Bile Acids LIV - Mass Spectra of Conjugated Bile Acids?

Roger Shaw and William H. EUiottj: Edward A. Doisy Department of Biochemistry, Saint Louis University School of Medicine, St Louis, Missouri 63 104, USA

The electron impact mass spectra of conjugated bile acids, their 5a-analogs and methyl esters of glyco conjugates were determined by direct insertion into the ion source and their fragmentation patterns were found to be basically similar to those of methyl esters of the free bile acids. The conjugates are additionally characterized by a significant loss of [NH,CH,CO,H] from the glycine moiety and [CH,=CHS03H] from the taurine group. Several 5a- and SP-isomers can be differentiated, but no general pattern of recognition is discernable. Field desorption mass spectra contain [M + Na]+ and [M + 2NaI" ions.

INTRODUCTION -

Bike acids are elaborated by the liver, conjugated with thic amino acids glycine or taurine and secreted as such in bile. Analyses of these derivatives are generally car- ried out following alkaline or enzymatic hydrolysis to thi: free acids. Alkaline hydrolysis provides artifacts and promotes loss of some products.' Enzymatic hydrolysis with Clostridial cholylglycine hydrolase' has been used, but either method fails to identify the nature of the conjugate. High pressure liquid chromatography (HPLC) has been investigated as a means of rapid separation of bile acids and their By means of a simple solvent system rapid separation of t h e components of rat or human bile has been

Since identification of these components is best obtained by mass spectrometry, a systematic examination of the mass spectra of these conjugated derivatives is desirable. Spectra derived from frag- mentation by electron impact have been reported for only a few conjugated Sp-bile acids, notably glyco- deoxycholic acid,7 glycocholic, ornithocholic, arginocholic and histidocholic acids and their deriva- t i v e ~ . ~ ' ~ This paper details the mass spectra of a series of naturally occurring glyco and tauro conjugates of 5p- and 5a-acids and their derivatives.

t Bile acids are derivatives of Sp- or Sa-cholanic acids; Sa- cholanic acids are referred to as allo acids. The bile acids used in these studies are cholic acid (C) (3a,7a, 12a-trihydroxy-SP-cholanic acid), chenodeoxycholic acid (CDC) (3a,7a-dihydroxy-S/3-~holanic acd), deoxycholic acid (DC) (3a,l2a-dihydroxy-SP-cholanic acid) and lithocholic acid (LC) (3a-hydroxy-5P-cholanic acid). These acids anti their Sa-analogs are normally conjugated with the amino acids glycine (G) or taurine (T) to provide derivatives such as taurocholic acid (TC). The allo or Sa-derivatives are identified in the abbreviated miinner by means of the capital A; thus, tauroallocholic acid is designated TAC. The abbreviation for taurolithocholate 3- 0-methyl ether is TLC-OMe; cholyl-N-6-aminovaleric acid is designated CV.

f Author to whom correspondence should be addressed.

EI Heyden & Son Ltd, 1978

R, = O H R , = H o r O H

R, = H or O H R4 = P H bile acid, or cuH allo bile acid

R, = NHCH2C02H glycine, or NHCH2CH2S03H taurine

EXPERIMENTAL

Materials

Sodium salts of conjugated bile acids were prepared" by a modification of the method of Lack et al." The acid forms of the glyco conjugates were obtained by collecting the precipitates of acidified aqueous or aqueous-ethanolic solutions of the sodium salts. Those of the tauro-conjugates were generated either by lyo- philization of acidified solutions of the sodium salts and dissolution in absolute ethanol, or in situ by mixing the salt with potassium h y d r o p phosphate prior to insertion into the ion source. The latter method was handicapped by the presence of water molecules during vaporization, unless a limited amount of phosphate was added. Methyl esters of the glyco conjugates were pre- pared by modification'3 of the method of Radin et all4

3a-Methoxy-5p-cholanic acid was prepared as follows: a solution of methyl lithocholate (70mg) in ether (dried over calcium chloride) was stirred with potassium t-butoxide (5 g) for 0.5 h. A large excess of methyl iodide (17 ml) was added and the stirring was continued for 4 h. The reaction mixture was poured into 2 N sulfuric acid (100 ml), and the aqueous layer

