METHOD FOR THE DETERMINATION OF SODIUM HYALURONATE CONTENT IN A HYDROGEL

Abstract

The present invention is directed to a method for the determination of the hyaluronic acid content of a hydrogel, the method comprising the following steps: a) preparing, as reagent A, a solution of sodium tetraborate in sulfuric acid; b) preparing reagent B by dissolving carbazole in ethanol; c) preparing test solutions by dissolving the hydrogel in an aqueous solution; d) treating the test solution with ultrasounds for a period of time sufficient to obtain a macroscopically homogeneous solution; e) preparing a reference stock solution by dissolving glucuronic acid, or a glucuronic acid-containing substance in an aqueous solution; f) preparing at least 3 reference solutions by dilution of the reference stock solution in aqueous solution, preferably at concentration comprised between 0.0005% w/v and 0.0100% w/v, preferably between 0.0010% w/v and 0.0050% w/v; g) preparing the test tubes by admixing reagent A, reagent B and one of the following: reference solution, test solution, aqueous solution (blank), and optionally solution for interference (crosslinker sample or additive sample); placing each test tube on a water bath for at least 5 min, then cool them to room temperature; h) reading the absorbance at a wavelength comprised between 500 and 580 nm, preferably at about 530 nm, against the blank and optionally the sample for interference.

Claims

1. A method for the determination of the hyaluronic acid content of a hydrogel, the method comprising the following steps: a. preparing, as reagent A, a solution of sodium tetraborate in sulfuric acid; b. preparing reagent B by dissolving carbazole in ethanol; c. preparing test solutions by dissolving the hydrogel in an aqueous solution; d. treating the test solution with ultrasounds for a period of time sufficient to obtain a macroscopically homogeneous solution; e. preparing a reference stock solution by dissolving glucuronic acid, or a glucuronic acid-containing substance in an aqueous solution; f. preparing at least 3 reference solutions by dilution of the reference stock solution in aqueous solution, preferably at concentration comprised between 0.0005% w/v and 0.0100% w/v, preferably between 0.0010% w/v and 0.0050% w/v; g. preparing the test tubes by admixing reagent A, reagent B and one of the following: reference solution, test solution, aqueous solution (blank), and optionally solution for interference (crosslinker sample or additive sample); place each test tube on a water bath for at least 5 min, then cool them to room temperature; h. reading the absorbance at a wavelength comprised between 500 and 580 nm, preferably at about 530 nm, against the blank and optionally the sample for interference.

2. The method according to claim 1 further comprising, after step c, the following step: c2. heating the test solutions to a temperature higher than room temperature, preferably between 75 and 85° C. for at least 15 minutes, preferably at least 2 hours, more preferably about 3 hours, preferably while continuously stirring.

3. The method of claim 1 wherein in step g, the water bath is a hot bath and the time is preferably about 15 minutes.

4. The method according to claim 1 wherein in step d. sonication is performed at a specific power between 0.5 W/kg and 50 W/kg.

5. The method of claim 1 wherein sonication is performed at a specific energy comprised between 500 J/kg and 30 kJ/kg.

6. The method according to claim 1 wherein reagent A is a solution 0.95% w/v of disodium tetraborate in sulfuric acid, and reagent B is a solution 0.125% w/v of carbazole in ethanol.

7. The method according to claim 1 wherein in step c. the test solution is prepared at least in double.

8. The method according to claim 1 wherein step g. is performed as follows: adding 5 parts of reagent A to each test tube, preferably previously cooled; layer each sample onto the reagent A in the test tube, as follows: Blank: 1 part of water solution; Reference solutions: 1 part of each dilution of the Reference stock solution; Samples: 1 part of each Test solution; shake the tubes to dissolve the double-phase created by the sample; put each sample in a water bath for at least 5 minutes, preferably in a boiling water bath for 15 minutes, then cool them down to room temperature; add to each tube 0.2 parts of reagent B, shake the tubes to dissolve the double-phase and put each tube in a warm water bath for at least 5 minutes, preferably in a boiling water bath for 15 minutes.

9. The method of claim 1 wherein, after step h., the percentage content of sodium hyaluronate is calculated by interpolating the experimental absorbance values with the reference standard curve.

