Determination of bile acids in human serum by on-line restricted access material–ultra high-performance liquid chromatography–mass spectrometry

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Abstract

This paper describes a new, fully automated on-line method combining restricted access material (RAM) extraction and ultra high-performance liquid chromatography (UHPLC) with mass spectrometric (MS) detection for determining congeners of bile acids (BAs) in human serum. In this method, low-pressure RAM and high-pressure UHPLC–MS are hyphenated by using a 2.5-mL loop-type interface. The compatibility problem between the large volume (1.2 mL) of strong solvent (methanol) used for RAM elution and the need for a weak solvent in UHPLC injection has been addressed by using an auxiliary pre-column cross-flow of 0.1% aqueous formic acid. In this way, the complete 2.5 mL loop volume can be injected into the UHPLC system, thereby maximizing sensitivity while maintaining good chromatographic performance. The optimised method allows the simultaneous analysis of 13 bile acids in a single run, including glycine- and taurine-conjugated bile acids, cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), and litocholic acid. The complete analysis of a 100-μL single serum sample is performed in 30 min, providing detection limits in the pg range (corresponding with clinically relevant concentration levels) for all of the analytes except lithocholic acid, intra-day precision values (%R.S.D.) below 4% (except ursodeoxycholic acid) and inter-day precision lower than 15% (except ursodeoxycholic, glycoursodeoxycholic acid (GUDCA) and lithocholic acid).

Introduction

Bile acids (BAs) are produced in the liver and, as the main components of bile, they are involved in several processes, including cholesterol homeostasis, absorption of dietary lipids and fat-soluble vitamins by formation of micelles, and both the excretion and recirculation of drugs and toxins. In total, about 20–30 g of bile acids are excreted daily into the small intestine via the bile duct and 95% are reabsorbed and recycled, so concentrations in the ng/g range are found in peripheral blood. During this recirculation, called enterohepatic circulation, BAs undergo multiple metabolic reactions. BAs produced by direct biosynthesis in the liver, called primary bile acids, are cholic acid (CA) and chenodeoxycholic acid (CDCA), whereas those produced by microbial flora are called secondary bile acids. These latter are less soluble in water and, therefore, toxic to cells. BAs are synthesized from cholesterol, and their major structural components are a steroid nucleus and a sidechain. In hepatocytes, BAs undergo amino acid conjugation at the sidechain with glycine, taurine or (less frequently), sulphate or glucuronide, which increases their water solubility.

The composition of bile and the concentrations of BAs in biological fluids, such as serum or urine, can provide indications regarding an individual's metabolism, and potentially useful information about hepatobiliary and intestinal disease states. In addition, since BAs play an important role in the elimination of cholesterol from the body, unusually low or high concentrations could be directly related to various hyper- or hypo-cholesterolemia diseases. Furthermore, since BAs have therapeutic applications, e.g. CDCA and ursodeoxycholic acid (UDCA) are used to dissolve cholesterol gallstones, their determination in serum provides a useful means for monitoring therapy progress.

Several analytical methods have been reported for the determination of BAs in serum, based on liquid chromatography (LC) with UV detection [1], [2], [3] or LC coupled to evaporative light scattering detection (ELSD) [4], [5] but they have limited sensitivity and are prone to interference by various components of biological interferences. Moreover, since BAs do not absorb UV radiation significantly, pre-column labelling is required to allow their detection by UV and fluorescence detection systems [6]. These problems can be overcome by using mass spectrometric (MS) detection. However, GC–MS analysis requires several steps – including preliminary group fractionation, hydrolysis of conjugates and preparation of volatile derivatives [7], [8], [9], [10] – which are labour intensive and time consuming. Therefore, LC–MS is the most suitable technique for determining BAs in serum because no derivatization steps are needed and it provides appropriate sensitivity [2], [9], [11], [12], [13], [14], [15], [16], [17] Prior to LC–MS, samples must be pre-treated, in order (inter alia) to remove proteins from them to avoid the system clogging and/or interfering substances affecting the detection. Serum samples are often cleaned by solid phase extraction (SPE), usually using C18 or anion-exchange cartridges [1], [2], [3], [4], [9], [11], [12], [13], [17]. The determination of BAs in serum, in clinical applications, requires analytical methods that are capable of handling very low volume samples while providing large sample throughputs. These requirements can be addressed using on-line methods but, to the best of our knowledge, no such method including LC–MS determination has previously been reported in the scientific literature for analysing BAs in serum.

