4,95-75%；1.4-2.2,75-75%；2.2-2.3,75-10%；2.3-3.1,10-
10%；3.1-3.2,10-95%；3.2-4.5,95-95%.
It is of the present invention based on before LC-MS/MS in Derivatization Determination human plasma Risedronic Acid method, plasma sample system
Standby process are as follows: acquisition ulnar vein blood 5mL to K2In-EDTA anticoagulant vacuum blood collection tube, need to abandon blood about 1.0mL every time before blood sampling,
10min, 1500 × g of centrifugal force are centrifuged at 2~8 DEG C immediately after, separated plasma freezes in -70 DEG C of refrigerators.
Risedronic Acid is performed the derivatization using this method, especially joined ammonium hydroxide in system, mainly obtains being five
Methylate, when reaction, do not need for water to be limited in very low amount or waterless operation, nor need to be protected from light and nitrogen protection,
Operation is extremely easy.And the predominantly tetramethyl product that literature method obtains, and since there are five methylation positions, but
Four position methylations are only needed, a variety of isomers can be generated, in order to separate with reaction raw materials and isomer, need to use
Solid Phase Extraction processing, has sample loss, leads to the problem of poor reproducibility often occur.
Compared with prior art, of the present invention based on Risedronic Acid in Derivatization Determination human plasma before LC-MS/MS
Method directly performs the derivatization reaction after passing through albumen precipitation, dramatically simplifies the Sample pretreatment mistake of Risedronic Acid
Journey, have the advantages that runing time it is short, selectivity good, high sensitivity, and can be applied to health volunteer take orally Risedronic Acid after
Pharmacokinetic research.
Figure of description
Fig. 1: level-one sweeps mass spectrogram entirely；Fig. 2: Risedronic Acid second order ms figure；Fig. 3: bare substrate extraction m/z 354.2 →
The peak m/z228.1 chromatogram；Fig. 4: bare substrate extracts 358.2 → m/z of m/z, 232.1 peak chromatogram；Fig. 5: determinand is quantitative
Lower limit extracts 354.2 → m/z of m/z, 228.1 chromatogram；Fig. 6: determinand lower limit of quantitation extracts 358.2 → m/z of m/z 232.1
Chromatogram；Fig. 7: subject 1 is oral to give the blood concentration figure in three periods after 5mg risedronate sodium piece.
Specific embodiment
Below with reference to specific embodiment to of the present invention based on benefit in Derivatization Determination human plasma before LC-MS/MS
The method and application for filling in phosphonic acids are described further, but the scope of protection of the present invention is not limited thereto, and each parameter can be identical
Ratio expand or adjust near marginal value.
Embodiment 1
1, drug and reagent
Risedronate sodium reference substance (lot number: 100613-201402, content 87.3%) is ground purchased from Chinese food drug assay
Study carefully institute, internal standard Risedronic Acid-D4 (lot number: 1287-010A1, content 99.2%) is purchased from Canada TLC Pharmaceutical
Standards Ltd, derivatization reagent trimethyl silicane diazomethane are purchased from Shanghai Aladdin biotech inc, color
It composes pure methanol and acetonitrile is purchased from Co., Ltd in Honeywell, analyze pure formic acid purchased from Fisher Co., Ltd, analyze pure
Trichloroacetic acid is purchased from Beijing Chemical Plant, analyzes pure ammonium acetate purchased from Shanghai Aladdin biotech inc, ultrapure water
It is prepared by Merck KGaA company MilliQ pure water meter；Blank plasma is provided by clinical pharmacology base.
2, instrument and condition
Instrument liquid chromatographic system be Japan Shimadzu liquid chromatographic system, including LC-20AD type binary infusion pump with
SIL-HTA type autosampler；Mass spectrometer system is the 5000 type triple quadrupole of API of U.S. Applied Biosystems company
Bar tandem mass spectrometer is equipped with electrospray ionisation source (Turbo Ionspray) and Analyst 1.6.3 data acquisition software；Match
More this scientific instrument (Beijing) Co., Ltd Secura 125-1CN type analysis balance of benefit；Changsha easily reaches TGL16W type desk type high speed
Refrigerated centrifuge.
