J. Mass Spectrom. Soc. Jpn. Vol. 51, No. 1, 2003 REGULAR PAPER Elimination Processes of Guest Molecules from the Inclusion Compounds of Deoxycholic Acid῍ Takayoshi K>BJG6,῏a) Yuichi K6H6>,a) Tomohide THJ?>BDID,a) Emi K6L6BJG6,a) Tadashi K6B>N6B6,a) and Hatsumi A@>b) (Received November 11, 2002; Accepted December 10, 2002) Thermal behaviors of the inclusion compounds of deoxycholic acid with methanol, ethanol, propane-1-ol, propane-2-ol, and butane-1-ol have been studied by TG-DTA-MS and DSC. Liberation processes of guest molecules from their inclusion compounds of methanol, ethanol were carried out in a single step. Those of propanols and butane-1-ol from the corresponding inclusion compounds proceed in complicated steps. The elimination temperatures, enthalpies and the activation enthalpies of eliminations have been determined by TG, DSC, and MS. 1. Introduction 2. Experimental Bile acids are synthesized from free cholesterol in the Deoxycholic acid (Sigma Chemical Co., Ltd.) was pur- liver and secreted into the small intestine via bile, ified by recrystallization using the purified acetone. All where they aggregate to form micelles in conjunction organic solvents (Kishida Chemical) used as guests with phospholipid or fatty acid. These mixed micelles were fractionally distilled over freshly activated molec- enable the solubilization of lipids and the absorption of ular sieves 4A.6) GLC results of purified solvents fat from the gastrointestinal tract. Also the bile acids showed no trace-impurity peak. Coulometric Karl- form various types of inclusion compounds with many Fischer’s method on a Moisturemeter (Mitsubishi Che- kinds of organic compounds.1) So those inclusion com- mical Ind., CA-02) gave the water content of each pounds might be performed ideal formulation. The sample to be 0.01 molῌ per cent or less. Water was crystal structures of the inclusion compounds of some purified as usual method.8) bile acid with alcohols, nitriles, ketones, esters and Inclusion compounds of deoxycholic acid with meth- others have been determined by X-ray di#raction.1)῎5) anol, ethanol, propane-1-ol, propane-2-ol, and butane-1- Thermal behaviours of cholic acid and its inclusion ol were obtained by recrystallization using the purified compounds with methanol, ethanol, and acetophenone guest solvents. The inclusion compounds synthesized were already investigated by TG-DTA, sample control were evacuated at 10 K below the first elimination thermogravimetry, DSC, and TG-MS.6), 7) temperature of each compound for 10 h to 20 h under In this paper, thermal behaviors of deoxycholic acid 0.01 Pa. BaCl2ῌ2H2O(Kishida Chemical) have purified and its inclusion compounds with methanol, ethanol, by recrystalization from water. And the crystal was propane-1-ol, propane-2-ol, and butane-1-ol have been evacuated at 420 K under 0.01 Pa for more than 6 studied by TG-DTA-MS and DSC. In order to clarify hours. Thermal properties have been determined by the behavior of elimination process of inclusion com- TG-DTA-MS (Rigaku, TG8101D), and DSC (Rigaku, pounds of deoxycholic acids, TG-MS method were DSC8240). Temperatures were scanned from 0.2 to 10 used. Kminῌ1 under the nitrogen gas flow of 50 mL minῌ1 To test the reliability of these behavioral characters for TG-DTA and DSC, and the helium gas flow of 100 ῌ1 by means of TG-MS, BaCl2ῌ2H2Ohave been measured mL min for TG-DTA-MS. by TG-MS, TG-DTA and DSC, and compared those 3. Results and Discussion results. In order to determine complicated elimination from the sample, TG-MS was powerful instruments for qual- itative analysis. The mass spectrometry and mass a) ῏ Department of Chemistry, Faculty of Science and Tech- chromatogram can identify the eliminated molecules ῎ ῎ nology, Kinki University (3 4 1 Kowakae, Higashi-Osaka from the sample by fragmentation analysis. An exam- 577῎8502, Japan) ple of curves of TG, DSC, and MS for BaCl ῌ2H Owas TEL: (81)῎6῎6721῎2332 ext 4112, FAX: (81)῎6῎6723῎ 2 2 2721 E-mail: [email protected] shown in Figs. 1, 2, and 3, respectively. The thermo- b) Faculty of Pharmaceutical Science, Fukuoka University (8῎ grams of TG, DSC, and MS were consistent for two- 19῎1Nanakuma, Jonan-ku, Fukuoka 814῎0180, Japan) step’s elimination behaviors of water from BaCl2ῌ2H2O. ῍ Congratulations on winning the 2002 Nobel Prize Hearty congratulation to you on the Nobel Prize. This must owe to your untiring and industry in your science, coupled with your extraordinary prominent ability. Looking forward to further success. ῍242῍ Elimination Processes of Guest Molecules from the Inclusion Compounds of Deoxycholic Acid Furthermore, to confirm