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Naphthyldiazomethane as a derivatizing agent for the high-performance liquid chromatography detection of bile acids
Analytica Chimica Acfu, 109 (1979) 161-164 0 Elsevier Scientific Publishing Company, Amsterdam
-
Printed in The Netherlands
Short Communication
NAPHTHYLDIAZOMETHANE AS A DERIVATIZING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY BLLE ACIDS*
DUANE
P. MATTHEES
Organic Analytical 20234 (U.S.A.) WLLLIAM
Research
Division.
National
Bureau
of Standards,
Washington.
D.C.
C. PURDY*
Department (Received
AGENT FOR THE DETECTION OF
of Chemistry. 6th February
;UcCill University,
Montreai,
Quebec,
H3A 2K6 (Canada)
1979)
Bileacidsare derivatized with l-naphthyldiazomethane and separated by h.p.1.c. on silica gel columns with a hexane--tetrahydrofuran--mcthanol solvent. Reactions proceed quickly at room temperature under mild conditions, to give strongly absorbing species for U.V. detection. Deoxycholic acid can be detected in cat feces.
Summary.
The bile acids play a vital role in digestion and cholesterol metabolism, and recent interest has centered on their correlation with cancer of the colon [ 1, 2 1. In addition, the identification and qu~tification of the various bile acids may be of value in the diagnosis of liver disease [3]. While there has been considerable effort to develop thin-layer and gas chromatographic methods of measurement, high-performance liquid chromatography (h.p.1.c.) appears to be particularly well suited for the measurement of bile acids, because it combines the advantages of mild separation conditions with fairly easy quantification, provided that a suitable detector is available. Underivatized bile acids have been detected by monitoring the column effluent in the far ultraviolet
[4, 5))
but derivatization
of the bile acids with chromo-
phores, such as the aromatic esters, has advantages. Derivatization of the carboxy1 group allows some degree of selectivity, especially in the analysis of biological materials where potential interferences (e.g., neutral sterols) are likely to be present. Furthermore, if the derivative absorbs strongly in the 250-280-nm region, highly sensitive fixed-wavelength detectors can be employed, and the choice of solvents is greater than when the effluent is monitored at 205-220 nm. Diazomethane has long been a favorite reagent for the formation of methyl esters of carboxylic acids because of its high reactivity under mild conditions [6] _ Similarly, the aryldiazoalkanes should readily form esters with strong TTaken 1978.
in part from
the Ph.D. Dissertation
of D. P. Matthees,
University
of Maryland,
162
chromophores for U.V. detection in h.p.I.c. For this study, l-naphthyldiazomethane was chosen as a derivatizing agent since it can be easily made from inexpensive, readily obtainable reagents, and the naphthyl group is an excellent chromophore [ 7]_ Moreover, a general advantage of the aryldiazoalkanes is their intense color, which fades as the reaction progresses. This color change makes it easier to determine whether enough reagent has been added, particularly when the exact amount of bile acid is unknown. Experimental Apparatus. The chromatograph equipment used was the same as described earlier with fixed-wavelength (254 nm) and variable-wavelength detectors [71. The column contained clPorasi1, 30 cm long; Waters Associates. Reagents. 1-Naphthaldehyde, hydrazine, mercury( II) oxide, and cholic and deoxycholic acids were obtained from common commercial sources. Other bile acids were prepared from cholic or deoxycholic acids. Solvents were of the usual reagent-grade redistilled from all-glass apparatus (rejecting the first and last 10%) and filtered before use. Bulk hexane was purified by refluxing with concentrated sulfuric acid, followed by water washing and drying over sodium hydroxide before redistillation. Preparation of reagent. l-Naphthyldiazomcthane was prepared by oxidation of 1-naphthaldehyde hvdrazone with mercury( II) oxide essentially as described by Nakaya et al. [ 81; magnetic stirring in a loosely stoppered flask was found to be more convenient than shaking in a pressure bottle. While l-naphthyldiazomcthane is soluble in many organic solvents, diethyl ether is most satisfactory for application to the bile acid derivatization. Solutions were prepared
as described earlier [7] _
