Method for detection and quantification of gadodiamide (gd-dtpa-bma) in pharmaceutical and biological samples and use

Abstract

Method for detection and quantification of gadodiamide (gd-dtpa-bma) in pharmaceutical and biological samples and use. The present invention describes an analytical method, by hydrophilic interaction liquid chromatography (HILIC) using a diode array detector (DAD) in a zwitterionic stationary phase, capable of detecting and quantifying gadodiamide (gd-dtpa-bma) in pharmaceutical and biological samples. This method can be used to detect and quantify gadodiamide (gd-dtpa-bma) in pharmaceutical and biological samples, such as liposomes and blood serum.

Description

1 / 25 “METHOD FOR DETECTION AND QUANTIFICATION OF GADODIAMIDE (Gd-DTPA-BMA) IN PHARMACEUTICAL AND BIOLOGICAL SAMPLES AND USE”

[001] The present invention describes an analytical method, by hydrophilic interaction liquid chromatography (HILIC) using a diode array detector (DAD) in a zwitterionic stationary phase, capable of detecting and quantifying gadodiamide (Gd-DTPA-BMA) in pharmaceutical and biological samples. This method can be used to detect and quantify gadodiamide (Gd-DTPA-BMA) in pharmaceutical and biological samples, such as liposomes and blood serum.

[002] Gd-DTPA-BMA is one of the most widely used contrast agents in magnetic resonance imaging (MRI) due to its low chemotoxicity. (MAIA, ALC et al. (2015). “Development and validation of high performance liquid chromatographic and derivative spectrophotometric methods for determination of gadodiamide in liposomal formulations”. Anal Methods, 7: 8315-8325). Several studies report the encapsulation of GdDTPA-BMA and other gadolinium (Gd) complexes in liposomes for diagnostic purposes, and to monitor in real time, through imaging, the biodistribution of drugs used in cancer treatment. The antitumor activity of this complex in liposomal form is also being investigated, since Gd-DTPA-BMA induces apoptosis of neoplastic cells through caspase-3 activation (MAIA, ALC et al., 2017. “Liposomes containing gadodiamide: preparation, physicochemical characterization, and in vitro cytotoxic evaluation”. Curr Drug Deliv, 14 (4): 566-574).

[003] The quantification of Gd-DTPA-BMA in environmental and biological samples has mostly been carried out using expensive techniques that require complex instrumentation, such as inductively coupled plasma optical emission spectrometry and Petition 870180045208, dated 05 / 28 / 2018, page 16 / 46 2 / 25 High-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). The method for quantifying Gd-DTPA-BMA described in the United States Pharmacopeia, as well as the method described by Hvattum et al., employs HPLC as a separation technique and post-column derivatization to enable detection of the complex in the visible spectrum region (HVATTUM, E. et al., 1995. “Detection and quantitation of gadolinium chelates in human serum and urine by high-performance liquid chromatography and post-column derivatization of gadolinium with Arsenazo III”. J Pharm Biomed Anal, 13 (7): 927-932; THE UNITED States Pharmacopeia. 34 ed. Rockville: The United States Pharmacopeial Convention, 2011. Gadodiamide Injection, p. 2934-2937). Derivatization, however, generally requires specialized instrumentation and a high consumption of reagents. Furthermore, the inclusion of this additional step makes the analysis more time-consuming.

[004] The determination of Gd-DTPA-BMA by HPLC using radioactivity detectors is also described (KINDBERG, GM et al., 2010. “The fate of Gd and chelate following intravenous injection of gadodiamide in rats”. Eur Radiol, 20 (7): 1636-1643). In this case, the main disadvantage is related to the requirement for prior radiolabeling of the complex.

[005] The detection of Gd-DTPA-BMA by micellar capillary electrokinetic chromatography is also described in the literature, however the detectability obtained by this technique is limited (ANDRÁSI, M. et al., 2011. “Determination of gadolinium-based magnetic resonance imaging contrast agents by micellar electrokinetic capillary chromatography”. Electrophoresis, 32 (16): 22232228).

[006] A method for identifying gadolinium complexes by potentiometric titration is reported in the literature (CACHERIS, WP et al., 1990. “The relationship between thermodynamics and the toxicity of gadolinium complexes”. Magn Reson Imaging, 8(4): 467-481). However, Petition 870180045208, dated 05 / 28 / 2018, page 17 / 46 3 / 25 This method is only appropriate for identifying complexes and evaluating their stability, and is not suitable for quantifying Gd-DTPA-BMA.

[007] In this context, the development of simpler, faster, and less expensive methods for the determination of Gd-DTPA-BMA can be extremely useful, especially for the determination of this complex in liposomal formulations. Liposome development can be laborious, complex, and involves several steps. Therefore, during this process, it can be advantageous to obtain rapid information about the influence of the formulation composition and the preparation method on the amount of encapsulated drug.

[008] Our research group recently developed two methods for the quantification of Gd-DTPA-BMA in liposomes by reversed-phase liquid chromatography (RP-LC) and derivative spectrophotometry (MAIA, ALC et al., 2015. “Development and validation of high performance liquid chromatographic and derivative spectrophotometric methods for determination of gadodiamide in liposomal formulations”. Anal Methods, 7: 8315-8325). In both methods, detection was performed in the ultraviolet region, without the need for a post-column derivatization step. Spectrophotometric analysis showed low detectability, while the retention of Gd-DTPA-BMA in RP-LC was a challenge due to its high polarity. Furthermore, the RP-LC method is not appropriate for the determination of this drug in more complex matrices, such as plasma, culture media, or protein-fortified buffers. In these cases, RP-LC does not provide adequate resolution.Furthermore, due to the impossibility of using any organic solvent in the mobile phase, optimization is limited. Comparing the present invention (HILIC method) with the method previously developed by our group (RP-LC method), the HILIC method showed five times greater detectability using the same type of detector (DAD). In addition, the HILIC method... Petition 870180045208, dated 05 / 28 / 2018, page 18 / 46 4 / 25 HILIC is suitable for determining this drug in more complex matrices such as fetal bovine serum.

