Method for determining hydroxychloroquine and deethylated hydroxychloroquine in human whole blood by online solid phase extraction-high performance liquid chromatography

Abstract

The invention discloses a method for determining hydroxychloroquine and deethylated hydroxychloroquine in human whole blood through online solid phase extraction-high performance liquid chromatography. The method is carried out by adopting an on-line solid phase extraction-high performance liquid chromatography combined system, and comprises the following steps: preparing a working solution, pre-treating a sample, analyzing and detecting the injected sample, and calculating the blood concentration. The method is accurate and good in reproducibility, target compounds are enriched and separated through online SPE, sample pretreatment steps are simplified, analysis efficiency is improved, and the concentrations of HCQ and DHCQ in whole blood of an SLE patient can be rapidly detected.

Description

Technical Field

[0001] The invention belongs to the technical field of detection and analysis, and particularly relates to a method for determining hydroxychloroquine and desethylhydroxychloroquine in human whole blood by online solid phase extraction-high performance liquid chromatography. Background Art

[0002] Systemic lupus erythematosus (SLE) is an autoimmune inflammatory connective tissue disease that is common in young women. Its main clinical features are multi-system and multi-organ involvement, repeated relapses, and the presence of a large number of autoantibodies in the body. If the treatment is not timely and standardized, it will cause irreversible damage to the affected organs and eventually lead to the death of the patient. Early treatment intervention can delay or prevent the occurrence and development of more serious sequelae. Currently, the drug treatment for this disease mainly uses hydroxychloroquine (HCQ), glucocorticoid (GC), immunosuppressants and biological agents.

[0003] HCQ is a 4-aminoquinoline antimalarial drug and one of the most valuable treatments for SLE. It has multiple benefits for the control of the disease itself and the prevention of related complications. According to some current literature reports, HCQ treatment of SLE can not only reduce disease activity, relapse and organ damage, reduce the incidence of lupus-related renal failure, improve pregnancy outcomes in SLE patients, but also reduce some risk factors for cardiovascular events. The whole blood drug concentration of HCQ and its main metabolite DHCQ is closely related to the control of SLE disease, adverse reactions (especially irreversible retinopathy) and patient compliance. Therefore, its therapeutic drug monitoring (TDM) is an issue that cannot be ignored by the majority of clinical workers. However, there is no perfect routine detection of HCQ and DHCQ blood drug concentration in clinical practice. Therefore, it is imperative to establish and promote an efficient, sensitive, simple, and clinically applicable HCQ and DHCQ blood drug concentration detection method, which can provide a more reasonable basis for clinical use of drugs, and is also of great significance for implementing individualized treatment plans, optimizing the therapeutic effect of HCQ, and reducing the occurrence of adverse reactions.

[0004] Methods for detecting HCQ whole blood concentration include HPLC, LC-MS / MS, UPLC-MS / MS, etc. Whole blood pretreatment methods mostly use protein precipitation method, or direct liquid-liquid extraction with ethyl acetate, followed by drying with nitrogen and re-dissolving with mobile phase, which is relatively complicated and time-consuming. There are few studies on the correlation between the simultaneous determination of HCQ and its metabolites, but due to their similar physical and chemical properties, the method for determining the concentration of HCQ metabolites is similar to that for measuring HCQ concentration. NoéG et al. established and verified the method for determining HCQ and its metabolites desethylchloroquine (DCQ) and DHCQ using UPLC-fluorescence method. The method is as follows: After adding chloroquine (internal standard) to the sample, protein precipitation is performed and the blood sample to be injected is obtained by filtration. The analytes are separated under isocratic elution on a U-HPLC RP18 column with a total running time of 7 min. The mobile phase consists of piperazine buffer (46.4 mM, pH = 9.8) and acetonitrile (68:32, V / V) with a flow rate of 0.4 mL / min. The fluorescence excitation and emission wavelengths were 335 nm and 390 nm, respectively. Results: HCQ and its two metabolites varied linearly in the range of 125 to 4000 ng / mL, and the lower limit of quantification of each analyte was 10 ng / mL. The limit of quantification of this method was in the same order of magnitude as that observed by LC-MS / MS, and the instrument was cheaper and more suitable for use in hospital laboratories. (Source: Noe G, Amoura Z, Combarel D, et al. Development and Validation of a Fast Ultra-High Performance Liquid Chromatography-Fluorescent Method for the Quantification of Hydroxychloroquine and Its Metabolites in Patients With Lupus[J]. Ther Drug Monit, 2019, 41(4): 476-82.)

