Determination of Free Bile Acids in Pharmaceutical Preparations by Packed Column Supercritical Fluid Chromatography

SANTO SCALIA**' AND DAVID E. GAMES* Received October 22, 1991. from the 'Mass Spectrometry Research Unit, Department of Chemistry, University College of Swansea, Singleton Park, Swansea SA2 8PP, U.K Universita' di Ferrara, via Fossato di Mortara 17- 19, 44 100 Ferrara, Italy.

Accepted for publication May 22, 1992. *Permanent address: Dipartimento di Scienze Farmaceutiche,

Abstract 17 A method was developed for the baseline separation of common free bile acids by supercritical fluid chromatography. A phenyl- bonded silica column, with UV detection at 21 0 nm, and carbon dioxide modified with methanol as the mobile phase were used. The influence of the stationary phase, modifier concentration, temperature, column pres- sure, and modifier identity on retention was studied. The separation obtained is at least eight times faster than those achieved for similar mixtures by the existing chromatographic techniques. This new proce- dure is applicable to the assay of ursodeoxycholic acid and chenode- oxycholic acid in capsule and tablet formulations. Individual dosage forms were disintegrated in methanol and an aliquot of the resulting suspension was filtered for the supercritical fluid chromatographic analysis. The method is rapid, accurate, and reproducible.

The commonly occurring bile acids in human fluids are cholic acid (CAI, chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA),ld which are present primarily as glycine or taurine conjugates. Whereas the more hydrophobic bile acids DCA and LCA are hepatotoxic2.4-6 and damaging to the gastric mucosa,7 CDCA and UDCA are widely used in capsule or tablet formulations for the dissolution of cholesterol gall- stones8,Sand in the therapy ofbile reflux gastritis.7 Moreover, the introduction of UDCA for the treatment of cholestatic liver diseases has made it possible for the first time to treat primary biliary cirrhosis, primary sclerosing cholangitis, and cystic fibrosis.10.11 The extensive use of UDCA and CDCA as therapeutic agents required the development of a rapid and precise procedure for the determination of the two bile acids in pharmaceutical preparations.

Published methods for the assay of CDCA and UDCA in dosage forms include calorimetry,l2 potentiometry,13 and voltammetry.14 However, for routine quality control, these

R R1

CA OH H

CDCA OH H

DCA H H

LCA H H

UDCA H OH

R2

OH

H

OH

H

H

techniques have distinct disadvantages such as the complex- ity of the apparatus or the procedure,l2J4 and unsatisfactory reproducibility and selectivity.1214 More recently, an im- proved method for the determination of CDCA and UDCA in commercial medicinal drugs,15 based on reversed-phase high- performance liquid chromatography (HPLC), has been devel- oped.

Supercritical fluid chromatography (SFC) with packed columns offers advantages over HPLC16 in terms of speed of analysis and method development and higher transparency of the eluant in the short UV wavelength region where com- pounds lacking a strong chromophore are normally detected. Although a large portion of the literature on SFC is devoted to the analysis of relatively nonpolar molecules, the addition of a polar modifier to low polarity supercritical fluids, such as carbon dioxide, has expanded the application of SFC to more polar analytes.17Js

A novel method is described in this study for the rapid and complete separation of the common free bile acids by packed- column SFC. The application of this procedure to the assay of UDCA and CDCA in pharmaceutical preparations is also reported.

Experimental Section Reagents-The sodium salts of CA, CDCA, UDCA, DCA, and LCA

were purchased from Sigma (St. Louis, MO). Their purity was checked by reversed-phase HPLC prior to use. Commercial medicinal drugs containing CDCA or UDCA were supplied by various manu- facturers (drug 1: lot A2201, Midy, Milan, Italy; Drug 2: lot 2614, ABC, Turin, Italy; Drug 3: lot 81240, Zambon Group, Milan, Italy).

Instrument-grade liquid carbon dioxide, supplied in cylinders with a dip tube, was obtained from BOC (London, U.K.). HPLC-grade methanol, acetonitrile, propyl alcohol, and water were from Fisons plc (Ipswich, U.K.). All other chemicals were of analyticalgrade (Sigma).

