Pharmaceutical Chemistry Journal

Vol. 39, No. 1, 2005

COMPOSITION OF GLUNAT PREPARATION STUDIED BY 13C NMR SPECTROSCOPY V. P. Panov1 and A. V. Panov2 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 1, pp. 51 – 53, January, 2005. Original article submitted January 21, 2003.

The composition of the blood plasma preparation Glunat from different producers and several model systems was studied by 13C NMR spectroscopy. It was established that the 13C NMR spectra of solutions of lyophilized samples of Glunat are identical to the spectrum of a dextrose solution autoclaved under the same conditions in sodium hydrocarbonate without addition of donor plasma. Partial condensation of the C1 – C4 and C1 – C6 types, rearrangement, and fragmentation of D-glucopyranose molecules was observed. The fraction of condensation products amounts to about 30%. The composition of Glunat preparation is not exhaustively characterized in the existing normative documentation. It is necessary to substantially revise the normative documentation, in particular, to add the section “Accompanying Compounds” and to determine the active component in this preparation.

The blood plasma preparation Glunat is intended for intramuscular injection. This preparation (used since 1972) was initially called “sterilized serum F” (Pharmacopoeial Article VFS 42-165-72) and given the present name in1986 (VFS 42-165-86). Glunat is injected for enhancing regenerative processes and stimulating the protective function. This treatment is used in cases of heavy skin ulceration, gastric and duodenal ulcers, pneumonia, etc. Commercial Glunat [1] is available in the form of solutions containing glucose (27.3 g/liter) and sodium hydrocarbonate (5.43 g/liter), and up to 25 wt.% of human blood plasma or serum. The solution is sterilized in an autoclave at 122.6°C for 70 min until reaching pH 6.0 – 7.0, cooled to room temperature, filtered under pressure through Sal’nikov and Schott filters, and distributed in 5.3-ml portions into ampules made of neutral glass. This preparation is presently manufactured according to the commercial technology at several hemotransfusion stations and plants of bactericidal preparations. The quality of Glunat according to the aforementioned standard (VFS 42-165-86) [2] is evaluated in terms of identity, appearance (coloration), protein content (according to Kjeldahl), glucose content, pyrogenicity, toxicity, and sterility [3]. The protein content is rated on a level of 1.5 – 2.0 g/100 ml, which is about one-third of the normal level for the donor plasma [4]. The content of glucose can vary within a very broad range, 1 2

10 – 20 g/liter, which implies that a considerable proportion (1/3 to 1/2) of glucose can be transformed in the course of autoclave treatment in alkaline medium into secondary products of unknown structure and composition. We have studied the composition of the blood plasma preparation Glunat from different manufacturers by 13C NMR spectroscopy. For the comparison, we also studied several model samples of glucose solutions without additives of donor plasma, which were autoclaved under the same conditions as Glunat (in a sodium hydrocarbonate medium). EXPERIMENTAL PART The 13C NMR spectra were measured using samples dissolved in D2O to a concentration of 10 % (0.05 g of lyophilized product in 0.45 ml of D2O). The measurements were performed on a high-resolution WP-200 (50.3 MHz) spectrometer (Bruker, Germany) with superconducting magnet and a broadband 1H/13C transducer. The signal was recorded in the regime of fully suppressed spin – spin interaction between 13C nuclei and protons. The chemical shifts of carbon nuclei were calculated relative to the Cb-1 signal of D-glucopyranose, which was identified using the SDBS database (No. 2023 CDS-07-025). The 13C NMR spectra were recorded in the regime of automated pulse sequence WALTZ-16 with the following parameters: pulse width PW = 4.1 msec (45°); spectral width SW = 10,000.0 Hz (200 ppm); spectral resolution Hz/Pt = 1.221; signal acquisi-

Hematological Research Center, Russian Academy of Medical Sciences, Moscow, Russia. Mendeleev University of Chemical Technology, Moscow, Russia.

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V. P. Panov and A. V. Panov Ca-2 Ca-5 Cb-4 Cb-3 Ca-4 Cb-5 Cb-2 Ca-3

Cb-1 C -1 a

Cb-6 Ca-6

à

b

c

90

80

70

60

50

40

30

d, ppm

Fig. 1. The typical 13C NMR spectra of D2O solutions of (a) glucose, (b ) glucose autoclaved in a weak alkaline medium, and (c) lyophilized Glunat preparation.

tion time AQ = 0.82 sec; number of scans NS = 13,000. The integral intensities of the resonance signals were determined using the Robend NMR [5, 6] data processing software. The assignment of signals in the 13C NMR spectra was made with reference to the spectrum of a D-glucopyranose solution in D2O. RESULTS AND DISCUSSION Figure 1 shows the typical 13C NMR spectra of (a) glucose solution, (b) glucose autoclaved in a sodium hydrocarbonate solution, and (c) a solution of lyophilized Glunat preparation in D2O. Figure 1a indicates the assignment of resonance signals from carbon nuclei according to [7]. The spectra of lyophilized Glunat preparations in D2O prepared using samples obtained from different hemotransfusion stations and plants of bactericidal preparations were practically identical. In the presence of a large set of minor components of plasma proteins with high molecular weights, the signals from carbon nuclei of the protein fractions in the 13C NMR spectra measured under the adopted conditions are not manifested, and only narrow signals due to low-molecular-weight bioorganic compounds are observed. The 13C NMR spectrum of a D2O solution of glucose treated in an autoclave in aqueous sodium hydrocarbonate for 70 min (to pH 7.0) at 122.6°C (Fig. 1b ) contains the sig-

