CN108445116B - Pretreatment method for free oxytocin in organism liquid sample - Google Patents
Pretreatment method for free oxytocin in organism liquid sample Download PDFInfo
- Publication number
- CN108445116B CN108445116B CN201810209364.9A CN201810209364A CN108445116B CN 108445116 B CN108445116 B CN 108445116B CN 201810209364 A CN201810209364 A CN 201810209364A CN 108445116 B CN108445116 B CN 108445116B
- Authority
- CN
- China
- Prior art keywords
- oxytocin
- organic solvent
- supernatant
- solution
- biological fluid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- 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
- G01N2030/062—Preparation extracting sample from raw material
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The application discloses a pretreatment method of free oxytocin in an organism liquid sample, which at least comprises the following steps: (1) carrying out oxytocin desorption treatment on a biological fluid sample to obtain a system I; (2) selectively enriching oxytocin and removing impurities from the system I in the step (1) to obtain a system II; (3) concentrating and secondarily desalting the system II in the step (2) to obtain a system III; (4) and (6) collecting oxytocin. The method can simply and efficiently enrich the free oxytocin from the biological body fluid, remove high-abundance proteins, inorganic salts, hydrophilic micromolecules, lipids and other interferents, and is used for the nano LC-MS analysis and quantification of the free oxytocin in the biological body fluid sample.
Description
Technical Field
The application relates to a pretreatment method of free oxytocin in an organism liquid sample, belonging to the field of analytical chemistry.
Background
Oxytocin is a neuropeptide that may be associated with complex emotional and social behaviors such as social cognition, stress buffering, aggression and trust. Oxytocin existing in a free state in biological body fluid is closely related to highly specific individual conditions such as age, sex, drug taking history and the like as a nerve regulator, and the accurate determination of the oxytocin is the key for understanding the action of the peptide in various neurophysiological processes and behaviors and has more value for clinical diagnosis. Accurate analysis of free oxytocin in biological fluids faces a huge challenge, and biological fluids, especially plasma, are complex in composition and incompatible with high-efficiency analysis techniques (such as liquid chromatography); the content of free oxytocin in biological fluids is very low, only in the order of pg/ml in plasma. Therefore, the pretreatment of the biological fluid sample, the removal of impurities and the enrichment of oxytocin are the primary steps of the analysis of free oxytocin. The efficient and rapid pretreatment method for the free oxytocin in the biological body fluid sample is established, plays an important role in realizing high-sensitivity and high-accuracy oxytocin analysis, and further promotes the progress of disease discovery and treatment.
At present, multi-step treatment methods such as a pretreatment method based on protein sedimentation and SPE, a pretreatment method based on protein sedimentation and ultrafiltration centrifugation and the like are common technologies for pretreatment of biological body fluid samples. However, the SPE processing steps are cumbersome and time consuming. Although on-line SPE sample pre-processing simplifies the overall processing process and reduces manual handling steps, on-line operation requires special equipment, maintenance and method set-up are complex, and often result in long chromatographic run times, and therefore, are rarely used in laboratory and clinical sample analysis. In addition, the extreme hydrophilicity of free oxytocin can also make it severely lost in SPE packings. The ultrafiltration and centrifugation operation is simple, but the retention rate of oxytocin with the molecular weight of only 1007.2Da is low, and the sample loss is serious. (Johnsen, E.; Leknes, S.; Wilson, S.R.; Lundanes, E.Sci.Rep.2015,5,9308.Zhang, G.; Zhang, Y.; Fast, D.M.; Lin, Z.; Steenwyk, R.anal.biochem.2011,416,45-52.)
Disclosure of Invention
The pretreatment process of biological body fluid (such as plasma) in the application, including protein denaturation, oxytocin extraction, protein precipitation, inorganic salt and hydrophilic small molecule removal, is completed in a one-step salting-out assisted liquid-liquid extraction (SALLE) system, and the nano LC-MS analysis can be realized through simple concentration and degreasing processes.
The pretreatment method for the free oxytocin in the biological fluid sample is characterized by at least comprising the following steps:
(1) carrying out oxytocin desorption treatment on a biological fluid sample to obtain a system I;
(2) selectively enriching oxytocin and removing impurities from the system I in the step (1) to obtain a system II;
(3) concentrating and secondarily desalting the system II in the step (2) to obtain a system III;
(4) and (6) collecting oxytocin.
Optionally, the oxytocin desorption treatment in step (1) comprises: and (3) carrying out protein denaturation treatment.
Optionally, the oxytocin desorption treatment comprises: adding a protein denaturant into the biological fluid sample to denature the protein, thereby realizing the desorption of the oxytocin which is non-covalently combined with the protein.
Optionally, the volume of the biological fluid sample is 100-1200 μ L, and the dosage of the protein denaturant is 0.2-1 times of the dosage of the biological fluid sample.
