RU2749566C1 - Method for determining amiodarone and its main metabolite desethylamiodarone in human blood serum - Google Patents

Abstract

FIELD: medicine; pharmacology.

SUBSTANCE: invention relates to medicine, namely to pharmacology, it can be used for sample preparation in the determination of amiodarone and its metabolite desethylamiodarone by high-performance liquid chromatography with mass spectrometric detection (HPLC-MS/MS) in human blood serum. In the pre-prepared calibration and analyzed samples, which are human blood serum, an effective amount of the internal standard is added, in form of a solution of cinacalcet at a concentration of 500 ng/ml. Next, an effective amount of the protein precipitator, which is undiluted acetonitrile, is added. It is then mixed to a homogeneous state, followed by centrifugation at a centrifugal acceleration of 15294 g until it is separated into solid and liquid phases, with further selection of the effective amount of the supernatant. Chromatographic separation of the sample components is performed in the gradient elution mode, followed by the detection of a signal with positive ionization for amiodarone, its metabolite desethylamiodarone, and the internal standard of cinacalcet. This method is used to determine amiodarone and its metabolite desethylamiodarone in human blood serum.

EFFECT: method makes it possible to perform sample preparation for the determination of amiodarone and desethylamiodarone in the combined presence in human blood serum by using a unified sample preparation scheme for all analyzed substances using undiluted acetonitrile, centrifuging of the sample once, using cinacalcet as a single internal standard for all analyzed substances, using gradient elution.

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Description

The present invention relates to pharmacy, pharmacology and clinical pharmacology, the study of biological materials, in particular to methods for the determination of amiodarone, its main metabolite desethylamiodarone in human serum by high performance liquid chromatography with mass spectrometric detection (HPLC-MS / MS) in therapeutic drug monitoring and pharmacokinetic studies.

State of the art

Amiodarone is the most effective antiarrhythmic drug available. In clinical practice, replacing the original amiodarone drug with reproduced ones often leads to a change in the concentration of the active substance and / or its active metabolite in the blood and serious clinical consequences, in the form of arrhythmogenic manifestations. Determination of the concentration of amiodarone and its active metabolite in the blood is important to ensure the effectiveness and safety of the use of generic amiodarone preparations. At the same time, there is no simple effective, sensitive, selective, accurate and reproducible method of sample preparation for the determination of amiodarone and its active metabolite desethylamiodarone in human serum.

From the prior art, a method is known for the determination of amiodarone and desethylamiodarone in human serum by high performance liquid chromatography with chemiluminescence detection [1]. In this work, acetonitrile is added to blood serum samples (1000 μl) as a protein precipitant (2000 μl), then shaken and centrifuged, after which the supernatant is taken and dried in a stream of nitrogen, the dry residue is dissolved in 250 μl of methanol. Chromatographic separation is carried out using a Mediterranea C18 column, 4.6 mm × 150 mm, sorbent particle diameter 5 μm (Teknokroma), elution is carried out in an isocratic mode with a mobile phase consisting of a mixture of methanol: 0.017 mol / L ammonium sulfate solution pH 6.8 ( 92: 8, v / v). For detection, an original setup is used that allows post-column derivatization of the sample and measurement of chemiluminescence with the participation of ruthenium complex compounds.

The disadvantages of this method are:

- use of specific equipment for chemiluminescence detection;

- a rare and expensive reagent for post-column derivatization is used (a complex compound of ruthenium);

- the use of isocratic elution mode lengthens the analysis time of samples up to 12 minutes;

- insufficient lower limit of quantitative determination (NPQD) of deethylamiodarone (0.5 μg / ml).

In another known method, the determination of amiodarone in human blood plasma is carried out by high performance liquid chromatography with mass spectrometric detection (HPLC MS / MS) [3]. Sample preparation is carried out by liquid-liquid extraction (LLE) with diethyl ether, followed by evaporation of the selected organic layer and reconstitution of the dry residue in methanol. Chromatographic separation is carried out in isocritical elution mode on a Hypersil GOLD column, 4.5 mm × 50 mm, sorbent particle diameter 1.9 μm with a mass spectrometric detector. The mobile phase consisted of two solutions: 10 mM ammonium acetate (solution A) and acetonitrile - 10 mM ammonium acetate (90:10) (solution B), taken in a ratio of 10%: 90%, respectively.

The disadvantages of this method include:

- the impossibility of determining desethylamiodarone together with amiodarone;

- use of diethyl ether in sample preparation;

- isocratic elution, increasing the analysis time up to 8 minutes;

- the need for equipment that ensures operation at ultra-high pressure due to the use of a sorbent with a particle diameter of 1.8 microns.

