Full-automatic detection device and detection method for free drug concentration in blood

Abstract

The invention provides a full-automatic detection device and method for the concentration of free drugs in blood. The full-automatic detection method adopts a full-automatic detection device and comprises the following steps: (1) loading a blood sample to a protein purification chromatographic column by adopting a pump 1 and an automatic sampler, removing protein components in the blood sample, and retaining free drug components on the protein purification chromatographic column; (2) enabling free drug components in the protein purification chromatographic column to flow into a trapping column and enriching the free drug components in the trapping column; and (3) enabling the free drug components in the trapping column to flow into an analytical column, separating, and detecting in a triple quadrupole mass spectrometer. Through the specific full-automatic detection device, the problems that total drug concentration monitoring cannot meet clinical treatment requirements, and the free drug pretreatment process is complex, long in time and high in cost are solved, clinical individualized drug administration can be assisted, the drug curative effect is improved, and the toxic and side effects of drugs are reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of blood sample detection, in particular to a full-automatic detection device and method for free drug concentration in blood. BACKGROUND

[0002] After the drug enters the blood circulation, part of it is combined with plasma protein, and the rest exists in the form of free drug. Only free drug has pharmacological activity and can produce therapeutic effect and adverse reactions. When the total concentration of the drug is within the therapeutic range, the high free drug concentration may cause toxic reactions. For example, in the pathological state of uremia, liver disease and hypoproteinemia, the protein binding rate of the drug decreases, and the free drug concentration increases, and the patient may have drug toxicity. Therefore, the free concentration can more truly reflect the efficacy and toxicity risk, and through free drug concentration monitoring, potential toxicity risks can be found in time to avoid adverse reactions caused by drug overdose. At the same time, free drug concentration monitoring can also ensure the effective concentration of the drug in the body and avoid insufficient efficacy due to low drug concentration.

[0003] Individualized drug administration can improve the efficacy of drugs, reduce the side effects of drugs, and reduce medical costs, so that patients can achieve the best treatment effect in the shortest time and with the least money. Individualized drug administration is becoming the focus of clinical treatment, and therapeutic drug monitoring (TDM) is an important means to achieve individualized drug administration, and free drug concentration detection is a key tool for optimizing individualized treatment, especially for high protein binding rate drugs, narrow therapeutic window drugs, special physiological / pathological states and multi-drug combination scenarios, such as immunosuppressant drugs, antiepileptic drugs, antitumor drugs, anticoagulants (warfarin), antibiotics (ceftriaxone).

[0004] At present, the drug concentration monitoring is still mainly total drug concentration, because the free drug concentration content is low, the instrument sensitivity requirement is high, the pretreatment process is complicated, and the cost is high, therefore, the free drug concentration detection is not widely applied to the clinic. The free drug concentration pretreatment method mainly includes equilibrium dialysis method, ultrafiltration centrifugation, solid phase extraction and the like. Yan Liu et al. apply the ultrafiltration centrifugation-LC-MS / MS method to detect the total mycophenolic acid and free mycophenolic acid concentration in blood plasma, and carry out the pharmacokinetic study in vivo of the renal transplant patients. Min Kan et al. establish the ultrafiltration centrifugation-LC-MS / MS method to detect the free cefoperazone concentration in the blood plasma of children, and apply to the age-dependent protein binding study of cefoperazone in children. Linlin Kong et al. use the protease-LC-MS / MS method to detect the total concentration and the apheresis complex concentration of apheresis, and obtain the free apheresis concentration by calculation, and apply to the pharmacokinetic study. The above methods all have problems of complicated pretreatment process, long time consumption and the like, and are not suitable for the clinical large sample detection. In view of the actual clinical significance and demand of the free drug concentration, it is necessary to develop and establish the free drug concentration detection method with high automation degree, fast analysis speed, suitable for clinical large sample analysis and rapid detection. SUMMARY

[0005] In view of the defects in the prior art, the full-automatic detection device and detection method for free drug concentration in blood are provided, which solve the problems that the total drug concentration monitoring cannot meet the clinical treatment demand, and the free drug pretreatment process is complicated, time-consuming, high-cost and not suitable for clinical sample detection.

