Method for simultaneously detecting two anti-HIV drugs in human plasma

Abstract

The invention relates to a method for simultaneously detecting the concentration of bicotegravir and emtricitabine in human plasma. The detection method comprises the following steps: pretreating a sample to be detected by an acetonitrile protein precipitation method, analyzing chromatographic column separation, and then detecting by an ultraviolet detector. The method has the advantages of less sample sampling, simple pretreatment, no need of expensive equipment and reagents, low cost, and simple and economical analysis and monitoring of the bitovin concentration change during AIDS treatment.

Description

Technical Field

[0001] The present invention belongs to the field of medical testing, relates to an analysis and determination method of drugs in vivo, and specifically relates to a method for simultaneously detecting the concentrations of bictegravir and emtricitabine in human plasma. Background Art

[0002] Since the human immunodeficiency virus (HIV) was first discovered in 1983, it has been a major global health problem. Over the past few decades, acquired immunodeficiency syndrome (AIDS) has caused nearly 40 million deaths. Due to the effective suppression of the virus by antiretroviral drugs, highly active antiviral therapy has become one of the most critical tools for treating the disease and controlling the spread of the virus. Therefore, more and more innovative drugs have been developed and approved. Currently, there are many classifications of antiretroviral drugs, such as nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), fusion inhibitors (FIs), CCR5 inhibitors, and integrase inhibitors (INIs). In routine clinical practice, once a patient is diagnosed, it is usually recommended to combine two NRTIs and another other type of drug. Results from multiple Phase III clinical trials have shown that the combination of bictegravir (BIC, 50 mg), emtricitabine (FTC, 200 mg) and tenofovir alafenamide (TAF, 25 mg) showed good safety and non-inferiority in the initial treatment of HIV infection for 48 weeks compared with other combinations such as dolutegravir (DTG) / FTC / TAF and DTG / abacavir / lamivudine. Therefore, in 2019, the China National Medical Products Administration (NMPA) approved Bituvi for the clinical treatment of HIV infection. In addition, according to the AIDS diagnosis and treatment guidelines, Bituvi has been used as a first-line drug for the treatment of patients who have not been treated before or who need alternative antiviral treatment options.

[0003] It has been reported that in long-term clinical treatment, interruption or intermittent drug intake will reduce drug concentrations, leading to insufficient viral suppression, which will prompt rapid mutation of HIV and produce irreversible drug resistance. Therefore, therapeutic drug monitoring (TDM) has become one of the most effective tools to identify insufficient drug concentrations, avoid or overcome irregular medication, thereby improving efficacy, reducing drug resistance, and minimizing public health problems. However, due to the lack of reliable, effective, simple and economical analytical methods, TDM is still not widely used in the treatment of Bitovit.

[0004] Currently, liquid chromatography-mass spectrometry (LC-MS) is the preferred platform for the determination of antiviral drugs in human plasma. However, the instruments and isotope-labeled reagents used in the analysis are expensive and difficult to obtain, which limits the widespread application of this method, especially in economically underdeveloped areas and laboratories with limited resources, where disease outbreaks or viral mutations caused by poor drug compliance or drug shortages are more likely to occur in these settings. Therefore, there is an urgent need to develop a simple and low-cost method to monitor the changes in biturbovir concentrations throughout AIDS treatment. Summary of the invention

[0005] The purpose of the present invention is to establish and validate a simple and economical LC-UV method for the simultaneous detection of bictegravir and emtricitabine in human plasma. The method can reliably, effectively, simply and economically analyze and monitor the changes in bictegravir concentration during the entire AIDS treatment period.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The method of the present invention for simultaneously detecting the concentrations of bictegravir and emtricitabine in human plasma comprises the following steps:

[0008] 1) Pre-treating plasma to obtain a test solution;

[0009] 2) The solution to be tested is injected into the LC-UV system for detection;

[0010] 3) Quantitative analysis of the test results.