Description

BRIEF DESCRIPTION OF THE FIGURE

[0013] FIG. 1 reports the R value and the standard curve's equation of the example.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention is directed to a method for the determination of HA content in a hydrogel, which method makes use of a sonication step. It has been surprisingly found that by sonicating a crosslinked hydrogel containing HA, it is possible to determine the HA content of the hydrogel in a reproducible way and with a low standard deviation.

[0015] The most commonly used method for the determination of HA in a water solution is defined in the European Pharmacopoeia 5.0 at pages 2434-7, herein incorporated by reference. Another method for the determination of glucuronic acid by reaction with carbazole was disclosed by T. Bitter and H. M. Muir (A modified Uronic Acid Carbazole Reaction, An. Biochem. 4, 330-334 (1962)), which is herein incorporated by reference. The present invention represents an important improvement over both the method of the European Pharmacopoeia and the method of Bitter et al., to be applied on hydrogels.

[0016] In a preferred embodiment of the invention, the method comprises the following steps: [0017] a. Preparing, as reagent A, a solution of Sodium tetraborate in Sulphuric acid. [0018] b. Preparing reagent B by dissolving Carbazole R in Ethanol. [0019] c. Preparing test solutions, at least in double, by dissolving the substance to be examined in an aqueous solution. [0020] c2. Optionally, heating the test solutions to a temperature higher than room temperature, preferably between 75° C. and 85° C., for at least 15 minutes, preferably for at least two hours, more preferably for about 3 hours, preferably while continuously stirring. [0021] d. Treating the test solution with ultrasounds for a period of time sufficient to obtain a macroscopically homogeneous solution; [0022] e. Preparing a reference stock solution by dissolving glucuronic acid, or a glucuronic acid-containing substance in an aqueous solution; preferably, the concentration of glucuronic acid or of the glucuronic acid-containing substance is comprised between 0.05 and 0.15 wt %. [0023] f. Preparing at least 3 reference solutions by dilution of the reference stock solution in aqueous solution, preferably at concentration comprised between 0.0005% w/v and 0.0100% w/v, preferably between 0.0010% w/v and 0.0050% w/v; [0024] g. Preparing the test tubes by admixing reagent A, reagent B and one of the following: reference solution, test solution, aqueous solution (blank), and optionally solution for interference (crosslinker sample or additive sample); place each test tube on a water bath for at least 5 min, then cool them to room temperature; preferably the water bath is a boiling water bath and the duration is of about 15 min. [0025] h. Reading the absorbance at a wavelength comprised between 500 and 580 nm, preferably at about 530 nm, against the blank and optionally the sample for interference.

[0026] The percentage content of sodium hyaluronate has been calculated by interpolating the experimental absorbance values with the reference standard curve, obtained starting from known concentrations of the substrate. Calculation details: [0027] the mean absorbance value of the blank sample is subtracted to the mean absorbance value of each sample; [0028] the mean value of the Cross-linker sample (if available) is subtracted to the mean absorbance value of each sample; [0029] the percentage content of sodium hyaluronate in the weighted sample is calculated by interpolating the experimental absorbance values of each replicate with the reference standard curve.

[0030] Concerning the interference of crosslinkers and additives, there are two possibilities. If the information concerning the composition of the hydrogel (including type of crosslinker), its quantity and the presence of other additives is available, then it is possible to prepare a solution which comprises additives and crosslinker in the amount in which they are present in the hydrogel (interference solution). In all other cases, the exact composition of the hydrogel cannot be known. Therefore, it is impossible to consider the interference of crosslinker and additives. The percent value of hyaluronic acid obtained at the end of the analysis will be probably somehow higher than the effective amount of HA in the sample.

[0031] The percent content of sodium hyaluronate in the final product is calculated based on the ratio (NaHA %×100)/ g of hydrogel. The percent value of each replicate is subjected to acceptance limits of ±20%. The modal value, the mean value and the standard deviation are calculated.

[0032] Step c2 introduces the step of heating the sample comprising the test solution for 3 h at 75-85° C. This step is not essential and could be removed; however, it is preferred because it helps to improve efficiency of the sonication step which follows immediately after.

[0033] Step d (sonication of the sample) represents the most important contribution to the methods of the prior art. In fact, it has been noticed that after sonication the sample is much more homogeneous than before, and this reflects in a higher reproducibility of the analysis. Although step c2 is useful, it is not necessary to achieve disruption of the hydrogel. However, if step c2 is not used, it is necessary to apply more severe sonication conditions to solubilize the hydrogel.