Chromatographic condition chromatographic column is Shim-pack GIST-HP C18 column (50 × 2.1mm I.D., 3 μm of partial sizes)；Flowing
Phase A phase is 10mM ammonium acetate solution (containing 0.5% formic acid), and B phase is methanol, using gradient elution program, is shown in Table 1.
1 HPLC gradient elution program of table
Mass Spectrometry Conditions ion source is electrospray ionisation source (source ESI)；Source injection electric is 5500V；Temperature is 550 DEG C；From
1 (N of source gas2) pressure be 60psi；2 (N of ion source gas2) pressure be 70psi；Curtain gas (N2) pressure be 25psi；Just
Ionic means detection；Removing cluster voltage (DP) is 70V；Scanning mode is multiple-reaction monitoring (MRM), and collision energy (CE) is
32eV；Ionic reaction for quantitative analysis is respectively 354.2 → m/z of m/z 228.1 (Risedronic Acid derivatization product) and m/
Z358.2 → m/z 232.1 (internal standard Risedronic Acid-D4 derivatization product)；It is 6 that collisional activation, which dissociates (CAD),；Sweep time is
50ms。
3, clinical trial protocol
12 health volunteers are assigned randomly to 3 order of administration groups according to 1: 1: 1 ratio, and every group each 4, respectively not
Reference preparation is taken with the period and by test preparation, compares the possible situation of the two equivalence.Wherein reference preparation (R) Risedronic Acid
Sodium piece, 5mg/ piece are produced by Procter&Gamble Pharmaceuticals Inc (Cleaning corporation)；By test preparation (T) benefit
Alendronate sodium tablet is filled in, 5mg/ piece is produced by sponsor and provided.Before administration 20min, 40min after (0h) and medicine, 1h, 1.5h,
2h, 3h, 4h, 6h, 8h, 10h, 12h, for 24 hours, 36h, 48h, 72h acquire ulnar vein blood 5mL to K respectively2- EDTA anticoagulant vacuum
In heparin tube (every time blood sampling before need abandon blood about 1.0mL, seal indwelling needle tubing up for safekeeping with appropriate physiological saline after blood sampling), immediately after in
It is centrifuged 10min (1500 × g) at 2~8 DEG C, separated plasma freezes to be measured in -70 DEG C of refrigerators.Dosage regimen such as the following table 2:
2 dosage regimen of table
4, the preparation of solution and sample
The preparation of Risedronic Acid stock solution and standard curve serial sample
Precision weighs risedronate sodium reference substance and is placed in glass sudden strain of a muscle bottle in right amount, is dissolved and is diluted with water, obtains concentration and is
The stock solution of 1.00mg/mL (in terms of Risedronic Acid)；10.0 μ L stock solutions are taken, 1.0mL is diluted with water to, being made into concentration is 10.0
The intermediate solution (Pre) of μ g/mL.Then with diluent (methanol/water, 1: 1) intermediate solution is diluted, obtain various concentration
Mark song working solution.Every part of mark 20.0 μ L of bent working solution is taken, it is dilute to bent working solution progress is marked that 980 μ L people blank plasmas are added
Release, prepare Risedronic Acid standard curve serial sample, process is shown in Table 3.
The preparation of 3 Risedronic Acid working solution of table and standard curve serial sample
The preparation of Risedronic Acid Quality Control solution and sample
Another precision weighs risedronate sodium reference substance and is placed in glass sudden strain of a muscle bottle in right amount, is dissolved and is diluted with water, obtains concentration
For 1.00mg/mL QC stock solution (in terms of alendronic acid)；10.0 μ L QC stock solutions are taken, 1.00mL is diluted with water to, is made into dense
Degree is the QC intermediate fluid (Pre-QC) of 10.0 μ g/mL.Then with diluent (methanol/water, 1: 1) QC intermediate fluid is diluted,
Obtain the QC working solution of various concentration.20.0 μ L of every part of QC working solution is taken, 980 μ L people blank plasmas are added to QC working solution
It is diluted, prepares to obtain Risedronic Acid QC serial, process is shown in Table 3.
