BIOMEDICAL CHROMATOGRAPHY, VOL. 6,255-257 (1Y92)
The Retention Behaviour of Conjugated Bile Acids in Reversed Phase High Performance Liquid Chromatography Y. K. Zhang*, N. Chen, R. H. Zao, T. Li, Z. Y. Wang and P. C. Lu National Chromatographic R. & A . Centrc, Dalian Institutc of Chemical Physics, Chinese Academy of Sciences. Dalian 116012, China
The retention behaviour of conjugated bile acids has been studied in a reversed phase high performance liquid chromatographic (RP-HPLC) system by using the mixture of methanol and aqueous phosphate buffer as the mobile phase. The retentions of the conjugates in RP-HPLC have been found to be mainly controlled by the glycine and taurine groups. The selectivity between five different glycine and taurine conjugated bile acids is a constant in RP-HPLC. This selectivity has been used for peak identification in the practical separation of conjugated bile acids.
INTRODUCTION Several attempts have been made to develop a reliable method for the identification and determination of individual bile acids and their conjugates in biological fluids. Reversed phase high performance liquid chromatography (RP-HPLC) with UV or fluorescence detection are the most widely used methods for determination of conjugated bile acids in various body fluids (Street and Setchell, 1989; Scalia, 1987; Zhou, 1989; Chen et al., 1992), but up until now there have been few studies on the retention behaviour of conjugated bile acids in RP-HPLC. As described in this paper, we have found that the retentions of conjugated bile acids in RP-HPLC are strongly controlled by the glycine and taurine groups. The selectivity between glycine and taurine conjugated bile acids in the RP-HPLC system by using a mixture of methanol and aqueous phosphate buffer as the mobile phase has been described and used for peak identification in the practical separations of the conjugates.
EXPERIMENTAL Materials and reagents. The conjugated bile acid standards, taurocholic acid (TC), taurochenodeoxycholic acid (TCDC), taurodeoxycholic acid (TDC), tauroursodeoxycholic acid (TUDC), taurolithocholic acid (TLC), glycocholic acid(GC), glycochenodeoxycholic acid(GCDC). glycodeoxycholic acid(GDC), glycoursodeoxycholic acid(GUDC) and glycolithocholic acid(GLC), were purchased from Sigma (St. Louis, MO, USA) and Cal-Biochem-Behring, La Jolla, CA, USA). A standard solution of the mixed conjugates was prepared by dissolving each bile acid in the mobile phase. Methanol (Beijing Chemical Reagent Factory, Beijing, China) and NaH2P0, were analytical grade. Sep-pak ClX cartridges were from Waters Associates Inc. (Milford, MA, USA).
* Author to whom correspondence should be addressed 0269-3879/92/050255-03 $06.50
01992 by John Wiley & Sons, Ltd
Apparatus. The RP-HPLC experiments were carried out using stainless steel columns, packed with RP packing materials of Nucleosil-3 ODS (3 vrn; Macherey-Nagel Gmbh. Duren, Germany) and YWG-C1X (10 pm; Tinjing Chemical Keagent Factory, Tinjing, China). The columns were packed by National Chromatographic R. & A. Centre (Dalian, China). The mobile phase was delivered by a BT-3000 Bitronik pump and the eluates wcre detectcd by a Knauer UV detector set at a 200nm. All the experiments were carried out at room temperature. Preparation of the human bile sample. Sep-pak C18 cartridges were used for the extraction of the conjugated bile acids from human bile. Sep-pak CIS cartridges were primed with methanol, followed by water. An aliquot of human bile was diluted with phosphate buffer, loaded into the cartridge which was then washed with 10% acetone in water. The conjugates were recovered by elution with 4 mL of methanol. The methanol was evaporated under nitrogen and the residue was dissolved in the mobile phase. 10- 50 pL aliquots were injected into the chromatograph.