[009] HILIC has been the technique of choice for the determination of polar compounds, mainly metal complexes. The increased use of HILIC may be related to its ability to overcome limitations of conventional chromatography, such as allowing the analysis of polar substances that exhibit low retention in RP-LC (GRECO, G.; LETZEL, T., 2013. “Main interactions and influences of the chromatographic parameters in HILIC separations”. J Chromatogr Sci, 51 (7): 684-693). In HILIC, several chromatographic parameters can interfere with the retention and separation of compounds. For this reason, the use of chemometric tools during method development can be an interesting approach (BUSZEWSKI, B.; NOGA, S., 2012. “Hydrophilic interaction liquid chromatography (HILIC) - a powerful separation technique. Anal Bioanal Chern, 402 (1): 231-247”).

[010] Several methods for determining Gd-DTPA-BMA in biological and environmental samples using HILIC are described in the literature. However, none of these studies reported the determination of Gd-DTPA-BMA in liposomes, or the use of DAD detection, making further investigations in this area necessary. Furthermore, there are no reported studies on the development of methods for determining Gd-DTPA-BMA using HILIC that employed a rational development approach. The methods for determining Gd-DTPA-BMA using HILIC described to date utilize MS detection. Despite the undeniable detectability provided by MS, the high cost of analyses and instrumentation justifies the development of simpler and less expensive methods.

[011] US patent document 9138721 (B2), with a priority date of January 28, 2011, entitled “Zwitterionic stationary phase for Petition 870180045208, dated 05 / 28 / 2018, page 19 / 46US9138721 (B2) describes a stationary phase for HILIC and its preparation method. This stationary phase has zwitterionic functional groups at the end of the bonded phase. The method proposed in US9138721 (B2) can be widely applied for the selective separation of various types of samples, mainly carbohydrates. The technology described in US9138721 (B2) does not show good results for the separation of metal complexes, such as Gd-DTPA-BMA. The frequent limitation observed in this type of stationary phase is that it has a high propensity for irreversible adsorptions due to the high reactivity of the amino group (BUSZEWSKI, B.; NOGA, S., 2012. “Hydrophilic interaction liquid chromatography (HILIC) - a powerful separation technique. Anal Bioanal Chern, 402 (1): 231-247”).Furthermore, this type of phase typically requires a longer baseline stabilization period, resulting in a more time-consuming analysis.

[012] The method described in the present invention proved to be simple, fast, and selective. Furthermore, it exhibited high detectability. Although this method was developed to determine only one analyte, it can be used to determine Gd-DTPA-BMA in more complex samples, such as fetal bovine serum. In this context, the use of Box-Behnken factorial design and response surface methodology were effective for the development of the method. This approach allowed for the evaluation of interactions between parameters and the obtaining of results that would likely not be obtained in a univariate analysis. The analytical method described in the present invention can be an advantageous alternative for professionals working with Gd-DTPA-BMA encapsulated in liposomes or in its traditional use as contrast agents. Petition 870180045208, dated 05 / 28 / 2018, page 20 / 46 6 / 25 BRIEF DESCRIPTION OF THE FIGURES

[013] Figure 1 represents the response surfaces for evaluating the dependent variable: signal-to-noise ratio. Graphs A, B, and C represent the pH results of the aqueous portion of the mobile phase versus the proportion of acetonitrile (ACN). These three graphs were obtained using a constant buffer concentration (graph A = 5 mmol / L, graph B = 15 mmol / L, and graph C = 25 mmol / L). Graphs D, E, and F represent the pH results of the aqueous portion of the mobile phase versus buffer concentration. In these three graphs, the fixed parameter was the proportion of ACN (graph D = 60%, graph E = 65%, and graph F = 70%). Graphs G, H, and I represent the results of the proportion of ACN versus buffer concentration. They were prepared keeping the pH values ​​of the aqueous portion of the mobile phase constant (graph G = 3.7, graph H = 4.2, and graph I = 4.7).

[014] Figure 2 represents the response surfaces for evaluating the dependent variable: resolution (Rs). Graphs A, B, and C represent the pH results of the aqueous portion of the mobile phase versus the acetonitrile (ACN) ratio. These three graphs were obtained using a constant buffer concentration (graph A = 5 mmol / L, graph B = 15 mmol / L, and graph C = 25 mmol / L). Graphs D, E, and F represent the pH results of the aqueous portion of the mobile phase versus buffer concentration. In these three graphs, the fixed parameter was the ACN ratio (graph D = 60%, graph E = 65%, and graph F = 70%). Graphs G, H, and I represent the ACN ratio results versus buffer concentration. They were created while keeping the pH values ​​of the aqueous portion of the mobile phase constant (graph G = 3.7, graph H = 4.2, and graph I = 4.7).