Optionally, the upper limit of the volume of the biological fluid sample is selected from 300 μ L, 600 μ L or 1200 μ L; the lower limit is selected from 100. mu.L, 300. mu.L or 600. mu.L.
Optionally, the upper limit of the volume ratio of the amount of the protein denaturant to the amount of the biological fluid sample is selected from 0.4:1 or 1: 1; the lower limit is selected from 0.2:1 or 0.4: 1.
Optionally, the protein denaturant is added to the biological fluid sample, vortexed, and desorbs free oxytocin non-covalently adsorbed to the protein.
Optionally, the time of the swirling is 25-35 seconds.
Optionally, the protein denaturant includes at least one of an acidic solution, a surfactant, and a basic detergent solution.
Optionally, the acidic solution is selected from at least one of phosphoric acid aqueous solution, trifluoroacetic acid aqueous solution and trichloroacetic acid aqueous solution; the surfactant is selected from at least one of sodium dodecyl sulfate aqueous solution, sodium dodecyl benzene sulfonate aqueous solution and sodium dodecyl sulfate aqueous solution; the alkaline detergent is at least one of urea aqueous solution and guanidine hydrochloride aqueous solution.
Optionally, the concentration of the acidic solution is 0.1-5 v%; the concentration of the surfactant was 4 wt%; the concentration of the alkaline detergent was 6M.
Optionally, the selective enrichment and impurity removal of oxytocin in the step (2) comprises: salting out to assist liquid-liquid extraction.
Optionally, the selective enrichment and impurity removal of oxytocin comprises: adding SALLE reagent into the system I in the step (1), shaking, whirling, centrifuging, and taking supernatant.
Optionally, the SALLE reagent comprises a hydrophilic organic solvent and a salt solution; wherein the hydrophilic organic solvent can be at least one of methanol, ethanol, acetonitrile and isopropanol; the salt solution is at least one of dipotassium hydrogen phosphate solution, sodium chloride solution and magnesium chloride solution.
Optionally, the solvent of the salt solution is water.
Optionally, the volume ratio of the hydrophilic organic solvent to the biological fluid sample is 20: 1-1: 1; the volume ratio of the salt solution to the hydrophilic organic solvent is 1: 1-1: 4.
Optionally, the upper limit of the volume ratio of the hydrophilic organic solvent to the biological fluid sample is selected from 20:1, 40:3, 10:1 or 20: 3; the lower limit is selected from 40:3, 10:1, 20:3 or 1: 1.
Optionally, the concentration of the salt solution is 0.7-4M.
Alternatively, the upper limit of the concentration of the salt solution is selected from 4M, 2.8M or 1.4M; the lower limit is selected from 0.7M, 1.4M or 2.8M.
Optionally, the adding of the SALLE reagent comprises: the salt solution was added first, vortexed, and the hydrophilic organic solvent was added.
Optionally, the number of oscillations is 10.
Optionally, the oscillation is followed by vortexing for 25-35 seconds.
Optionally, the time for vortexing after the oscillation is 25-35 seconds.
Alternatively, the centrifugation conditions are 3000rpm centrifugation for 10 minutes.
Optionally, the concentration and secondary desalination in step (3) comprises: drying and redissolving.
Optionally, the concentrating and secondary desalting comprises: and (3) drying the supernatant in the system II in the step (2), re-dissolving by using a hydrophilic organic solvent, centrifuging and taking the supernatant to realize concentration and secondary desalting of the sample.
Optionally, the volume of the hydrophilic organic solvent is 1-10 times of the volume of the biological fluid sample.
Optionally, the upper limit of the ratio of the volume of the hydrophilic organic solvent to the volume of the sample of biological fluid is selected from 10:1, 5:1, 8:3 or 5: 3; the lower limit is selected from 1.6:1, 5:3, 8:3 or 5: 1.
Optionally, the hydrophilic organic solvent is selected from at least one of methanol, ethanol, acetonitrile, isopropanol.
Optionally, the hydrophilic organic solvent redissolving comprises: adding hydrophilic organic solvent, ultrasonic treating, vortex, and collecting supernatant.
Optionally, the time of the ultrasonic treatment is 25-35 seconds; the vortex time is 2-35 seconds.
Optionally, the oxytocin collection in step (4) comprises: and (4) drying the supernatant in the system III in the step (3), adding a liquid chromatography mobile phase for redissolving, adding an organic solvent, and centrifuging to obtain the supernatant, namely the oxytocin enrichment component.
Optionally, the volume ratio of the liquid chromatography mobile phase to the organic solvent is 1: 1-5: 1.
Optionally, the upper limit of the volume ratio of the liquid chromatography mobile phase to the organic solvent is selected from 5:3, 4:1 or 5: 1; the lower limit is selected from 1:1, 5:3 or 4: 1.
Optionally, the volume fraction of the liquid chromatography mobile phase is 5-30%, and the addition amount is 1/20-1/5 times of the volume of the biological fluid sample.
