Method for the detection of amiodarone and desethylamiodarone in plasma

Abstract

The present invention provides a method for detecting amiodarone and desethylamiodarone in plasma, and relates to the technical field of drug detection. The detection method comprises detecting a pre-treated sample using high performance liquid chromatography, bringing the detection value into a standard curve, and calculating the content of amiodarone and desethylamiodarone in the plasma sample; the pre-treatment comprises extracting the sample to be tested using an extractant, and the extractant comprises methanol and acetonitrile. The detection method alleviates the problems of complex and time-consuming detection methods for amiodarone and desethylamiodarone in plasma in the prior art, and high detection costs.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, in particular to a method for detecting amiodarone and desethylamiodarone in plasma. Background Art

[0002] Arrhythmia is a common cardiovascular disease in clinical practice, with palpitations and dizziness as the main manifestations. If not treated in time, it can lead to syncope or even death, seriously threatening the patient's life. Amiodarone hydrochloride, also known as amiodarone, is a Class III antiarrhythmic drug widely used to treat and prevent ventricular and supraventricular arrhythmias.

[0003] Amiodarone is incompletely absorbed after oral administration, has a slow onset of action, and a long half-life; its bioavailability is about 50%, and its apparent distribution volume is about 60L / kg. It is mainly distributed in adipose tissue and fat-rich organs, followed by the heart, kidneys, lungs, liver, and lymph nodes. 62.1% of amiodarone is bound to proteins in plasma, and 33.5% may be bound to β-lipoprotein. Some studies have also shown that the plasma protein binding rate of amiodarone is 95%.

[0004] Intravenous infusion may cause phlebitis, and long-term use can easily cause the accumulation of amiodarone and its metabolite desethylamiodarone in the body. This adverse reaction caused by slow drug metabolism and drug accumulation can cause pulmonary poisoning, liver poisoning, neurotoxicity and other side effects (with large individual differences). Desethylamiodarone and the parent drug amiodarone have similar physiological activity and pharmacological effects. Therefore, monitoring amiodarone and desethylamiodarone can help guide their clinical application, improve drug efficacy, and avoid or reduce adverse reactions.

[0005] The detection methods of amiodarone mainly include high performance thin layer chromatography, high performance liquid chromatography, micellar capillary electrophoresis, ion chromatography, LCMSMS method and the like.

[0006] Liquid chromatography-mass spectrometry (LC-MS / MS) has very high sensitivity, specificity and accuracy in the determination of amiodarone. However, the LC-MS / MS method requires a time-consuming and complex pretreatment process and the complex equipment operation has higher requirements for operators. The expensive equipment and maintenance costs require greater capital investment. At the same time, due to the extremely high protein binding rate of amiodarone in plasma, this also increases the technical difficulty for accurate and sensitive detection of amiodarone in plasma.

[0007] Therefore, how to reduce detection costs, simplify operations, and at the same time ensure detection sensitivity, specificity, and accuracy is an issue that needs to be addressed urgently.

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] The purpose of the present invention is to provide a method for detecting amiodarone and desethylamiodarone in plasma, which alleviates the problems of complicated and time-consuming detection methods of amiodarone and desethylamiodarone in plasma and high detection costs in the prior art.

[0010] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0011] According to one aspect of the present invention, the present invention provides a method for detecting amiodarone and desethylamiodarone in plasma, comprising detecting a pre-treated sample using high performance liquid chromatography, inserting the detection value into a standard curve, and calculating the content of amiodarone and desethylamiodarone in the plasma sample;

[0012] The pretreatment includes extracting the sample to be tested using an extractant, and the extractant includes methanol and acetonitrile.

[0013] Preferably, the extractant is a mixed system of methanol and acetonitrile, containing 5-15% methanol and 85-95% acetonitrile by volume;

[0014] Preferably, the extractant contains 10% methanol and 90% acetonitrile by volume;

[0015] Preferably, the volume ratio of the sample to be tested to the extractant is 1:3 to 1:5; preferably 1:4.

[0016] Preferably, the pretreatment further comprises shaking the mixed system of the extractant and the sample to be tested;

[0017] Preferably, the shaking is carried out in a constant temperature oscillator for 3 to 10 minutes, preferably for 5 minutes;

[0018] Preferably, the mixed system of the extractant and the sample to be tested is centrifuged after shaking, the supernatant is taken into a new reaction container, and the mixture is centrifuged again and then subjected to high performance liquid chromatography detection;

[0019] Preferably, the pre-treatment step is performed in a 96-well plate.

[0020] Preferably, the mobile phase for HPLC detection includes mobile phase A and mobile phase B;

[0021] The mobile phase A is an aqueous phase, and the mobile phase A contains potassium dihydrogen phosphate and phosphoric acid;

[0022] The mobile phase B is an organic phase;

[0023] Preferably, the mobile phase A contains 5-10 mM potassium dihydrogen phosphate and 0.1-0.5% v / v concentrated phosphoric acid, wherein the concentrated phosphoric acid is HPLC grade phosphoric acid with a concentration of ≥85%;

[0024] Preferably, the mobile phase A contains 5 mM potassium dihydrogen phosphate and 0.2% v / v concentrated phosphoric acid;

[0025] Preferably, the mobile phase B is acetonitrile;

[0026] Preferably, the high performance liquid chromatography detection uses a Waters XBridge C18 chromatographic column.