[0005] At present, the technology of measuring hydroxychloroquine (HCQ) and desethylhydroxychloroquine (DHCQ) using ultra-high performance liquid chromatography (UPLC) and mass spectrometry (MS) has become increasingly mature, but due to its high equipment cost and low economic benefits, it has not been promoted in clinical use. In addition, the existing methods for detecting HCQ and DHCQ concentrations mostly use extraction or precipitation methods for the pretreatment of blood samples, and then dry them with nitrogen and then re-dissolve them, which is cumbersome and inefficient. Summary of the invention

[0006] In order to overcome the deficiencies in the prior art, the present invention aims to provide a method for determining hydroxychloroquine and desethylhydroxychloroquine in human whole blood by online solid phase extraction-high performance liquid chromatography.

[0007] The specific technical solutions of the present invention are as follows:

[0008] The invention provides a method for determining hydroxychloroquine and desethylhydroxychloroquine in human whole blood by an online solid phase extraction-high performance liquid chromatography method. The method is performed by adopting an online solid phase extraction-high performance liquid chromatography system. The system comprises an A pump, a B pump, an automatic sampler, a six-way valve, an online solid phase extraction column, an analytical column and a detector. The automatic sampler is connected in series to the downstream of the A pump in the system, the detector is connected in series to the downstream of the analytical column in the system, and the working state of the online solid phase extraction-high performance liquid chromatography system is switched by the six-way valve. In a first working state, the online solid phase extraction column is connected in series to the downstream of the automatic sampler in the system, the analytical column is connected in series to the downstream of the B pump in the system, and the online solid phase extraction column and the analytical column operate independently. In a second working state, the online solid phase extraction column is connected in series to the downstream of the B pump in the system, the analytical column is connected in series to the downstream of the online solid phase extraction column in the system, and the online solid phase extraction column and the analytical column operate in series. The method comprises the following steps:

[0009] 1) Preparation of working solution: Dissolve and dilute hydroxychloroquine sulfate and desethyl hydroxychloroquine with sterile water for injection to prepare a series of concentration mixed calibration working solutions;

[0010] 2) Sample pretreatment: Add a series of concentrations of mixed calibration working solution to the whole blood sample to be tested, mix well, add methanol-copper sulfate solution, vortex, centrifuge, and transfer the supernatant to an automatic sampler;

[0011] 3) Sampling analysis and detection: gradient elution and detection are performed according to the following chromatographic conditions, wherein the analytical column of the online solid phase extraction-high performance liquid chromatography coupling system is a C18 column, and the online solid phase extraction column is a C18 column; the mobile phase of the B pump is A1-B1, A1 is a 0.06% by volume phosphoric acid solution, pH is 2.95-3.05, and B1 is acetonitrile; the mobile phase of the A pump is A2-B2, A2 is a 10mM ammonium acetate solution, B2 is B1 is acetonitrile, the detector is a UV detector, and the gradient elution program is shown in the following table. The working state of the online solid phase extraction-high performance liquid chromatography coupling system is switched by a six-way valve, 0-1.5min in the first working state, 1.5-3.6min in the second working state, and 3.6-14min in the first working state;

[0012]

[0013] 4) Calculation of blood drug concentration: Take blank whole blood samples and analyze them according to the steps in 2) and 3) to obtain multiple groups of chromatographic peak areas Y. Respectively perform linear regression on the chromatographic peak areas Y of hydroxychloroquine sulfate and desethylhydroxychloroquine against the concentration X to obtain regression equations. Here, the blank whole blood sample refers to the whole blood sample without hydroxychloroquine sulfate and desethylhydroxychloroquine; Substitute the chromatographic peak area Y obtained from the whole blood sample to be tested into the regression equation to obtain the concentrations of hydroxychloroquine and desethylhydroxychloroquine in the whole blood sample to be tested.