Optionally, the upper volume fraction limit of the liquid chromatography mobile phase is selected from 15% or 30%; the lower limit is selected from 5%, 10% or 15%.
Optionally, the upper limit of the volume ratio of the liquid chromatography mobile phase to the biological fluid sample is selected from 1/12, 1/7.5 or 1/5; the lower limit is selected from 1/20, 1/12 or 1/7.5.
Optionally, the liquid chromatography mobile phase is acetonitrile with a volume fraction of 5% -30%, the solvent is water, and the addition amount is 1/20-1/5 times of the volume of the biological fluid sample.
Optionally, the organic solvent is at least one selected from dichloromethane and chloroform.
Optionally, after the organic solvent is added, performing ultrasonic treatment, vortexing and centrifuging, and taking supernatant to obtain the oxytocin-enriched component.
Optionally, the time of the ultrasonic treatment is 25-35 seconds; the vortex time is 25-35 seconds.
Optionally, the centrifugation condition is 15000rpm centrifugation for 20-60 minutes.
Alternatively, the oxytocin collected in step (4) is directly used for nanoLC-MS analysis.
Alternatively, the biological fluid sample may be obtained or collected according to common general knowledge in the art.
Optionally, the method comprises at least:
(a1) oxytocin desorption: adding an acidic solution or a surfactant or an alkaline detergent solution into the biological fluid sample to denature proteins, so as to realize the desorption of oxytocin non-covalently combined with the proteins in the biological fluid sample;
(a2) selective enrichment and impurity removal of oxytocin: adding a SALLE reagent into the sample treated in the step (a1), fully oscillating, vortexing, centrifuging to obtain supernatant, and simultaneously selectively enriching oxytocin and removing high-abundance proteins, inorganic salts and hydrophilic small molecular impurities in the biological fluid sample;
(a3) concentration and secondary desalting: drying the supernatant obtained in the step (a2), re-dissolving the supernatant in a hydrophilic organic solvent, centrifuging the re-dissolved hydrophilic organic solvent to obtain the supernatant, and concentrating and secondarily desalting the sample;
(a4) and (3) oxytocin collection: and (a3) drying the supernatant in the step (a3), adding acetonitrile aqueous solution with the volume concentration of 5-30% for redissolving, adding dichloromethane or trichloromethane, centrifuging and taking the supernatant to obtain the required oxytocin enrichment component.
Optionally, the oxytocin is endogenous free oxytocin or the sum of exogenous and endogenous free oxytocin after administration.
Optionally, the biological fluid comprises plasma, serum, cerebrospinal fluid or urine.
As a specific embodiment, the method at least comprises:
(b1) oxytocin desorption: adding an acidic solution or a surfactant or an alkaline detergent solution into the plasma to denature the protein, so as to realize the desorption of oxytocin non-covalently combined with the plasma protein;
(b2) selective enrichment and impurity removal of oxytocin: adding a SALLE reagent into the sample treated in the step (b1), fully oscillating, vortexing, centrifuging to obtain supernatant, and simultaneously performing selective enrichment of oxytocin and removal of high-abundance proteins, inorganic salts and hydrophilic small molecular impurities in plasma;
(b3) concentration and secondary desalting: drying the supernatant obtained in the step (b2), re-dissolving the supernatant in a hydrophilic organic solvent, centrifuging the re-dissolved hydrophilic organic solvent to obtain the supernatant, and concentrating and secondarily desalting the sample;
(b4) and (3) oxytocin collection: and (b3) drying the supernatant in the step (b), adding a low-concentration acetonitrile aqueous solution for redissolving, adding dichloromethane or trichloromethane, and centrifuging to obtain the supernatant, namely the required oxytocin enrichment component.
As a specific embodiment, the method at least comprises:
(c1) oxytocin desorption: adding an acidic solution (phosphoric acid, trifluoroacetic acid) or a surfactant (SDS) or an alkaline detergent (urea, guanidine hydrochloride) solution into the organism liquid sample, and vortexing for 30 seconds to desorb free oxytocin adsorbed by the noncovalent bond of the protein;
(c2) selective enrichment and impurity removal of oxytocin: and (c1) treating the plasma sample, adding a salt solution (dipotassium phosphate, sodium chloride, magnesium chloride and sodium carbonate) with the volume 2.5-10 times that of the plasma, swirling for 30 seconds, adding a hydrophilic organic solvent (methanol, ethanol, isopropanol and acetonitrile) with the volume 1-10 times that of the plasma, fully oscillating for 10 times, swirling for 30 seconds, centrifuging for 10 minutes at 3000rpm, and simultaneously performing selective enrichment of oxytocin and removal of high-abundance proteins, inorganic salts and hydrophilic small molecular impurities in the plasma sample.