As a prototype, a well-known method for the determination of amiodarone and its metabolite in human plasma and serum [2] is proposed. A scheme for sample preparation by the method of solid-phase extraction (SPE) is proposed. Chromatographic separation is carried out in a gradient elution mode with mobile phase A (solution of 0.1% formic acid and 2 mmol / L ammonium acetate in water) / B (solution of 0.1% formic acid and 2 mmol / L ammonium acetate in methanol) on column Phenomenex Hydro-RP 2 × 20 mm with a sorbent particle diameter of 2.5 μm. Amiodarone and its metabolite were determined using a mass spectrometric detector in the multiple reaction monitoring (MRM) mode.

The disadvantages of the prototype include:

- the use of an expensive and time-consuming method of sample preparation - solid-phase extraction;

- an excessive number of calibration samples and an unreasonably wide analytical range of up to 40 μg / ml, while the concentrations of amiodarone and desethylamiodarone are usually determined in blood plasma by an order of magnitude lower;

- the use of expensive deuterated analogs of amiodarone and deethylamiodarone as internal standards;

- insufficient chromatographic separation of analytes.

Thus, there is a need to develop a simple, effective, sensitive, selective, accurate and reproducible method for the determination of amiodarone and its active metabolite deethylamiodarone in blood serum by high performance liquid chromatography with mass spectrometric detection.

The essence of the invention

The technical objective of the claimed invention is to develop a simple method for sample preparation without the use of LLE, SPE and specific equipment, for the joint determination of amiodarone and deethylamiodarone in the range corresponding to the average therapeutic concentrations, to achieve high efficiency, accuracy and reproducibility in the determination of amiodarone, its active metabolite deethylamiodarone, the use of a single internal standard, as well as the expansion of the range of analytical methods suitable for the quantitative determination of the substances under study.

The technical solution of the proposed invention is that the claimed method is characterized by efficiency, selectivity, sensitivity, accuracy, with a wide linear range, allowing to determine the minimum amount of analytes in the joint presence in human serum.

In addition, the present technical solution is effective for use in the field of clinical pharmacology for therapeutic drug monitoring of amiodarone and deethylamiodarone by examining the patient's blood serum.

The technical result of the proposed solution is achieved due to the fact that:

- use a unified sample preparation scheme for all analyzed substances using a protein precipitant, which is undiluted acetonitrile;

- cinacalcet is used as a single internal standard for all analytes when determining them in blood serum, which has physicochemical properties similar to the analyzed substances and, as a consequence, retention on the sorbent;

using gradient elution adapted to the determination of highly lipophilic compounds in a short analysis time without compromising the efficiency of chromatographic separation;

- chromatographic separation is carried out on a sorbent with a particle diameter of 5 microns in order to reduce the working pressure, which makes it possible to expand the range of analytical methods suitable for the quantitative determination of amiodarone and deethylamiodarone in blood serum;

- during sample preparation, centrifuge the sample once;

- detected with positive ionization of the analyzed components.

Description of the invention.

In the claimed method for the determination of amiodarone and desethylamiodarone (Fig. 1-2) with their joint presence in human blood serum is carried out by the method of high performance liquid chromatography (hereinafter - HPLC MS / MS). To determine the concentrations, it is required to prepare a set of standard solutions of the analytes, that is, solutions with a precisely known concentration, for further preparation of calibration solutions from them, which are necessary for constructing a calibration curve by which the analyte concentrations of the test samples are determined.

Calibration samples are prepared by adding 5 μl of standard solutions of amiodarone and desethylamiodarone to 195 μl of intact blood serum to obtain concentrations in the range from 30 ng / ml to 3000 ng / ml.

Sample preparation of calibration and analyzed samples of human blood serum is carried out by the method of protein precipitation as follows: to 200 ml of a biological sample placed in centrifuge microtubes of the Eppendorf type with a capacity of 2 ml, add 10 μl of a working solution of an internal standard of cinacalcet with a concentration of 500.00 ng / ml, then add 1000 μl of neat acetonitrile from 99.9% wt. up to 100% wt .; stirred in a vortex mixer for at least 15 s until homogeneous (until complete mixing), then centrifuged until the contents are separated into solid and liquid phases, with a centrifugal acceleration of 15294 g for 10 minutes to 20 minutes, preferably for 15 minutes. Then the supernatant (supernatant), in a volume (amount) of 500 μl, is transferred into chromatographic vials, then placed in an automatic chromatograph sampler.