[0014] Further, dissolve and dilute hydroxychloroquine sulfate and desethylhydroxychloroquine with sterile injection water respectively to prepare a series of concentration mixed calibration working solutions containing 4.0×10 4 、2.0×10 4 、1.0×10 4 、5000.0, 2500.0, 1250.0, 625.0 ng / mL.

[0015] Further, the column temperature of the analytical column and the online solid-phase extraction column is 30 °C.

[0016] Further, the injection volume of the automatic sampler is 100 μL.

[0017] Further, when performing sample pretreatment, the volume ratio of the whole blood sample to be tested to the series of concentration mixed calibration working solutions is 10:1.

[0018] Further, when performing sample pretreatment, the volume ratio of the whole blood sample to be tested to the methanol-copper sulfate solution is 100:250.

[0019] Further, when performing sample pretreatment, the vortex time is 2 - 3 min.

[0020] Further, when performing sample pretreatment, the centrifugation speed is 12000 r / min and the centrifugation time is 10 - 15 min.

[0021] Further, when performing sample pretreatment, if there is precipitation floating or turbidity after centrifugation, perform secondary centrifugation and take the supernatant.

[0022] Further, the detection wavelength of the ultraviolet detector is 344 nm.

[0023] The beneficial effects of the present invention are:

[0024] The present invention provides an efficient, sensitive and simple detection method suitable for clinical testing of HCQ and DHCQ whole blood drug concentrations. The method is accurate and reproducible, and adopts online SPE to enrich and separate target compounds, simplifies the sample pretreatment steps, improves the analysis efficiency, can quickly detect the concentrations of HCQ and DHCQ in the whole blood of SLE patients, can further optimize the individualized medication of SLE patients, and has broad application prospects.

[0025] The present invention uses high performance liquid chromatography (HPLC) to simultaneously determine the concentration of HCQ and DHCQ in whole blood, uses an ultraviolet detector, and has a quantitative lower limit of 62.5 ng / mL. The linear relationship is good within the concentration range of 62.5-4000 ng / mL, and the clinical application requirements can be met with relatively cheap instruments and equipment. After simple protein precipitation and centrifugation, the whole blood sample is pre-treated and online solid phase extraction (online-SPE) is used, which not only reduces complicated manual operations, but also enriches and purifies the analyte, and also reduces the impurities entering the analytical column, thereby increasing the service life of the analytical column. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of online solid phase extraction-high performance liquid chromatography coupling system; Figure A: The valve is switched to position 1-6, and the analytical column and SPE column operate independently, completing column balance and sample purification and enrichment respectively, preparing for the next needle analysis or sample pretreatment; Figure B: The valve is switched to position 1-2, and the analytical column and SPE column operate in series, transferring the target compound enriched in the SPE column to the analytical column to achieve the separation and detection of HCQ and DHCQ. Pump A connects the autosampler and the SPE column, which is the extraction pump; Pump B connects the analytical column and the detector, which is the analysis pump; The solid line of the six-way valve indicates that the adjacent passages are connected, and the dotted line of the six-way valve indicates that the adjacent passages are disconnected.

[0027] Figure 2 is an online SPE-HPLC chromatogram of DHCQ and HCQ; A: blank whole blood; B: whole blood containing 62.5 ng / ml of DHCQ and HCQ; C: whole blood containing 500 ng / ml of DHCQ and HCQ; D: whole blood of a patient. DETAILED DESCRIPTION

[0028] In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0029] Example 1

[0030] This embodiment describes in detail the method for determining hydroxychloroquine and desethylhydroxychloroquine in human whole blood by online solid phase extraction-high performance liquid chromatography.

