CN100414293C - A kind of method for measuring mizoribine blood drug concentration - Google Patents

Abstract

The invention belongs to the field of medical examination and relates to an analysis and determination method of drugs in vivo, in particular to a method for determining the concentration of mizoribine in human blood plasma. In the method of the invention, the sample to be tested is pretreated by protein precipitation, isocratic eluted under acidic mobile phase conditions, separated by a chromatographic column, and detected by an ultraviolet detector. The method has a small amount of sample sampling, a simple, fast and sensitive pretreatment method, does not require expensive equipment and reagents, has wide application and low cost, and is suitable for the monitoring of routine clinical blood drug concentration.

Description

A kind of method for measuring mizoribine blood drug concentration

technical field

The invention belongs to the field of medical testing, and relates to an analysis and determination method of drugs in vivo. Specifically relates to a method for measuring the blood concentration of mizoribine.

Background technique

Mizoribine (MZR) is a purine nucleoside synthesis inhibitor, which can specifically inhibit the rapid growth of lymphocytes, thereby producing immunosuppressive effects. In recent years, it has been clinically used to suppress rejection during kidney transplantation, and is also used to treat autoimmune diseases such as lupus nephritis, rheumatoid arthritis and nephrotic syndrome. The pharmacokinetics of this drug varies greatly among individuals, and clinically, blood drug concentration monitoring is required to adjust the individualized dosage regimen.

At present, there are only relevant reports abroad about the determination method of MZR in biological samples. The existing technical methods have various defects such as low sensitivity (lowest limit of quantitation is 0.25 mg·L ^-1 ), cumbersome and time-consuming operation, low efficiency, high analysis cost, etc., and are not suitable for routine therapeutic drug concentration monitoring.

Contents of the invention

The purpose of the invention is to overcome the defects of the prior art and provide a simple, fast and sensitive method for measuring the blood concentration of mizoribine in the body.

The method does not require expensive equipment and reagents, has the advantages of simple and rapid operation, less plasma consumption, and low cost, and is suitable for routine blood drug concentration monitoring.

The technical scheme of the method is: after the sample to be tested is pretreated, it is separated on a chromatographic column through an acidic mobile phase, and detected by an ultraviolet detector.

Include the following steps:

1) Sample pretreatment

Take the sample to be tested, add quantitative organic solvent protein precipitation agent for protein precipitation,

Described organic solvent protein precipitation agent is selected from the mixture of methanol, acetonitrile or methanol and acetonitrile;

2) Sample separation and analysis

A general-purpose liquid chromatographic column is used, and its filler is Phenomenex Luna NH ₂ . The high-efficiency liquid phase system adopts a general-purpose ultraviolet detector and a high-pressure pump, and uses a mixture of methanol, acetonitrile and acidic aqueous solution (pH=2.1-2.5) as the mobile phase, isocratic elution;

The mobile phase of this method is preferably methanol-acetonitrile-0.1% trifluoroacetic acid (85 ± 2: 5 ± 1: 10 ± 1) mixed solution;

The peak area of MZR was detected by an ultraviolet detector at 280 ± 1 nm, and converted into concentration using the standard curve equation.

This method has the following advantages:

1. The pretreatment method is simple: one-step organic solvent protein precipitation method, suitable for routine detection.

2. Strong specificity: Use a chromatographic column filled with Phenomenex Luna NH ₂ as the stationary phase, a mixture of methanol, acetonitrile and acidic aqueous solution as the mobile phase, isocratic elution, and the determination is completed in 12 minutes. Endogenous substances and commonly used substances are combined Medication does not interfere with the determination of the above drugs.

3. High sensitivity: the lowest limit of quantification is 0.1 mg·L ^-1 , better than 0.25 mg·L ^-1 reported in the literature.

The method of the invention is fast and accurate, has high sensitivity, is easy to operate, and has low cost, and is suitable for routine clinical blood drug concentration monitoring. The linear range of MZR is 0.1-10 mg·L ^-1 . The mean method recoveries were 98.7%, respectively, and the intra-day and inter-day precision RSDs were both less than 9%.

Description of drawings

Figure 1 is a typical chromatogram,

Among them, A: plasma of kidney transplant patients who did not take mizoribine; B: blank plasma + standard product mizoribine (concentration: 0.1 mg·L ^-1 ); 1: mizoribine.

Figure 2 is the chromatogram of kidney transplant patients taking mizoribine (concentration: 0.26mg·L ^-1 ),

Wherein, 1: mizoribine.

