Focusing Review

Chromatography, Vol.25 No.1 (2004)

Focusing Review

Development of Highly Selective Analytical Systems for Biological Substances
Using Chromatography Combined with Mass Spectrometry−−With Special Reference to Bio−Analytical Studies of Bile Acids−−
Junichi Goto1,2*, Nariyasu Mano2 and Takaaki Goto2
1

Department of Pharmaceutical Sciences, Tohoku University Hospital, 1−1 Seiryo−machi, Aoba−ku, Sendai 980−8574, Japan
2

Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba−ku, Sendai, 980−8578, Japan
Received for review January 18, 2004. Accepted January 24, 2004

Summary
For the diagnosis of disease, reliable analytical methods for biologically important compounds are essential. Due to the existence of thesecompounds in complex matrices, such as blood, urine, or tissue, chromatographic procedures are preferable. In this article, we review highly
selective and sensitive chromatographic methods coupled with mass spectrometry for the analysis of biologically important materials, especially bile acids and related compounds. We also discuss immunoaffinity extraction employing highly specific antibodies.
Keywords: gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry, immunoaffinity extraction, bile acid, drug,
biological material, stable isotope labeled compounds, immobilized antibody

droxy−5 β−cholestanol derivative is generated. Further oxidation

1. Introduction
Bile acids are C24 carboxylic acid derivatives containing theof the side chain produces the cholestanoic acid derivative (so−steroid nucleus with a side chain at the C−17 β position. The com-called C27 bile acid). The cholestanoic acid is then transformed into

mon bile acids have an A/B−cis (5 β) configuration, a hydroxyl

CA and CDCA by the action of β−oxidation enzymes located ingroup at the 3 α, 7 α, 7 β and/or 12 α position, and a carboxylic

hepatic peroxisomes (Fig. 2).

acid group at the C−24 position, the terminus of the side chain. In

The bile acids are excreted into the small intestine via the bile

healthy human body fluids, five kinds of bile acids, cholic acidduct as their glycine and taurine conjugates. In the intestinal lumen,(CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA),

bile acids assist in the lipolysis and absorption of fats by forming

ursodeoxycholic acid (UDCA) and lithocholic acid (LCA), are usu-

mixed micelles, and then return to the liver through the ileum−

ally present along with their glycine and taurine conjugates (Fig.

proximal colon. During this enterohepatic circulation, bile acids are1). Among these acids, CA and CDCA are primary bile acids andtransformed into DCA, UDCA, and LCA by the action of intesti-

the others are secondary bile acids.

nal bacteria. This enterohepatic circulation is also responsible forBile acids are the main metabolites of cholesterol and the pri-the low concentration of bile acids in the peripheral blood. In con-mary bile acids are synthesized in the liver by hepatic enzymes.

trast, in hepatobiliary diseases, changes in the synthesis and clear-

The 7 α−hydroxylation of cholesterol is the first and the rate−limit-

ance of bile acids by the liver and absorption by the intestine cause

ing step in bile acid biosynthesis. The 7 α−hydroxycholesterol thenchanges in the level and metabolic profile of bile acids in biologi-

undergoes modification at the A and B rings, and a 3 α,7 α−dihy-

cal fluids. In turn, this leads to increases in the body fluid levels of*To whom corresponding should be addressed:
Department of Pharmaceutical Sciences, Tohoku University Hospital, 1−1 Seiryo−machi, Aoba−ku, Sendai 980−8574, Japan
Tel: 022−717−7525, Fax: 022−717−7545
E−mail: jun−goto@mail.pharm.tohoku.ac.jp or jun−goto@pharm.med.tohoku.ac.jp―１―Chromatography, Vol.25 No.1 (2004)

Figure 1. Structures of common bile acids.

Figure 2. Biosynthesis of primary bile acids.

sulfates, glucuronides, and N−acetylglucosaminides. In addition,

acids in biological fluids. Recently, it has been suggested that LC/

these changes result in the formation of uncommon bile acids con-

MS may be the most promising method for the separation and de-taining a double bond, a carbonyl group, an A/B−trans structure,termination of bile acids, because this technique can be applied to aand hydroxyl groups at the C−4 and 6 positions.

wide variety of bile acids including glucuronides, sulfates, andother conjugates.