It was unexpectedly found that for the determination of amiodarone and its metabolite desethylamiodarone in blood serum, it is advisable to use undiluted acetonitrile as a protein precipitant, which allows you to effectively extract the largest amount of analytes of analytes from human serum.

The use of undiluted acetonitrile as a protein precipitant for the determination of the analyzed group of analytes amiodarone and its metabolite deethylamiodarone leads to a more efficient, accurate and reproducible analysis, characterized by the ability to most effectively differentiate the analyzed group of analytes in human serum.

Also, it was unexpectedly found that the use of undiluted acetonitrile, as a protein precipitant, when amiodarone and its metabolite deethylamiodarone and cinacalcet solution are co-present in human serum, as an internal standard, lead to a decrease in sample preparation time, since they allow the use of a single sample preparation for human serum, and thus save time during the analysis.

In addition, it was unexpectedly found that the present technical solution with the claimed technical result is effective for use in the field of clinical pharmacology when conducting therapeutic drug monitoring by examining the patient's blood serum.

Conditions for chromatographic separation and detection The quantitative determination of amiodarone and deethylamiodarone is carried out on a high-performance liquid chromatograph equipped with a gradient pump, a column thermostat, a degasser, a thermostated automatic sampler and a tandem mass spectrometric detector (triple quadrupole). Primary data processing is carried out using appropriate software.

Column (stationary phase): for HPLC MS / MS, a column with a size of 150 × 2.1 mm with a C18 grafted phase from the Zorbax group and a sorbent with a particle diameter of 5 μm was chosen, with a universal guard column C 18.4 × 3.0 mm, which provides operating pressure in the system not more than 17 MPa.

Column oven temperature: 40 ° С.

Mobile phase:

- Eluent A: 0.1% (v / v) formic acid / deionized water solution

- Eluent B: 0.1% (v / v) formic acid / acetonitrile solution

Mobile phase flow rate: 0.75 ml / min.

The gradient mode of elution according to the composition of the mobile phase is presented in Table 1.

Injected sample volume: 1 μL.

Time of chromatogram registration by mass spectrometric detector: 0-3.5 min.

Retention time of cinacalcet: 0.79 ± 0.02 min

Amiodarone retention time: 1.83 ± 0.02 min

Retention time of deethylamiodarone: 1.38 ± 0.02 min

Ionization source parameters: atomizing gas 3 l / min, drying gas 20 l / min, heating unit 400 ° C, desolvation line 250 ° C, capillary voltage + 5 kV.

Signal detection conditions for amiodarone, its main metabolite deethylamiodarone and internal standard, in the form of cinacalcet solution, in the multiple reaction monitoring mode are presented in Table 2.

The obtained calibration curves are linear in the concentration range, for each analyte active substance from 30 ng / ml to 3000 ng / ml in human serum, with correlation coefficients of 0.997 and 0.999 for amiodarone and desethylamiodarone (Fig. 3-4), respectively. The accuracy of the results, which should be understood as the correctness and precision, are calculated in accordance with the requirements of the Guidelines for the examination of medicinal products [4]. The accuracy of measurements (E,%) does not exceed 3.92% for amiodarone and does not exceed 4.15% for desethylamiodarone. The relative standard deviation, which allows assessing the precision (RSD%) of the results, is no more than 5.47% for amiodarone and no more than 5.71% for desethylamiodarone.

The proposed method allows:

1. Refuse to use LLE and SPE during sample preparation and use the method of protein precipitation with acetonitrile.

2. Obtain analytical ranges for each analyte from 30 ng / ml to 3000 ng / ml due to the unification of sample preparation.

3. To increase the efficiency, sensitivity, selectivity, accuracy and reproducibility of the proposed method for the determination of amiodarone and desethylamiodarone in human serum.

4. Use cinacalcetz as a single and readily available internal standard.

5. To expand the range of analytical methods for the determination of amiodarone and deethylamiodarone in human blood serum through chromatographic separation on a sorbent with a particle size of 5 microns, which provides a working pressure in the system of no more than 17 MPa while maintaining the rate of analysis.

Brief description of drawings and other materials (Appendices 1-6).

FIG. 1. Structural formula of amiodarone.

FIG. 2. The structural formula of the active metabolite of amiodarone desethylamiodarone.

FIG. 3. Calibration dependence of the ratio of the amiodarone peak area to the cinacalcet peak area on the ratio of the amiodarone concentration to the cinacalcet concentration in the blood serum.

