DEVELOPMENT AND VALIDATION OF METHODS OF QUANTITATIVE DETERMINATION OF THE NEW ANTIDIABETIC DRUG IN THE BLOOD PLASMA OF RATS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH MASS SPECTROMETRIC DETECTION

We developed a method of quantification of the new antidiabetic drug 3-(1h-benzimidazole-2-yl)-1,2,2-trimethyl-cyclopentane-carbonic acid (c7070) in the blood plasma of rats by high-performance liquid chromatography with mass-spectrometric detection. The analytical range of the method was 0.02-3876.0 μg in 1 ml of blood plasma. The research was partially supported by the grant of the President of the Russian Federation No MD-4711.2015.7 and МК-6135.2016.4.

According to the State Register as of January 1, 2012, more than 3 million 540 thousand patients with diabetes mellitus (DM), 90% of themwith type 2 diabetes were registered in the Russian Federation.However, results of control and epidemiological studies of the Endocrinology Research Center, conducted in 2005-2010, as well as WHO's data for 2010 indicate that the actual number of diabetic patients exceeds the registered one by more than 3 times [1].
Considering this, the development of new and effective drugs for the treatment of DM is one of the urgent problems of modern medicine.As it is known, the development itself is impossible without studying the pharmacokinetics, which accurate assessment requires a sensitive and highly-selective method.Currently, the reference method for studying the pharmacokinetics is a high-performance liquid chromatography with mass spectrometry [1,3,4].Subject to the above, the objective of the study is to develop and validate a method of quantification of C7070 by high-performance liquid chromatography with mass-spectrometry (HPLC-MS/MS).
Experimental part.
Identification of C7070 in rat plasma was carried out with a liquid chromatograph UltiMate 3000 LC (Thermo Fisher Scientific, USA) equipped with a thermostated automatic dispenser, vacuum degasser, gradient pump, and column thermostat.Detection of the analyte was carried out with a mass-spectrometer Velos Pro (Thermo Scientific, USA) under ionization in the heated electrospray (H-ESI-II).

Sample preparation.
Preparation of C7070 solutions involved several stages.At the first stage, stock solution of C7070 was prepared in methanol at a concentration of 0.2%.At the second stage, C7070 solutions with methanol were prepared by a series of dilutions of stock solution to be further added to standard solutions and control solutions at a concentration of 0.00002%, 0.002% and 0.011%.
Fabomotizole solutions (internal standard) in methanol for introducing into standard and test solutions were prepared at the same concentration level -0.1% solution.

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The solutions for the construction of calibration curve, in accordance with modern requirements [5, 6, 7], were prepared at seven concentration levels (0.02 μg, 0.2 μg, 2.00 μg, 1.94 μg, 193.80 μg, 1938.00 μg and 3876.00 μg in 1 ml plasma).For this purpose, 100 μl of plasma were placed in 1.5 ml Eppendorf tubes, then added aliquots of stock solutions, 100 μl of internal standard solution and 100 μl of acetonitrile.Then the analyte was extracted in an ultrasonic bath for three minutes.The samples were then frozen at -70ºC.After defrosting, samples were centrifuged at 13,000 rev/min and a temperature of 4°C for 25 minutes.The supernatant was decanted and analyzed.
Test solutions were prepared similarly to the solutions for the calibration curve based on four levels of concentration in five repetitions: 0.02 μg (lower limit of quantification -LLOQ), 0.20 μg (lower quality control -LQC), 19.38 μg (mean control quality -MQC) and 1938.00 μg (upper quality control -UQC) in 1 ml of plasma.
Solutions for the determination of matrix effect were prepared similarly to test solutions, but with replacing plasma with water [8].
Chromatographic separation was performed on a 150×3.0mm column filled with converted-phase sorbent Zorbax Eclipce XDB C18 with particle size of 3.0 µm and with a 12.5×3.0mm guard column Zorbax Eclipce XDB C18 with a particle size of 5.0 µm at a temperature of 40°C.The chromatographic analysis was performed with the use of UltiMate 3000 LC system coupled with a mass spectrometric detector under the following chromatographic conditions:

Results and discussion:
We developed a highly-selective method with a wide range of linearity (0.02 to 3876.00 μg in 1 ml of plasma) for carrying out qualitative pre-clinical studies.The width of the range is due to the nature of the operations (dose adjustment and evaluation of the response linearity).Use of an internal standard ensured minimization of sample preparation errors and improved the accuracy of the determination.Since the pre-clinical studies in animals involve a large number of control points that need to be analyzed strictly in a particular analytical cycle, the method used a simple