[0011] In a preferred embodiment, the plasma sample is pre-treated by adding the plasma sample to a mixed solution of acetonitrile containing IS (0.5 μg / mL) at a volume ratio of 3 to 5:10, shaking, vortexing for 40 to 80 seconds, centrifuging at 10,000 to 15,000 rpm at 2-5°C, taking the supernatant, transferring the supernatant to a centrifuge tube, and evaporating to dryness under a nitrogen atmosphere. A further preferred plasma sample pre-treatment method is adding the plasma sample to a mixed solution of acetonitrile containing IS (0.5 μg / mL) at a volume ratio of 3:10, shaking, vortexing for 60 seconds, centrifuging at 10,000 at 4°C, and taking the supernatant.

[0012] In a preferred embodiment, the chromatographic column is a reverse phase analytical column, and the stationary phase of the chromatographic column is octadecyl bonded silica gel; for example, Symmetry Chromatographic separation was performed on a C18 column (150 mm × 4.6 mm × 5 μm), with a flow rate of 0.8-1.2 mL / min. The column temperature was set at 35-50 °C, and the injection volume was 30.0-50.0 μL. A further preferred solution, Symmetry Chromatographic separation was performed on a C18 column (150 mm × 4.6 mm × 5 μm) with a flow rate of 1.0 mL / min, a column temperature of 45°C, and an injection volume of 40.0 μL.

[0013] Mobile phase: Mobile phase A was 10 mM NaH2PO4-H3PO4 buffer (pH 3.3), and mobile phase B was methanol.

[0014] Mobile phase gradient: 0-10 min, 10%-100% (B); 10-15 min, 10% (B).

[0015] UV detector detection wavelength and program: 0-5min, 280nm; 5-15min, 260nm.

[0016] In the above determination method, within the range of 0.5-20.0 μg / mL, detection was performed using an ultraviolet detector, with the peak area ratios of the analytes bictegravir, emtricitabine and the internal standard (analyte / IS) as the ordinate and the nominal concentration of the analyte as the abscissa, to generate standard curves of bictegravir and emtricitabine, and fitting was performed using a linear regression model with a 1 / x2 weighting coefficient.

[0017] The beneficial effect of the present invention is that, in more and more clinical trials and real-world studies, Bituowei has shown strong efficacy and good safety. However, due to insufficient concentration or poor compliance, antiviral failure has always been a major challenge, which is closely related to the rapid mutation and increase of drug resistance of HIV. TDM is considered to be a powerful procedure for determining target concentration, resulting in greater effectiveness and less treatment failure. Compared with LC-MS, the LC-UV detection method provided by the present invention is much cheaper and easier to operate, and is particularly suitable for situations where expensive LC-MS equipment is limited or the instrument is damaged. In addition, the method established by the present invention only needs to add protein precipitation and 180 μL of plasma to complete a run within 15 minutes. Due to these analytical advantages, the method can meet the needs of clinical sample processing and result evaluation in a short time, which is particularly beneficial to TDM. In order to obtain the maximum signal intensity of the analyte, 280nm is selected for the quantification of emtricitabine, and 260nm is selected for the detection of bictegravir and IS. Due to the extremely complex nature of human plasma, in order to ensure complete chromatographic separation between the three analytes and between the analytes and endogenous substances, the components, gradients and types of separation columns of the mobile phase are also optimized. NaH2PO4-H3PO4 buffer (pH 3.3, 10 mM) and pure methanol were selected as mobile phases for BIC, FTC and IS elution. 18Reverse phase column chromatography column to obtain acceptable chromatographic behavior and peak shape, low interference and high signal response. The sample was prepared by acetonitrile protein precipitation method, which is simple to operate, low cost, high retention rate and negligible matrix interference. Under optimized conditions, simultaneous detection of bictegravir and emtricitabine in human plasma was achieved. Therefore, the present invention provides a method for simultaneous quantification of bictegravir and emtricitabine in human plasma by LC-UV with less sample sampling, simple operation, no need for expensive equipment and reagents, and low cost, and verifies the method for concentration monitoring or pharmacokinetic analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the LC-UV method for simultaneous quantification of bictegravir and emtricitabine in Bituowei in human plasma.