Detailed ways

Example 1

Chromatographic conditions

Chromatographic column: Phenomenex Luna NH ₂ (4.6mm×250mm, 5 μm, American Phenomenex Company); mobile phase: acetonitrile-methanol-0.1% trifluoroacetic acid (85:5:10); flow rate: 1.5mL·min ⁻¹ ; column Temperature: 30°C; detection wavelength, 280nm.

Plasma sample pretreatment

Take 100 μL of plasma sample, add 100 μL of acetonitrile solution, vortex mix for 20 s, centrifuge at 12000×g for 10 min at 4°C, and take 20 μL of supernatant for injection. The external standard method was used to quantify the peak area.

Exclusive

Blood samples from 10 kidney transplant patients who did not take MZR were taken from different sources, and measured according to the above sample pretreatment and measurement methods. The results showed that no endogenous substances were found to interfere with the above-mentioned components. The commonly used drug combination and non-prescription drug reference solution were injected. The results showed that cyclosporine, prednisone, tamoxix, sirolimus, ganciclovir, betaloc, acetaminophen, nifedipine , ibuprofen, acyclovir, etc. did not interfere with the determination of samples. Its typical retention time is 7.6min. The entire chromatographic analysis process takes 12 minutes.

linear test

Accurately weigh an appropriate amount of standard substance, dissolve it in distilled water to form a series of working solutions, and then add an appropriate amount of blank human plasma to prepare standard blood samples containing MZR 0.1, 0.5, 2.5, 5, 7.5, and 10 mg·L ^-1 . Take 100 μL of plasma, and operate according to the "plasma sample pretreatment" method. The external standard method uses MZR peak area (x) and MZR concentration (y) as weighted (1/x) linear regression, and the linear regression equation is: y=9171.3 x-21.6, (r=0.9996). The plasma concentration of MZR has a good linear relationship within the range of 0.1-10 mg·L ^-1 . The minimum quantitative concentration is 0.1mg·L ^-1 .

Accuracy and Precision

Accurately weigh an appropriate amount of standard substance, dissolve it in distilled water, dilute blank plasma to volume, and prepare low, medium and high quality control blood samples containing MZR 0.2, 4, 8 mg·L ^-1 . Take 100 μL of plasma, and operate according to the "plasma sample pretreatment" method to investigate the intra-day and inter-day precision and accuracy. Calculate the measured concentration according to the linear regression equation, calculate the measured average value and relative standard deviation (RSD) of each concentration, (C _measured /C _theory ) × 100% is the recovery rate, the experimental results show that: endogenous substances do not interfere Determination of analytes. In addition, the peak shapes of the components to be measured and the internal standard substance are symmetrical and well separated. Table 1 shows the intraday and interday precision and method recovery of mizoribine.

At the same time, this method has been verified in the determination of blood drug concentration in actual patients.

Table 1

Example 2

Chromatographic conditions

Chromatographic column: Phenomenex Luna NH ₂ (4.6mm×250mm, 5 μm, American Phenomenex Company); mobile phase: acetonitrile-methanol-0.1% trifluoroacetic acid (85:5:10); flow rate: 1.5mL·min ⁻¹ ; column Temperature: 30°C; detection wavelength, 280nm.

Plasma sample pretreatment

Take 100 μL of plasma sample, add 100 μL of a mixture of acetonitrile and methanol (1:1, v/v), vortex mix for 20 s, centrifuge at 12,000×g for 10 min at 4°C, and take 20 μL of supernatant for injection. The external standard method was used to quantify the peak area.

Exclusive

Blood samples were taken from 10 kidney transplant patients who did not take MZR, and were determined according to the above-mentioned sample pretreatment and determination methods. In addition, commonly used drug combination and over-the-counter drug reference solution were injected. Results showed endogenous substances, cyclosporine, prednisone, tamox, sirolimus, ganciclovir, betaloc, acetaminophen, nifedipine, ibuprofen, acyclovir etc. do not interfere with the sample determination. Its typical retention time is 7.6min. The entire chromatographic analysis process takes 12 minutes.

linear test

Accurately weigh an appropriate amount of standard substance, dissolve it in distilled water to form a series of working solutions, and then add an appropriate amount of blank human plasma to prepare standard blood samples containing MZR 0.1, 0.5, 2.5, 5, 7.5, and 10 mg·L ^-1 . Take 100 μL of plasma, and operate according to the "plasma sample pretreatment" method. The external standard method uses MZR peak area (x) and MZR concentration (y) as weighted (1/x) linear regression, and the linear regression equation is: y=9082.4 x-19.1, (r=0.9996). The plasma concentration of MZR has a good linear relationship within the range of 0.1-10 mg·L ^-1 . The minimum quantitative concentration is 0.1mg·L ^-1 . The minimum detection concentration is 0.3mg·L ^-1 .

