CN116482242B - LC-MS/MS method for determining alendronate concentration in biological sample - Google Patents
LC-MS/MS method for determining alendronate concentration in biological sample Download PDFInfo
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- OGSPWJRAVKPPFI-UHFFFAOYSA-N Alendronic Acid Chemical compound NCCCC(O)(P(O)(O)=O)P(O)(O)=O OGSPWJRAVKPPFI-UHFFFAOYSA-N 0.000 title claims abstract description 85
- 239000012472 biological sample Substances 0.000 title claims abstract description 58
- 229940062527 alendronate Drugs 0.000 title claims abstract description 48
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 38
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 45
- 238000001514 detection method Methods 0.000 claims abstract description 31
- 238000001212 derivatisation Methods 0.000 claims abstract description 24
- YXHKONLOYHBTNS-UHFFFAOYSA-N Diazomethane Chemical compound C=[N+]=[N-] YXHKONLOYHBTNS-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000006243 chemical reaction Methods 0.000 claims abstract description 18
- 238000004458 analytical method Methods 0.000 claims abstract description 15
- 239000012224 working solution Substances 0.000 claims abstract description 12
- 210000002700 urine Anatomy 0.000 claims abstract description 10
- 239000006228 supernatant Substances 0.000 claims abstract description 7
- 238000010790 dilution Methods 0.000 claims abstract description 6
- 239000012895 dilution Substances 0.000 claims abstract description 6
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 239000012470 diluted sample Substances 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000003960 organic solvent Substances 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 63
- 229960004343 alendronic acid Drugs 0.000 claims description 37
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 34
- 239000000523 sample Substances 0.000 claims description 32
- 239000000243 solution Substances 0.000 claims description 25
- 239000012071 phase Substances 0.000 claims description 23
- 238000000605 extraction Methods 0.000 claims description 18
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 17
- 235000019253 formic acid Nutrition 0.000 claims description 17
- 238000000746 purification Methods 0.000 claims description 17
- 150000002500 ions Chemical class 0.000 claims description 15
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 11
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 11
- 239000012498 ultrapure water Substances 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 10
- FUKUFMFMCZIRNT-UHFFFAOYSA-N hydron;methanol;chloride Chemical compound Cl.OC FUKUFMFMCZIRNT-UHFFFAOYSA-N 0.000 claims description 10
- 210000004369 blood Anatomy 0.000 claims description 9
- 239000008280 blood Substances 0.000 claims description 9
- 238000002347 injection Methods 0.000 claims description 9
- 239000007924 injection Substances 0.000 claims description 9
- -1 trimethyl aminopropyl Chemical group 0.000 claims description 8
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 claims description 7
- CHRJZRDFSQHIFI-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;styrene Chemical compound C=CC1=CC=CC=C1.C=CC1=CC=CC=C1C=C CHRJZRDFSQHIFI-UHFFFAOYSA-N 0.000 claims description 6
- 238000004949 mass spectrometry Methods 0.000 claims description 6
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- 238000004811 liquid chromatography Methods 0.000 claims description 4
- 101100348341 Caenorhabditis elegans gas-1 gene Proteins 0.000 claims description 3
- 101100447658 Mus musculus Gas1 gene Proteins 0.000 claims description 3
- 101100447665 Mus musculus Gas2 gene Proteins 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000002386 leaching Methods 0.000 claims description 3
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- 230000000155 isotopic effect Effects 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 29
- 210000002381 plasma Anatomy 0.000 description 28
- 230000000694 effects Effects 0.000 description 16
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- 239000003814 drug Substances 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 238000000926 separation method Methods 0.000 description 8
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- 229940079593 drug Drugs 0.000 description 6
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- 238000001121 post-column derivatisation Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 241001251200 Agelas Species 0.000 description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 4
- 239000005695 Ammonium acetate Substances 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 235000019257 ammonium acetate Nutrition 0.000 description 4
- 229940043376 ammonium acetate Drugs 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 229940122361 Bisphosphonate Drugs 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 150000004663 bisphosphonates Chemical class 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000004255 ion exchange chromatography Methods 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- UHNSRFWQBVXBSK-UHFFFAOYSA-N methanol;2,2,2-trifluoroacetic acid Chemical compound OC.OC(=O)C(F)(F)F UHNSRFWQBVXBSK-UHFFFAOYSA-N 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
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- 229910002027 silica gel Inorganic materials 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 208000001132 Osteoporosis Diseases 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000012156 elution solvent Substances 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- SVWLIIFHXFGESG-UHFFFAOYSA-N formic acid;methanol Chemical compound OC.OC=O SVWLIIFHXFGESG-UHFFFAOYSA-N 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
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- 238000002540 product ion scan Methods 0.000 description 2
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- 230000002829 reductive effect Effects 0.000 description 2
- 239000012088 reference solution Substances 0.000 description 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- PVOAHINGSUIXLS-UHFFFAOYSA-N 1-Methylpiperazine Chemical group CN1CCNCC1 PVOAHINGSUIXLS-UHFFFAOYSA-N 0.000 description 1
- 208000006386 Bone Resorption Diseases 0.000 description 1
- 229940078581 Bone resorption inhibitor Drugs 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- DBVJJBKOTRCVKF-UHFFFAOYSA-N Etidronic acid Chemical compound OP(=O)(O)C(O)(C)P(O)(O)=O DBVJJBKOTRCVKF-UHFFFAOYSA-N 0.000 description 1