[0019] Figure 2 Representative chromatograms of FTC, BIC, and IS. (a) Blank human plasma; (b) Blank human plasma supplemented with 1.5 μg / mL FTC, 1.5 μg / mL BIC, and 0.5 μg / mL IS; (c) 1.5 μg / mL FTC, 1.5 μg / mL BIC, and 0.5 μg / mL IS dissolved in water.

[0020] Figure 3 is the calibration curve of emtricitabine and bictegravir in human plasma.

[0021] Figure 4 Representative chromatograms of emtricitabine and bictegravir at LLOQ. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1: Determination of bictegravir and emtricitabine in volunteer plasma.

[0024] (1) Materials and reagents

[0025] BIC (>98%), LC grade acetonitrile and methanol were purchased from Fisher Scientific, USA. FTC (>98%), methyl 4-hydroxybenzoate (≥99%) and sodium dihydrogen phosphate (NaH2PO4) were purchased from Sigma-Aldrich, USA. Deionized water (resistivity >18 MΩ / cm) used in this study was from Millipore Purification Equipment (Billerica, MA, USA).

[0026] (2) Instruments

[0027] BIC and FTC were separated and quantified using a 1260-LC system (Agilent Technologies, USA) and a G 1314F UV detector (Agilent Technologies, USA). OpenLAB CDS ChemStation Edition (Agilent Technologies, USA) was used for data acquisition and peak integration.

[0028] (3) Chromatographic conditions

[0029] Using Symmetry Chromatographic separation was performed on a C18 column (150 mm × 4.6 mm × 5 μm). The flow rate was set at 1.0 mL / min. The column temperature was set at 45°C. The injection volume was 40.0 μL. Mobile phase A was 10 mM NaH2PO4-H3PO4 buffer (pH 3.3), and mobile phase B was methanol. The gradient was programmed as follows: 0-10 min, 10%-100% (B); 10-15 min, 10% (B). The following are the detection wavelengths and programs: 0-5 min, 280 nm; 5-15 min, 260 nm.

[0030] (4) Preparation of stock solution and working solution

[0031] Weigh BIC, FT and methyl 4-hydroxybenzoate (internal standard, IS) and dissolve them in acetonitrile to prepare a stock solution at a final concentration of 2.0 mg / mL. Dilute the working solutions used to prepare calibrants and quality control products with water in turn. Prepare an IS working solution with a concentration of 0.5 μg / mL in acetonitrile. All solutions were stored at -20°C for later use.

[0032] (5) Sample processing

[0033] To 180 μL of human plasma sample, add 20 μL of working solution containing different amounts of BIC and FTC to prepare calibration standards or mass spectrometers. Then add 600 μL of acetonitrile containing IS (0.5 μg / mL), vortex for 1 min, centrifuge at 10000 rpm at 4°C for 5 min, transfer the supernatant to a 1.5 mL centrifuge tube, and evaporate to dryness under a nitrogen atmosphere. Then dissolve the residue in 50 μL of water for LC analysis.

[0034] (6) Method validation

[0035] The established method was successfully validated according to the US Food and Drug Administration bioanalytical method validation guidelines, including selectivity, linearity, lower limit of quantitation (LLOQ), matrix effect, precision, accuracy, retention, and stability.

[0036] a. Selectivity

[0037] The selectivity of the method was evaluated using blank plasma samples from six individual donors treated with acetonitrile. The responses at the retention times of FTC (3.9 min), BIC (9.2 min) and IS (7.6 min) were compared with the signal at the LLOQ.

[0038] The selectivity of the established method was evaluated by using human plasma from six different donors, and representative chromatograms are shown in Figure 1 As shown. No visible peaks of endogenous or other components appeared in any of the six samples within the retention time of FTC (3.9 min), BIC (9.2 min) or IS (7.6 min), and the peaks corresponding to the target analytes appeared symmetrical. The results showed that the method had good selectivity, and at 0.5 μg / mL (LLOQ), the peak area of ​​interfering compounds accounted for less than 20% of the sample load and 5% of the IS.

