Article pubs.acs.org/crystal
Glucosamine Salts: Resolving Ambiguities over the Market-Based Compositions Published as part of the Crystal Growth & Design virtual special issue In Honor of Prof. G. R. Desiraju Subash Chandra Sahoo,† Anjana Tharalekshmy,‡ Seik Weng Ng,# and Panče Naumov*,†,⊥ †
New York University Abu Dhabi, P.O. Box 129188, Abu Dhabi, United Arab Emirates The Petroleum Institute, P.O. Box 2533, Abu Dhabi, United Arab Emirates # Department of Chemistry, University of Malaya, Kuala Lumpur, Malaysia ⊥ Institute for Chemical Research and the Hakubi Center for Advanced Research, Kyoto University, Uji, Kyoto 611−0011, Japan ‡
S Supporting Information *
ABSTRACT: The neutral form of glucosamine, C6H13NO5, one the most effective and most widely used over-the-counter health supplements for the relief of osteoarthritis, is very unstable in air. It is marketed as chloride and sulfate salts. Unlike the stable glucosamine chloride, direct use in pharmaceutical formulations of the sulfate, ostensibly the physiologically more active form, is hindered by its strong hygroscopicity. Copious patent literature exists describing methods for stabilization of the sulfate by converting it into double and/or mixed salts, usually with alkaline or earth alkaline sulfates and chlorides. Aiming to unravel the structures of the alleged double/mixed salts, we attempted synthesis of the stabilized forms of the sulfate following literature procedures. Our repeated attempts did not yield true glucosamine sulfate or any real (in the chemical sense) double or mixed salts. Instead, Fourier transform infrared spectroscopy, powder X-ray diffraction, thermogravimetric analysis, and elemental analyses consistently showed that physical mixtures of the stable glucosamine chloride, which has a strong propensity to crystallize out from solutions, and the respective alkaline salts are obtained in all cases. Expectedly, these mixtures were nonhygroscopic. The analysis of the commercially available sample of “glucosamine sulfate” showed that it is a mixture of glucosamine chloride and K2SO4, in accordance with the above conclusions. By using a simple ion exchange in glucosamine chloride, we devised a simple method to generate glucosamine sulfate. As anticipated, the latter is a very hygroscopic powder in the solid state and is chemically moderately unstable in solution. Along with the conclusions based on the products obtained following published procedures, reaction of this compound with alkali chlorides readily affords the (non-hygroscopic) glucosamine chloride in a mixture with the respective alkali sulfates. We are tempted to conclude that the alleged “stabilization” of glucosamine sulfate by formation of double/mixed salts is (in the chemical sense) misleading. We believe that these compounds have probably never been obtained, and the related published synthetic procedures should be reinvestigated. The conclusions of this study could have important implications on the effective amount of the active ingredient required to achieve physiological activity, because such “stabilized” mixtures contain less than the optimal amount of the physiologically active ingredient, which could also have some commercial implications.
1. INTRODUCTION Arthritis is the most common cause of long-term disability, with osteoarthritis (also known as degenerative joint disease, degenerative arthritis, and osteoarthrosis) being its most prevalent form.1,2 In 1969, the amino sugar glucosamine {(3R,4R,5S)-3-amino-6-(hydroxymethyl)oxane-2,4,5-triol, Scheme 1} was evaluated as the most successful therapeutic agent for osteoarthritis. In many countries, including UK and North America, it is available in the form of nonvitamin/ nonmineral dietary supplements for adults.3−5 Glucosamine is a common precursor in the biosynthesis of glycolipids, glycoproteins, glycosaminoglycans, proteoglycans, and hyaluronatebiogenic compounds that are involved in the structure and function of body joints.6,7 Being biosynthesized by chondrocytes (cartilage cells) from glucose and glutamine, it is one of the most abundant amino monosaccharides.8 Glucosamine acts as a stimulator in the production of mucin © 2012 American Chemical Society
Scheme 1. Chemical Formulas of Glucosamine (Left), and Its Sulfate and Chloride Salts (Right)
and mucous, which have lubricating and protective functions, provides strength and elasticity of joints, and repairs any existing joint cartilage damage and as a result progression of osteoarthritis decreases.9−11 As people age, the reduced ability Received: September 3, 2012 Published: September 10, 2012 5148
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and hygroscopicity of the sulfate and chloride of glucosamine, and to unravel the origin of the reported stabilization by formation of mixed and double salts with alkaline and earth alkaline halides. For comparison, in our experiments, we used glucosamine chloride and glucosamine sulfate from two different sources: salts as prepared by us, hereafter denoted GluCl and GluSO4, respectively, and commercially available salts, which for distinction will be denoted “GluCl” and “GluSO4”, respectively. The crystal structure of the chloride is known32,33 and it was refined with greater accuracy recently.34 With the intent to unravel the structure of the alleged mixed ionic salts, we attempted to synthesize GluSO4 and its stabilized forms by following procedures described in the patent literature. Specifically we were interested in elucidating the crystal structures of the mixed and double salts of glucosamine, which would represent interesting examples of hybrid organic− inorganic ionic salts, and could possibly provide some hints about the reasons behind the stabilizing effects of the metal halide. Being interested in alternative methods for stabilization of the hygroscopic sulfate, we went further on to investigate the behavior of these compounds and their compositions in air and during mechanical treatment.