RESULW AND DISCUSSION It has been widely accepted that the effect of the organic modifier concentrations ( C , ) in binary eluents on the logarithm of the capacity factor ( k ' ) values in RP-HPLC can be expressed as follows: (Snyder et al., 1979; Zhou et al., 1991; Zhang et al., 1990; Chen et al., 1988) In k'
= In kw
+ cC,
where parameter kw is the capacity factor obtained by extrapolation of retention data from binary eluents to pure water. Parameter c is mainly determined by the molecular interactions in the mobile phase. The selectivity a (G/T), which is usually called the conjugation selectivity between glycine and taurine conjugates in RP-HPLC, is defined as the ratio of the capacity factor of glycine conjugated bile acid to that of Receiued 28 February 1992 Arrepted I S April 1992
256
Y.K. ZHANG ET A L
2.0
-
1.6
;
.
(2
between In k' and the methanol concentration are also given in the table, where R is the regression coefficient. It can be seen that the linear regression of the experimental data in all cases is larger than 0.99, which strongly supports the validity of equation (1). The
0.8
1
L
0.8
1 I
2
3
4
5
Figure 1. The conjugation selectivity as a function of each pair of conjugated bile acids. 1 = GUDCITUDC, 2=GC/TC; 3 = GCDCITCDC, 4 = GDC/TDC, 5 = GLCITLC; 0.80, 0.75, 0.70 and 0.65 refer t o the methanol composition in the mobile phase. For analytical conditions, see Table 1.
taurine conjugated bile acid (Chen et uf., 1992) as is shown by equation (2).
from the table, the selectivity factors between five glycine and taurine conjugates are identical at the fixed proportion of methanol. The selectivity factor between glycine and taurine conjugates at 80%, 75% and 70% methanol varied by 1.58k 0.03, 1.47 f 0.02, 1.37 f 0.01, respectively (mean k SD). Figure 1 shows the conjugation selectivity as the function of each pair of conjugated bile acids. Table 2 summarizes the retention values of the 10 major conjugated bile acids and the selectivity factors between glycine and taurine bile acids on different RP-HPLC systems by using the mixture of methanol and aqueous phosphate buffer as the mobile phase at a
a(G/T) = k ' (G)/ k ' (T)
A
Table 1 lists the capacity factors of the 10 conjugated bile acids at different methanol concentrations in RP-HPLC. The results of the linear regression analysis Table2. The capacity factors of 10 major conjugated bile acids and the selectivity factor between glycine- and taurine-conjugated bile acids determined with methanol buffer as the mobile phase using different C18 stationary phases. For experimental conditions, see Experimental Section Bile acid
System l a
k'
System (I
k'
zb (I
1.76 1.31 1.67 1.32 GUDC 1.34 1.27 TUDC 2.47 1.32 2.70 1.32 GC 2.04 1.87 TC 5.07 1.33 5.39 1.34 GCDC 4.03 3.80 TCDC 6.22 1.34 5.80 1.32 GDC 4.63 4.40 TDC 12.22 1.36 12.0 1.33 G LC 8.97 9.00 TLC a System 1 : YWG-C18 (250 x 2.0 mrn i.d.1, methanol-0.01 M phosphate buffer, pH 5.8 (70:30). System 2: Nucleosil3C18, methanol-0.01 M phosphate buffer, pH 5.8 (70:30).
J
0
10
a
20
min
4
I
30
40
Figure 2. Separation of the standard mixture of the 10 conjugates using a C18 rnicrocolumn. YWG-C18 (250x 2.0 mrn i.d.1, methanol-0.01 M phosphate buffer, pH 5.8 (68:32). 1 =TUDC, 2 = GUDC, 3=TC, 4 = GC, 5 =TCDC, 6 =TDC, 7 = GCDC, 8 = GDC, 9=TLC, 10=GLC.