[015] Figure 3 represents the response surfaces for evaluating the dependent variable: asymmetry (As). Graphs A, B, and C represent the Petition 870180045208, dated 05 / 28 / 2018, page 21 / 46 7 / 25 pH results of the aqueous portion of the mobile phase versus the acetonitrile (ACN) ratio. These three graphs were obtained using a constant buffer concentration (graph A = 5 mmol / L, graph B = 15 mmol / L, and graph C = 25 mmol / L). Graphs D, E, and F represent the pH results of the aqueous portion of the mobile phase versus buffer concentration. In these three graphs, the fixed parameter was the ACN ratio (graph D = 60%, graph E = 65%, and graph F = 70%). Graphs G, H, and I represent the ACN ratio versus buffer concentration results. They were created while keeping the pH values ​​of the aqueous portion of the mobile phase constant (graph G = 3.7, graph H = 4.2, and graph I = 4.7). DETAILED DESCRIPTION OF THE TECHNOLOGY

[016] The present invention describes an analytical method, by hydrophilic interaction liquid chromatography (HILIC) using a diode array detector (DAD) in a zwitterionic stationary phase, capable of detecting and quantifying gadodiamide (Gd-DTPA-BMA) in pharmaceutical and biological samples. This method can be used to detect and quantify gadodiamide (Gd-DTPA-BMA) in pharmaceutical and biological samples, such as liposomes and blood serum.

[017] The method for detection and quantification of gadodiamide (Gd-DTPABMA) in pharmaceutical and biological samples comprises the following steps: a. Subject the sample to liquid chromatography on a hydrophilic interaction liquid chromatography (HILIC) column coupled to a diode array detector (DAD), where the stationary phase used in the HILIC column is a zwitterionic phase; b. Elute the sample using a mobile phase, with a flow rate of 0.6 mL / min to 1.0 mL / min, at a controlled temperature, preferably 30 °C; Petition 870180045208, dated 05 / 28 / 2018, page 22 / 46 8 / 25 c. Identify Gd-DTPA-BMA in the sample by means of detection in the ultraviolet region (200 to 250 nm, preferably 210 nm); d. Quantify Gd-DTPA-BMA in the sample using a calibration curve.

[018] In step “a”, the zwitterionic stationary phase can be selected from the group comprising ZIC®-HILIC, ZIC®-pHILIC and ZIC®-cHILIC.

[019] In step “b”, the mobile phase may consist of a mixture of an aprotic organic solvent and a buffer prepared from a salt; the aprotic organic solvent may comprise at least one of the solvents acetonitrile and acetone or a mixture of acetonitrile and acetone, preferably acetonitrile. The proportion of aprotic solvent should be in the range of 60 to 75%, preferably 60%. The salt should comprise at least one of the salts ammonium formate (NH4FA) and ammonium acetate (NH4Ac), preferably ammonium formate, at a concentration of 5 to 25 mmol / L, preferably 5 mmol / L. The pH of the buffer prepared from the salt is in the range of 3.7 to 5.8, preferably 4.5.

[020] The mobile phase can be composed of a mixture of aprotic organic solvent and buffer, such as acetonitrile / ammonium formate or acetonitrile / ammonium acetate, with detectability being superior when ammonium formate buffer is used.

[021] The proportion of aprotic organic solvent should preferably be 60 to 75%, with the analysis time increasing proportionally to the higher percentage of solvent.

[022] The buffer concentration should preferably be 5 to 25 mmol / L, with a minimum of 5 mmol / L required to obtain symmetrical peaks, and a maximum of 25 mmol / L to avoid solvent precipitation when it comes into contact with the organic solvent. Petition 870180045208, dated 05 / 28 / 2018, page 23 / 46 9 / 25

[023] The pH of ammonium formate buffer should preferably be between 3.7 and 4.7. Although the buffering range of ammonium formate is between pH 2.7 and 4.7, values ​​below 3.7 result in the degradation of Gd-DTPA-BMA.

[024] The pH of the ammonium acetate buffer should preferably be between 3.8 and 5.8.

[025] The combination of low buffer pH (3.7) and intermediate (15 mmol / L) or high (25 mmol / L) buffer concentrations should be avoided in the mobile phase composition, as it may result in inadequate resolution between the Gd-DTPA-BMA peak and the liposome peak.

[026] Detection can be performed from 200 to 250 nm, with maximum absorption obtained at 210 nm.

[027] The mobile phase flow rate should preferably be 0.6 mL / min to 1.0 mL / min, with lower flow rates resulting in greater efficiency, resolution, and detectability. However, lower flow rates make the analysis slower.

[028] The proposed method can be used for the determination of gadodiamide (Gd-DTPA-BMA) during the development of liposomes for use in cancer treatment or for use as a contrast agent; determination of Gd-DTPA-BMA during quality control of the production of injectable solutions for magnetic resonance imaging; determination of Gd-DTPA-BMA in samples of hospital waste and water and sewage treatment plants to investigate and control environmental contamination with Gd resulting from the use of contrast agents in magnetic resonance imaging; determination of Gd-DTPA-BMA in biological samples, for investigation of the pathogenicity mechanisms of Nephrogenic Systemic Fibrosis. Petition 870180045208, dated 05 / 28 / 2018, page 24 / 46 10 / 25

[029] The present invention can be better understood through the following examples, which are not limiting. EXAMPLE 1 - Development and optimization of an analytical method by HILIC for the determination of Gd-DTPA-BMA in liposomes

[030] The method was developed and optimized using three steps: (i) Variable screening; (ii) Response surface analysis using Box-Behnken factorial design; (iii) Van Deemter curve.