For the diagnosis of liver diseases, reliable analytical methods
for the bile acids in biological fluids are needed. For such purposes,

In this article, our work on bio−analytical studies of bile acids

chromatographic procedures combined with or without mass spec-

using GC/MS, pre−column derivatization LC, and LC/MS will betrometry (MS) are preferable. Although a reliable derivatization

briefly reviewed.

procedure to convert bile acids into their corresponding volatile
2. Gas chromatographic and mass spectrometric analysis

forms is needed, gas chromatography (GC) with MS is an effective
technique for the profile analysis of common and uncommon bile

Capillary gas chromatographic separation combined withacids in biological fluids and tissues. Liquid chromatography (LC)mass spectrometric detection is a powerful method for the profile

combined with the use of a pre−column derivatization technique or

analysis of bile acids in biological matrices. Commonly, after hy-

immobilized enzyme can also be applied to the analysis of uncon-

drolysis and/or solvolysis, the liberated bile acids are subjected tojugated, glycine−conjugated, and taurine−conjugated common bile

GC separation as methyl ester−trimethylsilyl (TMS) or dimeth-―２―

Chromatography, Vol.25 No.1 (2004)

Junichi Goto, Nariyasu Mano and Takaaki Goto

Figure 3. Structure of the dimethylethylsilyl ether−pentafluorobenzyl ester derivative of cholic acid.

Figure 4. Synthesis of stable isotope labeled cholic acid.

ylethylsilyl (DMES) ether derivatives and are then introduced into

rived by oxidation of the hydroxyl groups of the corresponding bilea mass spectrometer using an electron impact ionization (EI) mode.

acids are dissolved in H218O and heated at 90 C for 50 hr, followed

Unfortunately, this method has a detection limit of a few pg, and is

by further labeling with 2H by sodium borodeuteride reduction

therefore insufficient to sensitively determine bile acid levels in the(Fig. 4). Using this procedure, the target position can theoreticallyliver tissue. For this purpose, negative ion chemical ionization

be labeled with a stable isotope with high selectivity; therefore, la-(NICI) detection provides much higher sensitivity. Derivatization

beled compounds having high isotopic purity can easily be ob-of the carboxyl group into its pentafluorobenzyl (PFB) ester gener-

tained. For example, the ratios of the unlabeled species to the fully−−

ates characteristic carboxylate anions, [M−PFB] , which facilitateslabeled species in the stable isotope labeled mono−, di−, and tri−

the highly sensitive detection of approximately 2 fg (4 attomole) ofhydroxylated bile acids are about 1/500, 1/2000, and 1/10000, re-bile acids at a signal−to−noise ratio of more than 10 using the NICI

spectively, indicating high applicability to the trace analysis of bilemode (Fig. 3) [1]. This method is 1000 times more sensitive thanacids in tissue, blood, and urine specimens. These stable labeled

EI−MS, and permits the profile analysis of bile acids in the livercompounds can also be applied as tracers for biosynthetic andtissue [2] and the determination of C27 bile acids, which are specificmetabolic studies of bile acids. Indeed, the existence of a redox en-

diagnostic indicators of the cerebro−hepato−renal syndrome, Zell-zyme system for 3−oxo bile acids in human red blood cells [4−7]weger syndrome, in newborn infant urine [3]. The dimethylethyl-and an epimeric enzyme system for the C−25 position of C27 bilesilylation of the hydroxyl group on the steroid nucleus enables theacid−CoA thioester in the hepatic mitochondrial fraction have been

excellent GC resolution of common bile acids on a methylsilicone

identified using such compounds [8−11].

fused silica capillary column.

Uncommon bile acids having a vicinal −glycol structure can

The reversed isotope dilution technique in combination with

be found in biological specimens from patients with liver diseases

mass spectrometric detection provides high accuracy, specificity,and newborn infants. These uncommon bile acids are very difficultand sensitivity for the trace analysis of biologically important ma-

to identify and determine, even using capillary GC/MS or GC/MS−

terials. Although 2H labeled compounds are widely used for thisMS. The unique sililating agent, N, O−bis (diethylhydrogensilyl)

2

purpose, introduction of more than three H atoms into the target

trifluoroacetamide, may be very useful for this purpose. This re-compound with high isotope purity is somewhat difficult. In NICI−

agent reacts readily with an isolated hydroxyl group and a diaxial

MS, the base peaks of PFB esters of acidic compounds contain in-trans−glycol on the steroid nucleus to form the corresponding di-herent oxygen atoms, which originate from the hydroxyl and car-ethylhydrogensilyl (DEHS) ether. In addition, a diequatorial trans−

18boxyl groups. Introduction of an O atom into the hydroxyl groupglycol or an axial−equatorial cis−glycol can easily be transformed

can easily be achieved by an exchange reaction with the carbonyl

into the corresponding cyclic diethylsilylene (DES) derivative (Fig.

