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Order Description need annotated bibliography for this article i will attach with file. METHOD FOR QUANTITATIVE DETERMINATION OF VARIOUS FORMS OF WATER IN BIOLOGICALLY ACTIVE SUBSTANCES B. N. Boiko,1,* I. M. Kolpakov,1, 2 and A. A. Uminskii1 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 44, No. 10, pp. 46 – 52, October, 2010. Original article submitted June 24, 2009. The content of water in substances and preparations based on biologically active materials determines to a considerable extent their biopharmaceutical properties and stability. A new method has been developed for separate quantitative determination of water in various bound forms in biologically active substances isolated from raw plant materials. Dihydroquercetin is used as an example to demonstrate that the proposed method is capable of determining water fractions with various degrees of binding in a sample. Key words: water in biological substances, quantitative determination. Water contained in substances and preparations of biologically active compounds determines to a considerable extent the level of their biological activity and stability. Not only the total water content but also its distribution among fractions with various bond strengths to the active compound is important. Various methods and modes of drying influence the distribution and determine the quality of the resulting product. The goal of the present study was to develop a method for separate determination of the quantitative content of water with different bond strengths in biologically active substances obtained from raw plant material. Dihydroquercetin (DHQ) substance was selected as the studied sample. Let us differentiate fractions of free water, weakly bound water, and strongly bound water. The lack of uniform terminology obliges us to define these concepts for the present article. According to the common definition, free water will be considered the fraction capable of translocation in the sample under the influence of gravitational forces. The physical properties of this fraction correspond fully to those of water in condensation, evaporation, and crystallization processes. Strongly bound water is the fraction that is called in certain sources constitutional. We will consider it to be the fraction released only with molecular destruction of the principal compound, e.g., as a result of melting or thermal destruction. Weakly bound water will be considered those fractions for which the bond energy under normal conditions does not permit them to translate in the sample under the influence of gravitational forces or to be evaporated freely. However, the increase of their internal energy due to heat absorption upon heating destroys this bond even before the temperature of destruction or melting is reached. The physical nature of these fractions is different. However, they can be classified as follows with respect to thermodynamics. One of the principal modern experimental methods for studying the thermodynamic properties of substances and preparations of biologically active compounds is differential scanning calorimetry (DSC). This method is used in the present study. Interpretation of thermograms Figure 1 shows two DSC thermograms for DHQ samples that were obtained under different experimental conditions. Thermogram 1 was obtained at constant volume with samples packaged in hermetic containers. Thermogram 2 was obtained at constant pressure with samples packaged in non-hermetic containers that allowed free exchange of gas with the atmosphere. The thermograms were produced with subtraction of a baseline consisting of the sample heat capacity changes with temperature. A spline was used to interpolate this curve under the peak for the enthalpy change [1]. 574 0091-150X/11/4410-0574 © 2011 Springer Science+Business Media, Inc. Pharmaceutical Chemistry Journal Vol. 44, No. 10, 2011 1 Institute of Biological Instrumentation, RAS, Pushchino, Russia. 