Chromatography-A Hewlett-Packard 1084B high-performance liquid chromatograph (Hewlett-Packard, Avondale, PA), modified for SFC operation,lg was used. The carbon dioxide was introduced directly into the "A' pump of the chromatograph and the solvent modifier (usually methanol) was placed in the "B" pump, which was operated in the HPLC mode. The liquid carbon dioxide and the pump heads of the chromatograph were cooled to -20 "C with a Neslab RTEdZ refrigerated bath (Neslab Instruments, Newington, NH). To maintain supercritical conditions in the column, a Tescom mechan- ical back-pressure regulator (Tescom, Minneapolis, MN) was con- nected to the outlet of the flowcell. Samples were introduced onto the column via a Rheodyne 7125 injector fitted with a 10-pL sample loop (Rheodyne, Cotati, CAI. The column effluent was monitored by the built-in multiple wavelength UV-vis detector set at a wavelength of 210 nm and 0.05 absorbance units full scale. Separations were performed on 250 x 4.6-mm i.d. stainless-steel columns packed with octadecyl-, octyl-, cyanopropyl-, amino-, or phenyl-bonded 5-pm Zorbax phases (Hicrom, Reading, UK). A 5-pm Deltabond cyan0 column (250 x 4.6-mm i.d.; Keystone Scientific, State College, PA) was also used.

44 f Journal of Pharmaceuticai Sciences Vol. 82, No. 1, January 1993

0022-3549/93/0 I 00-0044$0Z.50/0 0 1993, American Pharmaceutical Association

The HPLC apparatus consisted of a Jasco chromatographic system (model 880-PU pump, model 880-02 ternary gradient unit, and model 875 W-vis detector; Jasco, Tokyo, Japan) linked to an injection valve with a 10-pL sample loop (Rheodyne) and a chromatographic data processor (Chromatopac C-R3A, Shimadzu, Kyoto, Japan). The de- tector was set at 210 nm and 0.08 absorbance units full scale. Separations were performed according to the method described earlier's with a 3-pm C-8 Ultrasphere column (75 x 4.6-mm i.d.; Beckman, Berkeley, CA) and isocratic elution with methanol:0.02 M aqueous sodium acetate (80:20, v/v) adjusted to pH 4.3 with phos- phoric acid. The mobile phase was filtered through type-HVLP filters (0.45 pm; Millipore S.A., Molsheim, France) and deaerated on-line with a model ERC-3311 solvent degasser (Erma, Tokyo, Japan). Chromatography was performed at ambient temperature at a flow rate of 1.0 mL/min. Areas under the chromatogram peaks (peak areas) were used for calculations.

Sample and Standard Preparation-Individual tablets were pow- dered and transferred to a 50-mL volumetric flask. Methanol was added, and the sample was dispersed by sonication (5 min) and then diluted to volume with methanol. A portion of the resulting suspen- sion was filtered through a 0.45-~m membrane filter (Millipore S.A.) for the SFC assay. Individual capsules were cut open and their contents rinsed into a 50-mL volumetric flask with methanol. The sample was sonicated for 5 min and then diluted to the 50-mL mark, and an aliquot was filtered for the SFC analysis.

Standard solutions were prepared by dissolving the labeled amounts of UDCA or CDCA reference material into a 50-mL volu- metric flask and diluting to volume with methanol. The obtained solutions were then assayed as outlined above.

Placebo preparations containing the excipients of the capsules ke., lactose, sodium starch glycolate, starch, talc, precipitated silica, magnesium stearate) or the tablet (i.e., lactose, starch, talc, magne- sium stearate, gum arabic) were spiked with the appropriate amounts of UDCA or CDCA in 50-mL volumetric flasks and subjected to the assay procedure.

Linearity, Accuracy, and Precision-Calibration curves of peak area versus concentration were generated with placebos spiked with known amounts of UDCA or CDCA corresponding to 50,75,100,125, and 150% of label.

The recovery of the assay was determined with placebos spiked with the labeled amounts of UDCA or CDCA. The percentage recovery was calculated by comparing the peak areas of UDCA and CDCA from the placebo preparations with those obtained by injec- tions of the standard solutions.

The reproducibility of the method was evaluated by replicate assays of placebo formulations spiked with the labeled amounts of UDCA or CDCA.