The preparation of 3 Risedronic Acid QC working solution of table and sample
The preparation of internal standard Risedronic Acid-D4 solution
Precision weighs risedronate sodium-D4 (content conversion) and is placed in glass sudden strain of a muscle bottle, solves concentration 1.00mg/mL with water-soluble
Internal standard stock solution, taking 10.0 μ L of inner mark solution to add diluent, (methanol/water, 1: 1) 100mL is mixed, and obtaining concentration is in 100ng/mL
Mark working solution.
5, plasma sample pre-processes
150 μ L of plasma sample is taken, 5.00 μ L internal standard working solutions are added, is mixed, 150 μ L, 10% trichloroacetic acid, oscillation is added
After 3min, 13000rpm is centrifuged 5min, and 100 μ L of supernatant is taken to add -25% ammonium hydroxide (50: 7, v/v) of 57.0 μ L methanol, vibrates 3min；
Then be added 400 μ L trimethylsilyldiazomwhiche whiches, 1600rpm vibrates derivative 4h, stand 2.00 μ L of Hou Qu lower layer solution into
Sample LC-MS/MS analysis.
6, methodology validation
Selective Risedronic Acid and interior target typical color spectrogram do not find endogenous as seen in figures 3-6, in blank plasma sample
The interference of property substance or impurity.
In the concentration range of 50.0~10000pg/mL, determinand response is dense with compound for standard curve and lower limit of quantitation
Spend linear related (r > 0.99).Three days methodology validation determinand standard curves and related coefficient are shown in Table 4, LLOQ concentration day
Interior precision is less than 8.19%, accuracy (table 4) between -6.80% and 17.0%.
Preci-sion and accuracy determinand QC sample withinday precision less than 7.95%, day to day precision less than 13.0%,
For bat between -1.80% and 0.12%, the accuracy of illustration method and reproducibility are good (table 5).
The rate of recovery using pooled plasma investigate low (LQC), in (MQC), high (HQC) three Quality Control concentration sample, each
6 parts of concentration operation repetitive.It (is mentioned after analyte and internal standard is added in bare substrate by calculating by the determinand peak area extracted
(Ext) is taken, with Quality Control sample preparation) (analyte and interior is added after extracting in bare substrate with un-extracted determinand peak area
Mark (UnExt), with matrix effect sample preparation containing matrix) ratio, calculate analyte and interior target extraction recovery.Determinand
The rate of recovery is between 91.2%~106%, and the interior target rate of recovery is between 97.1~102%, the precision of the rate of recovery
(RSD%) it is respectively less than 10.0%, shows that the rate of recovery is uniform and reappears.
Matrix effect uses at least 6 batches of bare substrates from different donors, measure low (LQC), in (MQC), high (HQC)
The matrix effect of three Quality Control concentration.Take 150 μ L blank plasmas (sample containing matrix) and 150 μ L deionized waters (without base respectively
Matter sample) in the centrifuge tube of 2mL, 5.00 μ L deionized waters are added, be vortexed concussion 30s；150 μ L, 10% trichloroacetic acid is added,
Vortex oscillation 3min, 13000rpm are centrifuged 5min；185 μ L of supernatant is taken to be added in the above-mentioned working solution of 10.0 μ L and 5.00 μ L after centrifugation
Mark working solution takes 100 μ L that -25% ammonium hydroxide (50: 7, v/v) of 57.0 μ L methanol is added after being vortexed, and be vortexed concussion 30s；Then it is added
600 μ L diazomethanes, 1600rpm shake 4h, stand Hou Qu lower layer solution, 2.00 μ L sample introductions.The matrix effect of determinand exists
Between 92.2%~120%, interior target matrix effect is between 90.6%~108%, and variation is less than between the individual of matrix effect
4.30%, show that negligible matrix effect influences.
This experiment of stability has been investigated after Risedronic Acid plasma sample is placed at room temperature for the stability of 2h, pretreatment puts in room temperature
Setting stability for 24 hours, plasma sample experience, the stability of freeze/thaw and -70 DEG C place 49 days stability three times.Surely
When qualitative investigation, prepare the plasma sample of low, high two concentration (Risedronic Acid plasma concentration is respectively 150 and 7500pg/mL).Often
When kind study on the stability, each concentration level carries out three sample analyses, is measured using LC-MS/MS method.The result shows that Risedronic Acid
Plasma sample is placed at room temperature for 2h and stablizes (RE is between -5.49~-2.67%), is placed at room temperature for that stable afterwards for 24 hours (RE exists after pretreatment
Between 5.00~6.67%), plasma sample stablizes (RE is between 0.80~6.67%) after undergoing freeze/thaw three times ,-