- 241000662429 Fenerbahce Species 0.000 description 1
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 230000024279 bone resorption Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000004821 effect on bone Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 229940009626 etidronate Drugs 0.000 description 1
- 239000002360 explosive Substances 0.000 description 1
- IRXSLJNXXZKURP-UHFFFAOYSA-N fluorenylmethyloxycarbonyl chloride Chemical compound C1=CC=C2C(COC(=O)Cl)C3=CC=CC=C3C2=C1 IRXSLJNXXZKURP-UHFFFAOYSA-N 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000004190 ion pair chromatography Methods 0.000 description 1
- 150000008040 ionic compounds Chemical class 0.000 description 1
- 238000004989 laser desorption mass spectroscopy Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- TZIHFWKZFHZASV-UHFFFAOYSA-N methyl formate Chemical compound COC=O TZIHFWKZFHZASV-UHFFFAOYSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- WRUUGTRCQOWXEG-UHFFFAOYSA-N pamidronate Chemical compound NCCC(O)(P(O)(O)=O)P(O)(O)=O WRUUGTRCQOWXEG-UHFFFAOYSA-N 0.000 description 1
- 229940046231 pamidronate Drugs 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000007847 structural defect Effects 0.000 description 1
- 238000003239 susceptibility assay Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
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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
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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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- G01N30/02—Column chromatography
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- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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Abstract
The invention discloses a method for determining alendronate concentration in a biological sample by LC-MS/MS, which comprises the following steps: step 1: mixing a biological sample to be tested with an internal standard working solution, and adding water for dilution; performing solid phase extraction on the diluted sample, eluting with an organic solvent, and volatilizing; step 2: redissolving the biological sample obtained in the step 1, adding trimethyl silanized diazomethane for derivatization reaction, and volatilizing after the reaction is finished; and 3, re-dissolving the biological sample obtained in the step 2, centrifuging, taking supernatant, and injecting the supernatant into a liquid chromatography-tandem mass spectrometry combined instrument for LC-MS/MS analysis to determine the alendronate concentration in the biological sample. The detection method provided by the invention can reach the detection sensitivity of LLOQ of 0.5000ng/ml when the plasma volume is only 200 mu L, is better than the prior art, has stronger universality, and is suitable for detecting the alendronate concentration in urine samples and plasma samples.
Description
Technical Field
The invention relates to the technical field of medicine analysis, in particular to a method for measuring alendronic acid concentration in a biological sample by liquid chromatography tandem mass spectrometry (LC-MS/MS).
Background
The alendronate sodium (Alendronate sodium) has a chemical name of 4-amino-1-hydroxybutylidene sodium diphosphate, has a structural formula shown below, is a third-generation bone resorption inhibitor, has strong affinity with intraosseous hydroxyapatite, can inhibit the activity of osteoclasts, and indirectly plays a role in inhibiting bone resorption through osteoblasts. The alendronate sodium has the characteristics of strong bone absorption resistance, no mineralization inhibition effect, avoidance of a plurality of defects and shortages of the first two generations of medicines, and inhibition effect on bone absorption is 1000 times of that of the first generation of etidronate, 10 times of that of the second generation of pamidronate, and is internationally considered as an osteoporosis medicine with the most potential in the twenty-first century, and is recommended as a therapeutic medicine for osteoporosis by the American FDA in 1995.
As is well known, the self structural characteristics of alendronate sodium determine that the content determination thereof becomes a technical problem: alendronate sodium is a strongly polar ionic compound that cannot be retained on reverse phase chromatography columns; the structure of the fluorescent probe has no chromophore and can not be directly detected by an ultraviolet detector and a fluorescence detector; multiple ionization states exist, interfering with mass spectrometry detection. The method is limited by the self structural characteristics of the alendronate sodium, and the prior art adopts ion chromatography or ion pair chromatography to detect the alendronate in the medicine, for example, the pharmacopoeia 2020 edition adopts ion chromatography to measure the content in the alendronate sodium tablet.
Compared with the determination of alendronate content in medicines, the determination of alendronate content in biological samples has higher requirements, and the influence of the following two aspects needs to be paid additional attention: (1) because alendronate has a similar structure to many endogenous substances in biological samples, pretreatment must be performed by using a suitable separation and purification step to eliminate interference of other endogenous substances on subsequent detection; (2) according to alendronate sodium tabletsThe specification states that the oral bioavailability of alendronate sodium is extremely low (about 0.6%), and after rapid distribution, the alendronate sodium is mainly excreted through urine, resulting in excessively low concentration of alendronate sodium in biological samples, especially blood plasma<5 ng/ml), a high sensitivity assay should be selectedThe method can only be detected.
Obviously, the ion chromatography method cannot meet the requirements of separation and purification and sensitivity of biological sample detection, and is unfavorable for detection. In order to improve the selectivity and sensitivity of the concentration detection of alendronic acid in biological samples, the alendronic acid in biological samples needs to be derivatized to overcome the structural defects, so that the method can be applied to detection methods with higher sensitivity.