0306-042~/78/0005-0433$03.00

BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 7, 1978 433

R. SHAW AND W. H. ELLIOTT

was extracted with ether. The combined organic phase was washed successively with water, saturated aqueous sodium bicarbonate and water, dried (MgSOJ, and finally evaporated to dryness. The residue was methyl- ated with 2,2-dimetho~ypropane,'~ purified by pre- parative layer chromatography (PLC) in acetone + benzene (1 : 19), and crystallized from acetone + water as colorless prisms (40 mg; 55% yield); m.p. 88-89 "C; Rf 0.45 (acetone+benzene 1 : 19); KRT 0.36 (3% OV- 210; relative to methyl deoxycholate); v max 1748, 1171 and 1106 cm-I; 6 0.66 (s; 3H), 0.93 (s), 2.88- 3.44 ( lH), 3.37 (s; 3H) and 3.68 ppm (s; 3H). The mass spectrum exhibited a molecular ion, m/e 404, and the characteristic fragment ions, m/e 372 [M- 32]+, 357 [M-(32+15)]+, 341 [M-(32+31)]+, 262 [M- (115+27)]+, 257 [M-(115+32)]', 230 [M- (1 15 + 32 + 27)]+ and 215 [M - (1 15 + 32 +42)]+.

The free acid was prepared from the methyl ester by treatment with aqueous methanolic potassium hydroxide; m.p. 139-146 "C ( r e p ~ r t e d ' ~ m.p. 140- 142 "C). Rf 0.82 (chloroform +methanol + acetic acid 80 : 6 : 2). The tauro derivative was prepared by modification" of the method of Lack et al."

Mass spectrometry

Electron impact mass spectra were obtained via direct probe with an LKB 9000 mass spectrometer at an ionizing voltage of 20 eV, accelerating voltage of 3.5 or

3.0 kV and an ion source temperature of 270 "<'. The temperature of the sample in the direct probe w ~ s raised until sufficient material had volatilized to provide a distinctive mass spectrum. Thus, spectra were recor- ded at the following temperatures: TC and TAC, 232 "C; TCDC and TACDC, 250 "C; TDC, 239 C arid TADC 225°C; TLC and TALC, 240°C; GC arid GAC, 162 "C; GCDC, 148 "C and GACDC, 178 "(3; GDC, 148°C and GADC, 155°C; GLC and GALC, 150 "C; and CV, 110-1 30 "C. More recently, tht. mass spectrometer was coupled with an on-line Logo\ Spec- trotek data aquisition system, Model DS/2000 WE: (Berkeley, California) which includes a Data General Nova 2/10 computer, a Western Dinex 11.6 megabyte dual disc system, a Wangco magnetic tape drive, a Tektronik Model 4010-1 CRT terminal, and a Vursatec Model 200A hard copy printer plotter.

-- RESULTS AND DISCUSSION

~~

The relative intensities and masses of importanr frag- ment ions of eight glyco conjugates and of CV acid are given in Table 1. Samples were inserted into the ion source via the direct inlet probe and spectra were obtained at probe temperatures stated abo\ e. Generally fragmentation was observed with loss of water molecules followed b fra ments derived from McLafferty rearrangement" gY of the g . sidechain; i x . loss

-- Table 1. Fragment ions and relative intensities" of glyco conjugated bile acids and CV

mie GC GAC

[MI+ 465 0.4 0.4 [M - nl 8]tb 429 8.0 8.0

411 8.8 5.1 [M - (n18+ 15)1+ 414 4.3 3.5

396 6.2 3.5 [M - ( n 1 8 + 75)1+ 390 2.3 3.3

372 12.3 19.7 354 13.7 6.2 336 2.9 2.2

[M - (n18+ 1 17)lt 312 6.8 8.3 294 6.3 4.5

[M- (n18+ 130)+ 299 5.0 5.2 281 7.5 3.7

[M - (n l8+ 158)]+ 271 90.6 100.0 253 100.0 50.0

[M-(n18+158+27)1+ 244 8.8 9.8 226 21.0 6.9 211 15.5 11.9

199 18.1 12.0 147 23.6 22.7 145 28.1 27.1 130 15.2 16.0 117 32.1 28.7

76 17.5 17.1 30 36.6 26.9

m/e GCDC GACDC GDC GADC

449 1.5 3.6 0.8 0.2 431 8.6 20.7 4.0 2.6 413 30.9 22.1 10.6 7.2 416 6.5 14.9 1.3 1.0 398 45.2 23.2 3.9 3.3

