ASSESSING THE HOMOGENEITY OF ACTIMASK® ACETAMINOPHEN AND N-ACETYL GLUCOSAMINE MIXTURE TO PREDICT FURTHER STEPS IN THE DEVELOPMENT OF ORODISPERSIBLE TABLETS

One of the main problems in the production of tablets, which has significant negative consequences, is the segregation of the tablet mixture leading to inhomogeneity of dosage units, material losses in the manufacturing process and improperness of the specified pharmaceutical technical characteristics of the mixture. The aim of the research. This work aims at the pharmaceutical technical study of the substances N-acetyl-D-glucosamine and Actimask® Acetaminophen and determination the uniformity of the powder mixture of active pharmaceutical ingredients (APIs) to predict the optimal technology for obtaining a pharmaceutical formulation with the acceptable properties. Materials and methods. N-acetyl-D-glucosamine (Zhejiang Candorly Pharmaceutical, China) and Actimask® Acetaminophen (SpiPharma, USA) were used. Scanning probe microscope Solver P47N-PRO («NT-MDT», Russia), optical microscope, flowability tester VP-12A, laser diffraction particle size analyzer SALD-2201 («Shimadzu», Japan), liquid chromatograph Agilent 1260 Infinity II with Diode Array Detector (Agilent Technologies, USA), spectrophotometers Shimadzu UV-1800 («Shimadzu», Japan) were used. The study of API pharmaceutical technical properties (microscopic characteristics, moisture absorption capacity, flowability, bulk volume and tapped volume, particle size distribution by sieve analysis and laser diffraction), as well as vibration simulation and following chromatographic study were carried out in this work. Results and discussion. The shape of the particles N-acetyl-D-glucosamine and Actimask® Acetaminophen, which was determined by microscopic analysis, demonstrated the possibility of N-acetyl-D-glucosamine particles to stick to Actimask® Acetaminophen ones. The experimental study allowed to reveal the hygroscopicity of both APIs; poor flowability, unsatisfactory Hausner ratio, and Carr index for N-acetyl-D-glucosamine; excellent flowability, Hausner ratio, and Carr index for Actimask®. Vibration caused segregation of the powder mixture. It was found that all layers do not meet the requirements and an excessive content of Actimask® is registered, which indicates the stratification of the powder mixture. Conclusions. The physical properties of the substances were determined and found to have significant differences in their particle size distribution. Segregation of the mixture after vibration was confirmed by laser diffraction and assay analysis. In order to solve the segregation problem, the granulation of N-acetyl-D-glucosamine may be proposed.