(c3) Concentration and secondary desalting: transferring the supernatant of step (c2) to a fresh centrifuge tube, transferring the centrifuge tube to a nitrogen blower, and drying; adding 0.5-2 ml of hydrophilic organic solvent (methanol and isopropanol), carrying out ultrasonic treatment for 30 seconds, carrying out vortex treatment for 30 seconds, taking the supernatant, transferring the supernatant into a nitrogen blowing instrument, and drying;
(c4) and (3) oxytocin collection: and (c3) adding a small-volume 1/20-1/5 low-concentration organic solvent (5-30% acetonitrile) compatible with liquid mass analysis into the dried sample, adding dichloromethane or trichloromethane to enable the volume ratio of the low-concentration organic solvent to the dichloromethane or trichloromethane to be 1: 1-5: 1, carrying out ultrasonic treatment for 30 seconds, carrying out vortex treatment for 30 seconds, centrifuging at 15000rpm for 40 minutes, and taking the supernatant to obtain the required oxytocin enrichment component.
The application relates to a pretreatment method of free oxytocin in biological fluid, which completes the extraction separation of the oxytocin, the denaturation and the precipitation of protein, the sample treatment process of removing inorganic salt and hydrophilic micromolecular impurities in one centrifugal tube, and can be used for subsequent high-efficiency liquid chromatography and mass spectrometry through simple concentration and degreasing processes. The method specifically comprises the following steps: (d1) adding a protein denaturant into the plasma, and uniformly mixing by vortex; (d2) sequentially adding a salt solution and a hydrophilic organic solvent, fully oscillating, uniformly mixing by vortex, centrifuging, taking a supernatant, and drying; (d3) ultrasonically dissolving the dried substance with hydrophilic organic solvent, centrifuging, collecting supernatant, and drying; (d4) adding chromatographic mobile phase and dichloromethane or chloroform into the dried substance, ultrasonic dissolving, centrifuging, and collecting supernatant. The invention can simply and efficiently enrich free oxytocin from biological body fluid, remove interferents such as high-abundance protein, inorganic salt, hydrophilic micromolecule, lipid and the like, and is used for nano LC-MS analysis.
The salting-out assisted liquid-liquid extraction System (SALLE) is an effective means for extracting and enriching polar compounds, is composed of a hydrophilic organic solvent and a salt solution, and has the advantages of mild conditions, simplicity in operation, multiple adjustable factors and the like.
In this application, "high abundance protein" refers to proteins in the mg/ml fraction.
In the present application, "nanoLC-MS" refers to nanoliter liquid chromatography-mass spectrometry.
In the present application, all conditions relating to a numerical range may be independently selected from any intermediate range within said numerical range.
In this application, all conditions relating to numerical ranges are inclusive of the endpoints unless specifically stated otherwise.
In the present application, a person skilled in the art can implement the technical solutions in the present application according to any specific numerical conditions in the range conditions (such as volume ratio, etc.) involved in the solutions, so as to achieve the effective effects described in the present application.
The beneficial effects that this application can produce include:
1. in the application, the extraction of oxytocin and the removal of impurities are completed in one step, oxytocin is enriched in an upper hydrophilic organic phase, high-abundance protein is in an intermediate solid layer, and inorganic salt and hydrophilic micromolecular interferent are in a lower solution layer, so that the intermediate transfer step is reduced, and the sample loss is reduced.
2. In the application, the oxytocin is enriched in the upper hydrophilic organic phase, so that the extraction liquid is easy to transfer, and the hydrophilic organic solvent has a low boiling point and is easy to carry out aftertreatment.
3. The hydrophilic organic solvent selected by the SALLE in the application can be completely miscible with water, so that a long or violent mixing step is not needed to promote the distribution of oxytocin and impurities between two phases, a long-time high-speed centrifugation is not needed to assist the phase separation of a system, the operation process is simple and quick, and the method is suitable for high-throughput analysis.
Drawings
FIG. 1 is a flow chart illustrating the pretreatment of plasma free oxytocin in the present application; wherein, 1: oxytocin resolution attached 2: selective enrichment and impurity removal of oxytocin 3: concentration and secondary desalination 4: collecting oxytocin;
FIG. 2 shows data of the nanolC-LTQ-Oritrap assay; base peak chromatograms of human plasma (BPC) obtained in positive mode by nano-LC-MS, scanning range 504.00-509.40 (fig. 2A); linear relationship (including regression equation and R2 value) between peak area ratio of extracted ion EIC of oxytocin/isotope-labeled oxytocin (OT/IS) in diluted plasma and initial mixing ratio (fig. 2B); the initial concentration ratio (OT/IS) was 2.500, and the EIC peak area ratio was 2.22 (FIG. 2B, panel C);
FIG. 3 extracted ion flow graph of oxytocin (dark line) in volunteer plasma (IS (light line) comprising 200pg/mL) (A and B);
FIG. 4 extracted ion flow graph of oxytocin (dark line) in rat plasma (including 200pg/mL IS) (A and B).