2 Pushchino State University, Pushchino, Russia. * e-mail: [email protected]; [email protected]; [email protected] Free and weakly bound water and traces of EtOH, which was used in the DHQ production process [2, 3], evaporated in both thermograms in the temperature range from the start to 122°C. Endothermic peak A corresponds to these processes. Exothermic peak B in the temperature range 134.8 – 148.3°C was studied before [4]. It was identified only in samples that were not heated above 40°C during drying and only for samples with non-hermetic packaging, i.e., under conditions of free gas exchange with the atmosphere. We suppose that the peak is present only in samples in which the molecules had their native structure in the preparation and retained it during drying. It corresponds to the energy of an oxidation reaction involving oxygen of air. This is consistent with the fact that this peak is missing for samples in hermetic packaging. The presence and specific energy of this peak can act as a quality criterion for both the DHQ preparation and the manufacturing process. Endothermic peak C in the rather narrow temperature range 220 – 258°C has a high specific energy and corresponds to DHQ melting (Table 1). The crystalline structure of DHQ is destroyed, releasing strongly bound water [5, 6]. The last broad endothermic peak D in the range 260 – 280°C is thermal destruction of the melt and release of gas from all destroyed DHQ constituents and certain impurities. The goal of the present study was to determine the physical properties of the fractions forming peak A (from 30 to 122°C). The shape of peak Ais complicated because the temperatures of the processes in the three fractions that it represents overlap. The shape of peak A obtained in thermogram 2 can be represented as the sum of three fundamental peaks. This is shown in Fig. 2. Peak 1 corresponds to evaporation of EtOH distributed throughout the sample. The shape of this peak is Gaussian for the model. Peak 2 reflects the difference between curve A and peaks 1 and 3. Its shape is an asymmetric Gaussian curve. It correlates well with evaporation of free water before the start of and during boiling. Peak 3 corresponds to evaporation of weakly bound water. The evaporation mechanism is determined by the type and energy of the bond. This peak also is represented well by a Gaussian curve. These models were used in the procedure for separating the peaks. The deviation of the curve obtained by summing peaks 1, 2, and 3 from that of peak A on the thermogram had a mean-square deviation of %, as will be shown in the results. The energy of peaks 1, 2, and 3 includes the phase-transition energy of the fraction into the gaseous state and the work for expansion of the resulting gas phase of this fraction. The processes are described by equations of the first law of thermodynamics and the Mendeleev – Clapeyron equation. EXPERIMENTAL PART DHQ substance of 98% purity from the bottom end of Siberian and Daur larch that was obtained from the Laboratory of Technology and Equipment for Complex Processing of Raw Plant Material, IBP, RAS (Pushchino, Russia) was used as the studied sample. The purity of the sample was confirmed by NMR methods on an AM-300 spectrometer (Bruker, Germany). PMR spectra were recorded in DMSO-d 6 . Resonances in PMR spectra of DHQ in DMSO-d 6 were assigned in the range 0.5 – 12 ppm [5]. The studies were carried out in a thermal analysis system consisting of a differential scanning calorimeter (DSC) (DSM-10MA, IBP, RAS), a computer, and DSMCALC applied programs that enabled the temperatures, energies, and areas of peaks recorded on thermograms to be determined. Metrological characteristics of the instrument were calibrated using certified standards for temperatures and energies of phase transitions for naphthalene (80.28 0.2°C, 150 J/g), indium (156.45 0.2°C, 28.44 J/g), and tin (231.75 0.3°C, 60.67 J/g) according to methodical instruc- Method for Quantitative Determination of Various Forms 575 20 34 48 62 76 90 104 118 132 140 160 174 188 202 216 230 244 258 272 286 endo exo 4 2 0 –2 –4 À À Â Ñ Ñ D D dP, mW 1 2 T, °C Fig. 1. DSC-thermograms for hermetic and non-hermetic sample packaging. tions MI 496–84. All thermograms for calibration and experimental samples were recorded at scan rate 8K/min and sensitivity range 1 for heat flux and temperature from 20 to 300°C. Weights were measured on a Sartorius R200D balance (Germany). The studied samples were divided into two groups: Group 1 included samples with similar weights of 5 0.25 mg. Samples were packaged in hermetic and non-hermetic containers; Group 2 included samples with weights from 1 to 10 mg in steps of 1 0.15 mg. Only samples packaged in non-hermetic containers were studied. All samples were sealed hermetically in aluminum containers after weighing in order to exclude absorption of moisture from the atmosphere before the start of the measurement. Containers with samples intended for non-hermetic measurement conditions were punctured immediately before the measurement. The results were reproducible if at least five punctures of ~0.5-mm diameter were made. All measurements of group 1 were made in series of at least 