CN108241026A - A kind of detection method of bisphosphonate class of drugs - Google Patents
A kind of detection method of bisphosphonate class of drugs Download PDFInfo
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- CN108241026A CN108241026A CN201611207400.5A CN201611207400A CN108241026A CN 108241026 A CN108241026 A CN 108241026A CN 201611207400 A CN201611207400 A CN 201611207400A CN 108241026 A CN108241026 A CN 108241026A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
Abstract
The present invention provides a kind of detection method of bisphosphonate class of drugs, including：Sample to be tested containing bisphosphonate class of drugs is pre-processed, obtains pretreated sample solution；The sample solution is detected using liquid chromatography tandem mass spectrometry, obtains the content of bisphosphonate class of drugs in sample to be tested, wherein, the chromatographic condition of the liquid chromatography tandem mass spectrometry includes：Using phenyl-pentafluoride pilum as chromatographic column, for mobile phase by A phases and B phase compositions, A phases are formic acid and the mixed aqueous solution of ammonium formate, and B phases are methanol.For polarity is strong, multiple ionization, the bisphosphonate class of drugs without chromophore in the present invention, it is detected with the liquid chromatography tandem mass spectrometry based on phenyl-pentafluoride pilum, it can avoid the derivatization treatment to sample to be tested, also it can avoid adding in expensive ion-pairing agent in mobile phase, it can realize efficient, the accurate qualitative and quantitative analysis to bisphosphonate class of drugs, solve the problems, such as bisphosphonate class of drugs detection difficult.
Description
Technical field
The present invention relates to biochemical analysis technical fields, and in particular to a kind of detection method of bisphosphonate class of drugs.
Background technology
Bisphosphonates are a kind of efficient bone resorption inhibitors, and clinically various relevant diseases are (such as on bone
Osteoporosis, scleromalacia and hypercalcemia of malignancy etc.) treatment.According to its chemical composition, bisphosphonate class of drugs can be divided into
Nitrogenous class and not nitrogenous class bisphosphonate class of drugs.Common nitrogenous class bisphosphonate class of drugs includes Sodium Pamidronate
(pamidronate), Alendronate sodium (alendronate), profit match Alendronate (risedronate) and zoledronic acid
(zoledronate) etc..By the analysis to bisphosphonate class of drugs (especially in biological sample) can be pharmacokinetic,
Medicine detects and the research of drug safety and validity provides effective evaluation measures.
It is following difficult to there is its analysis in special construction possessed by bisphosphonate class of drugs:(1) it is easily electric since its polarity is big
From reservation is difficult on nonpolar liquid-phase chromatographic column；It needs to add ion-pairing agent in mobile phase to improve it in chromatography
Reservation on column；(2) it can be detected with ion-exchange chromatography, but there is multiple ionization, mobile phases due to such drug
Strong acid or strong alkaline substance must be selected, and stationary phase is necessary for the anion-exchange column of strong alkali-acid resistance, so as to ensure it
Can single ionization form, the price is very expensive, and sensitivity and selectivity are also poor；(3) due to its difficult volatility, it is impossible to
It is directly analyzed with gas-chromatography, needs to perform the derivatization and be transformed into volatile derivatives and analyzed again；(4) most of two banks
Class drug does not have detectable chromophore, its skeleton structure can only be modified, by derivative means so as to change its physics and chemistry
Property, can be detected by conventional ultraviolet or fluorescence detector.Therefore, establish it is a kind of efficiently, efficiently detection side
Method is beneficial to the further investigation of bis-phosphonic acids, also establishes solid foundation for its further In vivo study.
Invention content
In consideration of it, the present invention provides it is a kind of efficiently, efficiently detection method, double phosphines that can be in Accurate Determining sample to be tested
Acid compounds content, to solve the problems, such as bisphosphonate class of drugs detection difficult in the prior art.
Specifically, the present invention provides a kind of detection method of bisphosphonate class of drugs, include the following steps：
Sample to be tested containing bisphosphonate class of drugs is pre-processed, obtains pretreated sample solution；
The sample solution is detected using Liquid Chromatography-Tandem Mass Spectrometry, obtains bis-phosphonic acids in sample to be tested
The content of drug, wherein, the chromatographic condition of the Liquid Chromatography-Tandem Mass Spectrometry includes：Made using phenyl-pentafluoride pilum (PFP columns)
For chromatographic column, for mobile phase by A phases and B phase compositions, A phases are formic acid and the mixed aqueous solution of ammonium formate, and B phases are methanol.
Wherein, in the A phases, the volume fraction of formic acid is 0.1~0.5%, a concentration of 5~20mM of ammonium formate.
The strong armaticity ring of PFP columns can be effectively retained highly polar bisphosphonate class of drugs, convenient for the separation in chromatography.
Wherein, the column internal diameter of the phenyl-pentafluoride pilum is 1~3mm, and column length is 50~150mm, and packing material size is 1~5 μm.
Wherein, the column temperature of the chromatographic column is 20~40 DEG C.
Wherein, the mobile phase uses gradient elution, and the program of the gradient elution is as follows：0~0.5min, A phase and B phases
Volume ratio be 95/5；0.5~1.5min, A phase and the volume ratio of B phases linearly become 50/50 from 95/5；1.5~2.5min,
The volume ratio for keeping A phases and B phases is 50/50；2.5~2.6min, A phase and the volume ratio of B phases linearly become 95/ again by 50/50
5；A phases and the volume ratio of B phases are kept into 5min for 95/5 later.