Derioatization. Dry, crystalline bile acid standards were dissolved in chloroform, with enough methanol added to bring them into solution; typically l-5 mg of each bile acid was dissolved in 0.5 ml of solvent. When a hydrolysate of bile salts was analyzed, it was acidified and extracted with ethyl acetate or butanol and a known aliquot taken. Several drops of the l-naphthyldiazoalkane solution were then mixed, in a l-ml graduated tube, with the bile acid solution to give a reddish-orange color. More reagent was added if the color disappeared within an hour as described for derivatization of fatty acids [ 7]_ After reaction, the excess of derivatizing agent could be decomposed by a drop of acetic acid, if desired. The mixture was then diluted to volume and an aliquot of 5 ~1 or so was taken for chromatography. Chromatographic conditions. There is a considerable volume of data on the separation of bile acids and their esters by thin-layer chromatography 191, and many of the solvent systems may be adapted to h.p.1.c. on silica gel columns. A 300: 120:8 mixture of hexane-tetrahydrofuran-methanol proved to be an excellent isocratic solvent for the separation of the l-naphthylmcthyl esters of the bile acids; flow rates of 1.0-1.5 ml min-’ were satisfactory. The amount of methanol in the system sharply affects the retention volumes of the bile acid derivatives, especially the polar acids, so that the solvent com-
163
position may be optimized for the polar or nonpolar acids. A 200:85:5 hexane-tetrahydrofuran-acetic acid solvent system gave similar behavior_ A number of different solvent systems were tested, with one of the most important criteria being the ability to resolve positional isomers. Results
and discussion
Figures 1A and 2 show separations of bile acid derivatives in solvent systems of hexane-tetrahydrofuran with a polar modifier of methanol or acetic acid. The dihydroxy acid derivatives, deoxycholic and chenodeoxycholic, were well resolved, in addition to acids with different numbers of hydroxyl groups. Figure 1B shows the separation of deoxycholic acid from other constituents of cat feces. A Soxhlet extract of cat feces with 1:1 chloroform-methanol was concentrated and a portion reacted with l-naphthyldiazomethanc. The 8
A
/
LJ
-*.0
--
_-•--
-
__.___ 20
IO Eluate
(ml)
i _-,.-
--.--+ 0
4
8 Eluate
‘-
12 ( ml
16
,-
20
1
Fig, 1. (A) Naphthyldiazomethane derivatives of some less polar bile acids. Mobile phase, 300 : 120 :8 hexane-tetrahydrofuran-methanot , flow rate, 1 .O ml min-’ ; 2% 1 injections containing about 15 pg of each bile acid; detection at 280 nm (Schoeffel). Peak identity: (1) lithocholic, (Z)deoxycholic,(3)chenodeoxycholic. (4) 3,7dihydroxy-12-ketocholanic acid. (B) Deoxycholic acid from cat feces. Conditions as for Fig. 1A; 5.~1 injection corresponding to the derivatized extract of 2 mg of feces (wet weight). Peak (1) is the deoxycholic acid derivative.
164
-r
I
OOIAU
Fig. 2. Naphthyldiazomethane derivatives of bile acids (about 20 pgof cholic, deoxycholic, and chenodeoxycholic acids and 5 ug of lithocholic and 3,7dihydroxy-12-ketocholanic acids). Mobile phase, 200:85:5 hexane-tetrahydrofuranacetic acid; flow rate, 1.0 ml min-‘; detection at 254 nm (Waters). Peak identity: (1) lithocholic. (2) deoxycholic, (3) chenodeoxycholic,
(4)
3,7dihydroxy-12-ketocholanic’acid,
cholic acid.
(5)
reaction mixture was injected directly onto the column and eluted. This sample contained sufficient bile acid to permit detection without additional cleanup or preconccntration. The other fecal constituents were eluted from
the silica gel column before the deoxycholate derivative. The aryldiazoalkanes react completely with carboxylic acids, so that the corresponding esters of the free bile acids and their glycine conjugates can be prepared. However, the taurinc conjugates, which are sulfonic acids, do not esterify. The detection limits for these derivatives depend on peak sharpness, retention volume, and other factors, but with the fixed-wavelength detector (254 nm), 20--30 ng of the bile acid should be detectable_ Since all the derivatives have the same chromophore, the detector response is proportional to the number of moles of acid derivatized. REFERENCE6 1 M. Winick, Nutrition and Aging, J. Wiley, New York, 1976, p. 161. 2 D. S. Reddy, A. Mastromarino and E. L. Wynder, Cancer Res., 35 (1975) 3 J. B. Carey,
Jr.,
in P. P. Nair
Press. New York, 1973. 4 S. Okuyama, D. Ucmura
and D. Kritchevsky
p_ 68. and Y. IIirata,
Chem.
(Eds.), Lett.,
(1977)
5 N. A. Parris, J. Chromatogr., 133 (1977) 273. 6 K. Blau and G. S. King (Eds.), Handbook of Derivatives
London,
1978,
7 D. P. Matthews
The
Bile Acids,
3403. Vol. 2, Plenum
679.
for Chromatography.
Heyden,
p. 49. and W. C. Purdy,
8 T. Nakaya, T. Tomomoto
Anal.
Chim.
Acta,
109 (1979)
61.
and M. Imoto, Bull. Chem. Sot. Jpn., 40 (1967) 691. 9 P. Eneroth, in G. V. Marinetti (Ed.), Lipid Chromatographic Analysis, Vol. 2, M. Dekker, New York, 1969, p_ 149.