[031] For the examples presented, thermosensitive formulations containing Gd-DTPA-BMA were prepared by the reversed-phase evaporation (RPE) method with a total lipid concentration of 40 mmol / L. For the preparation of the traditional thermosensitive liposome (TTSL-Gd), aliquots of DPPC, DSPC, and DSPE-PEG2000 chloroform solutions were used, in a lipid molar ratio of 80:15:5, respectively. For the preparation of the thermosensitive liposome containing lysophospholipid (LTSL-Gd), aliquots of DPPC, MSPC, and DSPE-PEG2000 chloroform solutions were used, in a lipid molar ratio of 85:10:5, respectively. After complete dissolution of the lipids, an aqueous solution of GdDTPA-BMA (250 μmol / mL) was added, respecting the aqueous phase:organic phase ratio of 1:3, respectively. The resulting dispersion was subjected to vigorous stirring at 3,000 rpm for 5 minutes, producing an oil-in-water emulsion.Subsequently, the W / O emulsion was subjected to evaporation under reduced pressure in order to eliminate the organic solvent, allowing the formation of LUV-type vesicles. To obtain traditional thermosensitive liposomes (TTSL) and thermosensitive liposomes containing "blank" lysophospholipids (LTSL), i.e., not containing Gd-DTPA-BMA, the same experimental protocol was performed, except for the drug addition step, which was replaced by the addition of HEPES buffer. The calibration of the obtained liposomes was performed using 10 extrusion cycles on polycarbonate membranes with pore sizes of 0.4, 0.2 and . Petition 870180045208, dated 05 / 28 / 2018, page 25 / 46 11 / 25 0.1 μm, respectively, under a nitrogen flow and at a temperature of 55 °C. Each cycle corresponds to the passage of the entire volume of the formulation through the extruder. The separation of unencapsulated Gd-DTPA-BMA, or HEPES buffer, was performed by ultracentrifugation at 350,000 x g, 4 °C for 2 hours. Before purification, the liposomes were diluted in purified water, using a dilution factor of 3. After ultracentrifugation, the pellet was reconstituted in HEPES buffer to obtain the same initial volume (before dilution).

[032] For variable screening, a literature review was initially conducted to determine the critical independent variables for the development of methods for the determination of Gd-DTPABMA by HILIC. To choose the detection wavelength, a spectral scan was performed between 200 and 400 nm of a Gd-DTPABMA sample with a concentration of 57 mg / mL. This concentration was chosen based on the identification test of Gd-DTPA-BMA by absorption spectrometry in the ultraviolet region, described in its official monograph (THE UNITED, 2011). The analyses were performed on a Shimadzu 1800 series UV-Vis spectrometer (Tokyo, Japan). From the obtained spectrum, it was observed that, due to the absence of extended chromophores in its structure, GdDTPA-BMA showed a maximum absorption at 210 nm.

[033] After defining the detection wavelength (210 nm), temperature (30 °C), and injection volume (20 μL), 11 experiments were performed to define the variables and investigate the range of variation and the levels at which the independent variables should be evaluated in a factorial design. In each experiment, nine determinations were performed: three determinations in a sample of Gd-DTPA-BMA 0.5 μmol / mL, three determinations in a sample of TTSL liposomes fortified with Gd-DTPA-BMA 0.5 pmol / mL (TTSL / Gd), and three determinations in a sample of LTSL liposomes fortified with Petition 870180045208, dated 05 / 28 / 2018, page 26 / 46 12 / 25 Gd-DTPA-BMA 0.5 μmol / mL (LTSL / Gd). The results of this step are expressed in Table 1. The mobile phase used in each experiment was: 1. ACN / NH4Ac 10 mmol / L, pH 5.8, (70:30 v / v); 2. ACN / NH4FA 10 mmol / L, pH 4.7, (70:30 v / v); 3. ACN / NH4FA 10 mmol / L, pH 4.7, (60:40 v / v); 4. ACN / NH4FA 10 mmol / L, pH 4.7, (70:30 v / v); 5. ACN / NH4FA 10 mmol / L, pH 4.7, (75:25 v / v); 6. ACN / NH4FA 5 mmol / L, pH 4.7, (70:30 v / v); 7. ACN / NH4FA 10 mmol / L, pH 4.7, (70:30 v / v); 8. ACN / NH4FA 15 mmol / L, pH 4.7, (70:30 v / v); 9. ACN / NH4FA 10 mmol / L, pH 2.7, (70:30 v / v); 10. ACN / NH4FA 10 mmol / L, pH 3.7, (70:30 v / v); 11. ACN / NH4FA 10 mmol / L, pH 4.7, (70:30 v / v). Petition 870180045208, dated 05 / 28 / 2018, page 27 / 46 13 / 25 Table 1 - Results of variable screening for the development of a method for determining Gd DTPA-BMA in liposomes by HILIC. Experiment Independent Variables Dependent Variables a Signal-to-noise ratio Ps As Height (mAU) Noise (mAU) N Width Area tr (min) k 1 NH4AC 2,586,043 11.8 1.44 9,852,825 3.81 3,687 0.76 218,788,085 8.9 4.6 2 NH4FA 8,389,613 6.9 1.44 10,990,393 1.31 4,106 0.72 233,993,993 8.6 4.4 3 ACN 60% 10,730,254 7.5 1.25 16,095,381 1.50 3,018 0.39 189,478,831 4.3 1.7 4 ACN 70% 8,389,613 6.9 1.44 10,990,393 1.31 4,106 0.72 233,993,993 8.6 4.4 5 ACN 75% NH4FA 5 9,169,528 15.6 1.71 6,326,974 0.69 4,610 1.37 235,782,267 16.2 9.2 6 mmol / L NH4FA 10 16,385,674 8.1 1.42 10,650,688 0.65 3,912 0.76 234,068,457 8.9 4.6 7 mmol / L NH4FA 15 8,389,613 6.9 1.44 10,990,393 1.31 4,106 0.72 233,993,993 8.6 4.4 8 mmol / L 3,766,828 7.8 1.45 10,170,436 2.70 4,255 0.73 212,774,504 8.9 4.6 9 b pH 2.7 - - - - 1.60 - - - - - 10 pH 3.7 7,788,154 7.0 1.44 10,436,127 1.34 3,377 0.72 239,358,814 8.8 4.6 11 pH 4.7 8,389,613 6.9 1.44 10,990,393 1.31 4,106 0.72 233,993,993 8.6 4.4 Petition 870180045208, dated 05 / 28 / 2018, page 28 / 46 14 / 25 Notes: aData expressed as mean (n= 3 samples, with 3 injections for each sample). bUnder these conditions, it was not possible to calculate most of the dependent variables analyzed, due to the degradation of Gd-DTPABMA.