Detailed Description
The present application will be described in detail with reference to examples, but the present application is not limited to these examples.
The raw materials in the examples of the present application were all purchased commercially, unless otherwise specified.
The analysis method in the examples of the present application is as follows:
NanoLC-LTQ-Oritrap assays were performed to quantify free oxytocin using LTQ-Orbitrap Discovery (Thermo Fisher Scientific, Bremen, Germany) in combination with nano-LC (Eksient Technologies, California, USA).
Example 1 plasma free oxytocin Pre-treatment
(1) Plasma collection
Human whole blood was collected using a commercial vacuum blood collection tube (BDTM P800, Becton, Dickinson and Company, USA), centrifuged at 3000rpm for 10min, and the supernatant was collected as plasma, which was either directly pre-treated or stored at-80 ℃ in a refrigerator.
(2) Denaturation of proteins
And (2) taking 600 mu L of the human plasma in the step (1), putting the human plasma in a 10mL centrifuge tube, adding an aqueous solution (with a final concentration of 200pg/mL) of oxytocin isotope internal standard (Pro [13C5,15N ] oxytocin, Invitrogen tracing Co., Ltd, Shanghai and China), adding 240 mu L of 5 v% phosphoric acid aqueous solution, and vortexing for 30 s.
(3) Oxytocin extraction and impurity removal
After the plasma sample is processed in step (2), 4mL of aqueous dipotassium phosphate (4M) is added, vortexed for 30 seconds, 4mL of isopropanol is added, fully shaken for 10 times, vortexed for 30 seconds, centrifuged at 3000rpm for 10 minutes, and 4mL of supernatant in the centrifuge tube is transferred to a new centrifuge tube.
(4) Concentration and secondary desalination
And (4) transferring the sample treated in the step (3) to a nitrogen blower, blowing nitrogen until the sample is dry, adding 1ml of isopropanol, ultrasonically dissolving for 30s, vortexing for 30s, centrifuging for 10 minutes at the rotating speed of 3000rpm, and transferring 0.9ml of supernate in a centrifuge tube to a new centrifuge tube.
(5) Oxytocin collection
And (3) transferring the sample treated in the step (4) into a nitrogen blowing instrument, blowing nitrogen to dry, adding 50 mu L of 15% acetonitrile (the volume fraction is 15%, the solvent is water) and 30 mu L of dichloromethane, carrying out ultrasonic treatment for 30 seconds, carrying out vortex for 30 seconds, centrifuging for 40min at the rotating speed of 15000rpm, and taking the supernatant, namely the required oxytocin enrichment component.
Example 2 plasma free oxytocin Pre-treatment
The pretreatment of plasma free oxytocin in this example was similar to that of example 1. Wherein:
in the protein denaturation step (2), "240. mu.L of 5 v% phosphoric acid aqueous solution" is replaced by "120. mu.L of 4 wt% sodium dodecyl sulfate aqueous solution";
in the step (3) of oxytocin extraction and impurity removal, the concentration of the dipotassium hydrogen phosphate aqueous solution is 0.7M;
in the concentration and secondary desalting in the step (4), '1 ml of isopropanol' is replaced by '0.6 ml of isopropanol';
in the oxytocin collection of step (5), "50 μ L of 15% acetonitrile (volume fraction 15%, solvent water)" was replaced with "30 μ L of 30% acetonitrile (volume fraction 30%, solvent water)".
The remaining operations and conditions were the same as in example 1, and the supernatant obtained was the desired oxytocin-enriched fraction.
Example 3 plasma free oxytocin Pre-treatment
(1) Plasma collection
Collecting SD rat whole blood with commercial vacuum blood collection tube (containing NaEDTA), centrifuging at 3000rpm for 10min, collecting supernatant to obtain blood plasma, and directly pretreating or storing at-80 deg.C in refrigerator.
(2) Denaturation of proteins
And (2) taking 300 mu L of the rat plasma in the step (1), putting the rat plasma in a 10mL centrifuge tube, adding an oxytocin isotope internal standard (Pro [13C5,15N ] oxytocin, Invitrogen tracing Co., Ltd, Shanghai and China) aqueous solution (the final concentration is 200pg/mL), adding 120 mu L of 5 v% phosphoric acid aqueous solution, and vortexing for 30 s.
(3) Oxytocin extraction and impurity removal
After the plasma sample is processed in step (2), 4mL of aqueous dipotassium phosphate (4M) is added, vortexed for 30 seconds, 4mL of isopropanol is added, fully shaken for 10 times, vortexed for 30 seconds, centrifuged at 3000rpm for 10 minutes, and 4mL of supernatant in the centrifuge tube is transferred to a new centrifuge tube.