374 6.8 27.9 2.1 1.0 356 7.9 7.8 4.9 2.7 338 3.7 2.4 2.2 2.4 314 15.1 25.1 4.6 3.1 296 7.7 21.6 3.9 3.4 301 2.5 2.4 1.7 1.1 283 3.8 3.4 2.9 2.2 273 18.6 45.1 30.9 43.6 255 44.7 27.6 100.0 100.0 246 8.2 19.0 2.3 1.2 228 14.2 18.4 4.5 2.6 213 31.6 39.4 8.5 5.1

201 18.1 14.5 7.9 8.4 147 24.8 35.3 21.6 21.6 145 21.6 27.7 15.3 11.3 130 46.3 54.8 32.1 28.5 117 100.0 100.0 45.6 39.2

- - - - -

a Intensities are expressed as per cent of base peak. n18 number of water molecules lost could be 0-3.

"m/e312or294=[M-(n18+159)1~.

76 48.1 58.7 23.7 19.5 30 33.8 44.8 21.1 15.3

m/ e

433 41 5 - -

400 - -

358 340 31 6 298 303 285 275 2 57 248 230 215

203 1 47 145 130 117

76 30

GLC GALC mle CV

2.9 7.8 507 3.4 17.0 34.1 471 6.2

- 453 4.9 - 456 2.7

6.0 17.2 438 4.8

- -

- - - __ 6.1 12.8 - __ 1.5 4.8 - 5.2 5.0 354 10.5 3.7 20.4 336 3.l 0.6 1.0 312' 4.2 1.4 2.9 294' 3.4 0.5 0.7 27Id 59.f 9.4 6.9 253d 56.0 1.9 6.4 244 3.5 6.8 5.1 226" 11.8

22.8 38.6 211 8.4

3.3 4.6 199 11.6 16.2 159 8.1 8.8 145

40.8 39.5 130 100.0 100.0 118

117 100

22.8 19.1 76 17.1 10.7 30

10.7' 80.6 19.6 3.2

22.4 9.5

100.0

31.1 _.

a m / e 271 or253=[M-(n18+158+42)1?. "m/e 226=[M(3~18+158+42+27)]+.

434 BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 7, 1978 @ Heyden & Son Ltd, 1978

CONJUGATED BILE ACIDS

of 117 [CH2=C(OH)NHCH2C02H] amu or of 75 [NH2CH2C02H] amu. Once the complete sidechain is lost, the fragment ions are those seen for the nucleus of the respective free bile The base peak for GCDC, GACDC, GLC and GALC was the ion m/e 11 7 [CH2=C+(OH)NHCH2C02H]. The ion m/e 75 was very small in all spectra, but the protonated spe- cies, m/e 76 [NH3CH2C02H]+ was more intense in all spectra, particularly in GCDC and GACDC. The base peitk for GDC and GADC was the ion m/e 255, but for the isomeric GCDC and GACDC derivatives these fragment ions showed relative intensities of less than 50%. The difference in intensities of ions m/e 374 (7 anti 23%0), 273 (19 and 45%) and 255 (45 and 28%) for GCDC and GACDC may be useful for identification. Spectra for the pair of trihydroxy derivatives, GC and GAC, indicated that the relative intensities of the ions ml'e 271 (91 and 100%) and 253 (100 and 50%) may also be useful for identification. It should be noted that GC' and GAC are separable'" in HPLC with the solvent system6 2-propanol+8.8 mM phosphate buffer (pH 2.5) (160: 340), as are GLC and GALC.

Myher et al.' noted that the mass spectra of ornitho- cholic and arginocholic acids contained a common fragment ion derived from the lactam of the a-amino acid moiety. To ascertain whether the chain length of the amino acid played a pronounced role in frag- mentation of the amide linkage found in conjugated bile acids, the spectrum of a sample of CV acid was examined. This substance is an analog of ornithocholic acid in which the a-amino group of ornithine is absent. Bale peak (Table 1) for this derivative is m/e 100 [CH2=CHCH2CH2CO2H]+; a number of small peaks result from loss of water and the fragment 117 [NH2CH2CH2CH2CH2C02H] amu, and loss of 159 [CH,=C(OH)NH(CH,>,CO,H] amu, or 172 [CHZCH2CONH(CH2)4C02H] amu. The ions m/e 118

(protonated m/e 117), 159 and 172 had relative intensities of 22, 81 and 6O%, respectively. Thus, this derivative fragments similarly to small peptides with cleavage at either side of the nitrogen atom. Loss of the fragment 100 amu provides an ion [R-C(OH)=NH]+ which can lose water to form the nitrile, or the ion m/e 59 [CH,=C-OH(NH,)]+. The relative intensity of the ion m/e 253 representing the nucleus of a trisubstituted bile acid or steroid was only 56% in comparison with GC ( 1 00%). Thus, the length of the amide moiety has a pronounced effect on the fragmentation pattern of cholyl derivatives.