to produce sufficient amounts of glucosamine results in degenerative joint diseases, causing arthritis. It is believed that glucosamine can also alleviate pain, and it can diminish dissipation and rebuild cartilaginous damage or osteoarthritic joints. The effects are similar to those of the polymer chondroitin. In pure form, glucosamine is chemically unstable. It is commercially available as a sulfate, hydrochloride, and N-acetyl derivative, occasionally combined with other supplements (chondroitin sulfate or methylsulfonylmethane). Of the three commonly available forms, only the sulfate and the chloride have been largely clinically tested. Comparative studies showed that both the purity and the content of glucosamine can vary among different manufacturers, and sometimes even among different batches from the same manufacturer.12,13 A noteworthy study by the U.S. National Institutes of Health overseeing the largest clinical trial on glucosamine and chondroitin for treatment of osteoarthritis could not find a satisfactory source with consistent composition from batch to batch, and thus the compounds had to be prepared.14 With the glucosamine being classified as dietary supplement instead of a drug, the manufacturers are not required to comply with the strict manufacturing regulations imposed on the pharmaceutical industry. Perhaps not surprisingly, a thorough review indicated that the effects of glucosamine supplements were highest in industry-funded studies and lowest in independent studies.15 The contradictory evidence for the glucosamine efficacy has even led to a debate among physicians on whether to recommend the chemical for treatment to their patients.16 A number of clinical trials in the 1980s and 1990s, all sponsored by the European patent-holder, demonstrated improvements, but these studies were criticized for methodological shortcomings.17,18 Unquestionably the biggest impediment with incorporating the allegedly more efficient glucosamine sulfate19 into supplement or drug formulations is its pronounced hygroscopicity. In air, the compound appears moist, and on contact with a press or during grinding and mixing it readily adheres to the walls of the container, thus rendering processing into dosage formulations practically impossible. The issues with processability are further augmented by the propensity of the amino group in solution for oxidation. These difficulties can be overcome by using the non-hygroscopic chloride salt; however, several reports have pointed out that the more stable chloride is physiologically less effective, although the relative efficacy of the two formulations in treating osteoarthritis remains unclear. Although it is very unlikely that the counterion plays a major role in the action and pharmacokinetics of glucosamine, it has been claimed that the enhanced activity of the sulfate salt is due to double action.20 Formation of stable mixed salts of glucosamine sulfate with alkaline or earth alkaline metal halides, including sodium and potassium chloride, was reported,21,22 but these compounds have remained poorly characterized and their existence as defined chemical compositions remains elusive. In attempt to stabilize the sulfate, a number of patents23−31 have been filed describing methods for stabilization, usually by converting it into double and/or mixed salts with alkaline and earth alkaline sulfates and chlorides. This study was initiated when one of the authors (S.W.N.) noticed that his relatives were consuming glucosamine sulfate whose cost differed greatly from one company to another. In an independent and objective study that spanned several years we set as our goal to clarify the reasons behind different stabilities