After the prior art search analysis, it was found that the derivatization treatment of alendronic acid can be roughly divided into two directions: (1) introducing a chromophore, for example, introducing 9-fluorenylmethyl chloroformate by derivatization, as in document 1, and detecting with a detection sensitivity of 3.5ng/ml by an HPLC method of fluorescence detection; (2) methyl derivatization, for example, document 2 and chinese patent CN111948307a, etc., are derivatized with Diazomethane (DAM) and detected using LC-MS/MS, the detection sensitivity of document 2 being 50pg/ml. In contrast, the former has lower sensitivity and is generally used for detecting alendronate in urine samples, while the latter has higher sensitivity and can be used for detecting alendronate in blood samples.
However, the derivatizing reagent DAM used in document 2 and chinese patent CN111948307a is unstable and explosive and has no universality; instead, trimethylsilylated diazomethane (TMS-DAM) may be considered a replacement as a safer replacement derivatizing reagent than DAM. For example, document 3 discloses a method for detecting alendronic acid in a biological sample by liquid chromatography-tandem mass spectrometry, which is first described in the following paragraphsHLB、/>WAX is subjected to solid phase extraction twice, TMS-DAM derivatization (namely post-column derivatization) is carried out, then 0.1% formic acid and 0.1% formic acid acetonitrile are taken as mixed mobile phases, gradient elution is carried out, LC-MS/MS detection is carried out, and the lower limit of quantification (LLOQ) in 0.5mL blood sample is 100ng/mL. Thus, although TMS-DAM can replace DAM, the technical disadvantage is that the detection sensitivity of alendronate in biological samples is obviously reduced, and the detection sensitivity is improved.
As described on pages 2940-2941 of document 4, the reason for the reduced LC-MS/MS detection sensitivity after derivatization of TMS-DAM may be: TMS-DAM derivatization has a slow reaction rate, is not suitable for a column derivatization method of DAM, and requires a reaction time of more than 30 minutes after extraction (namely, a post-column derivatization method), and the reaction formula is shown below.
In summary, for experimental safety, TMS-DAM is used as a derivatization reagent, and the TMS-DAM derivatization reaction can only completely adopt post-column derivatization, so that the technical problem of sensitivity reduction of detecting alendronic acid in biological samples by using LC-MS/MS is solved, which is an unsolved technical problem in the field.
The relevant literature relating to the background art is presented below:
document 1: ptacek P, et al determination of alendronate inhuman urine as 9-fluorenylmethyl derivative by high-performance liquid chromatography [ J ]. J chromatogrB, 2002,767 (1): 111-116.
Document 2: lee S.Zhu et al A general approach for the quantitative analysis of bisphosphonates in human serum and urine by high-performance liquid chromatography _ tandem mass spectrometry [ J ]. Rapid Commun.Mass Spectrom.2006,20:3421-3426.
Document 3: april s.y.wong et al liquid chromatography-mass spectrometry analysis of five bisphosphonates in equine urine and plasma [ J ]. Journal of Chromatography B,998-999,2015,1-7, document 4: veniamin N Lapko et al, quantitative analysis of bisphosphonates in biological samples [ J ]. Bioanalysis,2014,6 (21): 2931-2950.)
Disclosure of Invention
The invention aims to solve the technical problems that: on the premise of adopting solid phase extraction, TMS-DAM post-column derivatization and LC-MS/MS detection, the inventor team can further optimize the type of solid phase extraction filling material (trimethyl aminopropyl functionalized hydrophilic styrene-divinylbenzene), the type of eluting solvent (methanol solution of hydrochloric acid) and/or the type of mobile phase (0.5% formic acid solution) in the LC-MS/MS detection condition so as to improve the sensitivity of alendronic acid concentration detection in biological samples.
The technical problems to be solved by the invention are solved by the following technical scheme:
a method of LC-MS/MS determination of alendronate concentration in a biological sample comprising the steps of:
step 1: purification of biological samples
Mixing a biological sample to be tested with an internal standard working solution, and adding water for dilution; performing solid phase extraction on the diluted sample, eluting with an organic solvent, and volatilizing;
step 2: derivatization of biological samples
Redissolving the biological sample obtained in the step 1, adding trimethyl silanized diazomethane for derivatization reaction, and volatilizing after the reaction is finished;
step 3: liquid chromatography tandem mass spectrometry detection
And (3) re-dissolving the biological sample obtained in the step (2), centrifuging, taking supernatant, and injecting the supernatant into a liquid chromatography-tandem mass spectrometry combined instrument for LC-MS/MS analysis to determine the alendronate concentration in the biological sample.
In a preferred embodiment, the solid phase extraction in step 1 is performed using a PAX solid phase extraction plate or a PAX solid phase extraction column with a packing material of trimethylaminopropyl functionalized hydrophilic styrene-divinylbenzene.
In a preferred embodiment, the eluting solvent in step 1 is methanol hydrochloride.
More preferably, the hydrochloric acid concentration in step 1 is 0.02M to 0.4M.
In a more preferred embodiment, the hydrochloric acid concentration in step 1 is 0.1M. .