10 samples for each of the experimental conditions. The series had at least 3 samples for group 2. Some of the group 1 samples with non-hermetic packaging were scanned up to 122°C, after which they were weighed. The results were used to determine the total mass loss of the sample caused by the processes in the peak: [Equation (1), R. p. 48], m = m init – m 122 , (1) where m is the total mass loss of the sample (mg); m init , the initial mass of the sample (mg), m 122 , the mass of the same sample after scanning to 122°C (mg). The quantity m represents the total mass of fractions evaporated from the sample during scanning to 122°C: m = m 1 + m 2 + m 3 , (2) where m is the total mass of the fractions (mg); m 1 , the mass of the EtOH fraction (mg); m 2 , the mass of the free water fraction (mg); m 3 , the mass of the weakly bound water fraction (mg). The mass fraction i of each peak relative to the total mass loss m is: 576 B. N. Boiko et al. TABLE 1. Parameters of Group 1 Peaks for DHQ Samples with Hermetic and Non-hermetic Packaging Parameter Peak designation A(1) A(2) B(1) B(2) C(1) C(2) D(1) D(2) Melting point, °C 58.74 2 42.22 1.45 – 134.81 52* 241.409 0.11 240.09 0.67 259.89 0.54, 258.81 1.52 Peak maximum, °C 98.44 0.22 100.06 0.23 – 139.05 0.45 246.79 0.17 246.26 0.43 268.41 1.11 268.3 1.48 Specific heat of fusion, J/g 313.77 1.51 331.52 2.24 – 28.26 2.22 31.956** 134.497 1.52 141.603 2.24 161.149** 9.32 1.22 10.818 1.8 12.03** Note. Curve number is shown in parentheses. * Temperature of start of DHQ oxidation. ** Specific heat per DHQ dry weight (initial weight from which the total amount of free and weakly bound water was subtracted). 20 26 32 38 44 50 56 62 66 74 80 86 92 96 104 110 116 122 120 134 endo dP, mW 2 1 0 Peak 1 Peak 2 Peak 3 T, °C Fig. 2. Peak separation in a sample with non-hermetic container packaging. i m m m m 1 2 3 100%. (3) The mass of each fraction was determined by estimating the contribution of the fractions to the measured total energy of peak A by separating peaks according to the aforementioned interpretation. Considering that the energy distribution over the fractions is proportional to the distribution of the peak areas of these fractions on the thermogram, we obtain: Q QS S i i , (4) where Q is the total energy of peak A (mJ); S, the total area of peak A (area units); Q i , the energy of processes for fraction i (mJ); S i , the peak area of fraction i (area units). The mass of the EtOH fraction was calculated using its known specific heat of vaporization [7]. The small values of the relative temperature change in the examined EtOH evaporation process enable this process to be considered not only isobaric but also isothermal. The effect of the uncertainty of this assumption on the resulting estimates is small because the mass fraction of the determined fraction is small. Joint solution of the Mendeleev—Clapeyron equation and the first law of thermodynamics with respect to the mass gives: m Q q RT M 1 1 1 , (5) where m 1 is the mass fraction of EtOH (mg); Q 1 , the vaporization energy of the EtOH fraction (mJ); q 1 , the molar vaporization energy of the EtOH fraction (4.81 106 mJ/mol); R, the molar gas constant [8310 mJ/(mol·K)]; M, the molar mass of the EtOH fraction (46,069 mg/mol); T, the average temperature of the process (K). For the free water fraction, the temperature change and dependence of specific heat of vaporization on temperature, which is described by the empirical formula Eq. (6) [8], should be taken into account: q 2 (t ) = (25 – 0.024t ) 103, (6) where q 2 (t) is the specific heat of vaporization of the free water fraction (mJ/mg); t, the temperature at which the free water fraction evaporates (°C). Method for Quantitative Determination of Various Forms 577 TABLE 2. Physical Properties of Fractions Forming Peak A Peak Parameter Qi, mJ qi, J/g i, % Mole H2O/mole DHQ 1 91.339 839.9* 0.246 0.014 0.018** 2 946.173 varies from 2400 to 2269 using Eq. 6 9.428 0.31 0.911 3 455.365 3766.486 14.11 2.711 0.171 0.523 * Handbook value. ** Mole C 2 H 5 OH (peak 1 corresponds to evaporation of EtOH); i, peak number. 