Wherein, the flow velocity of the mobile phase is 0.2~0.4mL/min.
Wherein, the chromatographic condition of the Liquid Chromatography-Tandem Mass Spectrometry further includes：Sample size is 5~20 μ L.
Wherein, the Mass Spectrometry Conditions of the Liquid Chromatography-Tandem Mass Spectrometry include：Using electric spray ion source anion mould
Formula carries out data acquisition with the scan mode of multiple-reaction monitoring；Ion source temperature is 80~150 DEG C, capillary voltage for 2.5~
3.0kV, orifice potential are 10~50V, and desolvation temperature is 320~450 DEG C, and Desolvention gas velocity is 500~700L/h, is bored
Gas flow hole speed is 30~80L/h.
Wherein, the Mass Spectrometry Conditions of the Liquid Chromatography-Tandem Mass Spectrometry, further include：Impact energy is 5~40V；Detection from
Son is to for [M-H]-→[M–H–H3PO3]-。
Wherein, when the sample to be tested is cell culture fluid containing bisphosphonate class of drugs, the cell culture fluid is, with
Bisphosphonate class of drugs co-cultured after cell and culture medium mixture；The pretreatment includes：Draw the cell training
Supernatant in nutrient solution, and with milli-Q water cell, merges the supernatant and cleaning solution, and through diluting or concentrating, filter
Afterwards, the pretreated sample solution is obtained.
Wherein, the bisphosphonate class of drugs is included in Sodium Pamidronate, alendronic acid, profit match Alendronate and zoledronic acid
It is one or more.
Wherein, quantitative detection is carried out to the content of bisphosphonate class of drugs in sample to be tested using internal standard method, wherein, it is described interior
The step of mark method, includes：
The standard items of bisphosphonate class of drugs containing various concentration are prepared first and the hybrid standard product of same concentrations internal standard compound work
Solution, using the concentration ratio of standard items and internal standard compound as abscissa, using the chromatographic peak area of standard items and internal standard compound ratio as ordinate,
Establish standard curve；
The sample to be tested for having same concentrations internal standard compound to addition is detected, and sample to be tested is calculated according to the standard curve
The content of middle bisphosphonate class of drugs；Wherein, the internal standard compound is the isotopic label of bisphosphonate class of drugs or is to treat test sample
The other kinds of bisphosphonate class of drugs not contained in product.
For polarity is strong, multiple ionization, the bisphosphonate class of drugs without chromophore in the present invention, with liquid chromatography-tandem
Mass spectrography, and using PFP columns as chromatographic column, can avoid performing the derivatization processing to the object (bisphosphonate class of drugs) in sample,
Also can avoid adding in expensive ion-pairing agent in mobile phase, and can realize it is efficient, accurate to bisphosphonate class of drugs
Qualitative and quantitative analysis, solve the problems, such as bisphosphonate class of drugs detection difficult.
Advantages of the present invention will be illustrated partly in the following description, and a part is apparent according to specification
Or can be through the embodiment of the present invention implementation and know.
Description of the drawings
Fig. 1 is for the embodiment of the present invention using liquid chromatogram-triple quadrupole rods tandem mass spectrometries method to four kinds of nitrogenous bis-phosphonic acids
The extraction chromatography of ions figure of aqueous solution (concentration is 100 μ g/mL) based on pentafluorophenyl group chromatography post separation of pharmaceutical standards sample.
Fig. 2 is to Alendronate sodium and Li Sai Alendronate standard items using liquid chromatogram-triple quadrupole rods tandem mass spectrometries method
Aqueous solution (concentration is 100 μ g/mL) based on the extraction chromatography of ions figure in phenyl chromatographic column.
Fig. 3 is the extraction ion chromatography of Alendronate sodium and zoledronic acid in cell culture fluid to be measured in the embodiment of the present invention
Figure.
Specific embodiment
As described below is the preferred embodiment of the present invention, it is noted that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
An embodiment of the present invention provides a kind of detection methods of bisphosphonate class of drugs, include the following steps：
S10, the sample to be tested containing bisphosphonate class of drugs is pre-processed, obtains pretreated sample solution；
S20, the sample solution using Liquid Chromatography-Tandem Mass Spectrometry is detected, obtained in sample to be tested containing double
The content of phosphonic acid drug, wherein, the chromatographic condition of the Liquid Chromatography-Tandem Mass Spectrometry includes：Using phenyl-pentafluoride pilum
(PFP columns) as liquid-phase chromatographic column, mobile phase is by A phases and B phase compositions, and A phases are formic acid and the mixed aqueous solution of ammonium formate, B phases
For methanol.
In the present invention, the strong armaticity ring of PFP columns can be effectively retained highly polar bisphosphonate class of drugs (especially band virtue
The bisphosphonate class of drugs of fragrant ring, such as profit match Alendronate and zoledronic acid), convenient for the separation in chromatography.
In embodiment of the present invention, in step S10, the sample to be tested includes the cell culture fluid containing bisphosphonate class of drugs
Or blood.The cell culture fluid is the cell of gained after being co-cultured cell and bisphosphonate class of drugs and mixing for culture medium
Close object.