Introduction
Orally Disintegrating Tablets (hereinafter referred as ODTs) represent a modern direction in the development of solid dosage forms. Due to the rapid disintegration in a small amount of fluid, ODTs are characterized by better bioavailability, ease of administration, and less toxic effects of active pharmaceutical ingredients (APIs) on the liver due to partial alleviation of the first-pass effect.
One of the promising candidates for incorporation into the ODTs is Actimask ® Acetaminophen, an antipyretic and analgesic agent characterized by the absence of unpleasant taste sensations due to the gelatin coating applied. According to international recommendations (WHO, Medscape, German Society of general and family medicine, etc.), Acetaminophen is classified as a first-line drug for the relief of chronic pain [1,2]. It should be noted that in comparison with NSAIDs, also recommended for the treatment of chronic pain syndrome (metamizole, naproxen, ibuprofen, etc.), Acetaminophen is considered to be safer due to fewer side effects, primarily such as gastro-and cardiotoxicity [3,4]. However, a significant disadvantage of Acetaminophen is hepatotoxicity in high doses and/or with long-term treatment, which can especially occur in the long-term treatment of chronic diseases [5]. A possible approach to reducing or even avoiding the manifestation of Acetaminophen toxicity is its combined use along with N-acetyl-D-glucosamine. According to the literature, the combination of Acetaminophen with N-acetyl-D-glucosamine reduces hepatotoxicity and potentiates the analgesic effect of Acetaminophen [6]. However, the use of two substances that may differ in their physical and chemical properties (primarily morphology and size distribution) increases the risk of segregation of the tablet mixture representing one of the main problems in tablet preparation development and manufacturing process.
Segregation issues can cause improperness of the specified pharmaceutical technical characteristics of the mixture, material losses in the manufacturing process, and inconsistent tablet batches with unacceptable dosage variations. As a result, the amount of quality product created within a single batch can be reduced by up to 70 % of the total batch [7,8]. In addition, the segregation is responsible for unsatisfactory validation due to the non-reproducibility of the technological process of obtaining the product with an acceptable content of active and auxiliary substances [9,10].
Ensuring homogenous mixing of the tablet mass components is an important factor for obtaining high-quality products. The ideal mixture has a uniform distribution of all components. Uniformity in the traditional technological sense refers only to the distribution of APIs, provided that the excipients are also uniformly distributed.
The homogenization process occurs in competition with segregation hindering perfect mixing. Segregation can be described as the process of spontaneous separation of powder mixture components that was previously uniformly dispersed with each other [8,9]. Segregation occurs because during the movement of the powder mixture (which is a component of industrial production) fractions that differ in physical properties, such as particle size, shape, density, surface roughness, etc., manifest themselves differently [10,11].
The occurrence of segregation is driven by a set of physical and chemical properties of raw materials and production technological features. There are two main mechanisms of segregation in pharmaceutical manufacturing -sieving and fluidization. The first one is regarded as the most common and takes place when there is a large enough size variation of particles in a mixturetypically differences in the mean diameter of 3 times or more. In such case smaller particles move down through larger particles [12,13]. This mechanism is possible when vibration acts on the powder mixture, for instance, during pouring, tableting, etc. Fluidization segregation can occur when a mixture contains a large portion of light or fluffy free-flowing, fine component and a smaller portion of a relatively heavier component. The larger component easily penetrates the fluidized fines, pushing the fines layer to the top of the bin or vessel [14,15]. The fine powder particles can also remain suspended in the air stream, for example, when filling a hopper [16].
The ability of tablet mixture to segregate can be examined in several ways. One of them is based on the special devices that detect segregation in a matter of minutes. For instance, SPECTester © material segregation tester, which uses spectroscopic technology and can analyze a sample consisting of up to six individual components, provides a simple report indicating why and how much the mixture is segregated in terms of component concentrations, particle size differences, and product homogeneity [9,10]. However, such tools may be not available due to their expensiveness.

Pharmacology, Toxicology and Pharmaceutical Science
Another way involves testing the mixtures using the equipment able to simulate vibration and/or fluidization. Various tools are applicable for this purpose. L. Břenkováa et al. used the special device (pipe) made of stainless steel with a lid of length 250 mm, and an inner diameter of 25 mm. Mixture homogeneity testing on a laboratory scale was performed by selecting 5-7 samples along with the axial position of the segregation tube after vibrating the device along the axis (amplitude 40-300 mm, frequency 1-4 Hz) for 10-60 minutes. The quantitative composition was studied by HPLC [17]. M. Jaklič et al. used in their work a transparent pipe that was divided into two parts with a mechanically opening valve between them. The test substance was placed in the upper pipe, the sliding valve was opened and the mixture was poured down. A fluidized bed was formed due to dynamic air from the bottom up. Samples were taken from several layers and the composition was studied by laser diffraction. However, only monocomponent powders were investigated for segregation ability [18].
The aim of this work is to carry out the pharmaceutical technical study of N-acetyl-D-glucosamine and Actimask ® Acetaminophen and determine the segregation ability of their mixture in order to substantiate further steps in the development of pharmaceutical preparation with acceptable quality attributes.

Materials and methods
N-acetyl-D-glucosamine (Zhejiang Candorly Pharmaceutical, China) and Actimask ® Acetaminophen (SpiPharma, USA), which is acetaminophen substance coated with a gelatin shell (active ingredient content is 93.2 %), were used in this study.

Experimental procedures
Pharmaceutical technical properties were studied according to conventional methods: microscopic analysis, flowability, bulk density, tapped density, Carr index, Hausner ratio, and moisture absorption capacity [19].
Vibration. The vibration effect on the tablet mixture was simulated using a cone funnel with a closed bottom. The model mixture was prepared in the amount of 100 g by mixing Actimask ® acetaminophen and N-acetyl-D-glucosamine in a ratio of 4:1, respectively, based on 100 % of active substances. Immediately after mixing the homogeneous mixture was placed in a funnel connected to a vibrating device. After 10 min of vibrating the test samples were taken in three sites of the mixture -upper, middle, and lower ( Fig. 1). For quantitative determination, the API mixture from each site was compressed into tablets with a nominal weight of 500 mg.