(4) Concentration and secondary desalination
And (4) transferring the sample treated in the step (3) to a nitrogen blower, blowing nitrogen until the sample is dry, adding 0.8ml of isopropanol, dissolving by ultrasonic for 30s, vortexing for 30s, centrifuging for 10 minutes at the rotating speed of 3000rpm, and transferring 0.7ml of supernate in a centrifuge tube to a new centrifuge tube.
(5) Oxytocin collection
And (3) transferring the sample treated in the step (4) into a nitrogen blowing instrument, blowing nitrogen to dry, adding 40 mu L of 15% acetonitrile (the volume fraction is 15%, the solvent is water) and 10 mu L of dichloromethane, carrying out ultrasonic treatment for 30 seconds, carrying out vortex for 30 seconds, centrifuging for 40min at the rotating speed of 15000rpm, and taking the supernatant, namely the required oxytocin enrichment component.
Example 4 plasma free oxytocin Pre-treatment
The pretreatment of plasma free oxytocin in this example was similar to that of example 3. Wherein:
in the step (2) of protein denaturation, "120. mu.L of 5 v% aqueous phosphate solution" was replaced with "300. mu.L of 6M aqueous guanidine hydrochloride solution";
in the step (3) of oxytocin extraction and impurity removal, the concentration of the dipotassium hydrogen phosphate aqueous solution is 2.8M;
in the concentration and secondary desalting in the step (4), "0.8 ml of isopropanol" is replaced by "3 ml of isopropanol";
in the oxytocin collection of step (5), "40. mu.L of 15% acetonitrile (volume fraction of 15%, solvent of water) and 10. mu.L of dichloromethane" were replaced with "60. mu.L of 5% acetonitrile (volume fraction of 5%, solvent of water) and 12. mu.L of dichloromethane".
The remaining operations and conditions were the same as in example 3, and the supernatant obtained was the desired oxytocin-enriched fraction.
Example 5NanoLC-LTQ-Oritrap assay
(1) NanoLC-LTQ-Oritrap assay
The oxytocin-enriched fraction (individual plasma from 24 volunteers treated according to example 1; 2 batches of commercial plasma treated according to example 1; 11 rats treated according to example 3) was assayed using the nanoLC-LTQ-Oritrap. Analytical columns for the nanoLC separations used a PicoFrit series of capillary columns (New Objective, Woburn, MA, usa) 75 μm in internal diameter and 20cm in length with a 10 μm pin at one end, C18(3 μm,
dr, maisch GmbH, Ammerbuch-Entringen, germany) packing laboratory self-packing self-loading pre-column (3cm × 100 μm id), column tube PEEK column holder (Upchurch, Oak Harbor, WA, USA), packing C18AQ particles (5 μm,
Dr.Maisch GmbH, Ammerbuch-Entringen, Germany). The mass spectrometer analysis was performed in positive ion mode with spray voltage of 1.5-2.0V and heated capillary temperature of 320 ℃. Using Xcaliibur software to recordThe total ion flow diagram (FIG. 2A) was recorded with mass to charge ratios ranging from 504.0-509.4.
(2) Data analysis
EIC extraction is carried out on the collected atlas, and the quantitative ion of oxytocin is [ M +2H ]]2+Molecular weight 504.2270Da, oxytocin isotope internal standard (Pro [13C5, 15N)]oxocin) is [ M +2H]2+Molecular weight 507.2330, mass deviation 10 ppm. The limit of the method for the quantification of free oxytocin is 1 pg/ml. The standard recovery rate of 360pg/ml reaches 80 percent, and the matrix inhibits<18%, intra-and inter-day repetitive RSD<15 percent. The correction factor curve is Y-0.8251X +0.0762, R2The initial concentration ratio (OT/IS) was 2.500 and the EIC peak area ratio was 2.22 (fig. 2C) at 0.9993 (fig. 2B).
The oxytocin-enriched fraction obtained in examples 1 and 3 was assayed by the above-described assay method, and the EIC profiles of OT and IS in the plasma of some human individuals are shown in fig. 3(a and B), and the EIC profiles of OT and IS in the plasma sample of rat are shown in fig. 4(a and B). Table 1 gives detailed quantification results of free OT in plasma of human and rat individuals. Levels of free OT in individual human plasma ranged from 3 to 142pg/mL with a mean. + -. SD of 43.8. + -. 40.2 pg/mL. The OT levels in rat plasma were high, ranging from 166 to 2462pg/mL, with mean. + -. SD 837.4. + -. 798.1 pg/mL.
Table 124 free oxytocin content in volunteers, 2 batches of commercial plasma and 11 SD rat plasma
The test methods of example 2 and example 4 were the same as those described above, and the measurement results were similar to those of example 1 and example 3 described above.