Wherein, the bisphosphonate class of drugs is included in Sodium Pamidronate, alendronic acid, profit match Alendronate and zoledronic acid
It is one or more.
For sample to be tested (cell culture fluid or blood), bisphosphonate class of drugs type to be measured in sample
It is known.For example, as it is known which kind of bisphosphonate class of drugs is employed to be co-cultured with cell or known mammal
Which kind of bisphosphonate class of drugs (such as people, mouse) injects or has taken orally.External standard method may be used or internal standard method carries out bis-phosphonic acids
The quantitative test of drug.It later can be and double based on original addition before according to the content of bisphosphonate class of drugs in sample to be tested
The degree of being absorbed and utilized of cell or mammal to bisphosphonate class of drugs is calculated in the amount of phosphonic acid drug.
In an embodiment of the present invention, in step S10, the sample to be tested is the cell culture containing bisphosphonate class of drugs
Liquid, at this point, the pretreatment includes：It draws the supernatant in the cell culture fluid, and with milli-Q water cell, merges institute
It states supernatant and cleaning solution, and after diluting or concentrate, filter, obtains the pretreated sample solution.
Wherein, the filtering is that aperture is used to be filtered processing for 0.22 μm of filter membrane.
Specifically, the sample to be tested is obtained by following approach：It is carried out culture medium in cell inoculation to orifice plate, is added in
Culture, backward orifice plate hole in add in bisphosphonate class of drugs solution, at 30~45 DEG C co-cultivation 24~72h after, treated
Sample.
Wherein, the bisphosphonate class of drugs solution is as solvent using ultra-pure water or dimethyl sulfoxide (DMSO) (DMSO).It is described
The inoculum density of cell is (3~7) × 10 in orifice plate5A/mL.
In the present invention, the condition of culture (such as culture medium, temperature, time etc.) and cell and bis-phosphonic acids medicine of cell
The co-cultivation condition of object is determined according to the action time of specific cell category used, the drug to be investigated and organism.
In the embodiment of the invention, used cell is human osteosarcoma cell MG-63；Used medium is
The high glycoform DMEM fluid nutrient mediums of GIBCO companies, the glucose containing 4500mg/L, volume fraction are in the culture medium
10% fetal calf serum and volume fraction is 1% Pen .- Strep；The culture or co-cultivation are in 37 DEG C, volume point
Number is 5% CO2Incubator in carry out.
In another embodiment of the present invention, in step S10, the sample to be tested is the blood containing bisphosphonate class of drugs.
When bisphosphonate class of drugs is detected in blood sample, can the processing mode of routinely blood sample pre-processed (packet
Include albumen precipitation, extraction and purification), it does not need to perform the derivatization bisphosphonate class of drugs in sample to be tested.
In embodiment of the present invention, in step S20, key instrument is used by the Liquid Chromatography-Tandem Mass Spectrometry
Ultra performance liquid chromatography-electron spray ionisation of Waters-triple quadrupole bar tandem mass spectrometer (UPLC-ESI-MS/MS).
In embodiment of the present invention, in the A phases, the volume fraction of formic acid is 0.1~0.5%, a concentration of the 5 of ammonium formate
~20mM.Wherein, the selection of mobile phase can contribute to improve the chromatographic peak peak shape of object, wherein, ammonium formate can also avoid
The interference of signal during to later stage Mass Spectrometer Method.Mobile phase is the buffer solution of higher concentration, and mobile phase pH can be kept to stablize, and
Improve the peak shape of analyte (bisphosphonate class of drugs).
In embodiment of the present invention, the pH value of the A phases is 3.2~3.8.
In embodiment of the present invention, the flow velocity of the mobile phase is 0.2~0.4mL/min.
In embodiment of the present invention, the column internal diameter of the phenyl-pentafluoride pilum is 1~3mm, and column length is 50~150mm, filler
Grain size is 1~5 μm.Further, the packing material size is 1.5,1.6,1.7,1.8 or 2 μm.
In one embodiment of the invention, chromatographic column used is the model Acquity UPLC HSS PFP of Waters companies,
Its packing material size is 1.7 μm, and pillar size is 2.1 × 100mm.
In embodiment of the present invention, the column temperature of the chromatographic column is 20~40 DEG C.
In embodiment of the present invention, the chromatographic condition of the Liquid Chromatography-Tandem Mass Spectrometry further includes：Sample size for 5~
20μL。
In embodiment of the present invention, the mobile phase uses gradient elution, and the program of the gradient elution is as follows：0~
The volume ratio of 0.5min, A phase and B phases is 95/5；0.5~1.5min, A phase and the volume ratio of B phases linearly become 50/ from 95/5
50；1.5~2.5min, the volume ratio for keeping A phases and B phases is 50/50；The volume ratio of 2.5~2.6min, A phase and B phases is by 50/
50 linearly become 95/5 again；A phases and the volume ratio of B phases are kept into 5min for 95/5 later.
In embodiment of the present invention, the Mass Spectrometry Conditions of the liquid chromatography-mass spectrometry include：Ionization pattern for electron spray from
Component negative ion mode carries out data acquisition with the scan mode of multiple-reaction monitoring (MRM)；Ion source temperature is 80~150 DEG C,
Capillary voltage is 2.5~3.0kV, and orifice potential is 10~50V, and desolvation temperature is 320~450 DEG C, desolventizing air-flow
Speed is 500~700L/h, and taper hole gas velocity is 30~80L/h.