In a preferred embodiment, step 1 includes: mixing 200 mu L of biological sample with 20.00 mu L of internal standard working solution, adding ultrapure water for dilution, adding into a PAX 96 Kong Guxiang extraction plate activated and balanced by methanol and ultrapure water, sequentially adding ultrapure water and methanol for leaching, drying, adding hydrochloric acid methanol solution for eluting, and volatilizing the eluent.
In a preferred embodiment, step 2 includes: re-dissolving the biological sample obtained in the step 1 by 50-150 mu L of 50% methanol aqueous solution, adding 100-600 mu L of 2.0M trimethyl silanized diazomethane and 200 mu L of methanol, and volatilizing after derivatization reaction for 30-90 min.
In a more preferred embodiment, the 50% aqueous methanol solution in step 2 is used in an amount of 50. Mu.L, the trimethylsilylated diazomethane in an amount of 300. Mu.L, and the derivatization time is 60 minutes.
In a preferred embodiment, the liquid chromatography conditions in the LC-MS/MS detection of step 3 include: the chromatographic column is a C18 column, the mobile phase A is 0.5% formic acid aqueous solution, the mobile phase B is 0.5% formic acid acetonitrile solution, and the elution gradient is as follows:
| Time(min) | A(％) | B(％) |
| 0.00 | 91.0 | 9.0 |
| 0.50 | 91.0 | 9.0 |
| 1.00 | 0.0 | 100.0 |
| 3.00 | 0.0 | 100.0 |
| 3.10 | 91.0 | 9.0 |
| 4.50 | 91.0 | 9.0 |
in a preferred embodiment, the liquid chromatography conditions in the LC-MS/MS detection of step 3 further comprise: the column temperature was 40 ℃, the sample injection volume was 5.00. Mu.L, and the flow rate was 0.4mL/min.
In a preferred embodiment, the mass spectrometry conditions in the LC-MS/MS detection of step 3 are: ion mode: ESI (electronic service provider interface) + IS ion spray voltage: 4500v, gas1 atomizing gas: 45.00psi, gas2 assist gas: 45.00psi, ion source temperature: 500 ℃.
In a preferred embodiment, the isotopic internal standard of alendronic acid is alendronic acid-d 6.
In a preferred embodiment, the biological sample comprises a urine sample and a blood sample.
More preferably, the biological sample is selected from plasma.
Compared with the prior art, the method for measuring the alendronate concentration in the biological sample by using the LC-MS/MS provided by the invention has the following beneficial effects:
1) The application adds a purification step for the biological sample containing alendronic acid, adopts a solid phase extraction method, and preferably adopts a PAX solid phase extraction plate or a PAX solid phase extraction column of trimethyl aminopropyl functionalized hydrophilic styrene-divinylbenzene as a filling material so as to improve the recovery rate.
The specific filler material employed in the present application differs from the prior art in its effectiveness:
while literature 2 uses a SAX solid-phase extraction plate with a silica gel matrix to catalyze the derivatization reaction on the DAM column, the post-column derivatization reaction of TMS-DAM is not required to catalyze the SAX silica gel matrix, and example 3 of the application also shows that the separation and purification effects of SAX on alendronic acid are poor.
The effect of using a WAX solid phase extraction column in document 3 is extremely low, which is consistent with the effect of using a WAX solid phase extraction plate in example 4 of the present application, but unlike the method of adding two extractions of an HLB solid phase extraction column in document 3 to increase the recovery rate, the present application can increase the recovery rate to 25% or more by optimizing the solid phase extraction packing material for only one extraction.
2) The method for detecting alendronic acid in biological samples adopts reverse phase chromatography tandem mass spectrometry (LC-MS/MS) detection, optimizes the aqueous solution of formic acid with mobile phase A of 0.5% and the solution of formic acid-acetonitrile with mobile phase B of 0.5%; the elution mode is gradient elution, and the detection sensitivity is improved to 0.5000ng/ml.
The specific filler material employed in the present application differs from the prior art in its effectiveness:
in reference 3, an elution gradient system was employed in which mobile phase A was a 0.1% formic acid aqueous solution and mobile phase B was a 0.1% formic acid acetonitrile solution, and the detection sensitivity was low, which was only 100ng/mL. The concentration of formic acid is increased to 0.5%, and the addition of ammonium acetate is avoided to generate ion inhibition, so that the effects of improving sensitivity and improving peak shape are achieved.
3) The method adopts a LC-MS/MS method after solid phase extraction conditions and mobile phase optimization to detect the alendronate concentration in a biological sample, and can achieve detection sensitivity of LLOQ of 0.5000ng/ml when the plasma volume is only 200 mu L, the mass spectrum sample introduction volume is 5.0 mu L and the analysis time is 4.5 min; the plasma amount reported by the related literature is about 500 mu L, the mass spectrum sample injection amount is more than 10.0 mu L, the analysis time exceeds 5min, and the technical effect is inferior to the application.
In summary, the high sensitivity detection method provided by the present application is not only suitable for detecting alendronate concentration in urine samples, but even in blood samples (lower concentrations).