The mass spectra of the methyl esters of the glyco- conjugates (Table 2) show fragmentation patterns similar to those of the free acids, but generally with somewhat larger molecular ions. In accord with the observation of Anderson et al.,I7 the ion [M+ 11' of the esters was somewhat larger than attributable to isotopic abundances. The base peak for the methyl esters of GC and GAC was the fragment ion m/e 271 [M - (172 + 2 X 18)]', whereas for the other methyl esters the base peak was the ion m/e 131 [CH,=C(OH)NHCH,CO,CH,]t. Ramsdell et al. l8

have commented upon the derivation of the ion m/e 131 from methyl esters of N-acylglycines.

The ions m/e 390 (14 and 13%, respectively) apd m/e 200 (41 and 42%, respectively) (Table 2) may be diagnostic for the methyl esters of GLC and GALC. The ion m/e 390 or its equivalent does not appear to any appreciable extent in spectra of the other glyco conjugates, whereas the ion m/e 200 appears in GCDC and GACDC (25 and 35%, respectively), but only in minor amounts in spectra of the methyl esters of GDC, GADC, GC and GAC. The glycodeoxycholates show an ion m/e 265 (18 and 3O%, respectively) which should distinguish them from the glyco- chenodeoxycholates (GCDC and GACDC). GDC may

Table 2. Fragment ions and relative intensities^ of methyl esters of glyco conjugated bile acids

GDC GADC mle GLC GALC

[MI! 479 1.4 1.1 463 1.5 2.6 2.1 1.3 447 12.4 26.7 [M- n18]tb 443 8.2 74.8 445 2.2 5.4 7.5 6.8

425 6.9 8.6 427 10.0 10.5 14.3 5.7 429 30.0 21.5

410 4.9 4.0 412 22.1 11.8 5.4 4.4 414 17.6 16.5 iM - (n18+31)lf 412 2.7 2.5 414 2.6 0.9 3.3 4.7 416 13.9 15.3

394 1.9 1.4 396 2.9 0.2 1.8 0.9 398 0.5 2.2 390 14.2 12.8

[M- (n18+89)+ 354 1.9 1.8 356 1.5 1.1 3.1 5.7 358 11.2 8.2 336 3.0 2.1 338 2.1 1.3 3.4 1.8 340 4.6 4.7

[M - (n18+131)It 312 9.4 5.4 314 8.3 12.6 5.6 6.5 316 5.6 4.7 294 6.7 2.9

[M - (n18+172)]+ 289 7.5 3.4 271 100.0 100.0 273 8.0 15.4 29.3 99.1 275 0.5 0.4 253 83.1 61.0 255 22.1 10.5 98.4 53.2 257 17.5 7.5

[M -- (n18+ 172 + 27)1+ 244 3.0 8.8 246 2.8 3.1 1.4 2.2 248 1.7 5.4 226 7.1 4J 228 5.4 4.2 3.0 1.9 230 11.8 6.2

[M -(n18+172+42)1+ 229 6.0 5.9 231 2.6 3.3 2.6 3.1 233 2.5 16.0 211 12.0 10.4 213 18.6 20.0 9.7 10.0 215 58.0 49.0

265 4.9 3.1 265 2.2 2.1 18.1 30.0 265 3.5 1 .o 200 8.2 4.3 200 25.2 34.6 6.1 5.5 200 40.5 42.2 131 88.7 56.1 131 100.0 100.0 100.0 100.0 131 100.0 100.0

mle GC GAC mle GCDC GACDC

L - -

- - - [M- (n18+15)]+ 428 2.6 5.3 430 1.7 8.2 2.2 3.3

- - - - - - - -

- - - - - - - - - - - - - - - -

intensities are expressed as per cent of base peak. n18 number of water molecules lost could be 0-3.