Further, collision gas is argon gas, and collision gas flow velocity is 0.1mL/min.
Preferably, the ion source temperature is 100~130 DEG C.Such as it can be 100,110,120,130 DEG C.
Preferably, the orifice potential is 20~40V.
Preferably, desolvation temperature is 350~420 DEG C.Such as can be 360,370,380,390,400,410,420
℃。
In embodiment of the present invention, the Mass Spectrometry Conditions further include：The ion pair of detection is [M-H]-→[M–H–
H3PO3]-；Impact energy is 5~40V.Still optionally further, the impact energy is 10~20V.
Tandem mass spectrometry is that two mass spectrographs are together in series use (including first mass spectrometric instrument and second order ms instrument),
Series system is QqQ patterns, wherein entering the scanning of first mass spectrometric instrument by the sample after chromatographic isolation obtains untested compound
Molecular ion peak [M-H]-, then carry out the scanning of second order ms instrument and obtain corresponding feature daughter ion peak, what the application selected be [M-
H–H3PO3]-。
Further, when the bisphosphonate class of drugs is Sodium Pamidronate, parent ion m/z is 234, daughter ion m/z
It is 152；When the bisphosphonate class of drugs is alendronic acid, parent ion m/z is 248, and daughter ion m/z is 166；When described double
When phosphonic acid drug is profit match Alendronate, parent ion m/z is 282, and daughter ion m/z is 200；When the bisphosphonate class of drugs is
During zoledronic acid, parent ion m/z is 271, and daughter ion m/z is 189.
In the present invention, described in liquid chromatography-tandem mass spectrometry during sample to be tested, according to the mass spectrographic test result
(being compared with the mass spectrogram of predetermined standard items) can be determined as the bisphosphonate class of drugs of a certain concrete structure, then be based on
It is mass spectrographic as a result, with reference to extract the concrete structure bisphosphonate class of drugs chromatogram, according to bis-phosphonic acids in sample to be tested
The chromatographic peak retention time of drug is relatively come further qualitative compared with the retention time of respective standard product.Under same experimental conditions,
Corresponding retention time deviation within ± 2.5%, can determine whether sample in the retention time of test substance and standard solution in sample
Contain certain specific bisphosphonate class of drugs in product.In addition, external standard method or interior can be used in the present invention according to chromatographic peak peak area
Mark method carries out the quantitative detection of bisphosphonate class of drugs.
When the quantitative analysis that bisphosphonate class of drugs is carried out using external standard method, concrete operations are：In advance by contained two banks
Class drug standards prepare the standard serial solution of various concentration, draw the standard curve of chromatographic peak peak area-concentration.Analysis
When, under the conditions of identical chromatography-mass spectroscopy, into the testing sample solution of volume similary with standard specimen, according to gained peak area, from standard
The concentration of component to be measured is found on curve.If contained in sample to be tested there are many during bisphosphonate class of drugs, need for each
Bisphosphonate class of drugs makes standard curve.
When the quantitative analysis that bisphosphonate class of drugs is carried out using internal standard method, concrete operations are：Various concentration is prepared in advance
Object standard items standard serial solution, and add in internal standard compound into each standard serial solution, obtain hybrid standard product work
Make solution, and concentration of the internal standard compound in hybrid standard product working solution is made to remain unchanged (such as 0+C0、C1+C0、C2+C0、C3+
C0、C4+C0、C5+C0Working solution, C0It is the fixed concentration of internal standard compound).Using the concentration ratio of object standard items and internal standard compound as
Abscissa with the ratio between chromatographic peak area of each concentration point object standard film and internal standard compound for ordinate, draws standard curve.It surveys
During examination, internal standard compound is added in into sample to be tested, obtains sample introduction solution, the ultimate density for making internal standard compound remain unchanged (i.e. with it is above-mentioned
Concentration in working solution is identical, is C0), tested under the same test conditions, by object in sample to be tested with
The chromatographic peak area ratio of internal standard compound can obtain the content of object in sample to be tested.
In one embodiment of the invention, a concentration of 50~200 μ g/mL of the internal standard compound in sample introduction solution.
When wherein, using internal standard method, the other kinds of bisphosphonate class of drugs that can not be contained using in sample is in
It marks object or selects the isotopic label of bisphosphonate class of drugs as internal standard compound.
Embodiment 1
The assay method containing bisphosphonate class of drugs, includes the following steps in a kind of cell culture fluid：
(1) instrument parameter optimizes：With the ultra-pure water solution of the standard items of four kinds of common nitrogenous bisphosphonate class of drugs come into
Row optimization.