for quantitative analysis of rate process of elimination of water by means of TG, DSC, and MS, Ozawa method9), 10) were applied to the results of thermograms of BaCl2ῌ2H2OofTG, DSC, and MS. Elimination fractions of evolved gas from 10῎ to 90῎ were determined by integration of fragment ions and molecular ion in mass chromatogram. The loga- rithms of f at elimination fractions of 30῎,40῎,50῎, 60῎,and70῎ were plotted against inversed tempera- ture. Here f is scanning rate of 3 K minῌ1,5Kminῌ1, 7Kminῌ1,and10 K minῌ1.The activation energies determined from the Ozawa method by TG, DSC, and MS were summarized in Table 1. The three results were agreed within their error. Average values of Fig. 1. An example TG curves of BaCl ῌ2H O. three methods for activation energy of the first and 2 2 ῌ ῌ ῌ ῍ 1 Scanning rate: a, 3/K min 1;b,5/K min 1;c,7/ second elimination of water were (113 3) kJ mol ῌ1 Kminῌ1;d,10/K minῌ1. and (85῍2) kJ mol ,respectively. An example of the TG curves and the DSC curves of the inclusion compounds of the deoxycholic acid are shown in Figs. 4 and 5. TG curves of the inclusion compounds of deoxycholic acid with propanols and butane-1-ol showed clearly two or more steps during a Fig. 2. An example DSC curves of BaCl2ῌ2H2O. Scanning rate: a, 10/K minῌ1;b,5/K minῌ1;c, 2.5/K minῌ1;d,1.25/K minῌ1. Fig. 4. An example of DSC curves of DCA and their inclusion compounds. Scanning rate 5 K minῌ1: sample weight ca.10mg. Fig. 3. An example of mass chromatograms of BaCl2ῌ2 ῌ1 H2O. Scanning rate: a, 5/K min ;b,2.5/K minῌ1;c,1.25/K minῌ1. Fig. 5. An example of TG curves of DCA and their inclusion compounds. Scanning rate 2 K minῌ1: sample weight ca.10mg. Table 1. Activation Energy of Elimination of Water of BaCl2ῌ2H2ObyMeans of Three Techniques in kJ molῌ1 Technique TG DSC MS Elimination First Second First Second First Second ῌ1 Ea/kJ mol 112῍484.0῍3.7 111῍386.6῍3.1 117῍685.2῍1.8 ῍243῍ T. Kimura et al. low scanning rate below 3 K minῌ1.Deoxycholic acid ination temperatures and enthalpies were within 0.2 K showed no decomposition during the heating process and 0.02 kJ molῌ1,respectively. The elimination tem- in TG and DSC measurements with careful inert gas perature and the enthalpies of elimination of inclusion purge. However insu$cient inert gases (N2 and He) purge leads to some discoloration of deoxycholic acid. The color of the deoxycholic acid became a yellow to umber. Mass spectrum of those inclusion compounds showed that the evolved gases obtained from the inclu- sion compounds during all elimination steps were only guest alcohols from each inclusion compounds. All mass spectrum measured showed no water spectrum similarly to inclusion compounds of acetone and bile acids.11) The ion chromatograms as well as the TG curves of inclusion compounds were shown in Figs. 6῍ 10. Each number by the curve in ion chromatogram means mass-to-charge ratio of a gas detected. The inclusion compound of deoxycholic acid with metha- nol and ethanol showed a simple elimination process as shown in Figs. 6 and 7. The elimination process of guest molecules from the inclusion compounds with propanols and butane-1-ol showed two step-elimination under low scanning rates Fig. 8. An example of TG curve and mass chromato- in TG, and those behaviors similar to the cases of grams of the inclusion compound of DCAῌ inclusion compound of cholic acid.6), 7) propane-1-ol. The elimination temperatures and the enthalpies of elimination of guest molecules are summarized in Table 2. The experimental errors determined for elim- Fig. 9. An example of TG curve and mass chromato- grams of the inclusion compound of DCAῌ butane-1-ol. Fig. 6. An example of TG curve and mass chromato- grams of the inclusion compound of DCAῌ methanol. Fig. 7. An example of TG curve and mass chromato- Fig. 10. An example of TG curve and mass chromato- grams of the inclusion compound of DCAῌ grams of the inclusion compound of DCAῌ ethanol. propane-2-ol. ῌ244ῌ Elimination Processes of Guest Molecules from the Inclusion Compounds of Deoxycholic Acid Table 2. Thermal properties of inclusion compounds of alcohol-bile acid by TG and DSC Deoxycholic acid Cholic acida) Guest T1 T2 DH1 DH2 T1 T2 DH1 DH2 K K kJ molῌ1 kJ molῌ1 K K kJ molῌ1 kJ molῌ1 Methanol 306.9 0.798 377.7 20 Ethanol 365.5 4.87 384.4 33.1 Propane-1-ol 359.7 409.9 5.85 0.329 371.2 443 32.3 5.4 Propane-2-ol 361.6 378.1 0.92 1.24 Butane-1-ol 329.9 375.1 1.04 0.967 a) Cited values from T. Kimura et al.6), 7) compound of cholic acid with methanol,6) ethanol,6) and ol compound was larger than that for propane-2-ol propane-1-ol7) are also shown in the Table 2 for compar- compound.