Results and Discussion The objectives of this study were to evaluate the potential

of packed-column SFC with carbon dioxide and polar modi- fiers as the mobile phase for the analysis of the naturally occurring free bile acids. Pure carbon dioxide did not elute any of the compounds investigated from any of the columns used at 300 atm and 40 "C. This can be attributed to interactions between the solutes and active sites, such as residual silanol groups, present on the stationary phase and also to the low polarity of carbon dioxide. Consequently, a mobile phase modifier had to be applied.

The addition of 4% (v/v) methanol to carbon dioxide caused all the bile acids to elute from the nonpolar octadecyl and octyl stationary phases, but the compounds emerged as broad, little retained, and unresolved peaks. At lower methanol concen- tration (2%, v/v) no elution of CA occurred. Although optimal resolution for free bile acids is attained in HPLC by octadecyl packings,20-23 this stationary phase exhibits unsatisfactory retention and selectivity in SFC. An increase in column polarity to an amino-bonded phase produced greater retention but required the use of carbon dioxide modified with 25% methanol to obtain reasonable elution times. However, the column lacked selectivity as CDCA and UDCA coeluted. Moreover UDCA, CDCA, and CA exhibited tailed peaks. The analysis of free bile acids on the cyanopropyl column showed

a marked improvement in selectivity. To obtain reasonable elution times, mobile phases containing >5% modifier were required. The effect of the methanol concentration at 200 atm and 40°C on bile acid retention on the cyan0 column is illustrated in Figure 1. The addition of methanol to carbon dioxide reduced retention and improved peak shape consid- erably. This result is due to a combination of the deactivation of the stationary phase and to an enhancement of the solvent strength of the mobile phase.24 With carbon dioxide modified with 15% methanol, the influence of the column pressure and temperature on bile acid retention was investigated in the ranges 150-250 atm and 40-65 "C, respectively. The capacity factor (k') values decreased with increasing pressure at 40 "C (Figure 2A), although the retention changes were smaller than those obtained by varying the modifier concentration (Figure 1). Increasing the temperature from 40 to 65 "C at constant pressure (200 atm) increased retention (Figure 2B) without affecting the resolution significantly. Moreover, greater baseline instability was observed at higher temper- atures due to the low U V wavelength (210 nm) used for the detection of these compounds that lack a strong chromophore. Subsequent analyses were carried out at 200 atm and 40 "C because, under these conditions, satisfactory resolution was achieved within a shorter period of time. A chromatogram of a typical separation of the commonly occurring free bile acids on the cyanopropyl column, with 15% methanol in carbon dioxide as the mobile phase, is presented in Figure 3A. Under these conditions, the mobile phase is in a subcritical rather than a supercritical state. However, as reported in the literature,ls chromatographic results indicate that there is no difference in the solvent properties of a just supercritical dense gas or a just subcritical dense fluid, provided the densities of the two fluids are similar. The separation shown

20

15

L 10

0

CA

* UDCA

-+& CDCA

10 15 20 25

% MeOH Figure I-Effect of methanol concentration in carbon dioxide on the K of free bile acids. Operating conditions: column, cyanopropyl Zorbax; flow rate, 4 mUmin; inlet pressure, 200 atm; temperature, 40 "C; UV detection, 210 nm.

Journal of Pharmaceutical Sciences I 45 Vol. 82, No. 1, January 1993

10

12r B

+3- UDCA

- -% CDCA

'u

10

6 -

6 -

4 -

-

40 50 60 85 04 I

T Figure 2-Effect of column (A) pressure at 40 "C and (B) temperature at 200 atm on the K of free bile acids. The mobile phase was 15% methanol in carbon dioxide and the column and flow rate were as in Figure 1.

A

1 1 1 1 I I I l l 1 I 2 3 4 5 6 m i n 1 2 3 4 min

Figure *The SFC separation of a synthetic mixture of bile acids on (A) a cyanopropyl column and (B) a phenyl column. The mobile phase was 15% methanol in carbon dioxide and the other operating conditions were as in Figure 1. Peaks: (1) LCA, (2) DCA, (3) CDCA, (4) UDCA, and (5) CA.

in Figure 3A follows a normal-phase mechanism because the solutes elute in order of increasing polarity following the number of hydroxyl groups on the steroid nucleus (retention

increase in the order monohydroxy < dihydroxy < trihy- droxy). Although normal-phase systems for the resolution of free bile acids by HPLC have been reported,25 their require- ment for a preliminary derivatization step is a disadvantage. The retention sequence reported in Figure 3A is the reverse, with the exception of UDCA, of that obtained by the reversed- phase HPLC procedures used currently.20-23@