[0039] b. Linearity and lower limit of quantitation

[0040] In the range of 0.5-20.0 μg / mL, the peak area ratio (analyte / IS) of the analytes BIC and FTC was plotted against the nominal concentration of the analytes using a UV detector to generate standard curves for BIC and FTC, and the 1 / x 2 The linear regression model with weighted coefficients was fitted. The LLOQ was interpolated as the lowest concentration on the calibration curve. The accuracy and precision of BIC and FTC should be within the acceptable range (±20%).

[0041] The calibration curves of BIC and FTC showed good linearity in the range of 0.5-20.0 μg / mL, and the regression coefficients (R) were greater than 0.99 ( Figure 2The LLOQ was 0.5 μg / mL, meeting the acceptance criteria of S / N>10, and the CV (%) and RE (%) were within ±20% ( Figure 3 ).

[0042] c. Precision and accuracy

[0043] Five replicates of four quality control levels (LLOQ QC, LQC, MQC, and HQC) were analyzed in one run to assess intra-day precision and accuracy, and inter-day precision and accuracy were assessed on three different days. The concentration of each QC sample was calculated based on the completed calibration curve. Accuracy and precision defined the calculated relative error [RE% = [(concentration-nominal concentration / nominal concentration) × 100] and coefficient of variation [CV% = (standard deviation / mean) × 100]. The parameters for LLOQ QC should be within ±20%, and the parameters for the other three QC levels should be within ±15%.

[0044] The precision and accuracy of BIC and FTC quantification for LLOQ, LQC, MQC, and HQC were calculated, and the results are summarized in Table 1. The intra-day and intra-day precisions of the FTC method were less than 5.67% and 6.95%, respectively, and the intra-day and intra-day precisions of the BIC method were less than 5.11% and 9.76%, respectively. The Intra-day and Intre-day accuracies of FTC were -9.09% to 10.36% and -3.37% to 4.82%, respectively, and the intra-day and intra-day accuracies of BIC were -8.49% to 13.35% and 0.31% to 6.49%, respectively. The results showed that the precision and accuracy of the method met the recognized standards established by the U.S. Food and Drug Administration.

[0045] Table 1 Precision and accuracy of emtricitabine and bictegravir in human plasma

[0046]

[0047] d. Matrix Effects and Retention

[0048] Matrix effects were evaluated on blank plasma from six different individuals. The peak areas of blank extracts spiked with BIC, FTC, and IS were compared with those of standard solutions prepared in water at LQC and HQC levels, and the ratios were labeled as matrix factors. IS-normalized matrix factors were calculated as the ratio of the matrix factors of BIC to IS or FTC to IS.

[0049] Three replicates of LQC, MQC, and HQC were pretreated, and their peak areas were compared with the peak areas of the spiked supernatant at the same level to calculate the retention rate.

[0050] The matrix effects of FTC and BIC in human plasma were investigated at LQC and HQC levels. The corresponding results are shown in Table 2. It is noteworthy that there was no obvious signal suppression at both QC levels. At concentrations of 1.5 μg / mL, 7.5 μg / mL, and 15.0 μg / mL, the retention rates of FTC and BIC after sample preparation were greater than 85.0%, indicating that the pretreatment used combined with LC-UV analysis had a high retention rate.

[0051] Table 2 Matrix effects of emtricitabine and bictegravir in human plasma

[0052]

[0053] Table 3 Retention rate of emtricitabine and bictegravir in human plasma

[0054]

[0055] e. Stability

[0056] The stability of BIC and FTC in human plasma was evaluated at LQC and HQC for 24 h at room temperature and 7 d at -20 °C, respectively. In addition, the stability of BIC and FTC in the extraction matrix was evaluated at room temperature for 24 h. The stability was acceptable if the concentrations calculated from samples exposed to the above stability conditions were within 85-115% of the freshly prepared samples.

[0057] The stability of FTC and BIC in human plasma and extracts was evaluated to ensure the accuracy of the quantitative results after necessary sample collection and processing. The data in Table 4 show that BIC and FTC remain stable in human plasma after 24 hours at room temperature and 7 days at -20°C. In addition, the stability of FTC and BIC in extracts stored at room temperature for 24 hours is also acceptable.