[0027] Preferably, the elution procedure of the high performance liquid chromatography detection is:

[0028] 0.00 min, mobile phase A 50%, mobile phase B 50%;

[0029] 10.00 min, mobile phase A 40%, mobile phase B 60%;

[0030] 12.00 min, mobile phase A 15%, mobile phase B 85%;

[0031] 14.00 min, mobile phase A 15%, mobile phase B 85%;

[0032] 14.10 min, mobile phase A 50%, mobile phase B 50%;

[0033] 15.00 min, mobile phase A 50%, mobile phase B 50%.

[0034] Preferably, the plasma is from mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys or humans, preferably human plasma.

[0035] Preferably, the standard curve is the corresponding relationship between the content of the standard substance in the standard curve working solution and the detection value of the standard substance;

[0036] Preferably, the standard curve is subjected to linear regression using the chromatographic peak area of ​​the standard in the standard working solution as the dependent variable and the concentration of the standard in the standard working solution as the independent variable;

[0037] Preferably, the concentration ranges of amiodarone and desethylamiodarone in the standard curve working solution are independently 0.05 to 5.00 μg / mL;

[0038] Preferably, the matrix of the standard curve working solution is animal plasma; preferably bovine plasma; more preferably newborn calf plasma.

[0039] Preferably, the detection method further comprises detecting a quality control product.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The method for detecting amiodarone and desethylamiodarone in plasma provided by the present invention comprises detecting the pre-treated sample by high performance liquid chromatography, inserting the detection value into the standard curve, and calculating the content of amiodarone and desethylamiodarone in the plasma sample. The pre-treatment comprises extracting the sample to be tested by using an extractant, and the extractant comprises methanol and acetonitrile.

[0042] The present invention is aimed at the defects of the method for diagnosing amiodarone and deethylamiodarone in plasma on the market, and studies a high-performance liquid phase method that can accurately and high-throughput simultaneously detect amiodarone and deethylamiodarone in human plasma. The present invention improves the defect that amiodarone is easy to bind to plasma proteins in plasma and is difficult to separate by optimizing the composition of the extractant and the steps of pretreatment, thereby improving the detection effect of the subsequent high-performance liquid chromatography on the sample. After the sample is pretreated, it enters the liquid chromatograph for detection, which can achieve high-throughput detection, and the drug test of amiodarone and deethylamiodarone can be completed within 20 minutes, which is convenient and fast. The detection method can meet the requirements of linearity, repeatability, batch difference, accuracy, stability, etc. of amiodarone and deethylamiodarone drug detection in relevant laws and regulations, and plays an important role in monitoring patients' amiodarone and deethylamiodarone drugs.

[0043] This method uses high-performance liquid chromatography for high-throughput detection of amiodarone and desethylamiodarone in plasma. The detection results of amiodarone and desethylamiodarone can be obtained at the same time in one test, and has the characteristics of high sensitivity, good repeatability, high accuracy, good specificity, etc. The linear range of this method is 0.05-5.00μg / mL for amiodarone and 0.05-5.00μg / mL for desethylamiodarone, and the correlation coefficient is r≥0.999; the coefficient of variation (CV) of the repeatability of the low-value quality control product is ≤10%, the coefficient of variation (CV) of the repeatability of the mid-value quality control product is ≤10%, and the coefficient of variation (CV) of the repeatability of the high-value quality control product is ≤10%; the relative deviation (B) of accuracy is ≤±10%, and the spike recovery rate is 90% to 110%. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1 This is the peak diagram of deethylamiodarone and amiodarone under the conditions of the detection method of Example 1 of the present invention. DETAILED DESCRIPTION

[0046] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than 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.

[0047] According to one aspect of the present invention, the present invention provides a method for detecting amiodarone and desethylamiodarone in plasma, the method comprising detecting the pre-treated sample using high performance liquid chromatography, inserting the detection value into the standard curve, and calculating the content of amiodarone and desethylamiodarone in the plasma sample. The pre-treatment comprises extracting the sample to be tested using an extractant, and the extractant comprises methanol and acetonitrile.

[0048] It should be noted that the method for detecting amiodarone and desethylamiodarone in plasma provided by the present invention is not for diagnosis and treatment purposes.

[0049] The present invention does not limit the source of the plasma of the detection object, and the plasma sources include but are not limited to mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys or humans. Preferably, human plasma is detected.

[0050] The detection method provided by the present invention mainly extracts amiodarone and desethylamiodarone in a plasma sample by first using an extractant composed of methanol and acetonitrile, so that amiodarone in the plasma, especially amiodarone bound to plasma proteins, is separated from the plasma for subsequent high performance liquid chromatography detection. The present invention uses an extractant composed of methanol and acetonitrile to separate amiodarone and desethylamiodarone in a sample to be tested, and has a good separation effect, which alleviates the defect of inaccurate detection results caused by the binding of plasma proteins to amiodarone in plasma.

[0051] In some optional embodiments, the extractant is a mixed system of methanol and acetonitrile, containing 5-15% methanol and 85-95% acetonitrile by volume; the volume percentage of methanol can be, for example, but not limited to, 5%, 8%, 10%, 12% or 15%, and the volume percentage of acetonitrile can be, for example, but not limited to, 85%, 88%, 90%, 92% or 95%. The preferred ratio of the extractant is 10% methanol and 90% acetonitrile by volume.