The effect of different modifiers on the retention of the foregoing bile acids was also examined. Under the same experimental conditions, a change from methanol to n-propyl alcohol increased analysis time 1.6-fold and produced a broad and asymmetric CA peak. The bile acids were completely adsorbed on the cyan0 column when methanol was replaced with acetonitrile (15% v/v in carbon dioxide), which acted as a weak solvent, allowing larger injection volumes (>7 pL) compared with methanol.24 However, samples were dissolved in methanol because of the lower solubility of bile acids in acetonitrile.27

A deactivated cyano column (Deltabond) packed with poly- mer-coated porous silica particles was also evaluated for the separation of bile acids, with the same experimental condi- tions as those reported above. In comparison with the con- ventional cyano column, a decreased methanol concentration (8% v/v in carbon dioxide) was required to elute the com- pounds in similar retention times. Despite improved peak shape, the Deltabond column exhibited unsatisfactory selec- tivity, resulting in the coelution of DCA and CDCA.

At this point of the experimental work, a phenyl column became available. No significant difference in selectivity was observed between the cyanopropyl- and the phenyl-bonded phases (Figures 3A and 3B). However, with the optimized conditions developed for the cyano column, improved effi- ciency and resolution were attained with the phenyl column (Figure 3B) within a shorter analysis time (3.4 min). The separation of free bile acids (Figure 3B) is at least eight times faster than those typically obtained by the reversed-phase HPLC procedures reported in the literature.20-23 Even with a reversed-phase HPLC column for fast analysis (High Speed Ultrasphere), the complete resolution of the foregoing bile acids (chromatogram not shown) required much longer anal- ysis time (18.6 min) compared with the SFC procedure here developed.

The SFC system described (Figure 3B) was applied to the assay of UDCA and CDCA in pharmaceutical dosage forms. Representative chromatograms of a CDCA capsule formula- tion and of a UDCA tablet preparation are shown in Figures 4 and 5, respectively. No interference was observed from the excipients. Moreover the baseline resolution (Figure 3B) of UDCA and CDCA from the other bile acids (in particular, the hepatotoxic LCA and DCA) commonly present in the raw material of animal origin used for the production of these steroids further demonstrate the specificity of the method. Sample processing involves simply the addition of 50 mL of methanol to the formulation, dissolution, and filtration before injection onto the SFC column. This is the simplest possible sample preparation, circumventing extraction or evaporation steps, and an internal standard is not needed.15

Calibration curves (n = 6) were linear in the range 50-150% of the label claim (1.5-7.5 mg/mL). The average correlation coefficients and slopes were, respectively, 0.995 2 0.007 and 1.175 2 0.058 for UDCA, and 0.997 2 0.004 and 1.374 & 0.044 for CDCA. In no graphs was the intercept on the y-axis significantly different from zero at the 95% confidence interval.

The recovery of the two bile acids from the vehicle matrix was determined with spiked placebos at 100% of the label claim. The average recoveries (n = 10) for UDCA and CDCA were 100.2%, with a relative standard deviation (RSD) of 1.7% and 101.5% with a RSD of 2.2%, respectively.

46 I Journal of Pharmaceutical Sciences Vol. 82, No. 1, Januaty 1993

3 Table I-Assay Results with Individual Tablets and Capsules

Label Claim % Founda RSD Pharmaceutical Preparation

I I I I

1 2 3 4 min

Figure &The SFC chromatogram of a CDCA capsule. Operating conditions and peak identification were as in Figure 3B.

4

I I I I

Figure &The SFC chromatogram of a UDCA tablet. Operating condi- tions and peak identification were as in Figure 38.

1 2 3 4 min

The reproducibility of the method (see Experimental Sec- tion) was 1.4% RSD (n = 10) for UDCAand 1.2% RSD (n = 10) for CDCA.

Three different commercially available pharmaceutical preparations were assayed for UDCA and CDCA with the proposed SFC procedure (Table I). The obtained data confirm the precision of the method and show compliance with the label claim.