nals typical of D-glucopyranose and displays three additional signals in the region characteristic of the resonance of anomeric carbon nuclei. These signals have the chemical shifts d = 99.09, 95.11, and 94.74 ppm and a total intensity about 40% of that for the Cb signal. Numerous additional signals of relatively strong intensity appear in the region of resonance of the C(2) – C(6) nuclei, and two relatively weak signals appear in the region of strong fields at d = 38.08 and 24.57 ppm. The spectra of lyophilized Glunat preparations dissolved in D2O (Fig. 1c ) are much similar to the spectrum of plasma-free glucose solution treated under the same conditions. In addition to the signals typical of D-glucopyranose, the spectrum in Fig. 1c also displays three signals in the region characteristic of the resonance of anomeric carbon nuclei (d = 99.09, 95.11, and 94.74 ppm), signals corresponding to the resonance of C(2) – C(6) nuclei, and three relatively weak signals in the region of strong fields at d = 46.78, 38.08, and 24.57 ppm. The positions and intensities of these signals are close to those in the spectrum of the model glucose solution. Thus, the only significant difference is the presence of a relatively intense signal at d = 46.78 ppm (with intensity about twice that of the signal at 38.08 ppm) in the spectrum of Glunat preparations. The assignment of this signal requires additional investigation.

Composition of Glunat Preparation Studied by 13C NMR Spectroscopy The character of the 13C NMR spectra of the glucose solution and the solution treated at 122.6°C in a weakly alkaline medium, especially the presence of a large group of signals (four medium and twelve weak) in the region corresponding to the resonance of C(2) – C(6) nuclei, may be indicative of the partial condensation of D-glucopyranose molecules (of the C1 – C4 and C1 – C6 types), as well as of their rearrangement and fragmentation. For example, there might appear an equilibrium mixture of D-glucose with D-fructose and D-mannose [8, 9], and the later two may also be involved in the partial condensation reactions. Taking into account the relative integral intensities of signals from the products of condensation, rearrangement, and fragmentation of D-glucopyranose, their fraction in the mixture may exceed 30%. In the 13C NMR spectra of Glunat, signals from the possible products of condensation of glucose with donor plasma proteins are not manifested, probably because of the large number (i.e., low concentration) of minor components and because of large widths of the resonance lines of these biopolymers. The absence of signals in the region of resonance of the nuclei of carbon involved in double bonds indicates that a deep conversion of D-glucopyranose during the autoclave treatment in a weakly alkaline medium, with the formation of significant amounts of products such as hydroxymethylfurfurols, is unlikely. The optical spectra of Glunat solutions show rather weak absorption in the UV – VIS range, which indicates that the content of impurities absorbing in this spectral range is small. According to [8, 9], the formation of hydroxymethylfurfurols from D-glucose is typically observed in acid media. Thus, the results of our comparative study of Glunat and model solutions by 13C NMR spectroscopy showed that the composition of this preparation is still insufficiently studied. Data on the possible accompanying compounds are neither presented in the normative documentation not reported in the literature, although the content of such components (judging from the relative integral intensities of the 13C NMR signals of D-glucopyranose and its possible transformation products) may exceed 30%. It is still unclear how these compounds influence the physiological activity of Glunat and what side effects they can produce. Since the use of Glunat preparation involves the danger of potential viral infection, it is necessary to determine the role of accompanying compounds in

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the course of viral inactivation by sterilization in autoclave. The normative documentation for Glunat [2] does not stipulate characteristics of the viral safety. The main requirements to the quality, efficacy, and safety of blood preparations are formulated in [10, 11]. According to these requirements, the pharmacopoeial article for Glunat should be modified in the sections “Composition,” “Identity,” and “Glucose” and it should be supplemented with the new section “Accompanying Compounds.” The section “Composition” must restrict the content of accompanying compounds, the section “Identity” must stipulate the qualitative reactions for these components, and the section “Glucose” must specify its content within a narrow concentration interval. The section “Accompanying Compounds” must describe methods for the quantitative determination of these compounds and restrict their content. We believe that it is expedient to use HPLC for the identification and quantitative determination of accompanying compounds in Glunat. Otherwise, it is necessary to exclude this preparation from the State Register of Medicinal Preparations. REFERENCES 1. Hemotransfusion Preparations: Instructions and Methodological Recommendations [in Russian], S. P. Burenkov (ed.), Moscow (1976), p. 359. 2. Pharmacopoeial Article VFS 42-165-86. Glunat. 3. USSR State Pharmacopoeia, XIth Edition, Moscow (1989), Vols. I and II. 4. V. P. Panov and A. V. Karyakin, Advances in Transfusiology [in Russian], Moscow (2002), Issue 32, pp. 90 – 96. 5. V. P. Panov, V. I. Dubrovin, S. V. Tarabakin, et al., Khim.-Farm. Zh., 19(7), 899 – 883 (1987). 6. V. P. Panov, V. I. Dubrovin, S. V. Tarabakin, Robend Program, in: MOV Program Library “Abacus,” Germany (1992). 7. V. P. Panov and R. G. Zhbankov, Intra- and Intermoleclar Interactions in Mono- and Polysaccharides [in Russian], Nauka I Tekhnika, Minsk (1986), p. 359. 8. A. L. Lehninger, Biochemistry: The Molecular Basis of Cell Structure and Function, Worth, New York (1972). 9. A. L. Lehninger, D. L. Nelson, and M. M. Cox, Principles of Biochemistry, Worth, New York (1993), p. 298. 10. V. P. Panov, O. N. Petracheva, and L. N. Ermakova, Vestn. Sluzhby Krovi Ross., No. 4, 45 – 47 (19983). 11. State Standard OST 91500.05.0001-00. Standards of Quality for Medicinals. Main Stipulations.