1st, the optimization of chromatographic condition：
Chromatographic column is Waters PFP columns (phenyl-pentafluoride pilum) (100mm × 2.1mm, 1.7 μm)；The column temperature of chromatographic column is 25
DEG C, sample size is 10 μ L；
Mobile phase A is the mixed aqueous solution (pH=of the formic acid that volume fraction is 0.1% and the ammonium formate of a concentration of 10mM
3.3), Mobile phase B is methanol；The flow velocity of mobile phase is 0.2mL/min；
The gradient elution program of mobile phase：0~0.5min, A Phase Proportion are that 95%, B Phase Proportions are 5%；0.5~
By 95%, linearly be down to 50%, B Phase Proportions linearly rises to 50% to 1.5min, A Phase Proportion by 5%；1.5~2.5min maintains A phases
Ratio is that 50%, B Phase Proportions are 50%；The ratio of 2.5~2.6min, A phase and B phases is restored to start gradient, i.e., A Phase Proportions by
50% linear restoring to 95%, B Phase Proportion is by 50% linear restoring to 5%；2.6~7.5min, it is 50%, B to maintain A Phase Proportions
Phase Proportion is 50%.Four kinds of chromatograms for commonly using nitrogenous two banks pharmaceutical standards sample are as shown in Figure 1.
2nd, the optimization of tandem mass spectrum condition
Mass spectrographic ionization mode is electric spray ion source negative ion mode (ESI-), and scan pattern is multiple-reaction monitoring
(MRM)：Ion source temperature is 120 DEG C, capillary voltage 3.0kV, orifice potential 30V, and desolventizing temperature is 380 DEG C, is taken off
Solvent stream speed is 600L/h, taper hole gas velocity 50L/h；Collision gas is argon gas, pressure 7.0psi, impact energy 15V.Detection
Ion pair be：M/z 234 → 152 (Sodium Pamidronate), m/z 248 → 166 (Alendronate sodium), 271 → 189 (azoles of m/z
Carry out phosphonic acids).
(2) sample pre-treatments：
Sample preparation：By human osteosarcoma cell's MG-63 cells with 5 × 105The density of a/mL is seeded in six orifice plates,
In, culture medium dosage is 2mL in the every hole of six orifice plates, which is the high glycoform DMEM fluid nutrient mediums of GIBCO, is contained
4500mg/L glucose, the Pen .- Strep that the fetal calf serum and volume fraction that volume fraction is 10% are 1%.Cultivate 2h
Afterwards, the DMSO solution that 50 μ L, the Alendronate sodium of 500 μ g/mL and zoledronic acid are added in into every hole of six orifice plates carries out common training
It supports, wherein, the ultimate density of Alendronate sodium is 12.5 μ g/mL in every hole, and the ultimate density of zoledronic acid is 12.5 μ g/mL；
After 37 DEG C co-culture for 24 hours, sample to be tested is obtained, i.e., the mixing of cell and culture medium with Alendronate sodium and zoledronic acid
Object；
Culture supernatants in collecting per hole add in the ultra-pure water of 1mL into every hole to wash cell, wash 3 times, merge
The culture solution and cleaning solution, and the mixed solution after merging is diluted, the concentration of Allan sodium phosphate is about after dilution
The 1/10 of concentration, stores for future use in initial incubation liquid.
(3) method efficiency evaluation：
1. the range of linearity and detection limit：
Precision weighs each bisphosphonate class of drugs standard items (being to weigh Alendronate sodium or zoledronic acid in the present embodiment), with
Pure water is as solvent, the storing solution as each standard items.Precision weighs the internal standard compound of selection (with Sodium Pamidronate in the present embodiment
For internal standard), using pure water as solvent, as internal standard standard reserving solution.Accurate above-mentioned each standard items and the internal standard compound drawn respectively
Appropriate storing solution, and it is respectively C to be diluted to standard concentration with ultra-pure water1~C77 concentration levels hybrid standard product work
Solution, and the ultimate density of internal standard compound is fixed value C0(i.e. C1+C0、C2+C0、C3+C0、C4+C0、C5+C0、C6+C0、C7+C0Work
Make solution, C0It is the final concentration of internal standard compound, is 100 μ g/mL；C1~C7Final concentration for standard items).And to these hybrid standards
Product working solution repeats the analysis of UPLC/MS-MS loadings, with each standard items and the concentration ratio C of internal standard compoundi/C0For abscissa,
With the ratio between the chromatographic peak area of each concentration point object standard film and internal standard compound Si/S0For ordinate, standard curve is drawn.Each
Concentration level is parallel to make 3 samples, and the minimum concentration level of signal of more than 3 signal-to-noise ratio is obtained using 3 sample standard deviations as the object
The instrument detection limit of matter, the minimum concentration level that the signal of more than 10 signal-to-noise ratio is obtained using 3 sample standard deviations are determined as the substance
Measure lower limit.As a result as shown in following table table one.
Table one
The result explanation of table one, using detection method provided by the invention to the concentration of Alendronate sodium in 0.2~50 μ g/
In the range of mL or the concentration of zoledronic acid is linear preferable in the range of 0.5~10 μ g/mL, and related coefficient is full more than 0.99
Sufficient quantitative requirement.
2. the rate of recovery：
Culture medium solution is added in the orifice plate of not inoculating cell, later 2 horizontal Alendronate sodiums or azoles of middle addition
Carry out the standard solution of phosphonic acids, the wherein ultimate density of Alendronate sodium (or zoledronic acid) in the orifice plate is respectively 12.5,25 μ
g/mL.It is cultivated and is post-processed according to same steps with cell co-cultivation group.It is analyzed using aforementioned liquid chromatography-tandem mass spectrometry
Method calculates corresponding blank recycling concentration on the standard curve of upper one section structure.With the blank concentration and original common training
The ratio between concentration (can just be known as " exposure concentrations ") before supporting simultaneously is multiplied by the value that extension rate (such as 10 times) obtains and adds for blank
Mark the rate of recovery.