[034] For response surface analysis using Box-Behnken factorial design, the design was projected using three independent variables at three levels (-1, 0 and 1). The variables studied and their levels were: X1 = pH of the buffer, level -1 = 3.7, level 0 = 4.2 and level +1 = 4.7; X2 = proportion of ACN in the mobile phase (%), level -1 = 60, level 0 = 65, level +1 = 70; X3 = buffer concentration (mmol / L), level -1 = 5, level 0 = 15, level +1 = 25. The dependent variables evaluated as response were: signal-to-noise ratio, resolution (Rs) and skewness (As). The dependent variables were also evaluated: chromatographic peak height, baseline noise, number of theoretical plates (N), chromatographic peak width measured at 5% baseline height, chromatographic peak area, retention time (tr), and retention factor (k). Fifteen experiments were performed in random order, including three replicates of the central point.In each experiment, six determinations were performed: three determinations on a sample of 0.3 μmol / mL Gd-DTPA-BMA and three determinations on a sample of TTSL and LTSL liposomes fortified with 0.3 pmol / mL Gd-DTPA-BMA (TTSL / LTSL-Gd). The coefficients of determination (r²) and correlation (r) were obtained using the least squares method. The model was evaluated using analysis of variance (ANOVA), and the error estimate was calculated using experiments at the central point. The results were evaluated using the Statistica 7.0 software (StatSoft®, Tulsa, USA). In this stage of the study, a multivariate analysis employing factorial design was used. The variables chosen to compose the design, based on the initial screening stage, were: pH of the portion. Petition 870180045208, dated 05 / 28 / 2018, page 29 / 46 The aqueous mobile phase concentration (X1), ACN ratio (X2), and buffer concentration (X3) were determined. The remaining chromatographic conditions had their values ​​fixed: SeQuant® ZIC®-HILIC column (150 x 4.6 mm, 3.5 µm, 100 °C) (Merck, Darmstadt, Germany), isocratic elution at 1.0 mL / min, injection volume of 20 µL, temperature of 30 °C, and detection at 210 nm. The experimental conditions evaluated and the responses obtained are expressed in Table 2. Table 2 - Results of the Box-Behnken experimental design for the development and optimization of a method for the determination of Gd-DTPA-BMA in liposomes by HILIC. Levels Independent variables _________________________ -1 0 1 Xi pH 3.7 4.2 4.7 X2 ACN ratio (%) 60 65 70 X3 Buffer concentration 5 15 25 (mmol / L) Experiments Experimental conditions Dependent variables a X1 X2 X3 Signal-to-noise ratio Rs As 1 4.7 70 15 7,164,899 7.7 1.32 2 4.7 60 15 7,846,412 3.7 1.18 3 3.7 70 15 4,029,493 1.3 1.31 4 3.7 60 15 6,040,335 0.0 1.21 5 4.7 65 25 1,675,031 5.1 1.31 6 4.7 65 5 9,622,069 5.5 1.25 7 3.7 65 25 6,731,611 0.0 1.27 8 3.7 65 5 7,473,207 4.0 1.25 9 4.2 70 25 2,324,785 7.2 1.30 10 4.2 70 5 5,124,660 7.2 1.33 11 4.2 60 25 3,487,648 2.7 1.23 Petition 870180045208, dated 05 / 28 / 2018, pages 30 / 46 16 / 25 12 4.2 60 5 6.514.122 3.5 1.19 13 (central point) b 4.2 65 15 2.293.381 3.7 1.27 Notes: aAverage of three injections; bAverage of three samples, with three injections for each sample.

[035] In order to extrapolate the data obtained from the Box-Behnken matrix and calculate the optimum point corresponding to the values ​​that variables X1, X2, and X3 assume to generate the best responses, the data presented in Table 2 were used to construct mathematical models. Through the combination of the values ​​of the variables and the responses obtained, the coefficients of the equations describing the studied system were calculated (Table 3). These equations were developed from the effects of primary linear and quadratic interactions. Secondary interactions were excluded because they generated experimentally inconsistent optimum point results. The ANOVA data, r, r2, and the pure error calculated from the replicates of the central point, are described in Table 3. The r2 value obtained was satisfactory, since the closer the coefficient value is to 1, the better the model's fit to the observed responses.The optimum points, that is, the critical values ​​for obtaining the best signal-to-noise ratio responses, Rs and As, were calculated from the first derivative of the equation that describes the system. However, because they generated inconsistent results from an experimental point of view, only the slopes of the response surface plots were considered for choosing the best values ​​of X1, X2, and X3. Petition 870180045208, dated 05 / 28 / 2018, pages 31 / 46 17 / 25 Table 3 - Coefficients of the mathematical model obtained by Box-Behnken and summary of the ANOVA. Dependent variables Signal-to-noise ratio RsAs Independent Variables Coefficient p-value Coefficient p-value Coefficient p-value Xi 229221 0.03514 2.09589 0.00166 0.00169 0.10157 Xi2 - 1521253 0.00046 0.49960 0.01551 0.00319 0.01758 X2 - 680585 0.00418 1.69384 0.00254 0.05653 0.00011 X22 - 514916 0.00396 - 0.23383 0.06546 0.00610 0.00492 X3 - 1814373 0.00059 - 0.66067 0.01636 0.01092 0.00284 0.03022 0.00784 0.01088 Xi X3 - 1801360 0.00120 0.90450 0.01742 0.01104 0.00553 0.01400 0.00345 Intercept 5686189 0.00004 4.00106 0.00030 1.26278 0.00000 Pure error 155887 x 1050.0585495 0.0000027 r 0.9530 0.9729 0.9794 F 0.9082 0.9465 0.9592 Notes: X1, pH; X2, ACN proportion (%); X3, buffer concentration (mmol / L); p < 0.05 (ANOVA). Petition 870180045208, dated 05 / 28 / 2018, pages 32 / 46 18 / 25

[036] The response surfaces obtained are shown in Figures 1, 2, and 3. In these three-dimensional graphs, the independent variables were grouped in pairs to evaluate the influence of the interaction between them on the signal-to-noise ratio, R and As responses, respectively. In Figures 1, 2, and 3, graphs A, B, and C were obtained using a constant buffer concentration. In graphs D, E, and F, the fixed parameter was the ACN proportion. Graphs G, H, and I were constructed keeping the pH values ​​of the aqueous portion of the mobile phase constant.

[037] Analyzing the response surfaces obtained for signal-to-noise ratio evaluation, expressed in Figure 1, it can be observed through the scale of graphs 1A, 1B, and 1C that the lower the buffer concentration, the greater the response. Analyzing graph A in isolation, where the buffer was fixed at the minimum evaluated level (5 mmol / L), it is observed that higher signal-to-noise ratio values ​​are obtained when using pH > 4.4, regardless of the proportion of ACN used. The results expressed in graphs 1D, 1E, and 1F are in agreement with these observations. The values ​​of the scales of graphs 1G, 1H, and 1I demonstrate that the highest responses are obtained when the highest pH value of the NH4FA buffering range is used (graph 1I). Analyzing graph 1I in isolation, it can be seen that regardless of the ACN proportion, the highest signal-to-noise ratio is observed when the buffer concentration is equal to or less than 10 mmol / L.Based on the analysis of the nine response surfaces shown in Figure 1, it can be concluded that the pH of the aqueous portion of the mobile phase (values ​​between 4.4 and 4.7) and the buffer concentration (< 10 mmol / L) are the factors that most influence the signal-to-noise ratio.

[038] The Rs results were evaluated based on Figure 2. Analyzing the scale of graphs 2A, 2B, and 2C, it is observed that when the lowest buffer concentration is used (graph 2A), any combination of pH and ACN ratio results in a resolution > 2. The Petition 870180045208, dated 05 / 28 / 2018, pages 33 / 46 19 / 25 results expressed in graphs 2D, 2E, and 2F confirm these observations and reveal that high pH values ​​also influence the obtaining of higher Rs values. Analyzing the scales of graphs 2G, 2H, and 2I, the benefit of using high pH values ​​is confirmed, as the highest responses were found when the pH was fixed at 4.7 (graph 2I). In general, the analysis of the nine response surfaces in Figure 2 reveals that higher Rs values ​​are obtained when the following conditions are combined: low buffer concentration, high pH, ​​and a high proportion of ACN.

[039] The response surfaces shown in Figure 3 were used to evaluate As. Analysis of the scale of graphs 3A, 3B, and 3C reveals that lower As values ​​are obtained when lower buffer concentrations are used. Analyzing graph 3A in isolation, where the buffer concentration was kept fixed at the lowest level evaluated (5 mmol / L), it is observed that the combination of a low proportion of ACN and a high pH value results in lower As values. Analysis of the scale of graphs 3D, 3E, and 3F confirms this observation, since graph 3D, where the proportion of ACN was kept at the lowest level, showed the best responses. Analyzing graph 3D in isolation, there is a tendency to obtain lower As values ​​when the buffer concentration is < 6 mmol / L and the pH is > 4.4. In graphs 3G, 3H, and 3I, regardless of the pH used, the combination of a lower proportion of ACN and a low buffer concentration results in lower As values.The results of the response surface methodology are consistent with the data obtained from the Pareto charts, which allow us to identify the independent variables that influence the response, the extent of that influence, and the significant interactions between the variables investigated. Petition 870180045208, dated 05 / 28 / 2018, pages 34 / 46 20 / 25

[040] In this stage of the analytical method development, the following responses were also evaluated: chromatographic peak height, baseline noise, N, chromatographic peak width measured at 5% baseline height, chromatographic peak area, tre k. From the results of these analyses, expressed in Appendices B and C, it was possible to elaborate Table 4, which allowed the identification of the optimized conditions, considering the individual desirability of each parameter and the overall desirability for the proposed method. Table 4 - Results of the optimum point identified through the response surface obtained by Box-Behnken design for the determination of Gd-DTPA-BMA in liposomes by HILIC. Dependent Variables Optimal Value of Independent Variables pH ACN Proportion (%) Buffer Concentration (mmol / L) Signal-to-noise ratio > 4.4 little influence 5 Ks > 4.2 > 64 5 As > 4.4 60 < 6 Height (mAU) 4.2 - 4.5 60 > 6 Noise (mAU) > 4.5 60 5 N > 4.4 60 25 Width > 4.5 60 little influence Area 4.2 65 15 Optimized conditions 4.5 60 5

[041] In order to verify whether the defined optimized conditions result in optimal response values ​​for the chromatographic peak related to GdDTPA-BMA, a new experiment was carried out, employing a mobile phase composed of 60% ACN, NH4FA at a concentration of 5 mmol / L and pH of Petition 870180045208, dated 05 / 28 / 2018, pages 35 / 46 21 / 25 aqueous portion equal to 4.5. Under these conditions, the signal-to-noise ratio obtained was 9,594,265. This result is in agreement with the highest signal-to-noise ratio value found in the Box-Behnken design experiments (Experiment 6, Table 2). The Rs value obtained using the optimized conditions was 3.2. This result was considered adequate, as it is higher than the recommended value (Rs > 2) for obtaining satisfactory separation between the drug and possible interferents (US-FDA, 2000). The As result obtained after optimization of the chromatographic parameters was 1.11. This value corresponds to the best response obtained for this parameter, considering all the experiments performed. Furthermore, it is in accordance with the limits established by the FDA (US-FDA, 1994).