2. EXPERIMENTAL PROCEDURES 2.1. Materials and Methods Used for Characterization. All solvents were commercially available and were used as received. The commercially available “GluCl” and “GluSO4” were purchased from the manufacturer X35 and were used as received. Sodium sulfate, sodium chloride, and potassium chloride were from Sigma-Aldrich. The attenuated total reflection (ATR) FTIR spectra were recorded on Agilent Cary 670 spectrometer (codes used to describe the band intensities: s − strong, w − weak, br − broad). Plots of all IR spectra are deposited as Supporting Information, SI. The TGA measurements were carried out in the temperature range of 25−600 °C with a PerkinElmer STA600 TG-DTA analyzer in nitrogen atmosphere at a heating rate of 10 °C min−1. Elemental analysis was performed with PerkinElmer 2400 Series II analyzer. The milling experiments were performed with RETSCH MM 200 ball-mill. The samples were placed in a marble capsule and milled with stainless steel balls (6 mm or 9 mm). 2.2. Synthetic Procedures. Because the synthesis and product analysis of the reactions of these compounds are critically important for their identification and characterization, all synthetic and crystallization procedures will be described here in full detail. 2.2.1. New Method for Preparation of GluSO4. A solution of 2 g of AgNO3 in 5 mL of water and an equimolar amount of Na2SO4 (0.83 g) in minimum amount of water were mixed with stirring, whereupon intensely white precipitate of Ag2SO4 separated out (IR). The precipitated Ag2SO4 was filtered, thoroughly washed with water, and dried in a vacuum. 600 mg of the product was dissolved in 120 mL of water to obtain a clear solution. To this solution, 800 mg of “GluCl” dissolved in a minimum amount of water was added, whereupon bright white precipitate immediately formed. The mixture was stirred for 2 h and filtered. The filtrate (negative chloride test with AgNO3) was collected and evaporated to dryness to afford a semisolid white residue. After multiple washings with isopropanol, the product was dried in a vacuum and stored closed. After drying, the product was offwhite and strongly hygroscopic in air. Our repeated attempts to crystallize this compound remained without success. IR (cm−1): 3275 (br), 3097 (br), 2939 (br), 1616 (w), 1534 (w), 1027 (br). Melting point: 97−99 °C. El. analysis: w(%), calcd. for GluSO4·5H2O (closest hydrate composition): C, 26.37; H, 6.90; N, 5.12, found: C, 24.36; H, 5.48; N, 5.17. 2.2.2. Crystallization of “GluSO4”. Crystallization of “GluSO4” by Slow Diffusion of Alcohol Vapors. Twenty milligrams of “GluSO4” was dissolved in 2 mL of water and kept in a small vial. The vial was kept inside a closed tall glass bottle half-filled with methanol for slow 5149
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Mechanochemical Reaction of “GluCl” with Na2SO4. A mixture of “GluCl” (0.1 mg, 0.46 mmol) and Na2SO4 (0.064 mg, 0.46 mmol) was ball-milled for 3 h at frequency of 25 Hz. IR (cm−1): 3286 (s), 3090 (w), 3036 (w), 1616 (w), 1583 (w), 1538 (s), 1094 (s), 1033 (s), 773 (w), 617 (s). 2.3. X-ray Diffraction Analysis. All single crystal diffraction data were collected on a Bruker SMART APEX three-circle diffractometer equipped with a CCD area detector (Bruker Systems Inc.)36 operating at 1.5 kW power (50 kV, 30 mA) to generate Mo Kα radiation (λ = 0.71073 Å). The incident X-ray beam was focused and monochromated using Bruker Excalibur Göbel mirror optics. Crystals for characterization were mounted on nylon cryoloops (Hampton Research) with Paratone-N (Hampton Research). Data were integrated using Bruker SAINT software.37 The diffraction data were corrected for absorption with SADABS.38 All structures were solved by direct methods and refined with the SHELXTL 97 suite.38 The atoms were located from iterative examination of the difference F-maps following least-squares refinements of earlier models. Hydrogen atoms were placed in calculated positions and included as riding atoms with isotropic displacement parameters 1.2−1.5 times Ueq of the attached carbon atoms. All structures were examined using the AddSym subroutine of PLATON39 to confirm that no additional symmetry is applicable to the models. All ellipsoids in ORTEP diagrams are displayed at the 50% probability level, unless noted otherwise (Figures S17−S18). Since the structures have already been reported32−34 the crystallographic data were used only for identification.40 The powder X-ray diffraction patterns were recorded on Phillips PANalytical diffractometer with Cu Kα radiation (λ = 1.5406 Å), scan speed of 2° min−1 and a step size of 0.02° in 2θ.