Fig. 1. Sampling sites
Measurement of the size distribution of powder particles by laser diffraction. The particle size and size distribution were determined by laser diffraction (Ph.Eur. 2.9.31). For this test, samples of the mixture before and after vibration were used.
A mixture of ethyl acetate R -vaseline oil (1:2) was used as a medium to prepare the dispersion of the powder sample to be analyzed. 50 mg of the powder was placed into a 20 ml volumetric flask and thoroughly mixed with 10 ml of ethyl acetate R -vaseline oil (1:2) medium until the Pharmacology, Toxicology and Pharmaceutical Science powder particles were completely wetted. Then the volume of the dispersion was brought to the mark and placed in an ultrasonic bath with a power of 50-100 W for 1 min, thoroughly mixed and then the sample was immediately taken for the measurement.
Acetaminophen assay by spectrophotometry. The tests were performed by absorption spectrophotometry in the ultraviolet and visible range according to the requirements of Ph.Eur. 2.2.25.
Test solution. 10 tablets were weighed and the average weight of the tablets was determined. The weighed tablets were powdered and mixed thoroughly. To the precisely weighed amount of powdered tablets equivalent to 150 mg of acetaminophen, 80 ml of 0.01 M sodium hydroxide solution heated to about 60 °C was added, shaken for 20 min, and cooled. The volume of the solution was adjusted to 250.0 ml with the same solvent, stirred, and filtered through a 13 mm diame ter PTFE (hydrophilic) membrane filter with a pore size of 0.45 μm, discarding the first 3 ml of filtrate. The resulting solution was used to prepare a solution with a acetaminophen concentration of 6 μg/ml in 0.01 M sodium hydroxide solution.
Reference solution. The solution of acetaminophen pharmacopeial reference standard (PhRS) in 0.01 M sodium hydroxide solution with a concentration of 6 μg/ml acetaminophen was prepared.
Compensation solution. 0.01 M sodium hydroxide solution.
The optical density of the test solution and the reference solution was measured at a wavelength of 257 nm relative to the compensation solution.
The content of C 8 H 9 NO 2 in the tablet was calculated based on the average tablet weight and the declared content of C 8  Test solution. 10 tablets were weighed and the average weight of the tablets was determined. The weighed tablets were powdered and mixed thoroughly. To the precisely weighed amount of powdered tablets equivalent to 25 mg of N-acetyl D-glucosamine, 150 ml of water R was added and shaken for 20 min. The volume of the solution was adjusted to 250.0 ml with the same solvent, mixed, and filtered through a membrane PTFE (hydrophilic) filter with a diameter of 13 mm and pore size of 0.45 μm, discarding the first 3 ml of filtrate (concentration of N-acetyl D-glucosamine 100 μg/ml).
Reference solution. The solution of N-acetyl D-glucosamine PhRS in water R with a 100 μg/ml N-acetyl D-glucosamine concentration was prepared.
Phosphate buffer solution pH 6.0. 2,722 g of potassium dihydrogen phosphate R was dissolved in 900 ml of water for chromatography R. 1 M solution of potassium hydroxide was added to pH 6.0, and then the volume of the resulting solution was adjusted to 1000 ml with water for chromatography R and stirred.
The comparison solution and the test solution were chromatographed using a high-performance liquid chromatograph with diode array detector (Agilent Technologies, USA), obtaining the required number of chromatograms, under the following conditions: -column: Spherisorb Amino (NH 2 ), 80Å (P.N.: PSS832313), size 4.6 mm × 150 mm, with a particle size of 3 μm or similar, which comply with the requirements of the test «Checking the suitability of the chromatographic system»; -mobile phase: acetonitrile K1 -phosphate buffer solution pH 6.0 (75:25); -column oven temperature: 25 °С; -rate of mobile phase: 0.6 mL/min; -detection wavelength: 194 nm; -injection: 20 μl; -chromatography time: 2.5 times longer retention time of N-acetyl D-glucosamine. Suitability of the chromatographic system: -the number of theoretical plates calculated from the peak of N-acetyl-D-glucosamine on the chromatogram of the reference solution must be at least 600; -the symmetry factor calculated for the N-acetyl-D-glucosamine peak on the chromatogram of the reference solution must be not less than 0.8 and not more than 1.5; -the convergence calculated for the areas of the N-acetyl-D-glucosamine peaks, on the reference solutions chromatograms, should not exceed the values given in Table 1.