@ Hzyden &Son Ltd, 1978 BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 7, 1978 435

R. SHAW AND W. H. ELLIOTT

be distinguished from GADC by the intensities of the ions m/e 273 [M-(172+18)]+ (29 and 99%, respec- tively) and m/e 255 [M-(172+2x 18)]'(98 and 53%, respectively). The ion m/e 30 [NH2=CH2]+, (structure confirmed by Ramsdell et al.") was present in all the spectra of the glyco conjugates and of their methyl esters, but generally appeared with intensity less than

Since taurine and its N-acyl derivatives are strong acids (e.g. pKalg of TC= 1.85) and methyl esters are not formed by the usual methods of esterification, the tauro conjugates were examined only as the acids. Spectra could be obtained at vaporizing temperatures of 130-170°C in the probe, but only a very small fraction of total ionization was obtained, and identification was obscure. At probe temperatures above 220 "C the tauro conjugates generally gave rise to fragment ions characteristic of the respective free bile acids: loss of water, methyl groups, sidechain, and rings A and D.7,'6 Fragmentation of the tauro deriva- tives, as exemplified by taurocholic (TC) and tauroal- locholic (TAC) acids, proceeds characteristically with the loss of 108amu and 167amu represented by [ CH, =CHS03H] and [ CH2 =C( OH)NHCH2. CH2SO,H] respectively, as a result of McLafferty re- arrangements.

The relative intensity of the fragment ions derived by loss of 108 amu was less than 2% in all four epimeric pairs, but the loss of 126 [lo8 + 181 amu was detectable in 5p- and 5a-TCDC, 50- and 5a-TDC, and was significantly large in 5p- and Sa-TLC. An alternate rationalization of formation of the fragment of 126 amu (loss of water and carbons 19 and 1 through 6) was deemed unlikely from examination of the spectrum of taurolithocholate-3-0-methyl ether (TLC-OMe). The fragment ion m/e 389 [M-108]+ appeared with a relative intensity of 27% before the loss of C-3

50%.

methoxyl as methanol, [M-(108+32)]? (m/e 357, 60%). The fragment ion m/e 371 (9%).mdy be represented as [M-(108+ 18)]? with dehydratron of the amide residue of m/e 389. Loss of fragments I 108 +

18+32) is represented by m/e 339 (11%) (Tablc: 3). The amides of the nine tauro derivatives representd

in Table 3 by [M - (108 + n X 1 S)]? are each dehydrated to a nitrile, although the intensity of the ion mi e 3 3 7 for 5tr-TDC was very weak (0.90/,). Interpretation of the significance of the ubiquitous smaller fragment ions, m/e 59 and m/e 91, seems tenuous at present, hut the base peak of many spectra, m/e 59, may be derived in part from the structure [CH2=C(OH)NH2]+.

Scheme 1 shows a potential pathway of fragmen- tation of TC which appears to be applicable to thy other tauro derivatives with appropriate correction for fewer initial hydroxyl groups in the molecular ion. Loss of ti'le sulfur moiety as S03H2 with all hydroxyl groups as water molecules can result in the ion m/e 379. Loss of three molecules of water from the molecular ion and loss of the taurine moiety (108 amu) probably as [CH2=CHS03H]+ provides ion m/e 353, which may undergo loss of a methyl group to give m/e 338, or lcws of water to give m/e 335. If fragment ion mlie 353 undergoes a retro Diels-Alder reaction, the ion m / e 299 could result, which may undergo loss of w,ater to form the ion m/e 281. Alternately, a more likely route for the formation of ion m/e 299 is that involving a cleavage of the sidechain of the molecular ion at C20-C22 with concomitant loss of two molecules o f water. Loss of another molecule of water provides the ion m/e 281, which may be converted to the bast: peak, m/e 253, indicative of a trisubstituted steroid nucleus. Fragmentation to ion m/e 299 via m/e 353 or directly from m/e 515 cannot be ascertained here, since the route via m/e 353 suffers loss of (108+72+2x 18) amu which is equivalent to the loss of (180 x 2 x 18)

Table 3. Fragment ions and relative intensities' of tauro conjugated bile acids

[MI: [M - ( n 18b+82)]f [M - (n18+ 108)lt

[M- (n18+ 108+ 15)]+

[M - ( n 18+ 167)lf

[M-(n18+ 166)l' [M - (n 18 + 208)]+

[M- (n18+208+ 27)]+

[M - (n18+208+42)]+

mle TC TAC

515 0 0 379 2.8 3.0 353 16.7 16.7 335 6.9 10.0

338 6.3 7.8 320 4.9 8.2

299 5.9 1.3 281 11.7 6.0 312 0.6 0.4 294 5.0 3.6 295 2.8 1.8 271 0.9 1.3 253 100.0 100.0 244 0.9 0.7 226 6.1 6.3 211 6.4 6.4

