Crystal structure and characterization of pyrroloquinoline quinone disodium trihydrate
© Ikemoto et al.; licensee Chemistry Central Ltd. 2012
Received: 12 March 2012
Accepted: 6 June 2012
Published: 19 June 2012
Pyrroloquinoline quinone (PQQ), a tricarboxylic acid, has attracted attention as a growth factor, and its application to supplements and cosmetics is underway. The product used for these purposes is a water-soluble salt of PQQ disodium. Although in the past, PQQ disodiumpentahydrates with a high water concentration were used, currently, low hydration crystals of PQQ disodiumpentahydrates are preferred.
We prepared a crystal of PQQ disodium trihydrate in a solution of ethanol and water, studied its structure, and analyzed its properties. In the prepared crystal, the sodium atom interacted with the oxygen atom of two carboxylic acids as well as two quinones of the PQQ disodium trihydrate. In addition, the hydration water of the prepared crystal was less than that of the conventional PQQ disodium crystal. From the results of this study, it was found that the color and the near-infrared (NIR) spectrum of the prepared crystal changed depending on the water content in the dried samples.
The water content in the dried samples was restored to that in the trihydrate crystal by placing the samples in a humid environment. In addition, the results of X-ray diffraction (XRD) and X-ray diffraction-differential calorimetry (XRD-DSC) analyses show that the phase of the trihydrate crystal changed when the crystallization water was eliminated. The dried crystal has two crystalline forms that are restored to the original trihydrate crystals in 20% relative humidity (RH). This crystalline (PQQ disodium trihydrate) is stable under normal environment.
Previous studies showed that PQQ and reduced PQQ are antioxidative in nature [14, 15]. Their antioxidative function was observed in culture cells and rats [16, 17]. PQQ disodium of an ionic material is often used as a water-soluble salt because the tricarboxylic acid type has low solubility. In many cases, organic molecules exist in a crystalline state in several different forms such as polymorphs or hydrates. The crystalline form of an organic molecule is important from the viewpoint of its application in food and pharmaceutical products. However, the polymorphs of PQQ disodium crystalline have not been studied. Thus far, the crystal structures of an acetone adduct , a metal complex , an ester , and PQQ disodium pentahydrate  have been reported.
A single crystal of PQQ disodium pentahydrate was obtained by the slow evaporation of phosphate buffer at 20°C. However, the crystal of PQQ disodium pentahydrate obtained by this method is unstable because it contains a large amount of water. Therefore, owing to this drawback, the abovementioned method is not suitable for the industrial manufacture of crystals of PQQ disodium pentahydrate. On the other hand, a powder of PQQ disodium was industrially manufactured by adding ethanol (poor solvent) to a water solution of PQQ disodium . However, this product, which included ethanol, had low crystallinity.
We successfully prepared PQQ disodium trihydrate with low water content. The structure of the prepared crystal was analyzed by single crystal X-ray diffraction.
Further, we studied the effect of the water content on the crystalline form, color, and near-infrared (NIR) spectrum of the prepared crystal.
Results and discussion
Single crystal analysis
Crystal data for PQQdisodium trihydrate
Habit red, block
0.100 × 0.100 × 0.100 mm
Primitive, a = 7.6087(2) Å, b = 10.0963(2) Å, c = 11.4337(2) Å, α = 72.858(5) °, β = 88.015(7) °, γ = 82.627(6) °, V = 832.37(4) Å3
1.708 g/cm3, F000 436.00
The crystal obtained was nothing but the PQQ disodium crystal, but with a low amount of water. This low hydration is effective in suppressing the growth of microbe and increasing the stability of the material when it is used in food and drugs.
Drying and moisture absorption
Recently, near-infrared (NIR) analysis has been used for the quality control of food and drugs. An important point to note here is that the results of this method of analysis are also strongly influenced by the water content in a crystal. From the NIR spectrum of a crystal, it was found that a broad peak became sharp with a decrease in the water content (see Figure 3). The water content of the samples converged to approximately 12% when the samples were subjected to a humidity of 75% RH at 40°C for 1 day. After moisture absorption, the color and the NIR spectra of all the samples were identical to those of the original trihydrate crystal. It is interesting to note that a sample whose water content was 26.5% higher than that of the trihydrate crystal showed a water content of only 12%. This result indicated that the water outside the crystal evaporated and that the crystalline water remained. It is easy to change the weight of a very dry sample in a normal environment because the dried sample now becomes hygroscopic in nature.
Water content and powder X-rays analysis
Techniques for carrying out structure determination directly from powder X-ray diffraction (XRD) data clearly provide the structural properties of a polycrystalline product in solid-state transformation. We studied the structure of the crystal of PQQ disodium trihydrate (type I) by XRD when it was subjected to the drying process. The temperature was continuously changed to examine a decrease in the water content and a change in the crystal structure.
Amount of heat can be calculated from DSC curve. The analysis is shown below.