[0058] Table 4 Stability of emtricitabine and bictegravir in human plasma

[0059]

[0060] The above experimental results show that the LC-UV detection method of the present invention has a good linear relationship between bictegravir and emtricitabine in the range of 0.5-20.0 μg / mL. The LLOQ determined by the LC-UV detection method is 0.5 μg / mL. The LLOQ is sufficient to quantify the plasma concentration of BIC and FTC to determine insufficient drug uptake, thereby preventing irreversible drug resistance and controlling viral transmission.

[0061] For bictegravir and emtricitabine, the accuracy and precision of the LC-UV detection method did not exceed ±15%. In addition, the high retention rate of the method also gave it a satisfactory LLOQ.

[0062] Emtricitabine and bictegravir are stable in human plasma at room temperature for 24 hours and at -20°C for 7 days, which can meet the needs of clinical sample collection and storage. In addition, the stability of BIC and FTC in the extract at room temperature for 24 hours ensures the accuracy of quantitative results during the waiting period for injection.

[0063] The LC-UV detection method of the present invention still has certain limitations. TAF was not included in the analysis method due to its low dose (25 mg) and low plasma concentration (about 0.1 μg / mL). However, the simultaneous detection of bictegravir and emtricitabine from Bituowei can already meet the requirements of TDM, the purpose of which is insufficient drug uptake screening, thereby effectively controlling the disease.

[0064] In summary, the present invention has established a simple and economical LC-UV method for simultaneous quantification of bictegravir and emtricitabine in human plasma. Due to the advantages of easy availability and low price of instruments and reagents, the established method is widely used in TDM and pharmacokinetic studies, especially in resource-limited areas or laboratories.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for simultaneously detecting the concentrations of bictegravir and emtricitabine in human plasma, comprising the following steps: (1) Pre-treating a plasma sample to obtain a test solution; The method for pre-treating the plasma sample is as follows: adding the plasma sample to a mixed solution of acetonitrile containing 0.5 μg / mL internal standard at a volume ratio of 3 to 5:10, shaking, vortexing for 40 to 80 seconds, centrifuging at 10000 to 15000 rpm at 2 to 5° C., taking the supernatant, transferring the supernatant into a centrifuge tube, and evaporating to dryness under a nitrogen atmosphere; (2) The solution to be tested is injected into the LC-UV system for detection; The stationary phase of the chromatographic column is octadecyl bonded silica gel, the flow rate is 0.8-1.2mL / min, the column temperature is 35-50℃, and the injection volume is 30.0-50.0μL; Mobile phase: Mobile phase A was 10 mM NaH2PO4-H3PO4 buffer, pH 3.3, and mobile phase B was methanol; The mobile phase gradient was: 0-10 min, 10%-100% (B); 10-15 min, 10% (B); UV detector detection wavelength and program: 0-5min, 280nm; 5-15min, 260nm; (3) Quantitative analysis of test results; In the above determination method, within the range of 0.5-20.0 μg / mL, the UV detector was used for detection, and the peak area ratio of the analytes bictegravir and emtricitabine to the internal standard was used as the ordinate, and the nominal concentration of the analytes was used as the abscissa to generate the standard curves of bictegravir and emtricitabine, and 1 / x 2 A linear regression model with weighted coefficients was fitted.

2. The method according to claim 1, characterized in that: Plasma sample pretreatment method: add the plasma sample to a mixed solution of acetonitrile containing 0.5 μg / mL internal standard at a volume ratio of 3:10, shake it, vortex it for 60 seconds, centrifuge it at 10000 rpm at 4°C, and take the supernatant.

3. The method according to claim 1, characterized in that: The chromatographic column is Column 150mm×4.6mm×5μm.

4. The method according to claim 1, characterized in that: The flow rate was 1.0 mL / min.

5. The method according to claim 1, characterized in that: The column temperature was set at 45°C.

6. The method according to claim 1, characterized in that: The injection volume was 40.0 μL.

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