diffusion. After two days, rod-shaped crystals were grown, isolated, and dried. The same procedure was repeated with ethanol and isopropanol. By diffusion of ethanol two types of crystals were obtained, rods and blocks. The crystals were stable in air. Rod crystals, IR (cm−1): 3275 (br), 3097 (br), 2939 (br), 1616 (w), 1534 (w), 1027 (br). Plate crystals, IR (cm−1): 3275 (br), 3097 (br), 2939 (br), 1616 (w), 1534 (br), 1027 (br). Diffusion of isopropanol also afforded a mixture of airstable rod and blocky crystals. Rod crystals, IR (cm−1): 3275 (br), 3097 (br), 2939 (br), 1616 (w), 1534 (w), 1027 (br). Blocky crystals, IR (cm−1): 3275 (br), 3097 (br), 2939 (br), 1616 (w), 1534 (w), 1027 (br). 2.2.3. Wet Reactions of GluSO4 with KCl and NaCl. Reaction of GluSO4 with KCl or NaCl and Crystallization by Slow Diffusion of Isopropanol. 0.1 g of GluSO4 and 0.027 g of KCl were dissolved in 2 mL of water and kept in a 5 mL vial. The vial was placed inside a closed tall glass bottle half-filled with isopropanol for slow diffusion. Rod-like and blocky crystals were obtained, and were isolated and dried. The procedure was repeated by using 0.022 g of NaCl. Goodquality rod-like and blocky crystals were obtained in two days. 2.2.4. Wet Reactions of “GluSO4” with KCl and NaCl. Reaction of “GluSO4” with KCl or NaCl and Crystallization by Slow Evaporation. “GluSO4” (0.1 mg, 0.36 mmol) was dissolved in 1 mL of water and mixed with a solution of KCl (0.027 mg, 0.36 mmol) in 1 mL of water. The mixture was stirred 2 h in a water bath (50 °C). The heater was removed and the stirring was continued at room temperature (RT) for 3 h. The clear colorless mixture was stored at RT for slow evaporation. After 2 days, plate-like crystals were separated out from the solution. A small amount of a second phase was also isolated. All crystals were isolated and dried in a vacuum for analysis. IR (cm−1): 3284 (s), 3091 (w), 3034 (w), 1615 (w), 1581 (w), 1537 (s), 1092 (s), 1031 (s), 772 (w), 611 (s). The procedure was repeated by using NaCl (0.021 mg, 0.36 mmol). The mixture was stirred 2 h in a water bath (50 °C). The product was isolated and dried in a vacuum for analysis. IR (cm−1): 3283 (s), 3092 (w), 3035 (w), 1616 (w), 1582 (w), 1537 (s), 1093 (s), 1033 (s), 774 (w), 613 (s). Reaction of “GluSO4” with KCl or NaCl and Crystallization by Addition of Isopropanol. “GluSO4” (0.1 mg, 0.36 mmol) was dissolved in 1 mL of water and mixed with KCl (0.027 mg, 0.36 mmol) in 1 mL of water. The mixture was stirred in a water bath (50 °C) for 2 h and then cooled. Ten milliliters of isopropanol was added with continued stirring, whereupon a white precipitate was obtained. The white precipitate was isolated by filtration and dried in a vacuum. IR (cm−1): 1103 (s), 617 (s). The procedure was repeated by using NaCl (0.021 mg, 0.36 mmol). White precipitate was isolated by filtration and characterized. IR (cm−1): 3281 (w), 1537 (w), 1184 (s), 1094 (br), 616 (s). Yield: 60−70%. Reaction of “GluSO4” with KCl or NaCl and Crystallization by Addition of Acetone. “GluSO4” (0.1 mg, 0.36 mmol) was dissolved in 1 mL of water and mixed with KCl (0.027 mg, 0.36 mmol) in 1 mL of water. The mixture was stirred in water bath (40 °C) for 2 h. After cooling, 8−10 mL of acetone was added with continued stirring whereupon a white precipitate formed. The white precipitate was isolated by filtration and dried. IR (cm−1): 3282 (s), 3091 (w), 3035 (w), 1615 (w), 1581 (w), 1537 (s), 1092 (s), 1031 (s), 772 (w), 591 (br). The procedure was repeated by using NaCl (0.021 mg, 0.36 mmol) and stirring at 30 °C. Ten milliliters of acetone was added with continued stirring, whereupon a white precipitate formed, which was isolated by filtration and dried. IR (cm−1): 3282 (s), 3089 (w), 3033 (w), 1614 (w), 1581 (w), 1092 (s), 1031 (s), 772 (w), 609 (br). Yield: 50−60%. 