[042] For the Van Deemter curve, developed with the objective of determining the linear velocity (directly proportional to the mobile phase flow rate) at which the height equivalent to a theoretical plate (directly related to the broadening of the chromatographic peak) is minimal, the mobile phase flow rate was varied at the following values: 0.04; 0.06; 0.08; 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 1.0; 1.5 and 2.0 mL / min. For each flow rate value, a chromatographic peak was obtained, and a determined N and tr corresponded to it. The curve was obtained by plotting the height equivalent to a theoretical plate (H) as a function of the linear velocity of the mobile phase (U0). The maximum efficiency observed, using the chromatographic conditions of the developed method, occurred with an H value around 18 μm and a U0 close to 0.16 mm / s, which corresponds to a flow rate of 0.1 mL / min.This flow rate value is not feasible for use in the proposed method, since it results in a very long Gd-DTPA-BMA flow rate (tr = 44.15 minutes) to be used in routine analyses. Therefore, the optimization of the mobile phase flow rate was carried out by evaluating the chromatographic parameters found during the experiments performed for... Petition 870180045208, dated 05 / 28 / 2018, pages 36 / 46 22 / 25 Obtaining the Van Deemter curve. Based on the results obtained, a flow rate of 0.6 mL / min was selected for use in the developed method. The use of this new condition, compared to the use of a flow rate of 1.0 mL / mL, resulted in a higher tr of GdDTPA-BMA (tr = 7.1 min). However, this flow rate allowed for a 29% increase in efficiency (N) and a 12% increase in detectability and Rs of the proposed method. EXAMPLE 2 - Analytical method validation

[043] The chromatographic conditions used in this example were: Sequant® ZIC®-HILIC Merck column (150 x 4.6 mm, 3.5 µm, 100 °C) (Darmstadt, Germany), mobile phase composed of acetonitrile / ammonium formate 5 mmol / L, pH 4.5 (60:40, v / v), injection volume of 20 µL, temperature of 30 °C and detection at 210 nm. Elution was performed in isocratic mode with a flow rate of 0.6 mL / min. During the development of the method, the following were used: Box-Behnken factorial design, response surface methodology and Van Deemter curve in order to obtain high detectability and resolution. The method was validated according to the criteria established in Resolution RE No. 899 of May 29, 2003, of the National Health Surveillance Agency and in the ICH QR2 (R1) Analytical Procedure Validation Guide.HILIC analyses were performed using an Agilent Technologies 1260 series chromatograph (California, USA), equipped with a degasser, quaternary pump (G1311B), oven (G1316A), autosampler (G1329B) and DAD (G4212B), coupled to the EzChrom integration software.

[044] By superimposing the chromatograms of Gd-DTPA-BMA, TTSL / LTSL, isopropyl alcohol and mobile phase, no interfering peaks were observed at the tr of Gd-DTPA-BMA (tr = 7.1 min), demonstrating the selectivity of the method. The resolution obtained between Gd-DTPA-BMA and TTSL / LTSL was adequate (Rs = 3.6). Furthermore, the purity of the Gd-DTPA-BMA peak, calculated by DAD, was equal to 100% in all determinations. Petition 870180045208, dated 05 / 28 / 2018, pages 37 / 46 23 / 25

[045] The method proved to be linear for the determination of Gd-DTPA-BMA in the range between 40 and 120 nmol / mL. The equation of the line obtained was y = 803.100 x + 964.900. The calculated r² values ​​were satisfactory (> 0.999) (BRASIL, 2003). There was no significant difference between the slopes of the three calibration curves obtained (p <0.05).

[046] The determination of the limit of detection (LOD) and limit of quantification (LOQ) of Gd-DTPA-BMA was initially performed by evaluating the signal-to-noise ratio in order to include the LOQ as the lowest concentration level in the linear range of the analytical curve. Chromatograms were obtained for Gd-DTPA-BMA solutions at 10 nmol / mL and 40 nmol / mL, considered LOD and LOQ, respectively, according to this analysis. The Gd-DTPA-BMA peak was eluted in 7.1 min. After evaluating linearity, the theoretical LOD and LOQ values ​​were also calculated based on the linear regression parameters. The difference observed between the LOD and LOQ values ​​obtained by the two methods, signal-to-noise ratio (LOD = 10 nmol / mL, LOQ = 40 nmol / mL) and linear regression parameters (LOD = 4.56 nmol / mL, LOQ = 6.78 nmol / mL), is related to the particularities of each type of analysis.

[047] In a previous study by our research group, an analytical method for the determination of Gd-DTPA-BMA in liposomes was developed and validated using RP-LC / DAD (MAIA et al., 2015). This method showed a linear range between 100 and 500 nmol / mL. In the HILIC method, developed in the present invention, it was possible to include lower concentrations in the analytical curve (40 to 120 nmol / mL). Furthermore, comparing the LOD and LOQ values ​​obtained in the two studies, it is concluded that the HILIC method showed five times greater detectability, using the same type of detector (DAD) that was employed in the previous study. Petition 870180045208, dated 05 / 28 / 2018, pages 38 / 46 24 / 25

[048] The method developed demonstrated adequate intra-day and inter-day accuracy, with DPR values ​​in accordance with the specification established by RE 899, which recommends DPR < 5% as a criterion for evaluating these parameters.