Cell co-cultivation group：Culture medium is added in the orifice plate for being inoculated with cell to be cultivated, add later 2 it is horizontal
The ultimate density difference of Alendronate sodium or zoledronic acid standard solution, wherein Alendronate sodium (or zoledronic acid) in the orifice plate
For 12.5,25 μ g/mL, co-culture and post-process.Each concentration level is parallel makees 3 samples.Use aforementioned liquid chromatography-mass spectrography
Method calculates corresponding sample recycling concentration on the standard curve of upper one section structure.Concentration is recycled with the concentration and blank
The ratio between be used as sample recovery of standard addition.Specific measurement result is shown in Table two.
Table two
*Relative to blank recovery of standard addition
The result explanation of table two, is measured using detection method provided by the invention and existed to the recovery of standard addition of Alendronate sodium
Between 107~110%；To the recovery of standard addition of zoledronic acid between 86~106%, the RSD of 3 repetition experiments 2.3~
In the range of 7.6%.
(4) in sample to be tested two banks drug quantitative determination
Added in diluted sample to be tested into 1.0mL (two) 100 μ L, a concentration of 1.0mg/mL Sodium Pamidronate water
Solution is filtered using 0.2 μm of cellulose acetate sheets after vortex, it is (wherein, interior to obtain sample introduction sample liquid as internal standard compound
The ultimate density for marking object is 100 μ g/mL), in ultra performance liquid chromatography-triple quadrupole bar connection mass spectrograph (UPLC-MS/MS),
It is detected analysis.According to the standard curve of above-mentioned structure, quantitative calculating is carried out.
Fig. 3 comes to pass through Alendronate sodium and azoles in the cell culture fluid to be measured co-cultured with cell in the embodiment of the present invention 1
The extraction chromatography of ions figure of phosphonic acids.In Fig. 3, a is Alendronate sodium, and b is zoledronic acid.The retention time and Fig. 1 of a and b in Fig. 3
In their standard items retention time it is very close, retention time deviation further demonstrates to be measured thin within ± 2.5%
Contain Alendronate sodium and zoledronic acid in born of the same parents' culture solution.
By calculating, measure a concentration of 0.91 μ g/mL of the Alendronate sodium in sample introduction sample liquid, by extension rate and
After blank recovery of standard addition is corrected, Alendronate sodium is final concentration of in the original cell culture fluid to be measured after being co-cultured
13.5μg/mL.A concentration of 0.93 μ g/mL of the zoledronic acid in sample introduction sample liquid is measured, passes through extension rate and blank mark-on
After the rate of recovery is corrected, the final concentration of 10.8 μ g/mL of zoledronic acid in the original cell culture fluid to be measured after being co-cultured.
In addition, optimizing the selection aspect of chromatographic column in part in the embodiment of the present invention for instrument parameter, adopted to reduce
Derivatization treatment before being detached with liquid chromatogram compared reservation energy of the nitrogenous two banks drug in different type chromatographic column
Power.Wherein, using C18Chromatographic column is to need to add in ion-pairing agent, and four kinds of the retention time of the compound phases in mobile phase
Together, it is impossible to play the role of to different nitrogenous two banks medical separations, the present invention does not consider.
The present invention provides four kinds of common nitrogenous bisphosphonate class of drugs PFP columns and phenyl chromatographic column (Waters companies,
Model BEH Phenyl UPLC 2.1 × 50mm of column, 1.7 μm of packing material size) on separating effect, such as Fig. 1 and Fig. 2
It is shown.Wherein, in Fig. 1, A-D is respectively Sodium Pamidronate, alendronic acid, zoledronic acid and Li Sai Alendronates；In Fig. 2, a be Ah
Logical sequence Alendronate, b are profit match Alendronate.
As can be seen that when using phenyl chromatographic column, Alendronate sodium and Li Sai Alendronates are difficult to separate (see attached drawing 2), but
According to PFP columns, then the separating degree of four kinds of nitrogenous bisphosphonate class of drugs significantly improves (see attached drawing 1).More than comparative illustration uses
PFP columns can effectively improve the separating degree of this 4 kinds common nitrogenous bisphosphonate class of drugs.
Embodiment 2
The assay method containing bisphosphonate class of drugs, includes the following steps in a kind of cell culture fluid：
1st, sample to be tested is pre-processed by mode same as Example 1, obtains pretreated sample solution,
In add Alendronate sodium and Li Sai Alendronates in the cell culture fluid；
2nd, the sample solution is detected using Liquid Chromatography-Tandem Mass Spectrometry, obtains two banks in sample to be tested
The content of class drug makees internal standard using zoledronic acid, wherein, the chromatographic condition of the Liquid Chromatography-Tandem Mass Spectrometry includes：
Using phenyl-pentafluoride pilum (PFP columns) as chromatographic column, the column temperature of chromatographic column is 20 DEG C, and sample size is 5 μ L；Mobile phase
By A phases and B phase compositions, A phases are the mixed aqueous solution of the formic acid that volume fraction is 0.2% and the ammonium formate of a concentration of 12mM, B phases
For methanol, the sulfuric acid of mobile phase is 0.3mL/min, and mobile phase uses gradient elution, and elution program is 0~0.5min, A phases with
The volume ratio of B phases is 95/5；0.5~1.5min, A phase and the volume ratio of B phases linearly become 50/50 from 95/5；1.5~
2.5min, the volume ratio for keeping A phases and B phases is 50/50；The volume ratio of 2.5~2.6min, A phase and B phases by 50/50 linearly
Become 95/5 again；A phases and the volume ratio of B phases are kept into 5min for 95/5 later.