-
Printed in The Netherlands
Short Communication
NAPHTHYLDIAZOMETHANE AS A DERIVATIZING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY BLLE ACIDS*
DUANE
P. MATTHEES
Organic Analytical 20234 (U.S.A.) WLLLIAM
Research
Division.
National
Bureau
of Standards,
Washington.
D.C.
C. PURDY*
Department (Received
AGENT FOR THE DETECTION OF
of Chemistry. 6th February
;UcCill University,
Montreai,
Quebec,
H3A 2K6 (Canada)
1979)
Bileacidsare derivatized with l-naphthyldiazomethane and separated by h.p.1.c. on silica gel columns with a hexane--tetrahydrofuran--mcthanol solvent. Reactions proceed quickly at room temperature under mild conditions, to give strongly absorbing species for U.V. detection. Deoxycholic acid can be detected in cat feces.
Summary.
The bile acids play a vital role in digestion and cholesterol metabolism, and recent interest has centered on their correlation with cancer of the colon [ 1, 2 1. In addition, the identification and qu~tification of the various bile acids may be of value in the diagnosis of liver disease [3]. While there has been considerable effort to develop thin-layer and gas chromatographic methods of measurement, high-performance liquid chromatography (h.p.1.c.) appears to be particularly well suited for the measurement of bile acids, because it combines the advantages of mild separation conditions with fairly easy quantification, provided that a suitable detector is available. Underivatized bile acids have been detected by monitoring the column effluent in the far ultraviolet
[4, 5))
but derivatization
of the bile acids with chromo-
phores, such as the aromatic esters, has advantages. Derivatization of the carboxy1 group allows some degree of selectivity, especially in the analysis of biological materials where potential interferences (e.g., neutral sterols) are likely to be present. Furthermore, if the derivative absorbs strongly in the 250-280-nm region, highly sensitive fixed-wavelength detectors can be employed, and the choice of solvents is greater than when the effluent is monitored at 205-220 nm. Diazomethane has long been a favorite reagent for the formation of methyl esters of carboxylic acids because of its high reactivity under mild conditions [6] _ Similarly, the aryldiazoalkanes should readily form esters with strong TTaken 1978.
in part from
the Ph.D. Dissertation
of D. P. Matthees,
University
of Maryland,
162
chromophores for U.V. detection in h.p.I.c. For this study, l-naphthyldiazomethane was chosen as a derivatizing agent since it can be easily made from inexpensive, readily obtainable reagents, and the naphthyl group is an excellent chromophore [ 7]_ Moreover, a general advantage of the aryldiazoalkanes is their intense color, which fades as the reaction progresses. This color change makes it easier to determine whether enough reagent has been added, particularly when the exact amount of bile acid is unknown. Experimental Apparatus. The chromatograph equipment used was the same as described earlier with fixed-wavelength (254 nm) and variable-wavelength detectors [71. The column contained clPorasi1, 30 cm long; Waters Associates. Reagents. 1-Naphthaldehyde, hydrazine, mercury( II) oxide, and cholic and deoxycholic acids were obtained from common commercial sources. Other bile acids were prepared from cholic or deoxycholic acids. Solvents were of the usual reagent-grade redistilled from all-glass apparatus (rejecting the first and last 10%) and filtered before use. Bulk hexane was purified by refluxing with concentrated sulfuric acid, followed by water washing and drying over sodium hydroxide before redistillation. Preparation of reagent. l-Naphthyldiazomcthane was prepared by oxidation of 1-naphthaldehyde hvdrazone with mercury( II) oxide essentially as described by Nakaya et al. [ 81; magnetic stirring in a loosely stoppered flask was found to be more convenient than shaking in a pressure bottle. While l-naphthyldiazomcthane is soluble in many organic solvents, diethyl ether is most satisfactory for application to the bile acid derivatization. Solutions were prepared
as described earlier [7] _