20 34 48 62 76 90 104 118 132 146 160 174 188 202 216 230 244 258 272 286 endo exo 4 2 0 –2 –4 À À Â Ñ Ñ D D dP, mW 1 2 T, °C Fig. 3. DSC-thermograms of group 1 samples with hermetic and non-hermetic packaging. Therefore, Eq. (5) is valid for the small range of temperature changes in which the process can be considered to be isothermal with the specific heat of vaporization corresponding to this temperature. Taking into account the work of vapor expansion for this range, we obtain: m dQ q t R t M 2 2 2 27315 ( ) ( . ) , (7) where m 2 is the mass fraction of evaporated free water due to energy dQ at temperature t (mg); dQ 2 , the absorbed energy of the free water fraction at temperature t (mJ); R, the molar gas constant [8310 mJ/(mol·K)]; M, the molar mass of the free water fraction (18,000 mg/mol). The mass fraction of free water dm 2 was estimated by numerical integration of Eq. (6). Taking into account the work of vapor expansion gives a correction of the order of 0.03 mg for the mass of evaporated water (0.4 mg). We determine the mass of weakly bound water from the resulting values m 1 and m 2 according to Eq. (1): m 3 = m – m 1 – m 2 . (8) Two parameters for the weakly bound water fraction are interesting. These are the molar mass relative to the molar mass of DHQ, which indicates the amount of water bound to 578 B. N. Boiko et al. minit = 10,05 mg m = 9,02 init mg m = 8,41 init mg m = 7,13 init mg m = 6,14 init mg m = 3,91 init mg m = 4,95 init mg m = 3,19 init mg m = 1,03 init mg m = 1,97 init mg endo exo dP, ìÂò 6 4 2 0 Peak 1 Peak 2 Peak 3 20 26 32 38 44 50 56 62 66 74 80 86 92 98 104 110 116 122 128 134 T, °C Fig. 4. Separation of fractions forming peak A for group 2 samples with variable weight. one DHQ molecule, and the specific energy of vaporization, which enables the bond energy of this fraction to be estimated. In order to determine the specific energy of vaporization of weakly bound water, we subtract from energy Q 3 the work for expansion of the formed vapor corresponding to mass m 3 at the mean temperature of the peak: Am m RT M 3 3 , (9) where R and M are the same as for Eq. (5) and T is the average temperature for peak 3 (373 K). We obtain: q Q A m m 3 3 3 3 ( ) (10) RESULTS AND DISCUSSION The study of the water content in initial group 1 samples (Fig. 3) showed that DHQ contains 11.91% total water fractions. That amount exceeds the norm indicated in the pharmacopoeial article FS 42-3853-99 and SP XI (less than 7%). Table 2 presents results from the determination of the thermophysical parameters of DHQ group 1 samples. The above measurements for DHQ group 1 samples showed that the fractions forming peak A can be divided into fractions belonging to EtOH and free and weakly bound water, as was shown in Fig. 2. Endothermic peak 1 in the temperature range 46 – 82°C represents energy losses to evaporation. It is identified as EtOH with a maximum near 63°C that is shifted to lower temperature relative to the handbook boiling point of 78°C. This is probably a result of the fact that EtOH manages to evaporate before the start of boiling and is present as an impurity in large amounts of water and DHQ. DSC and NMR data for the quantitative content of EtOH showed about 0.3 and 0.5 mass %. The quantitative content of EtOH can be determined more accurately by DSC methods if it is considered that the vaporization energy of this fraction depends on the temperature at which it evaporates. Endothermic peak 2 in the temperature range 38 – 105°C represents energy losses for vaporization of the free water fraction with a maximum at 89.1°C. The experimental shape of this peak in this temperature range is very typical of free water [9]. DHQ contains mainly this type of water and, Method for Quantitative Determination of Various Forms 579 5000 4500 4000 3500 3000 2500 2000 1500 0 2 4 6 8 10 2 1 Weight, mg qi, J/g Fig. 5. Specific heat of vaporization of fractions as a function of weight change for group 2 samples: free water fraction (1) and weakly bound water fraction (2). TABLE 3. Physical Properties of Fractions Forming Peak A with Variable Weight Peak minit, mg Parameter Qi, mJ qi, J/g {roman mole roman {H sub {2} O}}over{roman mole roman DHQ} 1 1.03 24.592 839.9* 0.067** 1.97 33.671 0.058** 3.19 36.612 0.055** 3.91 55.675 0.042** 4.95 83.756 0.038** 6.14 85.602 0.036** 7.13 92.608 0.031** 8.41 115.342 0.03** 9.02 120.119 0.024** 10.05 126.156 0.021** 2 1.03 150.598 varies from 2400 to 2269 using Eq. 6 1.097 1.97 283.649 1.12 3.19 637.634 1.324 3.91 669.958 1.424 4.95 898.587 1.599 6.14 1264.654 1.411 7.13 1395.195 1.523 8.41 1405.256 1.543 9.02 1420.65 1.521 10.05 1596.63 1.54 3 1.03 58.339 2093.58 0.455 1.97 117.41 2798.062 0.545 3.19 303.943 3043.104 0.668 3.91 313.121 3072.723 0.717 4.95 438.756 3433.171 0.805 6.14 550.484 3636.445 0.868 7.13 718.453 3780.064 0.856 8.41 1070.953 4214.752 0.985 9.02 1900.943 4395.207 1.269 10.05 2292.181 4516.604 1.468 * Handbook value. ** Mole C 2 H 5 OH (peak 1 corresponds to evaporation of EtOH); i, peak number. therefore, it determines the total moisture of DHQ substance. The