[0031] 1. Construction of the method

[0032] 1.1 Preparation of working solution

[0033] Weigh 13.5 mg of hydroxychloroquine sulfate (equivalent to 10.5 mg of hydroxychloroquine) and 20.0 mg of deethyl hydroxychloroquine, dissolve them in sterile water for injection, prepare standard stock solutions of 10.5 mg / mL and 10.0 mg / mL, and store them at -20°C. Dilute them with sterile water for injection to prepare standard stock solutions containing 4.0×10 4 , 2.0×10 4 , 1.0×10 4 , 5000.0, 2500.0, 1250.0, 625.0 ng / mL of mixed calibration working solution, 2.0×10 4 , 5000.0 and 1250.0 ng / mL were used as a series of mixed quality control working solutions and stored at -20°C.

[0034] 1.2 Sample pretreatment

[0035] Take 100 μL of whole blood sample in a 1.5 mL EP tube, add 10 μL of water / various concentration mixed calibration working solution, mix for 10 seconds, then add 250 μL of methanol-copper sulfate solution (copper sulfate concentration is 6-7 mmol / L), vortex for 2 minutes, centrifuge at 12000 r / min for 10 minutes, take 150 μL of supernatant and transfer to the injection bottle. If there is floating precipitation or turbidity, take the supernatant after a second centrifugation.

[0036] 1.3 Chromatographic conditions

[0037] Analytical column: C18 column (4.6 mm × 150 mm, 5 μm), solid phase extraction column: C18 column (4 mm × 20 mm, 5 μm); mobile phase: analytical pump; 0.06% by volume phosphoric acid solution (adjusted to pH = 3, A1)-acetonitrile (B1), extraction pump: 10 mM ammonium acetate solution (A2)-acetonitrile (B2); detection wavelength: 344 nm; column temperature: 30 ° C; injection volume: 100 μL. Gradient elution program is shown in Table 1. Online solid phase extraction-high performance liquid chromatography coupling system is shown in Figure 1 .

[0038] Table 1 Gradient elution program of online HCQ and DHCQ

[0039]

[0040] Valve cut-off time:

[0041] 0-1.5min (valve position: 1-6), sample purification and enrichment;

[0042] 1.5-3.6 min (valve position: 1-2), transfer of target compound;

[0043] 3.6-14min (valve position: 1-6), balance the column and prepare for the next injection.

[0044] 1.4 Calculation of HCQ and DHCQ blood concentrations

[0045] Take 100 μL blank whole blood sample in a 1.5 mL EP tube and add 4.0×10 4 , 2.0×10 4 , 1.0×10 4 , 5000.0, 2500.0, 1250.0, 625.0 ng / mL series of mixed calibration working solutions are processed and analyzed according to "1.2 Sample Pretreatment". 7 groups of chromatographic peak areas (Y) can be obtained, and the concentration of the standard (X) is known. Substitute into the regression equation: Y = aX + b, and obtain the values ​​of a and b. Subsequent measurement of patient specimens to obtain the chromatographic peak area (Y) can be used to obtain the patient's blood drug concentration (X).

[0046] 2. Methodological Validation

[0047] 2.1 Specificity test

[0048] Take blank plasma, add HCQ and DHCQ to blank plasma, and process and analyze according to "1.2 Sample Pretreatment". The results are shown in Figure 2. The retention times of HCQ and DHCQ are 7.73min and 8.25min respectively, and the separation degree reaches 2.0. The endogenous substances in blank whole blood do not interfere with the determination results of HCQ and DHCQ.

[0049] 2.2 Investigation of linear relationship

[0050] Take 100 μL blank whole blood and add it into a series of mixed calibration working solutions, and process and analyze the samples according to "1.2 Sample Pretreatment".