Derioatization. Dry, crystalline bile acid standards were dissolved in chloroform, with enough methanol added to bring them into solution; typically l-5 mg of each bile acid was dissolved in 0.5 ml of solvent. When a hydrolysate of bile salts was analyzed, it was acidified and extracted with ethyl acetate or butanol and a known aliquot taken. Several drops of the l-naphthyldiazoalkane solution were then mixed, in a l-ml graduated tube, with the bile acid solution to give a reddish-orange color. More reagent was added if the color disappeared within an hour as described for derivatization of fatty acids [ 7]_ After reaction, the excess of derivatizing agent could be decomposed by a drop of acetic acid, if desired. The mixture was then diluted to volume and an aliquot of 5 ~1 or so was taken for chromatography. Chromatographic conditions. There is a considerable volume of data on the separation of bile acids and their esters by thin-layer chromatography 191, and many of the solvent systems may be adapted to h.p.1.c. on silica gel columns. A 300: 120:8 mixture of hexane-tetrahydrofuran-methanol proved to be an excellent isocratic solvent for the separation of the l-naphthylmcthyl esters of the bile acids; flow rates of 1.0-1.5 ml min-’ were satisfactory. The amount of methanol in the system sharply affects the retention volumes of the bile acid derivatives, especially the polar acids, so that the solvent com-
163
position may be optimized for the polar or nonpolar acids. A 200:85:5 hexane-tetrahydrofuran-acetic acid solvent system gave similar behavior_ A number of different solvent systems were tested, with one of the most important criteria being the ability to resolve positional isomers. Results
and discussion
Figures 1A and 2 show separations of bile acid derivatives in solvent systems of hexane-tetrahydrofuran with a polar modifier of methanol or acetic acid. The dihydroxy acid derivatives, deoxycholic and chenodeoxycholic, were well resolved, in addition to acids with different numbers of hydroxyl groups. Figure 1B shows the separation of deoxycholic acid from other constituents of cat feces. A Soxhlet extract of cat feces with 1:1 chloroform-methanol was concentrated and a portion reacted with l-naphthyldiazomethanc. The 8
A
/
LJ
-*.0
--
_-•--
-
__.___ 20
IO Eluate
(ml)
i _-,.-
--.--+ 0
4
8 Eluate
‘-
12 ( ml
16
,-
20
1
Fig, 1. (A) Naphthyldiazomethane derivatives of some less polar bile acids. Mobile phase, 300 : 120 :8 hexane-tetrahydrofuran-methanot , flow rate, 1 .O ml min-’ ; 2% 1 injections containing about 15 pg of each bile acid; detection at 280 nm (Schoeffel). Peak identity: (1) lithocholic, (Z)deoxycholic,(3)chenodeoxycholic. (4) 3,7dihydroxy-12-ketocholanic acid. (B) Deoxycholic acid from cat feces. Conditions as for Fig. 1A; 5.~1 injection corresponding to the derivatized extract of 2 mg of feces (wet weight). Peak (1) is the deoxycholic acid derivative.
164
-r
I
OOIAU
Fig. 2. Naphthyldiazomethane derivatives of bile acids (about 20 pgof cholic, deoxycholic, and chenodeoxycholic acids and 5 ug of lithocholic and 3,7dihydroxy-12-ketocholanic acids). Mobile phase, 200:85:5 hexane-tetrahydrofuranacetic acid; flow rate, 1.0 ml min-‘; detection at 254 nm (Waters). Peak identity: (1) lithocholic. (2) deoxycholic, (3) chenodeoxycholic,
(4)
3,7dihydroxy-12-ketocholanic’acid,
cholic acid.
(5)
reaction mixture was injected directly onto the column and eluted. This sample contained sufficient bile acid to permit detection without additional cleanup or preconccntration. The other fecal constituents were eluted from
the silica gel column before the deoxycholate derivative. The aryldiazoalkanes react completely with carboxylic acids, so that the corresponding esters of the free bile acids and their glycine conjugates can be prepared. However, the taurinc conjugates, which are sulfonic acids, do not esterify. The detection limits for these derivatives depend on peak sharpness, retention volume, and other factors, but with the fixed-wavelength detector (254 nm), 20--30 ng of the bile acid should be detectable_ Since all the derivatives have the same chromophore, the detector response is proportional to the number of moles of acid derivatized. REFERENCE6 1 M. Winick, Nutrition and Aging, J. Wiley, New York, 1976, p. 161. 2 D. S. Reddy, A. Mastromarino and E. L. Wynder, Cancer Res., 35 (1975) 3 J. B. Carey,
Jr.,
in P. P. Nair
Press. New York, 1973. 4 S. Okuyama, D. Ucmura
and D. Kritchevsky
p_ 68. and Y. IIirata,
Chem.
(Eds.), Lett.,
(1977)
5 N. A. Parris, J. Chromatogr., 133 (1977) 273. 6 K. Blau and G. S. King (Eds.), Handbook of Derivatives
London,
1978,
7 D. P. Matthews
The
Bile Acids,
3403. Vol. 2, Plenum
679.
for Chromatography.
Heyden,
p. 49. and W. C. Purdy,
8 T. Nakaya, T. Tomomoto
Anal.
Chim.
Acta,
109 (1979)
61.
and M. Imoto, Bull. Chem. Sot. Jpn., 40 (1967) 691. 9 P. Eneroth, in G. V. Marinetti (Ed.), Lipid Chromatographic Analysis, Vol. 2, M. Dekker, New York, 1969, p_ 149.