[0006] To achieve the above object, the technical scheme is as follows: According to the first aspect of the present application, a full-automatic detection device for free drug concentration in blood is provided, which comprises a two-position three-way valve, an automatic sampler, a pump 1, a protein purification chromatographic column, a six-way valve, a pump 2, a T-shaped mixer, a trapping column, an analysis column, a pump 3 and a triple quadrupole mass spectrometer. The automatic sampler is connected with the protein purification chromatographic column, the positions 2 and 3 of the two-position three-way valve are respectively connected with eluent and elution liquid, the position 1 of the two-position three-way valve is connected with the inlet of the protein purification chromatographic column through the pump 1, the outlet of the protein purification chromatographic column is connected with the position 1 of the six-way valve, the pump 2 is connected with the T-shaped mixer, the T-shaped mixer is connected with the trapping column, the trapping column is connected with the position 5 of the six-way valve, the pump 3 is connected with the position 3 of the six-way valve, the position 6 of the six-way valve is connected with a waste liquid collecting device, the position 4 of the six-way valve is connected with the analysis column, and the analysis column is connected with the triple quadrupole mass spectrometer. The position 1 of the two-position three-way valve is connected with the positions 1 and 2; The position 2 of the two-position three-way valve is connected with the positions 1 and 3; Position 1 of the six-way valve is connected with 1st and 6th positions, 2nd and 3rd positions, and 4th and 5th positions; Position 2 of the six-way valve is connected with 1st and 2nd positions, 3rd and 4th positions, and 5th and 6th positions.

[0007] Preferably, the protein purification chromatographic column is selected from Lichrospher ADs reverse phase chromatographic column, the bonded phase type is any one of C18, C8 and C4, and the column length is any one of 100 mm, 50 mm and 25 mm.

[0008] Preferably, the trapping column is selected from a silica gel matrix reverse phase chromatographic column, the bonded phase type is any one of C8 and C18, and the column length is any one of 25 mm, 10 mm and 5 mm.

[0009] Preferably, the analysis column is selected from a silica gel matrix reverse phase chromatographic column, the bonded phase type is any one of C8 and C18, the column inner diameter is any one of 2.1 mm and 3.0 mm, and the column length is any one of 50 mm, 100 mm and 150 mm.

[0010] According to the second aspect of the present application, a full-automatic detection method for free drug concentration in blood is provided, which adopts the full-automatic detection device, and comprises the following steps: Step 1, adjusting the two-position three-way valve to position 1 and the six-way valve to position 1, loading the blood sample into the protein purification chromatographic column by using pump 1 and the automatic sampler, eluting the protein component in the blood sample out of the chromatographic column by using the eluent, and entering the waste liquid collecting device, the free drug component remaining on the protein purification chromatographic column, and flushing and balancing the trapping column and the analysis column by using the mobile phase of pump 2 and pump 3; Step 2, adjusting the two-position three-way valve to position 2 and the six-way valve to position 2, flushing the free drug component out of the protein purification chromatographic column by using the eluent of pump 1, and enriching the free drug component in the trapping column under the dilution of the aqueous mobile phase of pump 2, and the organic phase of pump 3 is in a resting state; Step 3, when the free drug component completely enters the trapping column, switching the six-way valve to position 1, starting pump 3 to form a two-dimensional high-pressure gradient system with pump 2 and pump 3, flushing the free drug component from the trapping column to the analysis column, and finally entering the triple quadrupole mass spectrometer for detection after separation by the analysis column.

[0011] The present application adopts the protein purification chromatographic column to remove the protein component in the blood sample, realizes the full-automatic detection of the free drug concentration through the high-pressure pump mobile phase change, the trapping column negative electrode, the analysis column separation and the triple quadrupole mass spectrometer detection, and is suitable for drugs with high protein binding rate, narrow therapeutic window and large toxic side effects, such as antiepileptic drugs, immunosuppressants, antitumor drugs, anticoagulants or antibiotics.

[0012] Preferably, in step 1, the eluent is selected from a methanol aqueous solution or an acetonitrile aqueous solution, wherein the concentration of methanol in the methanol aqueous solution is 5-10%, and the concentration of acetonitrile in the acetonitrile aqueous solution is 3-6%.

[0013] Preferably, in step 1, the flow rate of the eluent is 0.2-0.4 mL / min. The flow phase of the pump 2 and the pump 3 is the initial flow phase in mass spectrometric detection, and the flow rate of the pump 2 and the pump 3 is the initial flow rate in mass spectrometric detection.

[0014] Preferably, in step 2, the eluent is selected from a methanol aqueous solution or an acetonitrile aqueous solution, wherein the concentration of methanol in the methanol aqueous solution is 90-100%, and the concentration of acetonitrile in the acetonitrile aqueous solution is 54-60%. The flow rate of the eluent is 0.1-0.2 mL / min, and the time is 2-3 min.