[0051] (1) Linear regression (weight coefficient: 1 / C) was performed on the HCQ chromatographic peak area (Y) and the concentration (X), and the regression equation was: Y = 7.18 × 10 -4 X-6.96×10 -3 (r=0.9983, n=5).

[0052] (2) Linear regression was performed on the DHCQ chromatographic peak area (Y) versus the concentration (X) (weight coefficient: 1 / C), and the regression equation was: Y = 6.97 × 10 -4 X+0.2×10-5 (r=9988, n=5).

[0053] HCQ and DHCQ showed good linear relationship in the concentration range of 62.5-4000 ng / mL, and the limit of quantification was 62.5 ng / mL (S / N>10).

[0054] 2.3 Extraction recovery test

[0055] Prepare standard drug-containing whole blood samples containing HCQ and DHCQ at concentrations of 125, 500, and 2000 ng / ml, respectively. Three parallel samples were processed and injected according to the "1.2 Sample Pretreatment" to obtain the peak area A 1 Sterile water for injection was used instead of whole blood to prepare standard drug-containing samples containing HCQ and DHCQ at concentrations of 125, 500, and 2000 ng / ml, respectively. Three parallel samples were processed and injected according to the "1.2 Sample Pretreatment" to obtain the peak area A 2 The extraction recovery rate is A 1 / A 2 ×100%, the results are shown in Table 2.

[0056] Table 2 Extraction recovery of HCQ and DHCQ

[0057]

[0058] 2.4 Accuracy and precision test

[0059] Prepare standard drug-containing whole blood samples with HCQ and DHCQ concentrations of 125, 500, and 2000 ng / ml, respectively, in parallel, and process the sample injection analysis according to "1.2 Sample Pretreatment", record the chromatographic peak area, substitute HCQ and DHCQ into the standard curve equation to calculate the HCQ and DHCQ concentrations of the quality control samples, and calculate the intra-batch precision of HCQ and DHCQ whole blood samples (n=5); for 3 consecutive days, measure three batches of quality control samples, record the chromatographic peak area, calculate the HCQ and DHCQ concentrations of the quality control samples according to the HCQ and DHCQ blood drug concentration standard curve, and calculate the inter-batch precision of HCQ and DHCQ whole blood samples (n=3). The results show that the RSD of the intra-batch and inter-batch precision of the quality control samples at low, medium, and high concentrations is less than 7.0%, and the accuracy is higher than 90.0%. See Table 3 for details.

[0060] Table 3 Precision and accuracy of HCQ and DHCQ content determination in human whole blood (n=5)

[0061]

[0062] 2.5 Stability test

[0063] Mixed standard drug-containing whole blood samples with HCQ and DHCQ mass concentrations of 125, 500, and 2000 ng / ml were prepared and placed under the following three conditions: room temperature (about 25°C) for 4 hours, refrigerated (about 4°C) for 24 hours, and repeatedly frozen and thawed and frozen for 1 week. The samples were processed and analyzed according to the "1.2 Sample Pretreatment" item. The measured values ​​were calculated from the standard curve newly configured on the same day. Five parallel tests were performed for each mass concentration under each condition. The results showed that HCQ and DHCQ had good stability under the conditions described, with an accuracy of 82.34%-99.48% for HCQ and RSDs of less than 8.08% for DHCQ and 83.76%-98.89% for DHCQ and RSDs of less than 7.29%.