Results
At the first stage, API microscopy was performed. The shapes of particles and their surface character are determined (Fig. 2). As it can be seen from the micrograph depicted in Fig. 2, a, N-acetyl-D-glucosamine is formed by particles of different sizes, but the same shape (the length is much longer than the width). The particle surface has small roughness, facilitating the adhesion of smaller particles to larger ones.
The shape of Actimask ® Acetaminophen particles is more uniform, as compared to N-acetyl-D-glucosamine, and close to spherical (Fig. 2, b). The particle surface has fragmentary small hollows where fine particles stick. From the micrographs, it can be supposed that the particles of the studied substances could form conglomerates due to sticking finer to coarser ones. The next step included the microscopy of Actimask ® -N-acetyl-D-glucosamine combination in a ratio of 4:1 (selected based on the previous pharmacological studies). As it can be seen from the micrograph depicted in Fig. 3, small particles of N-acetyl-D-glucosamine are layered on the roughness of the Actimask ® surface and form a conglomerate (N-acetyl-D-glucosamine is highlighted white on Actimask ® sphere). This indicates the possibility of direct mixing of the APIs to obtain a homogeneous mixture.

Pharmacology, Toxicology and Pharmaceutical Science
The bulk properties of the APIs and their mixture (4:1) are given in Table 2. As it can be seen from the above data, N-acetyl-D-glucosamine powder is a light, dust-forming substance with insufficient flowability. Actimask ® has excellent flowability, suggesting its easy dosing, mixing, and less material loss during the manufacturing process of tablets. Mixing 1 part of N-acetyl-D-glucosamine and 4 parts of Acetaminophen gives a practically homogenous blend. This fact may be explained by coating coarser particles of Acetaminophen with finer particles of N-acetyl-D-glucosamine. The pharmaceutical technical properties of the mixture are better than that of pure N-acetyl-D-glucosamine, and worse than that of Actimask ® . However, the combination study suggests that the flowability is satisfactory, and bulk and tapped densities are within acceptable limits.
The percentage of particle size fractions for both APIs was determined by sieve analysis. The results obtained are shown in Fig. 4. N-acetyl-D-glucosamine particles are quite different in their sizes, and this fact indicates the heterogeneity of the API (Fig. 4, a). The most percentage of 51.53 % is the fraction with particles from 1 to 2 mm. Actimask ® substance appears to be more homogenous in its particle size distribution: the dominant fraction (82.11 %) consists of particles ranging from 0.1 to 0.5 mm (Fig. 4, b). Due to the differences in the particle size distribution of N-acetyl-D-glucosamine and Actimask ® , stratification of the mixture is possible during mixture pouring and die filling in direct compression, which, in turn, can affect the uniformity of the APIs in dosage units. a -N-acetyl-D-glucosamine; b -Actimask ® Acetaminophen A further step was devoted to the determination of moisture absorption capacity of the substances being studied (Fig. 5).
The initial moisture contents in APIs were 1.99 and 0.40 % for Actimask ® and N-acetyl-D-glucosamine, respectively. Under relative humidity (RH) of 100 %, the Actimask ® moisture content raised to 9.74 % per 24 hours, while that of N-acetyl-D-glucosamine reached 4.63 %, Pharmacology, Toxicology and Pharmaceutical Science i.e., half as much. Under RH of 40 %, Actimask ® and N-acetyl-D-glucosamine absorbed water up to 2.18 % and 0.75 %, respectively. Therefore it is possible to conclude, that the substances are stable under normal humidity levels.
The particle size analysis was also performed by the laser diffraction method. At the first stage, APIs, Actimask ® Acetaminophen and N-acetyl-D-glucosamine, were studied separately.
According to Fig. 6 and Table 3, a median size of Actimask ® particles is 446.68 µm, while the particle size range is from 301.73 (10 %) to 874.69 (90 %) µm.   Pharmacology, Toxicology and Pharmaceutical Science The median particle size of N-acetyl-D-glucosamine is 44.67 µm ( Fig. 7 and Table 4). The particles range from 16.06 (10 %) to 391.76 (90 %) µm. The obvious discrepancy between sieve analysis and laser diffraction results can be explained by the electrostatic interaction of the substance with metal sieves which is attributable to the presence of an N-acetyl moiety. Apparently, this led to the remaining finer particles on the coarser mesh sieves. As a result, incorrect fractioning was observed. Taking into account the median particle size of APIs, it is supposed that the mixture of them cannot be homogeneous. To verify it laser diffraction study of the mixture (Actimask ® with N-acetyl-D-glucosamine in a 4:1 ratio) was performed directly after mixing and after exposure to vibration (simulating possible vibration and stratification during production). After vibration, samples were taken from three layers: upper, middle, and lower. The results were obtained by laser diffraction are shown in Fig. 8 and Table 5.