2.2.5. Dry Reactions of “GluSO 4 ” with KCl and NaCl. Mechanochemical Reaction of “GluSO4” with KCl and NaCl. A mixture of “GluSO4” (0.1 mg, 0.36 mmol) and KCl (0.027 mg, 0.36 mmol) was ball-milled for 3 h at frequency of 25 Hz. The color of the originally white mixture acquired yellow shade. IR (cm−1): 3286 (s), 3093 (w), 3037 (w), 1616 (w), 1583 (w), 1538 (s), 1121 (s), 1033 (s), 773 (w), 617 (s). The procedure was repeated by using NaCl (0.021 mg, 0.36 mmol). The (initially white) mixture had a yellow shade after the treatment. IR (cm−1): 3286 (s), 3094 (w), 3037 (w), 1616 (w), 1583 (w), 1538 (s), 1094 (s), 1033 (s), 773 (w), 618 (s).
3. RESULTS AND DISCUSSION 3.1. Characterization of the As-Received Glucosamine Chloride and Sulfate. At the beginning of this study, we used commercially available chloride and sulfate (“GluCl” and “GluSO4”, respectively). Although glucosamine sulfate has been described as a very hygroscopic compound,23 the asreceived “GluSO4” was a crystalline colorless solid that was stable in air and did not adsorb moisture at room temperature and ambient humidity with months (IR spectroscopy). Surprisingly, the IR spectra of “GluSO4” and “GluCl” were almost identical (Figure 1), with only minor differences in the relative intensity of three sharp bands around 1000−1200 cm−1. The TGA curves (Figure 2) were also very similar, and showed that the two compounds were free of adsorbed moisture and solvents, although the relative weight loss during the single decomposition step of “GluSO4” was smaller (40% for “GluSO4”, 55% for “GluCl”). The powder X-ray diffraction pattern of “GluCl” (Figure 3) matched perfectly the patterns calculated from the published structure.34 Structure determination from crystals of the commercial “GluCl” and GluCl obtained after recrystallization (see below) conformed to the published structure34 and confirmed the identity of the asreceived chloride. The diffraction pattern of “GluSO4”, however, contained all peaks of GluCl and additional peaks of unknown phase(s). While the elemental analysis of “GluCl” was consistent with its composition (w(%), calcd. C, 33.42; H, 6.54; N 6.49; found: C, 33.28; H, 6.80; N, 6.39%), that of “GluSO4” was very different from the expectations based on anhydrous salt (w(%), calcd. C, 31.57; H, 6.18; N 6.13; found: C, 24.36; H, 5.48; N, 4.62%). Taken together, these analytical data and the unexpected stability in air showed that “GluSO4” represents a mixture of GluCl and another phase with low carbon content. To resolve the identity of the secondary phase, we recrystallized “GluSO4” and “GluCl” separately by slow diffusion of alcohols (methanol, ethanol, and isopropanol) in 5150
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Figure 1. IR spectra of commercial glucosamine sulfate (“GluSO4”) and glucosamine chloride (“GluCl”). The spectra are surprisingly similar and question the identity of the sulfate.
Figure 3. X-ray powder diffraction patterns of the commercial glucosamine sulfate and chloride, and calculated patterns (based on the single crystal structures) of glucosamine chloride and potassium sulfate.