In conclusion, the first SFC method has been developed for the determination of the free bile acids in dosage forms. The procedure provides an alternative separation selectivity to the existing reversed-phase HPLC techniques with shorter analysis time. To the best of our knowledge, this is one of the first reports on the use of SFC for routine analyses of commercial pharmaceutical preparations. Because of its ra-

Drug 1 (capsule) 250rngCDCA 101.6 2.3

Drug 3 (tablet) 15OmgUDCA 102.9 2.7 Drug 2 (capsule) 250mgUDCA 101.0 1.9

a Mean of six determinations.

pidity, good accuracy, and reproducibility, the method is well suited to quality control assays of medicinal drugs containing UDCA and CDCA.

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3. Scalia, S. J. Pharm. Biomed. Anal. 1990, 8, 235-241. 4. Scholmerich, J.; Becher, M. S.; Schmidt, K.; Schubert, R.;

Kremer, B.; Feldhaus, S.; Gerok, W. Hepatology 1984,4,661-666. 5. Keane, R. M.; Gadacz, T. R.; Munster, A. M.; Birmingham, W.;

Winchurch, R. A. Surgery 1984,95, 439-443. 6. Whitney, J. 0. In Mass Spectrometry in Biomedical Research;

Gaskell, S., Ed.; John Wiley & Sons: Chichester, U.K., 1986 p 71. 7. Scalia, S.;. Pa&, P.; Stabellini, G.; GuarneA, M.. J. Pharm.

8. Danziger, R. G.; Hofmann, A. F.; Schoenfield, L. J.; Thistle, J. L. Biomed. Anal. 1988, 6, 911-917.

N. Engl. J. Med. 1972,286, 1-8. 9. Ward,A.; Brogden, R. N.; Heel, R. C.; Speight, T. M.; Avery, G. S.

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Darnis, F. Lancet 1987, 1, 83-36. 11. Colombo, C.; Setchell, K. D. R.; Podda, M.; Crosignani, A.; Roda,

A.; Curcio, L.; Ronchi, M.; Giunta, A. J. Pediatr. 1990, 117, 482-489.

12. Biader Ceipidor, U.; Curini, R.; D'Ascenzo, G.; Tomassetti, M. Thermochim. Acta 1981,46. 279-287.

13. Campanella, L.; Sorrentino; L.; Tomassetti, M. Analyst 1983, 108,1490-1494.

14. Ferri, T.; Campanella, L.; De Angelis G. Analyst 1984, 109,

15. Scalia, S.; Pazzi, P.; Guameri, M. Anal. Lett. 1989,22,915-927. 16. Games, D. E.; Berry, A. J.; Mylchreest, I. C.; Perkins, J. R.;

Pleasance, S. In Supercritical Fluid Chromatography; Smith, R. M., Ed.; Royal Society of Chemistry: London, U.K., 1988; pp

17. Berry, A. J.; Games, D. E.; Perkins, J. R. J. Chromatogr. 1986,

18. Berger, T. A.; Deve, J. F. J. Chromatopr. Sci. 1991.29.141-146.

923-925.

159-160.

363, 147-158.

19. GeG, D. R.; Board, 'R. D.; McManigil1;D. Anal. Chem: 1982,54, 736-740.

20. Reid, A. D.; Baker, P. R. J. Chromatogr. 1982,247, 149-156. 21. Elliott, W. H.; Shaw, R. In Methods in Enzymology; Law, J. H.;

Rilling, H. C., Eds.; Academic: London, U.K., 1985; Vol. 111, pp

22. Scalia, S. J. Chromatogr. 1988,431, 259-269. 23. Eckers, C.; Haskins, N. J.; Large, T. Biomed. Enuiron. Mass

24. Blilie, A. L.; Greibrokk, T. Anal. Chem. 1985, 57, 2239-2242. 25. Shaw, R.; Elliott, W. H. Lipids 1978, 13, 971-975. 26. Shaw, R.; Rivetna, M.; Elliott, W. H. J. Chromatogr. 1980,202,

27. Scalia, S. J. Liq. Chromatogr. 1987, 10, 2055-2080.

59-62.

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347-36 1.

Acknowledgments The authors thank the Science and Engineering Research Council

for assistance in the purchase of equipment used in these studies. Dr. Scalia thanks the Consiglio Nazionale delle Ricerche for financial support.

Journal of Pharmaceutical Sciences 1 47 Vol. 82, No. 1, January 1993