Restricted access material (RAM) are being increasingly often used in biological applications since they have useful combinations of size exclusion and in-pore reversed stationary phase properties and, furthermore, they can be conveniently packed in cartridges and used in on-line systems [18], [19], [20]. There are clear attractions for clinical applications in coupling such cartridges to ultra high-performance liquid chromatography (UHPLC) systems, in which stationary phases with smaller particles are used than those in “traditional” high-performance LC, providing faster run times and, hence, higher sample throughputs. However, direct hyphenation of UHPLC with any sample pre-treatment procedure is highly challenging, and no such coupled systems appear to have been previously described in the scientific literature. There are two main reasons for this. First, UHPLC has extreme pressure (up to 1000 bars) and low flow (usually less of 0.6 mL/min) requirements, which are not directly compatible with typical on-line extraction methods and, secondly, the large amounts (several mL) of strong solvent (typically methanol) needed for SPE or RAM elution cannot be directly introduced into UHPLC systems while maintaining retention and peak shape.

Therefore, this contribution has several aims. First, to design and test an automated on-line setup that can be used to hyphenate extraction (RAM, low-pressure) and separation-detection (UHPLC–MS, high-pressure) steps and to make compatible the respective flow requirements in terms of total volume, flow and solvent identity. Then, to use the developed setup to analyse bile acids in human serum in order to introduce an on-line method able to fulfil the analytical requirements (mainly sensitivity and sample throughput) needed for clinical analysis.

Section snippets

Chemicals and reagents

The following bile acids were included in the study: glycodeoxycholic acid (GDCA, purity 97%, Chemical Abstract Service identification number: 360-65-6), taurocholic acid (TCA, 97%, 345909-26-4), cholic acid (CA, 98%, 81-25-4), chenodeoxycholic acid (CDCA, 98%, 474-25-9), deoxycholic acid (DCA, 99%, 83-44-3), lithocholic acid (LCA, 99%, 434-13-9), glycocholic acid (GCA, 98% dried, 475-31-0), glycochenodeoxycholic acid (GCDA, 97%, 16564-43-5), taurochenodeoxycholic acid (TCDA, 95%, 6009-98-9),

On-line setup

As explained in the Introduction, one of the main aims of the work presented here was to develop an on-line analytical method to increase sample throughput. To achieve this goal, the sample pre-treatment steps had to be reduced. The use of a RAM cartridge allows the injection of serum samples into the system with very little pre-treatment, but it is highly recommended to remove cryoproteins and particulate impurities in order to prevent damage to the cartridge and clogging. Removal is performed

Conclusions

An on-line RAM–UHPLC–MS method has been developed for determining BAs in serum with reduced sample pre-treatment steps, allowing the complete processing of a single sample in 30 min, which could be further shortened by using a simultaneous sample treatment strategy.

The proposed method allows a low-pressure RAM extraction step to be coupled to an UHPLC–MS separation and detection step, by using a loop-type interface to collect the eluting flow and add a cross-flow to reduce the elution strength

Acknowledgements

This work was financed by funds awarded for the project PM-DGA-096-2006 and the GUIA Group T-10 from the Regional Government of Aragón. K. Bentayeb and R. Batlle gratefully acknowledge the Cuenca Villoro Patronato and the former Spanish Ministry of Science and Technology for personal funding. We are also very grateful to Dr. A.L. Otín and the Lipid Group from the University of Zaragoza for medical and clinical advice, and for providing samples.

References (21)

  • B.L. Lee et al.

    J. Chromatogr. B

    (1997)
  • M. Nobilis et al.

    J. Pharm. Biomed. Anal.

    (2001)
  • A. Criado et al.

    J. Chromatogr. B

    (2003)
  • A. Roda et al.

    J. Lipid Res.

    (1992)
  • M. Suzuki et al.

    J. Chromatogr.

    (1997)
  • A.K. Batta et al.

    J. Chromatogr. B

    (2002)
  • S. Perwaiz et al.

    J. Lipid Res.

    (2001)
  • T. Murai et al.

    J. Chromatogr. B

    (1997)
  • A. Roda et al.

    J. Chromatogr. B

    (1995)
  • E. Tessier et al.

    J. Chromatogr. B

    (2003)
There are more references available in the full text version of this article.

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