The Mass Spectrometry Conditions of the Liquid Chromatography-Tandem Mass Spectrometry include：
Ionization pattern be electric spray ion source negative ion mode (ESI-), with the scan mode of multiple-reaction monitoring (MRM) into
Row data acquire；Ion source temperature is 80 DEG C, capillary voltage 2.5kV, orifice potential 20V, desolvation temperature 320
DEG C, Desolvention gas velocity 500L/h, taper hole gas velocity 30L/h.Collision gas is argon gas, and collision gas flow velocity is 0.1mL/min,
Pressure 7.0psi, impact energy 10V.The ion pair of detection is m/z248 → 166 (Alendronate sodium), 271 → 189 (azoles of m/z
Carry out phosphonic acids), m/z 282 → 200 (profit match Alendronate).
Embodiment 3
The assay method of bisphosphonate class of drugs, includes the following steps in a kind of blood：
1st, the pretreatments such as albumen precipitation, extraction and purification are carried out to the blood sample containing bisphosphonate class of drugs, obtains pre- place
Sample solution after reason；
2nd, the sample solution is detected using Liquid Chromatography-Tandem Mass Spectrometry, obtains two banks in sample to be tested
The content of class drug, wherein, the chromatographic condition of the Liquid Chromatography-Tandem Mass Spectrometry includes：
Using phenyl-pentafluoride pilum (PFP columns) as chromatographic column, the column temperature of chromatographic column is 40 DEG C, and sample size is 20 μ L；Flowing
Mutually by A phases and B phase compositions, A phases are the mixed aqueous solution of the formic acid that volume fraction is 0.5% and the ammonium formate of a concentration of 20mM, B
Mutually for methanol, the sulfuric acid of mobile phase is 0.4mL/min, and mobile phase uses gradient elution, and elution program is 0~0.5min, A phases
Volume ratio with B phases is 95/5；0.5~1.5min, A phase and the volume ratio of B phases linearly become 50/50 from 95/5；1.5~
2.5min, the volume ratio for keeping A phases and B phases is 50/50；The volume ratio of 2.5~2.6min, A phase and B phases by 50/50 linearly
Become 95/5 again；A phases and the volume ratio of B phases are kept into 5min for 95/5 later.
The Mass Spectrometry Conditions of the Liquid Chromatography-Tandem Mass Spectrometry include：
Ionization pattern be electric spray ion source negative ion mode (ESI-), with the scan mode of multiple-reaction monitoring (MRM) into
Row data acquire；Ion source temperature is 150 DEG C, capillary voltage 2.8kV, orifice potential 40V, and desolvation temperature is
450 DEG C, Desolvention gas velocity 700L/h, taper hole gas velocity 50L/h.Collision gas is argon gas, impact energy 20V.Detection
Ion pair is [M-H]-→[M–H–H3PO3]-, wherein M is the molecular weight of a certain bisphosphonate class of drugs.
It should be noted that according to the above description the announcement of book and with illustrate, those skilled in the art in the invention also
The above embodiment can be changed and be changed.Therefore, the invention is not limited in specific realities disclosed and described above
Mode is applied, some equivalent modifications and change to the present invention should also be as within the scope of the claims of the present invention.This
Outside, although having used some specific terms in this specification, these terms merely for convenience of description, not to the present invention
Form any restrictions.
Claims (12)
1. a kind of detection method of bisphosphonate class of drugs, which is characterized in that include the following steps：
Sample to be tested containing bisphosphonate class of drugs is pre-processed, obtains pretreated sample solution；
The sample solution is detected using Liquid Chromatography-Tandem Mass Spectrometry, obtains bisphosphonate class of drugs in sample to be tested
Content, wherein, the chromatographic condition of the Liquid Chromatography-Tandem Mass Spectrometry includes：Using phenyl-pentafluoride pilum as chromatographic column, stream
Dynamic mutually by A phases and B phase compositions, A phases are formic acid and the mixed aqueous solution of ammonium formate, and B phases are methanol.
2. detection method as described in claim 1, which is characterized in that in the A phases, the volume fraction of formic acid for 0.1~
0.5%, a concentration of 5~20mM of ammonium formate.
3. detection method as described in claim 1, which is characterized in that the column internal diameter of the phenyl-pentafluoride pilum be 1~3mm, column
A length of 50~150mm, packing material size are 1~5 μm.
4. detection method as described in claim 1, which is characterized in that the mobile phase uses gradient elution, and the gradient is washed
De- program is as follows：The volume ratio of 0~0.5min, A phase and B phases is 95/5；The volume ratio of 0.5~1.5min, A phase and B phases by
95/5 linearly becomes 50/50；1.5~2.5min, the volume ratio for keeping A phases and B phases is 50/50；2.5~2.6min, A phase with
The volume ratio of B phases linearly becomes 95/5 again by 50/50；5min is kept as 95/5 using A phases and the volume ratio of B phases later.