- - -

- - -

91 19.4 19.7 59 9.9 7.4

mle TCDC TACDC TDC TADC

499 0 0 0 0 381 2.5 2.2 0.4 0.3 355 100.0 66.3 18.4 9.2 337 14.9 20.9 2.9 0.9

340 55.9 63.4 4.2 8.2 322 10.0 13.0 1.5 1.8

301 42.0 10.7 6.0 2.2 283 12.4 6.5 3.5 1.4 314 2.7 2.5 3.4 13.7 296 5.1 5.9 4.0 4.3 297 4.3 6.7 3.0 8.4 273 4.0 1.5 17.0 700.0 255 80.6 70.9 100.0 83.8 246 3.5 6.6 2.3 13.9 228 11.5 12.8 4.2 2.0 213 48.3 72.1 6.3 12.9

- - - - -

- - - - -

91 39.8 100.0 14.7 13.9 59 30.2 61.8 20.0 42.0

mle

483 383 357 339

342 324

303 285 316 298 299 275 257 248 230 21 5

91 59

-

-

TLC TALC

0 0 1.7 1.8

60.0 83.6 42.0 43.4

22.0 33.9 26.9 25.6

18.7 20.3 17.7 22.0 3.2 2.1 7.3 11.4

17.8 26.1 1.5 1.5

31.9 17.8 3.7 1.9

25.4 25.1 71.7 69.8

67.2 46.7 100.0 100.0

- -

- -

a intensities are expressed as per cent of base peak.

'Ions derived from combined losses of designated fragments and methanol. Number of water molecules lost could be 0-4.

Ions derived from combined losses of designated fragments and methanol; no water molecule was included.

mle TLC-OMe

497 0

389 274 371 8 9 339' 11 4 374 2 7 356 7 5 324d 4.6 317 0 6 285 4 0 330 90 298d 12 5 29gd 260 289 -- 257d 31.3 262 7.0 230d 38.0 215d 86.4

91 39.9 59 100.0

- __

436 BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 7, 1978 @ Heyden & Son Ltd, 197 3

CONJUGATED BILE ACIDS

H

7'

H m/e 353

1 - 1 8

H p9!! I ,;

1-18

C Z N

m/e 281 H

-NH,

lR i \ +

C Z N

& ' m/e335

y 5

m/e 320

Scheme 1

amu from the molecular ion. A study of taurocholic acid labeled with I5N would be useful to resolve this question.

The spectra of TC and TAC are strikingly similar, and analogous to the spectra of the methyl esters of the two free acids; the base peak (Table 3) is the ion m/e 253. The base peaks for TCDC and TACDC are the ion!; m/e 355 [M-(108+2x18)]+ and m/e 91, respectively, whereas for TDC and TADC the ions m/e 255 and 273 are the respective base peaks. The base peak for TLC, TALC and TLC-OMe is the ion m/e 59.

Bccause of the absence of molecular ions from these tauro conjugates, field desorption spectra of samples of sodium TACDC and sodium TAC were obtained and

H

1-28

@ m / e 253

H

1 &'+ H m/e226

found to be relatively simple. Readily discernible features for sodium TACDC included m/e 544 [M + Na]+ and m/e 283.5 [M + 2Na]*', respectively. For sodium TAC the main features were the ions m/e 560 [M+Na]+and m / e 291.5 [M+2Nal2+.

This study provides basic information relative to the mass spectra of glyco and tauro conjugates of 5p- and 5a-cholanic acids. With the development of means of separation of these conjugated bile acids from natural sources by HPLC,6,'0 these data will be useful for confirmation of the structure of these materials, in analogy to the combination of gas chromatography and

for the esters of the free bile

@ Hcyden &Son Ltd, 1978 BIOMEDICAL MASS SPECTROMETRY, YOL. 5, NO. 7, 1978 437

R. SHAW AND W. H. ELLIOTT

Acknowledgement

This investigation was supported by Grants HL-07878 and CA- 16375 through the National Large Bowel Cancer Project, and the Fannie Rippel Foundation. The sample of cholyl-baminovaleric acid

(CV) was kindly provided by Dr Leon Lack, Duke Univers,;ty, D u r - ham, North Carolina. The skilled assistance of Mr William Frasurt and Mr Er ic Stephenson is gratefully acknowledged. Field dcsorptior spectra were kindly provided by D r s Keith Olson and Kennetb L Rinehart, University of Illinois, Urbana.

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