[0015] Preferably, the flow phase of the pump 2 is ultrapure water or a 0.1% formic acid aqueous solution. The flow rate of the pump 2 is 0.3-0.4 mL / min.

[0016] Preferably, the blood sample is selected from serum, plasma or whole blood after red blood cells are broken.

[0017] The application provides a full-automatic detection device for free drug concentration in blood and a detection method thereof. (1) The full-automatic detection method for free drug concentration in blood provided by the application solves the problems that total drug concentration monitoring cannot meet the clinical treatment requirements, the free drug pretreatment process is complicated, time-consuming, high-cost and not suitable for clinical sample detection, and provides a rapid and accurate free drug concentration monitoring method for the clinic, provides accurate data basis for drugs with high protein binding rate, narrow treatment window and large toxic and side effects, assists clinical practice individualized drug administration, improves drug efficacy, reduces drug toxic and side effects, and achieves the best drug administration effect. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The connection schematic diagram for adjusting the two-position three-way valve to position 1 and the six-way valve to position 1; Figure 2 The connection schematic diagram for adjusting the two-position three-way valve to position 2 and the six-way valve to position 2; Figure 3 The connection schematic diagram for adjusting the two-position three-way valve to position 2 and the six-way valve to position 1. DETAILED DESCRIPTION

[0019] In order to better illustrate the content of the present application, the following specific examples are described. It should be understood that the following embodiments are only used to illustrate the present application, and not to limit the present application.

[0020] Example 1 The present application discloses a kind of automatic detection method of free drug concentration in blood, application this method detects free antiepileptic drug concentration in serum.

[0021] Antiepileptic drug includes phenobarbital, phenytoin, carbamazepine, lamotrigine, valproic acid, protein binding rate is respectively: 40-60%, 89-95%, 75-80%, 55%, 80-95%, all are high protein binding antiepileptic drug.

[0022] 1, detection method: (1) flow connection Automatic sampler is connected with protein purification chromatographic column, eluent and eluent are connected with two-position three-way valve, pump 1 is connected with two-position three-way valve, pump 1 is connected with protein purification chromatographic column inlet, protein purification chromatographic column outlet is connected with six-way valve 1, pump 2 is connected with T type mixer, T type mixer is connected with trapping column, trapping column is connected with six-way valve 5, pump 3 is connected with six-way valve 3, six-way valve 6 is connected with waste liquid collection device, six-way valve 4 is connected with analysis column, analysis column is connected with triple quadrupole mass spectrometer; Two-position three-way valve position 1 is connected with 1 and 2; Two-position three-way valve position 2 is connected with 1 and 3; Six-way valve position 1 is connected with 1 and 6, 2 and 3, 4 and 5; Six-way valve position 2 is connected with 1 and 2, 3 and 4, 5 and 6.

[0023] (2) protein removal Two-position three-way valve is switched to position 1, six-way valve is adjusted to position 1, as shown in Figure 1 Protein purification chromatographic column uses Lichrospher ADs C8 4.0×25 mm, eluent is 5% methanol / water, flow rate is 0.2ml / min, flushing time is 2.5min, serum dosage is 50ul, internal standard volume is 5ul, pump 2 mobile phase is 0.1% formic acid / water, pump 3 mobile phase is 0.1% formic acid / methanol.

[0024] (3) trapping column enrichment Two-position three-way valve is switched to position 2, six-way valve is switched to position 2, as shown in Figure 2The eluent is methanol, the flow rate is 0.1 ml / min, the time is 2 min, the mobile phase of pump 2 is ultrapure water, the flow rate is 0.3 ml / min, the time is 2 min, the free antiepileptic drug is washed out of the purification column to the trapping column, and is enriched in the trapping column. The trapping column is Kinetex C18 Guard Column 2.6 µm, 3×5 mm.

[0025] (4) Analysis column separation Keep two-position three-way valves in position 2, and six-way valve switches to position 1, as shown, free antiepileptic drugs are washed from the trapping column to the analysis column, and separated in the analysis column. Figure 3

[0026] Liquid phase: Waters ACQUITY UPLC I-Class Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in methanol Chromatographic column: Kinetex C18 (3×30mm, 2.6μm) Column temperature: 40 ℃ Flow rate: 0.4 mL / min The flow phase gradient is shown in Table 1:

[0027] (5) Mass spectrometry detection After the free drug component flows out of the analysis column, it enters the triple quadrupole mass spectrometer for detection.