oxygen atom [2]. Briefly, the potassium salts of oxo bile acids de-5) [12]. Accordingly, the profile analysis of common and uncom-―３―Chromatography, Vol.25 No.1 (2004)

ity between the 5 β− and 5 α−epimers. Bile acids containing equatorial hydroxyl groups exhibit smaller k values than corresponding
epimers having axial hydroxyl groups. Oxo bile acids show almost
the same k values as the corresponding bile acids which have equatorial hydroxyl groups. In bile acids, the β−face of the steroid nucleus participates in hydrophobic binding to the ODS stationary
phase. Therefore, introduction of a hydroxyl group on the α−facewould alter only the polarity of the molecule and, hence, its solubility in the aqueous mobile phase. On the other hand, the presenceof a β−hydroxyl or an oxo group on the steroid nucleus would reduce the hydrophobic surface area and increase the solubility i themobile phase, resulting in a decrease in the retention value. Because of the intramolecular steric interaction between the 12 α−hy-Figure 5. Derivatization of hydroxyl groups with N, O−bis (diethylhydrogensilyl) trifluoroacetamide.

droxyl group and the 24−carboxyl group in the dissociated form,
the resolution of two isomers, 3 α, 7 α−dihydroxylated CDCA and
3 α, 12 α−dihydroxylated DCA is influenced by the pH of the mo-

mon bile acids with efficient resolution can be achieved on capil-

bile phase. Unconjugated DCA has a larger k value than the corre-lary GC/MS. Moreover, the differences in methylene unit values

sponding CDCA in the higher pH region, whereas both exhibit al-

between TMS and DEHS or DES derivatives imply the number

most identical values in the lower pH region. On the other hand,and nature of the hydroxyl groups on the steroid nucleus of bile ac-

glycine− and taurine−conjugated DCAs exhibit larger k values thanids. This derivatization method with capillary GC/MS has been ap-

the corresponding CDCAs at pH between 4.0−7.5. The 3−sulfates

plied to the identification and determination of a novel 4 β−hy-

and 3−glucuronides of bile acids also show the same chroma-

droxylated bile acid in urine specimens from newborn infants [13−tographic behavior.
LC/MS is a reliable analytical tool for the separation and de-15].

termination of trace compounds in biological fluids. Among numerous interfaces, electrospray ionization (ESI) is more suitable

3. Liquid chromatographic and mass spectrometric analysis
High−performance liquid chromatography (HPLC) is a pow-

for the detection of ionic trace compounds. In the ESI mode, it iserful tool for the separation and determination of polar and ther-recommended that volatile electrolytes, such as ammonium acetate,mally unstable biological compounds. Therefore, this technique ap-

be included in the mobile phase to promote the proton transfer re-pears to be useful for the analysis of bile acids without prior hy-

action. It is generally accepted that the ESI dispersion of a sample

drolysis and/or solvolysis. A chemically bonded stationary phase,

solution from LC would result in the production of droplets con-an ODS column, has been widely used for the separation of bile ac-taining solute molecules. As the solvent evaporated from the drop-

ids and related compounds, and several factors affecting the chro-let, the solvent charge would be retained by the solute (Fig. 6).

matographic behavior of such compounds on the column have been

Therefore, the ionization efficiency of organic acid derivatives isclarified [16, 17]. The k value of an organic acid on an ODS col-markedly influenced by dissociation of electrolytes in a buffer so-

umn is dependent on the acidity of the eluent and can be estimatedlution. The use of buffer solution containing an organic acid havingfrom the pKa value of the organic acid and the pH of the eluent.

a pKa value relatively smaller than that of the acidic moiety in the

The pKa values of bile acids are as follows: 6.0 for unconjugatedtarget compound results in the depression of charge transfer fromacids, 4.5 for the glycine conjugates, and 1.5 for the taurine conju-

droplets to the acidic moiety to produce the corresponding ion [18].

gates. Therefore, in a mobile phase of pH 7.0−8.0, the unconju-

Based on these observations, simultaneous determinations of bile

gated, glycine−conjugated, and taurine−conjugated bile acids ex-acid 3−sulfates and 3−glucuronides [19] in biological fluids by LC/

hibit similar k values. Furthermore, the k values of the unconju-

ESI−MS have been developed.

gated and glycine−conjugated bile acids increase with a decrease in

It has also been demonstrated that ester−type bile acid glucu-

pH from 6.5 and 5.0, respectively. The k value of the taurine conju-

ronides, in which the glucuronic acid group is conjugated throughgate exhibits little change over the whole pH range.

the 24−carboxyl moiety, are biosynthesized as main glucuronides

The effect of hydroxyl groups on the retention value is de-when bile acids are incubated with rat hepatic microsomal glucu-

pendent on the position and increases in the sequence 3−OH = 6−

ronosyltransferases (Fig. 7) [20]. These acyl glucuronides have also

OH < 7−OH < 12−OH. There is no significant difference in mobil-

been identified and determined in human urine by the newly devel-―４―Chromatography, Vol.25 No.1 (2004)

Junichi Goto, Nariyasu Mano and Takaaki Goto

Figure 6. Ionization of the ionic compound under the ESI mode.