70 DEG C of placements, 49 days stabilizations (RE is between -2.37~2.67%), are shown in Table 6.
4 LC-MS/MS method of table measures Risedronic Acid standard curve parameter in human plasma sample
5 LC-MS/MS method of table measures the accuracy and precision of Risedronic Acid in human plasma sample
The stability of Risedronic Acid under each condition of storage of table 6
7, pharmacokinetic parameter calculates
Pharmacokinetic parameters are calculated with non-compartment model method with 7.4 software of WinNonlin of Pharsight company.Wherein reach
Cmax (Cmax) and peak time (Tmax) it is measured value；Elimination rate constant (ke) it is that Drug-time curve end carries out least square
Method linear regression fit；Half-life period is 0.693/ke；Area under the drug-time curve AUC0-tIt is calculated using trapezoidal method, AUC0-∞For
AUC0-t+Ct/ke, CtThe blood concentration at time point can be measured for the last one.
The bioequivalence Journal of Sex Research of risedronate sodium piece, subject point will be used for by the derivatization LC-MS/MS method of verifying
5mg is not given after by test preparation and reference preparation risedronate sodium, and the main pharmacokinetic parameters of acquisition are shown in Table 7-9, typical medicine
Dynamic curve of learning is shown in Fig. 7.Calculating SW is carried out to the intraindividual variation taken orally after giving risedronate sodiumR< 0.295, therefore use average
Bioequivalence evaluates it, and the two meets bioequivalence requirement, the results are shown in Table 10.
7 subject of table gives period 1 Risedronic Acid blood concentration (pg/mL) after risedronate sodium
8 subject of table gives second round Risedronic Acid blood concentration (pg/mL) after risedronate sodium
9 subject of table gives period 3 Risedronic Acid blood concentration (pg/mL) after risedronate sodium
Table 10 is through Logarithm conversion pharmacokinetic parameters two one-sided t tests, 90% confidence interval method result table
This research is established the durable derivatization of one kind and was operated using trimethyl silicane diazomethane as derivatization reagent
Journey, this method is easy to operate, favorable reproducibility, by directly performing the derivatization reaction after albumen precipitation, dramatically simplifies
The Sample pretreatment process of Risedronic Acid.This method is measured the Risedronic Acid after derivatization using LC-MS/MS, method
Runing time is short, selectivity is good, high sensitivity, and the Pharmacokinetic being applied to after the oral Risedronic Acid of health volunteer is ground
Study carefully.Reaction can be performed the derivatization to all bisphosphonate class of drugs using this method, and using corresponding LC-MS/MS condition to double
Phosphonate derivative is detected, to pervasively be used for the pharmacokinetic studies of bisphosphonate class of drugs.
Claims (4)
1. it is a kind of based on before LC-MS/MS in Derivatization Determination human plasma Risedronic Acid method, which is characterized in that specific steps
Are as follows:
(1) derivatization
150 μ L of plasma sample is taken, internal standard working solution is added, is mixed, the 8-12% trichloroacetic acid of 100-200 μ L is added, vibrates 1-
After 10min, 8000-20000rpm is centrifuged 1-15min, and 100 μ L of supernatant is taken to add 0.1-0.7 times of methanol for measuring volume: ammonium hydroxide=50:
7, vibrate 1-10min；Then 200-600 μ L trimethylsilyldiazomwhiche whiche is added, 1000-5000rpm vibrates derivative 1-
10h stands Hou Qu lower layer solution sample introduction LC-MS/MS analysis；
(2) Mass Spectrometry Conditions
Ion source is electrospray ionisation source；Source injection electric is 5000-6000V；Temperature is 500-600 DEG C；With N2For ion source gas
Body 1, pressure 50-70psi；With N2For ion source gas 2, pressure 60-80psi；With N2For curtain gas, pressure 20-