[0028] Mass spectrometer: Waters Xevo TQ-S ESI source: positive ion mode / negative ion mode Ion source parameters are shown in Table 2:

[0029] MRM parameters are shown in Table 3:

[0030] 2, Detection method verification (1) Linearity

[0031] (2) Precision

[0032]

[0033]

[0034] (3) Accuracy​

[0035] Example 2: This invention discloses a fully automated method for detecting the concentration of free drugs in blood, and applies this method to detect the concentration of free immunosuppressants in whole blood.

[0036] Immunosuppressants include cyclosporine and tacrolimus.

[0037] Cyclosporine has a protein binding rate of 90%-98%, while tacrolimus has a protein binding rate of 99%. Both drugs have very high protein binding rates and narrow therapeutic windows, requiring strict monitoring of blood drug concentrations. The detection of free drug concentrations is of great clinical significance.

[0038] 1. Detection Method (1) Flow path connection The autosampler is connected to the protein purification column. The eluent and elution buffer are connected to a two-position three-way valve. The two-position three-way valve is connected to pump 1. Pump 1 is connected to the inlet of the protein purification column. The outlet of the protein purification column is connected to position 1 of a six-way valve. Pump 2 is connected to a T-type mixer. The T-type mixer is connected to a trap column. The trap column is connected to position 5 of a six-way valve. Pump 3 is connected to position 3 of a six-way valve. Position 6 of the six-way valve is connected to a waste collection device. Position 4 of the six-way valve is connected to the analytical column. The analytical column is connected to a triple quadrupole mass spectrometer. The two-position three-way valve has position 1 connected to positions 1 and 2. The two-position three-way valve has position 2 connected to positions 1 and 3. The six-way valve has position 1 connected to positions 1 and 6, positions 2 and 3, and positions 4 and 5. The six-way valve has position 2 for connecting positions 1 and 2, positions 3 and 4, and positions 5 and 6.

[0039] (2) Remove protein Switch the two-position three-way valve to position 1, and adjust the six-way valve to position 1, as follows: Figure 1 As shown, the protein purification chromatography column used was a Lichrospher ADs C8 4.0×25 mm column, the eluent was 5% methanol / water, the flow rate was 0.2 ml / min, the rinsing time was 2.5 min, the whole blood injection volume was 50 μl, the internal standard volume was 5 μl, the mobile phase of pump 2 was 10 mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid), and the mobile phase of pump 3 was 0.1% formic acid / methanol.

[0040] (3) Trapping column enrichment Switch the six-way valve to position 2, then switch the six-way valve to position 1, as follows: Figure 2As shown, the mobile phase of pump 1 is methanol, the flow rate is 0.2 ml / min, the time is 2 min, the mobile phase of pump 2 is ultrapure water, the flow rate is 0.3 ml / min, the time is 2 min, the free immunosuppressants are flushed out of the purification column to the trapping column, enriched in the trapping column, and the trapping column is ACQUITY UPLC BEH C18 VanGuard, 1.7 μm, 2.1 x 5 mm.

[0041] (4) Analysis column separation Keep two-position three-way valve in position 2, six-way valve switch to position 1, as Figure 3 shown, the free immunosuppressants are flushed from the trapping column to the analysis column, and separated in the analysis column.

[0042] Chromatographic conditions: Liquid phase: Waters ACQUITY UPLC I-Class Mobile phase A: 10 mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid) Mobile phase B: 0.1% formic acid / methanol solution Chromatographic column: Waters Acquity UPLC BEH C18 (2.1 x 50 mm, 1.7 μm) Injection volume: 10 μL Flow rate: 0.5 mL / min Column temperature: 40 ℃ The mobile phase gradient is shown in Table 9:

[0043] Mass spectrometry conditions: Mass spectrometer: Waters Xevo TQ-S ESI source: positive ion mode Ion source parameters are shown in Table 10:

[0044] MRM parameters are shown in Table 11:

[0045] 2. Method validation (1) Linearity

[0046] (2) Precision

[0047]

[0048]

[0049] (3) Precision

[0050] While embodiments of the application have been shown and described, it is to be understood that the application is not limited to the details of the embodiments described, since various modifications can be made by those skilled in the art, without departing from the spirit and scope of the application, which are defined by the appended claims and their equivalents.

Claims

1. A fully automated device for detecting the concentration of free drugs in blood, characterized in that: Includes a two-position three-way valve, an autosampler, pump 1, a protein purification column, a six-way valve, pump 2, a T-shaped mixer, a trap column, an analytical column, pump 3, and a triple quadrupole mass spectrometer; The autosampler is connected to the protein purification column. Positions 2 and 3 of the two-position three-way valve are respectively supplied with eluent and elution solution. Position 1 of the two-position three-way valve is connected to the inlet of the protein purification column via pump 1. The outlet of the protein purification column is connected to position 1 of the six-way valve. Pump 2 is connected to the T-shaped mixer, which is connected to the trapping column. The trapping column is connected to position 5 of the six-way valve. Pump 3 is connected to position 3 of the six-way valve. Position 6 of the six-way valve is connected to a waste collection device. Position 4 of the six-way valve is connected to the analytical column, which is connected to the triple quadrupole mass spectrometer. The position 1 of the two-position three-way valve connects positions 1 and 2. The second position of the two-position three-way valve is connected between positions 1 and 3. The six-way valve has position 1, which connects position 1 to position 6, position 2 to position 3, and position 4 to position 5. The position 2 of the six-way valve connects positions 1 and 2, positions 3 and 4, and positions 5 and 6.

2. The fully automated detection device for free drug concentration in blood according to claim 1, characterized in that: The protein purification chromatographic column is selected from Lichrospher ADs reversed-phase chromatographic columns, and the bonded phase type is any one of C18, C8, and C4. The column length is any one of 100 mm, 50 mm, and 25 mm.

3. The fully automated detection device for free drug concentration in blood according to claim 1, characterized in that: The trapping column is selected from silica-based reversed-phase chromatography columns, the bonded phase type is either C8 or C18, and the column length is either 25 mm, 10 mm, or 5 mm.

4. The fully automated detection device for free drug concentration in blood according to claim 1, characterized in that: The analytical column is selected from silica-based reversed-phase chromatography columns, with the bonded phase type being either C8 or C18, the column inner diameter being either 2.1 mm or 3.0 mm, and the column length being either 50 mm, 100 mm, or 150 mm.

5. A fully automated method for detecting the concentration of free drug in blood, characterized in that: The fully automatic detection device according to any one of claims 1 to 4 includes the following steps: Step 1: Adjust the two-position three-way valve to position 1 and the six-way valve to position 1. Use pump 1 and autosampler to load the blood sample into the protein purification chromatography column. Use the eluent to elute the protein components in the blood sample from the chromatography column and enter the waste liquid collection device. The free drug components are retained on the protein purification chromatography column. Pumps 2 and 3 introduce the mobile phase to flush and balance the trapping column and the analytical column. Step 2: Adjust the three-way valve to position 2 and the six-way valve to position 2. Pump 1 pushes the eluent to wash the free drug components out of the protein purification chromatography column. Under the dilution of the aqueous mobile phase pushed by pump 2, the free drug components are enriched in the collection column, and the organic phase of pump 3 is in a residence state. Step 3: When the free drug component has completely entered the collection column, the six-way valve is switched to position 1, and pump 3 is started to form a two-dimensional high-pressure gradient system with pump 2 and pump 3, which washes the free drug component from the collection column to the analysis column. Finally, after separation by the analysis column, it enters the triple quadrupole mass spectrometer for detection.

6. The fully automated method for detecting the concentration of free drug in blood according to claim 5, characterized in that: In step 1, the rinsing solution is selected from methanol aqueous solution or acetonitrile aqueous solution, wherein the concentration of methanol in the methanol aqueous solution is 5-10%, and the concentration of acetonitrile in the acetonitrile aqueous solution is 3-6%.

7. The fully automated method for detecting the concentration of free drug in blood according to claim 5, characterized in that: In step 1, the flow rate of the rinsing solution is 0.2~0.4 mL / min; The mobile phase of both pump 2 and pump 3 is the initial mobile phase for mass spectrometry detection, and the flow rate of both pump 2 and pump 3 is the initial flow rate for mass spectrometry detection.

8. The fully automated method for detecting the concentration of free drug in blood according to claim 5, characterized in that: In step 2, the eluent is selected from methanol aqueous solution or acetonitrile aqueous solution, wherein the concentration of methanol in the methanol aqueous solution is 90-100%, and the concentration of acetonitrile in the acetonitrile aqueous solution is 54-60%. The flow rate of the eluent is 0.1~0.2 mL / min, and the time is 2~3 min.

9. The fully automated method for detecting the concentration of free drug in blood according to claim 5, characterized in that: The mobile phase of the pump 2 is ultrapure water or a 0.1% formic acid aqueous solution; The flow rate of pump 2 is 0.3~0.4 mL / min.

10. The fully automated method for detecting the concentration of free drug in blood according to claim 5, characterized in that: The blood sample is selected from serum, plasma, or whole blood after ruptured red blood cells.

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