Drawings
FIG. 1 is a schematic illustration of [ M+H ] after alendronate derivatization in example 1] + Is a product ion scan of (2);
FIG. 2 is a schematic representation of [ M+H ] after the derivation of alendronate-d 6 in example 1] + Is a product ion scan of (2);
FIG. 3 is a typical chromatogram of alendronate (left) and alendronate-d 6 (right) in a LLOQ sample of example 1;
FIG. 4 is a typical chromatogram of alendronate (left) and alendronate-d 6 (right) in a blank plasma sample of example 2;
FIG. 5 is a representative spectrum of the plasma sample standard curve of alendronate in example 2;
fig. 6 is a plasma drug-time profile of alendronate in a clinical sample test.
Detailed Description
The following examples are illustrative and are intended to provide further details of the invention to aid in understanding the nature of the invention. The scope of the invention includes but is not limited to these embodiments, and all changes and equivalents that do not depart from the spirit of the invention are intended to be included therein.
The test materials used in the examples below for the purposes of illustration are all conventional in the art and are commercially available.
Medicine and reagent:
alendronate sodium (lot number: 100901-202103, manufacturer: china food and drug inspection institute);
alendronate-d 6 (lot number 1029-046A1, manufacturer: TLC);
trimethylsilylated diazomethane (batch number: P2147214, manufacturer: adamas);
methanol (batch number: 219089, manufacturer: fisher);
acetonitrile (lot number: I1218030225, manufacturer: merck);
formic acid (batch number: K53610664133, manufacturer: merck)
Instrument apparatus:
an ultra-high performance liquid chromatography-tandem mass spectrometer (model: sciex Exion AD/TQ5500, manufacturer: applied biosystems, USA) is equipped with analysis 1.6.3 data acquisition software and Watson LIMS v7.5 data processing software;
a 96-well plate positive pressure apparatus (model: SPE-M96, manufacturer: agela Technologies);
a 96-well plate nitrogen blower (model: NDK200-1A, manufacturer: hangzhou Miou instruments Co., ltd.);
a bench-type refrigerated centrifuge (model: eppendorf 5810R, manufacturer: eppendorf);
ultrapure water instrument (model:pro, manufacturer: sartorius);
PAX 96 Kong Guxiang extraction plate (lot number: M02980, manufacturer: agela Technologies);
SAX96 Kong Guxiang extraction column (lot number: M04343, manufacturer: agela Technologies);
WAX96 Kong Guxiang extraction column (lot number: M04799, manufacturer: agela Technologies).
Example 1
A method for measuring alendronate concentration in biological sample by liquid chromatography tandem mass spectrometry (LC-MS/MS) comprises the following specific steps:
1. purification and derivatization of plasma samples
Purifying: 200. Mu.L of plasma to be measured is taken, 20.00. Mu.L of internal standard working solution is added, 1000. Mu.L of ultrapure water is added for dilution, and vortex is carried out at 2500rpm for 1min. And (3) adding the diluted sample into a corresponding hole of a PAX 96 Kong Guxiang extraction plate activated and balanced by 2mL of methanol and 2mL of ultrapure water, respectively and sequentially adding 4mL of ultrapure water and 4mL of methanol for leaching, and placing the leached sample on a 96-well plate positive pressure extraction instrument for drying. 1mL of 0.1M hydrochloric acid methanol solution was added for elution, and the eluate was nitrogen-blown at room temperature for 120 minutes until volatilized.
Derivatization: 50 μl of 50% aqueous methanol solution was added to the corresponding wells of the 96-well plate, and the plates were sealed and then reconstituted by vortexing at 2500rpm for 5 min. mu.L of derivatizing reagent (2.0M trimethylsilanized diazomethane, TMS-DAM) and 200. Mu.L of methanol were added, and after sealing the membrane, the mixture was placed on a vortex machine and reacted for 60min with gentle shaking at 1000 rpm. After the reaction was completed, nitrogen was blown at room temperature for 120 minutes until the reaction was volatilized.
2. Preparation of standard curve sample and quality control sample
Two parts of alendronate sodium reference substances are weighed, 30% of methanol aqueous solution is added for dissolution, then 30% of methanol aqueous solution is added for volume fixation, and alendronate stock solutions with the concentration of 122.1 mug/mL and 121.5 mug/mL are obtained respectively, one part is used for preparing standard curve working solution, and the other part is used for preparing quality control working solution. The alendronate standard curve stock solution and the quality control stock solution are taken, 30% methanol aqueous solution is taken as a diluent to prepare the alendronate standard curve working solution and the quality control working solution, and then blank plasma is used for diluting the standard curve working solution and the quality control working solution to prepare standard curve series samples with the concentration of 0.5000, 1.000, 2.500, 5.000, 10.00, 25.00 and 50.00ng/mL respectively and quality control samples with the concentration of 0.5000 (LLOQ), 1.500 (LQC), 4.500 (GMQC), 22.5 (MQC) and 37.50 (HQC) ng/mL respectively.