RP-HPLC OF CONJUGATED BILE ACIDS
257
4
Table3. The results of the peak identification of the glycine conjugates using the conjugation selectivity in RP-HPLC" Bile acid
0
10
rnin
2o
30
Figure 3. Chromatogram of the 10 conjugates in human bile i n patients suffering from gallstone diseases; for the experimental conditions see Fig. 2. 1 = TUDC, 2 = GUDC, 3 = TC, 4 GC, 5 = TCDC, 6 =TDC, 7 = GCDC, 8 = GDC, 9 = TLC, 10 = GLC. 7
3 1
TApre)
k' lexp)
a
(rnin)
k' Ipre)
GUDC 7.1 1.90 1.27 7.1 1.90 TUDC 6.1 1.50 GC 9.3 2.80 1.25 9.2 2.84 TC 7.9 2.24 16.5 5.76 1.29 16.0 5.67 GCDC TCDC 13.3 4.46 G DC 18.8 6.70 1.30 18.1 6.55 TDC 15.0 5.16 GLC 35.4 13.50 1.30 34.1 13.21 TLC 27.8 10.40 a YWG-C18 ( 2 5 0 ~ 2 . 0 mm i.d.), methanol-0.01 M phosphate buffer, pH 5.8 (68:32), where T,(exp), T,(pre), k'(exp) and k'(pre) are the experimental and predicted retention times and capacity factors, respectively.
1
I
0
TAexpI
(mid
5
10
min
15 .
Figure 4. Separation of the standard mixture of the 10 major conjugates using C18 packing materials with 3 pm particle size, Nucleosil-3ODS (150 x 4.6). methanol-phosphate buffer (pH=5.15) (76:24). 1 =TUDC, 2=GUDC, 3=TC, 4=GC, 5 = TCDC, 6 = TDC, 7 = GCDC, 8 = GDC, 9 = TLC, 10 = GLC.
specific composition. As is seen in the table, despite a considerable variation in retentions for each pair of glycine and taurine conjugates, the introduction of glycine and taurine groups into the conjugated bile acids results in a constant selectivity between glycine and taurine bile acids irrespective of whatever primary and secondary bile acids they are bonded to. Therefore the retentions of the glycine and taurine bile acids are mainly controlled by the glycine and taurine groups.
Figure 2 shows the separation of the standard of the 10 conjugated bile acids by using a C18 microbore column. Figure 3 shows a chromatogram of the 10 conjugated bile acids in human bile of patients suffering from heptatobiliary diseases. Good resolution of the 10 conjugates within 15 min has also been obtained by using C18 packing materials with 3 pm particle diameter which is illustrated in Fig. 4. The conjugation selectivity can be used to identify the peaks of the glycine or taurine conjugates in RP-HPLC. Peak identification is a relatively weak part of the analysis of the conjugated bile acids. The methods which have been commonly used are based on the retention of the standards. The conjugation selectivity can be used to predict the retentions of the conjugates and can thus identify the peaks of either glycine or taurine conjugates without any standards. Based on the retention behaviour of taurine conjugates, the retentions of the glycine conjugates were calculated by using the selectivity factor of the first eluted pair of GUDC and TUDC. Table 3 shows the results of the peak identification of glycine bile acids in the practical separation in Fig. 3 by using the conjugation selectivity. As can be seen from Table 3, the conjugation selectivity has proved to be a reliable method of peak identification in practical separation techniques.
REFERENCES
Chen. N., Zhang, Y.. Li, Y. and Lu, P. (1988). Chin. J. Chrornatogr. 6, 325. Chen, N., Zhang, Y. and Lu. P. (1992). J. Li9. Chromatogr., 15, 1523. Scalia, S . (1987). J. Liq. Chromatogr. 10, 2055. Snyder, L. R., Dolan, T. W. and Gant, J. R. (1979). J. Chromatogr. 165, 3.
Street, J. M. and Setchell. K. D. R. (1989). Biomed. Chrornatogr. 2,229. Zhang, Y., Zou, H. and Lu, P. (1990). J. Chromatogr. 515, 13. Zou, H. (1989). Doctorate Dissertation, Dalian Institute of Chemical Physics. Zou, H., Wang, Q., Gao, R., Yang, H., Yang, B.,Zhang, Y. and Lu, P. (1991). Chromatographia 31, 143.