(w(%), calcd C, 23.80; H, 4.66; N 4.62. Found: C, 23.36; H, 5.48; N, 4.62%). 3.2. Synthesis and Characterization of Glucosamine Sulfate (GluSO4). In the absence of a reference sample of glucosamine sulfate, we devised a method for preparation of this compound. The procedure includes exchange in aqueous solution of the chloride ion in GluCl with freshly prepared solution of Ag2SO4 (for details of the procedure, see the Experimental Procedures). After exchange of the anion and removal of AgCl (at 25 °C, Ksp(AgCl) = 1.8 × 10−10; Ksp(Ag2SO4) = 1.2 × 10−5), evaporation, washing and drying, GluSO4 was obtained as off-white solid. The material is strongly hygroscopic and readily turns into paste at ambient conditions. Its IR spectrum, TGA trace, and powder diffraction pattern were distinctly different from those of GluCl (Figures 4−6). In addition to the sharp glucosamine bands, the IR spectrum contains a very broad complex absorption around 3200 cm−1
Figure 2. TGA curves of the commercial glucosamine sulfate and chloride.
their aqueous solutions. A mixture of rod-like crystals and blocky crystals were obtained from the solution of “GluSO4”. The solution of “GluCl” afforded only blocky crystals. Identification of the crystals by single crystal X-ray diffraction proved that the blocky crystals were from GluCl, whereas the rod-like crystals were anhydrous K2SO4. An identical result was obtained by structure determination of randomly selected crystals from the as-received batch of “GluSO4”. We conclude that as-received “GluSO4” is a mixture of GluCl and K2SO4, along with the comparison of the powder diffraction patterns (Figure 3). The slight difference in intensity of the IR bands noted in the 1000−1200 cm−1 region (Figure 1) can now be explained as an effect of the overlapping ν(SO4) band from K2SO4 which is buried under the sharp glucosamine bands. The elemental analysis and the TGA data of the as-received sample conform to a composition of nearly 52% GluCl and 48% K2SO4
Figure 4. IR spectrum of glucosamine sulfate prepared by the new procedure. 5151
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To stabilize the hygroscopic GluSO4 as double or mixed salts we utilized several reported wet procedures, including precipitation with organic solvents (acetone and isopropanol) from aqueous solutions and crystallization by slow evaporation at ambient temperature (for details, see the Experimental Procedures). It should be noted that in practice isolation of the glucosamine salts by the precipitation method with organic solvents is burdened by numerous shortcomings, including (a) low yield (due to high solubility of both glucosamine and alkali salt in water), (b) difficulties with control over the ratio of the glucosamine in the final precipitate, which due to the difference in solubility of glucosamine and alkali salt in organic solvent can vary between 50% and 80%, (c) possible biotoxicity of even small amounts of organic solvents that may remain in pharmaceutical preparations, and (d) high cost of the procedures, which require a large quantity of pure organic solvents. Our analytical results and the FTIR spectra of the precipitated mixtures (Figures S3−S4) confirmed the reported low yields. To avoid the use of organic solvent, we tried to isolate the glucosamine salts by the slow evaporation method. As confirmed by FTIR, PXRD, and single crystal X-ray diffraction analysis (Figure S9), the repeated attempts always afforded a physical mixture of GluCl and the respective metal sulfates; no real chemical mixed or double salts with defined chemical composition could be isolated. Being composed of GluCl and stable sulfates, the product mixtures were nonhygroscopic. The GluCl has a strong propensity for crystallization. It separates well out of the other solute(s), either as a white powder (fast crystallization) or as colorless, well-shaped crystals (slow crystallization). We are tempted to conclude that the wet syntheses of mixed compositions claimed in the patent literature do not afford real mixed or double salts, at least not in the chemical sense of these terms; the products are probably physical mixtures, and from the chemical point of view, they are indistinguishable from the mixtures obtained by dry mixing of GluCl and metal sulfates. It is important that the metal sulfate in such a mixture does not alter the physicochemical and physiological properties of GluCl. Furthermore, we attempted to crystallize the products by slow diffusion of organic solvents (isopropanol, methanol, and ethanol) into mixed aqueous solutions of GluSO4 with either KCl or NaCl. By diffusing alcohols we obtained and isolated colorless block and rod crystals. Characterization with FTIR spectroscopy and X-ray diffraction revealed that in the presence of KCl or NaCl the glucosamine always crystallizes out from solution of GluSO4 in water as GluCl (blocky crystals) concomitantly with K2SO4 or Na2SO4 (rod crystals), in support of the above conclusions. 3.4. Dry Reactions of Glucosamine Sulfate with Alkali Chlorides. To avoid the exclusive formation of GluCl by the wet reaction method, we attempted synthesis of normal and mixed/double glucosamine salts by mechanochemical (dry ball milling) reaction, characterizing the reactants/products by FTIR spectroscopy and PXRD. The milling was performed during 6 h. In a set of preliminary experiments, we checked the effects of milling on pure “GluCl”. Except for the expected particle size effects, the salt proved stable on mechanical treatment and no new phases could be observed (Figure S11). Milling of “GluCl” with Na2SO4 did not show any reaction either; only the distinct sulfate peak became visible in the IR spectrum of the mixture after milling (Figure S7). Similarly, “GluSO4”, for which we confirmed that it consists of a mixture
Figure 5. TGA curves of the commercial glucosamine sulfate (“GluSO4”), before and after the milling, and the salt obtained by the new procedure. The newly synthesized material (GluSO4) undergoes a two-step weight loss corresponding to dehydration and decomposition.