[0064] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A method for determining hydroxychloroquine and desethylhydroxychloroquine in human whole blood by online solid phase extraction-high performance liquid chromatography, characterized in that, The method is carried out by using an online solid phase extraction-high performance liquid chromatography coupling system, which comprises an A pump, a B pump, an automatic sampler, a six-way valve, an online solid phase extraction column, an analytical column and a detector, wherein the automatic sampler is connected in series downstream of the A pump in the system, the detector is connected in series downstream of the analytical column in the system, and the working state of the online solid phase extraction-high performance liquid chromatography coupling system is switched by the six-way valve; in a first working state, the online solid phase extraction column is connected in series downstream of the automatic sampler in the system, the analytical column is connected in series downstream of the B pump in the system, and the online solid phase extraction column and the analytical column operate independently; in a second working state, the online solid phase extraction column is connected in series downstream of the B pump in the system, the analytical column is connected in series downstream of the online solid phase extraction column in the system, and the online solid phase extraction column and the analytical column operate in series; the method comprises the following steps: 1) Preparation of working solution: Dissolve and dilute hydroxychloroquine sulfate and desethyl hydroxychloroquine with sterile water for injection to prepare a series of concentration mixed calibration working solutions; 2) Sample pretreatment: Add a series of concentrations of mixed calibration working solution to the whole blood sample to be tested, mix well, add methanol-copper sulfate solution, vortex, centrifuge, and transfer the supernatant to an automatic sampler; 3) Sampling analysis and detection: gradient elution and detection are performed according to the following chromatographic conditions, wherein the analytical column of the online solid phase extraction-high performance liquid chromatography coupling system is a C18 column, and the online solid phase extraction column is a C18 column; the mobile phase of the B pump is A1-B1, A1 is a 0.06% by volume phosphoric acid solution, pH is 2.95-3.05, and B1 is acetonitrile; the mobile phase of the A pump is A2-B2, A2 is a 10mM ammonium acetate solution, B2 is B1 is acetonitrile, the detection wavelength of the detector is 344nm, and the gradient elution program is shown in the following table. The working state of the online solid phase extraction-high performance liquid chromatography coupling system is switched by a six-way valve, 0-1.5min in the first working state, 1.5-3.6min in the second working state, and 3.6-14min in the first working state; 4) Calculation of blood drug concentration: Take a blank whole blood sample and analyze it according to the steps of step 2) and step 3) to obtain multiple groups of chromatographic peak areas Y, and perform linear regression on the concentration X with the chromatographic peak areas Y of hydroxychloroquine sulfate and desethyl hydroxychloroquine, respectively, to obtain a regression equation, wherein the blank whole blood sample refers to a whole blood sample that does not contain hydroxychloroquine sulfate and desethyl hydroxychloroquine; substitute the chromatographic peak area Y obtained from the whole blood sample to be tested into the regression equation to obtain the concentrations of hydroxychloroquine and desethyl hydroxychloroquine in the whole blood sample to be tested.

2. The method according to claim 1, characterized in that Hydroxychloroquine sulfate and desethylhydroxychloroquine were dissolved and diluted with sterile water for injection to prepare 4.0×10 4 , 2.0×10 4 , 1.0×10 4 , 5000.0, 2500.0, 1250.0, 625.0 ng / mL series concentration mixed calibration working solution.

3. The method according to claim 1, characterized in that: The column temperature of the analytical column and the online solid phase extraction column is 30°C.

4. The method according to claim 1, characterized in that: The injection volume of the automatic sampler is 100 μL.

5. The method according to claim 1, characterized in that During sample pretreatment, the volume ratio of the whole blood sample to be tested to the series concentration mixed calibration working solution is 10:

1.

6. The method according to claim 1, characterized in that During sample pretreatment, the volume ratio of the whole blood sample to be tested to the methanol-copper sulfate solution is 100:

250.

7. The method according to claim 1, characterized in that During sample pretreatment, the vortexing time is 2-3 minutes.

8. The method according to claim 1, characterized in that During sample pretreatment, the centrifugal speed is 12000 r / min and the centrifugal time is 10-15 min.

9. The method according to claim 1, characterized in that: During sample pretreatment, if there is floating precipitation or turbidity after centrifugation, perform a second centrifugation and take the supernatant.

10. The method according to claim 1, characterized in that The detection wavelength of the ultraviolet detector is 344 nm.

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