The Retention Behaviour of Conjugated Bile Acids in Reversed Phase High Performance Liquid Chromatography Y. K. Zhang*, N. Chen, R. H. Zao, T. Li, Z. Y. Wang and P. C. Lu National Chromatographic R. & A . Centrc, Dalian Institutc of Chemical Physics, Chinese Academy of Sciences. Dalian 116012, China
The retention behaviour of conjugated bile acids has been studied in a reversed phase high performance liquid chromatographic (RP-HPLC) system by using the mixture of methanol and aqueous phosphate buffer as the mobile phase. The retentions of the conjugates in RP-HPLC have been found to be mainly controlled by the glycine and taurine groups. The selectivity between five different glycine and taurine conjugated bile acids is a constant in RP-HPLC. This selectivity has been used for peak identification in the practical separation of conjugated bile acids.
INTRODUCTION Several attempts have been made to develop a reliable method for the identification and determination of individual bile acids and their conjugates in biological fluids. Reversed phase high performance liquid chromatography (RP-HPLC) with UV or fluorescence detection are the most widely used methods for determination of conjugated bile acids in various body fluids (Street and Setchell, 1989; Scalia, 1987; Zhou, 1989; Chen et al., 1992), but up until now there have been few studies on the retention behaviour of conjugated bile acids in RP-HPLC. As described in this paper, we have found that the retentions of conjugated bile acids in RP-HPLC are strongly controlled by the glycine and taurine groups. The selectivity between glycine and taurine conjugated bile acids in the RP-HPLC system by using a mixture of methanol and aqueous phosphate buffer as the mobile phase has been described and used for peak identification in the practical separations of the conjugates.
EXPERIMENTAL Materials and reagents. The conjugated bile acid standards, taurocholic acid (TC), taurochenodeoxycholic acid (TCDC), taurodeoxycholic acid (TDC), tauroursodeoxycholic acid (TUDC), taurolithocholic acid (TLC), glycocholic acid(GC), glycochenodeoxycholic acid(GCDC). glycodeoxycholic acid(GDC), glycoursodeoxycholic acid(GUDC) and glycolithocholic acid(GLC), were purchased from Sigma (St. Louis, MO, USA) and Cal-Biochem-Behring, La Jolla, CA, USA). A standard solution of the mixed conjugates was prepared by dissolving each bile acid in the mobile phase. Methanol (Beijing Chemical Reagent Factory, Beijing, China) and NaH2P0, were analytical grade. Sep-pak ClX cartridges were from Waters Associates Inc. (Milford, MA, USA).
* Author to whom correspondence should be addressed 0269-3879/92/050255-03 $06.50
01992 by John Wiley & Sons, Ltd
Apparatus. The RP-HPLC experiments were carried out using stainless steel columns, packed with RP packing materials of Nucleosil-3 ODS (3 vrn; Macherey-Nagel Gmbh. Duren, Germany) and YWG-C1X (10 pm; Tinjing Chemical Keagent Factory, Tinjing, China). The columns were packed by National Chromatographic R. & A. Centre (Dalian, China). The mobile phase was delivered by a BT-3000 Bitronik pump and the eluates wcre detectcd by a Knauer UV detector set at a 200nm. All the experiments were carried out at room temperature. Preparation of the human bile sample. Sep-pak C18 cartridges were used for the extraction of the conjugated bile acids from human bile. Sep-pak CIS cartridges were primed with methanol, followed by water. An aliquot of human bile was diluted with phosphate buffer, loaded into the cartridge which was then washed with 10% acetone in water. The conjugates were recovered by elution with 4 mL of methanol. The methanol was evaporated under nitrogen and the residue was dissolved in the mobile phase. 10- 50 pL aliquots were injected into the chromatograph.