FIG. 4. Calibration dependence of the ratio of the area of the peak of desethylamiodarone to the area of the peak of cinacalcet on the ratio of the concentration of desethylamiodarone to the concentration of cinacalcet in the blood serum.

FIG. 5. Chromatogram of a patient's blood serum sample containing 601.50 ng / ml of amiodarone, 350.25 ng / ml of the active metabolite of deethylamiodarone, and a working standard solution of cinacalcet at a concentration of 25.0 ng / ml.

FIG. 6. Chromatogram of a sample of intact human blood serum not containing amiodarone, deethylamiodarone and cinacalcet.

The possibility of implementing the claimed invention is disclosed in the following examples.

Example 1. Determination of the content of amiodarone and desethylamiodarone in human serum.

The patient included in the study was on continuous therapy with amiodarone at a therapeutic effective dosage of 600 mg per day. Blood sampling was performed immediately before taking amiodarone to determine the value of the minimum steady-state concentration of amiodarone and desethylamiodarone in human serum.

Analysis.

1. Obtaining human blood serum. Blood sampling is performed in vacuum tubes. The blood taken from the patient is kept at room temperature for 30 minutes. After 30 minutes, the tubes are centrifuged for at least 15 minutes at 3000 rpm. Then an aliquot of the obtained blood serum is transferred into 2 ml Eppendorf Save Lock tubes for further sample preparation.

2. Calibration samples are prepared by adding 5 μl of standard solutions of amiodarone and desethylamiodarone to intact blood serum to obtain concentrations in the analyzed range from 30 ng / ml to 3000 ng / ml.

3. Sample preparation of calibration and analyzed blood serum samples is carried out by the method of protein precipitation as follows: to 200 μl of a biological sample placed in centrifuge microtubes of the Eppendorf type with a capacity of 2 ml, add 10 μl of a working solution of an internal standard of cinacalcet with a concentration of 500 ng / ml, then add 1000 μl of undiluted acetonitrile, mix in a vortex mixer until homogeneous (complete mixing) for at least 15 seconds, then centrifuge at 15294 g centrifugal acceleration for 10 minutes to 20 minutes, most preferably 15 minutes, until separation contents into solid and liquid phases. Then, 500 μL is transferred to the chromatographic vials and the supernatant is placed in the autosampler of the chromatograph.

4. The quantitative determination of amiodarone and desethylamiodarone is carried out on a high-performance liquid chromatograph equipped with a gradient pump, a column thermostat, a degasser, a thermostated automatic sampler and a tandem mass spectrometric detector (triple quadrupole). Primary data processing is carried out using appropriate software.

Column (stationary phase): for HPLC MS / MS, a column with a size of 150 × 2.1 mm with a C18 grafted phase from the Zorbax group and a sorbent with a particle diameter of 5 μm was selected, with a universal guard column C18, 4 × 3.0 mm, which provides operating pressure in the system, not exceeding 17 MPa.

Mobile phase flow rate: 0.75 ml / min.

Injected sample volume: 1 μL.

Time of chromatogram registration by mass spectrometric detector: 0-3.5 min.

Retention time of cinacalcet: 0.79 ± 0.02 min

Amiodarone retention time: 1.83 ± 0.02 min

Retention time of deethylamiodarone: 1.38 ± 0.02 min

Ionization source parameters: atomizing gas 3 l / min, drying gas 20 l / min, heating unit 400 ° C, desolvation line 250 ° C, capillary voltage + 5 kV.

The conditions for the detection of amiodarone, its main metabolite deethylamiodarone and cinacalcet taken as an internal standard in the multiple reaction monitoring mode are presented in Table 3.

Amiodarone 601.50 ng / ml and desethylamiodarone 350.25 ng / ml were detected (Fig. 5). From the moment of blood sampling to the provision of results, no more than 1 hour is spent.

This example demonstrates the possibility of determining amiodarone and its active metabolite desethylamiodarone, in the joint presence in human serum by the claimed method.

Example 2. Determination of the content of amiodarone and desethylamiodarone in intact blood serum.

Determination of the content of amiodarone and desethylamiodarone in intact human blood serum, which does not reliably contain these drugs, is carried out by the method described in Example 1.

On the chromatograms of the obtained samples there are no peaks (Fig. 6) corresponding to the retention time of amiodarone and desethylamiodarone.

Thus, this example demonstrates the effectiveness, selectivity of the claimed method and the absence of the risk of providing false positive results for the quantitative determination of amiodarone and desethylamiodarone in serum.