The method can obviously improve the pretreatment flux of the free oxytocin in the plasma sample, and simultaneously, the oxytocin is protected by the SALLE solvent in the first time, thereby avoiding the combination of the free oxytocin and the large protein and ensuring the accuracy and the repeatability of the quantification. In addition, the SALLE method can efficiently remove high-abundance proteins, inorganic salts and hydrophilic small molecule interferents which are incompatible with high-efficiency liquid chromatography and mass spectrometry in a plasma sample in one step, and can be used with subsequent nano LC-MS through simple concentration and fat removal steps.
Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.
Claims (17)
1. A method for pretreating free oxytocin in a biological fluid sample, which is characterized by comprising at least the following steps:
(1) carrying out protein denaturation treatment on a biological body fluid sample to obtain a system I;
(2) carrying out salting-out assisted liquid-liquid extraction on the system I in the step (1) to obtain a system II;
(3) drying the supernatant in the system II in the step (2), re-dissolving by using a hydrophilic organic solvent, centrifuging and taking the supernatant to realize concentration and secondary desalting of a sample to obtain a system III;
(4) and (3) drying the supernatant in the system III in the step (3), adding a liquid chromatography mobile phase for redissolving, adding an organic solvent, centrifuging and taking the supernatant to obtain the oxytocin enrichment component, wherein the liquid chromatography mobile phase is acetonitrile with the volume fraction of 5-30%, the solvent is water, the adding amount is 1/20-1/5 times of the volume of the biological fluid sample, and the organic solvent is at least one of dichloromethane and trichloromethane.
2. The method of claim 1, wherein the protein denaturation treatment comprises: adding a protein denaturant into the biological fluid sample to denature the protein, thereby realizing the desorption of the oxytocin which is non-covalently combined with the protein.
3. The method according to claim 1, wherein the volume of the biological fluid sample is 100 to 1200 μ L, and the amount of the protein denaturant is 0.2 to 1 times of the volume of the biological fluid sample.
4. The method of claim 2, wherein the protein denaturant includes at least one of an acidic solution, a surfactant, and a basic detergent solution.
5. The method according to claim 4, wherein the acidic solution is at least one selected from the group consisting of an aqueous phosphoric acid solution, an aqueous trifluoroacetic acid solution, and an aqueous trichloroacetic acid solution; the surfactant is selected from at least one of sodium dodecyl sulfate aqueous solution, sodium dodecyl benzene sulfonate aqueous solution and sodium dodecyl sulfate aqueous solution; the alkaline detergent is at least one of urea aqueous solution and guanidine hydrochloride aqueous solution.
6. The method according to claim 4, wherein the concentration of the acidic solution is 0.1 to 5% v; the concentration of the surfactant was 4 wt%; the concentration of the alkaline detergent was 6M.
7. The method of claim 1, wherein the salting-out assisted liquid-liquid extraction of oxytocin comprises: adding SALLE reagent into the system I in the step (1), shaking, whirling, centrifuging, and taking supernatant.
8. The method of claim 7, wherein the SALLE reagent comprises a hydrophilic organic solvent and a salt solution; wherein the volume ratio of the hydrophilic organic solvent to the biological fluid sample is 20: 1-1: 1; the volume ratio of the salt solution to the hydrophilic organic solvent is 1: 1-1: 4.
9. The method according to claim 8, wherein the hydrophilic organic solvent is at least one of methanol, ethanol, acetonitrile, isopropanol; the salt solution is at least one of dipotassium hydrogen phosphate solution, sodium chloride solution and magnesium chloride solution.
10. The method of claim 8, wherein the salt solution has a concentration of 0.7 to 4M.
11. The method according to claim 1, wherein the hydrophilic organic solvent is at least one selected from the group consisting of methanol, ethanol, acetonitrile, and isopropanol.
12. The method according to claim 11, wherein the volume of the hydrophilic organic solvent is 1 to 10 times the volume of the sample of biological fluid.
13. The method according to claim 1, wherein the volume ratio of the liquid chromatography mobile phase to the organic solvent in the step (4) is 1:1 to 5: 1.
14. The method of claim 1, wherein the oxytocin collected in step (4) is used directly in the nanoLC-MS analysis.
15. Method according to claim 1, characterized in that it comprises at least:
(a1) oxytocin desorption: adding an acidic solution or a surfactant or an alkaline detergent solution into the biological fluid sample to denature proteins, so as to realize the desorption of oxytocin non-covalently combined with the proteins in the biological fluid sample;
(a2) selective enrichment and impurity removal of oxytocin: adding a SALLE reagent into the sample treated in the step (a1), fully oscillating, vortexing, centrifuging to obtain supernatant, and simultaneously selectively enriching oxytocin and removing high-abundance proteins, inorganic salts and hydrophilic small molecular impurities in the biological fluid sample;
(a3) concentration and secondary desalting: drying the supernatant obtained in the step (a2), re-dissolving the supernatant in a hydrophilic organic solvent, centrifuging the re-dissolved hydrophilic organic solvent to obtain the supernatant, and concentrating and secondarily desalting the sample;
(a4) and (3) oxytocin collection: and (a3) drying the supernatant in the step (a3), adding acetonitrile aqueous solution with the volume concentration of 5-30% for redissolving, adding dichloromethane or trichloromethane, centrifuging and taking the supernatant to obtain the required oxytocin enrichment component.