[0034] In a preferred embodiment, the sonication step is performed using a power comprised between 0.5 W/kg and 50 W/kg, more preferably sonication is performed at a specific power of 0.1 to 10 W/kg. In terms of specific energy used during sonication, it is comprised between 500 J/kg and 30 kJ/kg, preferably between 2 kJ/kg and 20 kJ/kg.

[0035] These parameters are also influenced by the performance of step c2. When step c2 is performed, it is possible to use lower power or lower energy of the sonication treatment. Thus, when the method comprises step c2, the preferred range of energy is between 1 kJ/kg and 10 kJ/kg. When step c2 is not performed, the preferred energy range is between 3 kJ/kg and 30 kJ/kg.

[0036] Another important factor influencing the power and the duration of the treatment is the chemical nature of the crosslinked hydrogel. In general, with each new gel, it will be necessary to perform a few trials to determine the experimental conditions which produce a good dissolution of the gel.

[0037] Steps a and b are preferably performed according to the European Pharmacopoeia, i.e. dissolving 0.95 g of disodium tetraborate in 100.0 ml of sulfuric acid for Reagent A, and dissolving 0.125 g of carbazole in 100.0 ml of ethanol for reagent B.

[0038] In step e, the reference stock solution is prepared by dissolving 50.00 to 150.00 mg of glucuronic acid, or a glucuronic acid-containing substance in 100.00 g of water. As a glucuronic acid-containing substance, it is particularly preferred the use of hyaluronic acid.

Experimental Part

[0039] Analysis of a hydrogel

[0040] Reagent A was prepared by dissolving 0.95 g of Sodium tetraborate in 100.00 ml of Sulfuric acid.

[0041] Reagent B was prepared by dissolving 0.125 g of Carbazole in 100.00 ml of Ethanol.

[0042] Three independent samples of the hydrogel are prepared by dissolving an amount of about 0.170 g in water and bringing the total weight to 100 g with additional water. The hydrogel weight is reported: S1: 0.1723 g; S2: 0.1808 g; S3: 0.1728 g.

[0043] The hydrogel dispersions are heated at 85° C. for 3 hours. Then sonicated with a sonicator Q500 (Qsonica LLC.) at an amplification of 30% corresponding to 0.22 W. After sonication the hydrogel is dispersed in water.

[0044] A reference solution stock is prepared by dissolving 50 mg of HA in 100 g of water. From this solution, 5 solutions are prepared at the following concentrations (wt %): 0.001, 0.002, 0.003, 0.004, 0.005. In Table 1, the absorbance data, read at 530 nm, obtained from the standard curve are reported. The mean values and standard deviations are calculated for the replicates for each point of the standard curve.

TABLE-US-00001 TABLE 1 % of standard Mean absorbance Standard deviation 0 0.049 0.0009 0.001 0.062 0.004 0.002 0.070 0.001 0.003 0.079 0.002 0.004 0.088 0.002 0.005 0.098 0.002

[0045] The mean value obtained for the blank (0.000% of substance containing glucuronic acid) is subtracted to the mean value obtained for each point of the standard curve (Table 2). The R value and the standard curve's equation are calculated in FIG. 1.

TABLE-US-00002 TABLE 2 % of standard Absorbance 0 0 0.001 0.013 0.002 0.022 0.003 0.030 0.004 0.039 0.005 0.049

[0046] 5.0 ml of freshly prepared Reagent A were added to each test tube, previously cooled to 4° C. Each sample was carefully layered onto Reagent A in the test tube, as follows: Blank: 1.0 ml of water R; Reference solutions: 1.0 ml of each dilution of the Reference stock solution; Samples: 1.0 ml of each Test solution. The tubes were gently shaken to dissolve the double-phase created by the sample. To each test tube 0.20 ml of reagent B were added. The tubes were recapped, shacked, and put again on a water-bath for exactly 15 min. They were cooled to room temperature. Each sample was analyzed for the absorbance in double, each one for eight replicas. The mean absorbance value for the samples was calculated (Table3).