Example 3. Determination of the accuracy (correctness and precision) of the results of the determination of amiodarone and its metabolite desethylamiodarone in human serum. Accuracy was evaluated in the range from 30 ng / mg to 3000 ng / ml by analyzing quality control samples containing amiodarone and desethylamiodarone at levels of 30 ng / ml, 90 ng / ml, 900 ng / ml, 2100 ng / ml, 3000 ng / ml. The analysis was carried out within 3 cycles, which included 5 samples for each concentration level. Accuracy and precision were determined within the analytical run and between runs (Tables 3-5). The accuracy of measurements (E,%), and the relative standard deviation (RSD,%), which makes it possible to assess the precision of the results, corresponded to the norms.

This example reveals the possibility of determining amiodarone and desethylamiodarone in human serum with acceptable accuracy (accuracy and precision) at concentrations from 30 ng / ml to 3000 ng / ml, which corresponds to the stated linear analytical ranges.

The examples presented are not intended to limit the scope of the present invention and are for illustrative purposes only. Industrial applicability

All these examples confirm the efficiency, sensitivity, selectivity, accuracy and reproducibility of the claimed method.

Thus, the technical problem posed, namely, the development of an effective, sensitive, selective, accurate and reproducible method for the determination of amiodarone and its metabolite in blood serum, has been achieved, which is confirmed by the above examples.

The use of the claimed method in clinical pharmacology for conducting therapeutic drug monitoring by examining the patient's blood serum is effective.

Bibliography

1. Perez-Ruiz T., Martinez-Lozano C, Garcia-Martmez M. D. Simultaneous determination of amiodarone and its metabolite desethylamiodarone by high-performance liquid chromatography with chemiluminescent detection // Analytica chimica acta. - 2008. - T. 623. -No. l. -C. 89-95.

2. Abaimov DA et al. Open, randomized, crossover study of comparative pharmacokinetics and bioequivalence of drugs Santodarone and Cordaron // Pharmacokinetics and pharmacodynamics. - 2012. - No. 2.

3. Kuhn J., Gotting C, Kleesiek K. Simultaneous measurement of amiodarone and desethylamiodarone in human plasma and serum by stable isotope dilution liquid chromatography-tandem mass spectrometry assay // Journal of pharmaceutical and biomedical analysis.-2010.-T. 51.-no. 1. - C. 210-216.

4. Guidelines for the examination of medicines. Ed. prof. A.N. Mironov. Volume I. M .: Grif and K, 2013.S. 201-207.

5. Guidelines for the validation of analytical procedures for drug testing. Decision of the Board of the Eurasian Economic Commission dated July 17, 2018 No. 113.

Claims (9)

1. A method of sample preparation for the determination of amiodarone and its metabolite desethylamiodarone by high-performance liquid chromatography with mass spectrometric detection (HPLC-MS / MS) in human blood serum, characterized in that the previously prepared calibration and analyzed samples, which are human blood serum, are added an effective amount of an internal standard, in the form of a cinacalcet solution at a concentration of 500 ng / ml; then add an effective amount of a protein precipitant, which is undiluted acetonitrile, then mix until homogeneous, followed by centrifugation at centrifugal acceleration of 15294 g until separation into solid and liquid phases, with further selection of an effective amount of supernatant; followed by chromatographic separation of the sample components in the gradient elution mode, followed by detection of a signal with positive ionization for amiodarone, its metabolite desethylamiodarone, and cinacalcet internal standard. 2. The method according to claim 1, further characterized in that the analytical range for amiodarone and its metabolite desethylamiodarone is from 30 ng / ml to 3000 ng / ml. 3. The method according to claim 1, further characterized in that the proteins are precipitated with undiluted acetonitrile in a ratio of 5: 1 to serum. 4. The method according to claim 1, further characterized in that the previously prepared blood serum contains an effective amount of amiodarone and its metabolite desethylamiodarone. 5. The method according to claim 1, further characterized in that a sorbent from the Zorbax group with a grafted C 18 phase with a particle size of 5 μm is used. 6. The method according to claim 1, further characterized in that the sample is chromatographed at an operating pressure of not more than 17 MPa while maintaining the analysis rate. 7. The method according to claim 1, further characterized in that it is centrifuged for 15 minutes. 8. The method according to claim 1, further characterized in that the detected signal is obtained using a mass spectrometric detector in a multiple reaction monitoring mode. 9. Application of the method according to claim 1 for the determination of amiodarone and its metabolite desethylamiodarone in human serum.

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