quantitative content of the free water fraction is about 9.4%. Endothermic peak 3 represents energy losses to evaporation of the weakly bound water fraction with a maximum at 99°C in the temperature range 82 – 122°C. This type of water is stable and difficultly removed by nondestructive drying. However, it is less stable than strongly bound water, the destruction of which occurs only with melting and destruction of DHQ molecules. The quantitative content of the weakly bound water fraction is about 2.7%. Table 2 presents measurements of the physical characteristics of the fractions forming peak A. The measurements reflect well the behavior of the various fractions of peak A if the weights of the group 1 samples are similar enough. Figure 4 shows the separation of the fractions forming peak A for group 2 samples with variable weights. It can be seen that the maximum temperatures of peaks 1 and 2 undergo gradual and small shifts to higher temperatures as the weight increases whereas such behavior is not observed for peak 3. This is due to the fact that temperature gradients throughout the sample in the container and thermal resistance in the thermometer – container – sample chain do not increase in proportion to the weight increase (and the distribution of the sample within the container) [5, 10]. Table 3 presents measurements of the physical characteristics of the fractions forming peak A with variable weight. Figure 5 shows plots of the specific heats of vaporization of the free and weakly bound water fractions as functions of weight change. It can be seen that the specific heat of vaporization of the free water fraction is practically constant as the weight increases. The small trend can be explained by an increase in the evaporation area as the weight increases. A linear dependence is observed for the weakly bound water fraction. The resulting specific heats of evaporization are linearly related to the amount of substance. This dependence can be explained by the presence in DHQ of binding centers for water molecules. The substance first loses molecules from the upper outer layer. The work for release of water molecules from the inner layers is proportional to their path within the substance because they interact with vacated binding centers of higher lying layers, through which they must pass. The path within the substance for these molecules is proportional to the thickness of the substance layer inside the container, i.e., the weight. The parameters of the linear function can be used to estimate the bonding energy of water molecules to DHQ. To a first approximation, extrapolation of the line to zero weight can be used. This gives a value of 2125 J/g. The closest whole-number ratio for the number of DHQ molecules used per molecule of weakly bound water that corresponds to this estimate for the specific energy of weakly bound water would be two DHQ molecules per molecule of weakly bound water. REFERENCES 1. V. P. Egunov, Introduction to Thermal Analysis [in Russian], Samara (1996), p. 156. 2. E. E. Nifant?ev, M. P. Koroteev, G. Z. Kaznev, et al., Zh. Obshch. Khim., 76(1), 164 – 166 (2006). 3. RF Pat. No. 2,249,026 (2005). 4. I. M. Kolpakov, A. A. Radzion, and A. V. Tarasov, in: Abstracts of Papers from the 10 th Pushchino School-Conference of Young Scientists [in Russian], Pushchino (2006), p. 145. 5. I. M. Kolpakov, A. A. Uminsky, and B. N. Boyko, in: Abstracts of the 12 th International Congress Phytopharm 2008, St. Petersburg (2008), Abstr. 59. 6. M. Yu. Vyaznikova, S. S. Nikolaeva, V. A. Bykov, et al., Proceedings of the Scientific-Practical Convention Biomedical Technology [in Russian], 3, 18 – 25 (1996). 7. V. N. Stabnikov, Ethanol [in Russian], Pishchevaya Promyshlennost’, Moscow (1976), p. 142. 8. A. A. Aleksandrov and B. A. Grigor?ev, Tables of the Thermophysical Properties of Water and Steam, Handbook [in Russian], Gos. Sluzhba Standartnykh Spravochnykh Dannykh [State Service for Standard Handbook Data], GSSSD P-776 – 98, MEI, Moscow (1999), p. 28. 9. I. M. Kolpakov, L. M. Chailakhyan, B. N. Boiko, et al., in: Abstracts of Papers from the 11 th Pushchino School-Conference of Young Scientists “Biology, the Science of the XXIst Century” [in Russian], Pushchino (2007), p. 254. 10. B. N. Boiko, Applied Microcalorimetry: Domestic Instruments and Methods [in Russian], Nauka, Moscow (2006), p. 27. 580 B. N. Boiko et al. Copyright of Pharmaceutical Chemistry Journal is the property of Springer Science & Business Media B.V. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. 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