[0052] In some optional embodiments, the volume ratio of the sample to be tested and the extractant is 1:3 to 1:5; preferably 1:4, and specific embodiments may include but are not limited to 100 μL of the sample to be tested being extracted with 300 μL of the extractant; 100 μL of the sample to be tested being extracted with 400 μL of the extractant; or 100 μL of the sample to be tested being extracted with 500 μL of the extractant; preferably 100 μL of the sample to be tested being extracted with 400 μL of the extractant. The sample to be tested refers to a plasma sample, a standard or a quality control product.

[0053] In some optional embodiments, the pretreatment further includes shaking the mixed system of the extractant and the sample to be tested, and the preferred shaking condition is shaking in a constant temperature oscillator for 3 to 10 minutes, for example, but not limited to 3, 5, 8 or 10 minutes; the preferred shaking time is 5 minutes.

[0054] In some optional embodiments, the mixed system of the extractant and the sample to be tested is centrifuged after shaking, and the supernatant is taken into a new reaction container, and then centrifuged again and then detected by high performance liquid chromatography.

[0055] Among them, the centrifugation conditions for the first centrifugation, that is, centrifuging the mixed system of the extractant and the sample to be tested after shaking, are preferably: the centrifugation temperature is 0-10°C, for example, it can be but not limited to 0, 4, 5 or 10°C; the centrifugation speed is 3000-5000rmp, for example, it can be but not limited to 3000, 3500, 4000, 4500 or 5000rmp; the centrifugation time is 5-15min, for example, it can be but not limited to 5, 8, 10, 12 or 15min; preferably, the centrifugation conditions are 4°C, 4000rmp for 10min.

[0056] The second centrifugation, i.e., the centrifugation conditions before HPLC detection are preferably: the centrifugation temperature is 0-10°C, for example, it can be but not limited to 0, 4, 5 or 10°C; the centrifugation speed is 3000-5000rmp, for example, it can be but not limited to 3000, 3500, 4000, 4500 or 5000rmp; the centrifugation time is 3-10min, for example, it can be but not limited to 3, 5, 8, 10min; preferably, the centrifugation conditions are 4°C, 4000rmp for 5min.

[0057] In some optional embodiments, the pretreatment step is performed in a 96-well plate. Pretreatment in a 96-well plate can be performed using a dispenser to remove samples and reagents to achieve batch pretreatment.

[0058] In some optional embodiments, the provided method for detecting amiodarone and desethylamiodarone in plasma also optimizes chromatographic conditions.

[0059] In some optional embodiments, the mobile phase for HPLC detection is optimized. The mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A is an aqueous phase and the mobile phase B is an organic phase. The aqueous phase is preferably an aqueous solution containing potassium dihydrogen phosphate and phosphoric acid. Experiments have found that the addition of potassium dihydrogen phosphate and phosphoric acid can improve the peak shape of amiodarone, but the amount of potassium dihydrogen phosphate is too large, and the probability of potassium dihydrogen phosphate precipitation in the mobile phase increases, which will damage the chromatographic column. Therefore, the concentration of potassium dihydrogen phosphate in mobile phase A is preferably 5-10mM, for example, it can be but not limited to 5, 6, 7, 8, 9 or 10mM; the volume percentage of concentrated phosphoric acid is preferably 0.1-0.5%, for example, it can be but not limited to 0.1%, 0.2%, 0.3%, 0.4% or 0.5%, wherein concentrated phosphoric acid refers to a concentration of ≥85% HPLC grade phosphoric acid. The preferred composition of mobile phase A is potassium dihydrogen phosphate containing a concentration of 5mM and 0.2% v / v concentrated phosphoric acid. Mobile phase B is preferably acetonitrile.

[0060] In some optional embodiments, the HPLC detection uses a Waters XBridge C18 chromatographic column, and the detection results using the Waters XBridge C18 chromatographic column are more accurate than those using other chromatographic columns.

[0061] In some optional embodiments, the elution procedure for high performance liquid chromatography detection is:

[0062] 0.00 min, mobile phase A 50%, mobile phase B 50%;

[0063] 10.00 min, mobile phase A 40%, mobile phase B 60%;

[0064] 12.00 min, mobile phase A 15%, mobile phase B 85%;

[0065] 14.00 min, mobile phase A 15%, mobile phase B 85%;

[0066] 14.10 min, mobile phase A 50%, mobile phase B 50%;

[0067] 15.00 min, mobile phase A 50%, mobile phase B 50%.

[0068] In some optional embodiments, the standard curve is the corresponding relationship between the content of the standard in the standard curve working solution and the detection value of the standard, and the detection value can be, for example, but not limited to, peak height or peak area, preferably with the chromatographic peak area of ​​the standard in the standard curve working solution as the dependent variable, and the concentration of the standard in the standard working solution as the independent variable for linear regression. The concentration range of amiodarone in the standard curve working solution is 0.05-5.00 μg / mL, and the concentration of amiodarone is preferably distributed as 0.05, 0.10, 0.30, 0.50, 0.90, 1.80, 3.00 and 5.00 μg / mL; the concentration range of deethylamiodarone in the standard curve working solution is 0.05-5.00 μg / mL, and the concentration of deethylamiodarone is preferably distributed as 0.05, 0.10, 0.30, 0.50, 0.90, 1.80, 3.00 and 5.00 μg / mL. Since human samples are difficult to obtain, it is preferred to use animal plasma as a substitute matrix for human plasma to prepare the standard curve working solution, for example, bovine plasma, preferably newborn calf plasma, is used as the matrix of the standard curve working solution for each concentration point.