Figure 6. Powder X-ray diffraction patterns of the commercial glucosamine sulfate and the salt obtained according to the procedure described here.
due to the adsorbed and/or crystalline water, with which the ν(NH) bands are overlapped, and a distinct strong ν(SO4) band around 1023 cm−1. The TGA plot shows weight loss of 28% between 100 and 210 °C due to desorption of water (Figure 5). The elemental analysis revealed that the compound is hydrate close to a pentahydrate, GluSO4·xH2O (x ≈ 5), but establishing of the exact formula was burdened by the strong hygroscopicity and the difficulties with handling of the material. The PXRD pattern of GluSO4 is distinctly different from that of “GluSO4” and GluCl (Figure 6). The compound is only moderately stable in aqueous solution; the solution turns yellow within two days, presumably as a result of oxidation of the amino group. 3.3. Wet Reactions of Glucosamine Sulfate with Alkali Chlorides. Having pure GluSO4 and GluCl at hand, we went on to prepare the double and mixed salts with alkali chlorides. 5152
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pattern, the major peaks of the parent GluSO4 remained intact and additional peaks from the alkali salts appeared, thus confirming that a simple physical mixture has been obtained. Thus, similar to the wet methods described above, the dry method did not afford mixed/double salts. The milling, however, could be a good method for preparation of homogeneous physical mixtures of GluSO4 and other salts. The secondary salts could adsorb water irreversibly and alleviate the hygroscopicity of GluSO4.
of GluCl and K2SO4, yields a homogeneous mixture upon milling and shows a distinct sulfate peak at 1122 cm−1 in the IR spectrum (Figure S6). Contrary to GluCl, the mechanical treatment had a strong effect on the IR spectrum of GluSO4 (Figure 7). The bands that
4. CONCLUSIONS The “glucosamine sulfate case” is an interesting example of double ambiguity in the patent and pharmaceutical literature related to the same compound: ambiguity in wording and ambiguity in interpretation of results that can distinguish a true chemical compound of mixed composition from a physical mixture. Does a “mixed compound” (e.g., mixed salt) in a patent have the same meaning as it has to a chemist? Since glucosamine is classified as a supplement rather than a drug, as long as the final formulations containing alkali halides obtained following various proceduresare not hygroscopic and they serve their purpose, it might appear that this question is irrelevant in terms of its practical significance. Therefore, it is not surprising that there has been no detailed study to characterize the structures of its alleged mixed salts with alkali and earth alkaline halides. From a fundamental scientific viewpoint, however, there is a clear distinction between a pure substance and a mixturethe mixture is a physical combination of pure substances. Our analysis of a commercial sample of glucosamine sulfate (perhaps obtained following published procedures) revealed that the compound is a physical mixture of glucosamine chloride and potassium sulfate. It is not clear in which form is the supplement provided to the consumers, but it is likely that if it is used in the form of “stabilized” sulfate, it also is a mixture of the chloride and alkali sulfate(s). Such mixtures are not hygroscopic and the elemental analysis would give results similar to those expected for true mixed salts. Our attempts to prepare the true mixed compounds by employing reported procedures remained fruitless and in all cases physical mixtures were obtained. It is likely that double or mixed salts of glucosamine sulfate and chloride have never been obtained, and we believe that the related published synthetic procedures should be reinvestigated. Pure glucosamine sulfate, on the other hand, is a very hygroscopic compound which can only be partially stabilized by ball-milling. This limited stabilization effect is probably due to dehydration and might be also achieved by other means (e.g., heating). The conclusions of this study could have some implications on the effective amount of the active ingredient available to achieve physiological activity, because in such “stabilized” compositions only one portion of the actual amount is physiologically active.