RESULW AND DISCUSSION It has been widely accepted that the effect of the organic modifier concentrations ( C , ) in binary eluents on the logarithm of the capacity factor ( k ' ) values in RP-HPLC can be expressed as follows: (Snyder et al., 1979; Zhou et al., 1991; Zhang et al., 1990; Chen et al., 1988) In k'
= In kw
+ cC,
where parameter kw is the capacity factor obtained by extrapolation of retention data from binary eluents to pure water. Parameter c is mainly determined by the molecular interactions in the mobile phase. The selectivity a (G/T), which is usually called the conjugation selectivity between glycine and taurine conjugates in RP-HPLC, is defined as the ratio of the capacity factor of glycine conjugated bile acid to that of Receiued 28 February 1992 Arrepted I S April 1992
256
Y.K. ZHANG ET A L
2.0
-
1.6
;
.
(2
between In k' and the methanol concentration are also given in the table, where R is the regression coefficient. It can be seen that the linear regression of the experimental data in all cases is larger than 0.99, which strongly supports the validity of equation (1). The
0.8
1
L
0.8
1 I
2
3
4
5
Figure 1. The conjugation selectivity as a function of each pair of conjugated bile acids. 1 = GUDCITUDC, 2=GC/TC; 3 = GCDCITCDC, 4 = GDC/TDC, 5 = GLCITLC; 0.80, 0.75, 0.70 and 0.65 refer t o the methanol composition in the mobile phase. For analytical conditions, see Table 1.
taurine conjugated bile acid (Chen et uf., 1992) as is shown by equation (2).
from the table, the selectivity factors between five glycine and taurine conjugates are identical at the fixed proportion of methanol. The selectivity factor between glycine and taurine conjugates at 80%, 75% and 70% methanol varied by 1.58k 0.03, 1.47 f 0.02, 1.37 f 0.01, respectively (mean k SD). Figure 1 shows the conjugation selectivity as the function of each pair of conjugated bile acids. Table 2 summarizes the retention values of the 10 major conjugated bile acids and the selectivity factors between glycine and taurine bile acids on different RP-HPLC systems by using the mixture of methanol and aqueous phosphate buffer as the mobile phase at a
a(G/T) = k ' (G)/ k ' (T)
A
Table 1 lists the capacity factors of the 10 conjugated bile acids at different methanol concentrations in RP-HPLC. The results of the linear regression analysis Table2. The capacity factors of 10 major conjugated bile acids and the selectivity factor between glycine- and taurine-conjugated bile acids determined with methanol buffer as the mobile phase using different C18 stationary phases. For experimental conditions, see Experimental Section Bile acid
System l a
k'
System (I
k'
zb (I
1.76 1.31 1.67 1.32 GUDC 1.34 1.27 TUDC 2.47 1.32 2.70 1.32 GC 2.04 1.87 TC 5.07 1.33 5.39 1.34 GCDC 4.03 3.80 TCDC 6.22 1.34 5.80 1.32 GDC 4.63 4.40 TDC 12.22 1.36 12.0 1.33 G LC 8.97 9.00 TLC a System 1 : YWG-C18 (250 x 2.0 mrn i.d.1, methanol-0.01 M phosphate buffer, pH 5.8 (70:30). System 2: Nucleosil3C18, methanol-0.01 M phosphate buffer, pH 5.8 (70:30).
J
0
10
a
20
min
4
I
30
40
Figure 2. Separation of the standard mixture of the 10 conjugates using a C18 rnicrocolumn. YWG-C18 (250x 2.0 mrn i.d.1, methanol-0.01 M phosphate buffer, pH 5.8 (68:32). 1 =TUDC, 2 = GUDC, 3=TC, 4 = GC, 5 =TCDC, 6 =TDC, 7 = GCDC, 8 = GDC, 9=TLC, 10=GLC.