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RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY and express sample preparation -protein precipitation with acetonitrile, followed by ultrasound extraction [9,10,11].To ensure a satisfactory sample purity and stable detection, the method of chromatographic separation on an analytical column filled with sorbent Zorbax Eclipce XDB C18 was used.
Chromatographic system suitability parameters.
To determine the chromatographic system suitability, the solutions for calibration curve were analyzed, the solution at a concentration of 19.38 μg in 1.0 ml of plasma was chromatographed six times, the equation of the calibration curve was calculated, and the obtained amounts were subject to reverse recalculation.The chromatographic system was considered suitable when meeting the following generally accepted criteria [9]: -errors in the reverse recalculation should not exceed: for a point equal to the LLOQ not more than 20%; for all other points -not more than 15%; -relative standard deviation of the ratio of C7070 peaks area to the internal standard peak areas calculated on 6 consecutive chromatograms of solution at a concentration of 19.38 μg in 1.0 ml of plasma should not exceed 7%; -C7070 peak asymmetry factor at a concentration of 19.38 μg in 1.0 ml of plasma and the internal standard should not exceed 2.2%; -signal-to-noise ratio for the C7070 peak in the chromatogram of solution equal to the LLOQ must be at least 10:1.

Method validation.
Validation of the method for quantification of С7070 in rat plasma was conducted in accordance with the Guidelines for the examination of medicines [5], as well as Guidelines for the validation of bioanalytical FDA [8] and EMA practices [7] under the following characteristics: selectivity (specificity) linearity, accuracy and precision (within a series and between series), the limit of quantification, the sample stability, the matrix effect and the transferable residue.
To determine the selectivity (specificity) a pure rat plasma sample was analyzed, as well as pure plasma samples with the addition of the standard solutions C7070 and internal standard in the expected range of concentrations.
The chromatograms of pure plasma samples showed no peaks with retention time corresponding to retention time of C7070 and the internal standard (Fig. 1 (A-B)).Transferable residue.To determine the presence of transferable residue the pure sample of rat plasma was analyzed after administration of maximum concentration solution (solution for calibration curve, level 7).
The chromatograms of pure plasma samples after administration of maximum concentration solution showed no peaks with retention time corresponding to retention time of C7070 and the internal standard.This was achieved by satisfactory flushing of the input unit (syringe and needle were flushed with a 3 ml solution of water, acetonitrile, methanol, isopropanol and formic acid in a volumetric ratio of 24:25:25:25:1 prior to each administration).

Matrix Effect
The said indicator was studied in six samples of biological matrix from different donors.The values of the coefficient of variation (CV) of the normalized matrix factors for six different rat matrices on the lower and upper concentration levels did not exceed 15%.The calculation of the normalized matrix factor is presented in Table 1.None of the results exceeds allowable limits.

Limit of Quantitation (LOQ):
To find LOQ, a series of dilutions was prepared (from 1 to 25 ng in 1 ml of plasma).Based on the obtained results, the concentration with the signal/noise ratio for C7070 peak 10 and a coefficient of variation (CV) between six parallel definitions of not more than 10% was selected.LOQ was 20 ng in 1 ml of plasma.

Linearity
According to the obtained values, the calibration curve was constructed, shown in Fig. 2. To assess the acceptability of the obtained values the reverse calculation of C7070 amounts from those entered by the equation of the calibration curve was performed.
None of the values exceeded the allowable limits (for LOQ not more than 20%; for all other points -not more than 15%).Thus, the linear range of the method was 0.020 -3876.0μg in 1 ml of plasma.

Accuracy and precision (within a series and between series).
Accuracy and precision of the method was evaluated on rat plasma samples with the addition of known quantities of C7070 at four concentration levels (LLOQ, LQC, MQC and UQC), which were prepared separately from the solutions prepared to confirm the linearity of the method.Each level was prepared five-fold.The accuracy was expressed: within the series, as a percentage of a nominal content of C7070 (Recovery) and between the series in the form of CV of the obtained values for each level on different days, in percentage.The precision was expressed with CV between parallel determinations of each concentration level within the series, in percentage.The results are presented in Tables 2-3.Representative chromatograms of both C7070 and the internal standard (fabomotizole) at a MQC concentration in rat plasma (19.38 μg in 1 ml of rat plasma) are shown in Fig. 3. Thus, the developed method of quantification of C7070 in rat plasma by highperformance liquid chromatography with mass spectrometry is easy to implement, meets the requirements of the validation characteristics, and ensures reliable and accurate determination of C7070 in rat plasma in the concentration range of 0.02 μg/ml to 3876.00 μg/ml.
chromatogram of the solution for calibration curve.

Figure 1 .
Figure 1.Representative chromatograms in determining the selectivity.

Figure 2 .
Figure 2. The linear dependence of peak area С7070 on its concentration normalized to the internal standard.
Buzov A.A., Kulikov A.L., Avtina T.V., Pokrovskii M.V., Osipova O.A. Development and validation of methods of quantitative determination of the new antidiabetic drug in the blood plasma of rats by high performance liquid chromatography with mass spectrometric detection.Research result: pharmacology and clinical pharmacology.2016.Vol. 2, №1 (2): 52-57.56 RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY

Figure 3 .
Figure 3. Chromatography and mass spectra of both С7070 and internal standard (fabomotizol) in rat plasma.
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