5. detection method as described in claim 1, which is characterized in that the flow velocity of the mobile phase is 0.2~0.4mL/min.
6. detection method as described in claim 1, which is characterized in that the chromatographic condition of the Liquid Chromatography-Tandem Mass Spectrometry
It further includes：Sample size is 5~20 μ L.
7. detection method as described in claim 1, which is characterized in that the column temperature of the chromatographic column is 20~40 DEG C.
8. detection method as described in claim 1, which is characterized in that the Mass Spectrometry Conditions of the Liquid Chromatography-Tandem Mass Spectrometry
Including：Using electric spray ion source negative ion mode, data acquisition is carried out with the scan mode of multiple-reaction monitoring；Ion source temperature
It it is 80~150 DEG C, capillary voltage is 2.5~3.0kV, and orifice potential is 10~50V, and desolvation temperature is 320~450
DEG C, Desolvention gas velocity is 500~700L/h, and taper hole gas velocity is 30~80L/h.
9. detection method as claimed in claim 8, which is characterized in that the Mass Spectrometry Conditions of the Liquid Chromatography-Tandem Mass Spectrometry,
It further includes：Impact energy is 5~40V, and the ion pair of detection is [M-H]-→[M–H–H3PO3]-。
10. detection method as described in claim 1, which is characterized in that when the sample to be tested is containing bisphosphonate class of drugs
During cell culture fluid, the pretreatment includes：Draw the supernatant in the cell culture fluid, and with milli-Q water cell,
Merge the supernatant and cleaning solution, and after diluting or concentrate, filter, obtain the pretreated sample solution.
11. detection method as described in claim 1, which is characterized in that using internal standard method to bis-phosphonic acids medicine in sample to be tested
The content of object carries out quantitative detection, wherein, the step of internal standard method, includes：
The hybrid standard product working solution of the standard items of bisphosphonate class of drugs containing various concentration and same concentrations internal standard compound is prepared first,
Using the concentration ratio of standard items and internal standard compound as abscissa, using the chromatographic peak area of standard items and internal standard compound ratio as ordinate, establish
Standard curve；
The sample to be tested for having same concentrations internal standard compound to addition is detected, and is calculated according to the standard curve double in sample to be tested
The content of phosphonic acid drug；Wherein, the internal standard compound is the isotopic label of bisphosphonate class of drugs or is in sample to be tested
The other kinds of bisphosphonate class of drugs not contained.
12. such as claim 1-11 any one of them detection methods, which is characterized in that the bisphosphonate class of drugs includes pa rice
It is one or more in Alendronate, alendronic acid, profit match Alendronate and zoledronic acid.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110568114A (en) * | 2019-08-29 | 2019-12-13 | 福州佳宸生物科技有限公司 | Solid phase microextraction-high performance liquid chromatography on-line combined detection method for zoledronic acid and risedronic acid |
| CN111721748A (en) * | 2020-06-30 | 2020-09-29 | 重庆师范大学 | Fluorescent probe for detecting zoledronic acid, preparation method of fluorescent probe, fluorescent sensor, construction method of fluorescent sensor and application of fluorescent sensor |
| 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) |
| CN114929359A (en) * | 2019-12-16 | 2022-08-19 | 西根公司 | High performance liquid chromatography quantification of excipients |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105738493A (en) * | 2014-12-10 | 2016-07-06 | 人福医药集团股份公司 | Method for analyzing lapatinib ditosylate isomer impurities |
| CN105738544A (en) * | 2014-12-10 | 2016-07-06 | 人福医药集团股份公司 | Method for analyzing and detecting lapatinib ditosylate isomer impurities |
-
2016
- 2016-12-23 CN CN201611207400.5A patent/CN108241026A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105738493A (en) * | 2014-12-10 | 2016-07-06 | 人福医药集团股份公司 | Method for analyzing lapatinib ditosylate isomer impurities |
| CN105738544A (en) * | 2014-12-10 | 2016-07-06 | 人福医药集团股份公司 | Method for analyzing and detecting lapatinib ditosylate isomer impurities |
Non-Patent Citations (3)
| Title |
|---|
| M.A. POPOT，ET AL: "HPLC/ESI-MSn method for non-amino bisphosphonates: Application to the detection of tiludronate in equine plasma", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| WAGDY HA，ET AL: "A Validated HPLC Method for the Determination of Tiludronate Disodium on a Novel Brominated Stationary Phase", 《PHARMACEUTICA ANALYTICA ACTA》 * |
| 杨致一: "一种含氮双膦酸的非衍生化液相色谱-质谱分析方法", 《中国化学会第30届学术年会摘要集-第二十三分会：复杂样品分离分析》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110568114A (en) * | 2019-08-29 | 2019-12-13 | 福州佳宸生物科技有限公司 | Solid phase microextraction-high performance liquid chromatography on-line combined detection method for zoledronic acid and risedronic acid |
| CN114929359A (en) * | 2019-12-16 | 2022-08-19 | 西根公司 | High performance liquid chromatography quantification of excipients |
| CN111721748A (en) * | 2020-06-30 | 2020-09-29 | 重庆师范大学 | Fluorescent probe for detecting zoledronic acid, preparation method of fluorescent probe, fluorescent sensor, construction method of fluorescent sensor and application of fluorescent sensor |
| 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) |
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