TABLE-US-00003 TABLE 3 Sample S1 S1 S2 S2 S3 S3 0.100 0.099 0.088 0.080 0.081 0.095 0.098 0.099 0.084 0.080 0.079 0.095 0.097 0.099 0.085 0.082 0.080 0.094 0.100 0.099 0.085 0.082 0.082 0.095 0.100 0.096 0.087 0.083 0.084 0.093 0.100 0.097 0.087 0.083 0.079 0.093 0.098 0.098 0.091 0.082 0.079 0.095 0.097 0.098 0.087 0.083 0.084 0.094 Mean 0.0988 0.0981 0.0868 0.0819 0.0810 0.0943 absorbance value

[0047] For each absorbance value obtained for the samples, the mean absorbance value of the blank sample was subtracted (Table 4).

TABLE-US-00004 TABLE 4 Sample S1 S1 S2 S2 S3 S3 0.0513 0.0503 0.0393 0.0313 0.0323 0.0463 0.0493 0.0503 0.0353 0.0313 0.0303 0.0463 0.0483 0.0503 0.0363 0.0333 0.0313 0.0453 0.0513 0.0503 0.0363 0.0333 0.0333 0.0463 0.0513 0.0473 0.0383 0.0343 0.0353 0.0443 0.0513 0.0483 0.0383 0.0343 0.0303 0.0443 0.0493 0.0493 0.0423 0.0333 0.0303 0.0463 0.0483 0.0493 0.0383 0.0343 0.0353 0.0453

[0048] The percentage value referred to the NaHA present in the weighed hydrogel and diluted in 100 g of water, as reported in point C of the method, was calculated from the equation obtained by the standard curve for each absorbance value of the samples. E.g. 0.0513/9.9705=0.00514%.

[0049] The percentage of NaHA present in the final product was calculated, for each absorbance value of the samples (Table 5), with following ratio: A:100=B:x; x=B.Math.100/A

Where:

[0050] A=weight of the hydrogel used for the preparation of the sample;
B=percentage value of the NaHA present in the weighed hydrogel and diluted in water.
For the first sample S, for which it was calculated B=0.00514, we have x=0.00514x100/0.1723=2.98326

TABLE-US-00005 TABLE 5 Sample S1 S1 S2 S2 S3 S3 2.98326 2.92505 2.17733 1.73354 1.87184 2.68442 2.86684 2.92505 1.95544 1.73354 1.75576 2.68442 2.80863 2.92505 2.01091 1.84449 1.81379 2.62638 2.98326 2.92505 2.01091 1.84449 1.92988 2.68442 2.98326 2.75042 2.12186 1.89996 2.04597 2.56834 2.98326 2.80863 2.12186 1.89996 1.75576 2.56834 2.86684 2.86684 2.34375 1.84449 1.75576 2.68442 2.80863 2.86684 2.12186 1.89996 2.04597 2.62638 Average 2.91050 2.87412 2.10799 1.83756 1.87184 2.64089 value (%) Standard 0.08084 0.06554 0.12135 0.06914 0.12410 0.05145 deviation

[0051] The modal value is obtained and the acceptance limits (±20%) are calculated on the modal value. * values over the ±20% acceptance limit.

Modal value: 2.87%.

TABLE-US-00006 TABLE 6 NaHA (%) Acceptance limits 2.98 104.25 2.98 104.25 2.98 104.25 2.98 104.25 2.87 100.00 2.87 100.00 2.87 100.00 2.87 100.00 2.87 100.00 2.81 97.87 2.81 97.87 2.75 95.75 2.69 93.62 2.68 93.35 2.68 93.35 2.68 93.35 2.68 93.35 2.63 91.61 2.63 91.61 2.57 89.59 2.57 89.59 2.34 81.75 2.18 *75.95 2.12 *74.01 2.12 *74.01 2.12 *74.01 2.05 *71.37 2.05 *71.37 2.01 *70.14 2.01 *70.14 1.96 *68.21 1.93 *67.32 1.90 *66.27 1.90 *66.27 1.90 *66.27 1.87 *65.29 1.84 *64.34 1.84 *64.34 1.84 *64.34 1.81 *63.27 1.76 *61.24 1.76 *61.24 1.76 *61.24 1.73 *60.47 1.73 *60.47

[0052] The mean % value of NaHA and the standard deviation were calculated among the values gathered inside the acceptance interval. E.g.: mean value=2.76%; standard deviation: 0.16%.