[0069] In some optional embodiments, the detection method further includes detecting quality control products to verify whether the detection results are credible, and the quality control products preferably include at least two of low-value quality control products, median quality control products and high-value quality control products; the concentrations of amiodarone and desethylamiodarone in the low-value quality control products are independently 0.80 μg / mL; the concentrations of amiodarone and desethylamiodarone in the median quality control products are independently 2.50 μg / mL; the concentrations of amiodarone and desethylamiodarone in the high-value quality control products are independently 3.80 μg / mL.

[0070] The present invention is aimed at the defects of the method for diagnosing amiodarone and deethylamiodarone in plasma on the market, and studies a high-performance liquid phase method that can accurately and high-throughput simultaneously detect amiodarone and deethylamiodarone in human plasma. The present invention improves the defect that amiodarone is easy to bind to plasma proteins in plasma and is difficult to separate by optimizing the composition of the extractant and the steps of pretreatment, thereby improving the detection effect of the subsequent high-performance liquid chromatography on the sample. After the sample is pretreated, it enters the liquid chromatograph for detection, which can achieve high-throughput detection, and the drug test of amiodarone and deethylamiodarone can be completed within 20 minutes, which is convenient and fast. The detection method can meet the requirements of linearity, repeatability, batch difference, accuracy, stability, etc. of amiodarone and deethylamiodarone drug detection in relevant laws and regulations, and plays an important role in monitoring patients' amiodarone and deethylamiodarone drugs.

[0071] The technical solutions and beneficial effects of the present invention are further described below in conjunction with preferred embodiments.

[0072] The main reagents are shown in Table 1:

[0073] Table 1 Standards and reagents

[0074]

[0075] The main equipment is shown in Table 2:

[0076] Table 2 Instruments and equipment

[0077] Serial number Device Name Model / Specification 1 Liquid Chromatograph LC2300 2 96-well plate shaker MB100-4A 3 96-well plate centrifuge Xiangyi H2050R 4 10μL pipette 0.5-10μL 5 200μL pipette 20-200μL 6 1000μL pipette 100-1000μL 7 300μL 12-well pipette 30-300μL 8 96-well plate 2mL 9 96-well plate 1mL 10 Electronic balance One in 100,000

[0078] Example 1

[0079] (I) Reagent preparation

[0080] (1) Preparation of amiodarone and deethylamiodarone stock solutions

[0081] Preparation of amiodarone stock solution: Use a 1 / 100,000 electronic balance to accurately weigh 10 mg of amiodarone, place it in a 10 mL volumetric flask, dissolve it in methanol and dilute to the mark to prepare a 1 mg / mL stock solution, transfer it to a 15 mL brown glass bottle, label it, and store it at -20°C.

[0082] Preparation of deethylamiodarone stock solution: Use a 1 / 100,000 electronic balance to accurately weigh 10 mg of deethylamiodarone, place it in a 10 mL volumetric flask, dissolve it in methanol and dilute to the mark to prepare a 1 mg / mL stock solution, transfer it to a 15 mL brown glass bottle, label it, and store it at -20°C.

[0083] (2) Preparation of secondary stock solutions of amiodarone and desethylamiodarone

[0084] Preparation of secondary stock solutions of amiodarone and desethylamiodarone: Use a pipette to accurately transfer 1 mL of amiodarone stock solution and 1 mL of desethylamiodarone to a 10 mL volumetric flask, dilute to the mark with methanol to prepare a 100 μg / mL secondary stock solution, transfer to a 15 mL brown glass bottle, label, and store at -20°C.

[0085] (3) Preparation of calibrators S1 to S8

[0086] The standard products were prepared according to the high-low preparation mode, that is, after preparing S1 and S8, they were mixed in proportion to prepare S2 to S7 and LQC, MQC and HQC.

[0087] Preparation of calibrator S8: Use a pipette of appropriate specifications to transfer 500 μL of secondary stock solution of amiodarone and desethylamiodarone, place in a suitable HDPE tube, add 9.5 mL of newborn calf plasma, and prepare 5.00 μg / mL amiodarone and 5.00 μg / mL desethylamiodarone standard solutions. After thorough mixing, label and store at -20°C.

[0088] Preparation of calibrator S1: Use a pipette of appropriate specifications to transfer 7.5 μL of secondary stock solution of amiodarone and desethylamiodarone, place in a suitable HDPE tube, add 14992.5 μL of newborn calf plasma, and prepare 0.05 μg / mL amiodarone and 0.05 μg / mL desethylamiodarone standard solutions. After thorough mixing, label and store at -20°C.

[0089] Preparation of calibrator S2: Accurately pipette 2.94 mL of S1 standard and 0.03 mL of S8 standard, mix thoroughly to prepare 0.10 μg / mL amiodarone and 0.10 μg / mL deethylamiodarone standard solutions. After thorough mixing, label and store at -20°C.