Accuracy and Precision

Accurately weigh an appropriate amount of standard substance, dissolve it in distilled water, dilute blank plasma to volume, and prepare low, medium and high quality control blood samples containing MZR 0.2, 4, 8 mg·L ^-1 . Take 100 μL of plasma, operate according to the "plasma sample pretreatment" method, and investigate the intra-day and inter-day precision and accuracy (n=6). Calculate its measured concentration according to the linear regression equation, calculate the measured mean value and relative standard deviation (RSD) of each concentration, (C _measured /C _theory ) * 100% is the recovery rate. The experimental results show that the intra-day and inter-day precision of the method are both less than 10%, and the relative standard deviation is less than 8%.

Example 3

Chromatographic conditions

Chromatographic column: Phenomenex Luna NH ₂ (4.6mm×250mm, 5 μm, American Phenomenex Company); mobile phase: acetonitrile-methanol-0.1% trifluoroacetic acid (85:5:10); flow rate: 1.5mL·min ⁻¹ ; column Temperature: 30°C; detection wavelength, 280nm.

Plasma sample pretreatment

Take 100 μL of plasma sample, add 200 μL of methanol solution, vortex mix for 20 s, centrifuge at 12000×g for 10 min at 4°C, and take 30 μL of supernatant for injection. The external standard method was used to quantify the peak area.

Exclusive

Blood samples were taken from 10 kidney transplant patients who did not take MZR, and were determined according to the above-mentioned sample pretreatment and determination methods. In addition, commonly used drug combination and over-the-counter drug reference solution were injected. Results showed endogenous substances, cyclosporine, prednisone, tamox, sirolimus, ganciclovir, betaloc, acetaminophen, nifedipine, ibuprofen, acyclovir etc. do not interfere with the sample determination. Its typical retention time is 7.6min. The entire chromatographic analysis process takes 12 minutes.

linear test

Accurately weigh an appropriate amount of standard substance, dissolve it in distilled water to form a series of working solutions, and then add an appropriate amount of blank human plasma to prepare standard blood samples containing MZR 0.1, 0.5, 2.5, 5, 7.5, and 10 mg·L ^-1 . Take 100 μL of plasma, and operate according to the "plasma sample pretreatment" method. The external standard method uses MZR peak area (x) and MZR concentration (y) as weighted (1/x) linear regression, and the linear regression equation is: y=9174.8 x-27.6, (r=0.9994). The plasma concentration of MZR has a good linear relationship within the range of 0.1-10 mg·L ^-1 . The minimum quantitative concentration is 0.1mg·L ^-1 . The minimum detection concentration is 0.3mg·L ^-1 .

Accuracy and Precision

Accurately weigh an appropriate amount of standard substance, dissolve it in distilled water, dilute blank plasma to volume, and prepare low, medium and high quality control blood samples containing MZR 0.2, 4, 8 mg·L ^-1 . Take 100 μL of plasma, operate according to the "plasma sample pretreatment" method, and investigate the intra-day and inter-day precision and accuracy (n=6). Calculate its measured concentration according to the linear regression equation, calculate the measured mean value and relative standard deviation (RSD) of each concentration, (C _measured /C _theory ) * 100% is the recovery rate. The experimental results show that the intra-day and inter-day precision of the method are both less than 10%, and the relative standard deviation is less than 8%.

Claims (1)

1. method of measuring blood drug level of mizoribine, it is characterized in that the testing sample pre-service after, separate on chromatographic column through acid moving phase, detect with UV-detector, comprise the steps:

1) sample pretreatment

Get testing sample, add a kind of in quantitative methyl alcohol, acetonitrile or methyl alcohol and the acetonitrile potpourri, carry out albumen precipitation;

2) sample separation

Adopt universal liquid-phase chromatographic column, its filler is Phenomenex Luna NH ₂The high performance liquid chromatogram system adopts universal UV-detector and high-pressure pump, described acid moving phase adopts the mixed liquor of methyl alcohol-acetonitrile-0.1% trifluoroacetic acid aqueous solution, described methyl alcohol: acetonitrile: 0.1% trifluoroacetic acid aqueous solution is 85 ± 2: 5 ± 1: 10 ± 1, flow velocity is 1.5mL/min, isocratic elution;

3) UV-detector detects: the detection wavelength is 280 ± 1nm, surveys peak area, with typical curve equation conversion concentration.

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