30psi；Positive ion mode detection；Removing cluster voltage is 60-80V；Scanning mode is multiple-reaction monitoring, and collision energy is 25-
40eV；Distinguish for Risedronic Acid derivatization product and the ionic reaction of internal standard Risedronic Acid-D4 derivatization product quantitative analysis
For 354.2 → m/z of m/z 228.1 and 358.2 → m/z of m/z 232.1；Collisional activation is dissociated into 4-8；Sweep time is 40-
60ms。
2. it is according to claim 1 based on before LC-MS/MS in Derivatization Determination human plasma Risedronic Acid method, feature exists
In specific steps are as follows:
(1) derivatization
150 μ L of plasma sample is taken, 5.00 μ L internal standard working solutions are added, is mixed, 150 μ L10% trichloroacetic acids are added, vibrates 3min
Afterwards, 13000rpm is centrifuged 5min, and 100 μ L of supernatant is taken to add -25% ammonium hydroxide (50: 7, v/v) of 57.0 μ L methanol, vibrates 3min；Then
400 μ L trimethylsilyldiazomwhiche whiches are added, 1600rpm vibrates derivative 4h, stands 2.00 μ L sample introduction LC- of Hou Qu lower layer solution
MS/MS analysis；
(2) Mass Spectrometry Conditions
Ion source is electrospray ionisation source；Source injection electric is 5500V；Temperature is 550 DEG C；With N2For ion source gas 1, pressure
For 60psi；With N2For ion source gas 2, pressure 70psi；With N2For curtain gas, pressure 25psi；Positive ion mode inspection
It surveys；Removing cluster voltage is 70V；Scanning mode is multiple-reaction monitoring, and collision energy is 32eV；For Risedronic Acid derivatization product
And the ionic reaction of internal standard Risedronic Acid-D4 derivatization product quantitative analysis is respectively 354.2 → m/z of m/z 228.1 and m/z
358.2→m/z 232.1；Collisional activation is dissociated into 6；Sweep time is 50ms.
3. it is according to claim 1 based on before LC-MS/MS in Derivatization Determination human plasma Risedronic Acid method, feature exists
In liquid-phase condition is；Chromatographic column is Shim-packGIST-HPC18 column (50 × 2.1mm, 3 μm of partial sizes)；Mobile phase A is mutually
10mM ammonium acetate solution (contains 0.5% formic acid), and B phase is methanol, flow velocity 0.4mL/min, using gradient elution program, flowing
Phase A change procedure are as follows: 0-0.4min, 95-95%；0.4-1.4,95-75%；1.4-2.2,75-75%；2.2-2.3,75-
10%；2.3-3.1,10-10%；3.1-3.2,10-95%；3.2-4.5,95-95%.
4. it is according to claim 1 based on before LC-MS/MS in Derivatization Determination human plasma Risedronic Acid method, feature exists
In plasma sample preparation process are as follows: acquisition ulnar vein blood 5mL to K2In-EDTA anticoagulant vacuum blood collection tube, needed before blood sampling every time
Blood 1.0mL is abandoned, is centrifuged 10min at 2~8 DEG C immediately after, 1500 × g of centrifugal force, separated plasma is cold in -70 DEG C of refrigerators
Freeze.
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| CN116482242A (en) * | 2022-12-30 | 2023-07-25 | 杭州百杏生物技术有限公司 | LC-MS/MS method for determining alendronate concentration in biological sample |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112129855A (en) * | 2020-09-23 | 2020-12-25 | 浙江省疾病预防控制中心 | Method for measuring chloroacetic acid concentration in air by gas chromatography and application thereof |
| CN114113379A (en) * | 2021-11-12 | 2022-03-01 | 四川尚锐分析检测有限公司 | Method for detecting iban sodium phosphate in blood plasma by LC-MS (liquid chromatography-mass spectrometry) |
| CN114230772A (en) * | 2021-12-20 | 2022-03-25 | 内蒙古久泰新材料有限公司 | Non-metal catalyst for ring-opening polymerization of cyclic ester and application thereof |
| CN116482242A (en) * | 2022-12-30 | 2023-07-25 | 杭州百杏生物技术有限公司 | LC-MS/MS method for determining alendronate concentration in biological sample |
| CN116482242B (en) * | 2022-12-30 | 2024-02-23 | 杭州百杏生物技术有限公司 | LC-MS/MS method for determining alendronate concentration in biological sample |
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