Figure 7. Biosynthesis of bile acid ether and ester glucuronides.

oped analytical method employing LC/ESI−MS [21, 22]. The bile

ily controlled by pH and, hence, this functional group can be util-acid acyl glucuronides are readily non−enzymatically reactive withized as a novel reacting group for affinity labeling reagents [27].
As mentioned above, bile acids are synthesized in the liver

proteins through the amino group and produce protein bound bilefrom cholesterol by the action of hepatic enzymes. Recently, theacids, which may cause cytotoxicity and carcinogenesis [23].
It is well known that organic compounds having carboxylic

existence of unconjugated CA, DCA, and CDCA in the rat brainacid groups are transformed into acyl CoA thioesters prior to con-

was reported employing a unique pre−treatment procedure for thejugation with amino acids. It has also been demonstrated that, dur-

brain cytosolic fraction [28]. The concentration of CDCA stands

ing the activation of the carboxylic acid group of bile acid to theout among the three bile acids and is almost 10 times higher thancorresponding acyl CoA thioester, the acyl adenylate is formed in

the concentration of pregnenolone, a very abundant neurosteroid.

microsomal fractions and that acyl CoA synthetase may be respon-

Moreover, CDCA is only detected when high concentrations ofsible for this process (Fig. 8) [24−26]. The acyl adenylate is very

guanidine are added to the extract from the cytosolic fraction (Fig.

reactive with an amino group and was transformed into the amino9), suggesting that the CDCA is tightly bound to protein in theacid conjugate with taurine under a non−enzymatic condition. The

brain. It is not at all clear whether these bile acids are transferred

reactivity of the acyl adenylate toward the amino group can be eas-from the blood through the blood−brain barrier or biosynthesized―５―

Chromatography, Vol.25 No.1 (2004)

Figure 8. Biosynthesis of taurine conjugated cholic acid.

Figure 9. Procedure for the determination of bile acids in the rat brain soluble fraction.

biochemical and its related compounds. An antibody raised against

from cholesterol or related compounds in the brain.

a hapten molecule usually shows significant cross−reactivity withcompounds homologous around the bridge portion used for conju-

4. Immunoaffinity extraction employing highly specific anti-

gation with a carrier protein, such as bovine serum albumin. Ac-

bodiesAlthough chromatographic separation combined with mass

cordingly, an antibody should be prepared by immunization with

spectrometric detection is a powerful tool for the determination of

the corresponding antigen, where the hapten is conjugated to a car-trace compounds in biological matrices, tedious clean−up proce-rier protein through a position far from the discriminating structuredures are sometimes required just to remove interfering co−existingof the target compounds. For example, bufuralol, a potent non−se-

substances. However, LC alone is not necessarily effective for thelective β−adrenoseptor antagonist with β2 partial agonist properties,

simultaneous separation of racemates and their metabolites con-

has an asymmetric center at the C−1 position and mainly under-

taining plural asymmetric centers. These problems may be solved

goes aliphatic oxidation at C−1’, generating a new chiral center andby the use of immunoaffinity extraction, based on specific interac-yielding 1’−hydroxylated bufuralols (carbinols), which are further

tions between a matrix−bound antibody and soluble biochemicals

transformed into 1’−ketone derivatives (Fig. 10). To discriminate[29−34]. In this technique, the simultaneous extraction of biologi-

the C−1 position, (1 R )− and (1 S )−1’−oxobufurarols are trans-

cally important substances and their related compounds is at-formed into O−carboxymethyloxime derivatives, condensed withtempted employing an immunosorbent prepared by immobilization

BSA, and subcutaneously administered to rabbits with completeof an antibody having a broad affinity spectrum for both a parent

Freund’s adjuvant. The resulting anti−(1 R )−bufurarol antibody―６―

Chromatography, Vol.25 No.1 (2004)

Junichi Goto, Nariyasu Mano and Takaaki Goto

Figure 10. Metabolism of bufuralol.

shows high affinity to (1 R )−bufuralol and its 1’−oxygenated me-

Nambara (Tohoku University), Professor Shigeo Ikegawa (Kinki

tabolites. However, the reactivity toward the corresponding (1 S )−University), and Professor Norihiro Kobayashi (Kobe Pharmaceuti-

antipodes is significantly lower. A similar phenomenon is observed

cal University) for their valuable suggestions. The authors also

for the anti−(1 S )−bufuralol antibody. When these antibodies arethank their many co−workers for their assistance and discussions.

covalently coupled to agarose, the corresponding antipodes can be
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