Fig. 7. N-acetyl-D-glucosamine particle size
The mixture before vibration shows a medium particle size of 446.68 µm which is greater than that of N-acetyl-D-glucosamine and corresponds to the Actimask ® . But the fractional percentage has a larger range -from 194.43 (10 %) to 977.19 µm (90 %). This is due to the presence of N-acetyl-D-glucosamine, which is a fine powder. An increase in maximum size is possible due to the agglomeration of APIs. As it can be seen from Fig. 8, the upper and lower layers after vibration have similar particle size ranges -from 43.29 (10 %) to 506.25 (90 %) µm (for the upper layer) and 50.33 (10 %) to 508.92 (90 %) µm (for the lower layer). The mean particle size is 141.25 and 177.83 µm, respectively. At the same time, the middle layer of the mixture showed the mean particle size of 354.81 µm after vibration and particle size range from 142.99 (10 %) to 939.53 (90 %) µm.   Thus, according to the results obtained, it may be supposed the heterogeneity of the mixture. The upper and lower layers have more fine particles, i.e. N-acetyl-D-glucosamine particles. The middle layer has more agglomerated particles extending beyond 1000 µm. N-acetyl-D-glucosamine sticks to Actimask ® to form larger particles than pure substances. However, the adhesive properties of the substances appear to be insufficient for proper adhesion to each other.
In parallel, a chromatographic assay of APIs in tablets compressed from the mixture sampled before vibration and three layers formed after vibration was performed. The amount of acetaminophen and N-acetyl-D-glucosamine was determined experimentally in terms of 100 % of active substances. The results of chromatographic assay study are given in Table 6. The obtained data confirm the laser diffraction results on the occurrence of segregation in the API mixture. According to the requirements of European law [20] regarding the assay limits in medicines, the maximum acceptable deviation in the active substance content of the finished product shall Pharmacology, Toxicology and Pharmaceutical Science not exceed ± 5 % at the time of manufacture. That is, before vibration, the mixture met the abovementioned requirements, whereas exposing it to vibration has changed the quantitative ratio of APIs and the amount of Acetaminophen increased in all three layers. The lower and upper layers lost 35.53 and 31.05 % of N-acetyl-D-glucosamine, respectively, from its nominal content in tablets. The middle layer lost 12.73 % of N-acetyl-D-glucosamine, which, nevertheless, is also outside the acceptable limits.
It could be clearly seen that the percentage of N-acetyl-D-glucosamine did not increase in all three layers formed after vibration, but only reduced: to a greater extent in the upper and lower layer dosage units and a smaller extent -in the middle layer ones. Obviously, this fact could not be explained by the only mixture segregation in the tableting process. So, taking into account our previous results obtained in this work, especially those of sieve analysis and laser diffraction study, it is possible to conclude that inhomogeneity also occurs due to the sticking of N-acetyl-D-glucosamine to the metal vibrating cone. Sticking can be explained by electrostatic charging of glucosamine powder during friction against the funnel wall, most likely, due to amino group moiety (Fig. 9). Glucosamine has electrostatic properties that were demonstrated in the work of Yang Zhong et al. The paper describes the electrostatic activity of glucosamine particles between various substances in the liquid and gas phases [21]. Thus, it could be assumed that passing the mixture being studied through a funnel during vibration cause adhesion of N-acetyl-D-glucosamine to the metal and, accordingly, reduction its content in all three mixture layers after vibration. A possible solution to the segregation problem, as well as electrostatic adhesion of powder to metal surfaces, is coarsening N-acetyl-D-glucosamine particles up to the size of Actimask ® by means of granulation. The proposed approach is expected to reduce the electrification of N-acetyl-D-glucosamine.