RP-HPLC OF CONJUGATED BILE ACIDS
257
4
Table3. The results of the peak identification of the glycine conjugates using the conjugation selectivity in RP-HPLC" Bile acid
0
10
rnin
2o
30
Figure 3. Chromatogram of the 10 conjugates in human bile i n patients suffering from gallstone diseases; for the experimental conditions see Fig. 2. 1 = TUDC, 2 = GUDC, 3 = TC, 4 GC, 5 = TCDC, 6 =TDC, 7 = GCDC, 8 = GDC, 9 = TLC, 10 = GLC. 7
3 1
TApre)
k' lexp)
a
(rnin)
k' Ipre)
GUDC 7.1 1.90 1.27 7.1 1.90 TUDC 6.1 1.50 GC 9.3 2.80 1.25 9.2 2.84 TC 7.9 2.24 16.5 5.76 1.29 16.0 5.67 GCDC TCDC 13.3 4.46 G DC 18.8 6.70 1.30 18.1 6.55 TDC 15.0 5.16 GLC 35.4 13.50 1.30 34.1 13.21 TLC 27.8 10.40 a YWG-C18 ( 2 5 0 ~ 2 . 0 mm i.d.), methanol-0.01 M phosphate buffer, pH 5.8 (68:32), where T,(exp), T,(pre), k'(exp) and k'(pre) are the experimental and predicted retention times and capacity factors, respectively.
1
I
0
TAexpI
(mid
5
10
min
15 .
Figure 4. Separation of the standard mixture of the 10 major conjugates using C18 packing materials with 3 pm particle size, Nucleosil-3ODS (150 x 4.6). methanol-phosphate buffer (pH=5.15) (76:24). 1 =TUDC, 2=GUDC, 3=TC, 4=GC, 5 = TCDC, 6 = TDC, 7 = GCDC, 8 = GDC, 9 = TLC, 10 = GLC.
specific composition. As is seen in the table, despite a considerable variation in retentions for each pair of glycine and taurine conjugates, the introduction of glycine and taurine groups into the conjugated bile acids results in a constant selectivity between glycine and taurine bile acids irrespective of whatever primary and secondary bile acids they are bonded to. Therefore the retentions of the glycine and taurine bile acids are mainly controlled by the glycine and taurine groups.
Figure 2 shows the separation of the standard of the 10 conjugated bile acids by using a C18 microbore column. Figure 3 shows a chromatogram of the 10 conjugated bile acids in human bile of patients suffering from heptatobiliary diseases. Good resolution of the 10 conjugates within 15 min has also been obtained by using C18 packing materials with 3 pm particle diameter which is illustrated in Fig. 4. The conjugation selectivity can be used to identify the peaks of the glycine or taurine conjugates in RP-HPLC. Peak identification is a relatively weak part of the analysis of the conjugated bile acids. The methods which have been commonly used are based on the retention of the standards. The conjugation selectivity can be used to predict the retentions of the conjugates and can thus identify the peaks of either glycine or taurine conjugates without any standards. Based on the retention behaviour of taurine conjugates, the retentions of the glycine conjugates were calculated by using the selectivity factor of the first eluted pair of GUDC and TUDC. Table 3 shows the results of the peak identification of glycine bile acids in the practical separation in Fig. 3 by using the conjugation selectivity. As can be seen from Table 3, the conjugation selectivity has proved to be a reliable method of peak identification in practical separation techniques.
REFERENCES
Chen. N., Zhang, Y.. Li, Y. and Lu, P. (1988). Chin. J. Chrornatogr. 6, 325. Chen, N., Zhang, Y. and Lu. P. (1992). J. Li9. Chromatogr., 15, 1523. Scalia, S . (1987). J. Liq. Chromatogr. 10, 2055. Snyder, L. R., Dolan, T. W. and Gant, J. R. (1979). J. Chromatogr. 165, 3.
Street, J. M. and Setchell. K. D. R. (1989). Biomed. Chrornatogr. 2,229. Zhang, Y., Zou, H. and Lu, P. (1990). J. Chromatogr. 515, 13. Zou, H. (1989). Doctorate Dissertation, Dalian Institute of Chemical Physics. Zou, H., Wang, Q., Gao, R., Yang, H., Yang, B.,Zhang, Y. and Lu, P. (1991). Chromatographia 31, 143.