16. The method of claim 1, wherein said oxytocin is endogenous free oxytocin or the sum of exogenous and endogenous free oxytocin upon administration.
17. The method of claim 1, wherein the biological fluid comprises plasma, serum, cerebrospinal fluid, or urine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810209364.9A CN108445116B (en) | 2018-03-14 | 2018-03-14 | Pretreatment method for free oxytocin in organism liquid sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810209364.9A CN108445116B (en) | 2018-03-14 | 2018-03-14 | Pretreatment method for free oxytocin in organism liquid sample |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108445116A CN108445116A (en) | 2018-08-24 |
| CN108445116B true CN108445116B (en) | 2020-08-11 |
Family
ID=63194401
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810209364.9A Active CN108445116B (en) | 2018-03-14 | 2018-03-14 | Pretreatment method for free oxytocin in organism liquid sample |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108445116B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111141854A (en) * | 2018-11-06 | 2020-05-12 | 中国科学院大连化学物理研究所 | Method for simultaneously extracting polar and low-polar metabolites from biological sample |
| CN110987579A (en) * | 2019-12-24 | 2020-04-10 | 武汉生物技术研究院 | Method for rapidly enriching and purifying vitamin D from trace blood |
| CN113252807B (en) * | 2021-04-15 | 2022-05-06 | 远大生命科学(武汉)有限公司 | High performance liquid chromatography separation method for oxytocin raw material medicine related substances |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106146607A (en) * | 2015-04-09 | 2016-11-23 | 深圳华大基因研究院 | One peptide species method for extraction and purification and test kit |
| CN106831951A (en) * | 2017-01-11 | 2017-06-13 | 蚌埠丰原医药科技发展有限公司 | A kind of purification process of oxytocin |
-
2018
- 2018-03-14 CN CN201810209364.9A patent/CN108445116B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN108445116A (en) | 2018-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108445116B (en) | Pretreatment method for free oxytocin in organism liquid sample | |
| Fan et al. | Ionic liquid based dispersive liquid–liquid microextraction of aromatic amines in water samples | |
| Balchen et al. | Fast, selective, and sensitive analysis of low-abundance peptides in human plasma by electromembrane extraction | |
| Marchi et al. | Sample preparation development and matrix effects evaluation for multianalyte determination in urine | |
| Balchen et al. | Fundamental studies on the electrokinetic transfer of net cationic peptides across supported liquid membranes | |
| CN111141854A (en) | Method for simultaneously extracting polar and low-polar metabolites from biological sample | |
| JP2010519532A (en) | Mass spectrometric quantitative detection of methylmalonic acid and succinic acid using HILIC on zwitterionic stationary phase | |
| Cecala et al. | Sampling techniques for single-cell electrophoresis | |
| CN111624274A (en) | High-throughput rapid detection method for perfluoro and polyfluoro compounds in serum | |
| JP2023133382A (en) | Systems and methods for analyzing analyte extracted from sample using adsorbent material | |
| CN101339167B (en) | Active ingredient high throughput screen method based on target protein and selection | |
| CN112748198A (en) | Method and device for detecting antifungal drugs in serum by liquid chromatography tandem mass spectrometry technology | |
| Novotny | Capillary biomolecular separations | |
| CN101368890B (en) | Method for in-situ desalination and enrichment on trace amount of protein or polypeptide target | |
| CN103529159B (en) | Method for pretreating research sample in blood non-target metabonomics | |
| Zhang et al. | Salting‐out assisted liquid–liquid extraction (SALLE) in LC‐MS bioanalysis | |
| Heinzelman et al. | Shotgun proteomic profiling of bloodborne nanoscale extracellular vesicles | |
| McCool et al. | Top-down proteomics by capillary zone electrophoresis-tandem mass spectrometry for large-scale characterization of proteoforms in complex samples | |
| Long et al. | Simultaneous quantification of three iridoids in rat plasma after oral administration of Zhi‐zi‐chi decoction using LC‐MS | |
| JP2009294118A (en) | Biological sample separation method, biological sample detection system, biological sample separation system, and biological sample separation-detection system | |
| Cupp-Sutton et al. | RPLC-RPLC-MS/MS for Proteoform Identification | |
| Gu et al. | Determination of aristolochic acids in rat serum by high performance liquid chromatography-Q-TOF tandem mass spectrometry | |
| CN209858311U (en) | Online sample pretreatment device capable of being combined with mass spectrum | |
| Auclair et al. | Intact Mass Analysis of Biopharmaceuticals as its Own Unique Application | |
| RU2800474C1 (en) | Method of preparing urine samples based on the principles of micellar extraction for determining the content of adrenaline |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |