CN108445116B - Pretreatment method for free oxytocin in organism liquid sample - Google Patents

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

A kind of pretreatment method of free oxytocin in biological fluid sample

technical field

The present application relates to a method for pretreatment of free oxytocin in biological fluid samples, belonging to the field of analytical chemistry.

Background technique

Oxytocin is a neuropeptide that may be associated with complex emotional and social behaviors such as social cognition, stress buffering, aggression and trust. Oxytocin in free form in biological fluids as a neuromodulator is closely related to highly specific individual conditions such as age, gender and drug history. The key to clinical diagnosis is more valuable. The accurate analysis of free oxytocin in biological fluids faces great challenges. The composition of biological fluids, especially plasma, is complex and incompatible with high-efficiency analytical techniques (such as liquid mass analysis). pg/ml level. Therefore, the pretreatment of biological fluid samples, the removal of impurities and the enrichment of oxytocin are the first steps in the analysis of free oxytocin. The establishment of an efficient and fast pretreatment method for free oxytocin in biological fluid samples plays an important role in the realization of highly sensitive and accurate oxytocin analysis, thereby promoting the progress of disease discovery and treatment.

At present, multi-step treatment methods such as pretreatment methods based on protein sedimentation and SPE, and pretreatment methods based on protein sedimentation and ultrafiltration centrifugation are commonly used technologies for the pretreatment of biological fluid samples. However, the SPE processing steps are cumbersome and time-consuming. Although online SPE sample preparation simplifies the entire process and reduces manual steps, online operations require special equipment, complex maintenance and method setup, and often lead to longer chromatographic run times. Therefore, laboratory and clinical samples Rarely used in analysis. In addition, the extremely hydrophilic nature of free oxytocin also makes it severely lost in SPE fillers. Ultrafiltration centrifugation is simple, but the rejection rate of oxytocin with a molecular weight of only 1007.2 Da 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.)

SUMMARY OF THE INVENTION

The pretreatment process of biological fluids (such as plasma) described in this application, including protein denaturation, oxytocin extraction, protein precipitation, inorganic salts and hydrophilic small molecule removal, are all completed in a one-step salting-out assisted liquid-liquid extraction (SALLE) system , which can be analyzed by nanoLC-MS after a simple concentration and degreasing process.

The method for pretreatment of free oxytocin in the biological fluid sample is characterized in that at least comprising:

(1) biological fluid sample is carried out oxytocin desorption treatment, obtains system I;

(2) carrying out selective enrichment and impurity removal of oxytocin in system I in step (1) to obtain system II;

(3) system II in step (2) is concentrated and secondary desalination is carried out to obtain system III;

(4) Oxytocin collection.

Optionally, the oxytocin desorption treatment in step (1) includes: protein denaturation treatment.

Optionally, the oxytocin desorption treatment includes: adding a protein denaturant to the biological fluid sample, denaturing the protein, and realizing the desorption of oxytocin non-covalently bound to the protein.

Optionally, the volume of the biological fluid sample is 100-1200 μL, and the amount of the protein denaturant is 0.2-1 times the volume of the biological fluid sample.

Optionally, the upper limit of the volume of the biological fluid sample is selected from 300 μL, 600 μL or 1200 μL; the lower limit is selected from 100 μL, 300 μL or 600 μL.

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 desorbed free oxytocin non-covalently bound to the protein.

Optionally, the vortexing time is 25-35 seconds.

Optionally, the protein denaturing agent includes at least one of an acidic solution, a surfactant, and an alkaline 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 sodium dodecylsulfonate aqueous solution, dodecylbenzenesulfonic acid at least one of sodium aqueous solution and sodium lauryl sulfate aqueous solution; alkaline detergent is selected from at least one of urea aqueous solution and guanidine hydrochloride aqueous solution.

Optionally, the concentration of the acidic solution is 0.1v%-5v%; the concentration of the surfactant is 4wt%; the concentration of the alkaline detergent is 6M.

Optionally, the selective enrichment of oxytocin and the removal of impurities in step (2) include: salting out auxiliary liquid-liquid extraction.

Optionally, the selective enrichment of oxytocin and the removal of impurities include: adding the SALLE reagent to the system I in step (1), shaking, vortexing, centrifuging, and taking the supernatant.

Optionally, the SALLE reagent includes 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 selected from hydrogen phosphate At least one of dipotassium 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 to 1:1; the volume ratio of the salt solution to the hydrophilic organic solvent is 1:1 to 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.

Optionally, 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 includes: first adding a salt solution, vortexing, and then adding a hydrophilic organic solvent.

Optionally, the number of times of the oscillation is 10 times.

Optionally, vortexing is performed after the shaking, and the vortexing time is 25-35 seconds.

Optionally, the vortexing time is 25-35 seconds after the shaking.

Optionally, the conditions of the centrifugation are centrifugation at 3000 rpm for 10 minutes.

Optionally, the concentration and secondary desalting in step (3) include: drying-reconstitution.

Optionally, the concentration and secondary desalination include: drying the supernatant in the system II described in step (2), then redissolving in a hydrophilic organic solvent, and centrifuging the supernatant to achieve the concentration of the sample. and secondary desalination.

Optionally, the volume of the hydrophilic organic solvent is 1-10 times 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 biological fluid sample 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, and isopropanol.

Optionally, the reconstitution of the hydrophilic organic solvent includes: after adding the hydrophilic organic solvent, ultrasonication, vortexing, and taking the supernatant.

Optionally, the ultrasonic time is 25-35 seconds; the vortexing time is 2-35 seconds.

Optionally, the collection of oxytocin described in step (4) includes: drying the supernatant in system III described in step (3), adding liquid chromatography mobile phase to reconstitute, adding an organic solvent, and centrifuging to get the supernatant It is the oxytocin-enriched fraction.

Optionally, the volume ratio of the liquid chromatography mobile phase to the organic solvent is 1:1 to 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% to 30%, and the added amount is 1/20 to 1/5 times the volume of the biological fluid sample.

Optionally, the upper limit of the volume fraction 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% to 30%, the solvent is water, and the added amount is 1/20 to 1/5 times the volume of the biological fluid sample.

Optionally, the organic solvent is selected from at least one of dichloromethane and chloroform.

Optionally, after the organic solvent is added, ultrasonication, vortexing, and centrifugation are performed to obtain the supernatant to obtain an oxytocin-enriched fraction.

Optionally, the ultrasonic time is 25-35 seconds; the vortexing time is 25-35 seconds.

Optionally, the conditions of the centrifugation are centrifugation at 15000 rpm for 20-60 minutes.

Optionally, the oxytocin collected in step (4) is directly used for nanoLC-MS analysis.

Optionally, the biological fluid sample can be obtained or collected according to common knowledge in the art.

Optionally, the method includes at least:

(a1) Oxytocin desorption: adding an acidic solution or a surfactant or an alkaline detergent solution to the biological fluid sample to denature the protein to achieve the desorption of oxytocin non-covalently bound to the protein in the biological fluid sample;

(a2) Selective enrichment and impurity removal of oxytocin: add SALLE reagent to the sample processed in step (a1), fully shake, vortex, and centrifuge to take the supernatant, and simultaneously perform selective enrichment and biological enrichment of oxytocin. Removal of high-abundance proteins, inorganic salts, and hydrophilic small-molecule impurities in body fluid samples;

(a3) concentration and secondary desalting: drying the supernatant in step (a2), redissolving in a hydrophilic organic solvent, and centrifuging the supernatant to achieve concentration and secondary desalting of the sample;

(a4) Oxytocin collection: drying the supernatant in step (a3), adding an acetonitrile aqueous solution with a volume concentration of 5% to 30% to redissolve, adding dichloromethane or chloroform, and centrifuging to get the supernatant. The desired oxytocin enrichment fraction.

Optionally, the oxytocin is endogenous free oxytocin or the sum of exogenous and endogenous free oxytocin following administration.

Optionally, the biological fluid includes plasma, serum, cerebrospinal fluid or urine.

As a specific embodiment, the method includes at least:

(b1) Desorption of oxytocin: adding an acidic solution or a surfactant or an alkaline detergent solution to the plasma to denature the protein to achieve the desorption of oxytocin that is non-covalently bound to the plasma protein;

(b2) Selective enrichment and impurity removal of oxytocin: SALLE reagent is added to the sample processed in step (b1), fully shaken, vortexed, and centrifuged to take the supernatant, and at the same time, selective enrichment of oxytocin and plasma Removal of medium and high abundance proteins, inorganic salts, and small hydrophilic molecules;

(b3) concentration and secondary desalting: the supernatant in step (b2) is dried, redissolved in a hydrophilic organic solvent, and centrifuged to obtain the supernatant to realize concentration and secondary desalting of the sample;

(b4) Collection of oxytocin: drying the supernatant in step (b3), adding a low-concentration acetonitrile aqueous solution to redissolve, adding dichloromethane or chloroform, and centrifuging the supernatant to obtain the desired oxytocin enrichment group point.

As a specific embodiment, the method includes at least:

(c1) Oxytocin analysis and attachment: add acid solution (phosphoric acid, trifluoroacetic acid) or surfactant (SDS) or alkaline detergent (urea, guanidine hydrochloride) solution to the biological fluid sample, vortex for 30 seconds, dissolve Adsorption of free oxytocin that is non-covalently adsorbed to proteins;

(c2) Selective enrichment and impurity removal of oxytocin: After the plasma sample is processed in step (c1), a saline solution (dipotassium hydrogen phosphate, sodium chloride, magnesium chloride and sodium carbonate) with a volume of 2.5 to 10 times the plasma volume is added, Vortex for 30 seconds, add a hydrophilic organic solvent (methanol, ethanol, isopropanol, acetonitrile) that is 1 to 10 times the plasma volume, fully shake for 10 times, vortex for 30 seconds, centrifuge at 3000 rpm for 10 minutes, and select oxytocin at the same time Sexual enrichment and removal of high-abundance proteins, inorganic salts, and hydrophilic small-molecule impurities in plasma samples.

(c3) Concentration and secondary desalting: transfer the supernatant in step (c2) to a new centrifuge tube, and transfer the centrifuge tube to a nitrogen blower and dry; add 0.5-2ml of a hydrophilic organic solvent ( Methanol, isopropanol), sonicated for 30 seconds, vortexed for 30 seconds, took the supernatant, transferred it to a nitrogen blower, and dried;

(c4) Oxytocin collection: add a small volume of 1/20-1/5 of a low-concentration organic solvent (5-30% acetonitrile) compatible with liquid mass analysis to the dried sample in step (c3), add dichloromethane or trichloromethane Methyl chloride, the volume ratio of low-concentration organic solvent to dichloromethane or chloroform is 1:1 to 5:1, ultrasonic for 30 seconds, vortex for 30 seconds, centrifugation at 15000 rpm for 40 minutes, and the supernatant is the desired induction protein enriched fractions.

The present application relates to a method for the pretreatment of free oxytocin in biological fluids. The extraction and separation of oxytocin, the denaturation and precipitation of proteins, and the removal of inorganic salts and hydrophilic small molecule impurities are completed in one step in a centrifuge tube. , after a simple concentration and degreasing process, it can be used for subsequent high-performance liquid-mass analysis. Specifically: (d1) adding a protein denaturant to the plasma, vortexing and mixing; (d2) adding a salt solution and a hydrophilic organic solvent in sequence, fully shaking, vortexing and mixing, centrifuging the supernatant, and drying; (d3) The dried product is dissolved by ultrasonic wave in a hydrophilic organic solvent, the supernatant is collected after centrifugation, and dried; (d4) the dried product is added to the chromatographic mobile phase and dichloromethane or chloroform, dissolved by ultrasonic wave, and the supernatant is collected after centrifugation. The invention can simply and efficiently enrich free oxytocin from biological fluids, remove high-abundance proteins, inorganic salts, hydrophilic small molecules and lipids and other interfering substances for nanoLC-MS analysis.

The salting-out auxiliary liquid-liquid extraction system (SALLE) described in this application is an effective means for extracting and enriching polar compounds. It is composed of a hydrophilic organic solvent and a salt solution. Advantage.

In this application, "high-abundance protein" refers to mg/ml-level protein.

In this application, "nanoLC-MS" refers to nanoliter liquid chromatography-mass spectrometry.

In this application, all conditions referring to numerical ranges can be independently selected from any intermediate range within that numerical range.

In this application, all conditions referring to numerical ranges include endpoints unless otherwise stated.

In this application, those skilled in the art can complete the technical solutions in this application according to any specific numerical conditions in the range conditions (such as volume ratio, etc.) involved in the solution, and realize the effective effects described in this application. .

The beneficial effects that this application can produce include:

1. In this application, the extraction of oxytocin and the removal of impurities are completed in one step, oxytocin is enriched in the upper hydrophilic organic phase, high-abundance proteins are in the middle solid layer, and inorganic salts and hydrophilic small molecule interferers are in the lower solution layer. , reducing intermediate transfer steps and reducing sample loss.

2. In the present application, oxytocin is enriched in the upper hydrophilic organic phase, which is easy to transfer the extract, and the hydrophilic organic solvent has a low boiling point and is easy to handle.

3. The hydrophilic organic solvent selected by SALLE in this application can be completely miscible with water, so it does not need long or violent mixing steps to promote the distribution of oxytocin and impurities between the two phases, and does not require long-term high-speed centrifugation. The phase separation of the auxiliary system, the operation process is simple and fast, and it is suitable for high-throughput analysis.

Description of drawings

Fig. 1 is the pretreatment process flow of plasma free oxytocin in the application; wherein, 1: oxytocin analysis appendix 2: selective enrichment and impurity removal of oxytocin 3: concentration and secondary desalination 4: oxytocin collection;

Figure 2. NanoLC-LTQ-Oritrap detection data; base peak chromatogram (BPC) of human plasma obtained by nano-LC-MS in positive mode with a scan range of 504.00-509.40 (Figure 2A); induced labor in diluted plasma The linear relationship (including regression equation and R2 value) between the extracted ion (EIC) peak area ratio of oxytocin/isotope-labeled oxytocin (OT/IS) and the initial mixing ratio (Figure 2B); the initial concentration ratio (OT/IS) was 2.500, and its EIC peak area ratio is 2.22 (inset C in Figure 2B);

Figure 3. Extracted ion chromatogram of oxytocin (dark line) in volunteer plasma (including 200 pg/mL IS (light line)) (A and B);

Figure 4 Extracted ion chromatogram of oxytocin (dark line) in rat plasma (including 200 pg/mL IS) (A and B).

Detailed ways

The present application will be described in detail below with reference to the examples, but the present application is not limited to these examples.

Unless otherwise specified, the raw materials in the examples of this application are all purchased through commercial channels.

The analytical method in the embodiment of the application is as follows:

Free oxytocin was quantified by nanoLC-LTQ-Oritrap assay using LTQ-Orbitrap Discovery (Thermo Fisher Scientific, Bremen, Germany) combined with nano-LC (Eksigent Technologies, California, USA).

Example 1 Pretreatment of plasma free oxytocin

(1) Plasma collection

Human whole blood was collected with a commercial vacuum blood collection tube (BDTM P800, Becton, Dickinson and Company, USA), centrifuged at 3000 rpm for 10 min, and the supernatant was taken to obtain plasma, which could be directly pretreated or stored at -80°C.

(2) Protein denaturation

Take 600 μL of human plasma in step (1), put it in a 10ml centrifuge tube, add oxytocin isotope internal standard (Pro[13C5,15N]oxytocin, Invitrogen Trading Co., Ltd, Shanghai, China) aqueous solution (final concentration 200pg/ mL), add 240 μL of 5v% phosphoric acid in water and vortex for 30 s.

(3) Oxytocin extraction and impurity removal

After the plasma sample was processed in step (2), 4 mL of dipotassium hydrogen phosphate aqueous solution (4M) was added, vortexed for 30 seconds, 4 mL of isopropanol was added, vortexed for 10 times, vortexed for 30 seconds, and centrifuged at 3000 rpm for 10 minutes , transfer 4ml of the supernatant from the centrifuge tube to a new centrifuge tube.

(4) Concentration and secondary desalination

Transfer the sample processed in step (3) to a nitrogen blower, blow nitrogen to dryness, add 1 ml of isopropanol, dissolve by ultrasonic for 30s, vortex for 30 seconds, centrifuge at 3000rpm for 10 minutes, and transfer the supernatant in the centrifuge tube. Transfer 0.9ml of liquid to a new centrifuge tube.

(5) Oxytocin collection

Transfer the sample processed in step (4) to a nitrogen blower, blow nitrogen to dryness, add 50 μL of 15% acetonitrile (15% by volume, water as the solvent) and 30 μL of dichloromethane, sonicate for 30 seconds, and vortex for 30 Second, centrifuge at 15,000 rpm for 40 min, and take the supernatant as the desired oxytocin-enriched fraction.

Example 2 Pretreatment of plasma free oxytocin

The pretreatment method of plasma free oxytocin in this example is similar to that in Example 1. in:

In step (2) protein denaturation, "240μL 5v% phosphoric acid aqueous solution" was replaced with "120μL 4wt% sodium dodecyl sulfonate aqueous solution";

In step (3) oxytocin extraction and impurity removal, the concentration of dipotassium hydrogen phosphate aqueous solution is 0.7M;

In step (4) concentration and secondary desalination, "1ml isopropanol" is replaced with "0.6ml isopropanol";

In step (5) oxytocin collection, "50 μL of 15% acetonitrile (15% by volume, solvent is water)" is replaced with "30 μL of 30% acetonitrile (30% by volume, solvent is water)".

The remaining operations and conditions are the same as in Example 1, and the final supernatant obtained is the desired oxytocin-enriched component.

Example 3 Pretreatment of plasma free oxytocin

(1) Plasma collection

The whole blood of SD rats was collected with commercial vacuum blood collection tubes (containing NaEDTA), centrifuged at 3000 rpm for 10 min, and the supernatant was taken to obtain plasma, which could be directly pretreated or stored at -80°C.

(2) Protein denaturation

Take 300 μL of rat plasma in step (1), put it in a 10ml centrifuge tube, add oxytocin isotope internal standard (Pro[13C5,15N]oxytocin, Invitrogen Trading Co., Ltd, Shanghai, China) aqueous solution (final concentration 200pg) /mL), add 120 μL of 5v% phosphoric acid in water and vortex for 30 s.

(3) Oxytocin extraction and impurity removal

After the plasma sample was processed in step (2), 4 mL of dipotassium hydrogen phosphate aqueous solution (4M) was added, vortexed for 30 seconds, 4 mL of isopropanol was added, vortexed for 10 times, vortexed for 30 seconds, and centrifuged at 3000 rpm for 10 minutes , transfer 4ml of the supernatant from the centrifuge tube to a new centrifuge tube.

(4) Concentration and secondary desalination

Transfer the sample processed in step (3) to a nitrogen blower, blow nitrogen to dryness, add 0.8 ml of isopropanol, dissolve by ultrasonic for 30 s, vortex for 30 s, centrifuge at 3000 rpm for 10 minutes, and transfer the upper part of the centrifuge tube. Transfer 0.7ml of the supernatant to a new centrifuge tube.

(5) Oxytocin collection

Transfer the sample processed in step (4) to a nitrogen blower, blow nitrogen to dryness, add 40 μL of 15% acetonitrile (15% by volume, water as the solvent) and 10 μL of dichloromethane, sonicate for 30 seconds, and vortex for 30 Second, centrifuge at 15,000 rpm for 40 min, and take the supernatant as the desired oxytocin-enriched fraction.

Example 4 Pretreatment of plasma free oxytocin

The pretreatment method of plasma free oxytocin in this example is similar to that in Example 3. in:

In step (2), in protein denaturation, "120μL 5v% phosphoric acid aqueous solution" was replaced with "300μL 6M guanidine hydrochloride aqueous solution";

In step (3) oxytocin extraction and impurity removal, the concentration of dipotassium hydrogen phosphate aqueous solution is 2.8M;

In step (4) concentration and secondary desalination, "0.8ml isopropanol" is replaced by "3ml isopropanol";

In step (5) oxytocin collection, "40 μL of 15% acetonitrile (15% by volume, solvent is water) and 10 μL dichloromethane" is replaced with "60 μL of 5% acetonitrile (5% by volume, solvent is water)" ) and 12 μL of dichloromethane”.

The remaining operations and conditions are the same as in Example 3, and the final supernatant obtained is the desired oxytocin-enriched fraction.

Example 5 detection of nanoLC-LTQ-Oritrap

(1) nanoLC-LTQ-Oritrap detection

Dr.Maisch GmbH,Ammerbuch-Entringen,德国)填料实验室自装填。自装预柱(3cm×100μmID)，柱管PEEK column holder(Upchurch,Oak Harbor,WA,USA)，填料C18AQ particles(5μm,

Dr.Maisch GmbH,Ammerbuch-Entringen,Germany)。质谱仪分析在正离子模式下进行，喷雾电压为1.5-2.0V，加热毛细管温度为320℃。采用Xcalibur软件记录总离子流图(图2A)，质荷比范围为504.0-509.4。Oxytocin-enriched fractions (individual plasma were obtained from 24 volunteers, processed according to the method in Example 1; 2 batches of commercial plasma were processed according to the method in Example 1; 11 rats were processed according to the method in Example 3). processed) and detected using nanoLC-LTQ-Oritrap. The analytical column used for nanoLC separation was a PicoFrit series capillary column (New Objective, Woburn, MA, USA) with an inner diameter of 75 μm, a length of 20 cm, and a 10 μm spray needle at one end, C18 (3 μm,

Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) packing laboratory self-packing. Self-packing pre-column (3cm×100μmID), column tube PEEK column holder (Upchurch, Oak Harbor, WA, USA), packing C18AQ particles (5μm,

Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Mass spectrometer analysis was performed in positive ion mode with a spray voltage of 1.5-2.0 V and a heated capillary temperature of 320°C. Total ion chromatograms (Fig. 2A) were recorded using Xcalibur software with mass-to-charge ratios ranging from 504.0 to 509.4.

(2) Data analysis

The collected spectrum is extracted by EIC, the quantitative ion of oxytocin is [M+2H] ²⁺ , the molecular weight is 504.2270Da, and the quantitative ion of oxytocin isotope internal standard (Pro[13C5,15N]oxytocin) is [M+2H] ^{2 +} , molecular weight 507.2330, mass deviation 10ppm. The limit of quantification for free oxytocin was 1 pg/ml. 80% recovery at 360 pg/ml, matrix inhibition <18%, intra-day and inter-day reproducibility RSD <15%. The calibration factor curve was Y=0.8251X ⁺ 0.0762, R2=0.9993 (Fig. 2B), the initial concentration ratio (OT/IS) was 2.500, and its EIC peak area ratio was 2.22 (Fig. 2C).

The oxytocin-enriched fractions obtained in Examples 1 and 3 were detected by the above-mentioned test method. The EIC diagrams of OT and IS in the plasma of some human individuals are shown in Figure 3 (A and B), and the EIC of OT and IS in rat plasma samples Figures are shown in Figure 4(A and B). Table 1 presents the detailed quantitative results of free OT in human and rat individual plasma. Levels of free OT in individual human plasma ranged from 3 to 142 pg/mL, with a mean ± SD of 43.8 ± 40.2 pg/mL. Rat plasma OT levels were higher, ranging from 166 to 2462 pg/mL, with a mean ± SD of 837.4 ± 798.1 pg/mL.

Table 1 Free oxytocin content in plasma of 24 volunteers, 2 batches of commercial plasma and 11 SD rats

The test methods of Example 2 and Example 4 are the same as above, and the measurement results are similar to the test results of Example 1 and Example 3 above.

This method can significantly improve the pretreatment throughput of free oxytocin in plasma samples. At the same time, oxytocin is protected by SALLE solvent at the first time, avoiding the binding of free oxytocin to large proteins, and ensuring the accuracy and repeatability of quantification. In addition, the SALLE method can efficiently remove high-abundance proteins, inorganic salts, and hydrophilic small-molecule interferences in plasma samples that are not compatible with high-performance liquid-mass analysis in one step. use.

The above are only a few embodiments of the present application, and are not intended to limit the present application in any form. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Without departing from the scope of the technical solution of the present application, any changes or modifications made by using the technical content disclosed above are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims (17)

1. a method for pretreatment of free oxytocin in a biological fluid sample, is characterized in that at least comprising: (1) protein denaturation is carried out to biological fluid sample to obtain system I; (2) salting-out auxiliary liquid-liquid extraction of oxytocin is carried out in system I in step (1) to obtain system II; (3) drying the supernatant in the system II described in the step (2), then redissolving with a hydrophilic organic solvent, and centrifuging the supernatant to achieve concentration and secondary desalting of the sample to obtain system III; (4) drying the supernatant in the system III described in step (3), adding a liquid chromatography mobile phase to redissolve, adding an organic solvent, and centrifuging the supernatant to obtain the oxytocin-enriched component, and the liquid phase The chromatographic mobile phase is acetonitrile with a volume fraction of 5% to 30%, the solvent is water, the addition amount is 1/20 to 1/5 times the volume of the biological fluid sample, and the organic solvent is selected from dichloromethane and chloroform. at least one. 2 . The method according to claim 1 , wherein the protein denaturation treatment comprises: adding a protein denaturant to the biological fluid sample to denature the protein to achieve desorption of oxytocin non-covalently bound to the protein. 3 . 3 . The method according to claim 1 , wherein the volume of the biological fluid sample is 100-1200 μL, and the dosage of the protein denaturant is 0.2-1 times the volume of the biological fluid sample. 4 . 4. The method of claim 2, wherein the protein denaturing agent comprises at least one of an acidic solution, a surfactant, and an alkaline detergent solution. 5. The method according to claim 4, wherein the acidic solution is selected from at least one of phosphoric acid aqueous solution, trifluoroacetic acid aqueous solution, and trichloroacetic acid aqueous solution; and the surfactant is selected from dodecyl At least one of sodium sulfonate aqueous solution, sodium dodecylbenzenesulfonate aqueous solution, and sodium dodecyl sulfate aqueous solution; the alkaline detergent is selected from 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 v% to 5 v%; the concentration of the surfactant is 4 wt %; the concentration of the alkaline detergent is 6M. 7 . 7. method according to claim 1, is characterized in that, the salting-out auxiliary liquid-liquid extraction of described oxytocin comprises: SALLE reagent is added in the system 1 described in step (1), vibration, vortex, centrifugal , take the supernatant. 8. The method according to 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 and the hydrophilic organic solvent is 1:1 to 1:4. 9. method according to claim 8, is characterized in that, described hydrophilic organic solvent is at least one in methanol, ethanol, acetonitrile, isopropanol; Described salt solution is selected from dipotassium hydrogen phosphate solution, At least one of sodium chloride solution and magnesium chloride solution. 10. The method according to claim 8, wherein the concentration of the salt solution is 0.7-4M. 11. The method according to claim 1, wherein the hydrophilic organic solvent is selected from at least one of methanol, ethanol, acetonitrile, and isopropanol. 12 . The method according to claim 11 , wherein the volume of the hydrophilic organic solvent is 1-10 times the volume of the biological fluid sample. 13 . 13 . The method according to claim 1 , wherein the volume ratio of the liquid chromatography mobile phase to the organic solvent in step (4) is 1:1 to 5:1. 14 . 14. The method according to claim 1, wherein the oxytocin collected in step (4) is directly used for nanoLC-MS analysis. 15. The method of claim 1, wherein the method comprises at least: (a1) Oxytocin desorption: adding an acidic solution or a surfactant or an alkaline detergent solution to the biological fluid sample to denature the protein to achieve the desorption of oxytocin non-covalently bound to the protein in the biological fluid sample; (a2) Selective enrichment and impurity removal of oxytocin: add SALLE reagent to the sample processed in step (a1), fully shake, vortex, and centrifuge to take the supernatant, and simultaneously perform selective enrichment and biological enrichment of oxytocin. Removal of high-abundance proteins, inorganic salts, and hydrophilic small-molecule impurities in body fluid samples; (a3) concentration and secondary desalting: drying the supernatant in step (a2), redissolving in a hydrophilic organic solvent, and centrifuging the supernatant to achieve concentration and secondary desalting of the sample; (a4) Oxytocin collection: drying the supernatant in step (a3), adding an acetonitrile aqueous solution with a volume concentration of 5% to 30% to redissolve, adding dichloromethane or chloroform, and centrifuging to get the supernatant. The desired oxytocin enrichment fraction. 16. The method of claim 1, wherein the oxytocin is endogenous free oxytocin or the sum of administered exogenous and endogenous free oxytocin. 17. The method of claim 1, wherein the biological fluid comprises plasma, serum, cerebrospinal fluid or urine.

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