[049] The accuracy of the developed method was demonstrated. The average percentage recovery value found for the TTSL / LTSL formulations was 98.61%. Furthermore, the RSD value obtained among the determinations performed at the three levels evaluated was less than 5%.

[050] The robustness of the chromatographic method for the determination of GdDTPA-BMA in liposomes was evaluated using the Youden test. From the results obtained in the eight experiments, the effects of each variable and the major effect were calculated, allowing the influence of the modifications in the proposed method to be evaluated. According to the data obtained, it can be stated that the method was robust for all parameters evaluated, since the effects of each variable were smaller than the respective calculated major effects (Table 5). Table 5 - Evaluation of the effect of variables, in terms of content, Rs and As, on the determination of Gd-DTPA-BMA using the developed method. Variables Content (%) a Rs a Asa ACN ratio in mobile phase = -1.36 101.37 - 100.87 3.21 - 4.57 = 1.32 - 1.28 = 0.04 (A = 60%; a = 63%) 0.50 Apparent pH of mobile phase = -1.34 100.61 - 101.64 4.06 - 3.72 = 1.26 - 1.34 = 0.34 - 0.08 (B = 4.5; b = 4.7) - 1.03 Buffer concentration = -1.03 101.46 - 100.78 3.56 - 4.22 = 1.29 - 1.30 = (C = 5 mmol / L; c = 5.5 = -0.66 - 0.01 mmol / L) 0.68 Petition 870180045208, dated 05 / 28 / 2018, pp. 39 / 46 25 / 25 Column temperature (D = 30 °C; d = 33 °C) 101.35 - 100.90 0.45 3.80 - 3.98 = - 0.18 1.29 - 1.31 = - 0.02 Mobile phase flow rate (E = 0.6 mL / min; e = 0.7 mL / min) 101.16 - 101.09 0.07 4.37 - 3.41 = 0.96 1.28 - 1.31 = - 0.03 ACN brand 101.54 - 100.70 3.83 - 3.95 = 1.25 - 1.35 = (F = Tedia; f = JTBaker) 0.84 - 0.12 - 0.10 Buffer brand (G = Vetec; g = Spectrum) 101.26 - 100.98 0.28 4.05 - 3.73 = 0.32 1.26 - 1.34 = -0.08 Larger effect b 1.24 36.06 8.97 Notes: a) Average of the values ​​obtained under nominal conditions minus the average of the values ​​obtained under varied conditions; b) Difference between the values ​​obtained in the 8 experiments multiplied by the square root of 2. Petition 870180045208, dated 05 / 28 / 2018, pages 40 / 46

Claims

1 / 2 CLAIMS 1. A method for the detection and quantification of gadodiamide (GdDTPA-BMA) in pharmaceutical and biological samples, characterized by comprising the following steps: a. Subjecting the sample to liquid chromatography on a hydrophilic interaction liquid chromatography (HILIC) column coupled to a diode array detector (DAD), wherein the stationary phase used in the HILIC column is a zwitterionic phase; b. Eluting the sample using a mobile phase composed of a mixture of an aprotic organic solvent and a buffer prepared from a salt, wherein the proportion of aprotic solvent is 60 to 75%, with a flow rate of 0.6 mL / min to 1.0 mL / min, at a controlled temperature; c. Identifying Gd-DTPA-BMA in the sample by means of detection in the ultraviolet region (200 to 250 nm); d. Quantify Gd-DTPA-BMA in the sample using a calibration curve.

2. Method for detection and quantification of gadodiamide (GdDTPA-BMA) in pharmaceutical and biological samples, according to claim 1, characterized in that, in step “a”, the zwitterionic stationary phase is selected from the group comprising ZIC®-HILIC, ZIC®-pHILIC and ZIC®-cHILIC.

3. Method for detection and quantification of gadodiamide (GdDTPA-BMA) in pharmaceutical and biological samples, according to claim 1, characterized in that, in step “b”, the aprotic organic solvent comprises at least one of the solvents acetonitrile and acetone or a mixture of acetonitrile and acetone, preferably acetonitrile; and the temperature is controlled at 30 °C. Petition 870260069845, dated 07 / 14 / 2026, page 7 / 8 2 / 2 4. Method for detection and quantification of gadodiamide (GdDTPA-BMA) in pharmaceutical and biological samples, according to claim 1, characterized in that, in step “b”, the salt comprises at least one of the ammonium formate and ammonium acetate salts, preferably ammonium formate.

5. Method for detection and quantification of gadodiamide (GdDTPA-BMA) in pharmaceutical and biological samples according to claim 4, characterized by the salt concentration comprising a range between 5 and 25 mmol / L, preferably 5 mmol / L.

6. Method for detection and quantification of gadodiamide (GdDTPA-BMA) in pharmaceutical and biological samples according to claim 4, characterized in that the pH of the buffer prepared from the salt comprises a range between 3.7 and 5.8, preferably 4.

5.

7. Use of the method defined in claim 1, according to any of claims 2 to 6, characterized by being for the determination of Gd-DTPA-BMA during the development of liposomes for use in cancer treatment or for use as a contrast agent; determination of Gd-DTPA-BMA during quality control in the production of injectable solutions for magnetic resonance imaging; determination of Gd-DTPA-BMA in samples of hospital waste and water and sewage treatment plants to investigate and control environmental contamination with Gd resulting from the use of contrast agents in magnetic resonance imaging; determination of Gd-DTPA-BMA in biological samples for the investigation of the pathogenicity mechanisms of Nephrogenic Systemic Fibrosis. Petition 870260069845, dated 07 / 14 / 2026, p. 8 / 8

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