3. LC-MS/MS analysis
Adding 500 μl of 5% acetonitrile aqueous solution containing 0.5% formic acid, sealing, vortexing at 2500rpm for 5min for redissolution, centrifuging at 4deg.C for 10min, transferring 200 μl supernatant into another 96-well plate, sealing for sample injection, and performing LC-MS/MS analysis under the following chromatographic conditions and mass spectrometry conditions:
chromatographic conditions:
chromatographic column model: infinity Lab Poroshell 120SB-C18 (50X 3.0mm,2.7 μm); column temperature: 40 ℃; sample injection volume: 5.0. Mu.L; flow rate: 0.4mL/min, mobile phase A:0.5% formic acid in water, mobile phase B:0.5% formic acid-acetonitrile solution; the elution gradient was as follows:
| Time(min) | A(％) | B(％) |
| 0.00 | 91.0 | 9.0 |
| 0.50 | 91.0 | 9.0 |
| 1.00 | 0.0 | 100.0 |
| 3.00 | 0.0 | 100.0 |
| 3.10 | 91.0 | 9.0 |
| 4.50 | 91.0 | 9.0 |
mass spectrometry conditions:
ion mode: esi+; scanning mode: MRM (multiplex reaction monitoring); mass to charge ratio (m/z): the analyte alendronate: 348.000 (parent ion), 289.200 (child ion); internal standard alendronate-d 6:354.200 (parent ion), 168.200 (child ion); ion mode: esi+, IS (ion spray voltage): 4500V; gas1 (atomizing Gas): 45.00psi; gas2 (assist Gas): 45.00psi; ion source temperature: 500 ℃.
Typical mass spectra of the derivatized alendronate and alendronate-d 6 are shown in fig. 1 and 2, and typical chromatogram of LLOQ obtained after sample injection is shown in fig. 3 (left: alendronate; right: alendronate-d 6).
The method for determining alendronate concentration in biological sample by LC-MS/MS provided in this embodiment is applicable to not only plasma samples but also urine samples.
In the derivatization reaction provided in this example, the amount of 50% aqueous methanol solution may be selected from 50 to 150. Mu.L, including but not limited to 50. Mu.L; the amount of trimethylsilylated diazomethane used for the derivatizing reagent, including but not limited to 300 μl, can be selected from 100 to 600 μl; the time for the derivatization reaction, including but not limited to 60min, may be selected from 30 to 90 min; the lower limit of the amount is 0.5000ng/mL, and the peak shape is satisfactory.
Example 2 methodological verification
1. Selectivity of
Blank plasma and LLOQ samples were taken by the procedure of example 1, respectively, and then subjected to mass spectrometry analysis to evaluate the selectivity of the procedure.
The results show that endogenous substances do not interfere with the determination of alendronate and alendronate-d 6.
A typical blank plasma sample chromatogram is shown in fig. 3 and a typical LLOQ chromatogram is shown in fig. 4.
2. Standard curve
Linear regression calculation was performed with the theoretical concentration of alendronic acid as abscissa (x) and the peak area ratio of alendronic acid to internal standard alendronic acid-d 6 as ordinate (y) (weight factor w=1/x 2 )。
A typical regression equation for alendronic acid is y=64.3×x+0.00602 (r=0.9985), with alendronic acid having a good linear relationship between 0.5000 and 50.00 ng/mL.
A typical standard graph is shown in fig. 5.
3. Precision and accuracy
The method validates three analysis batches, each with 6 samples of lower limit of assay quantification (LLOQ: 0.5000 ng/mL), low concentration (LQC: 1.500 ng/mL), geometric mean concentration (GMQC: 4.500 ng/mL), medium concentration (MQC: 22.50 ng/mL) and high (HQC: 37.50 ng/mL) level QC. The precision and accuracy within and between batches were calculated.
The method has acceptable precision and accuracy in measuring alendronic acid, and the LLOQ of the alendronic acid is 0.5000ng/mL.
4. Recovery and matrix effects
And 6 samples with low, medium and high quality control are analyzed. Simultaneously taking 200 mu L of blank plasma and processing according to a plasma sample pretreatment method; and re-dissolving the sample with a re-solvent after the treatment, adding alendronate reference solution and internal standard solution to prepare low, medium and high quality control sample concentrations, derivatizing and sample injection analysis. The average peak area ratio of alendronate in 2 samples is the extraction recovery rate.
200 μl (n=6) of blank plasma from 6 different sources was taken and treated according to the plasma sample pretreatment method; and re-dissolving the sample with a re-solvent after the treatment, adding alendronate reference solution and internal standard solution to prepare low, medium and high quality control sample concentrations, derivatizing and sample injection analysis. And taking 100 mu L of ultrapure water, processing according to the steps, and analyzing by sample injection.
The results showed that the average recovery of extraction at the different concentration levels was 25.87%. The recovery of the internal standard was 28.05%. The mean values of internal standard normalized matrix effector factors of alendronic acid at the low, medium and high quality control levels are 1.03, 0.96 and 1.03 respectively, and the precision is less than 5.83%. It was shown that under the present test conditions the effect of the matrix effect on the alendronate assay can be neglected.
Examples 3-4 investigation of solid phase extraction plate species
Based on example 1, the types of solid phase extraction plates (PAX solid phase extraction plate, SAX solid phase extraction plate, WAX solid phase extraction plate) in the plasma sample purification step were examined, and the influence of the types of solid phase extraction plates on the recovery rate and sensitivity was examined. The investigation process is as follows:
example 3: the measurement method is the same as in example 1, and differs from example 1 in that: the purification step of the plasma samples used SAX96 Kong Guxiang extraction plates.
Example 4: the measurement method is the same as in example 1, and differs from example 1 in that: the plasma sample was purified using a WAX96 Kong Guxiang extraction plate.
TABLE 1 investigation of the type of solid phase extraction plate
| Example 1 | Example 3 | Example 4 | |
| Solid phase extraction plate variety | PAX 96 Kong Guxiang extraction plate | SAX96 Kong Guxiang extraction plate | WAX96 Kong Guxiang extraction plate |
| Recovery rate | 25.87％ | 3.17％ | 6.26％ |
| LLOQ | 0.5000ng/mL | 4.000ng/mL | 2.000ng/mL |
The results of examining the types of the solid-phase extraction plates are shown in table 1, and in examples 1, 3 and 4, alendronic acid in plasma is separated and purified by using an anion solid-phase extraction plate, but the separation and purification effects are obviously affected by different filling materials in the three anion solid-phase extraction plates.
As can be seen from table 1: the SAX solid phase extraction plate of the embodiment 3 (the matrix material is silica gel, the bonding functional group is trimethyl aminopropyl) or the WAX solid phase extraction plate of the embodiment 4 (the matrix material is N-vinyl pyrrolidone-divinylbenzene copolymer, the bonding functional group is methyl piperazine ring) has low recovery rate and sensitivity, and the alendronic acid in the plasma has poor separation and purification effects; only when the PAX solid phase extraction plate of the example 1 is adopted (the filling material is trimethyl aminopropyl functionalized hydrophilic styrene-divinylbenzene), the separating and purifying effect of the alendronic acid in the blood plasma is obviously improved.
In summary, it was determined that the present invention employs PAX 96 Kong Guxiang extraction plates with a packing material that is trimethylaminopropyl functionalized hydrophilic styrene-divinylbenzene.
EXAMPLES 5-6 investigation of the types of solid phase extraction elution solvents
On the basis of example 1, the solid phase extraction eluting solvent types (hydrochloric acid-methanol solution, formic acid-methanol solution, trifluoroacetic acid-methanol solution) in the plasma sample purification step were examined, and the influence of the solid phase extraction eluting solvent types on the recovery rate and sensitivity was examined, and the examination procedure was as follows:
example 5: the measurement method is the same as in example 1, and differs from example 1 in that: the solid phase extraction eluting solvent in the plasma sample purification step is methanol formate.
Example 6: the measurement method is the same as in example 1, and differs from example 1 in that: the solid phase extraction eluting solvent in the plasma sample purification step is methanol trifluoroacetate.
TABLE 2 investigation of the types of solid phase extraction elution solvents
| Example 1 | Example 5 | Example 6 | |
| Solid phase extraction eluting solvent species | Hydrochloric acid-methanol | Formic acid-methanol | Trifluoroacetic acid-methanol |
| Recovery rate | 25.87％ | 0.17％ | 16.30％ |
| LLOQ | 0.5000ng/mL | 75.00ng/mL | 1.000ng/mL |
The results of examining the types of the eluting solvents of the solid phase extraction plates are shown in table 2, and examples 1, 5 and 6 all use an acidic methanol solution with equal concentration to elute alendronic acid adsorbed by the PAX solid phase extraction plates, but it was unexpectedly found that different acid types have significant influence on the separation and purification effects.
As can be seen from table 2: the formic acid-methanol solution in the embodiment 5 is adopted for elution, so that the recovery rate and the sensitivity are low, and the separation and purification effect is poor; the trifluoroacetic acid-methanol solution of example 6 is adopted for elution, so that the recovery rate and the sensitivity are improved, but the detection requirement of LLOQ <1ng/ml is still not met; only when the hydrochloric acid-methanol solution of example 1 is adopted, the concentration of hydrochloric acid is 0.02M-0.4M, the recovery rate and the sensitivity are further improved, the detection requirements are met, and the separation and purification effects are good.
In conclusion, the invention is determined to use hydrochloric acid-methanol solution as a solid phase extraction eluting solvent.
Examples 7 to 8 examination of mobile phase species
On the basis of example 1, investigation of chromatographic condition mobile phase types (0.5% formic acid, 0.1% formic acid, 10mM ammonium acetate) in UPLC-MS/MS was performed, and the influence of the mobile phase types on peak shape and sensitivity was investigated as follows:
example 7: the measurement method is the same as in example 1, and differs from example 1 in that: mobile phase a was 0.1% aqueous formic acid and mobile phase B was 0.1% acetonitrile formic acid in chromatographic conditions.
Example 8: the measurement method is the same as in example 1, and differs from example 1 in that: mobile phase a was 0.1% formic acid in 10mM ammonium acetate and mobile phase B was 0.1% formic acid in acetonitrile under chromatographic conditions.
TABLE 3 investigation of mobile phase species
In chromatographic conditions of liquid chromatography tandem mass spectrometry, the results of examining the mobile phase types are shown in table 3, and examples 1, 7 and 8 use different mobile phases, which have a significant influence on the detection sensitivity of LC-MS/MS.
As can be seen from table 3: example 7 uses 0.1% formic acid, has poor peak shape and lower sensitivity; example 810 mM ammonium acetate was added to improve peak shape but detection sensitivity was still low; only example 1 achieves the dual effects of improving peak shape and improving sensitivity by increasing the formic acid concentration to 0.5%.
Example 9 clinical sample testing
This example is a bioequivalence study of clinical samples, performed between 12 healthy subjects, all given a dose of 70mg, two cycles of cross-administration of the test formulation (T) with the reference formulation (R). About 4mL of elbow venous blood was collected before and after administration for 10min, 20min, 30min, 45min, 1h, 1.25h, 1.5h, 1.75h, 2h, 2.33h, 2.67h, 3h, 3.5h, 4h, 5h, 6h, 8h, 10h, 12h, and about 0.5mL of blood was discarded before each blood collection. The collected 4mL elbow venous blood was placed in an anticoagulant tube containing an anticoagulant, centrifuged, and plasma was taken for drug concentration analysis.
The drug concentration versus time is plotted, see figure 6.
Claims (12)
1. A method for LC-MS/MS determination of alendronate concentration in a biological sample comprising the steps of:
step 1: purification of biological samples
Mixing a biological sample to be tested with an internal standard working solution, and adding water for dilution; performing solid phase extraction on the diluted sample, eluting with an organic solvent, and volatilizing;
step 2: derivatization of biological samples
Redissolving the biological sample obtained in the step 1, adding trimethyl silanized diazomethane for derivatization reaction, and volatilizing after the reaction is finished;
step 3: liquid chromatography tandem mass spectrometry detection
Re-dissolving the biological sample obtained in the step 2, centrifuging, taking supernatant, and injecting the supernatant into a liquid chromatography-tandem mass spectrometry combined instrument for LC-MS/MS analysis to determine the alendronate concentration in the biological sample;
the solid phase extraction in the step 1 adopts a PAX solid phase extraction plate or a PAX solid phase extraction column with a filling material of trimethyl aminopropyl functionalized hydrophilic styrene-divinylbenzene;
the eluting solvent in the step 1 is hydrochloric acid methanol solution.
2. The method of LC-MS/MS determination of alendronate concentration in biological sample according to claim 1, wherein the hydrochloric acid concentration of the hydrochloric acid methanol solution is 0.02M to 0.4M.
3. The method of LC-MS/MS determination of alendronate concentration in biological sample according to claim 2, wherein the hydrochloric acid concentration of the hydrochloric acid methanol solution is 0.1M.
4. The method of LC-MS/MS determination of alendronate concentration in a biological sample according to claim 1, wherein said step 1 comprises:
mixing 200 mu L of biological sample with 20.00 mu L of internal standard working solution, adding ultrapure water for dilution, adding into a PAX 96 Kong Guxiang extraction plate activated and balanced by methanol and ultrapure water, sequentially adding ultrapure water and methanol for leaching, drying, adding hydrochloric acid methanol solution for eluting, and volatilizing the eluent.
5. The method of LC-MS/MS determination of alendronate concentration in a biological sample according to claim 1, wherein said step 2 comprises:
re-dissolving the biological sample obtained in the step 1 by 50-150 mu L of 50% methanol aqueous solution, adding 100-600 mu L of 2.0M trimethyl silanized diazomethane and 200 mu L of methanol, and volatilizing after derivatization reaction for 30-90 min.
6. The method for determining the concentration of alendronate in a biological sample according to claim 5 by LC-MS/MS, wherein the 50% aqueous methanol solution in step 2 is used in an amount of 50 μl, the trimethylsilylated diazomethane is used in an amount of 300 μl, and the derivatization time is 60min.
7. The method for LC-MS/MS determination of alendronate concentration in a biological sample according to claim 1, wherein the liquid chromatography conditions in step 3 include:
the chromatographic column is a C18 column, the mobile phase A is 0.5% formic acid aqueous solution, the mobile phase B is 0.5% formic acid acetonitrile solution, and the elution gradient is as follows:
8. the method of LC-MS/MS determination of alendronate concentration in a biological sample according to claim 1, wherein the liquid chromatography conditions in step 3 further comprise: the column temperature was 40 ℃, the sample injection volume was 5.00. Mu.L, and the flow rate was 0.4mL/min.
9. The method of LC-MS/MS determination of alendronate concentration in a biological sample according to claim 1, wherein the mass spectrometry conditions in step 3 include: ion mode: esi+, IS ion spray voltage: 4500v, gas1 atomizing gas: 45.00psi, gas2 assist gas: 45.00psi, ion source temperature: 500 ℃.
10. The method of LC-MS/MS determination of alendronic acid concentration in a biological sample according to any one of claims 1 to 9, wherein the isotopic internal standard of alendronic acid is alendronic acid-d 6.
11. The method of LC-MS/MS determination of alendronate concentration in a biological sample according to any one of claims 1 to 9, wherein said biological sample comprises a urine sample and a blood sample.
12. The method of LC-MS/MS determination of alendronate concentration in a biological sample according to claim 11 wherein said biological sample is plasma.
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