[0090] Preparation of calibrator S3: Accurately pipette 2.82 mL of S1 standard and 0.15 mL of S8 standard, mix thoroughly to prepare 0.30 μg / mL amiodarone and 0.30 μg / mL deethylamiodarone standard solutions. After thorough mixing, label and store at -20°C.

[0091] Preparation of calibrator S4: Accurately pipette 2.70 mL of S1 standard and 0.27 mL of S8 standard, mix thoroughly to prepare 0.50 μg / mL amiodarone and 0.50 μg / mL deethylamiodarone standard solutions. After thorough mixing, label and store at -20°C.

[0092] Preparation of calibrator S5: Accurately pipette 2.46 mL of S1 standard and 0.51 mL of S8 standard, mix thoroughly to prepare amiodarone 0.90 μg / mL and deethylamiodarone 0.90 μg / mL standard solutions. After thorough mixing, label and store at -20°C.

[0093] Preparation of calibrator S6: Accurately pipette 1.92 mL of S1 standard and 1.05 mL of S8 standard, mix thoroughly to prepare amiodarone 1.80 μg / mL and desethylamiodarone 1.80 μg / mL standard solutions. After thorough mixing, label and store at -20°C.

[0094] Preparation of calibrator S7: Accurately pipette 1.20 mL of S1 standard and 1.77 mL of S8 standard, mix thoroughly to prepare amiodarone 3.00 μg / mL and desethylamiodarone 3.00 μg / mL standard solutions. After thorough mixing, label and store at -20°C.

[0095] (4) Preparation of quality control products

[0096] Preparation of low-concentration quality control LQC: Accurately pipette 2.52 mL of S1 standard and 0.45 mL of S6 standard, mix thoroughly to prepare amiodarone 0.80 μg / mL and deethylamiodarone 0.80 μg / mL standard solutions. After thorough mixing, label and store at -20°C.

[0097] Preparation of medium-concentration quality control product MQC: Accurately pipette 1.50 mL of S1 standard and 1.47 mL of S6 standard, mix thoroughly to prepare amiodarone 2.50 μg / mL and desethylamiodarone 2.50 μg / mL standard solutions. After thorough mixing, label and store at -20°C.

[0098] Preparation of high-concentration quality control HQC: Accurately pipette 0.72 mL of S1 standard and 2.25 mL of S6 standard, mix thoroughly to prepare amiodarone 3.80 μg / mL and deethylamiodarone 3.80 μg / mL standard solutions. After thorough mixing, label and store at -20°C.

[0099] (5) Sample extraction agent preparation:

[0100] Preparation of sample extractant stock solution: Pipette 100 mL of methanol into a 1000 mL solvent bottle, accurately add 900 mL of acetonitrile, mix thoroughly and set aside.

[0101] (6) Mobile phase preparation:

[0102] Preparation of mobile phase A: 5 mM potassium dihydrogen phosphate. Accurately weigh 0.68 g potassium dihydrogen phosphate using an electronic balance, add 1000 mL of pure water to prepare 1000 mL of 5 mmol / L potassium dihydrogen phosphate solution, add 200 μL of concentrated phosphoric acid, mix, filter and sonicate for later use.

[0103] Preparation of fluid B solution: acetonitrile solution, filter and degas by ultrasonication, and set aside.

[0104] (II) Pre-treatment process:

[0105] (1) Add the sample to be tested: Pipette 100 μL of the sample to be tested into a 96-well plate. The sample to be tested can be a plasma sample, a calibrator working solution, or a quality control product.

[0106] (2) Adding extraction agent: Pipette 400 μL of extraction agent and add it to the 96-well plate in (1) in sequence.

[0107] (3) Oscillate in a microplate constant temperature oscillator for 5 min.

[0108] (4) Centrifuge in a 96-well plate refrigerated centrifuge at 4000 rpm for 10 min at 4°C.

[0109] (5) Pipette 200 μl into a new 96-well plate, cover and wait for testing.

[0110] (6) Centrifuge in a 96-well plate refrigerated centrifuge at 4000 rpm at 4°C for 5 min.

[0111] (7) Place the 96-well plate in the instrument and use liquid phase LC 2300 for detection.

[0112] (III) Liquid phase test conditions

[0113] (1) Chromatographic column: Waters XBridge C18 5μm, 4.6×150mm

[0114] (2) Mobile phase: Phase A: water (containing 5 mM potassium dihydrogen phosphate and 0.2% concentrated phosphoric acid);

[0115] Phase B: Acetonitrile

[0116] (3) Elution gradient, as shown in Table 3:

[0117] Table 3 Gradient conditions

[0118]

[0119]

[0120] (4) Flow rate: 1.0 mL / min;

[0121] (5) Column temperature: 40°C;

[0122] (6) Automatic sampler temperature: 10°C;

[0123] (7) Needle washing solution: methanol: water (1:1 v / v);

[0124] (8) Injection volume: 20 μL.

[0125] Example 2

[0126] The difference between Example 2 and Example 1 is only the chromatographic column. The chromatographic column of this example is SHIMADZU Shim-pack GIST C18 (5 μm, 150 mm*3 mm).