Figure 7. IR spectra of GluSO4, synthesized according to the new method, before and after the ball milling procedure, and after aging. The dehydration is clearly observed during the milling as sharpening of the peaks The sample slowly acquires the water after exposure to air and the spectrum slowly reverts to that of the hydrated sample.
were originally broad due to the adsorbed moisture, sharpened and after the treatment the spectrum resembled closely the characteristic peaks of the glucosamine cation. This effect is apparently related to removal of the adsorbed and lattice water due to heating during the extended milling process. To confirm this, the ball-milled sample was aged at ambient conditions and humidity while monitoring its IR spectrum. As the sample was adsorbing water, the bands started to broaden. This recovery was slower than the hydration of the as-synthesized sample, and it proceeded with days. The ball milling of GluSO4 with NaCl and KCl resulted in distinct characteristic changes in the IR spectrum around 1120 cm−1, and the overall spectral appearance was improved relative to pure GluSO4 (Figures S2 and S14). In the powder diffraction
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ASSOCIATED CONTENT
S Supporting Information *
PXRD data, thermal stability and TGA plots, FTIR spectra, and ORTEP figure of GluCl. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. 5153
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Notes
(29) Chodekar, V. M.; McGough, M. Y.; Desai, H. S.; Schleck, J. G.; Redkar, S. N. U.S. Patent 7388001B1, 2008. (30) Chopdekar, V. M.; Redkar, S. N. U.S. Patent 7622576B1, 2009. (31) Chopdekar, V. M.; Redkar, S. N. U.S. Patent 7683042B1, 2010. (32) Chandrasekharan, R.; Mallikarjunan, M. Z. Kristallogr. 1969, 129, 29. (33) Chu, S. S. C.; Jeffery, G. A. Proc. R. Soc. London Ser. A 1965, 285, 470. (34) Harrison, W.T. A.; Yathirajan, H. S.; Narayana, B.; Sreevidya, T. V.; Sarojini, B. K. Acta Crystallogr. 2007, E63, o3248. (35) Our attempts to provide from the chemical supplier detailed analytical results to confirm the chemical identity and composition of the sulfate, even though a declaration was provided with the chemical, remained fruitless. Due to implications that the results presented here might have on the supplier, we will refrain from disclosing its identity here. (36) APEX2 (version 5.053), Bruker AXS Inc.: Madison, WI, 2005. (37) SAINT-Plus (version 7.03), Bruker AXS Inc.: Madison, WI, 2004. (38) Sheldrick, G. M. Acta Crystallogr. A 2008, 64, 112−122. (39) Spek, A. L. Acta Crystallogr. D 2009, 65, 148−155. (40) The structure of GluCl was reported in 1960s,32,33 and it was rerefined and described very briefly recently.34 GluCl crystallizes well as block-shape crystals by slow evaporation of aqueous solution or slow diffusion of organic solvents into the solution. Unit cell dimensions: a = 7.1218(9), b = 9.1808(8), c = 7.7101(9) Å, β = 112.330(14)o with final full matrix least-squares refinement on F2 converged to R1 = 0.0342 (F > 2σ(F)) and wR2 = 0.1022 (all data) with GOF = 1.047 and Flack parameter −0.05(7). The compound crystallizes in the monoclinic space group P21 and the structure corresponds to the formula C6H14NO5+Cl¯ (the unit cell dimensions are closely matching the reported values). The H atoms were clearly located showing that the amino group is protonated. There is one molecule of each glucosammonium ion and chloride ion per asymmetric unit. The configurations of the chiral C-atoms are C2 (R), C3 (S), C4 (R), C5 (R), and C6 (S) (Figure S17). A set of hydrogen bonding interactions, including O−H···O, N−H···O, O− H···Cl, and N−H···Cl bonds, results in a three-dimensional network where the intercation O−H···O and N−H···O bonds help to extend the molecular arrangement in a layer structure.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Rahul Banerjee and his group for making the Xray diffractometer available in their lab. This work was supported by the Kyoto University’s Hakubi Project (P.N.).
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DEDICATION This paper is dedicated to Professor Gautam Desiraju, on the occasion of his 60th birthday.
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REFERENCES
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dx.doi.org/10.1021/cg301276y | Cryst. Growth Des. 2012, 12, 5148−5154