In heat absorption, 187 J/g, the peak top temperature was 73°C, and crystal water was eliminated. A slight change of DSC was measured at 180°C, and the conversion in the phase occurred at 180°C. In the subsequent low-temperature process, the absorption of the heat and a change in the XRD profile were not observed. The peak obtained after the DSC of the dehydration of the crystalline water was in good agreement with that of the type II crystal obtained from the type I crystal of trihydrate. Furthermore, the type II crystal was changed to type III crystal at 180°C. The mixture of type I and type II crystals could be observed in air, but not in nitrogen. The crystal structure did not change under dry condition. When the temperature of the type III crystal was lowered to room temperature under nitrogen atmosphere, the structure was maintained. However, the type III crystal structure changed to type I in the air.
The type II crystal is an anhydride; the experiment performed in this study shows that the phase of the type II crystal changed at 180°C, at which point a new type III crystal was formed, with a structure that differed from that of the type II crystal. The hydration of the type III crystal at 35°C was measured by XRD-DSC in a moisture absorption process.
First, we thought that the change in the crystalline form advanced from trihydrates to mono- or di-hydrates in the type II crystal. It was thought that due to high absorbency, type III crystal was not observed in normal dry air. However, phase transition occurs in the type II crystal after a dehydration process on XRD-DSC in nitrogen. Moreover, the type II crystal was transformed to a type III crystal at a high temperature of 180°C. In other words, the type II crystal is an anhydride crystal, and the type III crystal is an anhydrous crystal. A mixture of a type I and type II crystal can be obtained in air with humidity. Details on non-aqueous crystal structures are not yet understood.
We expected that on elimination of the water, the crystals would become either amorphous or porous. However, contrary to our expectation, the crystals were not amorphous. The surface area of these crystals was measured by nitrogen absorption. From the results of this measurement, it was found that the surface area of all the crystals was less than the BET surface area of 2 m2/g. This result shows that the crystal structures are not porous and that they change considerably with the removal of water.
We also found that the water intercalated in to a crystal to result in the formation of trihydrate. The following conclusions can be drawn from the abovementioned results (Figure 7). The color, the NIR spectrum attributed to drying, and the structure of the type II trihydrate crystals change.
Furthermore, phase transition of the type II crystal occurs at 180°C—a temperature at which the type III crystal is formed. The crystal can either be of type I, a mixture of type I and type II, and type II, depending on the humidity in air. The type III crystal can also be obtained at room temperature by reducing the phase transition temperature of the type II crystal in the dry state. The trihydrates were found to be stable in a humid environment, whereas the type II and type III crystals were unstable in this environment in that they both returned to their original phase. Trihydrate crystals can easily absorb as much as 20% water from a humid environment, contributing to their stability under humidity. Therefore, a trihydrate crystal exhibits suitable properties such that it can be used as a commercial product.
A single crystal of PQQ disodium trihydrate was prepared. Two carboxylic acid and two quinone form ionic bonds with oxygen and sodium in this crystal. The water content of this crystal had a significant effect on the color and the change in the NIR absorption spectrum; the prepared crystal was found to undergo phase transition at different temperatures. This crystal exhibits a crystalline form that 180° or more different from that of an anhydride. The dried sample becomes a trihydrate crystal at 20% humidity, with the color and NIR spectrum same as trihydrate crystals. In case of this crystal, trihydrates are stable and their absorbency is low. For their analysis and stability of their color, extensive drying should be avoided. This crystalline (PQQ disodium trihydrate) is stable under normal environment.
Preparation of PQQ disodium crystals
The PQQ was prepared by fermentation and was purified by column chromatography. The PQQ trisodium solid was precipitated by adding NaCl at pH 7.5. A suspension of this solid was obtained with the addition of a 50% by volume ethanol-water solution. Then, hydrochloric acid was slowly added to this suspension, following which the suspension was crystallized at pH 3.5, and a PQQ disodium crystal was obtained. The samples with different content of crystallization water were prepared under different vacuum conditions.
Water content analysis
The sample was heated to 180°C, and the water that was eliminated from the crystal was analyzed by the Karl Fischer method using a Metrohm 831 KF coulometer.
Single crystal X-ray structure analysis
Rigaku R-AXIS RAPID, radiation Cu-Kα voltage/current: 40 kV/30 mA, 23.0°C. Crystallography data have been deposited additional files, Additional file 1: Check cif report, Additional file 2: Original X-ray analysis data by CIF format.
Powder X-ray diffractometry (XRD)
M18XCE, manufactured by MAC Science, Co. Ltd.
Cu-Kα 40 kV/100 mA, divergence slit: 1°, scattering slit: 1°, receiving slit: 0.3 mm, scan speed: 4.000°/min, sampling width: 0.02°.
Rigaku SmartLab (sample horizontal model multi-purpose X-ray diffraction)
High-speed one-dimensional detector D/teX Ultra
X-ray source Cu-Kα (40 kV/50 mA) with a scanning speed of 5°/min
0% RH, 30–180°C (3°C/min) Step 2: 35°C, 0–90% RH atmosphere dry-wet N2.
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