[0127] Example 3

[0128] The difference between Example 3 and Example 1 is only that the mobile phases are different. In this example, mobile phase A is water and mobile phase B is acetonitrile.

[0129] Comparative Example 1

[0130] The difference between Comparative Example 1 and Example 1 is only that the extractant is different, and the extractant in this comparative example is acetonitrile.

[0131] Effect Example 1

[0132] Selection of extraction agent:

[0133] A certain amount of standard was added to blank human plasma to prepare high concentration point and low concentration point samples. Acetonitrile (Comparative Example 1) and a mixed solution of acetonitrile and methanol (Example 1) were used to perform protein precipitation on the samples. The samples after protein precipitation were directly loaded for detection after centrifugation. The external standard method was used for calculation. The results are shown in the table.

[0134] Table 4. Accuracy results obtained using acetonitrile as the extraction agent (Comparative Example 1)

[0135]

[0136]

[0137] Table 5. Accuracy results obtained using a mixed solution of acetonitrile and methanol as an extractant (Example 1)

[0138]

[0139] By comparing Table 4 and Table 5, the results show that the recovery rate of amiodarone is significantly improved when a mixed solution of acetonitrile and methanol is used as a sample extractant (Example 1), so the extraction and detection effects of the technical solution provided in Example 1 are better. The experimental process shows that the protein particles formed after protein precipitation using acetonitrile as an extractant and using a mixed solution of acetonitrile and methanol as an extractant are different. The protein precipitated by the mixed solution of acetonitrile and methanol is suspended, and the protein precipitated by using acetonitrile alone as an extractant is in a block state.

[0140] Effect Example 2

[0141] Column selection:

[0142] A certain amount of standard was added to blank human plasma to prepare high concentration point and low concentration point samples. The samples were subjected to protein precipitation treatment using a mixed solution of acetonitrile and methanol. The protein precipitated samples were directly loaded for detection after centrifugation. Waters XBridge C18 (5 μm, 4.6×150 mm) chromatographic column (Example 1) and SHIMADZUShim-pack GIST C18 (5 μm, 150 mm*3 mm) chromatographic column (Example 2) were selected as analytical chromatographic columns, and the external standard method was used for calculation. The results are shown in Table 6. By comparing the recovery rate results of the two chromatographic columns, the results show that the technical effect of using the Waters XBridge C18 chromatographic column in Example 1 is better.

[0143] Table 6. Comparison of column test results

[0144]

[0145] Effect Example 3

[0146] Mobile phase selection:

[0147] When acetonitrile and water were tried as mobile phases (Example 3), the peak shape of amiodarone was severely deformed. However, the peak shape was significantly improved by adding potassium dihydrogen phosphate and 0.02% concentrated phosphoric acid (Example 1).

[0148] Table 7. Effect of different flow rates on the results

[0149]

[0150] Effect Example 4

[0151] Linear range test data:

[0152] (1) Verification method: The calibrants S1 to S8 of the tested products were treated according to the sample treatment method described in Example 1 for high throughput detection of amiodarone and desethylamiodarone in human plasma by high performance liquid chromatography, and the test was repeated for three days. The correlation coefficient r of linear regression can be calculated by referring to the formula, and the correlation coefficient r should be ≥ 0.990.

[0153]

[0154] r: linear regression correlation coefficient;

[0155] xi: concentration of S1 to S8;

[0156] yi: Peak area value of the calibrator in the corresponding concentration solution.

[0157] (2) Acceptance criteria: The linear regression correlation coefficients of amiodarone and desethylamiodarone were both ≥ 0.990.

[0158] (3) Experimental results:

[0159] Linear range: The linear range of desethylamiodarone is 0.05-5.00μg / mL, and the linear range of amiodarone is 0.05-5.00μg / mL. Under the test conditions, the correlation coefficient r is greater than 0.999, meeting the relevant requirements.

[0160] Table 8 Desethylamiodarone linear data

[0161]

[0162] Table 9. Amiodarone linearity data

[0163]

[0164] Effect Example 5

[0165] Precision test plan and data:

[0166] (1) Validation method: For intra-batch precision, at least one analytical batch of 3 concentrations is required, including low concentration level, medium concentration level, and high concentration level, with at least 5 samples for each concentration. For inter-batch precision, at least 3 analytical batches (at least 3 days) are required, including low concentration level, medium concentration level, and high concentration level, with at least 5 samples for each concentration, and the coefficient of variation is used to evaluate inter-batch precision.

[0167]

[0168] CV: coefficient of variation of repeatability;

[0169] The average of 5 measurements;

[0170] S: standard deviation of 5 measurements.

[0171] (2) Acceptance criteria: The intra-batch coefficient of variation shall generally not exceed 15%; the inter-batch coefficient of variation shall generally not exceed 15%.

[0172] (3) Experimental results:

[0173] Intra-batch precision of deethylamiodarone: After three days of testing, the highest coefficients of variation of deethylamiodarone at different concentrations were 4.65% at the low concentration level, 2.03% at the medium concentration level, and 3.63% at the high concentration level, which met the requirements.

[0174] Intra-batch precision of amiodarone: After three days of testing, the highest coefficients of variation of amiodarone at different concentrations were 3.63% at the low concentration level, 1.05% at the medium concentration level, and 3.53% at the high concentration level, which met the requirements.