Discussion
The results of our work convincingly evidence that the mixture of APIs with different particle sizes become segregated in the manufacturing process of the tablets. Although sticking of N-acetyl D-glucosamine particles to Actimask ® (as supposed due to electrostatic activity of the first ones) was observed, it appeared to be not sufficient to prevent segregation. It should be noted that the conclusion was made based on several methods used in this study. Thus, the laser diffraction technique showed particle size distribution which was discrepant from sieve fraction analysis results that are supposed to be linked to electrostatic properties of N-acetyl D-glucosamine. Anyway, laser diffraction was considered as preliminary data which have to be verified by assay methods. HPLC study of the mixture after vibration exposure strongly indicated that segregation of the API powders occurs, so our further work will be aimed at searching for an optimal way to prevent segregation of the mixture.
The problem of powder segregation is an important technological aspect in the development of solid dosage forms, which has been described in earlier scientific works as well. E.g., M. Jaklič et al. have conducted an experimental study inspecting the segregation of direct compression mixtures of fillers and API with different particle sizes. For this purpose, straight pipes with different bottomshermetic and non-hermetic -were used. Segregation was detected by fluidizing the mixture: large particles are moved downwards while small particles are displaced upwards and attached to the a b Pharmacology, Toxicology and Pharmaceutical Science surface of the device [18]. In another work, the authors applied the simulation of fluidization followed by sampling and HPLC analysis of the mixture to determine whether it underwent segregation. It was found that segregation possibility is associated not only with different sizes of the component particles, but also the flow rate of the mixture during its pouring out [11]. L. Devriendt et al. have found that segregation, although dependent on the size of individual particles, is also affected by the amounts and ratio of the components. That is, it is possible to eliminate segregation by selecting the «ideal» ratio between large and small particles [22]. However, this way is applicable only in the case of API-excipient mixture, when the proportion of the latest may be varied. Study limitations. Nevertheless, considering the findings of this study some limitations should be taken into account. In our opinion, the main limitation consists in the electrification of N-acetyl D-glucosamine particles as a result of the friction with metal parts of the tools. Other equipment that does not provoke electrification may be used to remove this restriction. However, on an industrial scale, metal equipment is commonly used, so in fact, this limitation reflects real manufacturing conditions. Another issue is that only vibration but not fluidization was applied to investigate segregation. However, the vibration as a way to cause segregation was chosen to take into account the smaller ratio of fine particles in the mixture and such a case sieving mechanism of segregation driven by vibration is most likely to occur.
The prospects for further research are to find a formulation approach allowing to obtain the homogenous mixture that will not be segregated in the process of tablet manufacture. A possible solution is to enlarge N-acetyl-D-glucosamine particles up to Actimask ® Acetaminophen by the granulation technique.

Conclusions
1. Microscopic analysis was used to describe the morphology of Actimask ® Acetaminophen and N-acetyl-D-glucosamine particles and their physical interaction with each other (sticking to each other). Surface roughness that can affect the pharmaceutical technical properties of the APIs and their mixture is revealed.
2. The pharmaceutical technical properties of APIs and their mixture, moisture absorption capacity, and the percentage of the particle size fractions are established. It is found that Actimask ® has excellent flowability, while N-acetyl-D-glucosamine, on the contrary, is characterized by very poor flow. However, mixing these components results in the averaged values of the indicators. Acetaminophen and N-acetyl-D-glucosamine can be regarded as moisture-stable substances, but having particles of different sizes, which is a factor of possible segregation of their mixture.
3. The homogeneity of the mixture was checked by laser diffraction and chromatographic examination. A vibration funnel was used to simulate the effect of vibration during the production process. Segregation of the mixture under the effect of vibration, which can occur in production, was confirmed. The reason for the stratification of the mixture is probably the difference in particle size between the APIs and the physical properties of N-acetyl-D-glucosamine (a fine powder is prone to the formation of an electrostatic charge and sticking to the metal surfaces of the manufacturing equipment). 4. A possible solution to the problem of segregation of the mixture is coarsening the N-acetyl-D-glucosamine particles up to the size of Actimask ® through granulation. The proposed approach is also expected to reduce the electrostatic activity of N-acetyl-D-glucosamine powder.