[0175] The inter-batch precision of desethylamiodarone: the maximum coefficient of variation at low concentration level was 6.40%, 2.88% at medium concentration level, and 3.35% at high concentration level, which met the requirements.

[0176] The inter-batch precision of amiodarone: the maximum coefficient of variation at the low concentration level is 3.87%, the medium concentration level (concentration near the middle of the standard curve) is 1.13%, and the high concentration level is 2.39%, which meets the requirements.

[0177] Table 10. Intra-batch and inter-batch precision data of desethylamiodarone

[0178]

[0179]

[0180] Table 11 Intra-batch and inter-batch precision data of amiodarone

[0181]

[0182]

[0183] Effect Example 6

[0184] Matrix effect evaluation:

[0185] (1) Experimental procedure: Use at least 6 batches of blank matrix from different donors. For each batch of matrix, the matrix factor of each analyte should be calculated by calculating the ratio of the peak area in the presence of matrix (measured by adding analyte after extraction of blank matrix) to the corresponding peak area without matrix (pure solution of analyte). Further, the matrix factor normalized by internal standard should be calculated based on the matrix factor of the analyte.

[0186] (2) Acceptance criteria:

[0187] The coefficient of variation of the internal standard normalized matrix factor calculated from 6 batches of matrix should all be less than 15%.

[0188] (3) Experimental results:

[0189] The coefficient of variation of the matrix factor of deethylamiodarone at low concentration point was 6.12%, and the coefficient of variation of the matrix factor of deethylamiodarone at high concentration point was 4.37%, which met the requirements.

[0190] The coefficient of variation of the matrix factor of amiodarone at low concentration point was 2.95%, and the coefficient of variation of the matrix factor of amiodarone at high concentration point was 5.66%, which met the requirements.

[0191] Table 12. Matrix Effects of Desethylamiodarone

[0192]

[0193] Table 13. Amiodarone matrix effects

[0194]

[0195] Effect Example 7

[0196] Residue studies:

[0197] (1) Experimental process: Carryover may affect the accuracy of sample determination, so carryover should be investigated and minimized during method development. Evaluate whether the carryover meets the requirements of performance evaluation. Use one analytical batch for validation. After injecting the high concentration sample (ULOQ), inject the blank sample and repeat 3 times.

[0198] (2) Acceptance criteria: The residue in the blank sample after the high concentration sample should not exceed 20% of the lower limit of quantitation.

[0199] (3) Experimental results: After the high concentration sample (ULOQ) was injected, the blank sample was injected three times. No related substances were detected in the blank samples of desethylamiodarone and amiodarone, meeting the residue acceptance criteria.

[0200] Table 14. Desethylamiodarone Residues

[0201]

[0202] Table 15. Amiodarone residues

[0203]

[0204]

[0205] Effect Example 8

[0206] Accuracy:

[0207] (1) Experimental process: This scheme evaluates accuracy by testing desethylamiodarone and amiodarone accuracy samples and calculating their recovery rates. Evaluate whether the accuracy meets the requirements of performance evaluation. Add a certain amount of the working solution of the test substance to the blank human matrix to prepare three concentrations of recovered samples LQC, MQC, and HQC (i.e., quality control samples). The concentration selection includes low concentration level, medium concentration level, and high concentration level. Take three analysis batches, repeat the measurement of 5 samples at each concentration, and test for three consecutive days.

[0208] (2) Acceptance criteria: Recovery rate: 85%-115%.

[0209] (3) Experimental results:

[0210] Accuracy of deethylamiodarone: After three days of testing, the accuracy of deethylamiodarone at different concentrations was 92.43%-102.70% at the low concentration level, 95.70%-100.51% at the medium concentration level, and 96.56%-102.10% at the high concentration level, which met the requirements.

[0211] Accuracy of amiodarone: After three days of testing, the accuracy of amiodarone at different concentrations was 91.23%-97.13% at the low concentration level, 94.66%-96.05% at the medium concentration level, and 95.48%-98.58% at the high concentration level, which met the requirements.

[0212] Table 16. Desethylamiodarone accuracy evaluation table

[0213]

[0214] Table 17. Amiodarone accuracy evaluation table

[0215]

[0216] Effect Example 9

[0217] Specificity:

[0218] (1) Experimental procedure: Six blank subject matrices were used for evaluation. One portion of each was prepared to the lower limit of quantitation (LLOQ) concentration level. Six blank plasmas and six human matrices with the lowest LLOQ concentration of the target analytes were obtained and validated using one analytical batch.

[0219] (2) Acceptance criteria: The compound peaks in the blank samples of the six subjects were all lower than 20% of the peaks in the LLQC samples, and the peak areas of the internal standards in the blank samples were all lower than 5% of the peaks in the internal standards in the LLOQ samples, indicating that the method can distinguish the target analytes from the endogenous components of the matrix or other components in the sample.

[0220] (3) Experimental results: The peak values ​​of desethylamiodarone and amiodarone compounds were not detected in the blank samples of the six subjects, which were lower than 20% of the peak value of the LLQC samples and met the acceptance criteria.

[0221] Table 18. Specificity results table

[0222] project Desethylamiodarone μg / mL Amiodarone μg / mL LLOQ1 0.751 0.930 LLOQ2 0.847 0.799 LLOQ3 0.902 0.827 LLOQ4 0.862 0.856 LLOQ5 0.898 0.837 LLOQ6 0.746 0.816 Human plasma 1 NA NA Human plasma 2 NA NA Human plasma 3 NA NA Human plasma 4 NA NA Human plasma 5 NA NA Human plasma 6 NA NA

[0223] 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 replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting amiodarone and desethylamiodarone in plasma, characterized in that: The method comprises using high performance liquid chromatography to detect the pre-treated sample, inserting the detection value into the standard curve, and calculating the content of amiodarone and desethylamiodarone in the plasma sample; the standard curve is the corresponding relationship between the content of the standard substance in the standard curve working solution and the detection value of the standard substance; The pretreatment includes extracting the sample to be tested using an extractant, wherein the extractant is a mixed system of methanol and acetonitrile, containing 5-15% methanol and 85-95% acetonitrile by volume; The mobile phase for the high performance liquid chromatography detection includes mobile phase A and mobile phase B; The mobile phase A is an aqueous phase, and the mobile phase A contains 5-10 mM potassium dihydrogen phosphate and 0.1-0.5% v / v concentrated phosphoric acid, wherein the concentrated phosphoric acid is HPLC grade phosphoric acid with a concentration of ≥85%; the mobile phase B is acetonitrile; The high performance liquid chromatography detection uses a Waters XBridge C18 chromatographic column; The elution procedure of the high performance liquid chromatography detection is: 0.00 min, mobile phase A 50%, mobile phase B 50%; 10.00 min, mobile phase A 40%, mobile phase B 60%; 12.00 min, mobile phase A 15%, mobile phase B 85%; 14.00 min, mobile phase A 15%, mobile phase B 85%; 14.10 min, mobile phase A 50%, mobile phase B 50%; 15.00 min, mobile phase A 50%, mobile phase B 50%.

2. The detection method according to claim 1, characterized in that: The extractant contains 10% methanol and 90% acetonitrile by volume.

3. The detection method according to claim 1, characterized in that: The volume ratio of the sample to be tested to the extractant is 1:3-1:

5.

4. The detection method according to claim 3, characterized in that: The volume ratio of the sample to be tested to the extractant is 1:

4.

5. The detection method according to claim 1, characterized in that: The pretreatment further comprises shaking the mixed system of the extractant and the sample to be tested.

6. The detection method according to claim 5, characterized in that: The shaking is carried out in a constant temperature oscillator for 3 to 10 minutes.

7. The detection method according to claim 6, characterized in that: The shaking is carried out in a constant temperature oscillator for 5 minutes.

8. The detection method according to claim 5, characterized in that: The mixed system of the extractant and the sample to be tested is centrifuged after shaking, and the supernatant is taken into a new reaction container. After centrifugation again, high performance liquid chromatography is performed.

9. The detection method according to claim 8, characterized in that The centrifugal conditions for centrifuging the mixed system of the extractant and the sample to be tested after shaking are 0-10° C. and 3000-5000 rpm for 5-15 minutes.

10. The detection method according to claim 9, characterized in that The centrifugation conditions for centrifuging the mixed system of the extractant and the sample to be tested after shaking are 4° C. and 4000 rpm for 10 minutes.

11. The detection method according to claim 8, characterized in that The centrifugation conditions before HPLC detection were 0-10°C, 3000-5000 rpm, and 3-10 min.

12. The detection method according to claim 11, characterized in that The centrifugation conditions before HPLC detection were 4°C, 4000 rpm for 5 min.

13. The detection method according to claim 1, characterized in that: The pretreatment steps were performed in 96-well plates.

14. The detection method according to claim 1, characterized in that: The mobile phase A contains 5 mM potassium dihydrogen phosphate and 0.2% v / v concentrated phosphoric acid.

15. The detection method according to claim 1, characterized in that: The sources of the plasma include mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys or humans.

16. The detection method according to any one of claims 1 to 15, characterized in that: The standard curve is subjected to linear regression with the chromatographic peak area of ​​the standard in the standard working solution as the dependent variable and the concentration of the standard in the standard working solution as the independent variable.

17. The detection method according to claim 16, characterized in that The concentration ranges of amiodarone and desethylamiodarone in the standard curve working solution are independently 0.05-5.00 μg / mL.

18. The detection method according to claim 16, characterized in that: The matrix of the standard curve working solution is animal plasma.

19. The detection method according to claim 18, characterized in that: The matrix of the standard curve working solution is bovine plasma.

20. The detection method according to claim 19, characterized in that: The matrix of the standard curve working solution is newborn calf plasma.

21. The detection method according to any one of claims 1 to 15, characterized in that: The detection method also includes detecting quality control products.

22. The detection method according to claim 21, characterized in that: The quality control products include at least two of low-value quality control products, medium-value quality control products and high-value quality control products; The concentrations of amiodarone and desethylamiodarone in the low-value quality control product are 0.80 μg / mL, respectively; the concentrations of amiodarone and desethylamiodarone in the mid-value quality control product are 2.50 μg / mL, respectively; and the concentrations of amiodarone and desethylamiodarone in the high-value quality control product are 3.80 μg / mL, respectively.

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